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Sample records for antigens reflect protection

  1. Demonstration of protective antigen carried by flagella of Clostridium chauvoei.

    PubMed

    Tamura, Y; Minamoto, N; Tanaka, S

    1984-01-01

    The protective antigen present on the flagella of Clostridium chauvoei was studied by the mouse protection test. A partially purified flagella preparation (PPF) showed protective antigenicity after two intraperitoneal injections of 2 micrograms as protein, while the protective antigenicity of nonflagellated mutants (NFM) was 100-fold less than that of the flagellated parent strain. Although the protective effect of antisera against the whole cells and PPF, in terms of ED50 values, was mostly lost after absorption with the parent strain, that of antisera after absorption with NFMs showed no appreciable loss. These results suggest that the flagella of Cl. chauvoei play some role in inducing protective immunity in mice.

  2. Protective cellular antigen of Clostridium chauvoei.

    PubMed

    Stevenson, J R; Stonger, K A

    1980-04-01

    Cellular antigens of Clostridium chauvoei, strain IRP-128, were demonstrated to be important in induction of immunity against this bacterium in guinea pigs. At least one major component of the cellular antigen complex was heat-labile. Acid extraction of the bacterial cells, followed by selective purification for flagella, led to the preparation of an acid extract antigen that possessed a high degree of immunogenicity. The acid extract antigen contained flagellar components and was resolved into two major and approximately five minor protein components by polyacrylamide-gel electrophoresis.

  3. Atomic structure of anthrax protective antigen pore elucidates toxin translocation.

    PubMed

    Jiang, Jiansen; Pentelute, Bradley L; Collier, R John; Zhou, Z Hong

    2015-05-28

    Anthrax toxin, comprising protective antigen, lethal factor, and oedema factor, is the major virulence factor of Bacillus anthracis, an agent that causes high mortality in humans and animals. Protective antigen forms oligomeric prepores that undergo conversion to membrane-spanning pores by endosomal acidification, and these pores translocate the enzymes lethal factor and oedema factor into the cytosol of target cells. Protective antigen is not only a vaccine component and therapeutic target for anthrax infections but also an excellent model system for understanding the mechanism of protein translocation. On the basis of biochemical and electrophysiological results, researchers have proposed that a phi (Φ)-clamp composed of phenylalanine (Phe)427 residues of protective antigen catalyses protein translocation via a charge-state-dependent Brownian ratchet. Although atomic structures of protective antigen prepores are available, how protective antigen senses low pH, converts to active pore, and translocates lethal factor and oedema factor are not well defined without an atomic model of its pore. Here, by cryo-electron microscopy with direct electron counting, we determine the protective antigen pore structure at 2.9-Å resolution. The structure reveals the long-sought-after catalytic Φ-clamp and the membrane-spanning translocation channel, and supports the Brownian ratchet model for protein translocation. Comparisons of four structures reveal conformational changes in prepore to pore conversion that support a multi-step mechanism by which low pH is sensed and the membrane-spanning channel is formed.

  4. Application of paramagnetic beads for purifying Bacillus anthracis protective antigen.

    PubMed

    Zarzecka, A; Bartoszcze, M

    2006-10-01

    Paramagnetic beads coated with Protein G and Tosylactivated-280 dynabeads have been used to purify Bacillus anthracis protective antigen from a liquid culture. The obtained protein was used in the enzyme-linked immunosorbent assay test to detect B. anthracis protective antigen antibodies in human sera collected from immunized individuals. The purification method using paramagnetic beads is very effective. It is fast, easy and may be carried out practically in any laboratory.

  5. Leishmania pifanoi amastigote antigens protect mice against cutaneous leishmaniasis.

    PubMed Central

    Soong, L; Duboise, S M; Kima, P; McMahon-Pratt, D

    1995-01-01

    In the search for a leishmaniasis vaccine, extensive studies have been carried out with promastigote (insect stage) molecules. Information in this regard on amastigote (mammalian host stage) molecules is limited. To investigate host immune responses to Leishmania amastigote antigens, we purified three stage-specific antigens (A2, P4, and P8) from in vitro-cultivated amastigotes of Leishmania pifanoi by using immunoaffinity chromatography. We found that with Corynebacterium parvum as an adjuvant, three intraperitoneal injections of 5 micrograms of P4 or P8 antigen provided partial to complete protection of BALB/c mice challenged with 10(5) to 10(7) L. pifanoi promastigotes. These immunized mice developed significantly smaller or no lesions and exhibited a 39- to 1.6 x 10(5)-fold reduction of lesion parasite burden after 15 to 20 weeks of infection. In addition, P8 immunization resulted in complete protection against L. amazonensis infection of CBA/J mice and partial protection of BALB/c mice, suggesting that this antigen provided cross-species protection of mice with different H-2 haplotypes. At different stages during infection, vaccinated mice exhibited profound proliferative responses to parasite antigens and increased levels of gamma interferon production, suggesting that a Th1 cell-mediated immune response is associated with the resistance in these mice. Taken together, the data in this report indicate the vaccine potential of amastigote-derived antigens. PMID:7642292

  6. Dendritic Cell Targeting of Bacillus anthracis Protective Antigen Expressed by Lactobacillus acidophilus Protects Mice from Lethal Challenge

    DTIC Science & Technology

    2008-10-28

    Dendritic cell targeting of Bacillus anthracis protective antigen expressed by Lactobacillus acidophilus protects mice from lethal challenge M...lethal chal- lenge. A vaccine strategy was established by using Lactobacillus acidophilus to deliver Bacillus anthracis protective antigen (PA) via...4. TITLE AND SUBTITLE Dendritic cell targeting of Bacillus anthracis protective antigen expressed by Lactobacillus acidophilus protects mice

  7. Partial purification of protective antigens from Nippostrongylus brasiliensis in mice.

    PubMed

    Rhalem, A; Bourdieu, C; Luffau, G; Pery, P

    1988-01-01

    The purification of antigens from Nippostrongylus brasiliensis, through their ability to provoke cellular proliferation of immune cells and through their recognition by antibodies, led to an antigenic preparation which was extracted from adult worms and which contained only two proteins (MW 14 and 43 Kd). Mice which were vaccinated by the oral route after the entrapment of these two proteins in liposomes were strongly protected.

  8. Expression of Bacillus anthracis Protective Antigen in Bacillus megaterium

    DTIC Science & Technology

    2004-03-01

    protective antigen in Bacillus megaterium B.J. Berger, K.E. Schwandt and C.L. Radford Defence R&D Canada - Suffield Technical Memorandum DISTRIBUTION...antigen in Bacillus megaterium B. J. Berger, K. E. Schwandt, and C. L. Radford Defence R&D Canada - Suffield Defence R&D Canada - Suffield Technical...expressed using Bacillus megaterium and a xylose-inducible heterologous expression system. After only 3.5 hours growth post-induction in Luria

  9. Antigen-binding site protection during radiolabeling leads to a higher immunoreactive fraction

    SciTech Connect

    Van den Abbeele, A.D.; Aaronson, R.A.; Daher, S.; Taube, R.A.; Adelstein, S.J.; Kassis, A.I. )

    1991-01-01

    It is generally accepted that the immunointegrity of an antibody (Ab) depends on the preservation of its antigen-binding sites. Our goal was to radiolabel an antibody at several iodine:antibody molar ratios under conditions protecting its combining site and to compare its immunoreactive fraction (IRF) and electrophoretic mobility with those of the same antibody radiolabeled without protection. The data indicate that an antibody radiolabeled while its antigen-binding site is occupied by its antigen had the same IRF, regardless of the number of iodine atoms per antibody molecule. On the other hand, even at an I:Ab ratio of 1:1, the IRF of the same antibody radiolabeled without protection was lower than that of a protected one and decreased with increasing I:Ab ratios. In addition, the iodination of these Ab changes their electrophoretic mobility; however, when the Ab is labeled in the protected state, the degree of change is less. The binding of an antibody to its antigen prior to radiolabeling, therefore, enhances its immuno-integrity and prevents major conformational changes as reflected by electrophoresis.

  10. F41 pili as protective antigens of enterotoxigenic Escherichia coli that produce F41, K99, or both pilus antigens.

    PubMed Central

    Runnels, P L; Moseley, S L; Moon, H W

    1987-01-01

    Pigs suckling dams that have been vaccinated with pilus antigen are protected against challenge with enterotoxigenic Escherichia coli (ETEC) strains that express the same pilus antigen. However, some ETEC strains express more than one pilus antigen. Pregnant swine were vaccinated either with E. coli HB101 that harbored a recombinant plasmid coding for F41 expression (F41+) or with the HB101 parent strain that carries the pHC79 vector (F41-). Suckling pigs born to vaccinated dams were challenged with ETEC that expressed either K99, F41, or both pilus antigens. Production of F41 in vivo was demonstrated by immunofluorescence assay of sections of ileum and by seroconversion against F41 antigen by pigs challenged with F41+ and K99+ F41+ ETEC strains. The F41+ vaccine protected against challenge with an F41+ ETEC strain. In contrast, F41+ vaccination did not protect against challenge with K99+ or K99+ F41+ ETEC strains. The F41- vaccine did not protect against challenge with any strain used. The results indicate that K99+ F41+ ETEC strains produce F41 antigen in the small intestine during disease and that F41+ vaccination can be a protective antigen if the challenge strain expresses only F41 antigen, but that F41+ vaccination may not protect against strains that produce both K99 and F41 antigens. PMID:2880807

  11. Genetic and antigenic diversity of the surface protective antigen proteins of Erysipelothrix rhusiopathiae.

    PubMed

    To, Ho; Nagai, Shinya

    2007-07-01

    The surface protective antigen (Spa) protein of Erysipelothrix rhusiopathiae has been shown to be highly immunogenic and is a potential candidate for a new vaccine against erysipelas. In this study, we cloned and sequenced spa genes from all E. rhusiopathiae serovar reference strains as well as from a serovar 18 strain which was not classified as any species in the genus Erysipelothrix. Sequence analysis revealed that the Spa proteins could be classified into three molecular species, including SpaA, which was previously found in serovars 1a and 2, and the newly designated SpaB and SpaC proteins. The SpaA protein is produced by E. rhusiopathiae serovars 1a, 1b, 2, 5, 8, 9, 12, 15, 16, 17, and N, the SpaB protein is produced by E. rhusiopathiae serovars 4, 6, 11, 19, and 21, and the SpaC protein is produced only by serovar 18. The amino acid sequence similarity was high among members of each Spa type (96 to 99%) but low between different Spa types ( approximately 60%). The greatest diversity in Spa proteins was found in the N-terminal half of the molecule (50 to 57% similarity), which was shown to be involved in immunoprotection. Coinciding with this, immunoblot analysis revealed that rabbit antisera specific to each Spa reacted strongly with the homologous Spa protein but weakly with heterologous Spa proteins. A mouse cross-protection study showed that the three recombinant Spa (rSpa) proteins elicited complete protection against challenge with homologous strains but that the level of protection against challenge with heterologous strains varied depending on the rSpa protein used for immunization. Our study is the first to demonstrate sequence and antigenic diversity in Spa proteins and to indicate that rSpaC may be the most promising antigen for use as a vaccine component because of its broad cross-protectiveness.

  12. Rabies virus glycoprotein as a carrier for anthrax protective antigen

    SciTech Connect

    Smith, Mary Ellen; Koser, Martin; Xiao Sa; Siler, Catherine; McGettigan, James P.; Calkins, Catherine; Pomerantz, Roger J.; Dietzschold, Bernhard; Schnell, Matthias J. . E-mail: matthias.schnell@jefferson.edu

    2006-09-30

    Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen was also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems.

  13. Duration of Protection of Rabbits after Vaccination with Bacillus anthracis Recombinant Protective Antigen Vaccine

    DTIC Science & Technology

    2005-12-27

    against an aerosol spore challenge with the Ames isolate of Bacillus anthracis at 6 and 12 months. At 6 months after the primary injection, survival...vaccine was examined against an aerosol spore challenge with the Ames isolate of Bacillus anthracis at 6 and 12 months. At 6 months after the...Vaccine 24 (2006) 2530–2536 Duration of protection of rabbits after vaccination with Bacillus anthracis recombinant protective antigen vaccine S.F

  14. Duration of Protection of Rabbits after Vaccination with Bacillus anthracis Recombinant Protective Antigen Vaccine

    DTIC Science & Technology

    2005-12-13

    against an aerosol spore challenge with the Ames isolate of Bacillus anthracis at 6 and 12 months. At 6 months after the primary injection, survival...rPA) vaccine was examined against an aerosol spore challenge with the Ames isolate of Bacillus anthracis at 6 and 12 months. At 6 months after the...Vaccine 24 (2006) 2530–2536 Duration of protection of rabbits after vaccination with Bacillus anthracis recombinant protective antigen vaccine S.F

  15. Protective antibody titres and antigenic competition in multivalent Dichelobacter nodosus fimbrial vaccines using characterised rDNA antigens.

    PubMed

    Raadsma, H W; O'Meara, T J; Egerton, J R; Lehrbach, P R; Schwartzkoff, C L

    1994-03-01

    The relationship between K-agglutination antibody titres and protection against experimental challenge with Dichelobacter nodosus, the effect of increasing the number of D. nodosus fimbrial antigens, and the importance of the nature of additional antigens in multivalent vaccines on antibody response and protection against experimental challenge with D. nodosus were examined in Merino sheep. A total of 204 Merino sheep were allocated to one of 12 groups, and vaccinated with preparations containing a variable number of rDNA D. nodosus fimbrial antigens. The most complex vaccine contained ten fimbrial antigens from all major D. nodosus serogroups, while the least complex contained a single fimbrial antigen. In addition to D. nodosus fimbrial antigens, other bacterial rDNA fimbrial antigens (Moraxella bovis Da12d and Escherichia coli K99), and bovine serum albumin (BSA) were used in some vaccines. Antibody titres to fimbrial antigens and BSA were measured by agglutination and ELISA tests, respectively. Antibody titres were determined on five occasions (Weeks 0, 3, 6, 8, and 11 after primary vaccination). All sheep were exposed to an experimental challenge with virulent isolates of D. nodosus from either serogroup A or B, 8 weeks after primary vaccination. For D. nodosus K-agglutinating antibody titres, a strong negative correlation between antibody titre and footrot lesion score was observed. This relationship was influenced by the virulence of the challenge strain. Increasing the number of fimbrial antigens in experimental rDNA D. nodosus fimbrial vaccines resulted in a linear decrease in K-agglutinating antibody titres to individual D. nodosus serogroups. Similarly, a linear decrease in protection to challenge with homologous serogroups was observed as the number of D. nodosus fimbrial antigens represented in the vaccine increased. The reduction in antibody titres in multicomponent vaccines is thought to be due to antigenic competition. The level of competition

  16. A Novel Protective Vaccine Antigen from the Core Escherichia coli Genome.

    PubMed

    Moriel, Danilo G; Tan, Lendl; Goh, Kelvin G K; Phan, Minh-Duy; Ipe, Deepak S; Lo, Alvin W; Peters, Kate M; Ulett, Glen C; Beatson, Scott A; Schembri, Mark A

    2016-01-01

    Escherichia coli is a versatile pathogen capable of causing intestinal and extraintestinal infections that result in a huge burden of global human disease. The diversity of E. coli is reflected by its multiple different pathotypes and mosaic genome composition. E. coli strains are also a major driver of antibiotic resistance, emphasizing the urgent need for new treatment and prevention measures. Here, we used a large data set comprising 1,700 draft and complete genomes to define the core and accessory genome of E. coli and demonstrated the overlapping relationship between strains from different pathotypes. In combination with proteomic investigation, this analysis revealed core genes that encode surface-exposed or secreted proteins that represent potential broad-coverage vaccine antigens. One of these antigens, YncE, was characterized as a conserved immunogenic antigen able to protect against acute systemic infection in mice after vaccination. Overall, this work provides a genomic blueprint for future analyses of conserved and accessory E. coli genes. The work also identified YncE as a novel antigen that could be exploited in the development of a vaccine against all pathogenic E. coli strains-an important direction given the high global incidence of infections caused by multidrug-resistant strains for which there are few effective antibiotics. IMPORTANCEE. coli is a multifaceted pathogen of major significance to global human health and an important contributor to increasing antibiotic resistance. Given the paucity of therapies still effective against multidrug-resistant pathogenic E. coli strains, novel treatment and prevention strategies are urgently required. In this study, we defined the core and accessory components of the E. coli genome by examining a large collection of draft and completely sequenced strains available from public databases. This data set was mined by employing a reverse-vaccinology approach in combination with proteomics to identify

  17. A Novel Protective Vaccine Antigen from the Core Escherichia coli Genome

    PubMed Central

    Moriel, Danilo G.; Tan, Lendl; Goh, Kelvin G. K.; Ipe, Deepak S.; Lo, Alvin W.; Peters, Kate M.

    2016-01-01

    ABSTRACT Escherichia coli is a versatile pathogen capable of causing intestinal and extraintestinal infections that result in a huge burden of global human disease. The diversity of E. coli is reflected by its multiple different pathotypes and mosaic genome composition. E. coli strains are also a major driver of antibiotic resistance, emphasizing the urgent need for new treatment and prevention measures. Here, we used a large data set comprising 1,700 draft and complete genomes to define the core and accessory genome of E. coli and demonstrated the overlapping relationship between strains from different pathotypes. In combination with proteomic investigation, this analysis revealed core genes that encode surface-exposed or secreted proteins that represent potential broad-coverage vaccine antigens. One of these antigens, YncE, was characterized as a conserved immunogenic antigen able to protect against acute systemic infection in mice after vaccination. Overall, this work provides a genomic blueprint for future analyses of conserved and accessory E. coli genes. The work also identified YncE as a novel antigen that could be exploited in the development of a vaccine against all pathogenic E. coli strains—an important direction given the high global incidence of infections caused by multidrug-resistant strains for which there are few effective antibiotics. IMPORTANCE E. coli is a multifaceted pathogen of major significance to global human health and an important contributor to increasing antibiotic resistance. Given the paucity of therapies still effective against multidrug-resistant pathogenic E. coli strains, novel treatment and prevention strategies are urgently required. In this study, we defined the core and accessory components of the E. coli genome by examining a large collection of draft and completely sequenced strains available from public databases. This data set was mined by employing a reverse-vaccinology approach in combination with proteomics

  18. Antigenic analysis of Clostridium chauvoei flagella with protective and non-protective monoclonal antibodies.

    PubMed

    Tamura, Y; Kijima, M; Ohishi, K; Takahashi, T; Suzuki, S; Nakamura, M

    1992-03-01

    Five monoclonal antibodies (mAbs) directed against the flagellin of Clostridium chauvoei were used to analyse the structural and antigenic characteristics on the bacterial flagellar surface. Immune electron microscopy showed that three protective mAbs recognized the surfaced-exposed epitopes on the flagellar filament of this bacteria. In contrast, two non-protective mAbs recognized internal epitopes of the flagellar filament. These findings have been confirmed by ELISA using mAbs absorbed with whole cells of C. chauvoei possessing flagella. Competitive binding assays showed that protective mAbs indicated reciprocal competition, while each of the non-protective mAbs had topographically distinct epitopes. Moreover, immunoblotting analysis with cyanogen-bromide-cleaved flagellin showed that protective mAbs may preferentially recognize conformational epitopes, whilst one of the non-protective mAbs may recognize a linear and conformation-independent epitope in the flagellin of C. chauvoei.

  19. Antigenic analysis of divergent genotypes human Enterovirus 71 viruses by a panel of neutralizing monoclonal antibodies: current genotyping of EV71 does not reflect their antigenicity.

    PubMed

    Chen, Yixin; Li, Chuan; He, Delei; Cheng, Tong; Ge, Shengxiang; Shih, James Wai-Kuo; Zhao, Qinjian; Chen, Pei-Jer; Zhang, Jun; Xia, Ningshao

    2013-01-02

    In recent year, Enterovirus 71 (EV71)-associated hand, foot and mouth disease (HFMD) has become an important public health issue in China. EV71 has been classified into genotypes A, B1-B5 and C1-C5. With such genetic diversity, whether the convalescent or recovery antibody responses can cross-protect infections from other genotypes remains a question. Understanding of the antigenicity of such diverse genetic EV71 isolates is crucial for the EV71 vaccine development. Here, a total of 186 clones anti-EV71 MAbs was generated and characterized with Western blot and cell-based neutralization assay. Forty neutralizing anti-EV71 MAbs were further used to analyze the antigenic properties of 18 recent EV71 isolates representing seven genotypes in neutralization assay. We found that most neutralizing anti-EV71 MAbs are specific to conformational epitopes. We also classified the 40 neutralizing anti-EV71 MAbs into two classes according to their reactivity patterns with 18 EV71 isolates. Class I MAb can neutralize all isolates, suggesting conserved epitopes are present among EV71. Class II MAb includes four subclasses (IIa-IId) and neutralizes only subgroups of EV71 strains. Conversely, 18 EV71 strains were grouped into antigenic types 1 and four antigenic subtypes (2.1-2.4). These results suggest that the current genotyping of EV71 does not reflect their antigenicity which may be important in the selection of EV71 vaccine strains. This panel of neutralizing anti-EV71 MAbs may be useful for the recognition of emerging antigenic variants of EV71 and vaccine development.

  20. Advax-Adjuvanted Recombinant Protective Antigen Provides Protection against Inhalational Anthrax That Is Further Enhanced by Addition of Murabutide Adjuvant

    PubMed Central

    Feinen, Brandon; Petrovsky, Nikolai; Verma, Anita

    2014-01-01

    Subunit vaccines against anthrax based on recombinant protective antigen (PA) potentially offer more consistent and less reactogenic anthrax vaccines but require adjuvants to achieve optimal immunogenicity. This study sought to determine in a murine model of pulmonary anthrax infection whether the polysaccharide adjuvant Advax or the innate immune adjuvant murabutide alone or together could enhance PA immunogenicity by comparison to an alum adjuvant. A single immunization with PA plus Advax adjuvant afforded significantly greater protection against aerosolized Bacillus anthracis Sterne strain 7702 than three immunizations with PA alone. Murabutide had a weaker adjuvant effect than Advax when used alone, but when murabutide was formulated together with Advax, an additive effect on immunogenicity and protection was observed, with complete protection after just two doses. The combined adjuvant formulation stimulated a robust, long-lasting B-cell memory response that protected mice against an aerosol challenge 18 months postimmunization with acceleration of the kinetics of the anamnestic IgG response to B. anthracis as reflected by ∼4-fold-higher anti-PA IgG titers by day 2 postchallenge versus mice that received PA with Alhydrogel. In addition, the combination of Advax plus murabutide induced approximately 3-fold-less inflammation than Alhydrogel as measured by in vivo imaging of cathepsin cleavage resulting from injection of ProSense 750. Thus, the combination of Advax and murabutide provided enhanced protection against inhalational anthrax with reduced localized inflammation, making this a promising next-generation anthrax vaccine adjuvanting strategy. PMID:24554695

  1. Advax-adjuvanted recombinant protective antigen provides protection against inhalational anthrax that is further enhanced by addition of murabutide adjuvant.

    PubMed

    Feinen, Brandon; Petrovsky, Nikolai; Verma, Anita; Merkel, Tod J

    2014-04-01

    Subunit vaccines against anthrax based on recombinant protective antigen (PA) potentially offer more consistent and less reactogenic anthrax vaccines but require adjuvants to achieve optimal immunogenicity. This study sought to determine in a murine model of pulmonary anthrax infection whether the polysaccharide adjuvant Advax or the innate immune adjuvant murabutide alone or together could enhance PA immunogenicity by comparison to an alum adjuvant. A single immunization with PA plus Advax adjuvant afforded significantly greater protection against aerosolized Bacillus anthracis Sterne strain 7702 than three immunizations with PA alone. Murabutide had a weaker adjuvant effect than Advax when used alone, but when murabutide was formulated together with Advax, an additive effect on immunogenicity and protection was observed, with complete protection after just two doses. The combined adjuvant formulation stimulated a robust, long-lasting B-cell memory response that protected mice against an aerosol challenge 18 months postimmunization with acceleration of the kinetics of the anamnestic IgG response to B. anthracis as reflected by ∼4-fold-higher anti-PA IgG titers by day 2 postchallenge versus mice that received PA with Alhydrogel. In addition, the combination of Advax plus murabutide induced approximately 3-fold-less inflammation than Alhydrogel as measured by in vivo imaging of cathepsin cleavage resulting from injection of ProSense 750. Thus, the combination of Advax and murabutide provided enhanced protection against inhalational anthrax with reduced localized inflammation, making this a promising next-generation anthrax vaccine adjuvanting strategy.

  2. Oral Vaccination Against Anthrax Using a Transgenic Plant Expressing Protective Antigen.

    DTIC Science & Technology

    1996-09-01

    Plant Expressing Protective Antigen DAMDI7-95-C-5102 6 . AUTHOR(S) Dr. Karen Oishi 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING...Bacillus anthracis protective antigen (PA), the Yersiniapestis V antigen modified to contain a His 6 epitope to facilitate affinity purification (His-Tag V...these reasons it has been very difficult to 6 develop more effective vaccines which will stimulate mucosal immunity. With the majority of vaccines

  3. Generation and Characterization of Human Monoclonal Antibodies Targeting Anthrax Protective Antigen following Vaccination with a Recombinant Protective Antigen Vaccine.

    PubMed

    Chi, Xiangyang; Li, Jianmin; Liu, Weicen; Wang, Xiaolin; Yin, Kexin; Liu, Ju; Zai, Xiaodong; Li, Liangliang; Song, Xiaohong; Zhang, Jun; Zhang, Xiaopeng; Yin, Ying; Fu, Ling; Xu, Junjie; Yu, Changming; Chen, Wei

    2015-05-01

    The anthrax protective antigen (PA) is the central component of the three-part anthrax toxin, and it is the primary immunogenic component in the approved AVA anthrax vaccine and the "next-generation" recombinant PA (rPA) anthrax vaccines. Animal models have indicated that PA-specific antibodies (AB) are sufficient to protect against infection with Bacillus anthracis. In this study, we investigated the PA domain specificity, affinity, mechanisms of neutralization, and synergistic effects of PA-specific antibodies from a single donor following vaccination with the rPA vaccine. Antibody-secreting cells were isolated 7 days after the donor received a boost vaccination, and 34 fully human monoclonal antibodies (hMAb) were identified. Clones 8H6, 4A3, and 22F1 were able to neutralize lethal toxin (LeTx) both in vitro and in vivo. Clone 8H6 neutralized LeTx by preventing furin cleavage of PA in a dose-dependent manner. Clone 4A3 enhanced degradation of nicked PA, thereby interfering with PA oligomerization. The mechanism of 22F1 is still unclear. A fourth clone, 2A6, that was protective only in vitro was found to be neutralizing in vivo in combination with a toxin-enhancing antibody, 8A7, which binds to domain 3 of PA and PA oligomers. These results provide novel insights into the antibody response elicited by the rPA vaccine and may be useful for PA-based vaccine and immunotherapeutic cocktail design.

  4. A study of recombinant protective H. pylori antigens

    PubMed Central

    Jiang, Zheng; Tao, Xiao-Hong; Huang, Ai-Long; Wang, Pi-Long

    2002-01-01

    AIM: To construct a recombinant vector which can express Mr26000 outer membrane protein (OMP) from Helicobacter pylori (H. pylori), and to obtain the vaccine protecting against H. pylori infection and a diagnostic reagent kit quickly detecting H. pylori infection. METHODS: The gene encoding the structural Mr26000 outer membrane protein of H. pylori was amplified from H. pylori chromosomal DNA by PCR, and inserted in the prokaryotic expression vector pET32a(+), which was transformed into the Top10 E. coli strain. Recombinant vector was selected, identified and transformed into BL-21(DE3) E. coli strain. The recombinant fusion proteins were expressed. The antigenicity of recombinant protein was studied by ELISA or immunoblotting and immunized Balb/c mice. RESULTS: The gene of Mr26000 OMP was amplified to be 594 base pairs, 1.1% of the cloned genes was mutated and 1.51% of amino acid residues was changed, but there was homogeneity between them. The recombinant fusion protein encoded objective polypeptides of 198 amino acid residues, corresponding to calculated molecular masses of Mr26000. The level of soluble expression products was about 38.96% of the total cell protein. After purification by Ni-NTA agarose resin columniation, the purity of objective protein became about 90%. The ELISA results showed that recombinant fusion protein could be recognized by patient serum infected with H. pylori and rabbit serum immunized with the recombinant protein. Furthermore, Balb/c mice immunized with the recombinant protein were protected against H. pylori infection. CONCLUSION: Mr26000 OMP may be a candidate vaccine preventing H. pylori infection. PMID:11925614

  5. Genetic mapping identifies novel highly protective antigens for an apicomplexan parasite.

    PubMed

    Blake, Damer P; Billington, Karen J; Copestake, Susan L; Oakes, Richard D; Quail, Michael A; Wan, Kiew-Lian; Shirley, Martin W; Smith, Adrian L

    2011-02-10

    Apicomplexan parasites are responsible for a myriad of diseases in humans and livestock; yet despite intensive effort, development of effective sub-unit vaccines remains a long-term goal. Antigenic complexity and our inability to identify protective antigens from the pool that induce response are serious challenges in the development of new vaccines. Using a combination of parasite genetics and selective barriers with population-based genetic fingerprinting, we have identified that immunity against the most important apicomplexan parasite of livestock (Eimeria spp.) was targeted against a few discrete regions of the genome. Herein we report the identification of six genomic regions and, within two of those loci, the identification of true protective antigens that confer immunity as sub-unit vaccines. The first of these is an Eimeria maxima homologue of apical membrane antigen-1 (AMA-1) and the second is a previously uncharacterised gene that we have termed 'immune mapped protein-1' (IMP-1). Significantly, homologues of the AMA-1 antigen are protective with a range of apicomplexan parasites including Plasmodium spp., which suggest that there may be some characteristic(s) of protective antigens shared across this diverse group of parasites. Interestingly, homologues of the IMP-1 antigen, which is protective against E. maxima infection, can be identified in Toxoplasma gondii and Neospora caninum. Overall, this study documents the discovery of novel protective antigens using a population-based genetic mapping approach allied with a protection-based screen of candidate genes. The identification of AMA-1 and IMP-1 represents a substantial step towards development of an effective anti-eimerian sub-unit vaccine and raises the possibility of identification of novel antigens for other apicomplexan parasites. Moreover, validation of the parasite genetics approach to identify effective antigens supports its adoption in other parasite systems where legitimate protective antigen

  6. Membrane Insertion by Anthrax Protective Antigen in Cultured Cells†

    PubMed Central

    Qa'dan, Maen; Christensen, Kenneth A.; Zhang, Lei; Roberts, Thomas M.; Collier, R. John

    2005-01-01

    The enzymatic moieties of anthrax toxin enter the cytosol of mammalian cells via a pore in the endosomal membrane formed by the protective antigen (PA) moiety. Pore formation involves an acidic pH-induced conformational rearrangement of a heptameric precursor (the prepore), in which the seven 2β2-2β3 loops interact to generate a 14-strand transmembrane β-barrel. To investigate this model in vivo, we labeled PA with the fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) at cysteine residues introduced into the 2β2-2β3 loop. Each labeled PA was bound to CHO cells, and NBD fluorescence was monitored over time in stirred cell suspensions or by confocal microscopy. A strong increase was observed with NBD at positions 305, 307, 309, and 311, sites where side chains are predicted to face the bilayer, and little change was seen at residues 304, 306, 308, 310, and 312, sites where side chains are predicted to face the pore lumen. The increase at position 305 was inhibited by membrane-restricted quenchers, low temperature, or various reagents known to affect toxin action. Of the 24 NBD attachment sites examined, all but three gave results qualitatively consistent with the β-barrel model. Besides supporting the β-barrel model of membrane insertion, our results describe the time course of insertion and identify PA residues where NBD gives a strong signal upon membrane insertion in vivo. PMID:15964805

  7. Rabbit immunoglobulin responses to the flagella, somatic, and protective antigens of a highly protective strain of Clostridium chauvoei.

    PubMed

    Chandler, H M

    1975-07-01

    The immunoglobulin response of rabbits to the flagells (H), somatic (O), and protective antigens of a highly protective strain of Clostridium chauvoei was studied using antisera that had been fractionated by Sephadex G-200 chromatography. The H antigen elicited the characteristic agglutinin response to a protein antigen--early production of 19S globulin followed by persistent 7S globulin production. The O antigen stimulated a transient agglutinin response which was detected in both the 19S and 7S serum fractions. Protective antibody was assayed by passive protection tests in mice. Using these tests the protective activity of the rabbit sera was found to be confined exclusively to the 7S serum fractions. Purified immunoglobulin G, prepared by DEAE-cellulose chromatography of the above sera, was also tested and found to confer considerable passive protection on mice. It is considered that either the protective antigen fails to stimulate an immunoglobulin M response or that immunoglobulin M is relatively ineffective in conferring protection against infection in the mouse passive protection tests.

  8. Reflective Coatings Protect People and Animals

    NASA Technical Reports Server (NTRS)

    2010-01-01

    Led by Marshall Space Flight Center, NASA engineers called upon National Metalizing of Cranbury, New Jersey, to help create a reflective sunshield to deploy on Skylab in place of a shield that was lost during launch in 1973. Years later, a former employee for National Metalizing founded Advanced Flexible Materials (AFM) Inc., of Petaluma, California, and utilized the radiant barrier technology in the public domain to produce a variety of products such as wraps to keep marathon finishers safe from hypothermia as well as a lining for mittens and vests. Recently, the material helped to keep manatees warm as they were lifted from the water as part of a tag-and-release program.

  9. Immunological fingerprinting of group B streptococci: from circulating human antibodies to protective antigens.

    PubMed

    Meinke, Andreas L; Senn, Beatrice M; Visram, Zehra; Henics, Tamás Z; Minh, Duc Bui; Schüler, Wolfgang; Neubauer, Christina; Gelbmann, Dieter; Noiges, Birgit; Sinzinger, Jan; Hanner, Markus; Dewasthaly, Shailesh; Lundberg, Urban; Hordnes, Knut; Masoud, Helga; Sevelda, Paul; von Gabain, Alexander; Nagy, Eszter

    2010-10-08

    Group B streptococcus is one of the most important pathogens in neonates, and causes invasive infections in non-pregnant adults with underlying diseases. Applying a genomic approach that relies on human antibodies we identified antigenic GBS proteins, among them most of the previously published protective antigens. In vitro analyses allowed the selection of conserved candidate antigens that were further evaluated in murine lethal sepsis models using several GBS strains. In active and passive immunization models, we identified four protective GBS antigens, FbsA and BibA, as well as two hypothetical proteins, all shown to contribute to virulence based on gene deletion mutants. These protective antigens have the potential to be components of novel vaccines or targets for passive immune prophylaxis against GBS disease.

  10. Production and Characterization of Monoclonal Antibodies Against the Protective Antigen Component of Bacillus anthracis Toxin

    DTIC Science & Technology

    1988-01-21

    F. Jaquet, P. Luethy, R. Huetter, and D. G. Braun. 1986. Characterization of mcnoclonal antibodies to a crystal protein of Bacillus thuringiensis ...AD-A192 855 UT FILE COPY Production and Characterization of Monoclonal Antibodies Against the Protective Antigen Component of Bacillus anthracis...Author Tel. No. 301-663-7341 1--ac",- 88 3 14 05 6 Krhirty-six monoclonal antibodies to the protective antigen protein of Bacillus anthracis exotoxin

  11. Sterile Protective Immunity to Malaria is Associated with a Panel of Novel P. falciparum Antigens*

    PubMed Central

    Trieu, Angela; Kayala, Matthew A.; Burk, Chad; Molina, Douglas M.; Freilich, Daniel A.; Richie, Thomas L.; Baldi, Pierre; Felgner, Philip L.; Doolan, Denise L.

    2011-01-01

    The development of an effective malaria vaccine remains a global public health priority. Less than 0.5% of the Plasmodium falciparum genome has been assessed as potential vaccine targets and candidate vaccines have been based almost exclusively on single antigens. It is possible that the failure to develop a malaria vaccine despite decades of effort might be attributed to this historic focus. To advance malaria vaccine development, we have fabricated protein microarrays representing 23% of the entire P. falciparum proteome and have probed these arrays with plasma from subjects with sterile protection or no protection after experimental immunization with radiation attenuated P. falciparum sporozoites. A panel of 19 pre-erythrocytic stage antigens was identified as strongly associated with sporozoite-induced protective immunity; 16 of these antigens were novel and 85% have been independently identified in sporozoite and/or liver stage proteomic or transcriptomic data sets. Reactivity to any individual antigen did not correlate with protection but there was a highly significant difference in the cumulative signal intensity between protected and not protected individuals. Functional annotation indicates that most of these signature proteins are involved in cell cycle/DNA processing and protein synthesis. In addition, 21 novel blood-stage specific antigens were identified. Our data provide the first evidence that sterile protective immunity against malaria is directed against a panel of novel P. falciparum antigens rather than one antigen in isolation. These results have important implications for vaccine development, suggesting that an efficacious malaria vaccine should be multivalent and targeted at a select panel of key antigens, many of which have not been previously characterized. PMID:21628511

  12. Properties of repeat domain found in a novel protective antigen, SpaA, of Erysipelothrix rhusiopathiae.

    PubMed

    Makino, S; Yamamoto, K; Murakami, S; Shirahata, T; Uemura, K; Sawada, T; Wakamoto, H; Morita, H; Morita, Y

    1998-08-01

    Erysipelothrix rhusiopathiae is a small gram-positive rod bacterium that causes erysipelas in swine and a variety of diseases in other animals and humans. Although live-attenuated or bacterin vaccines are effective in protecting against erysipelas, the genetic construction of their active antigen has not been identified. To clarify the surface antigen(s) involved in protective and arthritic response, using monoclonal antibody I2A against the surface proteins of E. rhusiopathiae, we identified a protective antigen, which consists of 606 amino acids. Analysis of deletion derivatives of the gene, spaA(surface protective antigen), showed that the SpaA protein binds tightly to the bacterial cell surface via eight repeat units with a GW-module consisting of 20 amino acids at the C-terminus. Although DeltaSpaA lacking their repeat units lost its ability to induce protection against E. rhusiopathiae infection, intact SpaA protein showed the protection. We conclude that the presence of repeat units is essential both for the binding of SpaA to the bacterial cell surface and for protection. We believe that the repeat region at the C-terminus should be a candidate for a subunit vaccine against erysipelas.

  13. Cross-protection provided by live Shigella mutants lacking major antigens.

    PubMed

    Szijártó, Valéria; Hunyadi-Gulyás, Eva; Emődy, Levente; Pál, Tibor; Nagy, Gábor

    2013-05-01

    The immune response elicited by Shigella infections is dominated by serotype-specific antibodies recognizing the LPS O-antigens. Although a marked antibody response to invasion plasmid antigens (Ipa-s) shared by all virulent strains is also induced, the varying level of immunity elicited by natural infections is serotype-restricted. Previous vaccines have tried to mimic and achieve this serotype-specific, infection-induced immunity. As, however, the four Shigella species can express 50 different types of O-antigens, current approaches with the aim to induce a broad coverage use a mixture of the most common O-antigens combined in single vaccines. In the current study we present data on an alternative approach to generate immunity protective against multiple serotypes. Mutants lacking both major immune-determinant structures (i.e. the Ipa and O-antigens) were not only highly attenuated, but, unlike their avirulent counterparts still expressing these antigens, elicited a protective immune response to heterologous serotypes in a murine model. Evidence is provided that protection was mediated by the enhanced immunogenic potential of minor conserved antigens. Furthermore, the rough, non-invasive double mutants triggered an immune response different from that induced by the smooth, invasive strains regarding the isotype of antibodies generated. These non-invasive, rough mutants may represent promising candidates for further development into live vaccines for the prophylaxis of bacillary dysentery in areas with multiple endemic serotypes.

  14. Identification of Two New Protective Pre-erythrocytic Malaria Vaccine Antigen Candidates

    DTIC Science & Technology

    2011-01-01

    Adenovirus-vectored Malaria Vaccines Encoding Plasmodium falciparum Circumsporozoite Protein (CSP) and Apical Membrane Antigen (AMA1) in Malaria -Naïve...Baisor M, Lorry K, Brown G, Pye D, Irving D, Smith T, Beck H, Alpers M: A recombinant blood-stage malaria vaccine reduces Plasmodium falciparum ...study, the efficacy of three pre-erythrocytic stage malaria antigens was evaluated in a Plasmodium yoelii/mouse protection model. Methods: Mice were

  15. A Chemically Synthesized Capture Agent Enables the Selective, Sensitive, and Robust Electrochemical Detection of Anthrax Protective Antigen

    DTIC Science & Technology

    2014-08-01

    A Chemically Synthesized Capture Agent Enables the Selective, Sensitive, and Robust Electrochemical Detection of Anthrax Protective Antigen...A Chemically Synthesized Capture Agent Enables the Selective, Sensitive, and Robust Electrochemical Detection of Anthrax Protective Antigen...AND SUBTITLE A Chemically Synthesized Capture Agent Enables the Selective, Sensitive, and Robust Electrochemical Detection of Anthrax Protective

  16. Does Antigen Masking by Ubiquitin Chains Protect from the Development of Autoimmune Diseases?

    PubMed Central

    Weil, Robert

    2014-01-01

    Autoimmune diseases are characterized by the production of antibodies against self-antigens and generally arise from a failure of central or peripheral tolerance. However, these diseases may develop when newly appearing antigens are not recognized as self by the immune system. The mechanism by which some antigens are “invisible” to the immune system is not completely understood. Apoptotic and complement system defects or autophagy imbalance can generate this antigenic autoreactivity. Under particular circumstances, cellular debris containing autoreactive antigens can be recognized by innate immune receptors or other sensors and can eventually lead to autoimmunity. Ubiquitination may be one of the mechanisms protecting autoreactive antigens from the immune system that, if disrupted, can lead to autoimmunity. Ubiquitination is an essential post-translational modification used by cells to target proteins for degradation or to regulate other intracellular processes. The level of ubiquitination is regulated during T cell tolerance and apoptosis and E3 ligases have emerged as a crucial signaling pathway for the regulation of T cell tolerance toward self-antigens. I propose here that an unrecognized role of ubiquitin and ubiquitin-like proteins could be to render intracellular or foreign antigens (present in cellular debris resulting from apoptosis, complement system, or autophagy defects) invisible to the immune system in order to prevent the development of autoimmunity. PMID:24917867

  17. Reactogenicity to major tuberculosis antigens absent in BCG is linked to improved protection against Mycobacterium tuberculosis.

    PubMed

    Aguilo, Nacho; Gonzalo-Asensio, Jesus; Alvarez-Arguedas, Samuel; Marinova, Dessislava; Gomez, Ana Belen; Uranga, Santiago; Spallek, Ralf; Singh, Mahavir; Audran, Regine; Spertini, François; Martin, Carlos

    2017-07-14

    MTBVAC is a live-attenuated Mycobacterium tuberculosis vaccine, currently under clinical development, that contains the major antigens ESAT6 and CFP10. These antigens are absent from the current tuberculosis vaccine, BCG. Here we compare the protection induced by BCG and MTBVAC in several mouse strains that naturally express different MHC haplotypes differentially recognizing ESAT6 and CFP10. MTBVAC induces improved protection in C3H mice, the only of the three tested strains reactive to both ESAT6 and CFP10. Deletion of both antigens in MTBVAC reduces its efficacy to BCG levels, supporting a link between greater efficacy and CFP10- and ESAT6-specific reactogenicity. In addition, MTBVAC (but not BCG) triggers a specific response in human vaccinees against ESAT6 and CFP10. Our results warrant further exploration of this response as potential biomarker of protection in MTBVAC clinical trials.

  18. Expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax.

    PubMed

    Aziz, Mohd Azhar; Singh, Samer; Anand Kumar, P; Bhatnagar, Rakesh

    2002-12-06

    Protective antigen (PA) is the most potent molecule for vaccination against anthrax. In the present study, we have successfully integrated protective antigen gene in nuclear genome of tobacco plants by Agrobacterium mediated leaf-disc transformation method. Expression of protective antigen gene was detected by immunoblot analysis using antisera raised against purified PA. A distinct band of approximately 83kDa lighted up in the protein extracted from transformed plants while there was no such band in untransformed plants. The plant expressed PA showed biological activity just like native PA, which was demonstrated by cytolytic assay on macrophage like cell lines with lethal factor. This study establishes for the first time expression of PA gene in a plant system and thus marks the first milestone towards developing edible vaccine against anthrax.

  19. New classes of orthopoxvirus vaccine candidates by functionally screening a synthetic library for protective antigens.

    PubMed

    Borovkov, Alexandre; Magee, D Mitch; Loskutov, Andrey; Cano, Jose A; Selinsky, Cheryl; Zsemlye, Jason; Lyons, C Rick; Sykes, Kathryn

    2009-12-05

    The licensed smallpox vaccine, comprised of infectious vaccinia, is no longer popular as it is associated with a variety of adverse events. Safer vaccines have been explored such as further attenuated viruses and component designs. However, these alternatives typically provide compromised breadth and strength of protection. We conducted a genome-level screening of cowpox, the ancestral poxvirus, in the broadly immune-presenting C57BL/6 mouse as an approach to discovering novel components with protective capacities. Cowpox coding sequences were synthetically built and directly assayed by genetic immunization for open-reading frames that protect against lethal pulmonary infection. Membrane and non-membrane antigens were identified that partially protect C57BL/6 mice against cowpox and vaccinia challenges without adjuvant or regimen optimization, whereas the 4-pox vaccine did not. New vaccines might be developed from productive combinations of these new and existing antigens to confer potent, broadly efficacious protection and be contraindicated for none.

  20. Protective-antigen (PA) based anthrax vaccines confer protection against inhalation anthrax by precluding the establishment of a systemic infection.

    PubMed

    Merkel, Tod J; Perera, Pin-Yu; Lee, Gloria M; Verma, Anita; Hiroi, Toyoko; Yokote, Hiroyuki; Waldmann, Thomas A; Perera, Liyanage P

    2013-09-01

    An intense effort has been launched to develop improved anthrax vaccines that confer rapid, long lasting protection preferably with an extended stability profile amenable for stockpiling. Protective antigen (PA)-based vaccines are most favored as immune responses directed against PA are singularly protective, although the actual protective mechanism remains to be unraveled. Herein we show that contrary to the prevailing view, an efficacious PA-based vaccine confers protection against inhalation anthrax by preventing the establishment of a toxin-releasing systemic infection. Equally importantly, antibodies measured by the in vitro lethal toxin neutralization activity assay (TNA) that is considered as a reliable correlate of protection, especially for PA protein-based vaccines adjuvanted with aluminum salts appear to be not absolutely essential for this protective immune response.

  1. Isolation of Protective Somatic Antigen from Vibrio cholerae (Ogawa) Ribosomal Preparations

    PubMed Central

    Jensen, Ronald; Gregory, Barry; Naylor, James; Actor, Paul

    1972-01-01

    Ribosomal preparations from Vibrio cholerae Ogawa and Inaba are protective immunogens for mice challenged with either serotype. Column chromatography of ribosomal fractions separated protective antigen from the ribosomes. The antigen is a heterogeneous colloid which contains protein, lipid, and carbohydrate in the ratio 3:1:1. Amino acid composition is that of a slightly acidic protein with no unusual complement of amino acids. The lipid component consists of several longchain fatty acids and of phosphatidyl ethanolamine. The major sugars identified were glucose and galactose. The colloid can be dissolved by treatment with chelating agents and sodium dodecyl sulfate. Purification of the solubilized material or of the colloid was unsuccessful. PMID:4654646

  2. Closeup view of the reflective insulation protecting the Crew Compartment ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Close-up view of the reflective insulation protecting the Crew Compartment bulkhead, orbiter structure and landing gear housing in the void created by the removal of the Forward Reaction Control System Module from the forward section of the Orbiter Discovery. This image was taken from the service platform in the Orbiter Processing Facility at Kennedy Space Center. - Space Transportation System, Orbiter Discovery (OV-103), Lyndon B. Johnson Space Center, 2101 NASA Parkway, Houston, Harris County, TX

  3. Subdominant outer membrane antigens in anaplasma marginale: conservation, antigenicity, and protective capacity using recombinant protein

    USDA-ARS?s Scientific Manuscript database

    Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a well- defined surface protein complex reproducibly induce protective immunity. However, there are seve...

  4. Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against experimental Neospora caninum infection

    PubMed Central

    Cho, Jung-Hwa; Chung, Woo-Suk; Song, Kyoung-Ju; Na, Byoung-Kuk; Kang, Seung-Won; Song, Chul-Yong

    2005-01-01

    Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against N. caninum infection was evaluated in vitro and in vivo. Two major immunodominant surface antigens (NcSAG1 and NcSRS2) and two dense granule proteins (NcDG1 and NcDG2) of N. caninum tachyzoites were expressed in E. coli, respectively. An in vitro neutralization assay using polyclonal antisera raised against each recombinant antigen showed inhibitory effects on the invasion of N. caninum tachyzoites into host cells. Separate groups of gerbils were immunized with the purified recombinant proteins singly or in combinations and animals were then challenged with N.caninum. Following these experimental challenges, the protective efficacy of each vaccination was determined by assessing animal survival rate. All experimental groups showed protective effects of different degrees against experimental infection. The highest protection efficacy was observed for combined vaccination with NcSRS2 and NcDG1. Our results indicate that combined vaccination with the N. caninum recombinant antigens, NcSRS2 and NcDG1, induces the highest protective effect against N. caninum infection in vitro and in vivo. PMID:15793355

  5. Proteolytic activation of receptor-bound anthrax protective antigen on macrophages promotes its internalization.

    PubMed

    Beauregard, K E; Collier, R J; Swanson, J A

    2000-06-01

    Immunofluorescence and other methods have been used to probe the self-assembly and internalization of the binary toxin, anthrax lethal toxin (LeTx), in primary murine macrophages. Proteolytic activation of protective antigen (PA; 83 kDa, the B moiety of the toxin) by furin was the rate-limiting step in internalization of LeTx and promoted clearance of PA from the cell surface. A furin-resistant form of PA remained at the cell surface for at least 90 min. Oligomerization of receptor-bound PA63, the 63 kDa active fragment of PA, was manifested by its conversion to a pronase-resistant state, characteristic of the heptameric prepore form in solution. That oligomerization of PA63 triggers toxin internalization is supported by the observation that PA20, the complementary 20 kDa fragment of PA, inhibited clearance of nicked PA. The PA63 prepore, with or without lethal factor (LF), cleared slowly from the cell surface. These studies show that proteolytic cleavage of PA, in addition to permitting oligomerization and LF binding, also promotes internalization of the protein. The relatively long period of activation and internalization of PA at the cell surface may reflect adaptation of this binary toxin that maximizes self-assembly.

  6. Antigens protected functional red blood cells by the membrane grafting of compact hyperbranched polyglycerols.

    PubMed

    Chapanian, Rafi; Constantinescu, Iren; Brooks, Donald E; Scott, Mark D; Kizhakkedathu, Jayachandran

    2013-01-02

    Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen, glucose, and ions. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane and mask RBC surface antigens.

  7. Prime-boost immunisation against tropical theileriosis with two parasite surface antigens: evidence for protection and antigen synergy.

    PubMed

    Gharbi, Mohamed; Darghouth, Mohamed Aziz; Weir, William; Katzer, Frank; Boulter, Nicky; Adamson, Rachel; Gilbert, Sarah C; Jongejan, Frans; Westbroek, Irene; Hall, Roger; Tait, Andrew; Shiels, Brian

    2011-09-02

    Current methods for control of tropical theileriosis in cattle suffer from several disadvantages that could be circumvented by development of an effective sub-unit vaccine. Previous work has utilised two major surface antigens (SPAG-1 and Tams1) and conventional adjuvants to provide partial protection against parasite challenge. In this study we have delivered these antigens using the prime-boost system and analysed whether a combination regime can enhance protection against lethal challenge. Delivery of the boost as recombinant protein or expressed from a recombinant MVA vector was also assessed. The results confirmed that immunisation with Tams1 alone could reduce the severity of several disease parameters compared to non-immunised controls and these effects were more marked when recombinant protein was used for boosting compared to MVA delivery. A similar outcome was obtained by immunisation with SPAG-1 alone. Significantly, delivery of SPAG-1 and Tams1 as a cocktail showed enhanced protection. This was manifest by significant improvement in a large range of clinical and parasitological parameters and, most dramatically, by the survival and recovery of 50% of the immunised animals compared to 0% of the controls. Analysis of the antibody response post-challenge showed that while there was a strong response to Tams1, no response to SPAG-1 was detected. In contrast, lymphoproliferation assays showed a significant enhancement of response at day 7 post-challenge in calves of the SPAG-1 group but a dramatic decrease of the proliferation activity in all three groups receiving Tams1. We conclude that immunisation with a cocktail of SPAG-1 and Tams1 generates a synergistic protective response that significantly improves the efficacy of recombinant vaccination against tropical theileriosis. Potential effector mechanisms that could mediate this response are discussed.

  8. Isolation of protective antigens from the gut of Boophilus microplus using monoclonal antibodies.

    PubMed Central

    Lee, R P; Opdebeeck, J P

    1991-01-01

    Monoclonal antibodies (mAb) were produced against midgut membrane (GM) antigens of Boophilus microplus. The isotypes of these mAb were established and their specificity characterized using double diffusion and Western blotting. GM antigens solubilized by Triton X-100 were precipitated by mAb QU13, and the precipitate was then injected into cattle to test for the presence of protective antigens. Vaccinated cattle challenged with 10-day-old larval ticks showed evidence of protection with a 62% reduction in eggs produced by ticks from vaccinated cattle compared to tick eggs from control cattle. In a second vaccine-challenge experiment, the dose of precipitate was increased and greater than 99% protection was provided to these vaccinated cattle following challenge (calculated from tick egg weights compared to the control group). The solubilized antigen(s) precipitated by QU13 were subjected to SDS-PAGE separation and the calculated sizes of these molecules were greater than 200,000, 80,000, 74,000, 62,000 57,000 and less than 30,000 MW. Images Figure 1 Figure 2 PMID:1997395

  9. Immunodominant Antigens of Leishmania chagasi Associated with Protection against Human Visceral Leishmaniasis

    PubMed Central

    Abánades, Daniel R.; Arruda, Leonardo V.; Arruda, Elaine S.; Pinto, José Roberto A. S.; Palma, Mario S.; Aquino, Dorlene; Caldas, Arlene J.; Soto, Manuel; Barral, Aldina; Barral-Netto, Manoel

    2012-01-01

    Background Protection and recovery from visceral leishmaniasis (VL) have been associated with cell-mediated immune (CMI) responses, whereas no protective role has been attributed to humoral responses against specific parasitic antigens. In this report, we compared carefully selected groups of individuals with distinct responses to Leishmania chagasi to explore antigen-recognizing IgG present in resistant individuals. Methodology and Principal Findings VL patients with negative delayed-type hypersensitivity (DTH) were classified into the susceptible group. Individuals who had recovered from VL and converted to a DTH+ response, as well as asymptomatic infected individuals (DTH+), were categorized into the resistant group. Sera from these groups were used to detect antigens from L. chagasi by conventional and 2D Western blot assays. Despite an overall reduction in the reactivity of several proteins after DTH conversion, a specific group of proteins (approximately 110–130 kDa) consistently reacted with sera from DTH converters. Other antigens that specifically reacted with sera from DTH+ individuals were isolated and tandem mass spectrometry followed by database query with the protein search engine MASCO were used to identify antigens. The serological properties of recombinant version of the selected antigens were tested by ELISA. Sera from asymptomatic infected people (DTH+) reacted more strongly with a mixture of selected recombinant antigens than with total soluble Leishmania antigen (SLA), with less cross-reactivity against Chagas disease patients' sera. Significance Our results are the first evidence of leishmania proteins that are specifically recognized by sera from individuals who are putatively resistant to VL. In addition, these data highlight the possibility of using specific proteins in serological tests for the identification of asymptomatic infected individuals. PMID:22724032

  10. Induction of Protection against Porcine Cysticercosis by Vaccination with Recombinant Oncosphere Antigens

    PubMed Central

    Flisser, Ana; Gauci, Charles G.; Zoli, André; Martinez-Ocaña, Joel; Garza-Rodriguez, Adriana; Dominguez-Alpizar, Jose Luis; Maravilla, Pablo; Rodriguez-Canul, Rossana; Avila, Guillermina; Aguilar-Vega, Laura; Kyngdon, Craig; Geerts, Stanny; Lightowlers, Marshall W.

    2004-01-01

    Two recombinant Taenia solium oncosphere antigens, designated TSOL18 and TSOL45-1A, were investigated as vaccines to prevent transmission of the zoonotic disease cysticercosis through pigs. Both antigens were effective in inducing very high levels of protection (up to 100%) in three independent vaccine trials in pigs against experimental challenge infection with T. solium eggs, which were undertaken in Mexico and Cameroon. This is the highest level of protection that has been achieved against T. solium infection in pigs by vaccination with a defined antigen. TSOL18 and TSOL45-1A provide the basis for development of a highly effective practical vaccine that could assist in the control and, potentially, the eradication of human neurocysticercosis. PMID:15322025

  11. A novel mimetic antigen eliciting protective antibody to Neisseria meningitidis.

    PubMed

    Granoff, D M; Moe, G R; Giuliani, M M; Adu-Bobie, J; Santini, L; Brunelli, B; Piccinetti, F; Zuno-Mitchell, P; Lee, S S; Neri, P; Bracci, L; Lozzi, L; Rappuoli, R

    2001-12-01

    Molecular mimetic Ags are of considerable interest as vaccine candidates. Yet there are few examples of mimetic Ags that elicit protective Ab against a pathogen, and the functional activity of anti-mimetic Abs has not been studied in detail. As part of the Neisseria meningitidis serogroup B genome sequencing project, a large number of novel proteins were identified. Herein, we provide evidence that genome-derived Ag 33 (GNA33), a lipoprotein with homology to Escherichia coli murein transglycosylase, elicits protective Ab to meningococci as a result of mimicking an epitope on loop 4 of porin A (PorA) in strains with serosubtype P1.2. Epitope mapping of a bactericidal anti-GNA33 mAb using overlapping peptides shows that the mAb recognizes peptides from GNA33 and PorA that share a QTP sequence that is necessary but not sufficient for binding. By flow cytometry, mouse antisera prepared against rGNA33 and the anti-GNA33 mAb bind as well as an anti-PorA P1.2 mAb to the surface of eight of nine N. meningitidis serogroup B strains tested with the P1.2 serosubtype. Anti-GNA33 Abs also are bactericidal for most P1.2 strains and, for susceptible strains, the activity of an anti-GNA33 mAb is similar to that of an anticapsular mAb but less active than an anti-P1.2 mAb. Anti-GNA Abs also confer passive protection against bacteremia in infant rats challenged with P1.2 strains. Thus, GNA33 represents one of the most effective immunogenic mimetics yet described. These results demonstrate that molecular mimetics have potential as meningococcal vaccine candidates.

  12. African swine fever virus serotype-specific proteins are significant protective antigens for African swine fever

    USDA-ARS?s Scientific Manuscript database

    African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available and progress is hindered by lack of knowledge concerning the extent of African swine fever virus (ASFV) strain diversity and the viral antigens conferring type specific protective im...

  13. The Influences of Glycosylation on the Antigenicity, Immunogenicity, and Protective Efficacy of Ebola Virus GP DNA Vaccines

    DTIC Science & Technology

    2006-11-22

    Microbiology. All Rights Reserved. Influences of Glycosylation on Antigenicity, Immunogenicity, and Protective Efficacy of Ebola Virus GP DNA Vaccines ...carbohydrates. We measured the influences of GP glycosylation on antigenicity, immu- nogenicity, and protection by testing DNA vaccines comprised of GP...may protect against filovirus infection (6, 39). Con- sistent with this, B-cell-deficient mice vaccinated with EBOV-like particles were not protected

  14. Combination of two candidate subunit vaccine antigens elicits protective immunity to ricin and anthrax toxin in mice.

    PubMed

    Vance, David J; Rong, Yinghui; Brey, Robert N; Mantis, Nicholas J

    2015-01-09

    In an effort to develop combination vaccines for biodefense, we evaluated a ricin subunit antigen, RiVax, given in conjunction with an anthrax protective antigen, DNI. The combination led to high endpoint titer antibody response, neutralizing antibodies, and protective immunity against ricin and anthrax lethal toxin. This is a natural combination vaccine, since both antigens are recombinant subunit proteins that would be given to the same target population.

  15. Identification of a 68-kilodalton protective protein antigen from Bordetella bronchiseptica.

    PubMed Central

    Montaraz, J A; Novotny, P; Ivanyi, J

    1985-01-01

    A 68-kilodalton (kd) outer membrane protein antigen of Bordetella bronchiseptica has been identified by using monoclonal antibodies that recognized two nonoverlapping determinants. Antibody BB05 also reacted with homologous proteins from Bordetella pertussis and Bordetella parapertussis but not with another 12 organisms from various bacterial genera. Passive injection of BB05 antibody protected mice from aerosol infection with B. bronchiseptica as shown by reduced mortality and reduced pathology of turbinate bones. The 68-kd B. bronchiseptica antigen was purified by BB05-based affinity chromatography and evaluated for its potency to immunize mice actively against either intraperitoneal or aerosol challenge with B. bronchiseptica. Immunization with the 68-kd antigen in incomplete Freund adjuvant significantly reduced the levels of mortality in intraperitoneally challenged mice. In the aerosol infection model, injection of the 68-kd antigen with complete or incomplete Freund adjuvant or saponin reduced the bacterial counts in the lungs of infected mice. These results suggest that the 68-kd protein may represent a potential "protective" antigen of B. bronchiseptica. Images PMID:3972452

  16. Antigen Export during Liver Infection of the Malaria Parasite Augments Protective Immunity

    PubMed Central

    Beigier-Bompadre, Macarena; Becker, Martina; Kroczek, Richard A.; Kaufmann, Stefan H. E.; Matuschewski, Kai

    2014-01-01

    ABSTRACT Protective immunity against preerythrocytic malaria parasite infection is difficult to achieve. Intracellular Plasmodium parasites likely minimize antigen presentation by surface-expressed major histocompatibility complex class I (MHC-I) molecules on infected cells, yet they actively remodel their host cells by export of parasite factors. Whether exported liver-stage proteins constitute better candidates for MHC-I antigen presentation to CD8+ T lymphocytes remains unknown. Here, we systematically characterized the contribution of protein export to the magnitude of antigen-specific T-cell responses against Plasmodium berghei liver-stage parasites in C57BL/6 mice. We generated transgenic sporozoites that secrete a truncated ovalbumin (OVA) surrogate antigen only in the presence of an amino-terminal protein export element. Immunization with live attenuated transgenic sporozoites revealed that antigen export was not critical for CD8+ T-cell priming but enhanced CD8+ T-cell proliferation in the liver. Upon transfer of antigen-specific CD8+ T cells, liver-stage parasites secreting the target protein were eliminated more efficiently. We conclude that Plasmodium parasites strictly control protein export during liver infection to minimize immune recognition. Strategies that enhance the discharge of parasite proteins into infected hepatocytes could improve the efficacy of candidate preerythrocytic malaria vaccines. PMID:25073641

  17. [Induction of protective antiamebic immunity in hamsters with heterologous antigens].

    PubMed

    Jiménez Cardoso, J M; Jiménez, E; de Jesús Bernal, M; Kumate, J

    1989-01-01

    Two hundred and twenty-five Syrian golden hamsters were used. Twenty five of them served as the control group. All other hamsters were intradermal immunized, once a week for four weeks, with a mixture of amebic proteins, mixed with complete Freund adjuvant, obtained from 5 x 10(5) homogenized dead amebic trophozoites from five different strains. Each group of hamsters (five groups of 40 animals each) were immunized with one of the following strains: E. histolytica HM-531, HJ-1, HM1-IMSS, E. chattoni PM-4 and PM-5. All hamsters, including those from the control group, were later inoculated with 0.2 mL equivalent to 1 x 10(5) live trophozoites from the different strains grown in axenic TYI-S-33 medium. Inoculation was performed by direct injection into the liver. The hamsters were sacrificed eight days later and their livers examined. All non-immunized animals showed extensive gross hepatic nodular abscesses. The liver of immunized hamsters showed mild to moderate lesions: the histopathological striking feature was non-specific granulomata. It is concluded that the immunized animals inoculated with homologous stock showed protective immunity to amebic infections. In other cases, immunity was seen though they were inoculated with a heterologous stock.

  18. Immunogenicity and protection efficacy of subunit-based smallpox vaccines using variola major antigens.

    PubMed

    Sakhatskyy, Pavlo; Wang, Shixia; Zhang, Chuanyou; Chou, Te-Hui; Kishko, Michael; Lu, Shan

    2008-02-05

    The viral strain responsible for smallpox infection is variola major (VARV). As a result of the successful eradication of smallpox with the vaccinia virus (VACV), the general population is no longer required to receive a smallpox vaccine, and will have no protection against smallpox. This lack of immunity is a concern due to the potential for use of smallpox as a biological weapon. Considerable progress has been made in the development of subunit-based smallpox vaccines resulting from the identification of VACV protective antigens. It also offers the possibility of using antigens from VARV to formulate the next generation subunit-based smallpox vaccines. Here, we show that codon-optimized DNA vaccines expressing three VARV antigens (A30, B7 and F8) and their recombinant protein counterparts elicited high-titer, cross-reactive, VACV neutralizing antibody responses in mice. Vaccinated mice were protected from intraperitoneal and intranasal challenges with VACV. These results suggest the feasibility of a subunit smallpox vaccine based on VARV antigen sequences to induce immunity against poxvirus infection.

  19. Antigen-specific T-cell lines transfer protective immunity against Trichinella spiralis in vivo.

    PubMed Central

    Riedlinger, J; Grencis, R K; Wakelin, D

    1986-01-01

    T-cell lines specific for infective muscle larvae antigens of the intestinal nematode Trichinella spiralis have been generated in vitro. These antigen-specific T-cell lines express the L3T4+ Ly2- phenotype and secrete the lymphokines IL-2, IL-3 and gamma-IFN. They are stable in culture for up to 15 weeks and are protective when adoptively transferred into naive recipients. As few as 2 x 10(5) T. spiralis-specific tract. In addition, intestinal mastocytosis and peripheral blood eosinophilia were accelerated after adoptive transfer of T. spiralis-specific T-cell lines. PMID:2423438

  20. Efficacy of a vaccine based on protective antigen and killed spores against experimental inhalational anthrax.

    PubMed

    Gauthier, Yves P; Tournier, Jean-Nicolas; Paucod, Jean-Charles; Corre, Jean-Philippe; Mock, Michèle; Goossens, Pierre L; Vidal, Dominique R

    2009-03-01

    Protective antigen (PA)-based anthrax vaccines acting on toxins are less effective than live attenuated vaccines, suggesting that additional antigens may contribute to protective immunity. Several reports indicate that capsule or spore-associated antigens may enhance the protection afforded by PA. Addition of formaldehyde-inactivated spores (FIS) to PA (PA-FIS) elicits total protection against cutaneous anthrax. Nevertheless, vaccines that are effective against cutaneous anthrax may not be so against inhalational anthrax. The aim of this work was to optimize immunization with PA-FIS and to assess vaccine efficacy against inhalational anthrax. We assessed the immune response to recombinant anthrax PA from Bacillus anthracis (rPA)-FIS administered by various immunization protocols and the protection provided to mice and guinea pigs infected through the respiratory route with spores of a virulent strain of B. anthracis. Combined subcutaneous plus intranasal immunization of mice yielded a mucosal immunoglobulin G response to rPA that was more than 20 times higher than that in lung mucosal secretions after subcutaneous vaccination. The titers of toxin-neutralizing antibody and antispore antibody were also significantly higher: nine and eight times higher, respectively. The optimized immunization elicited total protection of mice intranasally infected with the virulent B. anthracis strain 17JB. Guinea pigs were fully protected, both against an intranasal challenge with 100 50% lethal doses (LD(50)) and against an aerosol with 75 LD(50) of spores of the highly virulent strain 9602. Conversely, immunization with PA alone did not elicit protection. These results demonstrate that the association of PA and spores is very much more effective than PA alone against experimental inhalational anthrax.

  1. Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors.

    PubMed

    Wang, Hai-Chao; An, Huai-Jie; Yu, Yun-Zhou; Xu, Qing

    2015-02-01

    DNA vaccines require improvement for human use because they are generally weak stimulators of the immune system in humans. The efficacy of DNA vaccines can be improved using a viral replicon as vector to administer antigen of pathogen. In this study, we comprehensively evaluated the conventional non-viral DNA, viral replicon DNA or viral replicon particles (VRP) vaccines encoding different forms of anthrax protective antigen (PA) for specific immunity and protective potency against anthrax. Our current results clearly suggested that these viral replicon DNA or VRP vaccines derived from Semliki Forest virus (SFV) induced stronger PA-specific immune responses than the conventional non-viral DNA vaccines when encoding the same antigen forms, which resulted in potent protection against challenge with the Bacillus anthracis strain A16R. Additionally, the naked PA-expressing SFV replicon DNA or VRP vaccines without the need for high doses or demanding particular delivery regimens elicited robust immune responses and afforded completely protective potencies, which indicated the potential of the SFV replicon as vector of anthrax vaccines for use in clinical application. Therefore, our results suggest that these PA-expressing SFV replicon DNA or VRP vaccines may be suitable as candidate vaccines against anthrax.

  2. Disaccharides Protect Antigens from Drying-Induced Damage in Routinely Processed Tissue Sections

    PubMed Central

    Boi, Giovanna; Scalia, Carla Rossana; Gendusa, Rossella; Ronchi, Susanna; Cattoretti, Giorgio

    2015-01-01

    Drying of the tissue section, partial or total, during immunostaining negatively affects both the staining of tissue antigens and the ability to remove previously deposited antibody layers, particularly during sequential rounds of de-staining and re-staining for multiple antigens. The cause is a progressive loss of the protein-associated water up to the removal of the non-freezable water, a step which abolishes the immunoavailability of the epitope. In order to describe and prevent these adverse effects, we tested, among other substances, sugars, which are known to protect unicellular organisms from freezing and dehydration, and stabilize drugs and reagents in solid state form in medical devices. Disaccharides (lactose, sucrose) prevented the air drying-induced antigen masking and protected tissue-bound antigens and antibodies from air drying-induced damage. Complete removal of the bound antibody layers by chemical stripping was permitted if lactose was present during air drying. Lactose, sucrose and other disaccharides prevent air drying artifacts, allow homogeneous, consistent staining and the reuse of formalin-fixed, paraffin-embedded tissue sections for repeated immunostaining rounds by guaranteeing constant staining quality in suboptimal hydration conditions. PMID:26487185

  3. Yersinia outer proteins (YOPS) E, K and N are antigenic but non-protective compared to V antigen, in a murine model of bubonic plague.

    PubMed

    Leary, S E; Griffin, K F; Galyov, E E; Hewer, J; Williamson, E D; Holmström, A; Forsberg, A; Titball, R W

    1999-03-01

    The pathogenic Yersiniae produce a range of virulence proteins, encoded by a 70 kb plasmid, which are essential for infection, and also form part of a contact-dependent virulence mechanism. One of these proteins, V antigen, has been shown to confer a high level of protection against parenteral infection with Y. pestis in murine models, and is considered to be a protective antigen. In this study, the protective efficacy of V antigen has been compared in the same model with that of other proteins (YopE, YopK and YopN), which are part of the contact-dependent virulence mechanism. Mice immunised with two intraperitoneal doses of V antigen or each of the Yops, administered with either Alhydrogel or interleukin-12, produced high antigen-specific serum IgG titres. As shown in previous studies, V+Alhydrogel was fully protective, and 5/5 mice survived a subcutaneous challenge with 90 or 9x10(3) LD50's of Y. pestis GB. In addition, these preliminary studies also showed that V+IL-12 was partially protective: 4/5 or 3/5 mice survived a challenge with 90 or 9x10(3) LD50's, respectively. In contrast, none of the mice immunised with the Yops survived the challenges, and there was no significant delay in the mean time to death compared to mice receiving a control protein. These results show that using two different vaccine regimens, Yops E, K and N, failed to elicit protective immune responses in a murine model of plague, whereas under the same conditions, V antigen was fully or partially protective. Copyright 1999 Academic Press.

  4. Genetic and Physiological Control of Protective Antigen Synthesis by Bacillus Anthracis

    DTIC Science & Technology

    1981-12-01

    plasmid-negative variants of Bacillus pumilus . II. Is the presence of a plasmid or prophage related to toxin synthesis? (1) Isolation of plasmid-negative...genes and the control of spontaneous mutation. Ann. Rev. Genet. 10:135(1976). 5. Lovett, P.S. Plasmid in Bacillus pumilus and the enhanced sporulation...Unclassified AD REPORT NUMBER TWO GENETIC AND PHYSIOLOGICAL CONTROL OF PROTECTIVE ANTIGEN SYNTHESIS BY BACILLUS ANTHRACIS ANNUAL PROGRESS REPORT

  5. Identification of protective antigens by RNA interference for control of the lone star tick, Amblyomma americanum.

    PubMed

    de la Fuente, José; Manzano-Roman, Raúl; Naranjo, Victoria; Kocan, Katherine M; Zivkovic, Zorica; Blouin, Edmour F; Canales, Mario; Almazán, Consuelo; Galindo, Ruth C; Step, Douglas L; Villar, Margarita

    2010-02-17

    The lone star tick, Amblyomma americanum, vectors pathogens of emerging diseases of humans and animals in the United States. Currently, measures are not available for effective control of A. americanum infestations. Development of vaccines directed against tick proteins may reduce tick infestations and the transmission of tick-borne pathogens. However, the limiting step in tick vaccine development has been the identification of tick protective antigens. Herein, we report the application of RNA interference (RNAi) for screening an A. americanum cDNA library for discovery of tick protective antigens that reduce tick survival and weights after feeding. Four cDNA clones, encoding for putative threonyl-tRNA synthetase (2C9), 60S ribosomal proteins L13a (2D10) and L13e (2B7), and interphase cytoplasm foci protein 45 (2G7), were selected for vaccine studies in cattle, along with subolesin, a tick protective protein identified previously. In vaccinated cattle, an overall efficacy (E)>30% was obtained when considering the vaccine effect on both nymphs and adults, but only 2D10, 2G7 and subolesin affected both tick stages. The highest efficacy of control for adult ticks (E>55%) was obtained in cattle vaccinated with recombinant 2G7 or subolesin. These collective results demonstrated the feasibility of developing vaccines for the control of lone star tick infestations. The use of RNAi for identification of tick protective antigens proved to be a rapid and cost-effective tool for discovery of candidate vaccine antigens, and this approach could likely be applied to other parasites of veterinary and medical importance.

  6. Genetic and Physiological Control of Protective Antigen Synthesis by Bacillus anthracis

    DTIC Science & Technology

    1984-12-01

    acids (Difco) and 10 mg of thiamine hydrochloride . Minimal M: To Minimal I was added 10 mg of thiamine hydrochloride , 200 mg of glycine, and 40 mg of L...methionine and L-proline. Minimal 0: To Minimal I was added 10 mg of thiamine hydrochloride , 200 mg of glycine, and 40 mg of L-methionine, L-serine, L...Unclassified SAD M REPORT NUMBER FIVE A GENETIC AND PHYSIOLOGICAL CONTROL OF PROTECTIVE ANTIGEN SYNTHESIS BY BACILLUS ANTHRACIS ANNUAL PROGRESS

  7. Genetic and Physiological Control of Protective Antigen Synthesis by Bacillus anthracis

    DTIC Science & Technology

    1985-07-01

    Minimal I with 5 g of vitamin-free Casamino acids (Difco) and 10 mg of thiamine hydrochloride . Antisera. All antisera were kindly supplied by personnel of...Q0. Unclassified AD___________ REPORT NUM3ER SIX GENETIC AND PHYSIOLOGICAL CONTROL OF PROTECTIVE ANTIGEN SYNTHESIS BY BACILLUS ANTHRACIS ANNUAL...Proof that pX02 is involved in cap3ule synthesis came from experiments in which the plasmid was transferred by CP-51-mediated transduction and by a

  8. Genetic and Physiological Control of Protective Antigen Synthesis by Bacillus Anthracis

    DTIC Science & Technology

    1983-12-01

    with 5 g of vitamin-free Casamino acids (Difco) and 10 mg of thiamine hydrochloride . Minimal M: To Minimal I was added 10 mg of thiamine hvdrochloride...200 mg of glycine, and 40 mg of L-iaethionine and L-proline. Minimal 0: To Minimal I was added 10 mg of thiamine hydrochloride , 200 mg of glycine, and...AD GENETIC AND PHYSIOLOGICAL CONTROL OF PROTECTIVE ANTIGEN SYNTHESIS BY BACILLUS ANTHRACIS ANNUAL PROGRESS REPORT CURTIS B.THORNE SDECEMBER 1983

  9. Oral Administration of a Salmonella enterica-Based Vaccine Expressing Bacillus anthracis Protective Antigen Confers Protection against Aerosolized B. anthracis▿

    PubMed Central

    Stokes, Margaret G. M.; Titball, Richard W.; Neeson, Brendan N.; Galen, James E.; Walker, Nicola J.; Stagg, Anthony J.; Jenner, Dominic C.; Thwaite, Joanne E.; Nataro, James P.; Baillie, Leslie W. J.; Atkins, Helen S.

    2007-01-01

    Bacillus anthracis is the causative agent of anthrax, a disease that affects wildlife, livestock, and humans. Protection against anthrax is primarily afforded by immunity to the B. anthracis protective antigen (PA), particularly PA domains 4 and 1. To further the development of an orally delivered human vaccine for mass vaccination against anthrax, we produced Salmonella enterica serovar Typhimurium expressing full-length PA, PA domains 1 and 4, or PA domain 4 using codon-optimized PA DNA fused to the S. enterica serovar Typhi ClyA and under the control of the ompC promoter. Oral immunization of A/J mice with Salmonella expressing full-length PA protected five of six mice against a challenge with 105 CFU of aerosolized B. anthracis STI spores, whereas Salmonella expressing PA domains 1 and 4 provided only 25% protection (two of eight mice), and Salmonella expressing PA domain 4 or a Salmonella-only control afforded no measurable protection. However, a purified recombinant fusion protein of domains 1 and 4 provided 100% protection, and purified recombinant 4 provided protection in three of eight immunized mice. Thus, we demonstrate for the first time the efficacy of an oral S. enterica-based vaccine against aerosolized B. anthracis spores. PMID:17145938

  10. Identification of protective and broadly conserved vaccine antigens from the genome of extraintestinal pathogenic Escherichia coli.

    PubMed

    Moriel, Danilo Gomes; Bertoldi, Isabella; Spagnuolo, Angela; Marchi, Sara; Rosini, Roberto; Nesta, Barbara; Pastorello, Ilaria; Corea, Vanja A Mariani; Torricelli, Giulia; Cartocci, Elena; Savino, Silvana; Scarselli, Maria; Dobrindt, Ulrich; Hacker, Jörg; Tettelin, Hervé; Tallon, Luke J; Sullivan, Steven; Wieler, Lothar H; Ewers, Christa; Pickard, Derek; Dougan, Gordon; Fontana, Maria Rita; Rappuoli, Rino; Pizza, Mariagrazia; Serino, Laura

    2010-05-18

    Extraintestinal pathogenic Escherichia coli (ExPEC) are a common cause of disease in both mammals and birds. A vaccine to prevent such infections would be desirable given the increasing antibiotic resistance of these bacteria. We have determined the genome sequence of ExPEC IHE3034 (ST95) isolated from a case of neonatal meningitis and compared this to available genome sequences of other ExPEC strains and a few nonpathogenic E. coli. We found 19 genomic islands present in the genome of IHE3034, which are absent in the nonpathogenic E. coli isolates. By using subtractive reverse vaccinology we identified 230 antigens present in ExPEC but absent (or present with low similarity) in nonpathogenic strains. Nine antigens were protective in a mouse challenge model. Some of them were also present in other pathogenic non-ExPEC strains, suggesting that a broadly protective E. coli vaccine may be possible. The gene encoding the most protective antigen was detected in most of the E. coli isolates, highly conserved in sequence and found to be exported by a type II secretion system which seems to be nonfunctional in nonpathogenic strains.

  11. Identification of protective and broadly conserved vaccine antigens from the genome of extraintestinal pathogenic Escherichia coli

    PubMed Central

    Rappuoli, Rino; Serino, Laura

    2010-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) are a common cause of disease in both mammals and birds. A vaccine to prevent such infections would be desirable given the increasing antibiotic resistance of these bacteria. We have determined the genome sequence of ExPEC IHE3034 (ST95) isolated from a case of neonatal meningitis and compared this to available genome sequences of other ExPEC strains and a few nonpathogenic E. coli. We found 19 genomic islands present in the genome of IHE3034, which are absent in the nonpathogenic E. coli isolates. By using subtractive reverse vaccinology we identified 230 antigens present in ExPEC but absent (or present with low similarity) in nonpathogenic strains. Nine antigens were protective in a mouse challenge model. Some of them were also present in other pathogenic non-ExPEC strains, suggesting that a broadly protective E. coli vaccine may be possible. The gene encoding the most protective antigen was detected in most of the E. coli isolates, highly conserved in sequence and found to be exported by a type II secretion system which seems to be nonfunctional in nonpathogenic strains. PMID:20439758

  12. A study on the protection to relics and the related problems with diffuse reflectance spectroscopy.

    PubMed

    Wang, Liqin; Liang, Guozheng; Dang, Gaochao

    2005-03-01

    The application of diffuse reflectance spectroscopy to relic protection is studied by using a self-made fiber optics reflectance spectrophotometer. The major work done includes: (1) the composition of pigment on colored relics and its changes are identified; (2) the change on metal surface is monitored; (3) the reflectance spectrum characteristics of relic protection materials are studied. The results tell that diffuse reflectance spectroscopy is a new protection technique, characterized by its quickness and non-destructiveness to the relic.

  13. Influences of glycosylation on antigenicity, immunogenicity, and protective efficacy of ebola virus GP DNA vaccines.

    PubMed

    Dowling, William; Thompson, Elizabeth; Badger, Catherine; Mellquist, Jenny L; Garrison, Aura R; Smith, Jeffery M; Paragas, Jason; Hogan, Robert J; Schmaljohn, Connie

    2007-02-01

    The Ebola virus (EBOV) envelope glycoprotein (GP) is the primary target of protective immunity. Mature GP consists of two disulfide-linked subunits, GP1 and membrane-bound GP2. GP is highly glycosylated with both N- and O-linked carbohydrates. We measured the influences of GP glycosylation on antigenicity, immunogenicity, and protection by testing DNA vaccines comprised of GP genes with deleted N-linked glycosylation sites or with deletions in the central hypervariable mucin region. We showed that mutation of one of the two N-linked GP2 glycosylation sites was highly detrimental to the antigenicity and immunogenicity of GP. Our data indicate that this is likely due to the inability of GP2 and GP1 to dimerize at the cell surface and suggest that glycosylation at this site is required for achieving the conformational integrity of GP2 and GP1. In contrast, mutation of two N-linked sites on GP1, which flank previously defined protective antibody epitopes on GP, may enhance immunogenicity, possibly by unmasking epitopes. We further showed that although deleting the mucin region apparently had no effect on antigenicity in vitro, it negatively impacted the elicitation of protective immunity in mice. In addition, we confirmed the presence of previously identified B-cell and T-cell epitopes in GP but show that when analyzed individually none of them were neither absolutely required nor sufficient for protective immunity to EBOV. Finally, we identified other potential regions of GP that may contain relevant antibody or T-cell epitopes.

  14. Novel vaccine antigen combinations elicit protective immune responses against Escherichia coli sepsis.

    PubMed

    Mellata, Melha; Mitchell, Natalie M; Schödel, Florian; Curtiss, Roy; Pier, Gerald B

    2016-01-27

    Systemic infections caused by extraintestinal pathogenic Escherichia coli (ExPEC) have emerged as the most common community-onset bacterial infections and are major causes of nosocomial infections worldwide. The management of ExPEC infections has been complicated by the heterogeneity of ExPEC strains and the emergence of antibiotic resistance, thus their prevention through vaccination would be beneficial. The protective efficacy of four common ExPEC antigen candidates composed of common pilus antigens EcpA and EcpD and iron uptake proteins IutA and IroN, were tested by both active and passive immunization in lethal and non-lethal murine models of sepsis. Additionally, antibody raised to a synthetic form of a conserved surface polysaccharide, β-(1-6)-linked poly-N-acetylglucosamine (dPNAG) containing 9 monomers of (non-acetylated) glucosamine (9GlcNH2) conjugated to tetanus toxoid TT (9GlcNH2-TT) was tested in passive immunization protocols. Active immunization of mice with recombinant antigens EcpA, EcpD, IutA, or IroN elicited high levels of total IgG antibody of IgG1/IgG2a isotypes, and were determined to be highly protective against E. coli infection in lethal and non-lethal sepsis challenges. Moreover, passive immunization against these four antigens resulted in significant reductions of bacteria in internal organs and blood of the mice, especially when the challenge strain was grown in iron-restricted media. Inclusion of antibodies to PNAG increased the efficacy of the passive immunization under conditions where the challenge bacteria were grown in LB medium but not in iron-restricted media. The information and data presented are the first step toward the development of a broadly protective vaccine against sepsis-causing E. coli strains.

  15. Synthetic Long Peptide Derived from Mycobacterium tuberculosis Latency Antigen Rv1733c Protects against Tuberculosis

    PubMed Central

    Coppola, Mariateresa; van den Eeden, Susan J. F.; Wilson, Louis; Franken, Kees L. M. C.; Ottenhoff, Tom H. M.

    2015-01-01

    Responsible for 9 million new cases of active disease and nearly 2 million deaths each year, tuberculosis (TB) remains a global health threat of overwhelming dimensions. Mycobacterium bovis BCG, the only licensed vaccine available, fails to confer lifelong protection and to prevent reactivation of latent infection. Although 15 new vaccine candidates are now in clinical trials, an effective vaccine against TB remains elusive, and new strategies for vaccination are vital. BCG vaccination fails to induce immunity against Mycobacterium tuberculosis latency antigens. Synthetic long peptides (SLPs) combined with adjuvants have been studied mostly for therapeutic cancer vaccines, yet not for TB, and proved to induce efficient antitumor immunity. This study investigated an SLP derived from Rv1733c, a major M. tuberculosis latency antigen which is highly expressed by “dormant” M. tuberculosis and well recognized by T cells from latently M. tuberculosis-infected individuals. In order to assess its in vivo immunogenicity and protective capacity, Rv1733c SLP in CpG was administered to HLA-DR3 transgenic mice. Immunization with Rv1733c SLP elicited gamma interferon-positive/tumor necrosis factor-positive (IFN-γ+/TNF+) and IFN-γ+ CD4+ T cells and Rv1733c-specific antibodies and led to a significant reduction in the bacterial load in the lungs of M. tuberculosis-challenged mice. This was observed both in a pre- and in a post-M. tuberculosis challenge setting. Moreover, Rv1733c SLP immunization significantly boosted the protective efficacy of BCG, demonstrating the potential of M. tuberculosis latency antigens to improve BCG efficacy. These data suggest a promising role for M. tuberculosis latency antigen Rv1733c-derived SLPs as a novel TB vaccine approach, both in a prophylactic and in a postinfection setting. PMID:26202436

  16. Novel vaccine antigen combinations elicit protective immune responses against Escherichia coli sepsis

    PubMed Central

    Mellata, Melha; Mitchell, Natalie M.; Schödel, Florian; Curtiss, Roy; Pier, Gerald B.

    2015-01-01

    Systemic infections caused by extraintestinal pathogenic Escherichia coli (ExPEC) have emerged as the most common community-onset bacterial infections and are major causes of nosocomial infections worldwide. The management of ExPEC infections has been complicated by the heterogeneity of ExPEC strains and the emergence of antibiotic resistance, thus their prevention through vaccination would be beneficial. The protective efficacy of four common ExPEC antigen candidates composed of common pilus antigens EcpA and EcpD and iron uptake proteins IutA and IroN, were tested by both active and passive immunization in lethal and non-lethal murine models of sepsis. Additionally, antibody raised to a synthetic form of a conserved surface polysaccharide, β-(1–6)-linked poly-N-acetylglucosamine (dPNAG) containing 9 monomers of (non-acetylated) glucosamine (9GlcNH2) conjugated to tetanus toxoid TT (9GlcNH2-TT) was tested in passive immunization protocols. Active immunization of mice with recombinant antigens EcpA, EcpD, IutA or IroN elicited high levels of total IgG antibody of IgG1/IgG2a isotypes, and were determined to be highly protective against E. coli infection in lethal and non-lethal sepsis challenges. Moreover, passive immunization against these four antigens resulted in significant reductions of bacteria in internal organs and blood of the mice, especially when the challenge strain was grown in iron-restricted media. Inclusion of antibodies to PNAG increased the efficacy of the passive immunization under conditions where the challenge bacteria were grown in LB medium but not in iron-restricted media. The information and data presented are the first step toward the development of a broadly protective vaccine against sepsis-causing E. coli strains. PMID:26707217

  17. Antibodies to merkel cell polyomavirus T antigen oncoproteins reflect tumor burden in merkel cell carcinoma patients.

    PubMed

    Paulson, Kelly G; Carter, Joseph J; Johnson, Lisa G; Cahill, Kevin W; Iyer, Jayasri G; Schrama, David; Becker, Juergen C; Madeleine, Margaret M; Nghiem, Paul; Galloway, Denise A

    2010-11-01

    Merkel cell polyomavirus (MCPyV) is a common infectious agent that is likely involved in the etiology of most Merkel cell carcinomas (MCC). Serum antibodies recognizing the MCPyV capsid protein VP1 are detectable at high titer in nearly all MCC patients and remain stable over time. Although antibodies to the viral capsid indicate prior MCPyV infection, they provide limited clinical insight into MCC because they are also detected in more than half of the general population. We investigated whether antibodies recognizing MCPyV large and small tumor-associated antigens (T-Ag) would be more specifically associated with MCC. Among 530 population control subjects, these antibodies were present in only 0.9% and were of low titer. In contrast, among 205 MCC cases, 40.5% had serum IgG antibodies that recognize a portion of T-Ag shared between small and large T-Ags. Among cases, titers of T-Ag antibodies fell rapidly (∼8-fold per year) in patients whose cancer did not recur, whereas they rose rapidly in those with progressive disease. Importantly, in several patients who developed metastases, the rise in T-Ag titer preceded clinical detection of disease spread. These results suggest that antibodies recognizing T-Ag are relatively specifically associated with MCC, do not effectively protect against disease progression, and may serve as a clinically useful indicator of disease status.

  18. Immunization of Mice with Anthrax Protective Antigen Limits Cardiotoxicity but Not Hepatotoxicity Following Lethal Toxin Challenge.

    PubMed

    Devera, T Scott; Prusator, Dawn K; Joshi, Sunil K; Ballard, Jimmy D; Lang, Mark L

    2015-06-25

    Protective immunity against anthrax is inferred from measurement of vaccine antigen-specific neutralizing antibody titers in serum samples. In animal models, in vivo challenges with toxin and/or spores can also be performed. However, neither of these approaches considers toxin-induced damage to specific organ systems. It is therefore important to determine to what extent anthrax vaccines and existing or candidate adjuvants can provide organ-specific protection against intoxication. We therefore compared the ability of Alum, CpG DNA and the CD1d ligand α-galactosylceramide (αGC) to enhance protective antigen-specific antibody titers, to protect mice against challenge with lethal toxin, and to block cardiotoxicity and hepatotoxicity. By measurement of serum cardiac Troponin I (cTnI), and hepatic alanine aminotransferase (ALT), and aspartate aminotransferase (AST), it was apparent that neither vaccine modality prevented hepatic intoxication, despite high Ab titers and ultimate survival of the subject. In contrast, cardiotoxicity was greatly diminished by prior immunization. This shows that a vaccine that confers survival following toxin exposure may still have an associated morbidity. We propose that organ-specific intoxication should be monitored routinely during research into new vaccine modalities.

  19. A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge

    NASA Astrophysics Data System (ADS)

    Deliyannis, Georgia; Boyle, Jefferey S.; Brady, Jamie L.; Brown, Lorena E.; Lew, Andrew M.

    2000-06-01

    Improving the immunological potency, particularly the Ab response, is a serious hurdle for the protective efficacy and hence broad application of DNA vaccines. We examined the immunogenicity and protective efficacy of a hemagglutinin-based influenza DNA vaccine that was targeted to antigen-presenting cells (APCs) by fusion to CTLA4. The targeted vaccine was shown to induce an accelerated and increased Ab response (as compared with those receiving the nontargeted control) that was predominated by IgG1 and recognized conformationally dependent viral epitopes. Moreover, mice receiving the APC-targeted DNA vaccine had significantly reduced viral titers (100-fold) after a nonlethal virus challenge. The increased protective efficacy was most likely because of increased Ab responses, as cytotoxic T lymphocyte responses were not enhanced. Targeting was demonstrated by direct binding studies of CTLA4 fusion proteins to the cognate ligand (B7; expressed on APCs in vivo). In addition, a targeted protein was detected at 4-fold higher levels in draining lymph nodes within 2-24 h of administration. Therefore, this study demonstrates that targeting DNA-encoded antigen to APCs results in enhanced immunity and strongly suggests that this approach may be useful in improving the protective efficacy of DNA vaccines.

  20. A protective protein antigen of Rickettsia rickettsii has tandemly repeated, near-identical sequences.

    PubMed Central

    Anderson, B E; McDonald, G A; Jones, D C; Regnery, R L

    1990-01-01

    The nucleotide sequence of a Rickettsia rickettsii gene that encodes a high-molecular-mass surface antigen (190 kilodaltons), which elicits protective immunity, was determined. The 6,747-nucleotide gene coded for a 2,249-amino-acid protein with a calculated molecular weight of 224,321. A 3.8-kilobase PstI fragment proximal to the 5' end of the gene was found to consist of 13 highly related tandem repeats which constituted over 40% of the coding region. The repeated sequences could be divided into either a 225-nucleotide, 75-amino-acid unit (type I) or a 216-nucleotide, 72-amino-acid unit (type II), with extensive homology between the two types of repeating units. The deduced amino acid sequence for these repeat units, overall, was slightly hydrophobic with short hydrophilic domains. The carboxy-terminal (nonrepetitive) portion of the deduced protein sequence was hydrophilic, with potential surface-exposed epitopes. The full-length reading frame was reconstructed in Escherichia coli, and transient expression of the 190-kilodalton antigen was demonstrated; however, the protein appeared to be severely degraded by proteases and was apparently toxic to E. coli. The conservation of this unique repetitive gene structure, coupled with results from previous reports showing the protective properties of the 190-kilodalton antigen, suggests that this protein plays an important role in the pathogenesis of and immunity to Rocky Mountain spotted fever. Images PMID:2117568

  1. Pathogens Inactivated by Low-Energy-Electron Irradiation Maintain Antigenic Properties and Induce Protective Immune Responses

    PubMed Central

    Fertey, Jasmin; Bayer, Lea; Grunwald, Thomas; Pohl, Alexandra; Beckmann, Jana; Gotzmann, Gaby; Casado, Javier Portillo; Schönfelder, Jessy; Rögner, Frank-Holm; Wetzel, Christiane; Thoma, Martin; Bailer, Susanne M.; Hiller, Ekkehard; Rupp, Steffen; Ulbert, Sebastian

    2016-01-01

    Inactivated vaccines are commonly produced by incubating pathogens with chemicals such as formaldehyde or β-propiolactone. This is a time-consuming process, the inactivation efficiency displays high variability and extensive downstream procedures are often required. Moreover, application of chemicals alters the antigenic components of the viruses or bacteria, resulting in reduced antibody specificity and therefore stimulation of a less effective immune response. An alternative method for inactivation of pathogens is ionizing radiation. It acts very fast and predominantly damages nucleic acids, conserving most of the antigenic structures. However, currently used irradiation technologies (mostly gamma-rays and high energy electrons) require large and complex shielding constructions to protect the environment from radioactivity or X-rays generated during the process. This excludes them from direct integration into biological production facilities. Here, low-energy electron irradiation (LEEI) is presented as an alternative inactivation method for pathogens in liquid solutions. LEEI can be used in normal laboratories, including good manufacturing practice (GMP)- or high biosafety level (BSL)-environments, as only minor shielding is necessary. We show that LEEI efficiently inactivates different viruses (influenza A (H3N8), porcine reproductive and respiratory syndrome virus (PRRSV), equine herpesvirus 1 (EHV-1)) and bacteria (Escherichia coli) and maintains their antigenicity. Moreover, LEEI-inactivated influenza A viruses elicit protective immune responses in animals, as analyzed by virus neutralization assays and viral load determination upon challenge. These results have implications for novel ways of developing and manufacturing inactivated vaccines with improved efficacy. PMID:27886076

  2. Antigen Exposure History Defines CD8 T Cell Dynamics and Protection during Localized Pulmonary Infections

    PubMed Central

    Van Braeckel-Budimir, Natalija; Martin, Matthew D.; Hartwig, Stacey M.; Legge, Kevin L.; Badovinac, Vladimir P.; Harty, John T.

    2017-01-01

    Unlike systemic infections, little is known about the role of repeated localized infections on (re)shaping pathogen-specific memory CD8 T cell responses. Here, we used primary (1°) and secondary (2°) intranasal influenza virus infections of mice as a model to study intrinsic memory CD8 T cell properties. We show that secondary antigen exposure, relative to a single infection, generates memory CD8 T cell responses of superior magnitude in multiple tissue compartments including blood, spleen, draining lymph nodes, and lung. Unexpectedly, regardless of the significantly higher number of 2° memory CD8 T cells, similar degree of protection against pulmonary challenge was observed in both groups of mice containing 1° or 2° memory CD8 T cells. Mechanistically, using pertussis toxin-induced migration block, we showed that superior antigen-driven proliferation and ability to relocate to the site of infection allowed 1° memory CD8 T cells to accumulate in the infected lung during the first few days after challenge, compensating for the initially lower cell numbers. Taken together, the history of antigen exposures to localized pulmonary infections, through altering basic cell biology, dictates dynamic properties of protective memory CD8 T cell responses. This knowledge has important implications for a design of novel and an improvement of existing vaccines and immunization strategies. PMID:28191007

  3. Pathogens Inactivated by Low-Energy-Electron Irradiation Maintain Antigenic Properties and Induce Protective Immune Responses.

    PubMed

    Fertey, Jasmin; Bayer, Lea; Grunwald, Thomas; Pohl, Alexandra; Beckmann, Jana; Gotzmann, Gaby; Casado, Javier Portillo; Schönfelder, Jessy; Rögner, Frank-Holm; Wetzel, Christiane; Thoma, Martin; Bailer, Susanne M; Hiller, Ekkehard; Rupp, Steffen; Ulbert, Sebastian

    2016-11-23

    Inactivated vaccines are commonly produced by incubating pathogens with chemicals such as formaldehyde or β-propiolactone. This is a time-consuming process, the inactivation efficiency displays high variability and extensive downstream procedures are often required. Moreover, application of chemicals alters the antigenic components of the viruses or bacteria, resulting in reduced antibody specificity and therefore stimulation of a less effective immune response. An alternative method for inactivation of pathogens is ionizing radiation. It acts very fast and predominantly damages nucleic acids, conserving most of the antigenic structures. However, currently used irradiation technologies (mostly gamma-rays and high energy electrons) require large and complex shielding constructions to protect the environment from radioactivity or X-rays generated during the process. This excludes them from direct integration into biological production facilities. Here, low-energy electron irradiation (LEEI) is presented as an alternative inactivation method for pathogens in liquid solutions. LEEI can be used in normal laboratories, including good manufacturing practice (GMP)- or high biosafety level (BSL)-environments, as only minor shielding is necessary. We show that LEEI efficiently inactivates different viruses (influenza A (H3N8), porcine reproductive and respiratory syndrome virus (PRRSV), equine herpesvirus 1 (EHV-1)) and bacteria (Escherichia coli) and maintains their antigenicity. Moreover, LEEI-inactivated influenza A viruses elicit protective immune responses in animals, as analyzed by virus neutralization assays and viral load determination upon challenge. These results have implications for novel ways of developing and manufacturing inactivated vaccines with improved efficacy.

  4. Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen.

    PubMed

    Lopez, Jodie; Bittame, Amina; Massera, Céline; Vasseur, Virginie; Effantin, Grégory; Valat, Anne; Buaillon, Célia; Allart, Sophie; Fox, Barbara A; Rommereim, Leah M; Bzik, David J; Schoehn, Guy; Weissenhorn, Winfried; Dubremetz, Jean-François; Gagnon, Jean; Mercier, Corinne; Cesbron-Delauw, Marie-France; Blanchard, Nicolas

    2015-12-15

    Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV), resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN) of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation.

  5. Selection and evaluation of the immunogenicity of protective antigen mutants as anthrax vaccine candidates.

    PubMed

    Yan, Ming; Roehrl, Michael H; Basar, Emre; Wang, Julia Y

    2008-02-13

    Protective antigen (PA) is a central component of anthrax toxin and a major antigen in anthrax vaccines. However, the use of native PA as a vaccine is not optimal. If administered to people who have been freshly exposed to anthrax, PA may actually aid in anthrax toxin formation and thus may pose a serious safety concern for postexposure vaccination applications. A non-functional PA mutant may be a much safer alternative. To identify an improved anthrax vaccine antigen, we examined four non-functional mutants of PA, each being impaired in a critical step of the cellular intoxication pathway of PA. These mutants were Rec(-) (unable to bind PA-receptors), SSSR (resistant to activation by furin), Oligo(-) (unable to form oligomers), and DNI (Dominant Negative Inhibitory, unable to form endosomal transmembrane pores). When tested in mice and after three doses of immunization, all four mutants were highly potent in eliciting PA-specific, toxin-neutralizing antibodies, with immunogenicity increasing in the order of PAprotective epitopes of PA. Our study demonstrates that PA-based vaccines could be improved both in terms of safety and efficacy by strategic mutations that not only render PA non-functional but also simultaneously enhance its immunogenic potency. Recombinant PA mutants, particularly DNI, hold great promise as better and safer antigens than wild-type PA for use in postexposure

  6. Archaeosome Adjuvant Overcomes Tolerance to Tumor-Associated Melanoma Antigens Inducing Protective CD8+ T Cell Responses

    PubMed Central

    Krishnan, Lakshmi; Deschatelets, Lise; Stark, Felicity C.; Gurnani, Komal; Sprott, G. Dennis

    2010-01-01

    Vesicles comprised of the ether glycerolipids of the archaeon Methanobrevibacter smithii (archaeosomes) are potent adjuvants for evoking CD8+ T cell responses. We therefore explored the ability of archaeosomes to overcome immunologic tolerance to self-antigens. Priming and boosting of mice with archaeosome-antigen evoked comparable CD8+ T cell response and tumor protection to an alternate boosting strategy utilizing live bacterial vectors for antigen delivery. Vaccination with melanoma antigenic peptides TRP181-189 and Gp10025-33 delivered in archaeosomes resulted in IFN-γ producing antigen-specific CD8+ T cells with strong cytolytic capability and protection against subcutaneous B16 melanoma. Targeting responses against multiple antigens afforded prolonged median survival against melanoma challenge. Entrapment of multiple peptides within the same vesicle or admixed formulations were both effective at evoking CD8+ T cells against each antigen. Melanoma-antigen archaeosome formulations also afforded therapeutic protection against established B16 tumors when combined with depletion of T-regulatory cells. Overall, we demonstrate that archaeosome adjuvants constitute an effective choice for formulating cancer vaccines. PMID:21318177

  7. Novel antigen identification method for discovery of protective malaria antigens by rapid testing of DNA vaccines encoding exons from the parasite genome.

    PubMed

    Haddad, Diana; Bilcikova, Erika; Witney, Adam A; Carlton, Jane M; White, Charles E; Blair, Peter L; Chattopadhyay, Rana; Russell, Joshua; Abot, Esteban; Charoenvit, Yupin; Aguiar, Joao C; Carucci, Daniel J; Weiss, Walter R

    2004-03-01

    We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens.

  8. Protective Immunity Against a Lethal Respiratory Yersinia pestis Challenge Induced by V Antigen or the F1 Capsular Antigen Incorporated into Adenovirus Capsid

    PubMed Central

    Boyer, Julie L.; Sofer-Podesta, Carolina; Ang, John; Hackett, Neil R.; Chiuchiolo, Maria J.; Senina, Svetlana; Perlin, David

    2010-01-01

    Abstract The aerosol form of the bacterium Yersinia pestis causes pneumonic plague, a rapidly fatal disease that is a biothreat if deliberately released. At present, no plague vaccines are available for use in the United States, but subunit vaccines based on the Y. pestis V antigen and F1 capsular protein show promise when administered with adjuvants. In the context that adenovirus (Ad) gene transfer vectors have a strong adjuvant potential related to the ability to directly infect dendritic cells, we hypothesized that modification of the Ad5 capsid to display either the Y. pestis V antigen or the F1 capsular antigen on the virion surface would elicit high V antigen- or F1-specific antibody titers, permit boosting with the same Ad serotype, and provide better protection against a lethal Y. pestis challenge than immunization with equivalent amounts of V or F1 recombinant protein plus conventional adjuvant. We constructed AdYFP-pIX/V and AdLacZ-pIX/F1, E1–, E3– serotype 5 Ad gene transfer vectors containing a fusion of the sequence for either the Y. pestis V antigen or the F1 capsular antigen to the carboxy-terminal sequence of pIX, a capsid protein that can accommodate the entire V antigen (37 kDa) or F1 protein (15 kDa) without disturbing Ad function. Immunization with AdYFP-pIX/V followed by a single repeat administration of the same vector at the same dose resulted in significantly better protection of immunized animals compared with immunization with a molar equivalent amount of purified recombinant V antigen plus Alhydrogel adjuvant. Similarly, immunization with AdLacZ-pIX/F1 in a prime–boost regimen resulted in significantly enhanced protection of immunized animals compared with immunization with a molar-equivalent amount of purified recombinant F1 protein plus adjuvant. These observations demonstrate that Ad vaccine vectors containing pathogen-specific antigens fused to the pIX capsid protein have strong adjuvant properties and stimulate more robust

  9. Protection of gerbils from amebic liver abscess by immunization with a recombinant Entamoeba histolytica antigen.

    PubMed Central

    Zhang, T; Cieslak, P R; Stanley, S L

    1994-01-01

    Amebiasis, infection by the intestinal protozoan parasite Entamoeba histolytica, is a leading parasitic cause of death. As a step in the development of a recombinant antigen vaccine to prevent E. histolytica infection, we looked at the ability of a recombinant version of the serine-rich E. histolytica protein (SREHP) to elicit a protective immune response against invasive amebic disease. Gerbils, a standard model for amebic liver abscess, were immunized with either a recombinant SREHP/maltose-binding protein (MBP) fusion, recombinant MBP alone, or phosphate-buffered saline (PBS), all combined with complete Freund's adjuvant. In the first trial (group 1), gerbils received a primary and two booster immunizations intraperitoneally; in the second trial (group 2), gerbils were immunized by a single intradermal injection. SREHP/MBP-immunized gerbils in both groups produced antibody to native SHEHP and developed delayed-type hypersensitivity responses to recombinant SREHP. All gerbils were challenged by an intrahepatic injection with 5 x 10(4) virulent E. histolytica HM1-IMSS trophozoites. Complete protection from amebic liver abscess was seen in 64% of the SHEHP/MBP-immunized gerbils in group 1 and in 100% of the SREHP/MBP-immunized gerbils in group 2. There was no protection observed in MBP- or PBS-immunized gerbils in either group. Our results indicate that the SREHP molecule has potential as a vaccine to prevent amebic infection and demonstrate that successful vaccination of animals with recombinant E. histolytica antigen vaccines is possible. Images PMID:8132322

  10. Validation of the protective Ostertagia ostertagi ES-thiol antigens with different adjuvantia.

    PubMed

    Geldhof, P; Vercauteren, I; Vercruysse, J; Knox, D P; Van Den Broeck, W; Claerebout, E

    2004-01-01

    Intramuscular immunization of calves with an excretory-secretory antigen fraction enriched for cysteine proteinase activity (ES-thiol) and QuilA as adjuvant induces a protective immune response against the abomasal nematode Ostertagia ostertagi. The objectives of the present study were to confirm the protective capacity of ES-thiol in combination with QuilA, to test Al(OH)(3) as adjuvant for vaccination against O. ostertagi and to look for correlations between protection and immunological effector responses. Calves(seven animals/group) were vaccinated three times intramuscularly with 100 micro g antigen and/or adjuvant (ES-thiol with QuilA, ES-thiol with Al(OH)(3), QuilA alone and Al(OH)(3) alone) and subsequently challenged with a trickled oral infection of 25 000 infective larvae in total over 25 days. Faecal egg counts in the ES-thiol QuilA group were reduced by 56% during the two-month period of the trial compared to the QuilA control group (P < 0.002). Calves immunized with ES-thiol QuilA had significantly smaller adult worms (P < 0.002) and less eggs/female worm (P < 0.05) compared to the QuilA control group. No differences in egg output, worm counts or parameters of worm fitness were observed in the ES-thiol Al(OH)(3) group compared to the Al(OH)(3) control group. Although the protective immune mechanism against O. ostertagi remains unknown, protection in the ES-thiol QuilA group was associated with high levels of parasite-specific antibodies in the abomasal mucosa.

  11. Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection

    PubMed Central

    Tipper, Donald J.; Szomolanyi-Tsuda, Eva

    2016-01-01

    Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposes β-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%. PMID:27213160

  12. Protective Enterotoxigenic Escherichia coli Antigens in a Murine Intranasal Challenge Model

    PubMed Central

    Kumar, Amit; Hays, Mike; Lim, Francis; Foster, Leonard J.; Zhou, Mingxu; Zhu, Guoqiang; Miesner, Tracy; Hardwidge, Philip R.

    2015-01-01

    Enterotoxigenic Escherichia coli (ETEC) is an endemic health threat in underdeveloped nations. Despite the significant effort extended to vaccine trials using ETEC colonization factors, these approaches have generally not been especially effective in mediating cross-protective immunity. We used quantitative proteomics to identify 24 proteins that differed in abundance in membrane protein preparations derived from wild-type vs. a type II secretion system mutant of ETEC. We expressed and purified a subset of these proteins and identified nine antigens that generated significant immune responses in mice. Sera from mice immunized with either the MltA-interacting protein MipA, the periplasmic chaperone seventeen kilodalton protein, Skp, or a long-chain fatty acid outer membrane transporter, ETEC_2479, reduced the adherence of multiple ETEC strains differing in colonization factor expression to human intestinal epithelial cells. In intranasal challenge assays of mice, immunization with ETEC_2479 protected 88% of mice from an otherwise lethal challenge with ETEC H10407. Immunization with either Skp or MipA provided an intermediate degree of protection, 68 and 64%, respectively. Protection was significantly correlated with the induction of a secretory immunoglobulin A response. This study has identified several proteins that are conserved among heterologous ETEC strains and may thus potentially improve cross-protective efficacy if incorporated into future vaccine designs. PMID:26244636

  13. Discovering naturally processed antigenic determinants that confer protective T cell immunity

    PubMed Central

    Gilchuk, Pavlo; Spencer, Charles T.; Conant, Stephanie B.; Hill, Timothy; Gray, Jennifer J.; Niu, Xinnan; Zheng, Mu; Erickson, John J.; Boyd, Kelli L.; McAfee, K. Jill; Oseroff, Carla; Hadrup, Sine R.; Bennink, Jack R.; Hildebrand, William; Edwards, Kathryn M.; Crowe, James E.; Williams, John V.; Buus, Søren; Sette, Alessandro; Schumacher, Ton N.M.; Link, Andrew J.; Joyce, Sebastian

    2013-01-01

    CD8+ T cells (TCD8) confer protective immunity against many infectious diseases, suggesting that microbial TCD8 determinants are promising vaccine targets. Nevertheless, current T cell antigen identification approaches do not discern which epitopes drive protective immunity during active infection — information that is critical for the rational design of TCD8-targeted vaccines. We employed a proteomics-based approach for large-scale discovery of naturally processed determinants derived from a complex pathogen, vaccinia virus (VACV), that are presented by the most frequent representatives of four major HLA class I supertypes. Immunologic characterization revealed that many previously unidentified VACV determinants were recognized by smallpox-vaccinated human peripheral blood cells in a variegated manner. Many such determinants were recognized by HLA class I–transgenic mouse immune TCD8 too and elicited protective TCD8 immunity against lethal intranasal VACV infection. Notably, efficient processing and stable presentation of immune determinants as well as the availability of naive TCD8 precursors were sufficient to drive a multifunctional, protective TCD8 response. Our approach uses fundamental insights into T cell epitope processing and presentation to define targets of protective TCD8 immunity within human pathogens that have complex proteomes, suggesting that this approach has general applicability in vaccine sciences. PMID:23543059

  14. Enhanced Protective Efficacy of a Chimeric Form of the Schistosomiasis Vaccine Antigen Sm-TSP-2

    PubMed Central

    Pearson, Mark S.; Pickering, Darren A.; McSorley, Henry J.; Bethony, Jeffrey M.; Tribolet, Leon; Dougall, Annette M.; Hotez, Peter J.; Loukas, Alex

    2012-01-01

    The large extracellular loop of the Schistosoma mansoni tetraspanin, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. Moreover, Sm-TSP-2 is uniquely recognised by IgG1 and IgG3 from putatively resistant individuals resident in S. mansoni endemic areas in Brazil. In the present study, we expressed Sm-TSP-2 at high yield and in soluble form in E. coli without the need for a solubility enhancing fusion partner. We also expressed in E. coli a chimera called Sm-TSP-2/5B, which consisted of Sm-TSP-2 fused to the immunogenic 5B region of the hookworm aspartic protease and vaccine antigen, Na-APR-1. Sm-TSP-2 formulated with alum/CpG showed significant reductions in adult worm and liver egg burdens in two separate murine schistosomiasis challenge studies. Sm-TSP-2/5B afforded significantly greater protection than Sm-TSP-2 alone when both antigens were formulated with alum/CpG. The enhanced protection obtained with the chimeric fusion protein was associated with increased production of anti-Sm-TSP-2 antibodies and IL-4, IL-10 and IFN-γ from spleen cells of vaccinated animals. Sera from 666 individuals from Brazil who were infected with S. mansoni were screened for potentially deleterious IgE responses to Sm-TSP-2. Anti-Sm-TSP-2 IgE to this protein was not detected (also shown previously for Na-APR-1), suggesting that the chimeric antigen Sm-TSP-2/5B could be used to safely and effectively vaccinate people in areas where schistosomes and hookworms are endemic. PMID:22428079

  15. Cross-protective efficacy of dendritic cells targeting conserved influenza virus antigen expressed by Lactobacillus plantarum

    PubMed Central

    Yang, Wen-Tao; Shi, Shao-Hua; Yang, Gui-Lian; Jiang, Yan-Long; Zhao, Liang; Li, Yu; Wang, Chun-Feng

    2016-01-01

    Avian influenza virus (AIV) can infect birds and mammals, including humans, and are thus a serious threat to public health. Vaccination is vital for controlling AIV circulation. In this study, we generated a recombinant lactobacillus expressing the NP-M1-DCpep of H9N2 avian influenza virus and evaluated the activation effect of NC8-pSIP409-NP-M1-DCpep on dendritic cells (DCs) in a mouse model. The specific mucosal antibody responses and B and T cell responses in lymphoid tissues were also characterized. Importantly, we confirmed that specific CD8 T cells presented in vitro and antigen-specific cytotoxicity (activated the expression of CD107a) and in vivo antigen-specific cytotoxicity after vaccination. The adoptive transfer of NC8-pSIP409-NP-M1-DCpep-primed CD8+ T cells into NOD-SCID mice resulted in effective protection against mouse-adapted AIV infection. In addition, we observed protection in immunized mice challenged with mouse-adapted H9N2 AIV and H1N1 influenza virus, as evidenced by reductions in the lung virus titers, improvements in lung pathology, and weight loss and complete survival. Our data are promising for the generation of effective, non-traditional influenza vaccines against AIVs. PMID:28004787

  16. Heterosubtypic Protection against Pathogenic Human and Avian Influenza Viruses via In Vivo Electroporation of Synthetic Consensus DNA Antigens

    PubMed Central

    Laddy, Dominick J.; Yan, Jian; Kutzler, Michele; Kobasa, Darwyn; Kobinger, Gary P.; Khan, Amir S.; Greenhouse, Jack; Sardesai, Niranjan Y.; Draghia-Akli, Ruxandra; Weiner, David B.

    2008-01-01

    Background The persistent evolution of highly pathogenic avian influenza (HPAI) highlights the need for novel vaccination techniques that can quickly and effectively respond to emerging viral threats. We evaluated the use of optimized consensus influenza antigens to provide broad protection against divergent strains of H5N1 influenza in three animal models of mice, ferrets, and non-human primates. We also evaluated the use of in vivo electroporation to deliver these vaccines to overcome the immunogenicity barrier encountered in larger animal models of vaccination. Methods and Findings Mice, ferrets and non-human primates were immunized with consensus plasmids expressing H5 hemagglutinin (pH5HA), N1 neuraminidase (pN1NA), and nucleoprotein antigen (pNP). Dramatic IFN-γ-based cellular immune responses to both H5 and NP, largely dependent upon CD8+ T cells were seen in mice. Hemaggutination inhibition titers classically associated with protection (>1:40) were seen in all species. Responses in both ferrets and macaques demonstrate the ability of synthetic consensus antigens to induce antibodies capable of inhibiting divergent strains of the H5N1 subtype, and studies in the mouse and ferret demonstrate the ability of synthetic consensus vaccines to induce protection even in the absence of such neutralizing antibodies. After challenge, protection from morbidity and mortality was seen in mice and ferrets, with significant reductions in viral shedding and disease progression seen in vaccinated animals. Conclusions By combining several consensus influenza antigens with in vivo electroporation, we demonstrate that these antigens induce both protective cellular and humoral immune responses in mice, ferrets and non-human primates. We also demonstrate the ability of these antigens to protect from both morbidity and mortality in a ferret model of HPAI, in both the presence and absence of neutralizing antibody, which will be critical in responding to the antigenic drift that

  17. Antibodies to Plasmodium falciparum antigens predict a higher risk of malaria but protection from symptoms once parasitemic.

    PubMed

    Greenhouse, Bryan; Ho, Benjamin; Hubbard, Alan; Njama-Meya, Denise; Narum, David L; Lanar, David E; Dutta, Sheetij; Rosenthal, Philip J; Dorsey, Grant; John, Chandy C

    2011-07-01

    Associations between antibody responses to Plasmodium falciparum antigens and protection against symptomatic malaria have been difficult to ascertain, in part because antibodies are potential markers of both exposure to P. falciparum and protection against disease. We measured IgG responses to P. falciparum circumsporozoite protein, liver-stage antigen 1, apical-membrane antigen 1 (AMA-1), and merozoite surface proteins (MSP) 1 and 3, in children in Kampala, Uganda, and measured incidence of malaria before and after antibody measurement. Stronger responses to all 5 antigens were associated with an increased risk of clinical malaria (P < .01) because of confounding with prior exposure to P. falciparum. However, with use of another assessment, risk of clinical malaria once parasitemic, stronger responses to AMA-1, MSP-1, and MSP-3 were associated with protection (odds ratios, 0.34, 0.36, and 0.31, respectively, per 10-fold increase; P < .01). Analyses assessing antibodies in combination suggested that any protective effect of antibodies was overestimated by associations between individual responses and protection. Using the risk of symptomatic malaria once parasitemic as an outcome may improve detection of associations between immune responses and protection from disease. Immunoepidemiology studies designed to detect mechanisms of immune protection should integrate prior exposure into the analysis and evaluate multiple immune responses.

  18. Antibodies to Plasmodium falciparum Antigens Predict a Higher Risk of Malaria But Protection From Symptoms Once Parasitemic

    PubMed Central

    Hubbard, Alan; Njama-Meya, Denise; Narum, David L.; Lanar, David E.; Dutta, Sheetij; Rosenthal, Philip J.; Dorsey, Grant; John, Chandy C.

    2011-01-01

    (See the article by Bejon et al, on pages 9–18, and Bousema et al, on pages 1–3.) Background. Associations between antibody responses to Plasmodium falciparum antigens and protection against symptomatic malaria have been difficult to ascertain, in part because antibodies are potential markers of both exposure to P. falciparum and protection against disease. Methods. We measured IgG responses to P. falciparum circumsporozoite protein, liver-stage antigen 1, apical-membrane antigen 1 (AMA-1), and merozoite surface proteins (MSP) 1 and 3, in children in Kampala, Uganda, and measured incidence of malaria before and after antibody measurement. Results. Stronger responses to all 5 antigens were associated with an increased risk of clinical malaria (P < .01) because of confounding with prior exposure to P. falciparum. However, with use of another assessment, risk of clinical malaria once parasitemic, stronger responses to AMA-1, MSP-1, and MSP-3 were associated with protection (odds ratios, 0.34, 0.36, and 0.31, respectively, per 10-fold increase; P < .01). Analyses assessing antibodies in combination suggested that any protective effect of antibodies was overestimated by associations between individual responses and protection. Conclusions. Using the risk of symptomatic malaria once parasitemic as an outcome may improve detection of associations between immune responses and protection from disease. Immunoepidemiology studies designed to detect mechanisms of immune protection should integrate prior exposure into the analysis and evaluate multiple immune responses. PMID:21628654

  19. Role of surface protective antigen A in the pathogenesis of Erysipelothrix rhusiopathiae strain C43065.

    PubMed

    Borrathybay, Entomack; Gong, Feng-Juan; Zhang, Lei; Nazierbieke, Wulumuhan

    2015-02-01

    To clarify the role of surface protective antigen A (SpaA) in the pathogenesis of Erysipelothrix rhusiopathiae C43065 (serotype 2), the spaA deletion mutant of E. rhusiopathiae ΔspaA was constructed by homologous recombination. The virulence of the ΔspaA mutant decreased more than 76-fold compared with that of the wild-type strain C43065 in mice. The mutant strain was sensitive to the bactericidal action of swine serum, whereas the wild-type strain was resistant. The adhesion of wild-type strain to MEF cells was inhibited significantly by treatment with rabbit antiserum against recombinant SpaA (rSpaA) as compared with the treatment with normal rabbit serum, but the mutant strain was not affected. The mutant strain was readily taken up by mouse peritoneal macrophages in the normal rabbit serum, whereas the wild-type strain was resistant. Whereas the rabbit antiserum against rSpaA promoted the phagocytosis of wild-type strain by macrophages, the mutant strain was not affected. In addition, mice vaccinated with the formalin-killed mutant strain were provided 40% protection against challenge by the homologous virulent strain as compared with those with wild-type strain, NaOH-extracted antigen, or rSpaA, which provided more than 80% protection against the same infection. These suggested that SpaA has an important role in the pathogenesis of E. rhusiopathiae infection and could be a target for vaccination against swine erysipelas.

  20. Partial protective of chickens against Eimeria tenella challenge with recombinant EtMIC-1 antigen.

    PubMed

    Qi, N S; Wang, Y Y; Liao, S Q; Wu, C Y; Lv, M N; Li, J; Tong, Z X; Sun, M F

    2013-06-01

    Eimeria tenella microneme protein 1 (EtMIC-1) is highly conserved with TgMIC-2, which is involved in parasite binding specifically to host cells. Little is known about the immune responses and protective efficacy against E. tenella infection with EtMIC-1 antigen. In the present study, the recombinant proteins of E. tenella mature MIC-1 and adhesive domain (von Willebrand factor type A domain, EtMIC-1-VD) were obtained, protective efficacy against E.tenella infection and the mucosal immune response, which is induced in broilers was evaluated. The antibody levels and the transcription profiles of cytokine of chickens, such as interleukin-12 (IL-12) and interferon-γ (IFN-γ), were detected after being immunized three times with the recombinant EtMIC-1 and EtMIC-1-VD by ELISA assay and quantitative real-time PCR, respectively. The results showed that both groups of chickens, after being immunized with 100 μg EtMIC-1 or EtMIC-1-VD antigen, induced about tenfold higher IgG levels compared to the nonimmune groups. The transcription profiles of IL-12 and IFN-γ of the immunized groups were significantly higher than the control groups as well. The anticoccidial index of the group immunized with 100 μg EtMIC-1 and the group immunized with 100 μg EtMIC-1-VD were 167.2 and 165.5, respectively, which are significantly higher than low-dose immunized groups and challenged control groups. Our data suggests that VD domain is the key functional structure of EtMIC-1 that could trigger a significant humoral and cellular response against E. tenella infection, and EtMIC-1 had the potential in imparting partial protection in chickens against homologous challenge.

  1. Complement C3d Conjugation to Anthrax Protective Antigen Promotes a Rapid, Sustained, and Protective Antibody Response

    PubMed Central

    Kolla, Ravi V.; Chintalapati, Suresh; Sabet, Mojgan; Santelli, Eugenio; Liddington, Robert C.; David, Michael; Fierer, Joshua; Guiney, Donald; Rickert, Robert C.

    2007-01-01

    B. anthracis is the causative agent of anthrax. Pathogenesis is primarily mediated through the exotoxins lethal factor and edema factor, which bind protective antigen (PA) to gain entry into the host cell. The current anthrax vaccine (AVA, Biothrax™) consists of aluminum-adsorbed cell-free filtrates of unencapsulated B. anthracis, wherein PA is thought to be the principle target of neutralization. In this study, we evaluated the efficacy of the natural adjuvant, C3d, versus alum in eliciting an anti-PA humoral response and found that C3d conjugation to PA and emulsion in incomplete Freund's adjuvant (IFA) imparted superior protection from anthrax challenge relative to PA in IFA or PA adsorbed to alum. Relative to alum-PA, immunization of mice with C3d-PA/IFA augmented both the onset and sustained production of PA-specific antibodies, including neutralizing antibodies to the receptor-binding portion (domain 4) of PA. C3d-PA/IFA was efficacious when administered either i.p. or s.c., and in adolescent mice lacking a fully mature B cell compartment. Induction of PA-specific antibodies by C3d-PA/IFA correlated with increased efficiency of germinal center formation and plasma cell generation. Importantly, C3d-PA immunization effectively protected mice from intranasal challenge with B. anthracis spores, and was approximately 10-fold more effective than alum-PA immunization or PA/IFA based on dose challenge. These data suggest that incorporation of C3d as an adjuvant may overcome shortcomings of the currently licensed aluminum-based vaccine, and may confer protection in the early days following acute anthrax exposure. PMID:17940608

  2. A recombinant raccoon poxvirus vaccine expressing both Yersinia pestis F1 and truncated V antigens protects animals against lethal plague.

    USGS Publications Warehouse

    Rocke, Tonie E.; Kingstad-Bakke, B; Berlier, W; Osorio, J.E.

    2014-01-01

    In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307-a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis.. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas.

  3. A Recombinant Raccoon Poxvirus Vaccine Expressing both Yersinia pestis F1 and Truncated V Antigens Protects Animals against Lethal Plague

    PubMed Central

    Rocke, Tonie E.; Kingstad-Bakke, Brock; Berlier, Willy; Osorio, Jorge E.

    2014-01-01

    In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307—a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas. PMID:26344891

  4. A Recombinant Raccoon Poxvirus Vaccine Expressing both Yersinia pestis F1 and Truncated V Antigens Protects Animals against Lethal Plague.

    PubMed

    Rocke, Tonie E; Kingstad-Bakke, Brock; Berlier, Willy; Osorio, Jorge E

    2014-10-27

    In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307-a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas.

  5. Combination of the two schistosomal antigens Sm14 and Sm29 elicits significant protection against experimental Schistosoma mansoni infection.

    PubMed

    Ewaisha, Radwa E; Bahey-El-Din, Mohammed; Mossallam, Shereen F; Amer, Eglal I; Aboushleib, Hamida M; Khalil, Amal M

    2014-10-01

    Schistosomiasis continues to be a serious helminthic disease that is widespread in many regions in the world. Disease management relies mainly on early treatment with praziquantel, nevertheless, re-infection rates can still be high. An effective vaccine against Schistosoma mansoni is still lacking; a situation which hinders the efforts to eradicate the disease worldwide. Most investigators test S. mansoni antigens individually, rather than in combination, in their vaccine trials. A single-antigen vaccine is likely to elicit less protection against schistosomiasis than a multi-antigen vaccine. In the current study, we have selected two promising S. mansoni antigens, Sm14 and Sm29, and investigated their combination as a potential vaccine. Recombinant Sm14 and a truncated form of Sm29, designated TrSm29, were successfully expressed in Escherichiacoli. The two antigens were purified using affinity chromatography and administered to Swiss albino mice individually and in combination. Significant protection against S. mansoni infection was observed in mice immunized with the Sm14/TrSm29 combination in the presence/absence of the immunoadjuvant poly (I:C). The poly (I:C)-adjuvanted combination resulted in 40.3%, 68.2%, and 57.9% reduction in adult worm burden, liver egg burden and intestinal eggs, respectively. Granuloma size and count were also reduced besides improvement of the histopathological picture of livers of immunized mice. This study demonstrates the importance of using multi-antigen vaccines as an effective and simple approach to fulfill enhanced protection against schistosomiasis.

  6. Identification and characterization of Rhipicephalus (Boophilus) microplus candidate protective antigens for the control of cattle tick infestations.

    PubMed

    Almazán, Consuelo; Lagunes, Rodolfo; Villar, Margarita; Canales, Mario; Rosario-Cruz, Rodrigo; Jongejan, Frans; de la Fuente, José

    2010-01-01

    The cattle ticks, Rhipicephalus (Boophilus) spp., affect cattle production in tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The recombinant Rhipicephalus microplus Bm86 antigen has been shown to protect cattle against tick infestations. However, variable efficacy of Bm86-based vaccines against geographic tick strains has encouraged the research for additional tick-protective antigens. Herein, we describe the analysis of R. microplus glutathione-S transferase, ubiquitin (UBQ), selenoprotein W, elongation factor-1 alpha, and subolesin (SUB) complementary DNAs (cDNAs) by RNA interference (RNAi) in R. microplus and Rhipicephalus annulatus. Candidate protective antigens were selected for vaccination experiments based on the effect of gene knockdown on tick mortality, feeding, and fertility. Two cDNA clones encoding for UBQ and SUB were used for cattle vaccination and infestation with R. microplus and R. annulatus. Control groups were immunized with recombinant Bm86 or adjuvant/saline. The highest vaccine efficacy for the control of tick infestations was obtained for Bm86. Although with low immunogenic response, the results with the SUB vaccine encourage further investigations on the use of recombinant subolesin alone or in combination with other antigens for the control of cattle tick infestations. The UBQ peptide showed low immunogenicity, and the results of the vaccination trial were inconclusive to assess the protective efficacy of this antigen. These experiments showed that RNAi could be used for the selection of candidate tick-protective antigens. However, vaccination trials are necessary to evaluate the effect of recombinant antigens in the control of tick infestations, a process that requires efficient recombinant protein production and formulation systems.

  7. Generation of protective immune response against anthrax by oral immunization with protective antigen plant-based vaccine.

    PubMed

    Gorantala, Jyotsna; Grover, Sonam; Rahi, Amit; Chaudhary, Prerna; Rajwanshi, Ravi; Sarin, Neera Bhalla; Bhatnagar, Rakesh

    2014-04-20

    In concern with frequent recurrence of anthrax in endemic areas and inadvertent use of its spores as biological weapon, the development of an effective anthrax vaccine suitable for both human and veterinary needs is highly desirable. A simple oral delivery through expression in plant system could offer promising alternative to the current methods that rely on injectable vaccines extracted from bacterial sources. In the present study, we have expressed protective antigen (PA) gene in Indian mustard by Agrobacterium-mediated transformation and in tobacco by plastid transformation. Putative transgenic lines were verified for the presence of transgene and its expression by molecular analysis. PA expressed in transgenic lines was biologically active as evidenced by macrophage lysis assay. Intraperitoneal (i.p.) and oral immunization with plant PA in murine model indicated high serum PA specific IgG and IgA antibody titers. PA specific mucosal immune response was noted in orally immunized groups. Further, antibodies indicated lethal toxin neutralizing potential in-vitro and conferred protection against in-vivo toxin challenge. Oral immunization experiments demonstrated generation of immunoprotective response in mice. Thus, our study examines the feasibility of oral PA vaccine expressed in an edible plant system against anthrax. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Vaccinomics Approach to the Identification of Candidate Protective Antigens for the Control of Tick Vector Infestations and Anaplasma phagocytophilum Infection.

    PubMed

    Contreras, Marinela; Alberdi, Pilar; Fernández De Mera, Isabel G; Krull, Christoph; Nijhof, Ard; Villar, Margarita; De La Fuente, José

    2017-01-01

    Anaplasma phagocytophilum is an emerging tick-borne pathogen causing human granulocytic anaplasmosis (HGA), tick-borne fever (TBF) in small ruminants, and other forms of anaplasmosis in different domestic and wild animals. The main vectors of this pathogen are Ixodes tick species, particularly I. scapularis in the United States and I. ricinus in Europe. One of the main limitations for the development of effective vaccines for the prevention and control of A. phagocytophilum infection and transmission is the identification of effective tick protective antigens. The objective of this study was to apply a vaccinomics approach to I. scapularis-A. phagocytophilum interactions for the identification and characterization of candidate tick protective antigens for the control of vector infestations and A. phagocytophilum infection. The vaccinomics pipeline included the use of quantitative transcriptomics and proteomics data from uninfected and A. phagocytophilum-infected I. scapularis ticks for the selection of candidate protective antigens based on the variation in tick mRNA and protein levels in response to infection, their putative biological function, and the effect of antibodies against these proteins on tick cell apoptosis and pathogen infection. The characterization of selected candidate tick protective antigens included the identification and characterization of I. ricinus homologs, functional characterization by different methodologies including RNA interference, immunofluorescence, gene expression profiling, and artificial tick feeding on rabbit antibodies against the recombinant antigens to select the candidates for vaccination trials. The vaccinomics pipeline developed in this study resulted in the identification of two candidate tick protective antigens that could be selected for future vaccination trials. The results showed that I. scapularis lipocalin (ISCW005600) and lectin pathway inhibitor (AAY66632) and I. ricinus homologs constitute candidate protective

  9. Vaccinomics Approach to the Identification of Candidate Protective Antigens for the Control of Tick Vector Infestations and Anaplasma phagocytophilum Infection

    PubMed Central

    Contreras, Marinela; Alberdi, Pilar; Fernández De Mera, Isabel G.; Krull, Christoph; Nijhof, Ard; Villar, Margarita; De La Fuente, José

    2017-01-01

    Anaplasma phagocytophilum is an emerging tick-borne pathogen causing human granulocytic anaplasmosis (HGA), tick-borne fever (TBF) in small ruminants, and other forms of anaplasmosis in different domestic and wild animals. The main vectors of this pathogen are Ixodes tick species, particularly I. scapularis in the United States and I. ricinus in Europe. One of the main limitations for the development of effective vaccines for the prevention and control of A. phagocytophilum infection and transmission is the identification of effective tick protective antigens. The objective of this study was to apply a vaccinomics approach to I. scapularis-A. phagocytophilum interactions for the identification and characterization of candidate tick protective antigens for the control of vector infestations and A. phagocytophilum infection. The vaccinomics pipeline included the use of quantitative transcriptomics and proteomics data from uninfected and A. phagocytophilum-infected I. scapularis ticks for the selection of candidate protective antigens based on the variation in tick mRNA and protein levels in response to infection, their putative biological function, and the effect of antibodies against these proteins on tick cell apoptosis and pathogen infection. The characterization of selected candidate tick protective antigens included the identification and characterization of I. ricinus homologs, functional characterization by different methodologies including RNA interference, immunofluorescence, gene expression profiling, and artificial tick feeding on rabbit antibodies against the recombinant antigens to select the candidates for vaccination trials. The vaccinomics pipeline developed in this study resulted in the identification of two candidate tick protective antigens that could be selected for future vaccination trials. The results showed that I. scapularis lipocalin (ISCW005600) and lectin pathway inhibitor (AAY66632) and I. ricinus homologs constitute candidate protective

  10. Multivalent Chromosomal Expression of the Clostridium botulinum Serotype A Neurotoxin Heavy-Chain Antigen and the Bacillus anthracis Protective Antigen in Lactobacillus acidophilus

    PubMed Central

    Klaenhammer, Todd R.

    2016-01-01

    ABSTRACT Clostridium botulinum and Bacillus anthracis produce potent toxins that cause severe disease in humans. New and improved vaccines are needed for both of these pathogens. For mucosal vaccine delivery using lactic acid bacteria, chromosomal expression of antigens is preferred over plasmid-based expression systems, as chromosomal expression circumvents plasmid instability and the need for antibiotic pressure. In this study, we constructed three strains of Lactobacillus acidophilus NCFM expressing from the chromosome (i) the nontoxic host receptor-binding domain of the heavy chain of Clostridium botulinum serotype A neurotoxin (BoNT/A-Hc), (ii) the anthrax protective antigen (PA), and (iii) both the BoNT/A-Hc and the PA. The BoNT/A-Hc vaccine cassette was engineered to contain the signal peptide from the S-layer protein A from L. acidophilus and a dendritic-cell-targeting peptide. A chromosomal region downstream of lba0889 carrying a highly expressed enolase gene was selected for insertion of the vaccine cassettes. Western blot analysis confirmed the heterologous expression of the two antigens from plasmid and chromosome locations. Stability assays demonstrated loss of the vaccine cassettes from expression plasmids without antibiotic maintenance. RNA sequencing showed high expression of each antigen and that insertion of the vaccine cassettes had little to no effect on the transcription of other genes in the chromosome. This study demonstrated that chromosomal integrative recombinant strains are promising vaccine delivery vehicles when targeted into high-expression chromosomal regions. Levels of expression match high-copy-number plasmids and eliminate the requirement for antibiotic selective maintenance of recombinant plasmids. IMPORTANCE Clostridium botulinum and Bacillus anthracis produce potent neurotoxins that pose a biochemical warfare concern; therefore, effective vaccines against these bacteria are required. Chromosomal expression of antigens is

  11. Multivalent Chromosomal Expression of the Clostridium botulinum Serotype A Neurotoxin Heavy-Chain Antigen and the Bacillus anthracis Protective Antigen in Lactobacillus acidophilus.

    PubMed

    O'Flaherty, Sarah; Klaenhammer, Todd R

    2016-10-15

    Clostridium botulinum and Bacillus anthracis produce potent toxins that cause severe disease in humans. New and improved vaccines are needed for both of these pathogens. For mucosal vaccine delivery using lactic acid bacteria, chromosomal expression of antigens is preferred over plasmid-based expression systems, as chromosomal expression circumvents plasmid instability and the need for antibiotic pressure. In this study, we constructed three strains of Lactobacillus acidophilus NCFM expressing from the chromosome (i) the nontoxic host receptor-binding domain of the heavy chain of Clostridium botulinum serotype A neurotoxin (BoNT/A-Hc), (ii) the anthrax protective antigen (PA), and (iii) both the BoNT/A-Hc and the PA. The BoNT/A-Hc vaccine cassette was engineered to contain the signal peptide from the S-layer protein A from L. acidophilus and a dendritic-cell-targeting peptide. A chromosomal region downstream of lba0889 carrying a highly expressed enolase gene was selected for insertion of the vaccine cassettes. Western blot analysis confirmed the heterologous expression of the two antigens from plasmid and chromosome locations. Stability assays demonstrated loss of the vaccine cassettes from expression plasmids without antibiotic maintenance. RNA sequencing showed high expression of each antigen and that insertion of the vaccine cassettes had little to no effect on the transcription of other genes in the chromosome. This study demonstrated that chromosomal integrative recombinant strains are promising vaccine delivery vehicles when targeted into high-expression chromosomal regions. Levels of expression match high-copy-number plasmids and eliminate the requirement for antibiotic selective maintenance of recombinant plasmids. Clostridium botulinum and Bacillus anthracis produce potent neurotoxins that pose a biochemical warfare concern; therefore, effective vaccines against these bacteria are required. Chromosomal expression of antigens is preferred over plasmid

  12. Small Molecule Inhibitors of Bacillus anthracis Protective Antigen Proteolytic Activation and Oligomerization

    PubMed Central

    Wein, Alexander N.; Williams, Brian N.; Liu, Shihui; Ermolinsky, Boris; Provenzano, Daniele; Abagyan, Ruben; Orry, Andrew; Leppla, Stephen H.; Peredelchuk, Michael

    2012-01-01

    Protective antigen (PA), lethal factor, and edema factor, the protein toxins of Bacillus anthracis, are among its most important virulence factors and play a key role in infection. We performed a virtual ligand screen of a library of 10,000 members to identify compounds predicted to bind to PA and prevent its oligomerization. Four of these compounds slowed PA association in a FRET-based oligomerization assay, and two of those protected cells from intoxication at concentrations of 1–10 μM. Exploration of the protective mechanism by Western blot showed decreased SDS-resistant PA oligomer on cells, and surprisingly, decreased amounts of activated PA. In vitro assays showed that one of the inhibitors blocked furin-mediated cleavage of PA, apparently through its binding to the PA substrate. Thus, we have identified inhibitors that can independently block both PA’s cleavage by furin and its subsequent oligomerization. Lead optimization on these two backbones may yield compounds with high activity and specificity for the anthrax toxins. PMID:22954387

  13. Total Leishmania antigens with Poly(I:C) induce Th1 protective response.

    PubMed

    Sanchez, M V; Eliçabe, R J; Di Genaro, M S; Germanó, M J; Gea, S; García Bustos, M F; Salomón, M C; Scodeller, E A; Cargnelutti, D E

    2017-11-01

    Our proposal was to develop a vaccine based on total Leishmania antigens (TLA) adjuvanted with polyinosinic-polycytidylic acid [Poly(I:C)] able to induce a Th1 response which can provide protection against Leishmania infection. Mice were vaccinated with two doses of TLA-Poly(I:C) administered by subcutaneous route at 3-week interval. Humoral and cellular immune responses induced by the immunization were measured. The protective efficacy of the vaccine was evaluated by challenging mice with infective promastigotes of Leishmania (Leishmania) amazonensis into the footpad. Mice vaccinated with TLA-Poly(I:C) showed a high anti-Leishmania IgG titre, as well as increased IgG1 and IgG2a subclass titres compared with mice vaccinated with the TLA alone. The high IgG2a indicated a Th1 bias response induced by the TLA-Poly(I:C) immunization. Accordingly, the cellular immune response elicited by the formulation was characterized by an increased production of IFN-γ and no significant production of IL-4. The TLA-Poly(I:C) immunization elicited good protection, which was associated with decreased footpad swelling, a lower parasite load and a reduced histopathological alteration in the footpad. Our findings demonstrate a promising vaccine against cutaneous leishmaniasis that is relatively economic and easy to develop and which should be taken into account for preventing leishmaniasis in developing countries. © 2017 John Wiley & Sons Ltd.

  14. Preliminary protective capacity study of a Dicrocoelium dendriticum antigenic protein in hamsters.

    PubMed

    González-Lanza, C; Manga-González, M Y; Revilla-Nuín, B

    2006-11-01

    The purpose of this study was to evaluate the protective capacity of 130 kDa Dicrocoelium dendriticum protein in hamsters experimentally infected with this parasite. Forty hamsters divided into four groups of ten animals each were used: G1 (control), G2 (infected), G3 (immunized with Freund's adjuvant and infected), G4 (130 kDa protein vaccinated + adjuvant and infected). Infection with 40 metacercariae/hamster was carried out 4 weeks after the last immunization. Parasitological studies [number of eggs per gram (epg) and worm burden] and biochemical parameters (total proteins, albumin, and total bilirubin), hepatic enzymes [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)], and total IgG levels were determined. A reduction in epg in G3 and G4 was observed 16 weeks postinfection with the higher reduction percentage in the latter (25.2%). No statistically significant differences were detected in the number of recovered worms among groups, although the mean was slightly less in G4 (12.2 +/- 2.08, mean +/- SE) than in G2 (15.4 +/- 2.90). In G4, global protection was 20.9% and an increase in AST and ALT levels was observed. Total IgG levels were similar in the three infected groups. The protection obtained was inadequate, so the antigen dose, immunization-infection period, adjuvants, and immunization route must be optimized.

  15. Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity.

    PubMed

    Maynard, Jennifer A; Maassen, Catharina B M; Leppla, Stephen H; Brasky, Kathleen; Patterson, Jean L; Iverson, Brent L; Georgiou, George

    2002-06-01

    The tripartite toxin produced by Bacillus anthracis is the key determinant in the etiology of anthrax. We have engineered a panel of toxin-neutralizing antibodies, including single-chain variable fragments (scFvs) and scFvs fused to a human constant kappa domain (scAbs), that bind to the protective antigen subunit of the toxin with equilibrium dissociation constants (K(d)) between 63 nM and 0.25 nM. The entire antibody panel showed high serum, thermal, and denaturant stability. In vitro, post-challenge protection of macrophages from the action of the holotoxin correlated with the K(d) of the scFv variants. Strong correlations among antibody construct affinity, serum half-life, and protection were also observed in a rat model of toxin challenge. High-affinity toxin-neutralizing antibodies may be of therapeutic value for alleviating the symptoms of anthrax toxin in infected individuals and for medium-term prophylaxis to infection.

  16. Blocking anthrax lethal toxin at the protective antigen channel by using structure-inspired drug design.

    PubMed

    Karginov, Vladimir A; Nestorovich, Ekaterina M; Moayeri, Mahtab; Leppla, Stephen H; Bezrukov, Sergey M

    2005-10-18

    Bacillus anthracis secretes three polypeptides: protective antigen (PA), lethal factor (LF), and edema factor (EF), which interact at the surface of mammalian cells to form toxic complexes. LF and EF are enzymes that target substrates within the cytosol; PA provides a heptameric pore to facilitate LF and EF transport into the cytosol. Other than administration of antibiotics shortly after exposure, there is currently no approved effective treatment for inhalational anthrax. Here we demonstrate an approach to disabling the toxin: high-affinity blockage of the PA pore by a rationally designed low-molecular weight compound that prevents LF and EF entry into cells. Guided by the sevenfold symmetry and predominantly negative charge of the PA pore, we synthesized small cyclic molecules of sevenfold symmetry, beta-cyclodextrins chemically modified to add seven positive charges. By channel reconstitution and high-resolution conductance recording, we show that per-6-(3-aminopropylthio)-beta-cyclodextrin interacts strongly with the PA pore lumen, blocking PA-induced transport at subnanomolar concentrations (in 0.1 M KCl). The compound protected RAW 264.7 mouse macrophages from cytotoxicity of anthrax lethal toxin (= PA + LF). More importantly, it completely protected the highly susceptible Fischer F344 rats from lethal toxin. We anticipate that this approach will serve as the basis for a structure-directed drug discovery program to find new and effective treatments for anthrax.

  17. TSOL18/HP6-Tsol, an immunogenic Taenia solium oncospheral adhesion protein and potential protective antigen.

    PubMed

    Parkhouse, R Michael E; Bonay, Pedro; González, Luis Miguel; Ferrer, Elizabeth; Gárate, Teresa; Aguilar, Cruz M; Cortez A, Milagros M; Harrison, Leslie J S

    2008-04-01

    In this study, we employed Taenia solium mRNA extracted from a tapeworm of Venezuelan origin to clone express and test the recombinant protein of the T. solium homologue of the 18-kDa oncospheral adhesion molecule of Taenia saginata (HP6-Tsag/TSA18). We first confirm the conserved nature of the sequence of the T. solium homologue (TSOL18/HP6-Tsol) and demonstrate that the recombinant protein, which, as with its T. saginata homologue, is characterised by a fibronectin type III homology region, functions as an adhesion molecule. This emphasises the possible importance of TSOL18/HP6-Tsol in tissue invasion, thus providing a rational explanation for its efficacy as a vaccine. As protection against Taenia spp., oncospheres is antibody mediated, logically, therefore, TSOL18/HP6-Tsol may also serve as a diagnostic antigen, as is indeed the case for recombinant HP6-Tsag/TSA18.

  18. Mucosal Immunization with Iron Receptor Antigens Protects against Urinary Tract Infection

    PubMed Central

    Smith, Sara N.; Mobley, Harry L. T.

    2009-01-01

    Uncomplicated infections of the urinary tract, caused by uropathogenic Escherichia coli, are among the most common diseases requiring medical intervention. A preventive vaccine to reduce the morbidity and fiscal burden these infections have upon the healthcare system would be beneficial. Here, we describe the results of a large-scale selection process that incorporates bioinformatic, genomic, transcriptomic, and proteomic screens to identify six vaccine candidates from the 5379 predicted proteins encoded by uropathogenic E. coli strain CFT073. The vaccine candidates, ChuA, Hma, Iha, IreA, IroN, and IutA, all belong to a functional class of molecules that is involved in iron acquisition, a process critical for pathogenesis in all microbes. Intranasal immunization of CBA/J mice with these outer membrane iron receptors elicited a systemic and mucosal immune response that included the production of antigen-specific IgM, IgG, and IgA antibodies. The cellular response to vaccination was characterized by the induction and secretion of IFN-γ and IL-17. Of the six potential vaccine candidates, IreA, Hma, and IutA provided significant protection from experimental infection. In immunized animals, class-switching from IgM to IgG and production of antigen-specific IgA in the urine represent immunological correlates of protection from E. coli bladder colonization. These findings are an important first step toward the development of a subunit vaccine to prevent urinary tract infections and demonstrate how targeting an entire class of molecules that are collectively required for pathogenesis may represent a fundamental strategy to combat infections. PMID:19806177

  19. Protective activity of the purified protein antigen of Erysipelothrix rhusiopathiae in pigs.

    PubMed

    Yamazaki, Y; Sato, H; Sakakura, H; Shigeto, K; Nakano, K; Saito, H; Maehara, N

    1999-02-01

    We purified the protein antigen (P64), which contains 66 and 64 kDa proteins, from the alkaline extract (AE) of whole cells of Erysipelothrix rhusiopathiae strain Agata (serovar 5) to determine the protective activity of the antigen against E. rhusiopathiae infection in pigs. The serum titre of antibody against P64 rapidly increased in pigs immunized with 500 and 100 micrograms of P64 and reached maximum values at 3 weeks after the first immunization (1 week after the second immunization). However, the serum antibody titres were not increased in pigs immunized with 20 micrograms of P64 and in nonimmunized pigs. In the pigs immunized with live cell vaccine (acriflavin-fast attenuated strain Koganei 65-0.15), the serum titres of antibody against P64 also increased at 1-2 weeks after immunization. In a pig challenge test performed on immunized and nonimmunized pigs, all nonimmunized pigs showed typical clinical signs of swine erysipelas (fever, erysipeloid, arthritis), while all pigs immunized with 500 and 100 micrograms of P64 and live cell vaccine showed no clinical signs of this disease. In Western blot analysis, sera from pigs immunized with P64 and live cell vaccine strongly reacted with the 64 kDa protein. In contrast, the serum from nonimmunized pigs did not react with any proteins. From these results, it was suggested that a specific antibody against the 64 kDa protein could be increased in pigs immunized with P64 or live cell vaccine and that this anti-P64 antibody has a strong protective effect against E. rhusiopathiae infection in pigs.

  20. Breadth of humoral response and antigenic targets of sporozoite-inhibitory antibodies associated with sterile protection induced by controlled human malaria infection

    PubMed Central

    Peng, Kaitian; Goh, Yun Shan; Siau, Anthony; Franetich, Jean-François; Chia, Wan Ni; Ong, Alice Soh Meoy; Malleret, Benoit; Wu, Ying Ying; Snounou, Georges; Hermsen, Cornelus C.; Adams, John H.; Mazier, Dominique; Preiser, Peter R.; Sauerwein, Robert W.; Grüner, Anne-Charlotte; Rénia, Laurent

    2017-01-01

    The development of an effective malaria vaccine has remained elusive even until today. This is due to our incomplete understanding of the immune mechanisms that confer and/or correlate with protection. Human volunteers have been protected experimentally from a subsequent challenge by immunization with Plasmodium falciparum sporozoites under drug cover. Here, we demonstrate that sera from the protected individuals contain neutralizing antibodies against the pre erythrocytic stage. To identify the antigen(s) recognized by these antibodies, a newly developed library of P. falciparum antigens was screened with the neutralizing sera. Antibodies from protected individuals recognized a broad antigenic repertoire of which three antigens, PfMAEBL, PfTRAP and PfSEA1 were recognized by most protected individuals. As a proof of principle, we demonstrated that anti-PfMAEBL antibodies block liver stage development in human hepatocytes. Thus, these antigens identified are promising targets for vaccine development against malaria. PMID:27130708

  1. A few reflections on protective measurements and more.

    NASA Astrophysics Data System (ADS)

    Genovese, M.

    2017-08-01

    Quantum measurement represents the most severe and charming problem of foundations of quantum mechanics. Several issues ranging from the collapse of the wave function to the ontic meaning of the wave function remain unclarified. Here we briefly summarize some recent ideas about this problem, with particular attention to protective measurements and their meaning.

  2. A plant based protective antigen [PA(dIV)] vaccine expressed in chloroplasts demonstrates protective immunity in mice against anthrax.

    PubMed

    Gorantala, Jyotsna; Grover, Sonam; Goel, Divya; Rahi, Amit; Jayadev Magani, Sri Krishna; Chandra, Subhash; Bhatnagar, Rakesh

    2011-06-15

    The currently available anthrax vaccines are limited by being incompletely characterized, potentially reactogenic and have an expanded dosage schedule. Plant based vaccines offer safe alternative for vaccine production. In the present study, we expressed domain IV of Bacillus anthracis protective antigen gene [PA(dIV)] in planta (by nuclear agrobacterium and chloroplast transformation) and E. coli [rPA(dIV)]. The presence of transgene and the expression of PA(dIV) in planta was confirmed by molecular analysis. Expression levels up to 5.3% of total soluble protein (TSP) were obtained with AT rich (71.8% AT content) PA(dIV) gene in transplastomic plants while 0.8% of TSP was obtained in nuclear transformants. Further, we investigated the protective response of plant and E. coli derived PA(dIV) in mice by intraperitoneal (i.p.) and oral immunizations with or without adjuvant. Antibody titers of >10(4) were induced upon i.p. and oral immunizations with plant derived PA(dIV) and oral immunization with E. coli derived PA(dIV). Intraperitoneal injections with adjuvanted E. coli derived PA(dIV), generated highest antibody titers of >10(5). All the immunized groups demonstrated predominant IgG1 titers over IgG2a indicating a polarized Th2 type response. We also evaluated the mucosal antibody response in orally immunized groups. When fecal extracts were analyzed, low sIgA titer was demonstrated in adjuvanted plant and E. coli derived PA(dIV) groups. Further, PA(dIV) antisera enhanced B. anthracis spore uptake by macrophages in vitro and also demonstrated an anti-germinating effect suggesting a potent role at mucosal surfaces. The antibodies from various groups were efficient in neutralizing the lethal toxin in vitro. When mice were challenged with B. anthracis, mice immunized with adjuvanted plant PA(dIV) imparted 60% and 40% protection while E. coli derived PA(dIV) conferred 100% and 80% protection upon i.p. and oral immunizations. Thus, our study is the first attempt in

  3. Intranasal immunization with protective antigen of Bacillus anthracis induces a long-term immunological memory response.

    PubMed

    Woo, Sun-Je; Kang, Seok-Seong; Park, Sung-Moo; Yang, Jae Seung; Song, Man Ki; Yun, Cheol-Heui; Han, Seung Hyun

    2015-10-01

    Although intranasal vaccination has been shown to be effective for the protection against inhalational anthrax, establishment of long-term immunity has yet to be achieved. Here, we investigated whether intranasal immunization with recombinant protective antigen (rPA) of Bacillus anthracis induces immunological memory responses in the mucosal and systemic compartments. Intranasal immunization with rPA plus cholera toxin (CT) sustained PA-specific antibody responses for 6 months in lung, nasal washes, and vaginal washes as well as serum. A significant induction of PA-specific memory B cells was observed in spleen, cervical lymph nodes (CLNs) and lung after booster immunization. Furthermore, intranasal immunization with rPA plus CT remarkably generated effector memory CD4(+) T cells in the lung. PA-specific CD4(+) T cells preferentially increased the expression of Th1- and Th17-type cytokines in lung, but not in spleen or CLNs. Collectively, the intranasal immunization with rPA plus CT promoted immunologic memory responses in the mucosal and systemic compartments, providing long-term immunity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Reduced Human Leukocyte Antigen (HLA) Protection in Gulf War Illness (GWI)

    PubMed Central

    Georgopoulos, Apostolos P.; James, Lisa M.; Mahan, Margaret Y.; Joseph, Jasmine; Georgopoulos, Angeliki; Engdahl, Brian E.

    2015-01-01

    Background Gulf War Illness (GWI) is a disease of unknown etiology with symptoms suggesting the involvement of an immune process. Here we tested the hypothesis that Human Leukocyte Antigen (HLA) composition might differ between veterans with and without GWI. Methods We identified 144 unique alleles of Class I and II HLA genes in 82 veterans (66 with and 16 without GWI). We tested the hypothesis that a subset of HLA alleles may classify veterans in their respective group using a stepwise linear discriminant analysis. In addition, each participant rated symptom severity in 6 domains according to established GWI criteria, and an overall symptom severity was calculated. Findings We found 6 Class II alleles that classified participants 84.1% correctly (13/16 control and 56/66 GWI). The number of copies of the 6 alleles was significantly higher in the control group, suggesting a protective role. This was supported by a significant negative dependence of overall symptom severity on the number of allele copies, such that symptom severity was lower in participants with larger numbers of allele copies. Interpretation These results indicate a reduced HLA protection (i.e. genetic susceptibility) in veterans with GWI. Funding University of Minnesota and U.S. Department of Veterans Affairs. PMID:26870819

  5. Identification of new dominant-negative mutants of anthrax protective antigen using directed evolution.

    PubMed

    Wu, Gaobing; Feng, Chunfang; Cao, Sha; Guo, Aizhen; Liu, Ziduo

    2012-11-01

    The anthrax toxin is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema toxin (EF). The PA moiety carries EF and LF into the cytosol of mammalian cells via a mechanism that depends on the oligomerization of PA and transmembrane pore formation by the PA oligomer. Certain mutants of PA, termed dominant-negative (DN) mutants, can co-oligomerize with wild-type PA and disrupt the translocation ability of the pore. Here, we constructed a PA mutant library by introducing random mutations into domain II of PA and screened three new DN mutants of PA: V377E, T380S, and I432C. All the mutants inhibited the anthrax toxin action against sensitive cells. V377E had the strongest inhibitory effect and was further confirmed to be able to protect mice against a challenge with anthrax lethal toxin. Furthermore, we functionally characterized these mutants. The result showed that these mutations did not impair proteolytic activation or oligomer formation of PA, but impeded the prepore-pore conversion of the oligomer. These DN mutants of PA identified in our study may provide valuable information for elucidating the structure-function relationship of PA and for designing therapeutics for anthrax treatment.

  6. Metal oxide sunscreens protect skin by absorption, not by reflection or scattering.

    PubMed

    Cole, Curtis; Shyr, Thomas; Ou-Yang, Hao

    2016-01-01

    The inorganic metal oxide sunscreens titanium dioxide and zinc oxide have been considered to protect against sunburning ultraviolet radiation by physically reflecting/scattering the incident photons and thus protecting the skin. Earlier publications suggested, however, that the primary action of UV protection by these sunscreen agents is through absorption and not by reflection. The purpose of this work was to quantitate the contributions of each of these modes of action to the protection provided by inorganic UV sunscreen filters. An optical integrating sphere was used to measure the transmission and the reflectance of titanium dioxide and zinc oxide. The average range of reflection for zinc oxide and titanium dioxide throughout the UV range was only 4-5% (less than SPF 2), providing minimal UV protection via this mechanism. The remainder of the UV protection is provided by semiconductor band gap mediated absorbance of the UV photons. At wavelengths above the semiconductor band gap absorption energy levels (in the long UVA and visible wavelengths), they are predominantly reflectors of light (up to 60% reflection) and non-absorbing. Titanium dioxide and zinc oxide provide UV protection primarily via absorption of UV radiation and not through significant reflection or scattering. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Protective immunity against Taenia crassiceps murine cysticercosis induced by DNA vaccination with a Taenia saginata tegument antigen.

    PubMed

    Rosas, Gabriela; Fragoso, Gladis; Garate, Teresa; Hernández, Beatriz; Ferrero, Patricia; Foster-Cuevas, Mildred; Parkhouse, R Michael E; Harrison, Leslie J S; Briones, Sergio López; González, Luis Miguel; Sciutto, Edda

    2002-11-01

    This study investigated the protective capacity of the recombinant Taenia saginata Tso18 antigen administered as a DNA vaccine in the Taenia crassiceps murine model of cysticercosis. This Tso18 DNA sequence, isolated from a T. saginata oncosphere cDNA library, has homologies with Taenia solium and Echinococcus sp. It was cloned in the pcDNA3.1 plasmid and injected once intramuscularly into mice. Compared to saline-vaccinated control mice, immunization reduced the parasite burden by 57.3-81.4%, while lower levels of non-specific protection were induced in control mice injected with the plasmid pcDNA3.1 (18.8-33.1%) or a plasmid with irrelevant construct, pcDNA3.1/3D15 (33.4-38.8%). Importantly, significant levels of protection were observed between the pcDNA3.1/Tso18 plasmid and pcDNA3.1/3D15 plasmid immunized mice. Mice immunized with pTso18 synthesized low levels of, primarily IgG1 sub-class, antibodies. These antibodies were shown to recognize a 66 kDa antigen fraction of T. crassiceps and T. solium. Splenocytes enriched in both CD4+CD8- and CD4-CD8+ T cells from these vaccinated mice proliferated in vitro when exposed to antigens from both T. solium and T. crassiceps cestodes. Immunolocalization studies revealed the Tso18 antigen in oncospheres of T. saginata and T. solium, in the adult tapeworm and in the tegument of T. solium cysticerci. The protective capacity of this antigen and its extensive distribution in different stages, species and genera of cestodes points to the potential of Tso18 antigen for the possible design of a vaccine against cestodes.

  8. Recombinant Salmonella Expressing Burkholderia mallei LPS O Antigen Provides Protection in a Murine Model of Melioidosis and Glanders

    PubMed Central

    Moustafa, Dina A.; Scarff, Jennifer M.; Garcia, Preston P.; Cassidy, Sara K. B.; DiGiandomenico, Antonio; Waag, David M.; Inzana, Thomas J.; Goldberg, Joanna B.

    2015-01-01

    Burkholderia pseudomallei and Burkholderia mallei are the etiologic agents of melioidosis and glanders, respectively. These bacteria are highly infectious via the respiratory route and can cause severe and often fatal diseases in humans and animals. Both species are considered potential agents of biological warfare; they are classified as category B priority pathogens. Currently there are no human or veterinary vaccines available against these pathogens. Consequently efforts are directed towards the development of an efficacious and safe vaccine. Lipopolysaccharide (LPS) is an immunodominant antigen and potent stimulator of host immune responses. B. mallei express LPS that is structurally similar to that expressed by B. pseudomallei, suggesting the possibility of constructing a single protective vaccine against melioidosis and glanders. Previous studies of others have shown that antibodies against B. mallei or B. pseudomallei LPS partially protect mice against subsequent lethal virulent Burkholderia challenge. In this study, we evaluated the protective efficacy of recombinant Salmonella enterica serovar Typhimurium SL3261 expressing B. mallei O antigen against lethal intranasal infection with Burkholderia thailandensis, a surrogate for biothreat Burkholderia spp. in a murine model that mimics melioidosis and glanders. All vaccine-immunized mice developed a specific antibody response to B. mallei and B. pseudomallei O antigen and to B. thailandensis and were significantly protected against challenge with a lethal dose of B. thailandensis. These results suggest that live-attenuated SL3261 expressing B. mallei O antigen is a promising platform for developing a safe and effective vaccine. PMID:26148026

  9. Superoxide dismutase SodB is a protective antigen against Campylobacter jejuni colonisation in chickens

    PubMed Central

    Chintoan-Uta, Cosmin; Cassady-Cain, Robin L.; Al-Haideri, Halah; Watson, Eleanor; Kelly, David J.; Smith, David G.E.; Sparks, Nick H.C.; Kaiser, Pete; Stevens, Mark P.

    2015-01-01

    Campylobacter is the leading cause of foodborne diarrhoeal illness in the developed world and consumption or handling of contaminated poultry meat is the principal source of infection. Strategies to control Campylobacter in broilers prior to slaughter are urgently required and are predicted to limit the incidence of human campylobacteriosis. Towards this aim, a purified recombinant subunit vaccine based on the superoxide dismutase (SodB) protein of C. jejuni M1 was developed and tested in White Leghorn birds. Birds were vaccinated on the day of hatch and 14 days later with SodB fused to glutathione S-transferase (GST) or purified GST alone. Birds were challenged with C. jejuni M1 at 28 days of age and caecal Campylobacter counts determined at weekly intervals. Across three independent trials, the vaccine induced a statistically significant 1 log10 reduction in caecal Campylobacter numbers in vaccinated birds compared to age-matched GST-vaccinated controls. Significant induction of antigen-specific serum IgY was detected in all vaccinated birds, however the magnitude and timing of SodB-specific IgY did not correlate with lower numbers of C. jejuni. Antibodies from SodB-vaccinated chickens detected the protein in the periplasm and not membrane fractions or on the bacterial surface, suggesting that the protection observed may not be strictly antibody-mediated. SodB may be useful as a constituent of vaccines for control of C. jejuni infection in broiler birds, however modest protection was observed late relative to the life of broiler birds and further studies are required to potentiate the magnitude and timing of protection. PMID:26458797

  10. Antibody to a conserved antigenic target is protective against diverse prokaryotic and eukaryotic pathogens

    PubMed Central

    Cywes-Bentley, Colette; Skurnik, David; Zaidi, Tanweer; Roux, Damien; DeOliveira, Rosane B.; Garrett, Wendy S.; Lu, Xi; O’Malley, Jennifer; Kinzel, Kathryn; Zaidi, Tauqeer; Rey, Astrid; Perrin, Christophe; Fichorova, Raina N.; Kayatani, Alexander K. K.; Maira-Litràn, Tomas; Gening, Marina L.; Tsvetkov, Yury E.; Nifantiev, Nikolay E.; Bakaletz, Lauren O.; Pelton, Stephen I.; Golenbock, Douglas T.; Pier, Gerald B.

    2013-01-01

    Microbial capsular antigens are effective vaccines but are chemically and immunologically diverse, resulting in a major barrier to their use against multiple pathogens. A β-(1→6)–linked poly-N-acetyl-d-glucosamine (PNAG) surface capsule is synthesized by four proteins encoded in genetic loci designated intercellular adhesion in Staphylococcus aureus or polyglucosamine in selected Gram-negative bacterial pathogens. We report that many microbial pathogens lacking an identifiable intercellular adhesion or polyglucosamine locus produce PNAG, including Gram-positive, Gram-negative, and fungal pathogens, as well as protozoa, e.g., Trichomonas vaginalis, Plasmodium berghei, and sporozoites and blood-stage forms of Plasmodium falciparum. Natural antibody to PNAG is common in humans and animals and binds primarily to the highly acetylated glycoform of PNAG but is not protective against infection due to lack of deposition of complement opsonins. Polyclonal animal antibody raised to deacetylated glycoforms of PNAG and a fully human IgG1 monoclonal antibody that both bind to native and deacetylated glycoforms of PNAG mediated complement-dependent opsonic or bactericidal killing and protected mice against local and/or systemic infections by Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Neisseria meningitidis serogroup B, Candida albicans, and P. berghei ANKA, and against colonic pathology in a model of infectious colitis. PNAG is also a capsular polysaccharide for Neisseria gonorrhoeae and nontypable Hemophilus influenzae, and protects cells from environmental stress. Vaccination targeting PNAG could contribute to immunity against serious and diverse prokaryotic and eukaryotic pathogens, and the conserved production of PNAG suggests that it is a critical factor in microbial biology. PMID:23716675

  11. Superoxide dismutase SodB is a protective antigen against Campylobacter jejuni colonisation in chickens.

    PubMed

    Chintoan-Uta, Cosmin; Cassady-Cain, Robin L; Al-Haideri, Halah; Watson, Eleanor; Kelly, David J; Smith, David G E; Sparks, Nick H C; Kaiser, Pete; Stevens, Mark P

    2015-11-17

    Campylobacter is the leading cause of foodborne diarrhoeal illness in the developed world and consumption or handling of contaminated poultry meat is the principal source of infection. Strategies to control Campylobacter in broilers prior to slaughter are urgently required and are predicted to limit the incidence of human campylobacteriosis. Towards this aim, a purified recombinant subunit vaccine based on the superoxide dismutase (SodB) protein of C. jejuni M1 was developed and tested in White Leghorn birds. Birds were vaccinated on the day of hatch and 14 days later with SodB fused to glutathione S-transferase (GST) or purified GST alone. Birds were challenged with C. jejuni M1 at 28 days of age and caecal Campylobacter counts determined at weekly intervals. Across three independent trials, the vaccine induced a statistically significant 1 log10 reduction in caecal Campylobacter numbers in vaccinated birds compared to age-matched GST-vaccinated controls. Significant induction of antigen-specific serum IgY was detected in all vaccinated birds, however the magnitude and timing of SodB-specific IgY did not correlate with lower numbers of C. jejuni. Antibodies from SodB-vaccinated chickens detected the protein in the periplasm and not membrane fractions or on the bacterial surface, suggesting that the protection observed may not be strictly antibody-mediated. SodB may be useful as a constituent of vaccines for control of C. jejuni infection in broiler birds, however modest protection was observed late relative to the life of broiler birds and further studies are required to potentiate the magnitude and timing of protection. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Antigen Sparing and Enhanced Protection Using A Novel rOv-ASP-1 Adjuvant in Aqueous Formulation with Influenza Vaccines

    PubMed Central

    Jiang, Jiu; Fisher, Erin M.; Hensley, Scott E.; Lustigman, Sara; Murasko, Donna M.; Shen, Hao

    2014-01-01

    Influenza is one of the most common infectious diseases endangering the health of humans, especially young children and the elderly. Although vaccination is the most effective means of protection against influenza, frequent mutations in viral surface antigens, low protective efficacy of the influenza vaccine in the elderly, slow production process and the potential of vaccine supply shortage during a pandemic are significant limitations of current vaccines. Adjuvants have been used to enhance the efficacy of a variety of vaccines; however, no adjuvant is included in current influenza vaccines approved in the United States. In this study, we found that a novel adjuvant, rOv-ASP-1, co-administrated with inactivated influenza vaccine using an aqueous formulation, substantially improved the influenza-specific antibody response and protection against lethal infection in a mouse model. rOv-ASP-1 enhanced the magnitude of the specific antibody response after immunization with low doses of influenza vaccine, allowing antigen-sparring by 10-fold. The rOv-ASP-1 formulated vaccine induced a more rapid response and a stronger Th1-associated antibody response compared to vaccine alone and to the vaccine formulated with the adjuvant alum. Importantly, rOv-ASP-1 significantly enhanced cross-reactive antibody responses and protection against challenge with an antigenically distinct strain. These results demonstrate that rOv-ASP-1 is an effective adjuvant that: 1) accelerates and enhances the specific antibody response induced by influenza vaccine; 2) allows for antigen sparing; and 3) augments a Th1-biased and cross-reactive antibody response that confers protection against an antigenically distinct strain. PMID:24681229

  13. Antigen sparing and enhanced protection using a novel rOv-ASP-1 adjuvant in aqueous formulation with influenza vaccines.

    PubMed

    Jiang, Jiu; Fisher, Erin M; Hensley, Scott E; Lustigman, Sara; Murasko, Donna M; Shen, Hao

    2014-05-13

    Influenza is one of the most common infectious diseases endangering the health of humans, especially young children and the elderly. Although vaccination is the most effective means of protection against influenza, frequent mutations in viral surface antigens, low protective efficacy of the influenza vaccine in the elderly, slow production process and the potential of vaccine supply shortage during a pandemic are significant limitations of current vaccines. Adjuvants have been used to enhance the efficacy of a variety of vaccines; however, no adjuvant is included in current influenza vaccines approved in the United States. In this study, we found that a novel adjuvant, rOv-ASP-1, co-administrated with inactivated influenza vaccine using an aqueous formulation, substantially improved the influenza-specific antibody response and protection against lethal infection in a mouse model. rOv-ASP-1 enhanced the magnitude of the specific antibody response after immunization with low doses of influenza vaccine, allowing antigen-sparring by 10-fold. The rOv-ASP-1 formulated vaccine induced a more rapid response and a stronger Th1-associated antibody response compared to vaccine alone and to the vaccine formulated with the adjuvant alum. Importantly, rOv-ASP-1 significantly enhanced cross-reactive antibody responses and protection against challenge with an antigenically distinct strain. These results demonstrate that rOv-ASP-1 is an effective adjuvant that: (1) accelerates and enhances the specific antibody response induced by influenza vaccine; (2) allows for antigen sparing; and (3) augments a Th1-biased and cross-reactive antibody response that confers protection against an antigenically distinct strain.

  14. A novel mucosal vaccine targeting Peyer's patch M cells induces protective antigen-specific IgA responses.

    PubMed

    Shima, Hideaki; Watanabe, Takashi; Fukuda, Shinji; Fukuoka, Shin-Ichi; Ohara, Osamu; Ohno, Hiroshi

    2014-11-01

    Mucosal vaccines can induce mucosal immunity, including antigen-specific secretory IgA production, to protect from invasion by pathogens and to neutralize toxins at mucosal surfaces. We established an effective antigen-delivering fusion protein, anti-GP2-SA, as a mucosal vaccine. The anti-GP2-SA consists of streptavidin (SA) fused to the antigen-binding fragment region from a mAb against glycoprotein 2 (GP2), an antigen-uptake receptor specifically expressed on M cells. Anti-GP2-SA targets antigen-sampling M cells in the follicle-associated epithelium covering Peyer's patches. Immunofluorescence showed that anti-GP2-SA specifically bound to M cells. Orally administered biotinylated ovalbumin peptide (bOVA) conjugated with anti-GP2-SA more efficiently induced OVA-specific fecal IgA secretion compared with bOVA alone or bOVA conjugated with SA. Furthermore, mice immunized by oral administration of the biotinylated Salmonella enterica serovar Typhimurium (S. Typhimurium) lysate conjugated with anti-GP2-SA were significantly better protected from subsequent infection by virulent S. Typhimurium than mice treated with the bacterial lysate alone or conjugated with SA. These results suggest that anti-GP2-SA-based M-cell-targeting vaccines are a novel strategy for inducing efficient mucosal immunity.

  15. Influenza virus-like particles engineered by protein transfer with tumor-associated antigens induces protective antitumor immunity.

    PubMed

    Patel, Jaina M; Vartabedian, Vincent F; Kim, Min-Chul; He, Sara; Kang, Sang-Moo; Selvaraj, Periasamy

    2015-06-01

    Delivery of antigen in particulate form using either synthetic or natural particles induces stronger immunity than soluble forms of the antigen. Among naturally occurring particles, virus-like particles (VLPs) have been genetically engineered to express tumor-associated antigens (TAAs) and have shown to induce strong TAA-specific immune responses due to their nano-particulate size and ability to bind and activate antigen-presenting cells. In this report, we demonstrate that influenza VLPs can be modified by a protein transfer technology to express TAAs for induction of effective antitumor immune responses. We converted the breast cancer HER-2 antigen to a glycosylphosphatidylinositol (GPI)-anchored form and incorporated GPI-HER-2 onto VLPs by a rapid protein transfer process. Expression levels on VLPs depended on the GPI-HER-2 concentration added during protein transfer. Vaccination of mice with protein transferred GPI-HER-2-VLPs induced a strong Th1 and Th2-type anti-HER-2 antibody response and protected mice against a HER-2-expressing tumor challenge. The Soluble form of GPI-HER-2 induced only a weak Th2 response under similar conditions. These results suggest that influenza VLPs can be enriched with TAAs by protein transfer to develop effective VLP-based subunit vaccines against cancer without chemical or genetic modifications and thus preserve the immune stimulating properties of VLPs for easier production of antigen-specific therapeutic cancer vaccines.

  16. Genetic vaccination against the melanocyte lineage-specific antigen gp100 induces cytotoxic T lymphocyte-mediated tumor protection.

    PubMed

    Schreurs, M W; de Boer, A J; Figdor, C G; Adema, G J

    1998-06-15

    Melanocyte lineage-specific antigens, such as gp100, have been shown to induce both cellular and humoral immune responses against melanoma. Therefore, these antigens are potential targets for specific antimelanoma immunotherapy. A novel approach to induce both cellular and humoral immunity is genetic vaccination, the injection of antigen-encoding naked plasmid DNA. In a mouse model, we investigated whether genetic vaccination against the human gp100 antigen results in specific antitumor immunity. The results demonstrate that vaccinated mice were protected against a lethal challenge with syngeneic B16 melanoma-expressing human gp100, but not control-transfected B16. Both cytotoxic T cells and IgG specific for human gp100 could be detected in human gp100-vaccinated mice. However, only adoptive transfer of spleen-derived lymphocytes, not of the serum, isolated from protected mice was able to transfer antitumor immunity to nonvaccinated recipients, indicating that CTLs are the predominant effector cells. CTI, lines generated from human gp100-vaccinated mice specifically recognized human gp100. Interestingly, one of the CTL lines cross-reacted between human and mouse gp100, indicating the recognition of a conserved epitope. However, these CTLs did not appear to be involved in the observed tumor protection. Collectively, our results indicate that genetic vaccination can result in a potent antitumor response in vivo and constitutes a potential immunotherapeutic strategy to fight cancer.

  17. Protection against Nasopharyngeal Colonization by Streptococcus pneumoniae Is Mediated by Antigen-Specific CD4+ T Cells▿

    PubMed Central

    Trzciński, Krzysztof; Thompson, Claudette M.; Srivastava, Amit; Basset, Alan; Malley, Richard; Lipsitch, Marc

    2008-01-01

    CD4+ T-cell-dependent acquired immunity confers antibody-independent protection against pneumococcal colonization. Since this mechanism is poorly understood for extracellular bacteria, we assessed the antigen specificity of the induction and recall of this immune response by using BALB/c DO11.10Rag−/− mice, which lack mature B and T cells except for CD4+ T cells specific for the OVA323-339 peptide derived from ovalbumin. Serotype 6B Streptococcus pneumoniae strain 603S and unencapsulated strain Rx1ΔlytA were modified to express OVA323-339 as a fusion protein with surface protein A (PspA) (strains 603OVA1 and Rx1ΔlytAOVA1) or with PspA, neuraminidase A, and pneumolysin (Rx1ΔlytAOVA3). Whole-cell vaccines (WCV) were made of ethanol-killed cells of Rx1ΔlytA plus cholera toxin (CT) adjuvant, of Rx1ΔlytAOVA1 + CT (WCV-OVA1), and of Rx1ΔlytAOVA3 + CT (WCV-OVA3). Mice intranasally immunized with WCV-OVA1, but not with WCV or CT alone, were protected against intranasal challenge with 603OVA1. There was no protection against strain 603S in mice immunized with WCV-OVA1. These results indicate antigen specificity of both immune induction and the recall response. Effector action was not restricted to antigen-bearing bacteria since colonization by 603S was reduced in animals immunized with vaccines made of OVA-expressing strains when ovalbumin or killed Rx1ΔlytAOVA3 antigen was administered around the time of challenge. CD4+ T-cell-mediated protection against pneumococcal colonization can be induced in an antigen-specific fashion and requires specific antigen for effective bacterial clearance, but this activity may extend beyond antigen-expressing bacteria. These results are consistent with the recruitment and/or activation of phagocytic or other nonspecific effectors by antigen-specific CD4+ T cells. PMID:18391006

  18. Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding.

    PubMed

    Pavan, María Elisa; Pavan, Esteban Enrique; Cairó, Fabián Martín; Pettinari, María Julia

    2016-01-01

    Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenic protein that is the basis for both anthrax vaccines and diagnostic methods. Properly folded antigenic PA is necessary for these applications. In this study a high level of PA was obtained in recombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, which facilitated its efficient purification by simple washing steps; however, it could not be recognized by specific antibodies. Refolding conditions were subsequently analyzed in a high-throughput manner that enabled nearly a hundred different conditions to be tested simultaneously. The recovery of the ability of PA to be recognized by antibodies was screened by dot blot using a coefficient that provided a measure of properly refolded protein levels with a high degree of discrimination. The best refolding conditions resulted in a tenfold increase in the intensity of the dot blot compared to the control. The only refolding additive that consistently yielded good results was L-arginine. The statistical analysis identified both cooperative and negative interactions between the different refolding additives. The high-throughput approach described in this study that enabled overproduction, purification and refolding of PA in a simple and straightforward manner, can be potentially useful for the rapid screening of adequate refolding conditions for other overexpressed antigenic proteins. Copyright © 2015 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  19. Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe, Protective Vaccine Antigen

    PubMed Central

    Burman, Alison; Jackson, Terry; Ren, Jingshan; Loureiro, Silvia; Jones, Ian M.; Fry, Elizabeth E.; Stuart, David I.; Charleston, Bryan

    2013-01-01

    Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals. PMID:23544011

  20. Analysis of protective antigen peptide binding motifs using bacterial display technology

    NASA Astrophysics Data System (ADS)

    Sarkes, Deborah A.; Dorsey, Brandi L.; Stratis-Cullum, Dimitra N.

    2015-05-01

    In today's fast-paced world, a new biological threat could emerge at any time, necessitating a prompt, reliable, inexpensive detection reagent in each case. Combined with magnetic-activated cell sorting (MACS), bacterial display technology makes it possible to isolate selective, high affinity peptide reagents in days to weeks. Utilizing the eCPX display scaffold is also a rapid way to screen potential peptide reagents. Peptide affinity reagents for protective antigen (PA) of the biothreat Bacillus anthracis were previously discovered using bacterial display. Bioinformatics analysis resulted in the consensus sequence WXCFTC. Additionally, we have discovered PA binding peptides with a WW motif, one of which, YGLHPWWKNAPIGQR, can pull down PA from 1% human serum. The strength of these two motifs combined, to obtain a WWCFTC consensus, is assessed here using Fluorescence Activated Cell Sorting (FACS). While monitoring binding to PA, overall expression of the display scaffold was assessed using the YPet Mona expression control tag (YPet), and specificity was assessed by binding to Streptavidin R-Phycoerythrin (SAPE). The importance of high YPet binding is highlighted as many of the peptides in one of the three replicate experiments fell below our 80% binding threshold. We demonstrate that it is preferable to discard this experiment, due to questionable expression of the peptide itself, than to try to normalize for relative expression. The peptides containing the WWCFTC consensus were of higher affinity and greater specificity than the peptides containing the WW consensus alone, validating further investigation to optimize known PA binders.

  1. Protection against Taenia pisiformis larval infection induced by a recombinant oncosphere antigen vaccine.

    PubMed

    Chen, L; Yang, D Y; Xie, Y; Nong, X; Huang, X; Fu, Y; Gu, X B; Wang, S X; Peng, X R; Yang, G Y

    2014-02-13

    Taenia pisiformis larvae cause significant health problems to rabbits. At present, it is not known whether the recombinant antigen from the T. pisiformis oncosphere is able to confer protective immunity against T. pisiformis larval infection. The full-length cDNA was cloned into a pET32a (+) vector, and the recombinant protein was then expressed in BL21 (DE3) cells. Vaccination with the purified rTpUbc2 coupled with QuilA was carried out in New Zealand rabbits to evaluate the immunoprotective effect against T. pisiformis infection. The full-length open reading frame of the TpUbc2 gene was 444 bp, and encoded a 16.63-kDa protein. Finally, rTpUbc2 was used to evaluate the ability to induce immunoprotective responses in rabbits. A 79.3-90.8% reduction (P < 0.01) in the recovery of larvae was observed in the experimental group compared to the control group. Specific anti-rTpUbc2 antibodies from immunized rabbits had significantly higher levels of IgG (P < 0.01) compared to the control group; however, no significant difference in IgA levels was found between groups (P > 0.05). Our data support the use of rTpUbc2 as a potential candidate to develop a vaccine against T. pisiformis larvae.

  2. Phenylalanine-427 of anthrax protective antigen functions in both pore formation and protein translocation

    PubMed Central

    Sun, Jianjun; Lang, Alexander E.; Aktories, Klaus; Collier, R. John

    2008-01-01

    The protective antigen (PA) moiety of anthrax toxin forms a heptameric pore in endosomal membranes of mammalian cells and translocates the enzymatic moieties of the toxin to the cytosol of these cells. Phenylalanine-427 (F427), a solvent-exposed residue in the lumen of the pore, was identified earlier as being crucial for the transport function of PA. The seven F427 residues were shown in electrophysiological studies to form a clamp that catalyzes protein translocation through the pore. Here, we demonstrate by a variety of tests that certain F427 mutations also profoundly inhibit the conformational transition of the heptameric PA prepore to the pore and thereby block pore formation in membranes. Lysine, arginine, aspartic acid, or glycine at position 427 strongly inhibited this acidic pH-induced conformational transition, whereas histidine, serine, and threonine had virtually no effect on this step, but inhibited translocation instead. Thus, it is possible to inhibit pore formation or translocation selectively, depending on the choice of the side chain at position 427; and the net inhibition of the PA transport function by any given F427 mutation is the product of its effects on both steps. Mutations inhibiting either or both steps elicited a strong dominant-negative phenotype. These findings demonstrate the dual functions of F427 and underline its central role in transporting the enzymatic moieties of anthrax toxin across membranes. PMID:18334631

  3. Translocation of Non-Canonical Polypeptides into Cells Using Protective Antigen

    PubMed Central

    Rabideau, Amy E.; Liao, Xiaoli; Akçay, Gizem; Pentelute, Bradley L.

    2015-01-01

    A variety of pathogenic bacteria infect host eukaryotic cells using protein toxins, which enter the cytosol and exert their cytotoxic effects. Anthrax lethal toxin, for example, utilizes the membrane-spanning translocase, protective antigen (PA) pore, to deliver the protein toxin lethal factor (LF) from the endosome into the cytosol of cells. Previous work has investigated the delivery of natural peptides and enzymatic domains appended to the C-terminus of the PA-binding domain of lethal factor (LFN) into the cytosol via PA pore. Here, we move beyond natural amino acids and systematically investigate the translocation of polypeptide cargo containing non-canonical amino acids and functionalities through PA pore. Our results indicate translocation is not perturbed with alterations to the peptide backbone or side-chain. Moreover, despite their structural complexity, we found that the small molecule drugs, doxorubicin and monomethyl auristatin F (MMAF) translocated efficiently through PA pore. However, we found cyclic peptides and the small molecule drug docetaxel abrogated translocation due to their large size and structural rigidity. For cargos that reached the cytosol, we demonstrated that each remained intact after translocation. These studies show PA is capable of translocating non-canonical cargo provided it is in a conformational state conducive for passage through the narrow pore. PMID:26178180

  4. Cloning and expression of a protective antigen from the cattle tick Boophilus microplus.

    PubMed Central

    Rand, K N; Moore, T; Sriskantha, A; Spring, K; Tellam, R; Willadsen, P; Cobon, G S

    1989-01-01

    Glycoproteins located on the luminal surface of the plasma membrane of tick gut epithelial cells, when used to vaccinate cattle, are capable of stimulating an immune response that protects cattle against subsequent tick infestation. One such tick gut glycoprotein, designated Bm86, has been purified to homogeneity and the amino acid sequences of peptide fragments generated by endoproteinase Lys-C digestion have been determined. We report here the isolation and characterization of a cDNA that encodes Bm86. The nucleotide sequence of the cDNA contains a 1982-base-pair open reading frame and predicts that Bm86 contains 650 amino acids including a 19-amino acid signal sequence and a 23-amino acid hydrophobic region adjacent to the carboxyl terminus. The main feature of the deduced protein sequence is the repeated pattern of 6 cysteine residues, suggesting the presence of several epidermal growth factor-like domains. A fusion protein consisting of 599 amino acids of Bm86 and 651 amino acids of beta-galactosidase was expressed in Escherichia coli as inclusion bodies. Ticks engorging on cattle vaccinated with these inclusion bodies were significantly damaged as a result of the immune response against the cloned antigen. Images PMID:2690068

  5. Anthrax toxin: channel-forming activity of protective antigen in planar phospholipid bilayers.

    PubMed Central

    Blaustein, R O; Koehler, T M; Collier, R J; Finkelstein, A

    1989-01-01

    The three separate proteins that make up anthrax toxin--protective antigen (PA), edema factor (EF), and lethal factor (LF)--act in binary combinations to produce two distinct reactions in experimental animals: edema (PA + EF) and death (PA + LF). PA is believed to interact with a membrane receptor, and after proteolytic processing, to mediate endocytosis and subsequent translocation of EF or LF into the cytosol. PA can be separated, after mild trypsinolysis, into two fragments, PA65 (65 kDa) and PA20 (20 kDa). We demonstrate that trypsin-cleaved PA is capable of forming cation-selective channels in planar phospholipid bilayer membranes and that this activity is confined to the PA65 fragment; PA20, LF, and EF are devoid of channel-forming activity. These PA65 channels exhibit pH-dependent and voltage-dependent activity--a property reminiscent of the channels formed by the two-chain proteins diphtheria, tetanus, and botulinum toxins. Images PMID:2467303

  6. Protective antigen antibody augments hemodynamic support in anthrax lethal toxin shock in canines.

    PubMed

    Barochia, Amisha V; Cui, Xizhong; Sun, Junfeng; Li, Yan; Solomon, Steven B; Migone, Thi-Sau; Subramanian, G Mani; Bolmer, Sally D; Eichacker, Peter Q

    2012-03-01

    Anthrax-associated shock is closely linked to lethal toxin (LT) release and is highly lethal despite conventional hemodynamic support. We investigated whether protective antigen-directed monoclonal antibody (PA-mAb) treatment further augments titrated hemodynamic support. Forty sedated, mechanically ventilated, instrumented canines challenged with anthrax LT were assigned to no treatment (controls), hemodynamic support alone (protocol-titrated fluids and norepinephrine), PA-mAb alone (administered at start of LT infusion [0 hours] or 9 or 12 hours later), or both, and observed for 96 hours. Although all 8 controls died, 2 of 8 animals receiving hemodynamic support alone survived (median survival times 65 vs 85 hours, respectively; P = .03). PA-mAb alone at 0 hour improved survival (5 of 5 animals survived), but efficacy decreased progressively with delayed treatment (9 hours, 2 of 3 survived; 12 hours, 0 of 4 survived) (P = .004 comparing survival across treatment times). However, combined treatment increased survival irrespective of PA-mAb administration time (0 hours, 4 of 5 animals; 9 hours, 3 of 3 animals; and 12 hours, 4 of 5 animals survived) (P = .95 comparing treatment times). Compared to hemodynamic support alone, when combined over PA-mAb treatment times (0, 9, and 12 hours), combination therapy produced higher survival (P = .008), central venous pressures, and left ventricular ejection fractions, and lower heart rates, norepinephrine requirements and fluid retention (P ≤ .03). PA-mAb may augment conventional hemodynamic support during anthrax LT-associated shock.

  7. Human lymphocyte antigen DR7 protects against proliferative retinopathy with type II diabetes mellitus.

    PubMed

    Quiroz-Mercado, Hugo; Suárez-Licona, Ana; Fromow-Guerra, Jans; López-Carasa, Gabriela; Cárdenas-Hernández, Rubén; Ruiz-Morales, Jorge A; Portal-Celhay, Cynthia; Granados, Julio

    2002-01-01

    This study was undertaken in order to analyze the genetic incidence of human lymphocyte antigen diabetic retinopathy (HLA-DR) and its influence in proliferative diabetic retinopathy (PDR). We designed a case-control study in which 127 mestizo Mexican patients with DM II and diabetic retinopathy were studied. DNA was extracted and HLA-DR regions were amplified using PCR. Alleles were determined by DNA hybridization. Diagnosis was assessed clinically and by fluorescein angiography. Incidence of HLA-DR alleles in patients was compared with an ethnically matched control group of healthy subjects (n = 98). Statistical significance was established with non-parametric tests. Patients with diabetic retinopathy showed less frequency of HLA-D11 compared with the control group (p = 0.043). NPDR patients with 10 or more years of DM II showed an increase of HLA-DR7 (p = 0.01). Our results suggest that the presence of HLA-DR7 protects against the development of proliferative disease in the diabetic Mexican population.

  8. Protection of pigs against Taenia solium cysticercosis by immunization with novel recombinant antigens.

    PubMed

    Gauci, Charles G; Jayashi, César M; Gonzalez, Armando E; Lackenby, Julia; Lightowlers, Marshall W

    2012-06-06

    Recombinant antigens from the oncosphere stage of the parasite Taenia solium were expressed in Escherichia coli. The TSOL16, TSOL45-1A and TSOL45-1B recombinant antigens, each consisting of fibronectin type III (FnIII) domain S, were produced as fusion proteins with glutathione S-transferase (GST) and maltose binding protein (MBP). Groups of pigs were immunized twice with the GST fusions of the antigens and boosted a third time with the MBP fusions prior to receiving a challenge infection with T. solium eggs. The TSOL16 antigen was found to be capable of inducing high levels of immunity in pigs against a challenge infection with T. solium. Immunological investigations identified differences in immune responses in the pigs vaccinated with the various antigens. The results demonstrate that the TSOL16 antigen could be a valuable adjunct to current porcine vaccination approaches and may allow the further development of new vaccination strategies against T. solium cysticercosis.

  9. [Historical reflections on health protection and the condom].

    PubMed

    Forrai, J

    1991-12-01

    The condom was first mentioned in a 1564 writing by Gabriel Fallopius as a means of protection against syphilis describing his tests on 1100 people. The name itself has been ascribed to the Latin word condere, Cum Domino, the French city of Condom, and doctor Quondom, the physician of the English King Charles II. The Marquis de Sade and Casanova used it to avoid venereal diseases (VDs). In London condom manufacturing started in the 18th century. Later it became a symbol of prostitution and immorality. The material used consisted of fish bladder or animal intestines (calf, sheep). The discovery of the rubber tree and the invention of vulcanization by the American technician Goodyear in 1840 made possible large-scale production. In Hungary the 1st rubber manufacturing plant EMERGE started production in 1893 along with toys and other wares. IN 1895 the HUngarian medial association warned about the spread of syphilis facilitated by the activities of 15,400 syphilitic prostitutes in the country. 30% of hospital patients had syphilis. The use of the condom was limited, and illegitimate births increased by 10.5% during the millennium celebrations of Hungary's existence in 1896. EMERGE manufactured condoms called Nono which were mostly distributed to soldiers during World War I, yet they had little popularity. US soldiers did not use the condoms either, as 7 million active days were lost due to VDs during World War II. In the 1950's Anna Ratko was Minister of Health in Hungary who opposed promotion of condoms to increase the population. The invention of penicillin in 1942 also pushed the condom to the background, but in the 1980's the epidemic of AIDS has made its use widespread.

  10. Mimotope-Based Vaccines of Leishmania infantum Antigens and Their Protective Efficacy against Visceral Leishmaniasis

    PubMed Central

    Costa, Lourena Emanuele; Goulart, Luiz Ricardo; Pereira, Nathália Cristina de Jesus; Lima, Mayara Ingrid Sousa; Duarte, Mariana Costa; Martins, Vivian Tamietti; Lage, Paula Sousa; Menezes-Souza, Daniel; Ribeiro, Tatiana Gomes; Melo, Maria Norma; Fernandes, Ana Paula; Soto, Manuel; Tavares, Carlos Alberto Pereira; Chávez-Fumagalli, Miguel Angel; Coelho, Eduardo Antonio Ferraz

    2014-01-01

    Background The development of cost-effective prophylactic strategies to prevent leishmaniasis has become a high-priority. The present study has used the phage display technology to identify new immunogens, which were evaluated as vaccines in the murine model of visceral leishmaniasis (VL). Epitope-based immunogens, represented by phage-fused peptides that mimic Leishmania infantum antigens, were selected according to their affinity to antibodies from asymptomatic and symptomatic VL dogs' sera. Methodology/Main Findings Twenty phage clones were selected after three selection cycles, and were evaluated by means of in vitro assays of the immune stimulation of spleen cells derived from naive and chronically infected with L. infantum BALB/c mice. Clones that were able to induce specific Th1 immune response, represented by high levels of IFN-γ and low levels of IL-4 were selected, and based on their selectivity and specificity, two clones, namely B10 and C01, were further employed in the vaccination protocols. BALB/c mice vaccinated with clones plus saponin showed both a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with individual clones or L. infantum extracts. Additionally, these animals, when compared to control groups (saline, saponin, wild-type phage plus saponin, or non-relevant phage clone plus saponin), showed significant reductions in the parasite burden in the liver, spleen, bone marrow, and paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, mainly by CD8+ T cells, against parasite proteins. These animals also presented decreased parasite-mediated IL-4 and IL-10 responses, and increased levels of parasite-specific IgG2a antibodies. Conclusions/Significance This study describes two phage clones that mimic L. infantum antigens, which were directly used as immunogens in vaccines and presented Th1-type immune responses, and that significantly reduced the parasite burden. This

  11. Protective efficacy of oral immunization with heat-killed Shigella flexneri 2a in animal model: study of cross protection, immune response and antigenic recognition.

    PubMed

    Mukhopadhaya, A; Mahalanabis, D; Khanam, J; Chakrabarti, M K

    2003-06-20

    Oral immunization of rabbits with four doses of 10(11) heat-killed Shigella flexneri 2a showed 100% protection against challenge with virulent S. flexneri 2a. After orally immunizing Guinea pigs with four doses of heat-killed S. flexneri 2a 100% protection could be shown against ocular challenge with the same virulent S. flexneri 2a strain but this conferred no protection against challenge with Shigella dysenteriae type 1. In enzyme-linked immunosorbent assay (ELISA) and immunoblot experiments both whole cell lysate-envelope (WCL-E) fraction and outer membrane proteins (OMPs) were recognized by the antisera. Though protective mechanism in shigellosis is not established with certainty, outer membrane proteins (specially 38, 34, 23 and 20kDa proteins) may be the major antigens in the induction of protective immune responses as indicated by this observation.

  12. Variation in the cellular localization of host-protective oncospheral antigens in Taenia saginata and Taenia solium.

    PubMed

    Jabbar, A; Verástegui, M; Lackenby, J A; Walduck, A K; Gauci, C G; Gilman, R H; Lightowlers, M W

    2010-01-01

    Immunohistochemistry and immunofluorescence with confocal microscopy were used to localize the host-protective antigens of Taenia saginata (TSA9 and TSA18) and Taenia solium (TSOL16, TSOL18 and TSOL45). In nonactivated oncospheres, TSA9 and TSOL45 antigens were found primarily in the cytoplasm of the penetration gland type one (PG1) cell. A similar pattern of staining was seen for TSOL45 in oncospheres of T. solium that remained within the oncospheral membrane. In addition, there was less intense staining of TSA9 and TSOL45 in the quadri-nucleate penetration gland type 2 (PG2) cell. TSA18, TSOL16 and TSOL18 were predominantly found in the PG2 cell. In activated oncospheres that had escaped the oncospheral membrane, the antigens (other than TSA9) were seen both in the penetration gland cell locations and throughout the oncospheral parenchyma. Co-localization analyses revealed that only TSOL16 and TSOL18 antigens were co-localized in the PG2 cell of oncospheres that had not escaped the oncospheral membrane. However, in activated oncospheres that escaped the oncospheral membrane, all three antigens of T. solium were co-localized as they were present throughout the parenchyma. No positive staining was observed on the surface of nonactivated or recently activated oncospheres of T. saginata or T. solium.

  13. Surface plasmon resonance measurements of plasma antibody avidity during primary and secondary responses to anthrax protective antigen.

    PubMed

    Lynch, Heather E; Stewart, Shelley M; Kepler, Thomas B; Sempowski, Gregory D; Alam, S Munir

    2014-02-01

    Establishment of humoral immunity against pathogens is dependent on events that occur in the germinal center and the subsequent induction of high-affinity neutralizing antibodies. Quantitative assays that allow monitoring of affinity maturation and duration of antibody responses can provide useful information regarding the efficacy of vaccines and adjuvants. Using an anthrax protective antigen (rPA) and alum model antigen/adjuvant system, we describe a methodology for monitoring antigen-specific serum antibody concentration and avidity by surface plasmon resonance during primary and secondary immune responses. Our analyses showed that following a priming dose in mice, rPA-specific antibody concentration and avidity increases over time and reaches a maximal response in about six weeks, but gradually declines in the absence of antigenic boost. Germinal center reactions were observed early with maximal development achieved during the primary response, which coincided with peak antibody avidity responses to primary immunization. Boosting with antigen resulted in a rapid increase in rPA-specific antibody concentration and five-fold increase in avidity, which was not dependent on sustained GC development. The described methodology couples surface plasmon resonance-based plasma avidity measurements with germinal center analysis and provides a novel way to monitor humoral responses that can play a role in facilitating vaccine and adjuvant development.

  14. Antigenic and cross-protection studies of biotype 1 and biotype 2 isolates of Yersinia ruckeri in rainbow trout, Oncorhynchus mykiss (Walbaum).

    PubMed

    Tinsley, J W; Lyndon, A R; Austin, B

    2011-07-01

    The study investigated antigen characteristics of biotype (bt) 1 and bt 2 isolates of Yersinia ruckeri. The cell surface characteristics of Y. ruckeri were compared for their antigenic characteristics using polyclonal antibodies that revealed that both biotypes had a homogenous whole-cell protein antigenic profile. Notable differences in the antigenic properties were observed in the lipopolysaccharide profile of both biotypes. Two iron-regulated outer membrane proteins (IROMP) of c.90 and 100 kDa were shown to be major specific antigens. The results demonstrate for the first time differences in antigens between bt 1 and bt 2 isolates of serotype O1 isolates of Y. ruckeri. The protection induced in rainbow trout by a commercial monovalent, and bivalent inactivated vaccine was tested with the outcome that the ability of isolates to cause mortality in vaccinated fish varied with geographical location. In this context, vaccination studies suggested that the O antigen was the dominant immunogenic molecule involved in protection against the disease. The O antigen of Y. ruckeri was the dominant immunogenic molecule involved in the protection of rainbow trout against enteric redmouth disease. There are distinct phenotypic and antigenic differences in Y. ruckeri bt 1 and bt 2 with O antigen recognized as the dominant immunogenic molecule. The data have significance in explaining the lack of success of the earlier monovalent vaccine and demonstrate the effectiveness of the newer bivalent vaccine. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  15. Effect of particulate adjuvant on the anthrax protective antigen dose required for effective nasal vaccination.

    PubMed

    Bento, Dulce; Staats, Herman F; Borges, Olga

    2015-07-17

    Successful vaccine development is dependent on the development of effective adjuvants since the poor immunogenicity of modern subunit vaccines typically requires the use of potent adjuvants and high antigen doses. In recent years, adjuvant formulations combining both immunopotentiators and delivery systems have emerged as a promising strategy to develop effective and improved vaccines. In this study we investigate if the association of the mast cell activating adjuvant compound 48/80 (C48/80) with chitosan nanoparticles would promote an antigen dose sparing effect when administered intranasally. Even though the induction of strong mucosal immunity required higher antigen doses, incorporation of C48/80 into nanoparticles provided significant dose sparing when compared to antigen and C48/80 in solution with no significant effect on serum neutralizing antibodies titers. These results suggest the potential of this novel adjuvant combination to improve the immunogenicity of a vaccine and decrease the antigen dose required for vaccination.

  16. Comparative Vaccine Efficacy of Different Isoforms of Recombinant Protective Antigen Against Bacillus anthracis Spore Challenge in Rabbits

    DTIC Science & Technology

    2006-02-06

    bovine serum, 4 mM glutamine and 00 U of Penicillin G and 100 g of streptomycin per ml D-MEM complete) supplemented with 25 mM HEPES. ne -hundred...contains small, but varying quantities of other bacterial com- ponents. A second-generation vaccine currently undergoing clinical trials is a purified...Friedlander AM. Comparative safety and efficacy against Bacillus anthracis of protective antigen and live vaccines in mice. Microb Pathog 1988;5(2):127–39

  17. Production of a DNA Vaccine Specific for the 64 kDa Protective Antigen of Erysipelothrix rhusiopathiae

    DTIC Science & Technology

    2007-02-08

    cetacean antibody titers to E . rhusiopathiae ; and to test the DNA from the recombinant protein in vitro in cetacean specific cell lines for its ability...to transfect the cells. APPROACH: The gene for the protective antigen of E . rhusiopathiae will be inserted into a eukaryotic vector both for the...cetacean serum samples to E . rhusiopathiae . ACCOMPLISHMENTS: The identity of the U.S. Navy Space and Naval Warfare System Center (SPAWARSYSTENS) isolate of

  18. Structural and Immunological Analysis of Anthrax Recombinant Protective Antigen Adsorbed to Aluminum Hydroxide Adjuvant

    PubMed Central

    Wagner, Leslie; Verma, Anita; Meade, Bruce D.; Reiter, Karine; Narum, David L.; Brady, Rebecca A.; Little, Stephen F.

    2012-01-01

    New anthrax vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant. Intrinsic fluorescence emission spectra of tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on Alhydrogel. Using anti-PA monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two monoclonal antibodies that recognize epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-Alhydrogel formulations in mice was assessed by measuring the induction of toxin-neutralizing antibodies, as well as antibodies reactive to 12-mer peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-Alhydrogel formulations can lead to structural alteration of the protein and loss of the ability to elicit toxin-neutralizing antibodies. PMID:22815152

  19. Naturally acquired antibodies to Bacillus anthracis protective antigen in vultures of southern Africa.

    PubMed

    Turnbull, P C B; Diekmann, M; Kilian, J W; Versfeld, W; De Vos, V; Arntzen, L; Wolter, K; Bartels, P; Kotze, A

    2008-06-01

    Sera from 19 wild caught vultures in northern Namibia and 15 (12 wild caught and three captive bred but with minimal histories) in North West Province, South Africa, were examined by an enzyme-linked immunosorbent assay (ELISA) for antibodies to the Bacillus anthracis toxin protective antigen (PA). As assessed from the baseline established with a control group of ten captive reared vultures with well-documented histories, elevated titres were found in 12 of the 19 (63%) wild caught Namibian birds as compared with none of the 15 South African ones. There was a highly significant difference between the Namibian group as a whole and the other groups (P < 0.001) and no significant difference between the South African and control groups (P > 0.05). Numbers in the Namibian group were too small to determine any significances in species-, sex- or age-related differences within the raw data showing elevated titres in four out of six Cape Vultures, Gyps coprotheres, six out of ten White-backed Vultures, Gyps africanus, and one out of three Lappet-faced Vultures, Aegypius tracheliotus, or in five of six males versus three of seven females, and ten of 15 adults versus one of four juveniles. The results are in line with the available data on the incidence of anthrax in northern Namibia and South Africa and the likely contact of the vultures tested with anthrax carcasses. It is not known whether elevated titre indicates infection per se in vultures or absorption of incompletely digested epitopes of the toxin or both. The results are discussed in relation to distances travelled by vultures as determined by new tracking techniques, how serology can reveal anthrax activity in an area and the issue of the role of vultures in transmission of anthrax.

  20. Mechanistic Analysis of the Effect of Deamidation on the Immunogenicity of Anthrax Protective Antigen

    PubMed Central

    Verma, Anita; Ngundi, Miriam M.

    2016-01-01

    The spontaneous modification of proteins, such as deamidation of asparagine residues, can significantly affect the immunogenicity of protein-based vaccines. Using a “genetically deamidated” form of recombinant protective antigen (rPA), we have previously shown that deamidation can decrease the immunogenicity of rPA, the primary component of new-generation anthrax vaccines. In this study, we investigated the biochemical and immunological mechanisms by which deamidation of rPA might decrease the immunogenicity of the protein. We found that loss of the immunogenicity of rPA vaccine was independent of the presence of adjuvant. We assessed the effect of deamidation on the immunodominant neutralizing B-cell epitopes of rPA and found that these epitopes were not significantly affected by deamidation. In order to assess the effect of deamidation on T-cell help for antibody production elicited by rPA vaccine, we examined the ability of the wild-type and genetically deamidated forms of rPA to serve as hapten carriers. We found that when wild-type and genetically deamidated rPA were modified to similar extents with 2,4-dinitrophenyl hapten (DNP) and then used to immunize mice, higher levels of anti-DNP antibodies were elicited by wild-type DNP-rPA than those elicited by the genetically deamidated DNP-rPA, indicating that wild-type rPA elicits more T-cell help than the genetically deamidated form of the protein. These results suggest that a decrease in the ability of deamidated rPA to elicit T-cell help for antibody production is a possible contributor to its lower immunogenicity. PMID:26912784

  1. Erysipelothrix spp. genotypes, serotypes, and surface protective antigen types associated with abattoir condemnations.

    PubMed

    Bender, Joseph S; Irwin, Christa K; Shen, Hui-Gang; Schwartz, Kent J; Opriessnig, Tanja

    2011-01-01

    The objective of the current study was to investigate characteristics of Erysipelothrix spp. from slaughter condemnations. Specimens from 70 carcasses with lesions suspect for swine erysipelas were collected at an abattoir in Iowa from October 2007 to February 2009. Erysipelothrix spp. were isolated from 59 of 70 carcasses (84.3%). Abattoir inspectors classified lesions as acute, subacute, or chronic; 8 of 8 (100%) were acute cases, 31 of 32 (96.9%) were subacute cases, and 20 of 30 (66.6%) were chronic cases that were isolation positive. The following serotypes were identified: 1a (40.7%; 24/59), 2 (49.2%; 29/59), 7 (1/59), 10 (1/59), 11 (1/59), and untypeable (5.1%; 3/59). Serotypes 1a and 2 were identified in pigs with acute, subacute, or chronic clinical manifestations, whereas serotypes 7, 10, and 11 were only present in chronic cases. Fifty-seven of the 59 isolates were determined to belong to E. rhusiopathiae, and 2 of 59 of the isolates were determined to be E. tonsillarum by multiplex real-time polymerase chain reaction. Surface protective antigen (spa) A was detected in all E. rhusiopathiae isolates but not in E. tonsillarum serotypes 7 and 10. The results of the present study indicate that E. rhusiopathiae serotypes 1a and 2 continue to be commonly isolated from condemned pig carcasses and that spaA is the exclusive spa type in U.S. abattoir isolates. Interestingly, E. tonsillarum, thought to be avirulent for swine, was isolated from systemic sites from 3.4% of the carcasses that were negative for E. rhusiopathiae, indicating the potential importance of this genotype in erysipelas pathogenesis.

  2. Truncated surface protective antigen (SpaA) of Erysipelothrix rhusiopathiae serotype 1a elicits protection against challenge with serotypes 1a and 2b in pigs.

    PubMed

    Imada, Y; Goji, N; Ishikawa, H; Kishima, M; Sekizaki, T

    1999-09-01

    Erysipelothrix rhusiopathiae is a causal agent of swine erysipelas, which is of economic importance in the swine industry by virtue of causing acute septicemia, chronic arthritis, and endocarditis. However, little is known about the genetic properties of its protective antigens. Recently, a surface protective antigen (SpaA) gene was identified from serotype 2 in a mouse model. We cloned spaA from virulent strain Fujisawa (serotype 1a) and determined that the N-terminal 342 amino acids without C-terminal repeats of 20 amino acids have the ability to elicit protection in mice. Fusions of 342 amino acids of Fujisawa SpaA and histidine hexamer (HisSpa1.0) protected pigs against challenge with both serotype 1 and serotype 2, the most important serotypes in the swine industry. Pigs immunized with HisSpa1.0 reacted well with both HisSpa1.0 and intact SpaA by enzyme-linked immunosorbent assay and immunoblotting. Serum collected at the time of challenge from a pig immunized with HisSpa1. 0 markedly enhanced the in vitro phagocytic and killing activity of pig neutrophils against the bacteria. DNA sequences of protective regions of spaA genes from five strains of serotypes 1 and 2 were almost identical. The full DNA sequences also seemed to be conserved among strains of all 12 serotype reference strains harboring the spaA gene by restriction fragment length polymorphism analysis of PCR products. These results indicates that SpaA is a common protective antigen of serotypes 1 and 2 of E. rhusiopathiae in swine and will be a useful tool for development of new types of vaccines and diagnostic tools for effective control of the disease.

  3. Imperfect past and present progressive: beak color reflects early-life and adult exposure to antigen

    PubMed Central

    Naylor, Madeleine F.; Grindstaff, Jennifer L.

    2016-01-01

    Secondary sexual traits may convey information about individual condition. We assessed the capacity for immune challenge with lipopolysaccharide (LPS) or keyhole limpet hemocyanin (KLH) during the prenatal and early postnatal stages to impact beak color development and expression in captive zebra finches. In addition, we tested whether adult immune challenge impacted beak color, and if early-life experience was influential. Immune challenge with KLH early in life slowed development of red beak coloration, and males challenged with KLH as nestlings had reduced red coloration as adults. Following adult KLH challenge, males exhibited a decline in beak redness. Birds challenged with KLH during development produced more anti-KLH antibodies after adult challenge. There was a significant interaction between young treatment and anti-KLH antibody production; for males not challenged with KLH early in life, individuals that mounted a weaker antibody response lost more red coloration after challenge than males mounting a stronger antibody response. Based on models of avian vision, these differences in beak coloration should be detectable to the finches. In contrast to previous studies, we found no effect of early-life or adult challenge with LPS on any aspects of beak coloration. These results provide evidence that beak color reflects developmental and current conditions, and that the signal is linked to critical physiological processes. PMID:27656084

  4. Engineered erythrocytes covalently linked to antigenic peptides can protect against autoimmune disease

    PubMed Central

    Pishesha, Novalia; Bilate, Angelina M.; Wibowo, Marsha C.; Huang, Nai-Jia; Li, Zeyang; Dhesycka, Rhogerry; Bousbaine, Djenet; Li, Hojun; Patterson, Heide C.; Dougan, Stephanie K.; Maruyama, Takeshi; Lodish, Harvey F.; Ploegh, Hidde L.

    2017-01-01

    Current therapies for autoimmune diseases rely on traditional immunosuppressive medications that expose patients to an increased risk of opportunistic infections and other complications. Immunoregulatory interventions that act prophylactically or therapeutically to induce antigen-specific tolerance might overcome these obstacles. Here we use the transpeptidase sortase to covalently attach disease-associated autoantigens to genetically engineered and to unmodified red blood cells as a means of inducing antigen-specific tolerance. This approach blunts the contribution to immunity of major subsets of immune effector cells (B cells, CD4+ and CD8+ T cells) in an antigen-specific manner. Transfusion of red blood cells expressing self-antigen epitopes can alleviate and even prevent signs of disease in experimental autoimmune encephalomyelitis, as well as maintain normoglycemia in a mouse model of type 1 diabetes. PMID:28270614

  5. Engineered erythrocytes covalently linked to antigenic peptides can protect against autoimmune disease.

    PubMed

    Pishesha, Novalia; Bilate, Angelina M; Wibowo, Marsha C; Huang, Nai-Jia; Li, Zeyang; Dhesycka, Rhogerry; Bousbaine, Djenet; Li, Hojun; Patterson, Heide C; Dougan, Stephanie K; Maruyama, Takeshi; Lodish, Harvey F; Ploegh, Hidde L

    2017-03-21

    Current therapies for autoimmune diseases rely on traditional immunosuppressive medications that expose patients to an increased risk of opportunistic infections and other complications. Immunoregulatory interventions that act prophylactically or therapeutically to induce antigen-specific tolerance might overcome these obstacles. Here we use the transpeptidase sortase to covalently attach disease-associated autoantigens to genetically engineered and to unmodified red blood cells as a means of inducing antigen-specific tolerance. This approach blunts the contribution to immunity of major subsets of immune effector cells (B cells, CD4(+) and CD8(+) T cells) in an antigen-specific manner. Transfusion of red blood cells expressing self-antigen epitopes can alleviate and even prevent signs of disease in experimental autoimmune encephalomyelitis, as well as maintain normoglycemia in a mouse model of type 1 diabetes.

  6. Priming Cross-Protective Bovine Viral Diarrhea Virus-Specific Immunity Using Live-Vectored Mosaic Antigens

    PubMed Central

    Fang, Xin; Waghela, Suryakant D.; Bray, Jocelyn; Njongmeta, Leo M.; Herring, Andy; Abdelsalam, Karim W.; Chase, Christopher; Mwangi, Waithaka

    2017-01-01

    Bovine viral diarrhea virus (BVDV) plays a key role in bovine respiratory disease complex, which can lead to pneumonia, diarrhea and death of calves. Current vaccines are not very effective due, in part, to immunosuppressive traits and failure to induce broad protection. There are diverse BVDV strains and thus, current vaccines contain representative genotype 1 and 2 viruses (BVDV-1 & 2) to broaden coverage. BVDV modified live virus (MLV) vaccines are superior to killed virus vaccines, but they are susceptible to neutralization and complement-mediated destruction triggered by passively acquired antibodies, thus limiting their efficacy. We generated three novel mosaic polypeptide chimeras, designated NproE2123; NS231; and NS232, which incorporate protective determinants that are highly conserved among BVDV-1a, 1b, and BVDV-2 genotypes. In addition, strain-specific protective antigens from disparate BVDV strains were included to broaden coverage. We confirmed that adenovirus constructs expressing these antigens were strongly recognized by monoclonal antibodies, polyclonal sera, and IFN-γ-secreting T cells generated against diverse BVDV strains. In a proof-of-concept efficacy study, the multi-antigen proto-type vaccine induced higher, but not significantly different, IFN-γ spot forming cells and T-cell proliferation compared to a commercial MLV vaccine. In regards to the humoral response, the prototype vaccine induced higher BVDV-1 specific neutralizing antibody titers, whereas the MLV vaccine induced higher BVDV-2 specific neutralizing antibody titers. Following BVDV type 2a (1373) challenge, calves immunized with the proto-type or the MLV vaccine had lower clinical scores compared to naïve controls. These results support the hypothesis that a broadly protective subunit vaccine can be generated using mosaic polypeptides that incorporate rationally selected and validated protective determinants from diverse BVDV strains. Furthermore, regarding biosafety of using a

  7. Evaluation of the immunogenicity and protective efficacy of killed Leishmania donovani antigen along with different adjuvants against experimental visceral leishmaniasis.

    PubMed

    Thakur, Ankita; Kaur, Harpreet; Kaur, Sukhbir

    2015-08-01

    Visceral leishmaniasis (VL) caused by Leishmania donovani is a life-threatening disease involving uncontrolled parasitization of vital organs. Drugs to treat leishmaniasis have one or more limitations or insufficiencies in the long run. A safe and efficacious vaccine to control this disease is needed. Killed antigens that could be safer as vaccines have shown limited efficacy in clinical trials. Immunogenic enhancement with appropriate adjuvants may thus be required to elicit protective immunity based on antibodies and effector T-cell functions. Therefore, it is essential to search for adjuvant to enhance the immunogenicity of killed vaccines and to induce protection against leishmaniasis. So, the aim of the present study was to compare the effectiveness of four adjuvants, i.e. alum, saponin, monophosphoryl lipid A, cationic liposome in combination with Killed Leishmania donovani (KLD) antigen against murine VL. Animals were immunized subcutaneously thrice at an interval of 2 weeks with a final volume of 100 μl per dose. Challenge infection was given 2 weeks after last booster. Mice were sacrificed 15 days after last immunization and on 30, 60 and 90 post-infection/challenge days. The protective efficacy of vaccines was revealed by significant reduction in parasite burden and enhanced DTH responses in comparison with the infected controls. Immunized animals also generated significant levels of Th1 cytokines and increased production of IgG2a, thus indicating the generation of a protective Th1 response. All the adjuvants imparted significant protection, but liposomal formulation was most effective followed by KLD + MPL-A, KLD + saponin, KLD + alum and KLD antigen.

  8. Enhanced Protective Efficacy of Nonpathogenic Recombinant Leishmania tarentolae Expressing Cysteine Proteinases Combined with a Sand Fly Salivary Antigen

    PubMed Central

    Taheri, Tahereh; Taslimi, Yasaman; Doustdari, Fatemeh; Seyed, Negar; Torkashvand, Fatemeh; Meneses, Claudio; Papadopoulou, Barbara; Kamhawi, Shaden; Valenzuela, Jesus G.; Rafati, Sima

    2014-01-01

    Background Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. Methodology/Principal Findings Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. Conclusion/Significance The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis. PMID:24675711

  9. Priming Cross-Protective Bovine Viral Diarrhea Virus-Specific Immunity Using Live-Vectored Mosaic Antigens.

    PubMed

    Lokhandwala, Shehnaz; Fang, Xin; Waghela, Suryakant D; Bray, Jocelyn; Njongmeta, Leo M; Herring, Andy; Abdelsalam, Karim W; Chase, Christopher; Mwangi, Waithaka

    2017-01-01

    Bovine viral diarrhea virus (BVDV) plays a key role in bovine respiratory disease complex, which can lead to pneumonia, diarrhea and death of calves. Current vaccines are not very effective due, in part, to immunosuppressive traits and failure to induce broad protection. There are diverse BVDV strains and thus, current vaccines contain representative genotype 1 and 2 viruses (BVDV-1 & 2) to broaden coverage. BVDV modified live virus (MLV) vaccines are superior to killed virus vaccines, but they are susceptible to neutralization and complement-mediated destruction triggered by passively acquired antibodies, thus limiting their efficacy. We generated three novel mosaic polypeptide chimeras, designated NproE2123; NS231; and NS232, which incorporate protective determinants that are highly conserved among BVDV-1a, 1b, and BVDV-2 genotypes. In addition, strain-specific protective antigens from disparate BVDV strains were included to broaden coverage. We confirmed that adenovirus constructs expressing these antigens were strongly recognized by monoclonal antibodies, polyclonal sera, and IFN-γ-secreting T cells generated against diverse BVDV strains. In a proof-of-concept efficacy study, the multi-antigen proto-type vaccine induced higher, but not significantly different, IFN-γ spot forming cells and T-cell proliferation compared to a commercial MLV vaccine. In regards to the humoral response, the prototype vaccine induced higher BVDV-1 specific neutralizing antibody titers, whereas the MLV vaccine induced higher BVDV-2 specific neutralizing antibody titers. Following BVDV type 2a (1373) challenge, calves immunized with the proto-type or the MLV vaccine had lower clinical scores compared to naïve controls. These results support the hypothesis that a broadly protective subunit vaccine can be generated using mosaic polypeptides that incorporate rationally selected and validated protective determinants from diverse BVDV strains. Furthermore, regarding biosafety of using a

  10. Vaccination with TAT-antigen fusion protein induces protective, CD8(+) T cell-mediated immunity against Leishmania major.

    PubMed

    Kronenberg, Katharina; Brosch, Sven; Butsch, Florian; Tada, Yayoi; Shibagaki, Naotaka; Udey, Mark C; von Stebut, Esther

    2010-11-01

    In murine leishmaniasis, healing is mediated by IFN-γ-producing CD4(+) and CD8(+) T cells. Thus, an efficacious vaccine should induce Th1 and Tc1 cells. Dendritic cells (DCs) pulsed with exogenous proteins primarily induce strong CD4-dependent immunity; induction of CD8 responses has proven to be difficult. We evaluated the immunogenicity of fusion proteins comprising the protein transduction domain of HIV-1 TAT and the Leishmania antigen LACK (Leishmania homolog of receptors for activated C kinase), as TAT-fusion proteins facilitate major histocompatibility complex class I-dependent antigen presentation. In vitro, TAT-LACK-pulsed DCs induced stronger proliferation of Leishmania-specific CD8(+) T cells compared with DCs incubated with LACK alone. Vaccination with TAT-LACK-pulsed DCs or fusion proteins plus adjuvant in vivo significantly improved disease outcome in Leishmania major-infected mice and was superior to vaccination with DCs treated with LACK alone. Vaccination with DC+TAT-LACK resulted in stronger proliferation of CD8(+) T cells when compared with immunization with DC+LACK. Upon depletion of CD4(+) or CD8(+) T cells, TAT-LACK-mediated protection was lost. TAT-LACK-pulsed IL-12p40-deficient DCs did not promote protection in vivo. In summary, these data show that TAT-fusion proteins are superior in activating Leishmania-specific Tc1 cells when compared with antigen alone and suggest that IL-12-dependent preferential induction of antigen-specific CD8(+) cells promotes significant protection against this important human pathogen.

  11. White light reflectance spectroscopy biosensing system for fast quantitative prostate specific antigen determination in forensic samples.

    PubMed

    Koukouvinos, Georgios; Metheniti, Aristea; Karachaliou, Chrysoula-Evangelia; Goustouridis, Dimitrios; Livaniou, Evangelia; Misiakos, Konstantinos; Raptis, Ioannis; Kondili, Aikaterini; Miniati, Penelope; Petrou, Panagiota; Kakabakos, Sotirios

    2017-12-01

    A label-free biosensor based on white light reflectance spectroscopy for the determination of PSA as semen indicator in forensic samples is presented. The sensor is based on a two-step immunoassay which employs the same polyclonal anti-PSA antibody as capture and detection antibody followed by reaction with streptavidin as a signal enhancement step. The whole assay time was set to 10min; 5min reaction of immobilized antibody with the PSA calibrators or the samples, 3min reaction with the biotinylated anti-PSA antibody and 2min reaction with streptavidin. Following this protocol, a detection limit of 0.5ng/mL was achieved and the assay's linear response range extended up to 500ng/mL. Thus, taking into account the quantification limit of 1.0ng/mL and the average PSA concentration in semen (0.2-5.5mg/mL), semen quantities of a few nanoliters could be detected. The accuracy of the sensor developed was demonstrated through recovery (% recovery ranged from 89.6 to 106) and semen dilution experiments. A linear correlation was found for semen dilutions ranging from 5000 to 360,000. The lack of interference by other bodily fluids was confirmed by analysing stains of blood, urine and saliva prior to and after the addition of semen. Finally, the sensor was evaluated by analysing 51 forensic casework samples which were also analysed with a semi-quantitative membrane strip test (Seratec® PSA), through microscopic detection of spermatozoa, and male DNA identification through detection of Y chromosome. The results obtained with the sensor were in excellent agreement with those provided by an immunoradiometric assay kit (PSA-RIACT) and in complete agreement with the findings using the membrane strip assay, spermatozoa and Y chromosome detection. The excellent analytical performance and small size of the instrument make the sensor developed an attractive tool for use in forensic evidence screening for semen detection. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Attempts to protect severe combined immunodeficient (scid) mice with antibody enriched for reactivity to Cryptosporidium parvum surface antigen-1.

    PubMed

    Tatalick, L M; Perryman, L E

    1995-07-01

    Cryptosporidium parvum is a protozoal pathogen which infects the gastrointestinal epithelium of mammals causing diarrhoea, the duration and severity of which is determined by the immunocompetency of the host. Currently, there is no effective treatment or prevention. We evaluated the ability of surface antigen-1 (SA-1), defined as those antigens recognized by neutralizing mAb 17.41, to elicit a protective antibody response when used as an immunogen. A SA-1 enriched fraction was obtained by immunoaffinity chromatography and was used to immunize a naive Holstein calf. SA-1 immune serum from this calf detected C. parvum epitopes to a 1:10,000 dilution in a dot blot assay, and sporozoite surface epitopes at a 1:10,000 dilution in a live immunofluorescence assay. Western blot analysis showed that SA-1 immune bovine serum recognized a similar pattern of C. parvum antigens as the defining mAb 17.41. Oral passive transfer of SA-1 immune bovine serum did not protect severe combined immunodeficient (scid) mice or suckling BALB/c mice from initial infection with C. parvum, or terminate a persistent infection in scid mice.

  13. Animal and human antibodies to distinct Staphylococcus aureus antigens mutually neutralize opsonic killing and protection in mice

    PubMed Central

    Skurnik, David; Merighi, Massimo; Grout, Martha; Gadjeva, Mihaela; Maira-Litran, Tomas; Ericsson, Maria; Goldmann, Donald A.; Huang, Susan S.; Datta, Rupak; Lee, Jean C.; Pier, Gerald B.

    2010-01-01

    New prophylactic approaches are needed to control infection with the Gram-positive bacterium Staphylococcus aureus, which is a major cause of nosocomial and community-acquired infections. To develop these, greater understanding of protective immunity against S. aureus infection is needed. Human immunity to extracellular Gram-positive bacterial pathogens is primarily mediated by opsonic killing (OPK) via antibodies specific for surface polysaccharides. S. aureus expresses two such antigens, capsular polysaccharide (CP) and poly-N-acetyl glucosamine (PNAG). Here, we have shown that immunization-induced polyclonal animal antisera and monoclonal antibodies specific for either CP or PNAG antigens have excellent in vitro OPK activity in human blood but that when mixed together they show potent interference in OPK activity. In addition, reductions in antibody binding to the bacterial surface, complement deposition, and passive protection were seen in two mouse models of S. aureus infection. Electron microscopy, isothermal calorimetry, and surface plasmon resonance indicated that antibodies to CP and PNAG bound together via an apparent idiotype–anti-idiotype interaction. This interaction was also found in sera from humans with S. aureus bacteremia. These findings suggest that the lack of effective immunity to S. aureus infections in humans could be due, in part, to interference in OPK when antibodies to CP and PNAG antigens are both present. This information could be used to better design S. aureus vaccine components. PMID:20739753

  14. Topological phases protected by reflection symmetry and cross-cap states

    NASA Astrophysics Data System (ADS)

    Cho, Gil Young; Hsieh, Chang-Tse; Morimoto, Takahiro; Ryu, Shinsei

    2015-05-01

    Twisting symmetries provides an efficient method to diagnose symmetry-protected topological (SPT) phases. In this paper, edge theories of (2+1)-dimensional topological phases protected by reflection as well as other symmetries are studied by twisting reflection symmetry, which effectively puts the edge theories on an unoriented space-time, such as the Klein bottle. A key technical step taken in this paper is the use of the so-called cross-cap states, which encode entirely the unoriented nature of space-time, and can be obtained by rearranging the space-time geometry and exchanging the role of space and time coordinates. When the system is in a nontrivial SPT phase, we find that the corresponding cross-cap state is noninvariant under the action of the symmetries of the SPT phase, but acquires an anomalous phase. This anomalous phase, with a proper definition of a reference state, on which symmetry acts trivially, reproduces the known classification of (2+1)-dimensional bosonic and fermionic SPT phases protected by reflection symmetry, including in particular the Z8 classification of topological crystalline superconductors protected by reflection and time-reversal symmetries.

  15. Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs

    PubMed Central

    Petitdidier, Elodie; Pagniez, Julie; Papierok, Gérard; Vincendeau, Philippe; Lemesre, Jean-Loup; Bras-Gonçalves, Rachel

    2016-01-01

    Preventive vaccination is a highly promising strategy for interrupting leishmaniasis transmission that can, additionally, contribute to elimination. A vaccine formulation based on naturally excreted secreted (ES) antigens was prepared from L. infantum promastigote culture supernatant. This vaccine achieved successful results in Phase III trials and was licensed and marketed as CaniLeish. We recently showed that newly identified ES promastigote surface antigen (PSA), from both viable promastigotes and axenically-grown amastigotes, represented the major constituent and the highly immunogenic antigen of L. infantum and L. amazonensis ES products. We report here that three immunizations with either the recombinant ES LaPSA-38S (rPSA) or its carboxy terminal part LaPSA-12S (Cter-rPSA), combined with QA-21 as adjuvant, confer high levels of protection in naive L. infantum-infected Beagle dogs, as checked by bone marrow parasite absence in respectively 78.8% and 80% of vaccinated dogs at 6 months post-challenge. The parasite burden in infected vaccinated dogs was significantly reduced compared to placebo group, as measured by q-PCR. Moreover, our results reveal humoral and cellular immune response clear-cut differences between vaccinated and control dogs. An early increase in specific IgG2 antibodies was observed in rPSA/QA-21- and Cter-rPSA/QA-21-immunized dogs only. They were found functionally active in vitro and were highly correlated with vaccine protection. In vaccinated protected dogs, IFN-γ and NO productions, as well as anti-leishmanial macrophage activity, were increased. These data strongly suggest that ES PSA or its carboxy-terminal part, in recombinant forms, induce protection in a canine model of zoonotic visceral leishmaniasis by inducing a Th1-dominant immune response and an appropriate specific antibody response. These data suggest that they could be considered as important active components in vaccine candidates. PMID:27223609

  16. Oxidized multiwalled carbon nanotubes as antigen delivery system to promote superior CD8(+) T cell response and protection against cancer.

    PubMed

    de Faria, Paula Cristina Batista; dos Santos, Luara Isabela; Coelho, João Paulo; Ribeiro, Henrique Bücker; Pimenta, Marcos Assunção; Ladeira, Luiz Orlando; Gomes, Dawidson Assis; Furtado, Clascídia Aparecida; Gazzinelli, Ricardo Tostes

    2014-09-10

    Properties like high interfacial area with cellular membranes, unique ability to incorporate multiple functionalization, as well as compatibility and transport in biological fluids make carbon nanotubes (CNTs) useful for a variety of therapeutic and drug-delivery applications. Here we used a totally synthetic hybrid supramolecule as an anticancer vaccine formulation. This complex structure comprises CNTs as delivery system for the Cancer Testis Antigen named NY-ESO-1, allied to a synthetic Toll-Like Receptor agonist. The CNT constructs were rapidly internalized into dendritic cells, both in vitro and in vivo, and served as an intracellular antigen depot. This property favored the induction of strong CD4(+) T as well as CD8(+) T cell-mediated immune responses against the NY-ESO-1. Importantly, the vaccination significantly delayed the tumor development and prolonged the mice survival, highlighting the potential application of CNTs as a vaccine delivery system to provide superior immunogenicity and strong protection against cancer.

  17. A bacterial protease inhibitor protects antigens delivered in oral vaccines from digestion while triggering specific mucosal immune responses.

    PubMed

    Ibañez, Andrés Esteban; Coria, Lorena Mirta; Carabajal, Marianela Verónica; Delpino, María Victoria; Risso, Gabriela Sofía; Cobiello, Paula Gonzalez; Rinaldi, Jimena; Barrionuevo, Paula; Bruno, Laura; Frank, Fernanda; Klinke, Sebastián; Goldbaum, Fernando Alberto; Briones, Gabriel; Giambartolomei, Guillermo Hernán; Pasquevich, Karina Alejandra; Cassataro, Juliana

    2015-12-28

    We report here that a bacterial protease inhibitor from Brucella spp. called U-Omp19 behaves as an ideal constituent for a vaccine formulation against infectious diseases. When co-administered orally with an antigen (Ag), U-Omp19: i) can bypass the harsh environment of the gastrointestinal tract by inhibiting stomach and intestine proteases and consequently increases the half-life of the co-administered Ag at immune inductive sites: Peyer's patches and mesenteric lymph nodes while ii) it induces the recruitment and activation of antigen presenting cells (APCs) and increases the amount of intracellular Ag inside APCs. Therefore, mucosal as well as systemic Ag-specific immune responses, antibodies, Th1, Th17 and CD8(+) T cells are enhanced when U-Omp19 is co-administered with the Ag orally. Finally, this bacterial protease inhibitor in an oral vaccine formulation confers mucosal protection and reduces parasite loads after oral challenge with virulent Toxoplasma gondii.

  18. Host cell-induced components of the sulfate assimilation pathway are major protective antigens of Mycobacterium tuberculosis.

    PubMed

    Pinto, Rachel; Leotta, Lisa; Shanahan, Erin R; West, Nicholas P; Leyh, Thomas S; Britton, Warwick; Triccas, James A

    2013-03-01

    New therapies to control tuberculosis are urgently required because of the inability of the only available vaccine, BCG, to adequately protect against tuberculosis. Here we demonstrate that proteins of the Mycobacterium tuberculosis sulfate-assimilation pathway (SAP) represent major immunogenic targets of the bacillus, as defined by strong T-cell recognition by both mice and humans infected with M. tuberculosis. SAP proteins displayed increased expression when M. tuberculosis was resident within host cells, which may account in part for their ability to stimulate anti-M. tuberculosis host immunity. Vaccination with the first enzyme in this pathway, adenosine-5'-triphosphate sulfurylase, conferred significant protection against murine tuberculosis and boosted BCG-induced protective immunity in the lung. Therefore, we have identified SAP components as a new family of M. tuberculosis antigens, and we have demonstrated that these components are promising candidate for inclusion in new vaccines to control tuberculosis in humans.

  19. Host Cell–Induced Components of the Sulfate Assimilation Pathway Are Major Protective Antigens of Mycobacterium tuberculosis

    PubMed Central

    Pinto, Rachel; Leotta, Lisa; Shanahan, Erin R.; West, Nicholas P.; Leyh, Thomas S.; Britton, Warwick; Triccas, James A.

    2013-01-01

    New therapies to control tuberculosis are urgently required because of the inability of the only available vaccine, BCG, to adequately protect against tuberculosis. Here we demonstrate that proteins of the Mycobacterium tuberculosis sulfate-assimilation pathway (SAP) represent major immunogenic targets of the bacillus, as defined by strong T-cell recognition by both mice and humans infected with M. tuberculosis. SAP proteins displayed increased expression when M. tuberculosis was resident within host cells, which may account in part for their ability to stimulate anti-M. tuberculosis host immunity. Vaccination with the first enzyme in this pathway, adenosine-5′-triphosphate sulfurylase, conferred significant protection against murine tuberculosis and boosted BCG-induced protective immunity in the lung. Therefore, we have identified SAP components as a new family of M. tuberculosis antigens, and we have demonstrated that these components are promising candidate for inclusion in new vaccines to control tuberculosis in humans. PMID:23225904

  20. Protection against aerosolized Yersinia pestis challenge following homologous and heterologous prime-boost with recombinant plague antigens.

    PubMed

    Glynn, Audrey; Roy, Chad J; Powell, Bradford S; Adamovicz, Jeffrey J; Freytag, Lucy C; Clements, John D

    2005-08-01

    A Yersinia pestis-derived fusion protein (F1-V) has shown great promise as a protective antigen against aerosol challenge with Y. pestis in murine studies. In the current study, we examined different prime-boost regimens with F1-V and demonstrate that (i) boosting by a route other than the route used for the priming dose (heterologous boosting) protects mice as well as homologous boosting against aerosol challenge with Y. pestis, (ii) parenteral immunization is not required to protect mice against aerosolized plague challenge, (iii) the route of immunization and choice of adjuvant influence the magnitude of the antibody response as well as the immunoglobulin G1 (IgG1)/IgG2a ratio, and (iv) inclusion of an appropriate adjuvant is critical for nonparenteral immunization.

  1. Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection.

    PubMed

    Reed, Matthew D; Wilder, Julie A; Mega, William M; Hutt, Julie A; Kuehl, Philip J; Valderas, Michelle W; Chew, Lawrence L; Liang, Bertrand C; Squires, Charles H

    2015-01-01

    Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg.

  2. Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection

    PubMed Central

    Reed, Matthew D.; Wilder, Julie A.; Mega, William M.; Hutt, Julie A.; Kuehl, Philip J.; Valderas, Michelle W.; Chew, Lawrence L.; Liang, Bertrand C.; Squires, Charles H.

    2015-01-01

    Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg. PMID:26207820

  3. Adoptive transfer of helminth antigen-pulsed dendritic cells protects against the development of experimental colitis in mice.

    PubMed

    Matisz, Chelsea E; Leung, Gabriella; Reyes, Jose Luis; Wang, Arthur; Sharkey, Keith A; McKay, Derek M

    2015-11-01

    Infection with helminth parasites and treatment with worm extracts can suppress inflammatory disease, including colitis. Postulating that dendritic cells (DCs) participated in the suppression of inflammation and seeking to move beyond the use of helminths per se, we tested the ability of Hymenolepis diminuta antigen-pulsed DCs to suppress colitis as a novel cell-based immunotherapy. Bone marrow derived DCs pulsed with H. diminuta antigen (HD-DCs), or PBS-, BSA-, or LPS-DCs as controls, were transferred into wild-type (WT), interleukin-10 (IL-10) knock-out (KO), and RAG-1 KO mice, and the impact on dinitrobenzene sulphonic acid (DNBS)-induced colitis and splenic cytokine production assessed 72 h later. Mice receiving HD-DCs were significantly protected from DNBS-induced colitis and of the experimental groups only these mice displayed increased Th2 cytokines and IL-10 production. Adoptive transfer of HD-DCs protected neither RAG-1 nor IL-10 KO mice from DNBS-colitis. Furthermore, the transfer of CD4(+) splenocytes from recipients of HD-DCs protected naïve mice against DNBS-colitis, in an IL-10 dependent manner. Thus, HD-DCs are a novel anti-colitic immunotherapy that can educate anti-colitic CD4(+) T cells: mechanistically, the anti-colitic effect of HD-DCs requires that the host has an adaptive immune response and the ability to mobilize IL-10.

  4. Cloning and Characterization of Surface-Localized α-Enolase of Streptococcus iniae, an Effective Protective Antigen in Mice.

    PubMed

    Wang, Jun; Wang, Kaiyu; Chen, Defang; Geng, Yi; Huang, Xiaoli; He, Yang; Ji, Lili; Liu, Tao; Wang, Erlong; Yang, Qian; Lai, Weimin

    2015-06-25

    Streptococcus iniae is a major fish pathogen that can also cause human bacteremia, cellulitis and meningitis. Screening for and identification of protective antigens plays an important role in developing therapies against S. iniae infections. In this study, we indicated that the α-enolase of S. iniae was not only distributed in the cytoplasm and associated to cell walls, but was also secreted to the bacterial cell surface. The functional identity of the purified recombinant α-enolase protein was verified by its ability to catalyze the conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP), and both the recombinant and native proteins interacted with human plasminogen. The rabbit anti-rENO serum blockade assay shows that α-enolase participates in S. iniae adhesion to and invasion of BHK-21 cells. In addition, the recombinant α-enolase can confer effective protection against S. iniae infection in mice, which suggests that α-enolase has potential as a vaccine candidate in mammals. We conclude that S. iniae α-enolase is a moonlighting protein that also associates with the bacterial outer surface and functions as a protective antigen in mice.

  5. Analysis of H7 avian influenza viruses by antigenic cartography and correlation to protection by vaccination

    USDA-ARS?s Scientific Manuscript database

    The H7 hemagglutinin subtype one of the most common subtypes of avian influenza virus (AIV) in poultry world wide and since it has the potential to become highly pathogenic it is among the priority subtypes for vaccination. Selection of the optimal vaccine seed strains may now be aided by antigenic...

  6. Membrane insertion of anthrax protective antigen and cytoplasmic delivery of lethal factor occur at different stages of the endocytic pathway.

    PubMed

    Abrami, Laurence; Lindsay, Margaret; Parton, Robert G; Leppla, Stephen H; van der Goot, F Gisou

    2004-08-30

    The protective antigen (PA) of anthrax toxin binds to a cell surface receptor, undergoes heptamerization, and binds the enzymatic subunits, the lethal factor (LF) and the edema factor (EF). The resulting complex is then endocytosed. Via mechanisms that depend on the vacuolar ATPase and require membrane insertion of PA, LF and EF are ultimately delivered to the cytoplasm where their targets reside. Here, we show that membrane insertion of PA already occurs in early endosomes, possibly only in the multivesicular regions, but that subsequent delivery of LF to the cytoplasm occurs preferentially later in the endocytic pathway and relies on the dynamics of internal vesicles of multivesicular late endosomes.

  7. Enzyme-linked immunosorbent assay employing a recombinant antigen for detection of protective antibody against swine erysipelas.

    PubMed

    Imada, Yumiko; Mori, Yasuyuki; Daizoh, Masaji; Kudoh, Kazuma; Sakano, Tetsuya

    2003-11-01

    The specificities and sensitivities of five recombinant proteins of the surface protective antigen (SpaA) of Erysipelothrix rhusiopathiae were examined by indirect enzyme-linked immunosorbent assay (ELISA) with the aim of developing a reliable serological test for the detection of protective antibody against E. rhusiopathiae. Fully mature protein and the N-terminal 416 amino acids (SpaA416) showed sufficient antigenicities, and further examination was done with SpaA416 because of its higher yield. The antibody titers of pigs experimentally immunized with commercial live vaccine and two types of inactivated vaccines clearly increased after immunization, and all pigs were completely protected against challenge with virulent strains. On the other hand, the antibody titers of nonimmunized control pigs remained very low until they were challenged, and all showed severe symptoms or subsequently died. Interference with the production of antibody against live vaccine by maternal antibody or porcine respiratory and reproductive syndrome virus infection 1 week after vaccination was also clearly detected. Because the ELISA titer correlated well with the protection results, the specificity and sensitivity of the ELISA were further evaluated with sera collected from pigs reared on 1 farm on which animals had acute septicemia, 2 farms on which the animals were infected or free from infection, and 10 farms on which the animals were vaccinated with live vaccine, among others. The ELISA titers clearly revealed the conditions of the herds. These results indicate that the SpaA416 ELISA is an effective method not only for evaluating pigs for the presence of protective antibody levels resulting from vaccination or maternal antibody but also for detecting antibody produced by natural infection. This test has important potential for the effective control of swine erysipelas.

  8. Core ethical values of radiological protection applied to Fukushima case: reflecting common morality and cultural diversities.

    PubMed

    Kurihara, Chieko; Cho, Kunwoo; Toohey, Richard E

    2016-12-01

    The International Commission on Radiological Protection (ICRP) has established Task Group 94 (TG94) to develop a publication to clarify the ethical foundations of the radiological protection system it recommends. This TG identified four core ethical values which structure the system: beneficence and non-maleficence, prudence, justice, and dignity. Since the ICRP is an international organization, its recommendations and guidance should be globally applicable and acceptable. Therefore, first this paper presents the basic principles of the ICRP radiological protection system and its core ethical values, along with a reflection on the variation of these values in Western and Eastern cultural traditions. Secondly, this paper reflects upon how these values can be applied in difficult ethical dilemmas as in the case of the emergency and post-accident phases of a nuclear power plant accident, using the Fukushima case to illustrate the challenges at stake. We found that the core ethical values underlying the ICRP system of radiological protection seem to be quite common throughout the world, although there are some variations among various cultural contexts. Especially we found that 'prudence' would call for somewhat different implementation in each cultural context, balancing and integrating sometime conflicting values, but always with objectives to achieve the well-being of people, which is itself the ultimate aim of the radiological protection system.

  9. Macrophage migration inhibitory factor protects from nonmelanoma epidermal tumors by regulating the number of antigen-presenting cells in skin.

    PubMed

    Brocks, Tania; Fedorchenko, Oleg; Schliermann, Nicola; Stein, Astrid; Moll, Ute M; Seegobin, Seth; Dewor, Manfred; Hallek, Michael; Marquardt, Yvonne; Fietkau, Katharina; Heise, Ruth; Huth, Sebastian; Pfister, Herbert; Bernhagen, Juergen; Bucala, Richard; Baron, Jens M; Fingerle-Rowson, Guenter

    2017-02-01

    The response of the skin to harmful environmental agents is shaped decisively by the status of the immune system. Keratinocytes constitutively express and secrete the chemokine-like mediator, macrophage migration inhibitory factor (MIF), more strongly than dermal fibroblasts, thereby creating a MIF gradient in skin. By using global and epidermis-restricted Mif-knockout (Mif(-/-) and K14-Cre(+/tg); Mif(fl/fl)) mice, we found that MIF both recruits and maintains antigen-presenting cells in the dermis/epidermis. The reduced presence of antigen-presenting cells in the absence of MIF was associated with accelerated and increased formation of nonmelanoma skin tumors during chemical carcinogenesis. Our results demonstrate that MIF is essential for maintaining innate immunity in skin. Loss of keratinocyte-derived MIF leads to a loss of control of epithelial skin tumor formation in chemical skin carcinogenesis, which highlights an unexpected tumor-suppressive activity of MIF in murine skin.-Brocks, T., Fedorchenko, O., Schliermann, N., Stein, A., Moll, U. M., Seegobin, S., Dewor, M., Hallek, M., Marquardt, Y., Fietkau, K., Heise, R., Huth, S., Pfister, H., Bernhagen, J., Bucala, R., Baron, J. M., Fingerle-Rowson, G. Macrophage migration inhibitory factor protects from nonmelanoma epidermal tumors by regulating the number of antigen-presenting cells in skin. © FASEB.

  10. Effect of FMD vaccine antigen payload on protection, sub-clinical infection and persistence following needle challenge in sheep.

    PubMed

    Madhanmohan, Muthukrishnan; Nagendrakumar, Singanallur Balasubramanian; Narasu, Mangamoori Lakshmi; Srinivasan, Villuppanoor Alwar

    2010-12-01

    The relationship of Foot-and-Mouth Disease (FMD) virus antigen payload and single and double vaccinations in conferring protection against virus challenge in sheep was studied. Sheep vaccinated with half the cattle dose (1 ml) containing 15 and 3.75 μg of FMDV antigen with or without booster resisted virulent challenge on 21 days post vaccination or 7 days post booster. FMDV RNA could be detected in nasal secretions in 26% of vaccinated sheep (10(3.12) to 10(3.82) viral RNA copies) on day 35 post challenge. No live virus could be isolated after 5 days post challenge indicating that the risk of transmission of disease was probably very low. The finding showed that vaccines containing antigen payload of 1.88 μg may prevent or reduce the local virus replication at the oropharynx and shedding of virus from nasal secretions and thereby reduce the amount of virus released into the environment subsequent to exposure to live virus. Sheep with no vaccination or with poor sero conversion to vaccination can be infected without overt clinical signs and became carriers. Copyright © 2009 Elsevier Ltd. All rights reserved.

  11. Neutralizing antibody and functional mapping of Bacillus anthracis protective antigen-The first step toward a rationally designed anthrax vaccine.

    PubMed

    McComb, Ryan C; Martchenko, Mikhail

    2016-01-02

    Anthrax is defined by the Centers for Disease Control and Prevention as a Category A pathogen for its potential use as a bioweapon. Current prevention treatments include Anthrax Vaccine Adsorbed (AVA). AVA is an undefined formulation of Bacillus anthracis culture supernatant adsorbed to aluminum hydroxide. It has an onerous vaccination schedule, is slow and cumbersome to produce and is slightly reactogenic. Next-generation vaccines are focused on producing recombinant forms of anthrax toxin in a well-defined formulation but these vaccines have been shown to lose potency as they are stored. In addition, studies have shown that a proportion of the antibody response against these vaccines is focused on non-functional, non-neutralizing regions of the anthrax toxin while some essential functional regions are shielded from eliciting an antibody response. Rational vaccinology is a developing field that focuses on designing vaccine antigens based on structural information provided by neutralizing antibody epitope mapping, crystal structure analysis, and functional mapping through amino acid mutations. This information provides an opportunity to design antigens that target only functionally important and conserved regions of a pathogen in order to make a more optimal vaccine product. This review provides an overview of the literature related to functional and neutralizing antibody epitope mapping of the Protective Antigen (PA) component of anthrax toxin.

  12. An Antibody Screen of a Plasmodium vivax Antigen Library Identifies Novel Merozoite Proteins Associated with Clinical Protection.

    PubMed

    França, Camila T; Hostetler, Jessica B; Sharma, Sumana; White, Michael T; Lin, Enmoore; Kiniboro, Benson; Waltmann, Andreea; Darcy, Andrew W; Li Wai Suen, Connie S N; Siba, Peter; King, Christopher L; Rayner, Julian C; Fairhurst, Rick M; Mueller, Ivo

    2016-05-01

    Elimination of Plasmodium vivax malaria would be greatly facilitated by the development of an effective vaccine. A comprehensive and systematic characterization of antibodies to P. vivax antigens in exposed populations is useful in guiding rational vaccine design. In this study, we investigated antibodies to a large library of P. vivax entire ectodomain merozoite proteins in 2 Asia-Pacific populations, analysing the relationship of antibody levels with markers of current and cumulative malaria exposure, and socioeconomic and clinical indicators. 29 antigenic targets of natural immunity were identified. Of these, 12 highly-immunogenic proteins were strongly associated with age and thus cumulative lifetime exposure in Solomon Islanders (P<0.001-0.027). A subset of 6 proteins, selected on the basis of immunogenicity and expression levels, were used to examine antibody levels in plasma samples from a population of young Papua New Guinean children with well-characterized individual differences in exposure. This analysis identified a strong association between reduced risk of clinical disease and antibody levels to P12, P41, and a novel hypothetical protein that has not previously been studied, PVX_081550 (IRR 0.46-0.74; P<0.001-0.041). These data emphasize the benefits of an unbiased screening approach in identifying novel vaccine candidate antigens. Functional studies are now required to establish whether PVX_081550 is a key component of the naturally-acquired protective immune response, a biomarker of immune status, or both.

  13. Protection of HF Transmitters from Reflection Failure by Help of Semiconductor Isolators

    NASA Astrophysics Data System (ADS)

    Laurinavičius, L.

    2009-01-01

    The fundamental problem of the transmitters protection against microwave energy reflections from aircraft antenna is discussed. The paper presents some applications of active and passive isolators and circulators as a tool for reflections protection. It has been stated that electrical properties of ferrite isolators and circulators grow worse at lower operational frequencies (below 50 MHz) and temperatures (cryogenic). Semiconductor magnetoplasma exhibits gyrotropic effects similar to those of magnetized ferrites and can solve the problem non-reciprocity in the HF range from 3 to 30 MHz, which is important for HF communication. The semiconductor device realization, optimal parameters, and operational temperature range, based on minimum forward loss α and maximum reverse attenuation β, were chosen. Major electromagnetic parameters of semiconductor isolators were determined.

  14. Intramuscular delivery of adenovirus serotype 5 vector expressing humanized protective antigen induces rapid protection against anthrax that may bypass intranasally originated preexisting adenovirus immunity.

    PubMed

    Wu, Shipo; Zhang, Zhe; Yu, Rui; Zhang, Jun; Liu, Ying; Song, Xiaohong; Yi, Shaoqiong; Liu, Ju; Chen, Jianqin; Yin, Ying; Xu, Junjie; Hou, Lihua; Chen, Wei

    2014-02-01

    Developing an effective anthrax vaccine that can induce a rapid and sustained immune response is a priority for the prevention of bioterrorism-associated anthrax infection. Here, we developed a recombinant replication-deficient adenovirus serotype 5-based vaccine expressing the humanized protective antigen (Ad5-PAopt). A single intramuscular injection of Ad5-PAopt resulted in rapid and robust humoral and cellular immune responses in Fisher 344 rats. Animals intramuscularly inoculated with a single dose of 10⁸ infectious units of Ad5-PAopt achieved 100% protection from challenge with 10 times the 50% lethal dose (LD₅₀) of anthrax lethal toxin 7 days after vaccination. Although preexisting intranasally induced immunity to Ad5 slightly weakened the humoral and cellular immune responses to Ad5-PAopt via intramuscular inoculation, 100% protection was achieved 15 days after vaccination in Fisher 344 rats. The protective efficacy conferred by intramuscular vaccination in the presence of preexisting intranasally induced immunity was significantly better than that of intranasal delivery of Ad5-PAopt and intramuscular injection with recombinant PA and aluminum adjuvant without preexisting immunity. As natural Ad5 infection often occurs via the mucosal route, the work here largely illuminates that intramuscular inoculation with Ad5-PAopt can overcome the negative effects of immunity induced by prior adenovirus infection and represents an efficient approach for protecting against emerging anthrax.

  15. Expression of the ’Bacillus anthracis’ Protective Antigen Gene by Baculovirus and Vaccinia Virus Recombinants

    DTIC Science & Technology

    1990-02-01

    ANTIIRACIS PROTI-CTIVE ANTIGEN 367 in medium containing 10% fetal bovine serum. Vaccinia nitrocellulose filters . All cell lysate samples subjected...mM glycine. and 20% methanol at 4°C for 12 to 16 h. by CaCl. precipitation. A. cali irni’, nuclear polyhedrosis Nitrocellulose filters were washed in...and monoclonal antibodies used for these pl[XTJSCRilrr(Stratagcnc. La Jolla. Calif.I. pI3LSCRPPA studies. Filters were washed and incubated with

  16. Synthesis of type 2 Lewis antigens via novel regioselective glycosylation of an orthogonally protected lactosamine diol derivative.

    PubMed

    Yamazaki, Yuji; Sezukuri, Kyohei; Takada, Junko; Obata, Hiroaki; Kimura, Shunsaku; Ohmae, Masashi

    2016-03-03

    The novel and efficient synthesis of type 2 Lewis antigens is reported in this study. The rationally designed lactosamine-3,2'-diol derivative with an orthogonal set of protecting groups is efficiently glycosylated with a benzyl protected 1-thio-l-fucoside donor in a unique regioselective manner to produce Lewis x (Le(x)) and Lewis y (Le(y)) derivatives in good yields. These derivatives can be prepared not only exclusively but also synchronously by choosing the appropriate reaction temperature and donor-acceptor molar ratio. The Le(x) derivatives are easily converted into sulfated or non-sulfated Le(x) bearing a terminal azido functionalized oligo-(ethyleneoxide) linker; the Le(y) derivative having the same linker can also be prepared, all of which can be further used for the chemical modification of other compounds and materials. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Brucella abortus Omp19 recombinant protein subcutaneously co-delivered with an antigen enhances antigen-specific T helper 1 memory responses and induces protection against parasite challenge.

    PubMed

    Coria, Lorena M; Ibañez, Andrés E; Pasquevich, Karina A; Cobiello, Paula L González; Frank, Fernanda M; Giambartolomei, Guillermo H; Cassataro, Juliana

    2016-01-20

    The discovery of effective adjuvants for many vaccines especially those with limited commercial appeal, such as vaccines to poverty-related diseases, is required. In this work, we demonstrated that subcutaneous co-administration of mice with the outer membrane protein U-Omp19 from Brucella spp. plus OVA as antigen (Ag) increases Ag-specific T cell proliferation and T helper (Th) 1 immune responses in vitro and in vivo. U-Omp19 treated dendritic cells promote IFN-γ production by specific CD4(+) T cells and increases T cell proliferation. U-Omp19 co-administration induces the production of Ag specific effector memory T cell populations (CD4(+) CD44(high) CD62L(low) T cells). Finally, subcutaneous co-administration of U-Omp19 with Trypanosoma cruzi Ags confers protection against virulent parasite challenge, reducing parasitemia and weight loss while increasing mice survival. These results indicate that the bacterial protein U-Omp19 when delivered subcutaneously could be a suitable component of vaccine formulations against infectious diseases requiring Th1 immune responses.

  18. Novel protective antigens expressed by Trypanosoma cruzi amastigotes provide immunity to mice highly susceptible to Chagas' disease.

    PubMed

    Silveira, Eduardo L V; Claser, Carla; Haolla, Filipe A B; Zanella, Luiz G; Rodrigues, Mauricio M

    2008-08-01

    Earlier studies have demonstrated in A/Sn mice highly susceptible to Chagas' disease protective immunity against lethal Trypanosoma cruzi infection elicited by vaccination with an open reading frame (ORF) expressed by amastigotes. In our experiments, we used this mouse model to search for other amastigote-expressed ORFs with a similar property. Fourteen ORFs previously determined to be expressed in this developmental stage were individually inserted into a eukaryotic expression vector containing a nucleotide sequence that encoded a mammalian secretory signal peptide. Immunization with 13 of the 14 ORFs induced specific antibodies which recognized the amastigotes. Three of those immune sera also reacted with trypomastigotes and epimastigotes. After a lethal challenge with Y strain trypomastigotes, the vast majority of plasmid-injected mice succumbed to infection. In some cases, a significant delay in mortality was observed. Only two of these ORFs provided protective immunity against the otherwise lethal infection caused by trypomastigotes of the Y or Colombia strain. These ORFs encode members of the trans-sialidase family of surface antigens related to the previously described protective antigen amastigote surface protein 2 (ASP-2). Nevertheless, at the level of antibody recognition, no cross-reactivity was observed between the ORFs and the previously described ASP-2 from the Y strain. In immunofluorescence analyses, we observed the presence of epitopes related to both proteins expressed by amastigotes of seven different strains. In conclusion, our approach allowed us to successfully identify two novel protective ORFs which we consider interesting for future studies on the immune response to Chagas' disease.

  19. Novel Protective Antigens Expressed by Trypanosoma cruzi Amastigotes Provide Immunity to Mice Highly Susceptible to Chagas' Disease▿

    PubMed Central

    Silveira, Eduardo L. V.; Claser, Carla; Haolla, Filipe A. B.; Zanella, Luiz G.; Rodrigues, Mauricio M.

    2008-01-01

    Earlier studies have demonstrated in A/Sn mice highly susceptible to Chagas' disease protective immunity against lethal Trypanosoma cruzi infection elicited by vaccination with an open reading frame (ORF) expressed by amastigotes. In our experiments, we used this mouse model to search for other amastigote-expressed ORFs with a similar property. Fourteen ORFs previously determined to be expressed in this developmental stage were individually inserted into a eukaryotic expression vector containing a nucleotide sequence that encoded a mammalian secretory signal peptide. Immunization with 13 of the 14 ORFs induced specific antibodies which recognized the amastigotes. Three of those immune sera also reacted with trypomastigotes and epimastigotes. After a lethal challenge with Y strain trypomastigotes, the vast majority of plasmid-injected mice succumbed to infection. In some cases, a significant delay in mortality was observed. Only two of these ORFs provided protective immunity against the otherwise lethal infection caused by trypomastigotes of the Y or Colombia strain. These ORFs encode members of the trans-sialidase family of surface antigens related to the previously described protective antigen amastigote surface protein 2 (ASP-2). Nevertheless, at the level of antibody recognition, no cross-reactivity was observed between the ORFs and the previously described ASP-2 from the Y strain. In immunofluorescence analyses, we observed the presence of epitopes related to both proteins expressed by amastigotes of seven different strains. In conclusion, our approach allowed us to successfully identify two novel protective ORFs which we consider interesting for future studies on the immune response to Chagas' disease. PMID:18579696

  20. LptD is a promising vaccine antigen and potential immunotherapeutic target for protection against Vibrio species infection

    PubMed Central

    Zha, Zhenzhong; Li, Chuchu; Li, Weiyan; Ye, Zhicang; Pan, Jianyi

    2016-01-01

    Outer membrane proteins (OMPs) are unique to Gram-negative bacteria. Several features, including surface exposure, conservation among strains and ability to induce immune responses, make OMPs attractive targets for using as vaccine antigens and immunotherapeutics. LptD is an essential OMP that mediates the final transport of lipopolysaccharide (LPS) to outer leaflet. The protein in Vibrio parahaemolyticus was identified to have immunogenicity in our previous report. In this study, broad distribution, high conservation and similar surface-epitopes of LptD were found among the major Vibrio species. LptD was further revealed to be associated with immune responses, and it has a strong ability to stimulate antibody response. More importantly, it conferred 100% immune protection against lethal challenge by V. parahaemolyticus in mice when the mice were vaccinated with LptD, and this finding was consistent with the observation of efficient clearance of bacteria in vaccination mice. Strikingly, targeting of bacteria by the LptD antibody caused significant decreases in both the growth and LPS level and an increase in susceptibility to hydrophobic antibiotics. These findings were consistent with those previously obtained in lptD-deletion bacteria. These data demonstrated LptD is a promising vaccine antigens and a potential target for antibody-based therapy to protect against Vibrio infections. PMID:27922123

  1. Binding to histo-blood group antigen-expressing bacteria protects human norovirus from acute heat stress

    PubMed Central

    Li, Dan; Breiman, Adrien; le Pendu, Jacques; Uyttendaele, Mieke

    2015-01-01

    This study aims to investigate if histo-blood group antigen (HBGA) expressing bacteria have any protective role on human norovirus (NoV) from acute heat stress. Eleven bacterial strains were included, belonging to Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Clostridium difficile, Bifidobacterium adolescentis, and B. longum. HBGA expression of the bacteria as well as binding of human NoV virus-like particles (VLPs, GI.1, and GII.4 strains) to the bacteria were detected by flow cytometry. NoV VLPs pre-incubated with HBGA expressing or non-HBGA expressing bacteria were heated and detected by both direct ELISA and porcine gastric mucin-binding assay. The NoV-binding abilities of the bacteria correlated well with their HBGA expression profiles. Two HBGA expressing E. coli (LMG8223 and LFMFP861, both GI.1 and GII.4 binders) and one non-HBGA expressing E. coli (ATCC8739, neither GI.1 nor GII.4 binder) were selected for the heat treatment test with NoV VLPs. Compared with the same cell numbers of non-HBGA expressing E. coli, the presence of HBGA-expressing E. coli could always maintain higher antigen integrity, as well as mucin-binding ability of NoV VLPs of both GI.1 and GII.4 after heat-treatment at 90°C for 2 min. These results indicate that HBGA-expressing bacteria may protect NoVs during the food processing treatments, thereby facilitating their transmission. PMID:26191052

  2. Presentation of peptides from Bacillus anthracis protective antigen on Tobacco Mosaic Virus as an epitope targeted anthrax vaccine.

    PubMed

    McComb, Ryan C; Ho, Chi-Lee; Bradley, Kenneth A; Grill, Laurence K; Martchenko, Mikhail

    2015-11-27

    The current anthrax vaccine requires improvements for rapidly invoking longer-lasting neutralizing antibody responses with fewer doses from a well-defined formulation. Designing antigens that target neutralizing antibody epitopes of anthrax protective antigen, a component of anthrax toxin, may offer a solution for achieving a vaccine that can induce strong and long lasting antibody responses with fewer boosters. Here we report implementation of a strategy for developing epitope focused virus nanoparticle vaccines against anthrax by using immunogenic virus particles to present peptides derived from anthrax toxin previously identified in (1) neutralizing antibody epitope mapping studies, (2) toxin crystal structure analyses to identify functional regions, and (3) toxin mutational analyses. We successfully expressed two of three peptide epitopes from anthrax toxin that, in previous reports, bound antibodies that were partially neutralizing against toxin activity, discovered cross-reactivity between vaccine constructs and toxin specific antibodies raised in goats against native toxin and showed that antibodies induced by our vaccine constructs also cross-react with native toxin. While protection against intoxication in cellular and animal studies were not as effective as in previous studies, partial toxin neutralization was observed in animals, demonstrating the feasibility of using plant-virus nanoparticles as a platform for epitope defined anthrax vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Heat gain from thermal radiation through protective clothing with different insulation, reflectivity and vapour permeability.

    PubMed

    Bröde, Peter; Kuklane, Kalev; Candas, Victor; Den Hartog, Emiel A; Griefahn, Barbara; Holmér, Ingvar; Meinander, Harriet; Nocker, Wolfgang; Richards, Mark; Havenith, George

    2010-01-01

    The heat transferred through protective clothing under long wave radiation compared to a reference condition without radiant stress was determined in thermal manikin experiments. The influence of clothing insulation and reflectivity, and the interaction with wind and wet underclothing were considered. Garments with different outer materials and colours and additionally an aluminised reflective suit were combined with different number and types of dry and pre-wetted underwear layers. Under radiant stress, whole body heat loss decreased, i.e., heat gain occurred compared to the reference. This heat gain increased with radiation intensity, and decreased with air velocity and clothing insulation. Except for the reflective outer layer that showed only minimal heat gain over the whole range of radiation intensities, the influence of the outer garments' material and colour was small with dry clothing. Wetting the underclothing for simulating sweat accumulation, however, caused differing effects with higher heat gain in less permeable garments.

  4. Expression in Escherichia coli of a high-molecular-weight protective surface antigen found in nontypeable and type b Haemophilus influenzae.

    PubMed Central

    Thomas, W R; Callow, M G; Dilworth, R J; Audesho, A A

    1990-01-01

    An Escherichia coli clone producing a high-molecular-weight surface antigen of Haemophilus influenzae type b (Hib) was isolated from a library of Hib DNA fragments cloned as lysogens in a lambda replacement vector. The antigen is found in sarcosyl-insoluble outer membrane protein preparations and was produced by all 36 H. influenzae isolates tested. Absorption studies indicated that the antigen is a surface determinant on all isolates tested. Antibodies to the antigen (D15) were found in eight of nine convalescent-phase sera from children with invasive Hib infection. Affinity-purified antibodies prepared against the cloned antigen gave protection against the development of bacteremia in a rat pup model. Images PMID:2187812

  5. Immunoglobulin G Subclass-Specific Responses against Plasmodium falciparum Merozoite Antigens Are Associated with Control of Parasitemia and Protection from Symptomatic Illness▿ †

    PubMed Central

    Stanisic, Danielle I.; Richards, Jack S.; McCallum, Fiona J.; Michon, Pascal; King, Christopher L.; Schoepflin, Sonja; Gilson, Paul R.; Murphy, Vincent J.; Anders, Robin F.; Mueller, Ivo; Beeson, James G.

    2009-01-01

    Substantial evidence indicates that antibodies to Plasmodium falciparum merozoite antigens play a role in protection from malaria, although the precise targets and mechanisms mediating immunity remain unclear. Different malaria antigens induce distinct immunoglobulin G (IgG) subclass responses, but the importance of different responses in protective immunity from malaria is not known and the factors determining subclass responses in vivo are poorly understood. We examined IgG and IgG subclass responses to the merozoite antigens MSP1-19 (the 19-kDa C-terminal region of merozoite surface protein 1), MSP2 (merozoite surface protein 2), and AMA-1 (apical membrane antigen 1), including different polymorphic variants of these antigens, in a longitudinal cohort of children in Papua New Guinea. IgG1 and IgG3 were the predominant subclasses of antibodies to each antigen, and all antibody responses increased in association with age and exposure without evidence of increasing polarization toward one subclass. The profiles of IgG subclasses differed somewhat for different alleles of MSP2 but not for different variants of AMA-1. Individuals did not appear to have a propensity to make a specific subclass response irrespective of the antigen. Instead, data suggest that subclass responses to each antigen are generated independently among individuals and that antigen properties, rather than host factors, are the major determinants of IgG subclass responses. High levels of AMA-1-specific IgG3 and MSP1-19-specific IgG1 were strongly predictive of a reduced risk of symptomatic malaria and high-density P. falciparum infections. However, no antibody response was significantly associated with protection from parasitization per se. Our findings have major implications for understanding human immunity and for malaria vaccine development and evaluation. PMID:19139189

  6. Identification of broadly conserved cross-species protective Leishmania antigen and its responding CD4+ T cells.

    PubMed

    Mou, Zhirong; Li, Jintao; Boussoffara, Thouraya; Kishi, Hiroyuki; Hamana, Hiroshi; Ezzati, Peyman; Hu, Chuanmin; Yi, Weijing; Liu, Dong; Khadem, Forough; Okwor, Ifeoma; Jia, Ping; Shitaoka, Kiyomi; Wang, Shufeng; Ndao, Momar; Petersen, Christine; Chen, Jianping; Rafati, Sima; Louzir, Hechmi; Muraguchi, Atsushi; Wilkins, John A; Uzonna, Jude E

    2015-10-21

    There is currently no clinically effective vaccine against leishmaniasis because of poor understanding of the antigens that elicit dominant T cell immunity. Using proteomics and cellular immunology, we identified a dominant naturally processed peptide (PEPCK335-351) derived from Leishmania glycosomal phosphoenolpyruvate carboxykinase (PEPCK). PEPCK was conserved in all pathogenic Leishmania, expressed in glycosomes of promastigotes and amastigotes, and elicited strong CD4(+) T cell responses in infected mice and humans. I-A(b)-PEPCK335-351 tetramer identified protective Leishmania-specific CD4(+) T cells at a clonal level, which comprised ~20% of all Leishmania-reactive CD4(+) T cells at the peak of infection. PEPCK335-351-specific CD4(+) T cells were oligoclonal in their T cell receptor usage, produced polyfunctional cytokines (interleukin-2, interferon-γ, and tumor necrosis factor), and underwent expansion, effector activities, contraction, and stable maintenance after lesion resolution. Vaccination with PEPCK peptide, DNA expressing full-length PEPCK, or rPEPCK induced strong durable cross-species protection in both resistant and susceptible mice. The effectiveness and durability of protection in vaccinated mice support the development of a broadly cross-species protective vaccine against different forms of leishmaniasis by targeting PEPCK. Copyright © 2015, American Association for the Advancement of Science.

  7. Wave interaction with a partially reflecting vertical wall protected by a submerged porous bar

    NASA Astrophysics Data System (ADS)

    Zhao, Yang; Liu, Yong; Li, Huajun

    2016-08-01

    This study gives an analytical solution for wave interaction with a partially reflecting vertical wall protected by a submerged porous bar based on linear potential theory. The whole study domain is divided into multiple sub-regions in relation to the structures. The velocity potential in each sub-region is written as a series solution by the separation of variables. A partially reflecting boundary condition is used to describe the partial reflection of a vertical wall. Unknown expansion coefficients in the series solutions are determined by matching velocity potentials among different sub-regions. The analytical solution is verified by an independently developed multi-domain boundary element method (BEM) solution and experimental data. The wave run-up and wave force on the partially reflecting vertical wall are estimated and examined, which can be effectively reduced by the submerged porous bar. The horizontal space between the vertical wall and the submerged porous bar is a key factor, which affects the sheltering function of the porous bar. The wave resonance between the porous bar and the vertical wall may disappear when the vertical wall has a low reflection coefficient. The present analytical solution may be used to determine the optimum parameters of structures at a preliminary engineering design stage.

  8. A CpG-Ficoll Nanoparticle Adjuvant for Anthrax Protective Antigen Enhances Immunogenicity and Provides Single-immunization Protection against Inhaled Anthrax in Monkeys1

    PubMed Central

    Kachura, Melissa A.; Hickle, Colin; Kell, Sariah A.; Sathe, Atul; Calacsan, Carlo; Kiwan, Radwan; Hall, Brian; Milley, Robert; Ott, Gary; Coffman, Robert L.; Kanzler, Holger; Campbell, John D.

    2015-01-01

    Nanoparticulate delivery systems for vaccine adjuvants, designed to enhance targeting of secondary lymphoid organs and activation of APCs, have shown substantial promise for enhanced immunopotentiation. We investigated the adjuvant activity of synthetic oligonucleotides containing CpG-rich motifs (CpG-ODN) linked to the sucrose polymer Ficoll, forming soluble 50 nm particles (DV230-Ficoll), each containing over 100 molecules of the TLR9 ligand, DV230. DV230-Ficoll was evaluated as an adjuvant for a candidate vaccine for anthrax using a recombinant form of protective antigen (rPA) from Bacillus anthracis. A single immunization with rPA plus DV230-Ficoll induced 10-fold higher titers of toxin-neutralizing antibodies in cynomolgus monkeys at 2 weeks compared with animals immunized with equivalent amounts of monomeric DV230. Monkeys immunized either once or twice with rPA plus DV230-Ficoll were completely protected from challenge with 200 LD50 aerosolized anthrax spores. In mice, DV230-Ficoll was more potent than DV230 for the induction of innate immune responses at the injection site and draining lymph nodes. DV230-Ficoll was preferentially co-localized with rPA in key antigen-presenting cell populations and induced greater maturation marker expression (CD69 and CD86) on these cells and stronger germinal center B and T cell responses, relative to DV230. DV230-Ficoll was also preferentially retained at the injection site and draining lymph nodes and produced fewer systemic inflammatory responses. These findings support the development of DV230-Ficoll as an adjuvant platform, particularly for vaccines such as for anthrax, for which rapid induction of protective immunity and memory with a single injection is very important. PMID:26608924

  9. Expression of Bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop

    PubMed Central

    Watson, Jennifer; Koya, Vijay; Leppla, Stephen H.; Daniell, Henry

    2012-01-01

    The Centers for Disease Control (CDC) lists Bacillus anthracis as a category A agent and estimates the cost of an anthrax attack to exceed US$ 26 billion per 100,000 exposed individuals. Concerns regarding anthrax vaccine purity, a requirement for multiple injections, and a limited supply of the protective antigen (PA), underscore the urgent need for an improved vaccine. Therefore, the 83 kDa immunogenic Bacillus anthracis protective antigen was expressed in transgenic tobacco chloroplasts. The PA gene (pag) was cloned into a chloroplast vector along with the psbA regulatory signals to enhance translation. Chloroplast integration of the transgenes was confirmed by PCR and Southern blot analyses. Crude plant extracts contained up to 2.5 mg full length PA/g of fresh leaf tissue and this showed exceptional stability for several months in stored leaves or crude extracts. Maximum levels of expression were observed in mature leaves under continuous illumination. Co-expression of the ORF2 chaperonin from Bacillus thuringiensis did not increase PA accumulation or induce folding into cuboidal crystals in transgenic chloroplasts. Trypsin, chymotrypsin and furin proteolytic cleavage sites present in PA were protected in transgenic chloroplasts because only full length PA 83 was observed without any degradation products. Both CHAPS and SDS detergents extracted PA with equal efficiency and PA was observed in the soluble fraction. Chloroplast-derived PA was functionally active in lysing mouse macrophages when combined with lethal factor (LF). Crude leaf extracts contained up to 25 μg functional PA/ml. With an average yield of 172 mg of PA per plant using an experimental transgenic cultivar grown in a greenhouse, 400 million doses of vaccine (free of contaminants) could be produced per acre, a yield that could be further enhanced 18-fold using a commercial cultivar in the field. PMID:15474731

  10. Protective Efficacy of Coccidial Common Antigen Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) against Challenge with Three Eimeria Species

    PubMed Central

    Tian, Lu; Li, Wenyu; Huang, Xinmei; Tian, Di; Liu, Jianhua; Yang, Xinchao; Liu, Lianrui; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui; Song, Xiaokai

    2017-01-01

    Coccidiosis is an intestinal disorder of poultry and often caused by simultaneous infections of several Eimeria species. GAPDH is one of the immunogenic common antigens among Eimeria tenella, E. acervulina, and E. maxima identified in our previous study. The present study was performed to further evaluate its immunogenicity and protective efficacy. The genes of GAPDH cloned from E. acervulina and E. maxima were named as EaGAPDH and EmGAPDH, respectively. The immunogenicity of recombinant proteins of EaGAPDH and EmGAPDH were analyzed by Western blot. The transcription and expression of pVAX-EaGAPDH and pVAX-EmGAPDH in the injected muscles were detected by reverse transcription PCR (RT-PCR) and Western blot, respectively. GAPDH-induced changes of T lymphocytes subpopulation, cytokines production, and antibody were determined using flow cytometry, quantitative real-time PCR (qPCR), and ELISA, respectively. Finally, the protective efficacies of pVAX-EaGAPDH and pVAX-EmGAPDH were evaluated by vaccination and challenge experiments. The results revealed that the recombinant GAPDH proteins reacted with the corresponding chicken antisera. The EaGAPDH genes were successfully transcribed and expressed in the injected muscles. Vaccination with pVAX-EaGAPDH and pVAX-EmGAPDH significantly increased the proportion of CD4+ and CD8+ T lymphocytes, the cytokines productions of IFN-γ, IL-2, IL-4 et al., and IgG antibody levels compared to controls. The vaccination increased the weight gains, decreased the oocyst outputs, alleviate the enteric lesions compared to controls, and induced moderate anti-coccidial index (ACI). In conclusion, the coccidial common antigen of GAPDH induced significant humoral and cellular immune response and effective protection against E. tenella, E. acervulina, E. maxima, and mixed infection of the three Eimeria species. PMID:28769877

  11. Phase Variable O Antigen Biosynthetic Genes Control Expression of the Major Protective Antigen and Bacteriophage Receptor in Vibrio cholerae O1

    PubMed Central

    Seed, Kimberley D.; Faruque, Shah M.; Mekalanos, John J.; Calderwood, Stephen B.; Qadri, Firdausi; Camilli, Andrew

    2012-01-01

    The Vibrio cholerae lipopolysaccharide O1 antigen is a major target of bacteriophages and the human immune system and is of critical importance for vaccine design. We used an O1-specific lytic bacteriophage as a tool to probe the capacity of V. cholerae to alter its O1 antigen and identified a novel mechanism by which this organism can modulate O antigen expression and exhibit intra-strain heterogeneity. We identified two phase variable genes required for O1 antigen biosynthesis, manA and wbeL. manA resides outside of the previously recognized O1 antigen biosynthetic locus, and encodes for a phosphomannose isomerase critical for the initial step in O1 antigen biosynthesis. We determined that manA and wbeL phase variants are attenuated for virulence, providing functional evidence to further support the critical role of the O1 antigen for infectivity. We provide the first report of phase variation modulating O1 antigen expression in V. cholerae, and show that the maintenance of these phase variable loci is an important means by which this facultative pathogen can generate the diverse subpopulations of cells needed for infecting the host intestinal tract and for escaping predation by an O1-specific phage. PMID:23028317

  12. Partial purification and characterization of protection-inducing antigens from the muscle larva of Trichinella spiralis by molecular sizing chromatography and preparative flatbed isoelectric focusing.

    PubMed

    Despommier, D D

    1981-01-01

    The soluble portion of a large particle fraction which was derived from the muscle larva of T. spiralis was subjected to molecular sizing column chromatography using Sephacryl S-200. Five major peaks of 280 nm absorbing material were obtained. Analysis by immunoelectrophoresis revealed that each peak contained antigens, with the majority of them occurring in peaks 3, 4 and 5. Preliminary studies indicated that peak 4(mol. wt range 20 000--10 000) contained protection-inducing antigens. Crossed-immunoelectrophoretic and single-dimension electrophoretic analysis of peak 4 revealed a minimum of 10 antigens, while analytical isoelectric focusing demonstrated the presence of proteins with widely different pl, ranging from 4.0 to 9.0. Peak 4 was fractionated by preparative flatbed isoelectric focusing (PIEF) using two gradients: one from 3.5 to 9.5 and the other from 3.5 to 5.5. Fused rocket immunoelectrophoretic (FRIEP) analysis of both runs indicated that several antigens were separated from the others: one at pl 4.0 and the other at pl 9.0. The remaining antigens focused between pl 4.3 and 4.9. One hundred micrograms of whole peak 4, pl 9.0 antigen and the group of antigens at pl 4.3--4.9 were each separately injected, along with Freund's complete adjuvant, into mice. In addition, a portion of the pl 4.0 antigen was also assayed for protection. All antigenic preparations induced significant levels of protection. The pl 4.0 was further analysed on high-performance liquid chromatography (HPLC). Two sharp peaks of antigen, as detected by FRIEP, were eluted isocratically with 65% acetonitrile from a C-18 (aliphatic) column. Both peaks of antigen showed complete cross-reactivity on FRIEP and absorbed at 220 nm. Amino acid analysis of each HPLC peak revealed no detectable differences in composition. Each peak contained predominance of aspartic (13 mol%) and glutamic (18 mol%) acid. This antigen did not contain significant quantities of aromatic amino acids, and absorbed

  13. Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity

    PubMed Central

    Vasconcelos, Jose R.; Motz, R. Geoffrey; Sullivan, Nicole L.; Gohara, David W.; Blase, Jennifer R.; Di Cera, Enrico; Hoft, Daniel F.

    2016-01-01

    Trypanosoma cruzi infection is controlled but not eliminated by host immunity. The T. cruzi trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4+ and CD8+ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against T. cruzi challenge. We generated a new TS vaccine in which the immunodominant TS CD8+ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8+ T cell epitope enhance subdominant pathogen-specific CD8+ T cell responses. More specifically, CD8+ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8+ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for T. cruzi vaccine development. Of broader significance, we demonstrate that increasing breadth of T

  14. Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity.

    PubMed

    Eickhoff, Christopher S; Zhang, Xiuli; Vasconcelos, Jose R; Motz, R Geoffrey; Sullivan, Nicole L; O'Shea, Kelly; Pozzi, Nicola; Gohara, David W; Blase, Jennifer R; Di Cera, Enrico; Hoft, Daniel F

    2016-09-01

    Trypanosoma cruzi infection is controlled but not eliminated by host immunity. The T. cruzi trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4+ and CD8+ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against T. cruzi challenge. We generated a new TS vaccine in which the immunodominant TS CD8+ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8+ T cell epitope enhance subdominant pathogen-specific CD8+ T cell responses. More specifically, CD8+ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8+ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for T. cruzi vaccine development. Of broader significance, we demonstrate that increasing breadth of T

  15. Dose of incorporated immunodominant antigen in recombinant BCG impacts modestly on Th1 immune response and protective efficiency against Mycobacterium tuberculosis in mice.

    PubMed

    Ma, Hui; Wu, Kang; Liu, Fang; Yang, Hua; Kang, Han; Chen, Ning-Ning; Yuan, Qin; Zhou, Wen-Jiang; Fan, Xiao-Yong

    2014-01-01

    One approach for improving BCG efficacy is to utilize BCG as vehicle to develop recombinant BCG (rBCG) strains overexpressing Mycobacterium tuberculosis (M. tb) antigens. Also expression level of a candidate antigen should impact the final T cell responses conferred by rBCG. In this study, based on our previously constructed differential expression system, we developed two rBCG strains overexpressing M. tb chimeric antigen Ag856A2 (coding a recombinant ag85a with 2 copies of esat-6 inserted at Acc I site of ag85a) at differential levels under the control of the subtly modified furA promoters. These two rBCG strains were used to vaccinate C57BL/6 mice and exploit dose of incorporated antigen in rBCG to optimize immune response and protective efficiency against M. tb challenge in mouse model. The results showed that rBCG strains overexpressing Ag856A2 at differential levels induced different antigen-specific IFN-γ production and comparable number of M. tb-specific CD4 T cells expressing IL-2. M. tb challenge experiment showed that rBCG strains afforded enhanced but comparable immune protection characterized by reduced bacillary load, lung pathology, and inflammation. These results suggested that the dose of antigens incorporated in rBCG can impact T cell immune responses but imposed no significantly differential protective efficacies.

  16. Poor memory B cell generation contributes to non-protective responses to DTaP vaccine antigens in otitis-prone children.

    PubMed

    Basha, S; Pichichero, M E

    2015-12-01

    We recently identified a cohort of children with recurrent episodes of acute otitis media (AOM) who fail to generate protective antibody titres to otopathogens and several vaccine antigens. In this study we determined the antibody levels against DTaP vaccine antigens, diphtheria toxoid (DT), tetanus toxoid (TT) and acellular pertussis toxoid (PT) in sera from 15 stringently defined otitis-prone (sOP) children and 20 non-otitis-prone (NOP) children. We found significantly lower concentrations of immunoglobulin (Ig)G antibodies against vaccine antigens in the serum of sOP children compared to age-matched NOP children. To elucidate immunological cellular responses to the vaccines in these children, we investigated memory B cell responses to DTaP vaccination. We used fluorescently conjugated vaccine antigens to label antigen receptors on the surface of memory B cells and examined the frequency of antigen-specific CD19(+) CD27(+) memory B cells in the peripheral blood. sOP children showed a significantly lower percentage of antigen-specific CD19(+) CD27(+) memory B cells than NOP children. We also found a linear correlation between the frequencies of memory B cells and circulating IgG titres for DT, TT and PT proteins. To our knowledge, this is the first study to show significant differences in memory B cell responses to DTaP vaccine antigens and their correlation with the circulating antibodies in young children with recurrent AOM. © 2015 British Society for Immunology.

  17. An Antibody Screen of a Plasmodium vivax Antigen Library Identifies Novel Merozoite Proteins Associated with Clinical Protection

    PubMed Central

    França, Camila T.; Hostetler, Jessica B.; Sharma, Sumana; White, Michael T.; Lin, Enmoore; Kiniboro, Benson; Waltmann, Andreea; Darcy, Andrew W.; Li Wai Suen, Connie S. N.; Siba, Peter; King, Christopher L.; Rayner, Julian C.; Fairhurst, Rick M.; Mueller, Ivo

    2016-01-01

    Background Elimination of Plasmodium vivax malaria would be greatly facilitated by the development of an effective vaccine. A comprehensive and systematic characterization of antibodies to P. vivax antigens in exposed populations is useful in guiding rational vaccine design. Methodology/Principal Findings In this study, we investigated antibodies to a large library of P. vivax entire ectodomain merozoite proteins in 2 Asia-Pacific populations, analysing the relationship of antibody levels with markers of current and cumulative malaria exposure, and socioeconomic and clinical indicators. 29 antigenic targets of natural immunity were identified. Of these, 12 highly-immunogenic proteins were strongly associated with age and thus cumulative lifetime exposure in Solomon Islanders (P<0.001–0.027). A subset of 6 proteins, selected on the basis of immunogenicity and expression levels, were used to examine antibody levels in plasma samples from a population of young Papua New Guinean children with well-characterized individual differences in exposure. This analysis identified a strong association between reduced risk of clinical disease and antibody levels to P12, P41, and a novel hypothetical protein that has not previously been studied, PVX_081550 (IRR 0.46–0.74; P<0.001–0.041). Conclusion/Significance These data emphasize the benefits of an unbiased screening approach in identifying novel vaccine candidate antigens. Functional studies are now required to establish whether PVX_081550 is a key component of the naturally-acquired protective immune response, a biomarker of immune status, or both. PMID:27182597

  18. Structures of synthetic O-antigen fragments from serotype 2a Shigella flexneri in complex with a protective monoclonal antibody

    PubMed Central

    Vulliez-Le Normand, B.; Saul, F. A.; Phalipon, A.; Bélot, F.; Guerreiro, C.; Mulard, L. A.; Bentley, G. A.

    2008-01-01

    The anti-LPS IgG mAb F22-4, raised against Shigella flexneri serotype 2a bacteria, protects against homologous, but not heterologous, challenge in an experimental animal model. We report the crystal structures of complexes formed between Fab F22-4 and two synthetic oligosaccharides, a decasaccharide and a pentadecasaccharide that were previously shown to be both immunogenic and antigenic mimics of the S. flexneri serotype 2a O-antigen. F22-4 binds to an epitope contained within two consecutive 2a serotype pentasaccharide repeat units (RU). Six sugar residues from a contiguous nine-residue segment make direct contacts with the antibody, including the nonreducing rhamnose and both branching glucosyl residues from the two RUs. The glucosyl residue, whose position of attachment to the tetrasaccharide backbone of the RU defines the serotype 2a O-antigen, is critical for recognition by F22-4. Although the complete decasaccharide is visible in the electron density maps, the last four pentadecasaccharide residues from the reducing end, which do not contact the antibody, could not be traced. Although considerable mobility in the free oligosaccharides can thus be expected, the conformational similarity between the individual RUs, both within and between the two complexes, suggests that short-range transient ordering to a helical conformation might occur in solution. Although the observed epitope includes the terminal nonreducing residue, binding to internal epitopes within the polysaccharide chain is not precluded. Our results have implications for vaccine development because they suggest that a minimum of two RUs of synthetic serotype 2a oligosaccharide is required for optimal mimicry of O-Ag epitopes. PMID:18621718

  19. A Multiple Antigenic Peptide Mimicking Peptidoglycan Induced T Cell Responses to Protect Mice from Systemic Infection with Staphylococcus aureus

    PubMed Central

    Wang, Xiang-Yu; Huang, Zhao-Xia; Chen, Yi-Guo; Lu, Xiao; Zhu, Ping; Wen, Kun; Fu, Ning; Liu, Bei-Yi

    2015-01-01

    Due to the enormous capacity of Staphylococcus aureus to acquire antibiotic resistance, it becomes imperative to develop vaccines for decreasing the risk of its life-threatening infections. Peptidoglycan (PGN) is a conserved and major component of S. aureus cell wall. However, it has not been used as a vaccine candidate since it is a thymus-independent antigen. In this study, we synthesized a multiple antigenic peptide, named MAP27, which comprised four copies of a peptide that mimics the epitope of PGN. After immunization with MAP27 five times and boosting with heat-inactivated bacterium one time, anti-MAP27 serum bound directly to S. aureus or PGN. Immunization with MAP27 decreased the bacterial burden in organs of BALB/c mice and significantly prolonged their survival time after S. aureus lethal-challenge. The percentage of IFN-γ+CD3+ T cells and IL-17+CD4+ T cells in spleen, as well as the levels of IFN-γ, IL-17A/F and CCL3 in spleen and lung, significantly increased in the MAP27-immunized mice after infection. Moreover, in vitro incubation of heat-inactivated S. aureus with splenocytes isolated from MAP27-immunized mice stimulated the production of IFN-γ and IL-17A/F. Our findings demonstrated that MAP27, as a thymus-dependent antigen, is efficient at eliciting T cell-mediated responses to protect mice from S. aureus infection. This study sheds light on a possible strategy to design vaccines against S. aureus. PMID:26317210

  20. Vaccination with cyclin-dependent kinase tick antigen confers protection against Ixodes infestation.

    PubMed

    Gomes, Helga; Moraes, Jorge; Githaka, Naftaly; Martins, Renato; Isezaki, Masayoshi; Vaz, Itabajara da Silva; Logullo, Carlos; Konnai, Satoru; Ohashi, Kazuhiko

    2015-07-30

    Among arthropods, ticks lead as vectors of animal diseases and rank second to mosquitoes in transmitting human pathogens. Cyclin-dependent kinases (CDK) participate in cell cycle control in eukaryotes. CDKs are serine/threonine protein kinases and these catalytic subunits are activated or inactivated at specific stages of the cell cycle. To determine the potential of using CDKs as anti-tick vaccine antigens, hamsters were immunized with recombinant Ixodes persulcatus CDK10, followed by a homologous tick challenge. Though it was not exactly unexpected, IpCDK10 vaccination significantly impaired tick blood feeding and fecundity, which manifested as low engorgement weights, poor oviposition, and a reduction in 80% of hatching rates. These findings may underpin the development of more efficacious anti-tick vaccines based on the targeting of cell cycle control proteins.

  1. Rhipicephalus (Boophilus) microplus aquaporin as an effective vaccine antigen to protect against cattle tick infestations.

    PubMed

    Guerrero, Felix D; Andreotti, Renato; Bendele, Kylie G; Cunha, Rodrigo C; Miller, Robert J; Yeater, Kathleen; Pérez de León, Adalberto A

    2014-10-12

    Vaccination as a control method against the cattle tick, Rhipicephalus (Boophilus) microplus has been practiced since the introduction of two products in the mid-1990s. There is a need for a vaccine that could provide effective control of R. microplus in a more consistent fashion than existing products. During our transcriptome studies of R. microplus, several gene coding regions were discovered to encode proteins with significant amino acid similarity to aquaporins. A cDNA encoding an aquaporin from the cattle tick, Rhipicephalus microplus, was isolated from transcriptomic studies conducted on gut tissues dissected from fully engorged adult female R. microplus. Bioinformatic analysis indicates this aquaporin, designated RmAQP1, shows greatest amino acid similarity to the human aquaporin 7 family. Members of this family of water-conducting channels can also facilitate the transport of glycerol in addition to water. The efficacy of this aquaporin as an antigen against the cattle tick was explored in cattle vaccine trials conducted in Brazil. A cDNA encoding a significant portion of RmAQP1 was expressed as a recombinant protein in Pichia pastoris, purified under native conditions using a polyhistidine C-terminus tag and nickel affinity chromatography, emulsified with Montanide adjuvant, and cattle vaccinated intramuscularly. The recombinant protein provided 75% and 68% efficacy in two cattle pen trials conducted in Campo Grande, Brazil on groups of 6 one year old Holstein calves. The effectiveness of this vaccine in reducing the numbers of adult female ticks shows this aquaporin antigen holds promise as an active ingredient in cattle vaccines targeted against infestations of R. microplus.

  2. A molecular assembly system that renders antigens of choice highly repetitive for induction of protective B cell responses.

    PubMed

    Jegerlehner, Andrea; Tissot, Alain; Lechner, Franziska; Sebbel, Peter; Erdmann, Iris; Kündig, Thomas; Bächi, Thomas; Storni, Tazio; Jennings, Gary; Pumpens, Paul; Renner, Wolfgang A; Bachmann, Martin F

    2002-08-19

    Virus like particles (VLPs) are known to induce potent B cell responses in the absence of adjuvants. Moreover, epitope-specific antibody responses may be induced by VLPs that contain peptides inserted in their immunodominant regions. However, due to steric problems, the size of the peptides capable of being incorporated into VLPs while still permitting capsid assembly, is rather limited. While peptides genetically fused to either the N- or C-terminus of VLPs present fewer assembly problems, the immune responses obtained against such epitopes are often limited, most likely because the epitopes are not optimally exposed. In addition, such particles may be less stable in vivo. Here, we show that peptides and proteins engineered to contain a free cys can be chemically coupled to VLPs formed from the hepatitis B core antigen (HBcAg) containing a lys in the immuno-dominant region. By using this approach steric hindrance of capsid assembly is abrogated. Peptides or protein coupled to VLPs in an oriented fashion are shown to induce strong and protective B cell responses even against self-epitopes in the absence of adjuvants. This molecular assembly system may be used to induce strong B cell responses against most antigens.

  3. Enterobacterial Common Antigen Mutants of Salmonella enterica Serovar Typhimurium Establish a Persistent Infection and Provide Protection against Subsequent Lethal Challenge

    PubMed Central

    Gilbreath, Jeremy J.; Colvocoresses Dodds, Jennifer; Rick, Paul D.; Soloski, Mark J.

    2012-01-01

    Infection with Salmonella spp. is a significant source of disease globally. A substantial proportion of these infections are caused by Salmonella enterica serovar Typhimurium. Here, we characterize the role of the enterobacterial common antigen (ECA), a surface glycolipid ubiquitous among enteric bacteria, in S. Typhimurium pathogenesis. Construction of a defined mutation in the UDP-N-acetylglucosamine-1-phosphate transferase gene, wecA, in two clinically relevant strains of S. Typhimurium, TML and SL1344, resulted in strains that were unable to produce ECA. Loss of ECA did not affect the gross cell surface ultrastructure, production of lipopolysaccharide (LPS), flagella, or motility. However, the wecA mutant strains were attenuated in both oral and intraperitoneal mouse models of infection (P < 0.001 for both routes of infection; log rank test), and virulence could be restored by complementation of the wecA gene in trans. Despite the avirulence of the ECA-deficient strains, the wecA mutant strains were able to persistently colonize systemic sites (spleen and liver) at moderate levels for up to 70 days postinfection. Moreover, immunization with the wecA mutant strains provided protection against a subsequent lethal oral or intraperitoneal challenge with wild-type S. Typhimurium. Thus, wecA mutant (ECA-negative) strains of Salmonella may be useful as live attenuated vaccine strains or as vehicles for heterologous antigen expression. PMID:22025511

  4. Hepatitis B Surface Antigen Vector Delivers Protective Cytotoxic T-Lymphocyte Responses to Disease-Relevant Foreign Epitopes†

    PubMed Central

    Woo, Wai-Ping; Doan, Tracy; Herd, Karen A.; Netter, Hans-Jürgen; Tindle, Robert W.

    2006-01-01

    Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen- and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases. PMID:16571814

  5. Cationic liposomes containing soluble Leishmania antigens (SLA) plus CpG ODNs induce protection against murine model of leishmaniasis.

    PubMed

    Heravi Shargh, Vahid; Jaafari, Mahmoud Reza; Khamesipour, Ali; Jalali, Seyed Amir; Firouzmand, Hengameh; Abbasi, Azam; Badiee, Ali

    2012-07-01

    Development of an effective vaccine against leishmaniasis is possible due to the fact that individuals cured from cutaneous leishmaniasis (CL) are protected from further infection. First generation Leishmania vaccines consisting of whole killed parasites reached to phase 3 clinical trials but failed to show enough efficacies mainly due to the lack of an appropriate adjuvant. In this study, an efficient liposomal protein-based vaccine against Leishmania major infection was developed using soluble Leishmania antigens (SLA) as a first generation vaccine and cytidine phosphate guanosine oligodeoxynucleotides (CpG ODNs) as an immunostimulatory adjuvant. 1, 2-Dioleoyl-3-trimethylammonium-propane was used as a cationic lipid to prepare the liposomes due to its intrinsic adjuvanticity. BALB/c mice were immunized subcutaneously (SC), three times in 2-week intervals, with Lip-SLA-CpG, Lip-SLA, SLA + CpG, SLA, or HEPES buffer. As criteria for protection, footpad swelling at the site of challenge and spleen parasite loads were assessed, and the immune responses were evaluated by determination of IFN-γ and IL-4 levels of cultured splenocytes, and IgG subtypes. The group of mice that received Lip-SLA-CpG showed a significantly smaller footpad swelling, lower spleen parasite burden, higher IgG2a antibody, and lower IL-4 level compared to the control groups. It is concluded that cationic liposomes containing SLA and CpG ODNs are appropriate to induce Th1 type of immune response and protection against leishmaniasis.

  6. Protection of rats against Mycoplasma arthritidis-induced arthritis by active and passive immunizations with two surface antigens.

    PubMed

    Washburn, L R; Weaver, E J

    1997-05-01

    We previously identified two surface-exposed Mycoplasma arthritidis protein antigens, designated MAA1 and MAA2, that may be involved in cytadherence. Since adherence to host tissues is an important first step in most bacterial infections, we suggest that MAA1 and MAA2 may be virulence factors for M. arthritidis. In order to provide evidence for such a role, we conducted a series of experiments in which rats were actively immunized with each of these proteins purified from sodium dodecyl sulfate-polyacrylamide gels or passively immunized with poly- or monoclonal antibodies against MAA1 and MAA2. In each case, immunity against MAA1 and MAA2 conferred at least partial protection against M. arthritidis-induced disease. The greatest protection was achieved by passive immunization with monoclonal antibody A9a, directed against a surface-exposed epitope of putative adhesin MAA1. Because protective immunity in most bacterial infections is directed against major virulence factors, these results suggest that MAA1 and MAA2 may play a role in the pathogenesis of M. arthritidis-induced arthritis of rats, possibly by mediating initial colonization of joint tissues.

  7. Protective effect of intranasal immunization with Neospora caninum membrane antigens against murine neosporosis established through the gastrointestinal tract

    PubMed Central

    Ferreirinha, Pedro; Dias, Joana; Correia, Alexandra; Pérez-Cabezas, Begoña; Santos, Carlos; Teixeira, Luzia; Ribeiro, Adília; Rocha, António; Vilanova, Manuel

    2014-01-01

    Neospora caninum is an Apicomplexa parasite that in the last two decades was acknowledged as the main pathogenic agent responsible for economic losses in the cattle industry. In the present study, the effectiveness of intranasal immunization with N. caninum membrane antigens plus CpG adjuvant was assessed in a murine model of intragastrically established neosporosis. Immunized mice presented a lower parasitic burden in the brain on infection with 5 × 107 tachyzoites, showing that significant protection was achieved by this immunization strategy. Intestinal IgA antibodies raised by immunization markedly agglutinated live N. caninum tachyzoites whereas previous opsonization with IgG antibodies purified from immunized mice sera reduced parasite survival within macrophage cells. Although an IgG1 : IgG2a ratio < 1 was detected in the immunized mice before and after infection, indicative of a predominant T helper type 1 immune response, no increased production of interferon-γ was detected in the spleen or mesenteric lymph nodes of the immunized mice. Altogether, these results show that mucosal immunization with N. caninum membrane proteins plus CpG adjuvant protect against intragastrically established neosporosis and indicate that parasite-specific mucosal and circulating antibodies have a protective role against this parasitic infection. PMID:24128071

  8. Hepatitis B surface antigen vector delivers protective cytotoxic T-lymphocyte responses to disease-relevant foreign epitopes.

    PubMed

    Woo, Wai-Ping; Doan, Tracy; Herd, Karen A; Netter, Hans-Jürgen; Tindle, Robert W

    2006-04-01

    Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen- and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases.

  9. Multiple antigens of Yersinia pestis delivered by live recombinant attenuated Salmonella vaccine strains elicit protective immunity against plague.

    PubMed

    Sanapala, Shilpa; Rahav, Hannah; Patel, Hetal; Sun, Wei; Curtiss, Roy

    2016-05-05

    Based on our improved novel Salmonella vaccine delivery platform, we optimized the recombinant attenuated Salmonella typhimurium vaccine (RASV) χ12094 to deliver multiple Yersinia pestis antigens. These included LcrV196 (amino acids, 131-326), Psn encoded on pYA5383 and F1 encoded in the chromosome, their synthesis did not cause adverse effects on bacterial growth. Oral immunization with χ12094(pYA5383) simultaneously stimulated high antibody titers to LcrV, Psn and F1 in mice and presented complete protection against both subcutaneous (s.c.) and intranasal (i.n.) challenges with high lethal doses of Y. pestis CO92. Moreover, no deaths or other disease symptoms were observed in SCID mice orally immunized with χ12094(pYA5383) over a 60-day period. Therefore, the trivalent S. typhimurium-based live vaccine shows promise for a next-generation plague vaccine. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Asymmetric Cryo-EM Structure of Anthrax Toxin Protective Antigen Pore with Lethal Factor N-Terminal Domain.

    PubMed

    Machen, Alexandra J; Akkaladevi, Narahari; Trecazzi, Caleb; O'Neil, Pierce T; Mukherjee, Srayanta; Qi, Yifei; Dillard, Rebecca; Im, Wonpil; Gogol, Edward P; White, Tommi A; Fisher, Mark T

    2017-09-22

    The anthrax lethal toxin consists of protective antigen (PA) and lethal factor (LF). Understanding both the PA pore formation and LF translocation through the PA pore is crucial to mitigating and perhaps preventing anthrax disease. To better understand the interactions of the LF-PA engagement complex, the structure of the LFN-bound PA pore solubilized by a lipid nanodisc was examined using cryo-EM. CryoSPARC was used to rapidly sort particle populations of a heterogeneous sample preparation without imposing symmetry, resulting in a refined 17 Å PA pore structure with 3 LFN bound. At pH 7.5, the contributions from the three unstructured LFN lysine-rich tail regions do not occlude the Phe clamp opening. The open Phe clamp suggests that, in this translocation-compromised pH environment, the lysine-rich tails remain flexible and do not interact with the pore lumen region.

  11. Helper T cells induced by a purified 28-kilodalton antigen of Schistosoma mansoni protect rats against infection.

    PubMed Central

    Auriault, C; Balloul, J M; Pierce, R J; Damonneville, M; Sondermeijer, P; Capron, A

    1987-01-01

    Schistosoma mansoni adult worm 28-kilodalton (kDa) proteins were separated on polyacrylamide slab gels, recovered by electrophoretic elution, and used to immunize Fischer rats. After the second or third injection, inguinal lymph node T lymphocytes were propagated in vitro for 4 weeks in the presence of syngeneic antigen-presenting cells and adult worm antigens in medium containing interleukin-2. After this period of culture, 99% of the cells expressed the W3/13+ surface phenotype and 93% of the cells expressed the W3/25+ surface phenotype. These cells were then tested for their in vivo functional activity after transfer to Fischer rats that had been either infected with S. mansoni cercariae or immunized with the 28-kDa purified protein. In each case, an increase of S. mansoni-specific antibodies was observed. Whereas anti-28-kDa antibodies were only detectable at day 40 postinfection in controls injected with normal T lymphocytes, they appeared as early as day 13 postinfection when the animals received 28-kDa protein-specific T lymphocytes. This led to an effective protection of infected rats (45 to 85%) which correlated with the increase in S. mansoni-specific antibodies. These results therefore demonstrate that the 28-kDa protein possesses epitopes capable of activating helper T cells, which confer a strong protective immunity by enhancing the production of cytotoxic antibodies. The stimulation of the 28-kDa-specific T cells with recombinant proteins suggests that the major epitopes are located toward the carboxylic end of the molecule. Images PMID:2952594

  12. Secretion of Protective Antigens by Tissue-Stage Nematode Larvae Revealed by Proteomic Analysis and Vaccination-Induced Sterile Immunity

    PubMed Central

    Hewitson, James P.; Ivens, Al C.; Harcus, Yvonne; Filbey, Kara J.; McSorley, Henry J.; Murray, Janice; Bridgett, Stephen; Ashford, David; Dowle, Adam A.; Maizels, Rick M.

    2013-01-01

    Gastrointestinal nematode parasites infect over 1 billion humans, with little evidence for generation of sterilising immunity. These helminths are highly adapted to their mammalian host, following a developmental program through successive niches, while effectively down-modulating host immune responsiveness. Larvae of Heligmosomoides polygyrus, for example, encyst in the intestinal submucosa, before emerging as adult worms into the duodenal lumen. Adults release immunomodulatory excretory-secretory (ES) products, but mice immunised with adult H. polygyrus ES become fully immune to challenge infection. ES products of the intestinal wall 4th stage (L4) larvae are similarly important in host-parasite interactions, as they readily generate sterile immunity against infection, while released material from the egg stage is ineffective. Proteomic analyses of L4 ES identifies protective antigen targets as well as potential tissue-phase immunomodulatory molecules, using as comparators the adult ES proteome and a profile of H. polygyrus egg-released material. While 135 proteins are shared between L4 and adult ES, 72 are L4 ES-specific; L4-specific proteins correspond to those whose transcription is restricted to larval stages, while shared proteins are generally transcribed by all life cycle forms. Two protein families are more heavily represented in the L4 secretome, the Sushi domain, associated with complement regulation, and the ShK/SXC domain related to a toxin interfering with T cell signalling. Both adult and L4 ES contain extensive but distinct arrays of Venom allergen/Ancylostoma secreted protein-Like (VAL) members, with acetylcholinesterases (ACEs) and apyrase APY-3 particularly abundant in L4 ES. Serum antibodies from mice vaccinated with L4 and adult ES react strongly to the VAL-1 protein and to ACE-1, indicating that these two antigens represent major vaccine targets for this intestinal nematode. We have thus defined an extensive and novel repertoire of H

  13. Cross-Species Protection Mediated by a Bordetella bronchiseptica Strain Lacking Antigenic Homologs Present in Acellular Pertussis Vaccines▿

    PubMed Central

    Sukumar, Neelima; Sloan, Gina Parise; Conover, Matt S.; Love, Cheraton F.; Mattoo, Seema; Kock, Nancy D.; Deora, Rajendar

    2010-01-01

    The Bordetella species are Gram-negative bacterial pathogens that are characterized by long-term colonization of the mammalian respiratory tract and are causative agents of respiratory diseases in humans and animals. Despite widespread and efficient vaccination, there has been a world-wide resurgence of pertussis, which remains the leading cause of vaccine-preventable death in developed countries. It has been proposed that current acellular vaccines (Pa) composed of only a few bacterial proteins may be less efficacious because of vaccine-induced antigenic shifts and adaptations. To gain insight into the development of a newer generation of vaccines, we constructed a Bordetella bronchiseptica strain (LPaV) that does not express the antigenic homologs included in any of the Pa vaccines currently in use. This strain also lacks adenylate cyclase toxin, an essential virulence factor, and BipA, a surface protein. While LPaV colonized the mouse nose as efficiently as the wild-type strain, it was highly deficient in colonization of the lower respiratory tract and was attenuated in induction of inflammation and injury to the lungs. Strikingly, to our surprise, we found that in an intranasal murine challenge model, LPaV elicited cross-species protection against both B. bronchiseptica and Bordetella pertussis. Our data suggest the presence of immunogenic protective components other than those included in the pertussis vaccine. Combined with the whole-genome sequences of many Bordetella spp. that are available, the results of this study should serve as a platform for strategic development of the next generation of acellular pertussis vaccines. PMID:20176797

  14. Influence of polymer architecture on antigens camouflage, CD47 protection and complement mediated lysis of surface grafted red blood cells.

    PubMed

    Chapanian, Rafi; Constantinescu, Iren; Rossi, Nicholas A A; Medvedev, Nadia; Brooks, Donald E; Scott, Mark D; Kizhakkedathu, Jayachandran N

    2012-11-01

    Hyperbranched polyglycerol (HPG) and polyethylene glycol (PEG) polymers with similar hydrodynamic sizes in solution were grafted to red blood cells (RBCs) to investigate the impact of polymer architecture on the cell structure and function. The hydrodynamic sizes of polymers were calculated from the diffusion coefficients measured by pulsed field gradient NMR. The hydration of the HPG and PEG was determined by differential scanning calorimetry analyses. RBCs grafted with linear PEG had different properties compared to the compact HPG grafted RBCs. HPG grafted RBCs showed much higher electrophoretic mobility values than PEG grafted RBCs at similar grafting concentrations and hydrodynamic sizes indicating differences in the structure of the polymer exclusion layer on the cell surface. PEG grafting impacted the deformation properties of the membrane to a greater degree than HPG. The complement mediated lysis of the grafted RBCs was dependent on the type of polymer, grafting concentration and molecular size of grafted chains. At higher molecular weights and graft concentrations both HPG and PEG triggered complement activation. The magnitude of activation was higher with HPG possibly due to the presence of many hydroxyl groups per molecule. HPG grafted RBCs showed significantly higher levels of CD47 self-protein accessibility than PEG grafted RBCs at all grafting concentrations and molecular sizes. PEG grafted polymers provided, in general, a better shielding and protection to ABO and minor antigens from antibody recognition than HPG polymers, however, the compact HPGs provided greater protection of certain antigens on the RBC surface. Our data showed that HPG 20 kDa and HPG 60 kDa grafted RBCs exhibited properties that are more comparable to the native RBC than PEG 5 kDa and PEG 10 kDa grafted RBCs of comparable hydrodynamic sizes. The study shows that small compact polymers such as HPG 20 kDa have a greater potential in the generation of functional RBC for therapeutic

  15. Label-free and real-time detection of antigen-antibody interactions by Oblique-incidence Reflectivity Difference (OIRD) method

    NASA Astrophysics Data System (ADS)

    He, LiPing; Sun, Yue; Dai, Jun; Wang, JingYi; Lü, HuiBin; Wang, ShuFang; Jin, KuiJuan; Zhou, YueLiang; Yang, GuoZhen

    2012-09-01

    We label-free and real-time detected three interaction processes of antigen-antibodies, Human Immunoglobulin G (IgG), Rabbit IgG, and Mouse IgG as the targets, and Goat Anti-human IgG, Goat Anti-rabbit IgG, and Goat Anti-mouse IgG as the probe, by the Oblique-incidence Reflectivity Difference (OIRD) method. The interaction dynamic curves of the OIRD signal, corresponding to the interaction processes of antigen-antibodies, are generated. The reaction times from beginning to equilibrium state are about 1800, 900, and 1200 s for Human IgG, Rabbit IgG, and Mouse IgG, respectively. The experimental results demonstrate that the OIRD method not only can distinguish biomolecular interactions, but also can be used in real-time detection of interactions and dynamic processes of biomolecules.

  16. [In vitro comparative study of the protective effect of different theophyllines on the leucocytes' histamine-release induced by antigen (author's transl)].

    PubMed

    Biot, N; Grosclaude, M; Kofman, J; Perrin-Fayolle, M

    1980-01-01

    The recent discovery of the protective action of xanthic bases towards histamine-release consecutive to the antigen-antibody reaction led the authors to study the importance of this protection induced by various theophylline derivatives. The technique used was that described by LICHTENSTEIN and OSLER: isolation of leucocytes, measurement of histamine liberated in the presence of antigen, same measurement done in the presence of antigen and theophylline. The antigens used were: house dust, Dermatophagoides pteronyssimus (DP), graminaceae pollens. The tested theophyllines were: the theophylline base, aminophylline, bamiphylline, diprophylline, thio theo, piperazine acefilinate. All these substances studied brought a reduction of the histamine-release but according to a variable frequency and intensity: the thio theo, the diprophylline and the piperazine acefilinate gave the best results. Besides the latters are clearer when the antigen was dust or DP. This study enables the confirmation of the inhibiting action of theophyllines on histamine-release induced by antigen, but complementary studies on a larger number of cases seem necessary in order to determine precisely the most efficient derivatives.

  17. Mycobacterium tuberculosis multistage antigens confer comprehensive protection against pre- and post-exposure infections by driving Th1-type T cell immunity

    PubMed Central

    Fan, Xionglin; Yu, Qi; Jing, Yukai; Wang, Weihua; Li, Li; Zhou, Zijie

    2016-01-01

    There is an urgent need for a vaccine against tuberculosis (TB) that is more effective than the current sole licensed option. However, target antigens of Mycobacterium tuberculosis with the vaccine potential remain elusive. Five immunodominant antigens with characteristic expressions at the stages of primary infection (Ag85A), the regulation of nutrition and metabolism when transferring from rapid growth to latency (PhoY2 and Rv3407), latency (Rv2626c), and reactivation (RpfB) were selected to construct the fusion polyprotein WH121, which has better immunogenicity and protection than each multistage antigen. DMT adjuvanted WH121 vaccinated C57BL/6 mice could confer persistent and significant protection against the respiratory challenge with 80 CFU of virulent M. tuberculosis H37Rv at 9 and 18 weeks after immunization, as the BCG vaccine did. Moreover, WH121/DMT could boost the BCG primed mice against post-exposure infection, and more significantly inhibit the growth of M. tuberculosis in the spleen than BCG repeat vaccination. The protection elicited by WH121/DMT is attributed to the WH121-specific Th1-type biased immune responses, characterized by increased antigen-specific IgG2a/IgG1 ratio and high levels of IFN-γ secreted by the splenocytes of vaccinated mice. In particular, high levels of IFN-γ+ TEM cells in the spleen are an effective biomarker for the vaccine-induced early protection, and the persistent protection mainly depends on the increasing IL-2+IFN-γ+CD4+ and CD8+ T cells, especially IL-2+ TCM cells. These findings demonstrate that multistage-specific antigens might be promising targets for the next generation TB vaccine, and a combination of these antigens such as WH121/DMT is required for further preclinical evaluation. PMID:27566581

  18. Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice.

    PubMed

    Moayeri, Mahtab; Tremblay, Jacqueline M; Debatis, Michelle; Dmitriev, Igor P; Kashentseva, Elena A; Yeh, Anthony J; Cheung, Gordon Y C; Curiel, David T; Leppla, Stephen; Shoemaker, Charles B

    2016-01-06

    Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 μg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 μg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 μg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  19. Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice

    PubMed Central

    Moayeri, Mahtab; Tremblay, Jacqueline M.; Debatis, Michelle; Dmitriev, Igor P.; Kashentseva, Elena A.; Yeh, Anthony J.; Cheung, Gordon Y. C.; Curiel, David T.; Leppla, Stephen

    2016-01-01

    Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 μg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 μg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 μg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors. PMID:26740390

  20. rBCG30-Induced Immunity and Cross-Protection against Mycobacterium leprae Challenge Are Enhanced by Boosting with the Mycobacterium tuberculosis 30-Kilodalton Antigen 85B

    PubMed Central

    Gillis, Thomas P.; Tullius, Michael V.

    2014-01-01

    Leprosy remains a major global health problem and typically occurs in regions in which tuberculosis is endemic. Vaccines are needed that protect against both infections and do so better than the suboptimal Mycobacterium bovis BCG vaccine. Here, we evaluated rBCG30, a vaccine previously demonstrated to induce protection superior to that of BCG against Mycobacterium tuberculosis and Mycobacterium bovis challenge in animal models, for efficacy against Mycobacterium leprae challenge in a murine model of leprosy. rBCG30 overexpresses the M. tuberculosis 30-kDa major secretory protein antigen 85B, which is 85% homologous with the M. leprae homolog (r30ML). Mice were sham immunized or immunized intradermally with BCG or rBCG30 and challenged 2.5 months later by injection of viable M. leprae into each hind footpad. After 7 months, vaccine efficacy was assessed by enumerating the M. leprae bacteria per footpad. Both BCG and rBCG30 induced significant protection against M. leprae challenge. In the one experiment in which a comparison between BCG and rBCG30 was feasible, rBCG30 induced significantly greater protection than did BCG. Immunization of mice with purified M. tuberculosis or M. leprae antigen 85B also induced protection against M. leprae challenge but less so than BCG or rBCG30. Notably, boosting rBCG30 with M. tuberculosis antigen 85B significantly enhanced r30ML-specific immune responses, substantially more so than boosting BCG, and significantly augmented protection against M. leprae challenge. Thus, rBCG30, a vaccine that induces improved protection against M. tuberculosis, induces cross-protection against M. leprae that is comparable or potentially superior to that induced by BCG, and boosting rBCG30 with antigen 85B further enhances immune responses and protective efficacy. PMID:25001602

  1. rBCG30-induced immunity and cross-protection against Mycobacterium leprae challenge are enhanced by boosting with the Mycobacterium tuberculosis 30-kilodalton antigen 85B.

    PubMed

    Gillis, Thomas P; Tullius, Michael V; Horwitz, Marcus A

    2014-09-01

    Leprosy remains a major global health problem and typically occurs in regions in which tuberculosis is endemic. Vaccines are needed that protect against both infections and do so better than the suboptimal Mycobacterium bovis BCG vaccine. Here, we evaluated rBCG30, a vaccine previously demonstrated to induce protection superior to that of BCG against Mycobacterium tuberculosis and Mycobacterium bovis challenge in animal models, for efficacy against Mycobacterium leprae challenge in a murine model of leprosy. rBCG30 overexpresses the M. tuberculosis 30-kDa major secretory protein antigen 85B, which is 85% homologous with the M. leprae homolog (r30ML). Mice were sham immunized or immunized intradermally with BCG or rBCG30 and challenged 2.5 months later by injection of viable M. leprae into each hind footpad. After 7 months, vaccine efficacy was assessed by enumerating the M. leprae bacteria per footpad. Both BCG and rBCG30 induced significant protection against M. leprae challenge. In the one experiment in which a comparison between BCG and rBCG30 was feasible, rBCG30 induced significantly greater protection than did BCG. Immunization of mice with purified M. tuberculosis or M. leprae antigen 85B also induced protection against M. leprae challenge but less so than BCG or rBCG30. Notably, boosting rBCG30 with M. tuberculosis antigen 85B significantly enhanced r30ML-specific immune responses, substantially more so than boosting BCG, and significantly augmented protection against M. leprae challenge. Thus, rBCG30, a vaccine that induces improved protection against M. tuberculosis, induces cross-protection against M. leprae that is comparable or potentially superior to that induced by BCG, and boosting rBCG30 with antigen 85B further enhances immune responses and protective efficacy.

  2. Liposome-Antigen-Nucleic Acid Complexes Protect Mice from Lethal Challenge with Western and Eastern Equine Encephalitis Viruses

    PubMed Central

    Phillips, Aaron T.; Schountz, Tony; Toth, Ann M.; Rico, Amber B.; Jarvis, Donald L.; Powers, Ann M.

    2014-01-01

    Alphaviruses are mosquito-borne viruses that cause significant disease in animals and humans. Western equine encephalitis virus (WEEV) and eastern equine encephalitis virus (EEEV), two New World alphaviruses, can cause fatal encephalitis, and EEEV is a select agent of concern in biodefense. However, we have no antiviral therapies against alphaviral disease, and current vaccine strategies target only a single alphavirus species. In an effort to develop new tools for a broader response to outbreaks, we designed and tested a novel alphavirus vaccine comprised of cationic lipid nucleic acid complexes (CLNCs) and the ectodomain of WEEV E1 protein (E1ecto). Interestingly, we found that the CLNC component, alone, had therapeutic efficacy, as it increased survival of CD-1 mice following lethal WEEV infection. Immunization with the CLNC-WEEV E1ecto mixture (lipid-antigen-nucleic acid complexes [LANACs]) using a prime-boost regimen provided 100% protection in mice challenged with WEEV subcutaneously, intranasally, or via mosquito. Mice immunized with LANACs mounted a strong humoral immune response but did not produce neutralizing antibodies. Passive transfer of serum from LANAC E1ecto-immunized mice to nonimmune CD-1 mice conferred protection against WEEV challenge, indicating that antibody is sufficient for protection. In addition, the LANAC E1ecto immunization protocol significantly increased survival of mice following intranasal or subcutaneous challenge with EEEV. In summary, our LANAC formulation has therapeutic potential and is an effective vaccine strategy that offers protection against two distinct species of alphavirus irrespective of the route of infection. We discuss plausible mechanisms as well the potential utility of our LANAC formulation as a pan-alphavirus vaccine. PMID:24257615

  3. Studies on the protective efficacy of freeze thawed promastigote antigen of Leishmania donovani along with various adjuvants against visceral leishmaniasis infection in mice.

    PubMed

    Thakur, Ankita; Kaur, Harpreet; Kaur, Sukhbir

    2015-09-01

    Visceral leishmaniasis (VL) caused by Leishmania donovani persists as a major public health issue in tropical and subtropical areas of the world. Current treatment of this disease relies on use of drugs. It is doubtful that chemotherapy can alone eradicate the disease, so there is a need for an effective vaccine. Killed antigen candidates remain a good prospect considering their ease of formulation, stability, low cost and safety. To enhance the efficacy of killed vaccines suitable adjuvant and delivery system are needed. Therefore, the current study was conducted to determine the protective efficacy of freeze-thawed L. donovani antigen in combination with different adjuvants against experimental infection of VL. For this, BALB/c mice were immunized thrice at an interval of two weeks. Challenge infection was given two weeks after last immunization. Mice were sacrificed after last immunization and on different post challenge/infection days. Immunized mice showed significant reduction in parasite burden, enhanced DTH responses with increased levels of Th1 cytokines and lower levels of Th2 cytokines, thus indicating the development of a protective Th1 response. Maximum protection was achieved with liposome encapsulated freeze thawed promastigote (FTP) antigen of L. donovani and it was followed by group immunized with FTP+MPL-A, FTP+saponin, FTP+alum and FTP antigen (alone). The present study highlights greater efficacy of freeze thawed promastigote antigen as a potential vaccine candidate along with effective adjuvant formulations against experimental VL infection.

  4. Screening and Molecular Cloning of a Protective Antigen from the Midgut of Haemaphysalis longicornis

    PubMed Central

    Hu, Yonghong; Zhang, Jincheng; Yang, Shujie; Wang, Hui; Zeng, Hua; Zhang, Tiantian

    2013-01-01

    Vaccination is considered a promising alternative for controlling tick infestations. Haemaphysalis longicornis midgut proteins separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane were screened for protective value against bites. The western blot demonstrated the immunogenicity of 92 kDa protein (P92). The analysis of the P92 amino acid sequence by LC-MS/MS indicated that it was a H. longicornis paramyosin (Hl-Pmy). The full lenghth cDNA of Hl-Pmy was obtained by rapid amplification of cDNA ends (RACE) which consisted of 2,783 bp with a 161 bp 3' untranslated region. Sequence alignment of tick paramyosin (Pmy) showed that Hl-Pmy shared a high level of conservation among ticks. Comparison with the protective epitope sequence of other invertebrate Pmy, it was calculated that the protective epitope of Hl-Pmy was a peptide (LEEAEGSSETVVEMNKKRDTE) named LEE, which was close to the N-terminal of Hl-Pmy protein. The secondary structure analysis suggested that LEE had non-helical segments within an α-helical structure. These results provide the basis for developing a vaccine against biting H. longicornis ticks. PMID:23864744

  5. Protective effect of Bifidobacterium pseudocatenulatum CECT7765 against induced bacterial antigen translocation in experimental cirrhosis.

    PubMed

    Moratalla, Alba; Gómez-Hurtado, Isabel; Santacruz, Arlette; Moya, Ángela; Peiró, Gloria; Zapater, Pedro; González-Navajas, José M; Giménez, Paula; Such, José; Sanz, Yolanda; Francés, Rubén

    2014-07-01

    Intervention in the gut ecosystem is considered as a potential strategy to treat liver diseases and their complications. We have evaluated the effects of Bifidobacterium pseudocatenulatum CECT7765 on bacterial translocation and the liver status in experimental cirrhosis. Liver damage was induced in Balb/c mice by weight-controlled oral administration of carbon tetrachloride. Laparotomies were performed at week 12. One week prior to laparotomy, animals received B. pseudocatenulatum CECT7765 (10(9) cfu/daily) or placebo intragastrically. All animals received Escherichia coli (10(7) cfu/single dose) intragastrically 24 hours before laparotomy. A group of naïve non-treated animals was included as control. Liver tissue specimens, mesenteric lymph nodes, intestinal content and blood were collected. Liver histology, profibrogenic genes expression, bacterial DNA translocation, serum endotoxaemia and liver cytokine levels were measured. Bifidobacterium pseudocatenulatum CECT7765 showed no significant effect on structural liver damage, as determined by histological evaluation, alpha-smooth muscle actin distribution, profibrogenic gene expression levels, total hydroxyproline levels and malon dialdehyde production compared with mice receiving placebo. Interestingly, bacterial DNA translocation and serum endotoxin levels were significantly decreased in mice receiving the Bifidobacterium strain compared with placebo. Gut barrier integrity markers were up-regulated in mice receiving B. pseudocatenulatum CECT7765 and quantitatively correlated with intestinal gene copy numbers of the bifidobacterial strain. Gene expression levels of several anti-inflammatory mediators were also increased in mice receiving B. pseudocatenulatum CECT7765 compared with placebo. Oral administration of B. pseudocatenulatum CECT7765 is associated with improved gut barrier integrity and shows a beneficial effect against induced bacterial antigen translocation in the CCl4 -model of cirrhosis. © 2013 John

  6. Protective antibody responses elicited by a meningococcal outer membrane vesicle vaccine with overexpressed genome-derived neisserial antigen 1870.

    PubMed

    Hou, Victor C; Koeberling, Oliver; Welsch, Jo Anne; Granoff, Dan M

    2005-08-15

    Background. Meningococcal outer membrane vesicle (OMV) vaccines are efficacious in humans but have serosubtype-specific serum bactericidal antibody responses directed at the porin protein PorA and the potential for immune selection of PorA-escape mutants.Methods. We prepared an OMV vaccine from a Neisseria meningitidis strain engineered to overexpress genome-derived neisserial antigen (GNA) 1870, a lipoprotein discovered by genome mining that is being investigated for use in a vaccine.Results. Mice immunized with the modified GNA1870-OMV vaccine developed broader serum bactericidal antibody responses than control mice immunized with a recombinant GNA1870 protein vaccine or an OMV vaccine prepared from wild-type N. meningitidis or a combination of vaccines prepared from wild-type N. meningitidis and recombinant protein. Antiserum from mice immunized with the modified GNA1870-OMV vaccine also elicited greater deposition of human C3 complement on the surface of live N. meningitidis bacteria and greater passive protective activity against meningococcal bacteremia in infant rats. A N. meningitidis mutant with decreased expression of PorA was more susceptible to bactericidal activity of anti-GNA1870 antibodies.Conclusions. The modified GNA1870-OMV vaccine elicits broader protection against meningococcal disease than recombinant GNA1870 protein or conventional OMV vaccines and also has less risk of selection of PorA-escape mutants than a conventional OMV vaccine.

  7. Protective Antibody Responses Elicited by a Meningococcal Outer Membrane Vesicle Vaccine with Overexpressed Genome-Derived Neisserial Antigen 1870

    PubMed Central

    Hou, Victor C.; Koeberling, Oliver; Welsch, Jo Anne; Granoff, Dan M.

    2008-01-01

    Background Meningococcal outer membrane vesicle (OMV) vaccines are efficacious in humans but have serosubtype-specific serum bactericidal antibody responses directed at the porin protein PorA and the potential for immune selection of PorA-escape mutants. Methods We prepared an OMV vaccine from a Neisseria meningitidis strain engineered to overexpress genome-derived neisserial antigen (GNA) 1870, a lipoprotein discovered by genome mining that is being investigated for use in a vaccine. Results Mice immunized with the modified GNA1870-OMV vaccine developed broader serum bactericidal antibody responses than control mice immunized with a recombinant GNA1870 protein vaccine or an OMV vaccine prepared from wild-type N. meningitidis or a combination of vaccines prepared from wild-type N. meningitidis and recombinant protein. Antiserum from mice immunized with the modified GNA1870-OMV vaccine also elicited greater deposition of human C3 complement on the surface of live N. meningitidis bacteria and greater passive protective activity against meningococcal bacteremia in infant rats. A N. meningitidis mutant with decreased expression of PorA was more susceptible to bactericidal activity of anti-GNA1870 antibodies. Conclusions The modified GNA1870-OMV vaccine elicits broader protection against meningococcal disease than recombinant GNA1870 protein or conventional OMV vaccines and also has less risk of selection of PorA-escape mutants than a conventional OMV vaccine. PMID:16028126

  8. Cross-protective effect of a novel multi-antigen-chimeric vaccine against Streptococcus and Staphylococcus aureus infection in mice.

    PubMed

    Yu, Liquan; Fan, Ziyao; Ma, Jinzhu; Tong, Chunyu; Song, Baifen; Zhu, Zhanbo; Cui, Yudong

    2014-12-01

    Staphylococcal and streptococcal species are the most common pathogens that cause bovine mastitis. Induction of a broad-spectrum protective immunity against staphylococci and streptococci by combining multiple antigens into a single vaccine is highlighted. To develop a universal vaccine candidate, a GapC1-tIsdB-TRAP (GIT) construct was generated. The GIT contained the truncated GapC from Streptococcus dysgalactiae, and truncated IsdB and full-length TRAP from Staphylococcus aureus. The humoral and cellular immune responses elicited by GIT were evaluated in mice. Antibody levels against GIT displayed a consistent tendency with antibody levels against GapC, IsdB and TRAP. The level of IFN-γ was higher in the GIT group than in the IsdB group (P<0.05), and the level of IL-4 was higher in the GIT group than in the GapC or TRAP groups (P<0.05). The GIT group showed an improved protection against Streptococcus in comparison with GapC group. A significant difference in S. aureus challenge test was detected between the GIT group and the IsdB or TRAP groups (P<0.05) in per cent survival of mice, and a synergistic immunoprotection against S. aureus or S. dysgalactiae was produced in the GIT group. These results suggested that the GIT would be a promising common vaccine candidate against S. aureus and Streptococcus. © 2014 The Authors.

  9. Clinical and parasitological protection in a Leishmania infantum-macaque model vaccinated with adenovirus and the recombinant A2 antigen.

    PubMed

    Grimaldi, Gabriel; Teva, Antonio; Porrozzi, Renato; Pinto, Marcelo A; Marchevsky, Renato S; Rocha, Maria Gabrielle L; Dutra, Miriam S; Bruña-Romero, Oscar; Fernandes, Ana-Paula; Gazzinelli, Ricardo T

    2014-06-01

    Visceral leishmaniasis (VL) is a severe vector-born disease of humans and dogs caused by Leishmania donovani complex parasites. Approximately 0.2 to 0.4 million new human VL cases occur annually worldwide. In the new world, these alarming numbers are primarily due to the impracticality of current control methods based on vector reduction and dog euthanasia. Thus, a prophylactic vaccine appears to be essential for VL control. The current efforts to develop an efficacious vaccine include the use of animal models that are as close to human VL. We have previously reported a L. infantum-macaque infection model that is reliable to determine which vaccine candidates are most worthy for further development. Among the few amastigote antigens tested so far, one of specific interest is the recombinant A2 (rA2) protein that protects against experimental L. infantum infections in mice and dogs. Primates were vaccinated using three rA2-based prime-boost immunization regimes: three doses of rA2 plus recombinant human interleukin-12 (rhIL-12) adsorbed in alum (rA2/rhIL-12/alum); two doses of non-replicative adenovirus recombinant vector encoding A2 (Ad5-A2) followed by two boosts with rA2/rhIL-12/alum (Ad5-A2+rA2/rhIL12/alum); and plasmid DNA encoding A2 gene (DNA-A2) boosted with two doses of Ad5-A2 (DNA-A2+Ad5-A2). Primates received a subsequent infectious challenge with L. infantum. Vaccines, apart from being safe, were immunogenic as animals responded with increased pre-challenge production of anti-A2-specific IgG antibodies, though with some variability in the response, depending on the vaccine formulation/protocol. The relative parasite load in the liver was significantly lower in immunized macaques as compared to controls. Protection correlated with hepatic granuloma resolution, and reduction of clinical symptoms, particularly when primates were vaccinated with the Ad5-A2+rA2/rhIL12/alum protocol. The remarkable clinical protection induced by A2 in an animal model that is

  10. Clinical and Parasitological Protection in a Leishmania infantum-Macaque Model Vaccinated with Adenovirus and the Recombinant A2 Antigen

    PubMed Central

    Grimaldi, Gabriel; Teva, Antonio; Porrozzi, Renato; Pinto, Marcelo A.; Marchevsky, Renato S.; Rocha, Maria Gabrielle L.; Dutra, Miriam S.; Bruña-Romero, Oscar; Fernandes, Ana-Paula; Gazzinelli, Ricardo T.

    2014-01-01

    Background Visceral leishmaniasis (VL) is a severe vector-born disease of humans and dogs caused by Leishmania donovani complex parasites. Approximately 0.2 to 0.4 million new human VL cases occur annually worldwide. In the new world, these alarming numbers are primarily due to the impracticality of current control methods based on vector reduction and dog euthanasia. Thus, a prophylactic vaccine appears to be essential for VL control. The current efforts to develop an efficacious vaccine include the use of animal models that are as close to human VL. We have previously reported a L. infantum-macaque infection model that is reliable to determine which vaccine candidates are most worthy for further development. Among the few amastigote antigens tested so far, one of specific interest is the recombinant A2 (rA2) protein that protects against experimental L. infantum infections in mice and dogs. Methodology/Principal Findings Primates were vaccinated using three rA2-based prime-boost immunization regimes: three doses of rA2 plus recombinant human interleukin-12 (rhIL-12) adsorbed in alum (rA2/rhIL-12/alum); two doses of non-replicative adenovirus recombinant vector encoding A2 (Ad5-A2) followed by two boosts with rA2/rhIL-12/alum (Ad5-A2+rA2/rhIL12/alum); and plasmid DNA encoding A2 gene (DNA-A2) boosted with two doses of Ad5-A2 (DNA-A2+Ad5-A2). Primates received a subsequent infectious challenge with L. infantum. Vaccines, apart from being safe, were immunogenic as animals responded with increased pre-challenge production of anti-A2-specific IgG antibodies, though with some variability in the response, depending on the vaccine formulation/protocol. The relative parasite load in the liver was significantly lower in immunized macaques as compared to controls. Protection correlated with hepatic granuloma resolution, and reduction of clinical symptoms, particularly when primates were vaccinated with the Ad5-A2+rA2/rhIL12/alum protocol. Conclusions/Significance The

  11. Label-Free and Real-Time Detection of Antigen-Antibody Capture Processes Using the Oblique-Incidence Reflectivity Difference Technique

    NASA Astrophysics Data System (ADS)

    He, Li-Ping; Dai, Jun; Sun, Yue; Wang, Jing-Yi; Lü, Hui-Bin; Wang, Shu-Fang; Jin, Kui-Juan; Zhou, Yue-Liang; Yang, Guo-Zhen

    2012-07-01

    We successfully label-free and real-time detect the capture processes of human immunoglobulin G (IgG)/goat anti-human IgG and mouse IgG/goat anti-mouse IgG antigen-antibody pairs with different concentrations using the oblique-incidence reflectivity difference (OIRD) method, and obtain the interaction kinetics curves and the interaction times. The experimental results prove that the OIRD method is a promising technique for label-free and real-time detection of the biomolecular interaction processes and achieving the quantitative information of interaction kinetics.

  12. Vaccination with the T cell antigen Mtb 8.4 protects against challenge with Mycobacterium tuberculosis.

    PubMed

    Coler, R N; Campos-Neto, A; Ovendale, P; Day, F H; Fling, S P; Zhu, L; Serbina, N; Flynn, J L; Reed, S G; Alderson, M R

    2001-05-15

    The development of an effective vaccine against Mycobacterium tuberculosis is a research area of intense interest. Mounting evidence suggests that protective immunity to M. tuberculosis relies on both MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells. By purifying polypeptides present in the culture filtrate of M. tuberculosis and evaluating these molecules for their ability to stimulate PBMC from purified protein derivative-positive healthy individuals, we previously identified a low-m.w. immunoreactive T cell Ag, Mtb 8.4, which elicited strong Th1 T cell responses in healthy purified protein derivative-positive human PBMC and in mice immunized with recombinant Mtb 8.4. Herein we report that Mtb 8.4-specific T cells can be detected in mice immunized with the current live attenuated vaccine, Mycobacterium bovis-bacillus Calmette-Guérin as well as in mice infected i.v. with M. tuberculosis. More importantly, immunization of mice with either plasmid DNA encoding Mtb 8.4 or Mtb 8.4 recombinant protein formulated with IFA elicited strong CD4(+) T cell and CD8(+) CTL responses and induced protection on challenge with virulent M. tuberculosis. Thus, these results suggest that Mtb 8.4 is a potential candidate for inclusion in a subunit vaccine against TB.

  13. Assessment of protective immune responses against hydatid disease in sheep by immunization with synthetic peptide antigens.

    PubMed

    Woollard, D J; Heath, D D; Lightowlers, M W

    2000-08-01

    Four synthetic peptides which comprise the immunodominant linear epitopes of the EG95 recombinant protein, were investigated for their ability to induce host-protective immunity against Echinococcus granulosus in sheep. Sheep were immunized with either free peptide or peptide conjugated to diphtheria toxoid and challenge infected with E. granulosus eggs. All of the peptides elicited specific antibody, but these did not kill the parasite in in vitro culture assays, nor did the peptides induce protection against challenge infection. In contrast, anti-EG95 antibodies affinity purified against each of the 4 peptides were lethal to the parasite in in vitro culture. These affinity-purified antibodies were shown to contain specific antibody to both peptide and EG95. In in vitro inhibition assays, the peptides did not diminish anti-EG95 antibody binding to EG95 or parasite lysis in oncosphere killing assays. These results suggest that the fine specificities of antibodies raised against the recombinant protein are different to those raised against the peptide immunogens and that the majority of the antibody induced by vaccination with EG95 is raised against conformational determinants.

  14. Subdominant antigens in bacterial vaccines: Am779 is subdominant in the anaplasma marginale outer membrane vaccine but does not associate with protective immunity

    USDA-ARS?s Scientific Manuscript database

    Identification of specific antigens responsible for the ability of complex immunogens to induce protection is a major goal in development of bacterial vaccines. Much of the investigation has focused on highly abundant and highly immunodominant outer membrane proteins. Recently however, genomic and p...

  15. A human monoclonal antibody targeting the conserved staphylococcal antigen IsaA protects mice against Staphylococcus aureus bacteremia.

    PubMed

    van den Berg, Sanne; Bonarius, Hendrik P J; van Kessel, Kok P M; Elsinga, Goffe S; Kooi, Neeltje; Westra, Hans; Bosma, Tjibbe; van der Kooi-Pol, Magdalena M; Koedijk, Danny G A M; Groen, Herman; van Dijl, Jan Maarten; Buist, Girbe; Bakker-Woudenberg, Irma A J M

    2015-01-01

    Due to substantial therapy failure and the emergence of antibiotic-resistant Staphylococcus aureus strains, alternatives for antibiotic treatment of S. aureus infections are urgently needed. Passive immunization using S. aureus-specific monoclonal antibodies (mAb) could be such an alternative to prevent and treat severe S. aureus infections. The invariantly expressed immunodominant staphylococcal antigen A (IsaA) is a promising target for passive immunization. Here we report the development of the human anti-IsaA IgG1 mAb 1D9, which was shown to bind to all 26 S. aureus isolates tested. These included both methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA, respectively). Immune complexes consisting of IsaA and 1D9 stimulated human as well as murine neutrophils to generate an oxidative burst. In a murine bacteremia model, the prophylactic treatment with a single dose of 5 mg/kg 1D9 improved the survival of mice challenged with S. aureus isolate P (MSSA) significantly, while therapeutic treatment with the same dose did not influence animal survival. Neither prophylactic nor therapeutic treatment with 5 mg/kg 1D9 resulted in improved survival of mice with S. aureus USA300 (MRSA) bacteremia. Importantly, our studies show that healthy S. aureus carriers elicit an immune response which is sufficient to generate protective mAbs against invariant staphylococcal surface antigens. Human mAb 1D9, possibly conjugated to for example another antibody, antibiotics, cytokines or chemokines, may be valuable to fight S. aureus infections in patients.

  16. A non-glycosylated, plant-produced human monoclonal antibody against anthrax protective antigen protects mice and non-human primates from B. anthracis spore challenge.

    PubMed

    Mett, Vadim; Chichester, Jessica A; Stewart, Michelle L; Musiychuk, Konstantin; Bi, Hong; Reifsnyder, Carolyn J; Hull, Anna K; Albrecht, Mark T; Goldman, Stanley; Baillie, Les W J; Yusibov, Vidadi

    2011-01-01

    The health and economic burden of infectious diseases in general and bioterrorism in particular necessitate the development of medical countermeasures. One proven approach to reduce the disease burden and spread of pathogen is treatment with monoclonal antibodies (mAb). mAbs can prevent or reduce severity of the disease by variety of mechanisms, including neutralizing pathogen growth, limiting its spread from infected to adjacent cells, or by inhibiting biological activity of toxins, such as anthrax lethal toxin. Here, we report the production of glycosylated (pp-mAb (PA) ) and non-glycosylated (pp-mAb (PANG) ) versions of a plant-derived mAb directed against protective antigen (PA) of Bacillus anthracis in Nicotiana benthamiana plants using agroinfiltration. Both forms of the antibody were able to neutralize anthrax lethal toxin activity in vitro and protect mice against an intraperitoneal challenge with spores of B. anthracis Sterne strain. A single 180 µg intraperitoneal dose of pp-mAb (PA) or pp-mAb (PANG) provided 90% and 100% survival, respectively. When tested in non-human primates, pp-mAb (PANG) was demonstrated to be superior to pp-mAb (PA) in that it had a significantly longer terminal half-life and conferred 100% protection against a lethal dose of aerosolized anthrax spore challenge after a single 5 mg/kg intravenous dose compared to a 40% survival rate conferred by pp-mAb (PA) . This study demonstrates the potential of a plant-produced non-glycosylated antibody as a useful tool for the treatment of inhalation anthrax.

  17. Live recombinant Lactococcus lactis vaccine expressing immobilization antigen (i-Ag) for protection against Ichthyophthirius multifiliis in goldfish.

    PubMed

    Yao, Jia-Yun; Yuan, Xue-Mei; Xu, Yang; Yin, Wen-Lin; Lin, Ling-Yun; Pan, Xiao-Yi; Yang, Gui-Lian; Wang, Chun-Feng; Shen, Jin-Yu

    2016-11-01

    The parasite Ichthyophthirius multifiliis (Ich) has been reported in various freshwater fishes worldwide and results in severe losses to both food and aquarium fish production. Lactobacillus strains have a number of properties that make them attractive candidates as delivery vehicles for the presentation to the mucosa of compounds with pharmaceutical interest, in particular vaccines. Here, the present study was conducted to evaluate a live recombinant Lactococcus lactis vaccine expressing immobilization antigen (IAG-52X) in protection against I. multifiliis. A 1266 bp gene fragment containing a potential antigenic epitope of the 48 kDa immobilization antigen of I. multifiliis was assembled from six synthetic ohgonucleotides and cloned into pSIP409 and electrotransformed into Lactobacillus plantarum NC8. The recombinant vaccine candidate was then orally fed into goldfish. The expression of immune-related genes: complement component 3 (C3), MHC I, IgM gene in blood from goldfish at different time points after immunization were evaluated. Immunized fish were than challenged with a lethal dose of infectious I. multifiliis. The cumulative mortality and relative percentage survival (RPS) were also determined. Our results showed that the antibody level in the blood and skin of the immunized fish was statistically significant (P < 0.05) in relation to the control groups. Goldfish orally immunized with NC8-pSIP409- IAG-52X had high serum antibody titers that ranged from 32 to 256 after 28d post immunization, while fish fed with NC8-pSIP409 or PBS had no detectable immobilizing antibody response. Expression of IgM, C3, MHC I genes in the group immunized with IAG-52X were significantly (P < 0.05) up regulated as compared with control group, indicating that different immune cells were actively involved in cellular immune response. The results showed that the average survival rate of fish orally immunized with 10(8) and 10(6)NC8-pSIP409-IAG-52X was 60% and 50

  18. Histamine release factor from Dermanyssus gallinae (De Geer): characterization and in vitro assessment as a protective antigen.

    PubMed

    Bartley, Kathryn; Nisbet, Alasdair J; Offer, Jill E; Sparks, Nicholas H C; Wright, Harry W; Huntley, John F

    2009-03-01

    A cDNA encoding a 174-amino-acid orthologue of a tick histamine release factor (HRF) was identified from the haematophagous poultry red mite Dermanyssus gallinae. The predicted D. gallinae HRF protein (Dg-HRF-1) sequence is highly conserved with the tick HRFs (identity 52-54%) and to a lesser degree with translationally controlled tumour proteins (TCTP) from mammals and other invertebrates (range 38-47%). Phylogenetically, Dg-HRF-1 partitions with the tick HRF clade suggesting a shared linage and potentially similar function(s). A recombinant Dg-HRF-1 protein (rDg-HRF-1) was produced and shown to induce degranulation of rat peritoneal mast cells in vitro, confirming conservation of the histamine-releasing function in D. gallinae. Polyclonal antibodies were generated in rabbits and hens to rDg-HRF-1. Western blotting demonstrated that native Dg-HRF is a soluble protein and immunohistochemical staining of mite sections revealed that the distribution of Dg-HRF, although ubiquitous, is more common in mite reproductive, digestive and synganglion tissues. A survey of hens housed continuously in a mite-infested commercial poultry unit failed to identify IgY specific for recombinant or native Dg-HRF, indicating that Dg-HRF is not exposed to the host during infestation/feeding and may therefore have potential as a vaccine using the concealed antigen approach. To test the protective capability of rDg-HRF-1, fresh heparinised chicken blood was enriched with yolk-derived anti-Dg-HRF IgY antibodies and fed to semi-starved mites using an in vitro feeding system. A statistically significant increase in mortality was shown (P=0.004) in mites fed with anti-Dg-HRF IgY after just one blood meal. The work presented here demonstrates, to our knowledge for the first time, the feasibility of vaccinating hens with recombinant D. gallinae antigens to control mite infestation and the potential of rDg-HRF-1 as a vaccine antigen.

  19. Effective Protective Immunity to Yersinia pestis Infection Conferred by DNA Vaccine Coding for Derivatives of the F1 Capsular Antigen

    PubMed Central

    Grosfeld, Haim; Cohen, Sara; Bino, Tamar; Flashner, Yehuda; Ber, Raphael; Mamroud, Emanuelle; Kronman, Chanoch; Shafferman, Avigdor; Velan, Baruch

    2003-01-01

    Three plasmids expressing derivatives of the Yersinia pestis capsular F1 antigen were evaluated for their potential as DNA vaccines. These included plasmids expressing the full-length F1, F1 devoid of its putative signal peptide (deF1), and F1 fused to the signal-bearing E3 polypeptide of Semliki Forest virus (E3/F1). Expression of these derivatives in transfected HEK293 cells revealed that deF1 is expressed in the cytosol, E3/F1 is targeted to the secretory cisternae, and the nonmodified F1 is rapidly eliminated from the cell. Intramuscular vaccination of mice with these plasmids revealed that the vector expressing deF1 was the most effective in eliciting anti-F1 antibodies. This response was not limited to specific mouse strains or to the mode of DNA administration, though gene gun-mediated vaccination was by far more effective than intramuscular needle injection. Vaccination of mice with deF1 DNA conferred protection against subcutaneous infection with the virulent Y. pestis Kimberley53 strain, even at challenge amounts as high as 4,000 50% lethal doses. Antibodies appear to play a major role in mediating this protection, as demonstrated by passive transfer of anti-deF1 DNA antiserum. Taken together, these observations indicate that a tailored genetic vaccine based on a bacterial protein can be used to confer protection against plague in mice without resorting to regimens involving the use of purified proteins. PMID:12496187

  20. Bovine leukocyte antigen major histocompatibility complex class II DRB3*2703 and DRB3*1501 alleles are associated with variation in levels of protection against Theileria parva challenge following immunization with the sporozoite p67 antigen.

    PubMed

    Ballingall, Keith T; Luyai, Anthony; Rowlands, G John; Sales, Jill; Musoke, Anthony J; Morzaria, Subash P; McKeever, Declan J

    2004-05-01

    Initial laboratory trials of an experimental subunit vaccine against Theileria parva based on the 67-kDa major sporozoite surface antigen revealed a range of responses to challenge. We have analyzed convergence in seven sets of monozygotic twins which suggests that genetic factors may have an influence in determining the degree of protection provided by p67 immunization. In addition, we have examined whether allelic diversity at major histocompatibility complex class II loci influences protection. Analysis of bovine leukocyte antigen DRB3 diversity in 201 animals identified significant associations with vaccine success (DRB3*2703; P = 0.027) and vaccine failure (DRB3*1501; P = 0.013). Furthermore, DRB3*2703 was associated with the likelihood of immunized animals showing little to no clinical signs of disease following challenge. We discuss the acquired and innate immune mechanisms that may be behind the associations described here.

  1. Vaccination with liposomal leishmanial antigens adjuvanted with monophosphoryl lipid-trehalose dicorynomycolate (MPL-TDM) confers long-term protection against visceral leishmaniasis through a human administrable route.

    PubMed

    Ravindran, Rajesh; Maji, Mithun; Ali, Nahid

    2012-01-01

    The development of a long-term protective subunit vaccine against visceral leishmaniasis depends on antigens and adjuvants that can induce an appropriate immune response. The immunization of leishmanial antigens alone shows limited efficacy in the absence of an appropriate adjuvant. Earlier we demonstrated sustained protection against Leishmania donovani with leishmanial antigens entrapped in cationic liposomes through an intraperitoneal route. However, this route is not applicable for human administration. Herein, we therefore evaluated the immune response and protection induced by liposomal soluble leishmanial antigen (SLA) formulated with monophosphoryl lipid-trehalose dicorynomycolate (MPL-TDM) through a subcutaneous route. Subcutaneous immunization of BALB/c mice with SLA entrapped in liposomes or with MPL-TDM elicited partial protection against experimental visceral leishmaniasis. In contrast, liposomal SLA adjuvanted with MPL-TDM induced significantly higher levels of protection in liver and spleen in BALB/c mice challenged 10 days post-vaccination. Protection conferred by this formulation was sustained up to 12 weeks of immunization, and infection was controlled for at least 4 months of the challenge, similar to liposomal SLA immunization administered intraperitoneally. An analysis of cellular immune responses of liposomal SLA + MPL-TDM immunized mice demonstrated the induction of IFN-γ and IgG2a antibody production not only 10 days or 12 weeks post-vaccination but also 4 months after the challenge infection and a down regulation of IL-4 production after infection. Moreover, long-term immunity elicited by this formulation was associated with IFN-γ production also by CD8⁺ T cells. Taken together, our results suggest that liposomal SLA + MPL-TDM represent a good vaccine formulation for the induction of durable protection against L. donovani through a human administrable route.

  2. Protection of chickens to antigenically variant avian influenza virus challenge after immunization with two antigenically unrelated strains of the same subtype

    USDA-ARS?s Scientific Manuscript database

    The antigenic diversity of avian influenza virus (AIV) within a subtype has been well established and is believed to be driven by the selection of immunologic escape mutants. In regions where vaccination against AIV has been implemented for prolonged periods (e.g. Vietnam and Egypt), vaccines which...

  3. Enhanced and durable protective immune responses induced by a cocktail of recombinant BCG strains expressing antigens of multistage of Mycobacterium tuberculosis.

    PubMed

    Liang, Jinping; Teng, Xindong; Yuan, Xuefeng; Zhang, Ying; Shi, Chunwei; Yue, Tingting; Zhou, Lei; Li, Jianrong; Fan, Xionglin

    2015-08-01

    Although Bacillus Calmette-Guérin (BCG) vaccine confers protection from Mycobacterium tuberculosis infection in children, its immune protection gradually wanes over time, and consequently leads to an inability to prevent the reactivation of latent infection of M. tuberculosis. Therefore, improving BCG for better control of tuberculosis (TB) is urgently needed. We thus hypothesized that recombinant BCG overexpressing immunodominant antigens expressed at different growth stages of M. tuberculosis could provide a more comprehensive protection against primary and latent M. tuberculosis infection. Here, a novel cocktail of recombinant BCG (rBCG) strains, namely ABX, was produced by combining rBCG::85A, rBCG::85B, and rBCG::X, which overexpressed respective multistage antigens Ag85A, Ag85B, and HspX of M. tuberculosis. Our results showed that ABX was able to induce a stronger immune protection than individual rBCGs or BCG against primary TB infection in C57BL/6 mice. Mechanistically, the immune protection was attributed to stronger antigen-specific CD4(+) Th1 responses, higher numbers of IFN-γ(+) CD4(+) TEM and IL-2(+) CD8(+) TCM cells elicited by ABX. These findings thus provide a novel strategy for the improvement of BCG efficacy and potentially a promising prophylactic TB vaccine candidate, warranting further investigation.

  4. Non-responsiveness of antigen-experienced CD4 T cells reflects more stringent co-stimulatory requirements.

    PubMed Central

    Hamel, M E; Noteboom, E; Kruisbeek, A M

    1998-01-01

    We recently reported that previously activated T cells, irrespective of the nature of the first stimulus they encountered, are unable to respond to Staphylococcal enterotoxin B (SEB), nor to soluble anti-CD3 monoclonal antibody (mAb) presented by splenic antigen-presenting cells (APC). Such previously activated T cells are, however, fully capable of responding to plate-bound anti-CD3 plus splenic APC. These data suggest differential integration of the T-cell receptor (TCR) and co-stimulatory signalling pathways in naive versus antigen-experienced T cells. Consistent with this hypothesis, anti-CD28 mAb restores the proliferative capacity of resting ex vivo CD45RBlo CD4+ T cells (representing previously activated T cells) to both soluble anti-CD3 mAb and SEB. Interestingly, mAb-mediated engagement of cytotoxic T-lymphocyte antigen-4 (CTLA-4) completely negates the rescue effects mediated by anti-CD28 mAb in CD45RBlo cells. Nevertheless, the non-responsiveness of CD45RBlo CD4+ T cells cannot be reversed by anti-CTLA-4 Fab fragments, indicating that it is not related to negative regulatory effects of CTLA-4 engagement itself. Interestingly, the addition of interleukin-2 (IL-2) restores the proliferative capacity of CD45RBlo CD4+ T cells to SEB and soluble anti-CD3 mAb. Moreover, when rescued by IL-2, the cells are less susceptible to the negative regulatory effects of CTLA-4 engagement. Together, these findings suggest that the non-responsiveness of CD45RBlo CD4+ T cells to certain stimuli may be related to inadequate TCR signalling, primarily affecting IL-2 production. Images Figure 1 PMID:9640247

  5. Non-responsiveness of antigen-experienced CD4 T cells reflects more stringent co-stimulatory requirements.

    PubMed

    Hamel, M E; Noteboom, E; Kruisbeek, A M

    1998-03-01

    We recently reported that previously activated T cells, irrespective of the nature of the first stimulus they encountered, are unable to respond to Staphylococcal enterotoxin B (SEB), nor to soluble anti-CD3 monoclonal antibody (mAb) presented by splenic antigen-presenting cells (APC). Such previously activated T cells are, however, fully capable of responding to plate-bound anti-CD3 plus splenic APC. These data suggest differential integration of the T-cell receptor (TCR) and co-stimulatory signalling pathways in naive versus antigen-experienced T cells. Consistent with this hypothesis, anti-CD28 mAb restores the proliferative capacity of resting ex vivo CD45RBlo CD4+ T cells (representing previously activated T cells) to both soluble anti-CD3 mAb and SEB. Interestingly, mAb-mediated engagement of cytotoxic T-lymphocyte antigen-4 (CTLA-4) completely negates the rescue effects mediated by anti-CD28 mAb in CD45RBlo cells. Nevertheless, the non-responsiveness of CD45RBlo CD4+ T cells cannot be reversed by anti-CTLA-4 Fab fragments, indicating that it is not related to negative regulatory effects of CTLA-4 engagement itself. Interestingly, the addition of interleukin-2 (IL-2) restores the proliferative capacity of CD45RBlo CD4+ T cells to SEB and soluble anti-CD3 mAb. Moreover, when rescued by IL-2, the cells are less susceptible to the negative regulatory effects of CTLA-4 engagement. Together, these findings suggest that the non-responsiveness of CD45RBlo CD4+ T cells to certain stimuli may be related to inadequate TCR signalling, primarily affecting IL-2 production.

  6. Adaptation of the Endogenous Salmonella enterica Serovar Typhi clyA-Encoded Hemolysin for Antigen Export Enhances the Immunogenicity of Anthrax Protective Antigen Domain 4 Expressed by the Attenuated Live-Vector Vaccine Strain CVD 908-htrA

    PubMed Central

    Galen, James E.; Zhao, Licheng; Chinchilla, Magaly; Wang, Jin Yuan; Pasetti, Marcela F.; Green, Jeffrey; Levine, Myron M.

    2004-01-01

    Bacterial live-vector vaccines aim to deliver foreign antigens to the immune system and induce protective immune responses, and surface-expressed or secreted antigens are generally more immunogenic than cytoplasmic constructs. We hypothesize that an optimum expression system will use an endogenous export system to avoid the need for large amounts of heterologous DNA encoding additional proteins. Here we describe the cryptic chromosomally encoded 34-kDa cytolysin A hemolysin of Salmonella enterica serovar Typhi (ClyA) as a novel export system for the expression of heterologous antigens in the supernatant of attenuated Salmonella serovar Typhi live-vector vaccine strains. We constructed a genetic fusion of ClyA to the reporter green fluorescent protein and showed that in Salmonella serovar Typhi CVD 908-htrA, the fusion protein retains biological activity in both domains and is exported into the supernatant of an exponentially growing live vector in the absence of detectable bacterial lysis. The utility of ClyA for enhancing the immunogenicity of an otherwise problematic antigen was demonstrated by engineering ClyA fused to the domain 4 (D4) moiety of Bacillus anthracis protective antigen (PA). A total of 11 of 15 mice immunized intranasally with Salmonella serovar Typhi exporting the protein fusion manifested fourfold or greater rises in serum anti-PA immunoglobulin G, compared with only 1 of 16 mice immunized with the live vector expressing cytoplasmic D4 (P = 0.0002). In addition, the induction of PA-specific gamma interferon and interleukin 5 responses was observed in splenocytes. This technology offers exceptional versatility for enhancing the immunogenicity of bacterial live-vector vaccines. PMID:15557633

  7. Dendritic cells presenting equine herpesvirus-1 antigens induce protective anti-viral immunity.

    PubMed

    Steinbach, F; Borchers, K; Ricciardi-Castagnoli, P; Ludwig, H; Stingl, G; Elbe-Bürger, A

    1998-12-01

    Equine herpesvirus-1 (EHV-1) causes rhinopneumonitis, abortion and CNS disorders in horses. Using intranasal inoculation, the mouse model of this disease mimics the major pathogenic and clinical features of the equine disease. The aim of this study was to investigate whether murine dendritic cells (DC) can be infected with EHV-1 and whether they can be used as cellular vaccines for the induction of prophylactic anti-EHV-1 immunity. It was found that the DC lines FSDC, D2SC1, 18 (all H-2d) and 80/1 (H-2k), when incubated with the Ab4 strain of EHV-1, do not change their morphology and phenotype but sustain virus replication and induce proliferation of naive, syngeneic T cells. An even stronger proliferation of T cells was seen when DC were used that had been pre-exposed to heat-inactivated virus. DC lines were therefore pulsed with inactivated virus and were then administered intranasally to either BALB/c or C3H mice on days -25, -15 and -5. Control groups received either medium, unpulsed DC or inactivated virus only. Animals were challenged with EHV-1. Whereas mice of control panels showed clinical signs of EHV-1 disease and 27% died, animals immunized with the pulsed DC lines showed only subtle clinical symptoms, lost significantly less weight, exhibited a reduced virus load in their lungs and CNS and did not succumb to the disease during the observation period. These results show that murine DC can present EHV-1 and initiate a protective anti-viral immunity in vivo.

  8. IDO1 and TGF-β Mediate Protective Effects of IFN-α in Antigen-Induced Arthritis

    PubMed Central

    Pallotta, Maria Teresa; Narendra, Sudeep Chenna; Carlsson, Björn; Iacono, Alberta; Namale, Joanitah; Boon, Louis; Grohmann, Ursula; Magnusson, Mattias

    2016-01-01

    IFN-α prevents Ag-induced arthritis (AIA), and in this study we investigated the role of IDO1 and TGF-β signaling for this anti-inflammatory property of IFN-α. Arthritis was induced by methylated BSA (mBSA) in mBSA-sensitized wild-type (WT), Ido1−/−, or Ifnar−/− mice, treated or not with IFN-α or the IDO1 product kynurenine (Kyn). Enzymatic IDO1 activity, TGF-β, and plasmacytoid dendritic cells (pDC) were neutralized by 1-methyltryptophan and Abs against TGF-β and pDC, respectively. IDO1 expression was determined by RT-PCR, Western blot, and FACS, and enzymatic activity by HPLC. Proliferation was measured by 3H-thymidine incorporation and TGF-β by RT-PCR and ELISA. WT but not Ido1−/− mice were protected from AIA by IFN-α, and Kyn, the main IDO1 product, also prevented AIA, both in WT and Ifnar−/− mice. Protective treatment with IFN-α increased the expression of IDO1 in pDC during AIA, and Ab-mediated depletion of pDC, either during mBSA sensitization or after triggering of arthritis, completely abrogated the protective effect of IFN-α. IFN-α treatment also increased the enzymatic IDO1 activity (Kyn/tryptophan ratio), which in turn activated production of TGF-β. Neutralization of enzymatic IDO1 activity or TGF-β signaling blocked the protective effect of IFN-α against AIA, but only during sensitization and not after triggering of arthritis. Likewise, inhibition of the IDO1 enzymatic activity in the sensitization phase, but not after triggering of arthritis, subdued the IFN-α–induced inhibition of mBSA-induced proliferation. In conclusion, presence of IFN-α at Ag sensitization activates an IDO1/TGF-β–dependent anti-inflammatory program that upon antigenic rechallenge prevents inflammation via pDC. PMID:27647832

  9. Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge

    PubMed Central

    Magini, Diletta; Giovani, Cinzia; Mangiavacchi, Simona; Maccari, Silvia; Cecchi, Raffaella; Ulmer, Jeffrey B.; De Gregorio, Ennio; Geall, Andrew J.; Brazzoli, Michela; Bertholet, Sylvie

    2016-01-01

    Current hemagglutinin (HA)-based seasonal influenza vaccines induce vaccine strain-specific neutralizing antibodies that usually fail to provide protection against mismatched circulating viruses. Inclusion in the vaccine of highly conserved internal proteins such as the nucleoprotein (NP) and the matrix protein 1 (M1) was shown previously to increase vaccine efficacy by eliciting cross-reactive T-cells. However, appropriate delivery systems are required for efficient priming of T-cell responses. In this study, we demonstrated that administration of novel self-amplifying mRNA (SAM®) vectors expressing influenza NP (SAM(NP)), M1 (SAM(M1)), and NP and M1 (SAM(M1-NP)) delivered with lipid nanoparticles (LNP) induced robust polyfunctional CD4 T helper 1 cells, while NP-containing SAM also induced cytotoxic CD8 T cells. Robust expansions of central memory (TCM) and effector memory (TEM) CD4 and CD8 T cells were also measured. An enhanced recruitment of NP-specific cytotoxic CD8 T cells was observed in the lungs of SAM(NP)-immunized mice after influenza infection that paralleled with reduced lung viral titers and pathology, and increased survival after homologous and heterosubtypic influenza challenge. Finally, we demonstrated for the first time that the co-administration of RNA (SAM(M1-NP)) and protein (monovalent inactivated influenza vaccine (MIIV)) was feasible, induced simultaneously NP-, M1- and HA-specific T cells and HA-specific neutralizing antibodies, and enhanced MIIV efficacy against a heterologous challenge. In conclusion, systemic administration of SAM vectors expressing conserved internal influenza antigens induced protective immune responses in mice, supporting the SAM® platform as another promising strategy for the development of broad-spectrum universal influenza vaccines. PMID:27525409

  10. Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

    NASA Astrophysics Data System (ADS)

    Luo, Zhang; Liu, Zhixin; Fu, Jianping; Zhang, Qiusheng; Huang, Bei; Nie, Pin

    2016-11-01

    Flavobacterium columnare causes columnaris disease in freshwater fish. In the present study, the antigenic regions of five outer membrane proteins (OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 kDa, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purified recombinant protein was used to vaccinate the grass carp, Ctenopharyngodon idella. Following vaccination of the fish their IgM antibody levels were examined, as was the expression of IgM, IgD and IgZ immunoglobulin genes and other genes such as MHC Iα and MHC IIβ, which are also involved in adaptive immunity. Interleukin genes ( IL), including IL-1β, IL-8 and IL-10, and type I and type II interferon ( IFN) genes were also examined. At 3 and 4 weeks post-vaccination (wpv), significant increases in IgM antibody levels were observed in the fish vaccinated with the recombinant fusion protein, and an increase in the expression levels of IgM, IgD and IgZ genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fish. At four wpv, the fish were challenged with F. columnare, and the vaccinated fish showed a good level of protection against this pathogen, with 39% relative percent survival (RPS) compared with the control group. It can be concluded, therefore, that the five OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fish and protection against F. columnare.

  11. Use of the protective antigen of Erysipelothrix rhusiopathiae in the enzyme-linked immunosorbent assay and latex agglutination.

    PubMed

    Sato, H; Yamazaki, Y; Tsuchiya, K; Aoyama, T; Akaba, N; Suzuki, T; Yokoyama, A; Saito, H; Maehara, N

    1998-09-01

    To establish a safe and convenient serodiagnostic method for swine erysipelas, a purified protective protein antigen of Erysipelothrix rhusiopathiae, which included a large amount of protective protein (64 kDa protein), was used for enzyme-linked immunosorbent assay (ELISA) and the latex agglutination (LA) test. In the ELISA, the antisera to four different serovars (1a, 2, 5 and 20) of E. rhusiopathiae exhibit a positive reaction, while antisera to other species of bacteria (Listeria monocytogenes, Staphylococcus aureus, Streptococcus suis, Rhodococcus equi and Corynebacterium pseudotuberculosis) exhibit a negative reaction. In the LA test, the antisera to three different serovars (1a, 2 and 5) of E. rhusiopathiae reacted with P64-sensitized latex beads, while the antiserum to serovar 20 (2553 strain) did not. Moreover, the antisera to other species of bacteria (Listeria monocytogenes, Staphylococcus aureus, Streptococcus suis, Rhodococcus equi and Corynebacterium pseudotuberculosis) did not in this test. Comparing the results of the growth agglutination (GA), ELISA and LA tests of 284 swine sera, there was a high degree of correlation among the results. The detection of anti-E. rhusiopathiae antibodies in the GA, ELISA and LA tests were compared using sera from pigs immunized with P64, alkaline extract (AE) and live-cell vaccine (LV). In all three tests, anti-E. rhusiopathiae antibodies could be detected 1 week after immunization. The serum antibody titre as determined by the LA test increased moderately, as did that by the GA test, while that determined by ELISA increased rapidly. These results suggested that ELISA could be used to monitor changes in anti-E. rhusiopathiae antibody titre and the LA test could be used in the screening test for swine erysipelas.

  12. Immunogenicity and protective efficacy of recombinant Modified Vaccinia virus Ankara candidate vaccines delivering West Nile virus envelope antigens.

    PubMed

    Volz, Asisa; Lim, Stephanie; Kaserer, Martina; Lülf, Anna; Marr, Lisa; Jany, Sylvia; Deeg, Cornelia A; Pijlman, Gorben P; Koraka, Penelope; Osterhaus, Albert D M E; Martina, Byron E; Sutter, Gerd

    2016-04-07

    West Nile virus (WNV) cycles between insects and wild birds, and is transmitted via mosquito vectors to horses and humans, potentially causing severe neuroinvasive disease. Modified Vaccinia virus Ankara (MVA) is an advanced viral vector for developing new recombinant vaccines against infectious diseases and cancer. Here, we generated and evaluated recombinant MVA candidate vaccines that deliver WNV envelope (E) antigens and fulfil all the requirements to proceed to clinical testing in humans. Infections of human and equine cell cultures with recombinant MVA demonstrated efficient synthesis and secretion of WNV envelope proteins in mammalian cells non-permissive for MVA replication. Prime-boost immunizations in BALB/c mice readily induced circulating serum antibodies binding to recombinant WNV E protein and neutralizing WNV in tissue culture infections. Vaccinations in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice elicited WNV E-specific CD8+ T cell responses. Moreover, the MVA-WNV candidate vaccines protected C57BL/6 mice against lineage 1 and lineage 2 WNV infection and induced heterologous neutralizing antibodies. Thus, further studies are warranted to evaluate these recombinant MVA-WNV vaccines in other preclinical models and use them as candidate vaccine in humans.

  13. Employing Escherichia coli-derived outer membrane vesicles as an antigen delivery platform elicits protective immunity against Acinetobacter baumannii infection

    NASA Astrophysics Data System (ADS)

    Huang, Weiwei; Wang, Shijie; Yao, Yufeng; Xia, Ye; Yang, Xu; Li, Kui; Sun, Pengyan; Liu, Cunbao; Sun, Wenjia; Bai, Hongmei; Chu, Xiaojie; Li, Yang; Ma, Yanbing

    2016-11-01

    Outer membrane vesicles (OMVs) have proven to be highly immunogenic and induced an immune response against bacterial infection in human clinics and animal models. We sought to investigate whether engineered OMVs can be a feasible antigen-delivery platform for efficiently inducing specific antibody responses. In this study, Omp22 (an outer membrane protein of A. baumannii) was displayed on E. coli DH5α-derived OMVs (Omp22-OMVs) using recombinant gene technology. The morphological features of Omp22-OMVs were similar to those of wild-type OMVs (wtOMVs). Immunization with Omp22-OMVs induced high titers of Omp22-specific antibodies. In a murine sepsis model, Omp22-OMV immunization significantly protected mice from lethal challenge with a clinically isolated A. baumannii strain, which was evidenced by the increased survival rate of the mice, the reduced bacterial burdens in the lung, spleen, liver, kidney, and blood, and the suppressed serum levels of inflammatory cytokines. In vitro opsonophagocytosis assays showed that antiserum collected from Omp22-OMV-immunized mice had bactericidal activity against clinical isolates, which was partly specific antibody-dependent. These results strongly indicated that engineered OMVs could display a whole heterologous protein (~22 kDa) on the surface and effectively induce specific antibody responses, and thus OMVs have the potential to be a feasible vaccine platform.

  14. Employing Escherichia coli-derived outer membrane vesicles as an antigen delivery platform elicits protective immunity against Acinetobacter baumannii infection

    PubMed Central

    Huang, Weiwei; Wang, Shijie; Yao, Yufeng; Xia, Ye; Yang, Xu; Li, Kui; Sun, Pengyan; Liu, Cunbao; Sun, Wenjia; Bai, Hongmei; Chu, Xiaojie; Li, Yang; Ma, Yanbing

    2016-01-01

    Outer membrane vesicles (OMVs) have proven to be highly immunogenic and induced an immune response against bacterial infection in human clinics and animal models. We sought to investigate whether engineered OMVs can be a feasible antigen-delivery platform for efficiently inducing specific antibody responses. In this study, Omp22 (an outer membrane protein of A. baumannii) was displayed on E. coli DH5α-derived OMVs (Omp22-OMVs) using recombinant gene technology. The morphological features of Omp22-OMVs were similar to those of wild-type OMVs (wtOMVs). Immunization with Omp22-OMVs induced high titers of Omp22-specific antibodies. In a murine sepsis model, Omp22-OMV immunization significantly protected mice from lethal challenge with a clinically isolated A. baumannii strain, which was evidenced by the increased survival rate of the mice, the reduced bacterial burdens in the lung, spleen, liver, kidney, and blood, and the suppressed serum levels of inflammatory cytokines. In vitro opsonophagocytosis assays showed that antiserum collected from Omp22-OMV-immunized mice had bactericidal activity against clinical isolates, which was partly specific antibody-dependent. These results strongly indicated that engineered OMVs could display a whole heterologous protein (~22 kDa) on the surface and effectively induce specific antibody responses, and thus OMVs have the potential to be a feasible vaccine platform. PMID:27849050

  15. Identification of small molecules that inhibit the interaction of TEM8 with anthrax protective antigen using a FRET assay

    PubMed Central

    Cryan, Lorna M.; Habeshian, Kaiane A.; Caldwell, Thomas P.; Morris, Meredith T.; Ackroyd, P. Christine; Christensen, Kenneth A.; Rogers, Michael S.

    2013-01-01

    Tumor marker endothelial 8 (TEM8) is a receptor for the Protective Antigen (PA) component of anthrax toxin. TEM8 is upregulated on endothelial cells lining the blood vessels within tumors, compared to normal blood vessels. A number of studies have demonstrated a pivotal role for TEM8 in developmental and tumor angiogenesis. We have also shown that targeting the anthrax receptors with a mutated form of PA inhibits angiogenesis and tumor formation in vivo. Here we describe the development and testing of a high-throughput fluorescence resonance energy transfer assay to identify molecules that strongly inhibit the interaction of PA and TEM8. The assay we describe is sensitive and robust, with a Z-prime value of 0.8. A preliminary screen of 2310 known bioactive library compounds identified ebselen and thimerosal as inhibitors of the TEM8-PA interaction. These molecules each contain a cysteine-reactive transition metal, and complimentary studies indicate that their inhibition of interaction is due to modification of a cysteine residue in the TEM8 extracellular domain. This is the first demonstration of a high-throughput screening assay that identifies inhibitors of TEM8, with potential application for anti-anthrax and anti-angiogenic diseases. PMID:23479355

  16. Anti-Protective Antigen IgG Enzyme-Linked Immunosorbent Assay for Diagnosis of Cutaneous Anthrax in India

    PubMed Central

    Ghosh, N.

    2012-01-01

    Anthrax caused by Bacillus anthracis is a public health problem in several developing countries whose main source of income is farming. Anthrax is a disease of herbivorous animals, and humans can be infected by handling infected animals or contaminated animal products. Specific diagnostic tests are unavailable in India for the detection and confirmation of cutaneous anthrax in humans. Here, we describe the development of an enzyme-linked immunosorbent assay (ELISA) for detection of serum antibodies against Bacillus anthracis protective antigen in the Indian population. A total of 405 serum samples collected from different groups were tested by the developed ELISA. The assay provided a specificity of 99.41% (95% confidence interval [CI], 97.89 to 99.93) and a sensitivity of 100% (CI, 94.4 to 100) using a cutoff value of 0.29 ELISA unit (EU). The positive predictive value (PPV) and negative predictive value (NPV) of the assay were 97% and 100%, respectively. The efficiency and J index for the reliability of the assay were 99.5% and 0.994, respectively. The assay can be a very useful tool for surveillance as well as for diagnosis of cutaneous anthrax cases in India. PMID:22718130

  17. Effect of delayed anthrax vaccine dose on Bacillus anthracis protective antigen IgG response and lethal toxin neutralization activity.

    PubMed

    Pittman, Phillip R; Fisher, Diana; Quinn, Xiaofei; Schmader, Trevor; Barrera-Oro, Julio G

    2013-10-17

    We describe the Bacillus anthracis protective antigen IgG antibody response and the B. anthracis lethal toxin neutralization activity to a delayed dose of anthrax vaccine adsorbed (AVA, BioThrax(®)) using validated assays. 373 individuals received 1, 2, or 3 priming doses, 18-24 months afterward, they received a delayed dose of AVA. Overall, 23.6% of subjects showed detectable anti-PA IgG before the boost, compared to 99.2% (P<0.0001) 28 days after the boost. Geometric mean anti-PA IgG concentration (GMC) was 1.66 μg/mL before and 887.82 μg/mL after the boost (P<0.0001). The proportion of individuals with four-fold increase in GMC following the boost ranged from 93.8% to 100%. Robust anti-PA IgG levels and B. anthracis lethal toxin neutralization activity are induced when an AVA dose is delayed as long as two years. These data support continuing with the vaccination schedule when a dose is delayed as long as two years rather than restarting the series. Published by Elsevier Ltd.

  18. A Dual TLR Agonist Adjuvant Enhances the Immunogenicity and Protective Efficacy of the Tuberculosis Vaccine Antigen ID93

    PubMed Central

    Orr, Mark T.; Beebe, Elyse A.; Hudson, Thomas E.; Moon, James J.; Fox, Christopher B.; Reed, Steven G.; Coler, Rhea N.

    2014-01-01

    With over eight million cases of tuberculosis each year there is a pressing need for the development of new vaccines against Mycobacterium tuberculosis. Subunit vaccines consisting of recombinant proteins are an attractive vaccine approach due to their inherent safety compared to attenuated live vaccines and the uniformity of manufacture. Addition of properly formulated TLR agonist-containing adjuvants to recombinant protein vaccines enhances the antigen-specific CD4+ T cell response characterized by IFN-γ and TNF, both of which are critical for the control of TB. We have developed a clinical stage vaccine candidate consisting of a recombinant fusion protein ID93 adjuvanted with the TLR4 agonist GLA-SE. Here we examine whether ID93+GLA-SE can be improved by the addition of a second TLR agonist. Addition of CpG containing DNA to ID93+GLA-SE enhanced the magnitude of the multi-functional TH1 response against ID93 characterized by co-production of IFN-γ, TNF, and IL-2. Addition of CpG also improved the protective efficacy of ID93+GLA-SE. Finally we demonstrate that this adjuvant synergy between GLA and CpG is independent of TRIF signaling, whereas TRIF is necessary for the adjuvant activity of GLA-SE in the absence of CpG. PMID:24404140

  19. A dual TLR agonist adjuvant enhances the immunogenicity and protective efficacy of the tuberculosis vaccine antigen ID93.

    PubMed

    Orr, Mark T; Beebe, Elyse A; Hudson, Thomas E; Moon, James J; Fox, Christopher B; Reed, Steven G; Coler, Rhea N

    2014-01-01

    With over eight million cases of tuberculosis each year there is a pressing need for the development of new vaccines against Mycobacterium tuberculosis. Subunit vaccines consisting of recombinant proteins are an attractive vaccine approach due to their inherent safety compared to attenuated live vaccines and the uniformity of manufacture. Addition of properly formulated TLR agonist-containing adjuvants to recombinant protein vaccines enhances the antigen-specific CD4(+) T cell response characterized by IFN-γ and TNF, both of which are critical for the control of TB. We have developed a clinical stage vaccine candidate consisting of a recombinant fusion protein ID93 adjuvanted with the TLR4 agonist GLA-SE. Here we examine whether ID93+GLA-SE can be improved by the addition of a second TLR agonist. Addition of CpG containing DNA to ID93+GLA-SE enhanced the magnitude of the multi-functional TH1 response against ID93 characterized by co-production of IFN-γ, TNF, and IL-2. Addition of CpG also improved the protective efficacy of ID93+GLA-SE. Finally we demonstrate that this adjuvant synergy between GLA and CpG is independent of TRIF signaling, whereas TRIF is necessary for the adjuvant activity of GLA-SE in the absence of CpG.

  20. Evaluation of protective effect of multi-epitope DNA vaccine encoding six antigen segments of Toxoplasma gondii in mice.

    PubMed

    Liu, Shan; Shi, Lin; Cheng, Yan-bin; Fan, Gui-xiang; Ren, Hui-xun; Yuan, Yu-kang

    2009-07-01

    To investigate the vaccine potential of multi-epitope vaccines against toxoplasmosis, a multi-epitope DNA vaccine, eukaryotic plasmid pcDNA3.1/T-ME expressing six antigen segments (SAG1(238-256), SAG1(281-320), GRA1(170-193), GRA4(331-345), GRA4(229-245), and GRA2(171-185)) of Toxoplasma gondii was constructed. We investigated the efficacy of pcDNA3.1/T-ME with or without co-administration of a CpG-oligodeoxynucleotide (CpG-ODN) as an adjuvant to protect mice (BALB/c and C57BL/6) against toxoplasmosis. High survival rates were observed in mice immunized with pcDNA3.1/T-ME when challenged with T. gondii RH strain. Lymphocyte proliferation assays, cytokine, and antibody determinations show that mice immunized with pcDNA3.1/T-ME produced stronger humoral and Th1-type cellular immune responses compared to untreated mice or those immunized with empty plasmids. However, co-immunization with CpG-ODN resulted in impaired immune responses. Our data demonstrates that multi-epitope DNA vaccination is a potential strategy for the control of toxoplasmosis and paves the way for further investigations into producing a multi-epitope anti-T. gondii DNA vaccine.

  1. Bacillus subtilis spores expressing the VP28 antigen: a potential oral treatment to protect Litopenaeus vannamei against white spot syndrome.

    PubMed

    Nguyen, Anh T V; Pham, Cuong K; Pham, Huong T T; Pham, Hang L; Nguyen, Anh H; Dang, Lua T; Huynh, Hong A; Cutting, Simon M; Phan, Tuan-Nghia

    2014-09-01

    The envelope protein VP28 of white spot syndrome virus (WSSV) is considered a candidate antigen for use in a potential vaccine to this important shrimp pathogen (the cause of white spot syndrome, WSS). Here, we used spores of Bacillus subtilis to display VP28 on the spore surface. Trials were conducted to evaluate their ability to protect shrimps against WSSV infection. The gene cotB-vp28 was integrated into the chromosome of the laboratory strain B. subtilis PY79, and expression of CotB-VP28 was detected by Western blotting and immunofluorescence. Expression of CotB-VP28 was equivalent to 1000 molecules per spore. PY79 and CotB-VP28 spores were mixed with pellets for feeding of whiteleg shrimps (Litopenaeus vannamei), followed by WSSV challenge. Superoxidase dismutase (SOD), phenoloxidase activities and mortality rates of the two shrimp groups were evaluated. Groups fed with PY79 and CotB-VP28 spores at day 7 had increased SOD activities of 29% and increased phenoloxidase activities of 15% and 33%, respectively, compared to those of the control group. Fourteen days postchallenge, 35% of vaccinated shrimps had died compared to 49% of those fed naked spores (PY79) and 66% untreated, unchallenged animals. These data suggest that spores expressing VP28 have potential as a prophylactic treatment of WSS.

  2. Laboratory evolution of artificially expanded DNA gives redesignable aptamers that target the toxic form of anthrax protective antigen

    PubMed Central

    Biondi, Elisa; Lane, Joshua D.; Das, Debasis; Dasgupta, Saurja; Piccirilli, Joseph A.; Hoshika, Shuichi; Bradley, Kevin M.; Krantz, Bryan A.; Benner, Steven A.

    2016-01-01

    Reported here is a laboratory in vitro evolution (LIVE) experiment based on an artificially expanded genetic information system (AEGIS). This experiment delivers the first example of an AEGIS aptamer that binds to an isolated protein target, the first whose structural contact with its target has been outlined and the first to inhibit biologically important activities of its target, the protective antigen from Bacillus anthracis. We show how rational design based on secondary structure predictions can also direct the use of AEGIS to improve the stability and binding of the aptamer to its target. The final aptamer has a dissociation constant of ∼35 nM. These results illustrate the value of AEGIS-LIVE for those seeking to obtain receptors and ligands without the complexities of medicinal chemistry, and also challenge the biophysical community to develop new tools to analyze the spectroscopic signatures of new DNA folds that will emerge in synthetic genetic systems replacing standard DNA and RNA as platforms for LIVE. PMID:27701076

  3. Bacillus anthracis protective antigen kinetics in inhalation spore-challenged untreated or levofloxacin/ raxibacumab-treated New Zealand white rabbits.

    PubMed

    Corey, Alfred; Migone, Thi-Sau; Bolmer, Sally; Fiscella, Michele; Ward, Chris; Chen, Cecil; Meister, Gabriel

    2013-01-14

    Inhaled Bacillus anthracis spores germinate and the subsequent vegetative growth results in bacteremia and toxin production. Anthrax toxin is tripartite: the lethal factor and edema factor are enzymatic moieties, while the protective antigen (PA) binds to cell receptors and the enzymatic moieties. Antibiotics can control B. anthracis bacteremia, whereas raxibacumab binds PA and blocks lethal toxin effects. This study assessed plasma PA kinetics in rabbits following an inhaled B. anthracis spore challenge. Additionally, at 84 h post-challenge, 42% of challenged rabbits that had survived were treated with either levofloxacin/placebo or levofloxacin/raxibacumab. The profiles were modeled using a modified Gompertz/second exponential growth phase model in untreated rabbits, with added monoexponential PA elimination in treated rabbits. Shorter survival times were related to a higher plateau and a faster increase in PA levels. PA elimination half-lives were 10 and 19 h for the levofloxacin/placebo and levofloxacin/raxibacumab groups, respectively, with the difference attributable to persistent circulating PA-raxibacumab complex. PA kinetics were similar between untreated and treated rabbits, with one exception: treated rabbits had a plateau phase nearly twice as long as that for untreated rabbits. Treated rabbits that succumbed to disease had higher plateau PA levels and shorter plateau duration than surviving treated rabbits.

  4. Bacillus anthracis Protective Antigen Kinetics in Inhalation Spore-Challenged Untreated or Levofloxacin/Raxibacumab-Treated New Zealand White Rabbits

    PubMed Central

    Corey, Alfred; Migone, Thi-Sau; Bolmer, Sally; Fiscella, Michele; Ward, Chris; Chen, Cecil; Meister, Gabriel

    2013-01-01

    Inhaled Bacillus anthracis spores germinate and the subsequent vegetative growth results in bacteremia and toxin production. Anthrax toxin is tripartite: the lethal factor and edema factor are enzymatic moieties, while the protective antigen (PA) binds to cell receptors and the enzymatic moieties. Antibiotics can control B. anthracis bacteremia, whereas raxibacumab binds PA and blocks lethal toxin effects. This study assessed plasma PA kinetics in rabbits following an inhaled B. anthracis spore challenge. Additionally, at 84 h post-challenge, 42% of challenged rabbits that had survived were treated with either levofloxacin/placebo or levofloxacin/raxibacumab. The profiles were modeled using a modified Gompertz/second exponential growth phase model in untreated rabbits, with added monoexponential PA elimination in treated rabbits. Shorter survival times were related to a higher plateau and a faster increase in PA levels. PA elimination half-lives were 10 and 19 h for the levofloxacin/placebo and levofloxacin/raxibacumab groups, respectively, with the difference attributable to persistent circulating PA-raxibacumab complex. PA kinetics were similar between untreated and treated rabbits, with one exception: treated rabbits had a plateau phase nearly twice as long as that for untreated rabbits. Treated rabbits that succumbed to disease had higher plateau PA levels and shorter plateau duration than surviving treated rabbits. PMID:23344456

  5. KSAC, a Defined Leishmania Antigen, plus Adjuvant Protects against the Virulence of L. major Transmitted by Its Natural Vector Phlebotomus duboscqi

    PubMed Central

    Gomes, Regis; Teixeira, Clarissa; Oliveira, Fabiano; Lawyer, Phillip G.; Elnaiem, Dia-Eldin; Meneses, Claudio; Goto, Yasuyuki; Bhatia, Ajay; Howard, Randall F.; Reed, Steven G.; Valenzuela, Jesus G.; Kamhawi, Shaden

    2012-01-01

    Background Recombinant KSAC and L110f are promising Leishmania vaccine candidates. Both antigens formulated in stable emulsions (SE) with the natural TLR4 agonist MPL® and L110f with the synthetic TLR4 agonist GLA in SE protected BALB/c mice against L. major infection following needle challenge. Considering the virulence of vector-transmitted Leishmania infections, we vaccinated BALB/c mice with either KSAC+GLA-SE or L110f+GLA-SE to assess protection against L. major transmitted via its vector Phlebotomus duboscqi. Methods Mice receiving the KSAC or L110f vaccines were challenged by needle or L. major-infected sand flies. Weekly disease progression and terminal parasite loads were determined. Immunological responses to KSAC, L110f, or soluble Leishmania antigen (SLA) were assessed throughout vaccination, three and twelve weeks after immunization, and one week post-challenge. Results Following sand fly challenge, KSAC-vaccinated mice were protected while L110f-vaccinated animals showed partial protection. Protection correlated with the ability of SLA to induce IFN-γ-producing CD4+CD62LlowCCR7low effector memory T cells pre- and post-sand fly challenge. Conclusions This study demonstrates the protective efficacy of KSAC+GLA-SE against sand fly challenge; the importance of vector-transmitted challenge in evaluating vaccine candidates against Leishmania infection; and the necessity of a rapid potent Th1 response against Leishmania to attain true protection. PMID:22509423

  6. Evaluation of immune response to recombinant potential protective antigens of Mycoplasma hyopneumoniae delivered as cocktail DNA and/or recombinant protein vaccines in mice.

    PubMed

    Chen, Austen Y; Fry, Scott R; Daggard, Grant E; Mukkur, Trilochan K S

    2008-08-12

    Intramuscular immunization of mice with DNA cocktail vaccines, comprising potential protective antigens P36, P46, NrdF, and P97or P97R1 of Mycoplasma hyopneumoniae, induced strong Th1-polarized immune responses against each antigen, with only P46 eliciting a serum IgG response. Subcutaneous immunization with protein cocktail vaccines, surprisingly, induced both Th1-polarized immune response as well as antibody response whereas mice immunized with DNA cocktail vaccines followed by boosting with protein cocktail vaccines generated strong Th1-polarized and humoral immune responses. P97 was not recognized by serum antibodies from commercial bacterin-immunized mice indicating potential lack of expression of this important antigen in inactivated whole-cell vaccines.

  7. Multifunctional T Cell Response to DosR and Rpf Antigens Is Associated with Protection in Long-Term Mycobacterium tuberculosis-Infected Individuals in Colombia

    PubMed Central

    Arroyo, Leonar; Rojas, Mauricio; Franken, Kees L. M. C.; Ottenhoff, Tom H. M.

    2016-01-01

    Multifunctional T cells have been shown to be protective in chronic viral infections. In mycobacterial infections, however, evidence for a protective role of multifunctional T cells remains inconclusive. Short-term cultures of peripheral blood mononuclear cells stimulated with the Mycobacterium tuberculosis RD1 antigens 6-kDa early secretory antigenic target (ESAT6) and 10-kDa culture filtrate antigen (CFP10), which are induced in the early infection phase, have been mainly used to assess T cell multifunctionality, although long-term culture assays have been proposed to be more sensitive than short-term assays for assessment of memory T cells, which are essential for long-term immunity. Here we used a long-term culture assay system to study the T cell immune responses to the M. tuberculosis latency-associated DosR antigens and reactivation-associated Rpf antigens, compared to ESAT6 and CFP10, in patients with pulmonary tuberculosis (PTB) and household contacts of PTB patients with long-term latent tuberculosis infection (ltLTBI), in a community in which M. tuberculosis is endemic. Our results showed that the DosR antigens Rv1737c (narK2) and Rv2029c (pfkB) and the Rv2389c (rpfD) antigen of M. tuberculosis induced higher frequencies of CD4+ or CD8+ mono- or bifunctional (but not multifunctional) T cells producing interferon gamma (IFN-γ) and/or tumor necrosis alpha (TNF-α) in ltLTBI, compared to PTB. Moreover, the frequencies of CD4+ and/or CD8+ T cells with a CD45RO+ CD27+ phenotype were higher in ltLTBI than in PTB. Thus, the immune responses to selected DosR and Rpf antigens may be associated with long-term latency, correlating with protection from M. tuberculosis reactivation in ltLTBI. Further study of the functional and memory phenotypes may contribute to further discrimination between the different states of M. tuberculosis infections. PMID:27489136

  8. Multifunctional T Cell Response to DosR and Rpf Antigens Is Associated with Protection in Long-Term Mycobacterium tuberculosis-Infected Individuals in Colombia.

    PubMed

    Arroyo, Leonar; Rojas, Mauricio; Franken, Kees L M C; Ottenhoff, Tom H M; Barrera, Luis F

    2016-10-01

    Multifunctional T cells have been shown to be protective in chronic viral infections. In mycobacterial infections, however, evidence for a protective role of multifunctional T cells remains inconclusive. Short-term cultures of peripheral blood mononuclear cells stimulated with the Mycobacterium tuberculosis RD1 antigens 6-kDa early secretory antigenic target (ESAT6) and 10-kDa culture filtrate antigen (CFP10), which are induced in the early infection phase, have been mainly used to assess T cell multifunctionality, although long-term culture assays have been proposed to be more sensitive than short-term assays for assessment of memory T cells, which are essential for long-term immunity. Here we used a long-term culture assay system to study the T cell immune responses to the M. tuberculosis latency-associated DosR antigens and reactivation-associated Rpf antigens, compared to ESAT6 and CFP10, in patients with pulmonary tuberculosis (PTB) and household contacts of PTB patients with long-term latent tuberculosis infection (ltLTBI), in a community in which M. tuberculosis is endemic. Our results showed that the DosR antigens Rv1737c (narK2) and Rv2029c (pfkB) and the Rv2389c (rpfD) antigen of M. tuberculosis induced higher frequencies of CD4(+) or CD8(+) mono- or bifunctional (but not multifunctional) T cells producing interferon gamma (IFN-γ) and/or tumor necrosis alpha (TNF-α) in ltLTBI, compared to PTB. Moreover, the frequencies of CD4(+) and/or CD8(+) T cells with a CD45RO(+) CD27(+) phenotype were higher in ltLTBI than in PTB. Thus, the immune responses to selected DosR and Rpf antigens may be associated with long-term latency, correlating with protection from M. tuberculosis reactivation in ltLTBI. Further study of the functional and memory phenotypes may contribute to further discrimination between the different states of M. tuberculosis infections.

  9. Interleukin-25 (IL-25) Promotes Efficient Protective Immunity against Trichinella spiralis Infection by Enhancing the Antigen-Specific IL-9 Response

    PubMed Central

    Srimanote, Potjanee; Wang, Yui-Hsi; Pootong, Anek; Sakolvaree, Yuwaporn; Pattanapanyasat, Kovit; Chaicumpa, Wanpen; Chaiyaroj, Sansanee; Dong, Chen

    2013-01-01

    Mammalian hosts often develop distinct immune response against the diverse parasitic helminths that have evolved for immune evasion. Interleukin-25 (IL-25), an IL-17 cytokine family member, plays a key role in initiating the protective immunity against several parasitic helminths; however, the involvement and underlying mechanisms by which IL-25 mediates immune response against Trichinella spiralis infection have not been investigated. Here we showed that IL-25 functions in promoting protective immunity against T. spiralis infection. Mice treated with IL-25 exhibited a lower worm burden and fewer muscle larvae in the later stage of T. spiralis infection. In contrast, mice treated with neutralizing antibody against IL-25 failed to expel T. spiralis effectively. During T. spiralis infection, intestinal IL-25 expression was rapidly elevated before the onset of IL-4 and IL-9 induction. While antigen-specific Th2 and Th9 immune responses were both developed during T. spiralis infection, an antigen-specific Th9 response appeared to be transiently induced in the early stage of infection. Mice into which antigen-specific T cells deficient in IL-9 were transferred were less effective in worm clearance than those given wild-type T cells. The strength of the antigen-specific Th9 immune response against T. spiralis could be enhanced or attenuated after treatment with IL-25 or neutralizing antibody against IL-25, respectively, correlating positively with the levels of intestinal mastocytosis and the expression of IL-9-regulated genes, including mast cell- and Paneth cell-specific genes. Thus, our study demonstrates that intestinal IL-25 promotes protective immunity against T. spiralis infection by inducing antigen-specific Th9 immune response. PMID:23897610

  10. Directed selection of influenza virus produces antigenic variants that match circulating human virus isolates and escape from vaccine-mediated immune protection.

    PubMed

    DeDiego, Marta L; Anderson, Christopher S; Yang, Hongmei; Holden-Wiltse, Jeanne; Fitzgerald, Theresa; Treanor, John J; Topham, David J

    2016-06-01

    Influenza vaccination does not provide 100% protection from infection, partly due to antigenic drift of the haemagglutinin (HA) protein. Low serum antibody titres increase the risk of infection. To determine whether there were additional correlates of risk, we examined the relationship between human serum immunity and antigenic variation in seasonal H3N2 influenza viruses. Seasonal H3N2 vaccine strains grown in the presence of heterogeneous human or mono-specific ferret antisera selected variants with mutations in the HA antigenic sites. Surprisingly, circulating strains infecting human subjects in the same seasons displayed mutations in the same positions, although only in one case did the change correspond to the same amino acid. Serum antibody titres were lower against both the in vitro selected and clinical isolates compared with the vaccine strains, suggesting that the mutations are relevant to vaccine failure. Antibody titres were also significantly lower in sera from infected subjects than in non-infected subjects, suggesting relatively poor responses to vaccination in the infected subjects. Collectively, the data suggest that risk from influenza infection is a result of poor response to vaccination, as well as encounter with drifted seasonal influenza virus antigenic variants. The results also show that directed selection under human immune pressure could reveal antigenic variants relevant to real-world drifted viruses, helping in annual vaccine re-formulation. © 2016 The Authors. Immunology Published by John Wiley & Sons Ltd.

  11. Prior infection with influenza virus but not vaccination leaves a long-term immunological imprint that intensifies the protective efficacy of antigenically drifted vaccine strains.

    PubMed

    Kim, Jin Hyang; Liepkalns, Justine; Reber, Adrian J; Lu, Xiuhua; Music, Nedzad; Jacob, Joshy; Sambhara, Suryaprakash

    2016-01-20

    The role of pre-existing immunity for influenza vaccine responses is of great importance for public health, and thus has been studied in various contexts, yet the impact of differential priming on vaccine responses in the midst of antigenic drift remains to be elucidated. To address this with antigenically related viruses, mice were first primed by either infection or immunization with A/Puerto Rico/8/34 (PR8) virus, then immunized with whole-inactivated A/Fort Monmouth/1/47 (FM1) virus. The ensuing vaccine responses and the protective efficacy of FM1 were superior in PR8 infection-primed mice compared to PR8 immunization-primed or unprimed mice. Increased FM1-specific Ab responses of PR8 infection-primed mice also broadened cross-reactivity against contemporary as well as antigenically more drifted strains. Further, prior infection heightened the protective efficacy of antigenically distant strains, such as A/Brisbane/59/2006 infection followed by immunization with split pandemic H1N1 vaccine (A/California/07/2009). Therefore, influenza infection is a significant priming event that intensifies future vaccine responses against drift strains. Published by Elsevier Ltd.

  12. Recombinant Adenovirus Delivery of Calreticulin–ESAT-6 Produces an Antigen-Specific Immune Response but no Protection Against a Mycobacterium Tuberculosis Challenge

    PubMed Central

    Esparza-González, S. C.; Troy, A.; Troudt, J.; Loera-Arias, M. J.; Villatoro-Hernández, J.; Torres-López, E.; Ancer-Rodríguez, J.; Gutiérrez-Puente, Y.; Muñoz-Maldonado, G.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.; Izzo, A.

    2015-01-01

    Bacillus Calmette–Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell-mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT-6 to calreticulin and constructed a recombinant replication-deficient adenovirus to express the resulting fusion protein (AdCRT–ESAT-6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT-6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon-γ and tumour necrosis factor-α in response to ESAT-6. This immune response was not improved by the addition of CFP-10 to the CRT-ESAT-6 fusion protein (AdCRT–ESAT-6–CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low-dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis. PMID:22010821

  13. Haldane insulator protected by reflection symmetry in the doped Hubbard model on the three-legged ladder

    NASA Astrophysics Data System (ADS)

    Nourse, H. L.; McCulloch, I. P.; Janani, C.; Powell, B. J.

    2016-12-01

    We demonstrate the existence of an insulating phase in the three-legged Hubbard ladder at two-thirds filling. In this phase chargons are bound because the physics within a unit cell favors the formation of triplets. The resultant moments lead to a ground state in the Haldane phase, a symmetry protected topological state of matter. In this purely fermionic model, reflection is protecting but time-reversal and dihedral symmetries are not, in contrast to spin models.

  14. [Characterization of surface antigens of the nematode parasite Trichinella spiralis: study of its role in protection mechanisms and their usefulness in the diagnosis of trichinosis].

    PubMed

    Ortega-Pierres, M G

    1995-01-01

    Among the most important aspects in the study of trichinosis are the development of specific and sensitive diagnostic methods for the detection of the infection by the parasite as well as the characterization of antigens from Trichinella spiralis that induce protection in the host. In the context, the characterization of surface stichosome and excretory secretory antigens of T. spiralis muscle larvae has been an important issue due to the high immunogenicity of such components in most hosts so far studied. In this work, we have been able to identify and characterize molecules from both compartments of muscle larvae. These components have been used in assays for specific detection of T. spiralis infections particularly in pigs, as well as in assays to evaluate their role in the induction of protection in mice.

  15. Simultaneous multicolor detection system of the single-molecular microbial antigen by total internal reflection fluorescence microscopy with fluorescent nanocrystal quantum dots

    NASA Astrophysics Data System (ADS)

    Hoshino, Akiyoshi; Fujioka, Kouki; Yamamoto, Mayu; Manabe, Noriyoshi; Yasuhara, Masato; Suzuki, Kazuo; Yamamoto, Kenji

    2005-11-01

    Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystals QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD-conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD-conjugated antibodies. Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high-sensitivity detection systems.

  16. A Bivalent Typhoid Live Vector Vaccine Expressing both Chromosome- and Plasmid-Encoded Yersinia pestis Antigens Fully Protects against Murine Lethal Pulmonary Plague Infection

    PubMed Central

    Wang, Jin Yuan; Carrasco, Jose A.; Lloyd, Scott A.; Mellado-Sanchez, Gabriela; Diaz-McNair, Jovita; Franco, Olga; Buskirk, Amanda D.; Nataro, James P.; Pasetti, Marcela F.

    2014-01-01

    Live attenuated bacteria hold great promise as multivalent mucosal vaccines against a variety of pathogens. A major challenge of this approach has been the successful delivery of sufficient amounts of vaccine antigens to adequately prime the immune system without overattenuating the live vaccine. Here we used a live attenuated Salmonella enterica serovar Typhi strain to create a bivalent mucosal plague vaccine that produces both the protective F1 capsular antigen of Yersinia pestis and the LcrV protein required for secretion of virulence effector proteins. To reduce the metabolic burden associated with the coexpression of F1 and LcrV within the live vector, we balanced expression of both antigens by combining plasmid-based expression of F1 with chromosomal expression of LcrV from three independent loci. The immunogenicity and protective efficacy of this novel vaccine were assessed in mice by using a heterologous prime-boost immunization strategy and compared to those of a conventional strain in which F1 and LcrV were expressed from a single low-copy-number plasmid. The serum antibody responses to lipopolysaccharide (LPS) induced by the optimized bivalent vaccine were indistinguishable from those elicited by the parent strain, suggesting an adequate immunogenic capacity maintained through preservation of bacterial fitness; in contrast, LPS titers were 10-fold lower in mice immunized with the conventional vaccine strain. Importantly, mice receiving the optimized bivalent vaccine were fully protected against lethal pulmonary challenge. These results demonstrate the feasibility of distributing foreign antigen expression across both chromosomal and plasmid locations within a single vaccine organism for induction of protective immunity. PMID:25332120

  17. A bivalent typhoid live vector vaccine expressing both chromosome- and plasmid-encoded Yersinia pestis antigens fully protects against murine lethal pulmonary plague infection.

    PubMed

    Galen, James E; Wang, Jin Yuan; Carrasco, Jose A; Lloyd, Scott A; Mellado-Sanchez, Gabriela; Diaz-McNair, Jovita; Franco, Olga; Buskirk, Amanda D; Nataro, James P; Pasetti, Marcela F

    2015-01-01

    Live attenuated bacteria hold great promise as multivalent mucosal vaccines against a variety of pathogens. A major challenge of this approach has been the successful delivery of sufficient amounts of vaccine antigens to adequately prime the immune system without overattenuating the live vaccine. Here we used a live attenuated Salmonella enterica serovar Typhi strain to create a bivalent mucosal plague vaccine that produces both the protective F1 capsular antigen of Yersinia pestis and the LcrV protein required for secretion of virulence effector proteins. To reduce the metabolic burden associated with the coexpression of F1 and LcrV within the live vector, we balanced expression of both antigens by combining plasmid-based expression of F1 with chromosomal expression of LcrV from three independent loci. The immunogenicity and protective efficacy of this novel vaccine were assessed in mice by using a heterologous prime-boost immunization strategy and compared to those of a conventional strain in which F1 and LcrV were expressed from a single low-copy-number plasmid. The serum antibody responses to lipopolysaccharide (LPS) induced by the optimized bivalent vaccine were indistinguishable from those elicited by the parent strain, suggesting an adequate immunogenic capacity maintained through preservation of bacterial fitness; in contrast, LPS titers were 10-fold lower in mice immunized with the conventional vaccine strain. Importantly, mice receiving the optimized bivalent vaccine were fully protected against lethal pulmonary challenge. These results demonstrate the feasibility of distributing foreign antigen expression across both chromosomal and plasmid locations within a single vaccine organism for induction of protective immunity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. Expression of VP7, a Bluetongue Virus Group Specific Antigen by Viral Vectors: Analysis of the Induced Immune Responses and Evaluation of Protective Potential in Sheep

    PubMed Central

    Bouet-Cararo, Coraline; Contreras, Vanessa; Caruso, Agathe; Top, Sokunthea; Szelechowski, Marion; Bergeron, Corinne; Viarouge, Cyril; Desprat, Alexandra; Relmy, Anthony; Guibert, Jean-Michel; Dubois, Eric; Thiery, Richard; Bréard, Emmanuel; Bertagnoli, Stephane; Richardson, Jennifer; Foucras, Gilles; Meyer, Gilles; Schwartz-Cornil, Isabelle; Zientara, Stephan; Klonjkowski, Bernard

    2014-01-01

    Bluetongue virus (BTV) is an economically important Orbivirus transmitted by biting midges to domestic and wild ruminants. The need for new vaccines has been highlighted by the occurrence of repeated outbreaks caused by different BTV serotypes since 1998. The major group-reactive antigen of BTV, VP7, is conserved in the 26 serotypes described so far, and its role in the induction of protective immunity has been proposed. Viral-based vectors as antigen delivery systems display considerable promise as veterinary vaccine candidates. In this paper we have evaluated the capacity of the BTV-2 serotype VP7 core protein expressed by either a non-replicative canine adenovirus type 2 (Cav-VP7 R0) or a leporipoxvirus (SG33-VP7), to induce immune responses in sheep. Humoral responses were elicited against VP7 in almost all animals that received the recombinant vectors. Both Cav-VP7 R0 and SG33-VP7 stimulated an antigen-specific CD4+ response and Cav-VP7 R0 stimulated substantial proliferation of antigen-specific CD8+ lymphocytes. Encouraged by the results obtained with the Cav-VP7 R0 vaccine vector, immunized animals were challenged with either the homologous BTV-2 or the heterologous BTV-8 serotype and viral burden in plasma was followed by real-time RT-PCR. The immune responses triggered by Cav-VP7 R0 were insufficient to afford protective immunity against BTV infection, despite partial protection obtained against homologous challenge. This work underscores the need to further characterize the role of BTV proteins in cross-protective immunity. PMID:25364822

  19. The Detection of Protective Antigen (PA) Associated with Spores of Bacillus Anthracis and the Effects of Anti-PA Antibodies on Spore Germination and Macrophage Interactions

    DTIC Science & Technology

    2005-04-22

    unlimited 13. SUPPLEMENTARY NOTES The original document contains color images . 14. ABSTRACT The protective antigen (PA) component of the anthrax...air dried before inserted in a JEOL Jem 1010 transmission electron microscope. Images were captured with a Hamamatsu CCD camera aided with AMT 12-HR...Bacillus anthracis. Schweiz Z Allgem Pathol Bakteriol 1959;22:630–40. [36] Kramer MJ, Roth IL. Ultrastructural differences in the exosporium of the

  20. Identification of a Receptor-Binding Region within Domain 4 of the Protective Antigen Component of Anthrax Toxin

    PubMed Central

    Varughese, Mini; Teixeira, Avelino V.; Liu, Shihui; Leppla, Stephen H.

    1999-01-01

    Anthrax toxin from Bacillus anthracis is a three-component toxin consisting of lethal factor (LF), edema factor (EF), and protective antigen (PA). LF and EF are the catalytic components of the toxin, whereas PA is the receptor-binding component. To identify residues of PA that are involved in interaction with the cellular receptor, two solvent-exposed loops of domain 4 of PA (amino acids [aa] 679 to 693 and 704 to 723) were mutagenized, and the altered proteins purified and tested for toxicity in the presence of LF. In addition to the intended substitutions, novel mutations were introduced by errors that occurred during PCR. Substitutions within the large loop (aa 704 to 723) had no effect on PA activity. A mutated protein, LST-35, with three substitutions in the small loop (aa 679 to 693), bound weakly to the receptor and was nontoxic. A mutated protein, LST-8, with changes in three separate regions did not bind to receptor and was nontoxic. Toxicity was greatly decreased by truncation of the C-terminal 3 to 5 aa, but not by their substitution with nonnative residues or the extension of the terminus with nonnative sequences. Comparison of the 28 mutant proteins described here showed that the large loop (aa 704 to 722) is not involved in receptor binding, whereas residues in and near the small loop (aa 679 to 693) play an important role in receptor interaction. Other regions of domain 4, in particular residues at the extreme C terminus, appear to play a role in stabilizing a conformation needed for receptor-binding activity. PMID:10085028

  1. Detection of anthrax protective antigen (PA) using europium labeled anti-PA monoclonal antibody and time-resolved fluorescence ◊

    PubMed Central

    Stoddard, Robyn A.; Quinn, Conrad P.; Schiffer, Jarad M.; Boyer, Anne E.; Goldstein, Jason; Bagarozzi, Dennis A.; Soroka, Stephen D.; Dauphin, Leslie A.; Hoffmaster, Alex R.

    2015-01-01

    Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response. The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63 × 10−6 μM (0.551 ng/ml) for PA83 and 2.51 × 10−5 μM (1.58 ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n=7), inhalation (n=2), and gastrointestinal (n=1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through use of monoclonal antibodies to detect PA and LF in the lethal toxin complex. PMID:24857756

  2. Detection of anthrax protective antigen (PA) using europium labeled anti-PA monoclonal antibody and time-resolved fluorescence.

    PubMed

    Stoddard, Robyn A; Quinn, Conrad P; Schiffer, Jarad M; Boyer, Anne E; Goldstein, Jason; Bagarozzi, Dennis A; Soroka, Stephen D; Dauphin, Leslie A; Hoffmaster, Alex R

    2014-06-01

    Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response. The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63×10(-6)μM (0.551ng/ml) for PA83 and 2.51×10(-5)μM (1.58ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n=7), inhalation (n=2), and gastrointestinal (n=1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through the use of monoclonal antibodies to detect PA and LF in the lethal toxin complex. Published by Elsevier B.V.

  3. Anthrax lethal factor (LF) mediated block of the anthrax protective antigen (PA) ion channel: effect of ionic strength and voltage.

    PubMed

    Neumeyer, Tobias; Tonello, Fiorella; Dal Molin, Federica; Schiffler, Bettina; Orlik, Frank; Benz, Roland

    2006-03-07

    The anthrax toxin complex consists of three different molecules, protective antigen (PA), lethal factor (LF), and edema factor (EF). The activated form of PA, PA(63), forms heptamers that insert at low pH in biological membranes forming ion channels and that are necessary to translocate EF and LF in the cell cytosol. LF and EF are intracellular active enzymes that inhibit the host immune system promoting bacterial outgrowth. Here, PA(63) was reconstituted into artificial lipid bilayer membranes and formed ion-permeable channels. The heptameric PA(63) channel contains a binding site for LF on the cis side of the channel. Full-size LF was found to block the PA(63) channel in a dose- and ionic-strength-dependent way with half-saturation constants in the nanomolar concentration range. The binding curves suggest a 1:1 relationship between (PA(63))(7) and bound LF that blocks the channel. The presence of a His(6) tag at the N-terminal end of LF strongly increases the affinity of LF toward the PA(63) channel, indicating that the interaction between LF and the PA(63) channel occurs at the N terminus of the enzyme. The LF-mediated block of the PA(63)-induced membrane conductance is highly asymmetric with respect to the sign of the applied transmembrane potential. The result suggested that the PA(63) heptamers contain a high-affinity binding site for LF inside domain 1 or the channel vestibule and that the binding is ionic-strength-dependent.

  4. A protective antigen mutation increases the pH threshold of anthrax toxin receptor 2-mediated pore formation.

    PubMed

    Dennis, Melissa K; Mogridge, Jeremy

    2014-04-08

    Anthrax toxin protective antigen (PA) binds cellular receptors and self-assembles into oligomeric prepores. A prepore converts to a protein translocating pore after it has been transported to an endosome where the low pH triggers formation of a membrane-spanning β-barrel channel. Formation of this channel occurs after some PA-receptor contacts are broken to allow pore formation, while others are retained to preserve receptor association. The interaction between PA and anthrax toxin receptor 1 (ANTXR1) is weaker than its interaction with ANTXR2 such that the pH threshold of ANTXR1-mediated pore formation is higher by 1 pH unit. Here we examine receptor-specific differences in toxin binding and pore formation by mutating PA residue G342 that selectively abuts ANTXR2. Mutation of G342 to valine, leucine, isoleucine, or tryptophan increased the amount of PA bound to ANTXR1-expressing cells and decreased the amount of PA bound to ANTXR2-expressing cells. The more conservative G342A mutation did not affect the level of binding to ANTXR2, but ANTXR2-bound PA-G342A prepores exhibited a pH threshold higher than that of wild-type prepores. Mixtures of wild-type PA and PA-G342A were functional in toxicity assays, and the pH threshold of ANTXR2-mediated pore formation was dictated by the relative amounts of the two proteins in the hetero-oligomers. These results suggest that PA subunits within an oligomer do not have to be triggered simultaneously for a productive membrane insertion event to occur.

  5. Dynamic Phenylalanine Clamp Interactions Define Single-Channel Polypeptide Translocation through the Anthrax Toxin Protective Antigen Channel.

    PubMed

    Ghosal, Koyel; Colby, Jennifer M; Das, Debasis; Joy, Stephen T; Arora, Paramjit S; Krantz, Bryan A

    2017-03-24

    Anthrax toxin is an intracellularly acting toxin where sufficient detail is known about the structure of its channel, allowing for molecular investigations of translocation. The toxin is composed of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). The toxin's translocon, PA, translocates the large enzymes, LF and EF, across the endosomal membrane into the host cell's cytosol. Polypeptide clamps located throughout the PA channel catalyze the translocation of LF and EF. Here, we show that the central peptide clamp, the ϕ clamp, is a dynamic site that governs the overall peptide translocation pathway. Single-channel translocations of a 10-residue, guest-host peptide revealed that there were four states when peptide interacted with the channel. Two of the states had intermediate conductances of 10% and 50% of full conductance. With aromatic guest-host peptides, the 50% conducting intermediate oscillated with the fully blocked state. A Trp guest-host peptide was studied by manipulating its stereochemistry and prenucleating helix formation with a covalent linkage in the place of a hydrogen bond or hydrogen-bond surrogate (HBS). The Trp peptide synthesized with ʟ-amino acids translocated more efficiently than peptides synthesized with D- or alternating D,ʟ-amino acids. HBS stapled Trp peptide exhibited signs of steric hindrance and difficulty translocating. However, when mutant ϕ clamp (F427A) channels were tested, the HBS peptide translocated normally. Overall, peptide translocation is defined by dynamic interactions between the peptide and ϕ clamp. These dynamics require conformational flexibility, such that the peptide productively forms both extended-chain and helical states during translocation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Oligomerization of PcrV and LcrV, Protective Antigens of Pseudomonas aeruginosa and Yersinia pestis*S⃞

    PubMed Central

    Caroline, Gébus; Eric, Faudry; Bohn, Yu-Sing Tammy; Sylvie, Elsen; Attree, Ina

    2008-01-01

    Protective antigens of Pseudomonas aeruginosa (PcrV) and Yersinia pestis (LcrV) are key elements of specialized machinery, the type III secretion system (T3SS), which enables the injection of effector molecules into eukaryotic cells. Being positioned at the injectisome extremity, V proteins participate in the translocation process across the host cell plasma membrane. In this study, we demonstrate the assembly of V proteins into oligomeric doughnut-like complexes upon controlled refolding of the proteins in vitro. The oligomeric nature of refolded PcrV was revealed by size exclusion chromatography, native gel electrophoresis, and native mass spectrometry, which ascertain the capacity of the protein to multimerize into higher-order species. Furthermore, transmission electron microscopy performed on oligomers of both PcrV and LcrV revealed the presence of distinct structures with approximate internal and external diameters of 3-4 and 8-10 nm, respectively. The C-terminal helix, α12, of PcrV and notably the hydrophobic residues Val255, Leu262, and Leu276 located within this helix, were shown to be crucial for oligomerization. Moreover, the corresponding mutant proteins produced in P. aeruginosa were found to be non-functional in in vivo type III-dependent cytotoxicity assays by directly affecting the correct assembly of PopB/D translocon within the host cell membranes. The detailed understanding of structure-function relationships of T3SS needle tip proteins will be of value in further developments of new vaccines and antimicrobials. PMID:18583342

  7. Epitope structure of the Bordetella pertussis protein P.69 pertactin, a major vaccine component and protective antigen.

    PubMed

    Hijnen, Marcel; Mooi, Frits R; van Gageldonk, Pieter G M; Hoogerhout, Peter; King, Audrey J; Berbers, Guy A M

    2004-07-01

    Bordetella pertussis is reemerging in several countries with a traditionally high vaccine uptake. An analysis of clinical isolates revealed antigenic divergence between vaccine strains and circulating strains with respect to P.69 pertactin. Polymorphisms in P.69 pertactin are mainly limited to regions comprised of amino acid repeats, designated region 1 and region 2. Region 1 flanks the RGD motif, which is involved in adherence. Although antibodies against P.69 pertactin are implicated in protective immunity, little is known about the structure and location of its epitopes. Here we describe the identification by pepscan analysis of the locations of mainly linear epitopes recognized by human sera and mouse monoclonal antibodies (MAbs). A total of 24 epitopes were identified, and of these only 2 were recognized by both MAbs and human antibodies in serum. A number of immunodominant epitopes were identified which were recognized by 78 to 93% of the human sera tested. Blocking experiments indicated the presence of high-avidity human antibodies against conformational epitopes. Human antibodies against linear epitopes had much lower avidities, as they were unable to block MAbs. Pepscan analyses revealed several MAbs which bound to both region 1 and region 2. The two regions are separated by 289 amino acids in the primary structure, and we discuss the possibility that they form a single conformational epitope. Thus, both repeat regions may serve to deflect the immune response targeted to the functional domain of P.69 pertactin. This may explain why the variation in P.69 pertactin is so effective, despite the fact that it is limited to only two small segments of the molecule.

  8. O-mannosylation of the Mycobacterium tuberculosis adhesin Apa is crucial for T cell antigenicity during infection but is expendable for protection.

    PubMed

    Nandakumar, Subhadra; Kannanganat, Sunil; Dobos, Karen M; Lucas, Megan; Spencer, John S; Fang, Sunan; McDonald, Melissa A; Pohl, Jan; Birkness, Kristin; Chamcha, Venkateswarlu; Ramirez, Melissa V; Plikaytis, Bonnie B; Posey, James E; Amara, Rama Rao; Sable, Suraj B

    2013-01-01

    Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

  9. O-mannosylation of the Mycobacterium tuberculosis Adhesin Apa Is Crucial for T Cell Antigenicity during Infection but Is Expendable for Protection

    PubMed Central

    Dobos, Karen M.; Lucas, Megan; Spencer, John S.; Fang, Sunan; McDonald, Melissa A.; Pohl, Jan; Birkness, Kristin; Chamcha, Venkateswarlu; Ramirez, Melissa V.; Plikaytis, Bonnie B.; Posey, James E.; Amara, Rama Rao

    2013-01-01

    Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis. PMID:24130497

  10. Protection of mice from Brucella infection by immunization with attenuated Salmonella enterica serovar typhimurium expressing A L7/L12 and BLS fusion antigen of Brucella.

    PubMed

    Zhao, Zhongpeng; Li, Min; Luo, Deyan; Xing, Li; Wu, Shuo; Duan, Yueqiang; Yang, Penghui; Wang, Xiliang

    2009-08-20

    This study describes the potential use of attenuated Salmonella enterica serovar Typhimurium Strains (S. typhimurium) to express and deliver a L7/L12 and BLS fusion antigen of Brucella as a vaccination strategy to prevent Brucella infection in mice. S. typhimurium X4072 that contained a pTrc99A-BLS-L7/L12 plasmid, designated X4072bl, can deliver a L7/L12 and BLS fusion antigen expressed by the bacterium itself, while S. typhimurium X4550 that contained an asd-pVAX1-BLS-L7/L12 (asd-pBL) plasmid, designated X4550bl, can deliver the antigen to be expressed in target eukaryotic cells. When orally administered to BALB/c mice, both attenuated carrier strains were able to elicit mucosal and systemic immunity, which induced protection against B. abortus 544 infection in mice. The immunogenicity and protective efficacy of X4072bl and X4550bl were compared with a recombinant BLS-L7/L12 fusion protein vaccine (rBL) and a pVAX1-BLS-L7/L12 DNA vaccine (pBL) in this study. When rBL and pBL were intramuscularly injected into mice, both vaccines could also elicit comparable humoral and cellular immune responses, but not mucosal immunity, which therefore induced lower protection. Taken together, Salmonella-based subunit vaccines are a promising vaccine strategy in the prevention of Brucella infection.

  11. Protection against Invasive Amebiasis by a Single Monoclonal Antibody Directed against a Lipophosphoglycan Antigen Localized on the Surface of Entamoeba histolytica

    PubMed Central

    Marinets, Alexandra; Zhang, Tonghai; Guillén, Nancy; Gounon, Pierre; Bohle, Barbara; Vollmann, Ute; Scheiner, Otto; Wiedermann, Gerhard; Stanley, Samuel L.; Duchêne, Michael

    1997-01-01

    A panel of monoclonal antibodies was raised from mice immunized with a membrane preparation from Entamoeba histolytica, the pathogenic species causing invasive amebiasis in humans. Antibody EH5 gave a polydisperse band in immunoblots from membrane preparations from different E. histolytica strains, and a much weaker signal from two strains of the nonpathogenic species Entamoeba dispar. Although the exact chemical structure of the EH5 antigen is not yet known, the ability of the antigen to be metabolically radiolabeled with [32P]phosphate or [3H]glucose, its sensitivity to digestion by mild acid and phosphatidylinositol-specific phospholipase C, and its specific extraction from E. histolytica trophozoites by a method used to prepare lipophosphoglycans from Leishmania showed that it could be classified as an amebal lipophosphoglycan. Confocal immunofluorescence and immunogold labeling of trophozoites localized the antigen on the outer face of the plasma membrane and on the inner face of internal vesicle membranes. Antibody EH5 strongly agglutinated amebas in a similar way to concanavalin A (Con A), and Con A bound to immunoaffinity-purified EH5 antigen. Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A. The protective ability of antibody EH5 was tested in a passive immunization experiment in a severe combined immunodeficient (SCID) mouse model. Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess. Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas. PMID:9348313

  12. Signaling When (and When Not) to Be Cautious and Self-Protective: Impulsive and Reflective Trust in Close Relationships

    PubMed Central

    Murray, Sandra L.; Pinkus, Rebecca T.; Holmes, John G.; Harris, Brianna; Gomillion, Sarah; Aloni, Maya; Derrick, Jaye L.; Leder, Sadie

    2011-01-01

    A dual process model is proposed to explain how automatic evaluative associations to the partner (i.e., impulsive trust) and deliberative expectations of partner caring (i.e., reflective trust) interact to govern self-protection in romantic relationships. Experimental and correlational studies of dating and marital relationships supported the model. Subliminally conditioning more positive evaluative associations to the partner increased confidence in the partner’s caring, suggesting that trust has an impulsive basis. Being high on impulsive trust (i.e., more positive evaluative associations to the partner on the IAT) also reduced the automatic inclination to distance in response to doubts about the partner’s trustworthiness. It similarly reduced self-protective behavioral reactions to these reflective trust concerns. The studies further revealed that the effects of impulsive trust depend on working memory capacity: Being high on impulsive trust inoculated against reflective trust concerns for people low on working memory capacity. PMID:21443370

  13. A live attenuated BCG vaccine overexpressing multistage antigens Ag85B and HspX provides superior protection against Mycobacterium tuberculosis infection.

    PubMed

    Yuan, Xuefeng; Teng, Xindong; Jing, Yukai; Ma, Jilei; Tian, Maopeng; Yu, Qi; Zhou, Lei; Wang, Ruibo; Wang, Weihua; Li, Li; Fan, Xionglin

    2015-12-01

    Tuberculosis (TB) remains one of the most menacing infectious diseases, although attenuated Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine has been widely used to protect children against primary TB. There are increasing evidences that rapid growing and dormant Mycobacterium tuberculosis (M. tuberculosis) coexist in vivo after infection. However, BCG vaccine only elicits cell-mediated immune responses to secretory antigens expressed by rapid growing pathogen. BCG vaccine is thus unable to thwart the reactivation of latent tuberculosis infection (LTBI), and its protection wanes over age after neonatal immunization. In order to extend its ability for a durable protection, a novel recombinant BCG (rBCG) strain, named rBCG::XB, was constructed by overexpressing immunodominant multistage antigens of Ag85B and HspX, which are expressed by both rapid replicating and dormant M. tuberculosis. Long-term protective effect and immunogenicity of rBCG::XB were compared with the parental BCG in vaccinated C57BL/6 mice. Our results demonstrated that rBCG::XB provided the stronger and long-lasting protection against M. tuberculosis H37Rv intranasal infection than BCG. The rBCG::XB not only elicited the more durable multistage antigen-specific CD4(+)Th1-biased immune responses and specific polyfunctional CD4(+)T cells but also augmented the CD8(+) CTL effects against Ag85B in vivo. In particular, higher levels of CD4(+) TEM and CD8(+) TCM cells, dominated by IL2(+) CD4(+) and CD8(+) TCM cells, were obtained in the spleen of rBCG::XB vaccinated mice. Therefore, our findings indicate that rBCG::XB is a promising candidate to improve the efficacy of BCG.

  14. Cholera toxin-B (ctxB) antigen expressing Salmonella Typhimurium polyvalent vaccine exerts protective immune response against Vibrio cholerae infection.

    PubMed

    Vishwakarma, Vikalp; Sahoo, Sushree Sangita; Das, Susmita; Ray, Shilpa; Hardt, Wolf-Dietrich; Suar, Mrutyunjay

    2015-04-08

    Live attenuated vaccines are cost effective approach for preventing a broad range of infectious diseases, and thus are of great interest. However, immune-defects can predispose the patient to infections by the vaccine candidate itself. So far, few live vaccine candidates have been designed specifically for immune compromised individuals. Recently, we reported a new Salmonella Typhimurium Z234-vaccine strain (Periaswamy et al., PLoS ONE 2012;7:e45433), which was specifically attenuated in the NADPH-oxidase deficient host. In the present study, the Z234-vaccine strain was further engineered to express heterologous antigen (Vibrio cholerae toxin antigen subunit-B, i.e. CtxB) with the intention of creating a vector for simultaneous protection against Cholera and Salmonellosis. The primary aim of this study was to ensure the expression of CtxB antigen by the recombinant vaccine strain Z234-pMS101. The antigen CtxB was expressed through Z234 as a fusion protein with N-terminal signal sequence of Salmonella outer protein (SopE), an effector protein from Salmonella under the control of SopE promoter. The CtxB-expressing plasmid construct pMS101 (pM968-pSopE-ctxB) was found to be stable both in vitro and in vivo. In an oral mouse infection model, the vaccine strain Z234-pMS101 efficiently colonized the host gut. The extent of protection was confirmed after challenging the immunized hosts with live V. cholerae. Vaccinated mice showed reduced gut colonization by V. cholerae. Further assessment of immunological parameters supported the possibility of conferring effective immune response by Z234-pMS101 vaccine strain. Overall, the Z234-pMS101 vaccine strain showed potential as a promising polyvalent vaccine candidate to protect against S. Typhimurium and V. cholerae infection simultaneously.

  15. Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice.

    PubMed

    Soto, Manuel; Corvo, Laura; Garde, Esther; Ramírez, Laura; Iniesta, Virginia; Bonay, Pedro; Gómez-Nieto, Carlos; González, Víctor M; Martín, M Elena; Alonso, Carlos; Coelho, Eduardo A F; Barral, Aldina; Barral-Netto, Manoel; Iborra, Salvador

    2015-05-01

    Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs). Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid. The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasis.

  16. Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice

    PubMed Central

    Corvo, Laura; Garde, Esther; Ramírez, Laura; Iniesta, Virginia; Bonay, Pedro; Gómez-Nieto, Carlos; González, Víctor M.; Martín, M. Elena; Alonso, Carlos; Coelho, Eduardo A. F.; Barral, Aldina; Barral-Netto, Manoel

    2015-01-01

    Background Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs). Methodology/Principal Findings Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid. Conclusion/Significance The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasis. PMID:25955652

  17. Discovery of a Protective Rickettsia prowazekii Antigen Recognized by CD8+ T Cells, RP884, Using an In Vivo Screening Platform

    PubMed Central

    Goez, Yenny; Cespedes, Maria A.; Hidalgo, Marylin; Correa, Paula; Valbuena, Gustavo

    2013-01-01

    Rickettsia prowazekii has been tested for biological warfare due to the high mortality that it produces after aerosol transmission of very low numbers of rickettsiae. Epidemic typhus, the infection caused by these obligately intracellular bacteria, continues to be a threat because it is difficult to diagnose due to initial non-specific symptoms and the lack of commercial diagnostic tests that are sensitive and specific during the initial clinical presentation. A vaccine to prevent epidemic typhus would constitute an effective deterrent to the weaponization of R. prowazekii; however, an effective and safe vaccine is not currently available. Due to the cytoplasmic niche of Rickettsia, CD8+ T-cells are critical effectors of immunity; however, the identification of antigens recognized by these cells has not been systematically addressed. To help close this gap, we designed an antigen discovery strategy that uses cell-based vaccination with antigen presenting cells expressing microbe's proteins targeted to the MHC class I presentation pathway. We report the use of this method to discover a protective T-cell rickettsial antigen, RP884, among a test subset of rickettsial proteins. PMID:24146844

  18. Pretreatment with antibody to eosinophil major basic protein prevents hyperresponsiveness by protecting neuronal M2 muscarinic receptors in antigen-challenged guinea pigs.

    PubMed Central

    Evans, C M; Fryer, A D; Jacoby, D B; Gleich, G J; Costello, R W

    1997-01-01

    In antigen-challenged guinea pigs there is recruitment of eosinophils into the lungs and to airway nerves, decreased function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lungs, and airway hyperresponsiveness. A rabbit antibody to guinea pig eosinophil major basic protein was used to determine whether M2 muscarinic receptor dysfunction, and the subsequent hyperresponsiveness, are due to antagonism of the M2 receptor by eosinophil major basic protein. Guinea pigs were sensitized, challenged with ovalbumin and hyperresponsiveness, and M2 receptor function tested 24 h later with the muscarinic agonist pilocarpine. Antigen-challenged guinea pigs were hyperresponsive to electrical stimulation of the vagus nerves compared with controls. Likewise, loss of M2 receptor function was demonstrated since the agonist pilocarpine inhibited vagally-induced bronchoconstriction in control but not challenged animals. Pretreatment with rabbit antibody to guinea pig eosinophil major basic protein prevented hyperresponsiveness, and protected M2 receptor function in the antigen-challenged animals without inhibiting eosinophil accumulation in the lungs or around the nerves. Thus, hyperresponsiveness is a result of inhibition of neuronal M2 muscarinic receptor function by eosinophil major basic protein in antigen-challenged guinea pigs. PMID:9410903

  19. Studies of reflectivity degradation of retroreflectors in LHD and mitigation of impurity deposition using shaped diagnostic ducts and protective windows

    NASA Astrophysics Data System (ADS)

    Akiyama, T.; Yoshida, N.; Kawahata, K.; Tokitani, M.; Iwakiri, H.; Okajima, S.; Nakayama, K.

    2012-06-01

    Maintaining the reflectivity of first mirrors is indispensable in future fusion devices. While a retroreflector (corner cube mirror) is useful for laser diagnostics, impurities tend to accumulate and form a thick deposition layer in the central region, which causes degradation of reflectivity, due to the hollow shape of the retroreflector. Two mirror structures are tested to retain the reflectivity in the Large Helical Device (LHD). One is a bending mirror structure with a protective cylinder with fins and it could maintain the reflectivity over a three-month experimental campaign. The other is a cover window just in front of the reflector. Candidates of the window materials were exposed to the LHD plasmas and the degradation of the transmissivity of ZnSe and silicon, which are used for infrared and far infrared laser light, respectively, were small.

  20. Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

    PubMed

    Ingavle, Ganesh C; Baillie, Les W J; Zheng, Yishan; Lis, Elzbieta K; Savina, Irina N; Howell, Carol A; Mikhalovsky, Sergey V; Sandeman, Susan R

    2015-05-01

    Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0

  1. Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II, and CFA/IV

    PubMed Central

    Ruan, Xiaosai; Knudsen, David E.; Wollenberg, Katie M.

    2014-01-01

    Diarrhea is the second leading cause of death in children younger than 5 years and continues to be a major threat to global health. Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacteria causing diarrhea in developing countries. ETEC strains are able to attach to host small intestinal epithelial cells by using bacterial colonization factor antigen (CFA) adhesins. This attachment helps to initiate the diarrheal disease. Vaccines that induce antiadhesin immunity to block adherence of ETEC strains that express immunologically heterogeneous CFA adhesins are expected to protect against ETEC diarrhea. In this study, we created a CFA multiepitope fusion antigen (MEFA) carrying representative epitopes of CFA/I, CFA/II (CS1, CS2, and CS3), and CFA/IV (CS4, CS5, and CS6), examined its immunogenicity in mice, and assessed the potential of this MEFA as an antiadhesin vaccine against ETEC. Mice intraperitoneally immunized with this CFA MEFA exhibited no adverse effects and developed immune responses to CFA/I, CFA/II, and CFA/IV adhesins. Moreover, after incubation with serum of the immunized mice, ETEC or E. coli strains expressing CFA/I, CFA/II, or CFA/IV adhesins were significantly inhibited in adherence to Caco-2 cells. Our results indicated this CFA MEFA elicited antibodies that not only cross-reacted to CFA/I, CFA/II and CFA/IV adhesins but also broadly inhibited adherence of E. coli strains expressing these seven adhesins and suggested that this CFA MEFA could be a candidate to induce broad-spectrum antiadhesin protection against ETEC diarrhea. Additionally, this antigen construction approach (creating an MEFA) may be generally used in vaccine development against heterogenic pathogens. PMID:24351757

  2. Multiepitope fusion antigen induces broadly protective antibodies that prevent adherence of Escherichia coli strains expressing colonization factor antigen I (CFA/I), CFA/II, and CFA/IV.

    PubMed

    Ruan, Xiaosai; Knudsen, David E; Wollenberg, Katie M; Sack, David A; Zhang, Weiping

    2014-02-01

    Diarrhea is the second leading cause of death in children younger than 5 years and continues to be a major threat to global health. Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacteria causing diarrhea in developing countries. ETEC strains are able to attach to host small intestinal epithelial cells by using bacterial colonization factor antigen (CFA) adhesins. This attachment helps to initiate the diarrheal disease. Vaccines that induce antiadhesin immunity to block adherence of ETEC strains that express immunologically heterogeneous CFA adhesins are expected to protect against ETEC diarrhea. In this study, we created a CFA multiepitope fusion antigen (MEFA) carrying representative epitopes of CFA/I, CFA/II (CS1, CS2, and CS3), and CFA/IV (CS4, CS5, and CS6), examined its immunogenicity in mice, and assessed the potential of this MEFA as an antiadhesin vaccine against ETEC. Mice intraperitoneally immunized with this CFA MEFA exhibited no adverse effects and developed immune responses to CFA/I, CFA/II, and CFA/IV adhesins. Moreover, after incubation with serum of the immunized mice, ETEC or E. coli strains expressing CFA/I, CFA/II, or CFA/IV adhesins were significantly inhibited in adherence to Caco-2 cells. Our results indicated this CFA MEFA elicited antibodies that not only cross-reacted to CFA/I, CFA/II and CFA/IV adhesins but also broadly inhibited adherence of E. coli strains expressing these seven adhesins and suggested that this CFA MEFA could be a candidate to induce broad-spectrum antiadhesin protection against ETEC diarrhea. Additionally, this antigen construction approach (creating an MEFA) may be generally used in vaccine development against heterogenic pathogens.

  3. Foot-and-mouth disease marker vaccine: cattle protection with a partial VP1 G-H loop deleted virus antigen.

    PubMed

    Fowler, V L; Bashiruddin, J B; Maree, F F; Mutowembwa, P; Bankowski, B; Gibson, D; Cox, S; Knowles, N; Barnett, P V

    2011-10-26

    Contrary to the dogma that the VP1 G-H loop is essential for FMD vaccine efficacy, it has been previously shown that foot-and-mouth disease 146s antigen containing heterologous VP1 G-H loops confers complete protection in pigs and cattle. Moreover, serological evaluation of cattle vaccinated with an antigen lacking a large proportion of the VP1 G-H loop indicated that these animals should be protected against infection with FMD. Absence of this loop provides opportunity for the development of an FMD negative marker vaccine, allowing infection to be detected by antibodies against this missing region. Cattle vaccinated with this negative marker vaccine were fully protected following virus challenge 28 days post vaccination as determined by the absence of generalised lesions on their feet. Furthermore, use of our improved differentiation ELISA identified animals exposed to infection as early as 7 days post-challenge. We thus demonstrate, for the first time, the ability of this FMD negative marker vaccine to fully protect cattle from experimental challenge and rapidly distinguish animals that are subsequently exposed to infection.

  4. Antigenicity and immunogenicity of a multiple peptidic construction of the Schistosoma mansoni Sm-28 GST antigen in rat, mouse, and monkey. 1. Partial protection of Fischer rat after active immunization.

    PubMed

    Wolowczuk, I; Auriault, C; Bossus, M; Boulanger, D; Gras-Masse, H; Mazingue, C; Pierce, R J; Grezel, D; Reid, G D; Tartar, A

    1991-03-15

    Among the schistosome proteins characterized as vaccine candidates, an Ag of 28 kDa (Sm-28-GST) has received considerable attention. It was shown to be antigenic in humans and protective in mice, rats, hamsters, and baboons. Synthetic peptides derived from its sequence have been used to characterize the immune response to the molecule and one of these, comprising aminoacids 115-131 has been shown to incorporate both T and B cell recognition sites in a variety of experimental models. An octameric ("octopus") construction of the 115-131 peptide has been synthesized and its antigenicity and immunogenicity have been examined. The octopus construct is immunogenic in rats, mice and baboons in the presence of CFA (for rodents) and Bacille-Calmette-Guérin vaccine (for primates) as adjuvants. This clearly indicates that the construction allowed the conservation of the immune sites of the cognate protein. Moreover, anti-octopus sera from immunized Fischer rats were able to mediate platelet-, macrophage-, and eosinophil-dependent cytotoxicity toward schistosomula. Rats immunized with the 115-131 octopus were partially protected against a challenge infection with Schistosoma mansoni cercariae and this was paralleled by an increased level of IgG and more importantly, of IgE Sm-28-GST-specific antibodies.

  5. Expression, purification, immunogenicity, and protective efficacy of a recombinant Tc24 antigen as a vaccine against Trypanosoma cruzi infection in mice.

    PubMed

    Martinez-Campos, Viridiana; Martinez-Vega, Pedro; Ramirez-Sierra, Maria Jesus; Rosado-Vallado, Miguel; Seid, Christopher A; Hudspeth, Elissa M; Wei, Junfei; Liu, Zhuyun; Kwityn, Cliff; Hammond, Molly; Ortega-López, Jaime; Zhan, Bin; Hotez, Peter J; Bottazzi, Maria Elena; Dumonteil, Eric

    2015-08-26

    The Tc24 calcium binding protein from the flagellar pocket of Trypanosoma cruzi is under evaluation as a candidate vaccine antigen against Chagas disease. Previously, a DNA vaccine encoding Tc24 was shown to be an effective vaccine (both as a preventive and therapeutic intervention) in mice and dogs, as evidenced by reductions in T. cruzi parasitemia and cardiac amastigotes, as well as reduced cardiac inflammation and increased host survival. Here we developed a suitable platform for the large scale production of recombinant Tc24 (rTc24) and show that when rTc24 is combined with a monophosphoryl-lipid A (MPLA) adjuvant, the formulated vaccine induces a Th1-biased immune response in mice, comprised of elevated IgG2a antibody levels and interferon-gamma levels from splenocytes, compared to controls. These immune responses also resulted in statistically significant decreased T. cruzi parasitemia and cardiac amastigotes, as well as increased survival following T. cruzi challenge infections, compared to controls. Partial protective efficacy was shown regardless of whether the antigen was expressed in Escherichia coli or in yeast (Pichia pastoris). While mouse vaccinations will require further modifications in order to optimize protective efficacy, such studies provide a basis for further evaluations of vaccines comprised of rTc24, together with alternative adjuvants and additional recombinant antigens. Copyright © 2015. Published by Elsevier Ltd.

  6. Levels of antibodies to Plasmodium falciparum sporozoite surface antigens reflect malaria transmission rates and are persistent in the absence of reinfection.

    PubMed Central

    Druilhe, P; Pradier, O; Marc, J P; Miltgen, F; Mazier, D; Parent, G

    1986-01-01

    Antibodies reacting with Plasmodium falciparum sporozoite surface antigens were measured by an immunofluorescence assay using wet preparations of sporozoites attached to poly-L-lysine-treated glass slides, a procedure which was found to be more specific than one using glutaraldehyde-treated and dried preparations. Subjects recovering from a first attack were found to be negative. In two African villages which differed in the level at which mosquitoes transmit the disease (1 and 100 infective bites per year and per individual), both the prevalence by age group and the levels of anti-sporozoite antibodies differed markedly, as follows. In the low-transmission area, these antibodies were not detected in subjects aged 2 to 10 years; thereafter, prevalence increased gradually with the age of the subject and reached 90% in subjects aged 50 to 80 years. In the high-transmission area, all of the subjects studied, including the younger ones, were positive. Anti-sporozoite antibody levels were independent of the levels of antibodies directed against blood stages. On average, the mean antibody titers were equal to 1/16 in the first village and 1/1,650 in the second one. These results suggest that stage-specific antibodies reflect the cumulative number of sporozoites inoculated in humans by mosquitoes and may therefore have useful epidemiological applications. In addition, the presence of stage-specific antibodies in the sera of African adults collected at different times after departure from the endemic area indicates that they may last for several years. During the course of this study, we observed a heterogeneity of immunofluorescence labeling in parasite populations prepared from mosquito salivary glands. This raises the question of possible qualitative or quantitative antigenic differences or both between one sporozoite and the other. PMID:3525412

  7. Combining Cationic Liposomal Delivery with MPL-TDM for Cysteine Protease Cocktail Vaccination against Leishmania donovani : Evidence for Antigen Synergy and Protection

    PubMed Central

    Das, Amrita; Ali, Nahid

    2014-01-01

    Background With the paucity of new drugs and HIV co-infection, vaccination remains an unmet research priority to combat visceral leishmaniasis (VL) requiring strong cellular immunity. Protein vaccination often suffers from low immunogenicity and poor generation of memory T cells for long-lasting protection. Cysteine proteases (CPs) are immunogenic proteins and key mediators of cellular functions in Leishmania. Here, we evaluated the vaccine efficacies of CPs against VL, using cationic liposomes with Toll like receptor agonists for stimulating host immunity against L. donovani in a hamster model. Methodology/Principal Findings Recombinant CPs type I (cpb), II (cpa) and III (cpc) of L. donovani were tested singly and in combination as a triple antigen cocktail for antileishmanial vaccination in hamsters. We found the antigens to be highly immunoreactive and persistent anti-CPA, anti-CPB and anti-CPC antibodies were detected in VL patients even after cure. The liposome-entrapped CPs with monophosphoryl lipid A-Trehalose dicorynomycolate (MPL-TDM) induced significantly high nitric oxide (up to 4 fold higher than controls) mediated antileishmanial activity in vitro, and resulted in strong in vivo protection. Among the three CPs, CPC emerged as the most potent vaccine candidate in combating the disease. Interestingly, a synergistic increase in protection was observed with liposomal CPA, CPB and CPC antigenic cocktail which reduced the organ parasite burden by 1013–1016 folds, and increased the disease-free survival of >80% animals at least up to 6 months post infection. Robust secretion of IFN-γ and IL-12, along with concomitant downregulation of Th2 cytokines, was observed in cocktail vaccinates, even after 3 months post infection. Conclusion/Significance The present study is the first report of a comparative efficacy of leishmanial CPs and their cocktail using liposomal formulation with MPL-TDM against L. donovani. The level of protection attained has not been

  8. Montanide™ ISA 71 VG adjuvant enhances antibody and cell-mediated immune responses to profilin subunit antigen vaccination and promotes protection against Eimeria acervulina and Eimeria tenella.

    PubMed

    Jang, Seung I; Lillehoj, Hyun S; Lee, Sung Hyen; Lee, Kyung Woo; Lillehoj, Erik P; Bertrand, François; Dupuis, Laurent; Deville, Sébastien

    2011-01-01

    The present study was conducted to investigate the immunoenhancing effects of Montanide™ ISA 71 VG adjuvant on profilin subunit antigen vaccination. Broiler chickens were immunized subcutaneously with a purified Eimeria acervulina recombinant profilin protein, either alone or mixed with ISA 71 VG, and host immune responses were evaluated. After secondary immunization, antigen-specific antibody and T-cell responses were higher in the group which received profilin plus ISA 71 VG compared with the other groups. Furthermore, body weight gains and fecal oocyst shedding were evaluated following oral challenge infection with live E. acervulina or Eimeria tenella oocysts. Vaccination with profilin plus ISA 71 VG reduced oocyst shedding compared with animals immunized with profilin alone. These results demonstrate that the recombinant profilin subunit vaccine, when given in combination with Montanide™ ISA 71 VG, augments protective immunity against E. acervulina and E. tenella. Published by Elsevier Inc.

  9. Challenge with Bovine viral diarrhea virus by exposure to persistently infected calves: protection by vaccination and negative results of antigen testing in nonvaccinated acutely infected calves

    PubMed Central

    Johnson, Bill J.; Briggs, Robert E.; Ridpath, Julia F.; Saliki, Jeremiah T.; Confer, Anthony W.; Burge, Lurinda J.; Step, Douglas L.; Walker, Derek A.; Payton, Mark E.

    2006-01-01

    Abstract Calves persistently infected (PI) with Bovine viral diarrhea virus (BVDV) represent an important source of infection for susceptible cattle. We evaluated vaccine efficacy using calves PI with noncytopathic BVDV2a for the challenge and compared tests to detect BVDV in acutely or transiently infected calves versus PI calves. Vaccination with 2 doses of modified live virus vaccine containing BVDV1a and BVDV2a protected the calves exposed to the PI calves: neither viremia nor nasal shedding occurred. An immunohistochemistry test on formalin-fixed ear notches and an antigen-capture enzyme-linked immunosorbent assay on fresh notches in phosphate-buffered saline did not detect BVDV antigen in any of the acutely or transiently infected calves, whereas both tests had positive results in all the PI calves. PMID:16639944

  10. Immunochemical characterization and purification of Sm-97 a Schistosoma manosin antigen monospecifically recognized by antibodies from mice protectively immunized with a nonliving vaccine

    SciTech Connect

    Pearce, E.J.; James, S.L.; Dalton, J.; Barrall, A.; Ramos, C.; Strand, M.; Sher, A.

    1986-12-01

    Mice protected against Shistosoma mansoni infection by intradermal (i.d.) vaccination with nonliving schistosomula or soluble extracts of larval or adult schistosomes (SCHLARP and SWAP, respectively) produce antibodies that react by Western blot analysis with one antigen of M/sub r/ (x 10/sup -3/) 97 in SWAP prepared in the presence of protease inhibitors and two antigens of M/sub r/ (x 10/sup -3/) 95 and 78 in SWAP prepared in their absence. Vaccine antibodies also immunoprecipitated a single 97k molecule, with a pI of 5.5, from detergent extracts of (/sup 35/S)methionine-labeled schistosomes. Three hybridomas, produced from spleen cells of i.d. immunized mice, all recognized both the 95k/78k doublet and one 97k antigen, indicating that the two lower M/sub r/ components are degradation products of the same 97k molecule. /sup 125/I-concanavalin a bound weakly to purified Sm-97, indicating that this antigen is minimally glycosylated. Competitive radioimmunoassays performed with /sup 125/I-labeled monoclonal antibodies and purified antigen defined at least two distinct epitopes on Sm-97. Antibodies from i.d. vaccinated mice recognized both monoclonal antibody-defined epitopes, whereas anti-Sm-97 antibodies in chronic infection sera recognized neither. Finally, purified Sm-97 was shown to elicit delayed-type hypersensitivity in i.d. vaccinated mice, suggesting that this molecule is also capable of evoking cell-mediated responses, a finding consistent with its proposed function as a vaccine immunogen.

  11. Fusion of antigen to a dendritic cell targeting chemokine combined with adjuvant yields a malaria DNA vaccine with enhanced protective capabilities.

    PubMed

    Luo, Kun; Zhang, Hong; Zavala, Fidel; Biragyn, Arya; Espinosa, Diego A; Markham, Richard B

    2014-01-01

    Although sterilizing immunity to malaria can be elicited by irradiated sporozoite vaccination, no clinically practical subunit vaccine has been shown to be capable of preventing the approximately 600,000 annual deaths attributed to this infection. DNA vaccines offer several potential advantages for a disease that primarily affects the developing world, but new approaches are needed to improve the immunogenicity of these vaccines. By using a novel, lipid-based adjuvant, Vaxfectin, to attract immune cells to the immunization site, in combination with an antigen-chemokine DNA construct designed to target antigen to immature dendritic cells, we elicited a humoral immune response that provided sterilizing immunity to malaria challenge in a mouse model system. The chemokine, MIP3αCCL20, did not significantly enhance the cellular infiltrate or levels of cytokine or chemokine expression at the immunization site but acted with Vaxfectin to reduce liver stage malaria infection by orders of magnitude compared to vaccine constructs lacking the chemokine component. The levels of protection achieved were equivalent to those observed with irradiated sporozoites, a candidate vaccine undergoing development for further large scale clinical trial. Only vaccination with the combined regimen of adjuvant and chemokine provided 80-100% protection against the development of bloodstream infection. Treating the immunization process as requiring the independent steps of 1) attracting antigen-presenting cells to the site of immunization and 2) specifically directing vaccine antigen to the immature dendritic cells that initiate the adaptive immune response may provide a rational strategy for the development of a clinically applicable malaria DNA vaccine.

  12. Fusion of Antigen to a Dendritic Cell Targeting Chemokine Combined with Adjuvant Yields a Malaria DNA Vaccine with Enhanced Protective Capabilities

    PubMed Central

    Luo, Kun; Zhang, Hong; Zavala, Fidel; Biragyn, Arya; Espinosa, Diego A.; Markham, Richard B.

    2014-01-01

    Although sterilizing immunity to malaria can be elicited by irradiated sporozoite vaccination, no clinically practical subunit vaccine has been shown to be capable of preventing the approximately 600,000 annual deaths attributed to this infection. DNA vaccines offer several potential advantages for a disease that primarily affects the developing world, but new approaches are needed to improve the immunogenicity of these vaccines. By using a novel, lipid-based adjuvant, Vaxfectin, to attract immune cells to the immunization site, in combination with an antigen-chemokine DNA construct designed to target antigen to immature dendritic cells, we elicited a humoral immune response that provided sterilizing immunity to malaria challenge in a mouse model system. The chemokine, MIP3αCCL20, did not significantly enhance the cellular infiltrate or levels of cytokine or chemokine expression at the immunization site but acted with Vaxfectin to reduce liver stage malaria infection by orders of magnitude compared to vaccine constructs lacking the chemokine component. The levels of protection achieved were equivalent to those observed with irradiated sporozoites, a candidate vaccine undergoing development for further large scale clinical trial. Only vaccination with the combined regimen of adjuvant and chemokine provided 80–100% protection against the development of bloodstream infection. Treating the immunization process as requiring the independent steps of 1) attracting antigen-presenting cells to the site of immunization and 2) specifically directing vaccine antigen to the immature dendritic cells that initiate the adaptive immune response may provide a rational strategy for the development of a clinically applicable malaria DNA vaccine. PMID:24599116

  13. Evaluation of protective potential of Yersinia pestis outer membrane protein antigens as possible candidates for a new-generation recombinant plague vaccine.

    PubMed

    Erova, Tatiana E; Rosenzweig, Jason A; Sha, Jian; Suarez, Giovanni; Sierra, Johanna C; Kirtley, Michelle L; van Lier, Christina J; Telepnev, Maxim V; Motin, Vladimir L; Chopra, Ashok K

    2013-02-01

    Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1(-) strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1(-) mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1(-) CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains.

  14. Evaluation of Protective Potential of Yersinia pestis Outer Membrane Protein Antigens as Possible Candidates for a New-Generation Recombinant Plague Vaccine

    PubMed Central

    Erova, Tatiana E.; Rosenzweig, Jason A.; Sha, Jian; Suarez, Giovanni; Sierra, Johanna C.; Kirtley, Michelle L.; van Lier, Christina J.; Telepnev, Maxim V.; Motin, Vladimir L.

    2013-01-01

    Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1− strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1− mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1− CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains. PMID:23239803

  15. Recombinant parvovirus-like particles as an antigen carrier: A novel nonreplicative exogenous antigen to elicit protective antiviral cytotoxic T cells

    PubMed Central

    Sedlik, C.; Saron, M.-F.; Sarraseca, J.; Casal, I.; Leclerc, C.

    1997-01-01

    To develop a strategy that promotes efficient antiviral immunity, hybrid virus-like particles (VLP) were prepared by self-assembly of the modified porcine parvovirus VP2 capsid protein carrying a CD8+ T cell epitope from the lymphocytic choriomeningitis virus nucleoprotein. Immunization of mice with these hybrid pseudoparticles, without adjuvant, induced strong cytotoxic T lymphocyte (CTL) responses against both peptide-coated- or virus-infected-target cells. This CD8+ class I-restricted cytotoxic activity persisted in vivo for at least 9 months. Furthermore, the hybrid parvovirus-like particles were able to induce a complete protection of mice against a lethal lymphocytic choriomeningitis virus infection. To our knowledge, this study represents the first demonstration that hybrid nonreplicative VLP carrying a single viral CTL epitope can induce protection against a viral lethal challenge, in the absence of any adjuvant. These recombinant particles containing a single type of protein are easily produced by the baculovirus expression system and, therefore, represent a promising and safe strategy to induce strong CTL responses for the elimination of virus-infected cells. PMID:9207121

  16. The effect of different adjuvants on immune parameters and protection following vaccination of sheep with a larval-specific antigen of the gastrointestinal nematode, Haemonchus contortus.

    PubMed

    Piedrafita, David; Preston, Sarah; Kemp, Joanna; de Veer, Michael; Sherrard, Jayne; Kraska, Troy; Elhay, Martin; Meeusen, Els

    2013-01-01

    It has recently been recognised that vaccine adjuvants play a critical role in directing the nature of a vaccine induced effector response. In the present study, several adjuvants were evaluated for their ability to protect sheep after field vaccination with the larval-specific Haemonchus contortus antigen, HcsL3. Using a suboptimal antigen dose, aluminium adjuvant was shown to reduce the cumulative faecal egg counts (cFEC) and worm burden by 23% and 25% respectively, in agreement with a previous study. The addition of Quil A to the aluminium-adjuvanted vaccine brought cFEC back to control levels. Vaccination with the adjuvant DEAE-dextran almost doubled the protection compared to the aluminium-adjuvanted vaccine resulting in 40% and 41% reduction in cFEC and worm counts compared to controls. Examination of skin responses following i.d. injection of exsheathed L3, revealed that cFEC was negatively correlated with wheal size and tissue eosinophils for the DEAE-dextran and aluminium-adjuvanted groups respectively. These studies have for the first time shown the potential of DEAE-dextran adjuvant for helminth vaccines, and discovered significant cellular correlates of vaccine-induced protection.

  17. Presence of Human T-Cell Responses to the Mycobacterium leprae 45-Kilodalton Antigen Reflects Infection with or Exposure to M. leprae

    PubMed Central

    Macfarlane, Anne; Mondragon-Gonzalez, Rafael; Vega-Lopez, Francisco; Wieles, Brigitte; de Pena, Josefina; Rodriguez, Obdulia; Suarez y de la Torre, Raul; de Vries, Rene R. P.; Ottenhoff, Tom H. M.; Dockrell, Hazel M.

    2001-01-01

    The ability of the 45-kDa serine-rich Mycobacterium leprae antigen to stimulate peripheral blood mononuclear cell (PBMC) proliferation and gamma interferon (IFN-γ) production was measured in leprosy patients, household contacts, and healthy controls from areas of endemicity in Mexico. Almost all the tuberculoid leprosy patients gave strong PBMC proliferation responses to the M. leprae 45-kDa antigen (92.8%; n = 14). Responses were lower in lepromatous leprosy patients (60.6%; n = 34), but some responses to the 45-kDa antigen were detected in patients unresponsive to M. leprae sonicate. The proportion of positive responses to the M. leprae 45-kDa antigen was much higher in leprosy contacts (88%; n = 17) than in controls from areas of endemicity (10%; n = 20). None of 15 patients with pulmonary tuberculosis gave a positive proliferation response to the 45-kDa antigen. The 45-kDa antigen induced IFN-γ secretion similar to that induced by the native Mycobacterium tuberculosis 30/31-kDa antigen in tuberculoid leprosy patients and higher responses than those induced by the other recombinant antigens (M. leprae 10- and 65-kDa antigens, thioredoxin, and thioredoxin reductase); in patients with pulmonary tuberculosis it induced lower IFN-γ secretion than the other recombinant antigens. These results suggest that the M. leprae 45-kDa antigen is a potent T-cell antigen which is M. leprae specific in these Mexican donors. This antigen may therefore have diagnostic potential as a new skin test reagent or as an antigen in a simple whole-blood cytokine test. PMID:11329466

  18. Optimizing Immunization Strategies for the Induction of Antigen-Specific CD4 and CD8 T Cell Responses for Protection against Intracellular Parasites.

    PubMed

    Hofmeyer, Kimberly A; Duthie, Malcolm S; Laurance, John D; Favila, Michelle A; Van Hoeven, Neal; Coler, Rhea N; Reed, Steven G

    2016-09-01

    Immunization strategies that generate either CD4 or CD8 T cell responses are relatively well described, but less is known with regard to optimizing regimens to induce both CD4 and CD8 memory T cells. Considering the importance of both CD4 and CD8 T cells in the control of intracellular pathogens such as Leishmania donovani, we wanted to identify vaccines that could raise both CD4 and CD8 T cell responses and determine how to configure immunization strategies to generate the best combined protective T cell response. We examined responses generated against the Leishmania vaccine antigen F3 following its administration in either recombinant form with the Toll-like receptor 4 (TLR4) agonist-containing adjuvant formulation GLA-SE (F3+GLA-SE) or as a gene product delivered in an adenoviral vector (Ad5-F3). Homologous immunization strategies using only F3+GLA-SE or Ad5-F3 preferentially generated either CD4 or CD8 T cells, respectively. In contrast, heterologous strategies generated both antigen-specific CD4 and CD8 T cells. Administration of F3+GLA-SE before Ad5-F3 generated the greatest combined CD4 and CD8 responses. Cytotoxic CD8 T cell responses were highest when Th1 cells were generated prior to their induction by Ad5-F3. Finally, a single immunization with a combination of F3+GLA-SE mixed with Ad5-F3 was found to be sufficient to provide protection against experimental L. donovani infection. Taken together, our data delineate immunization regimens that induce antigen-specific CD4 and CD8 T cell memory responses, and identify a single immunization strategy that could be used to rapidly provide protection against intracellular pathogens in regions where access to health care is limited or sporadic. Copyright © 2016 Hofmeyer et al.

  19. Optimizing Immunization Strategies for the Induction of Antigen-Specific CD4 and CD8 T Cell Responses for Protection against Intracellular Parasites

    PubMed Central

    Hofmeyer, Kimberly A.; Laurance, John D.; Favila, Michelle A.; Van Hoeven, Neal; Coler, Rhea N.; Reed, Steven G.

    2016-01-01

    Immunization strategies that generate either CD4 or CD8 T cell responses are relatively well described, but less is known with regard to optimizing regimens to induce both CD4 and CD8 memory T cells. Considering the importance of both CD4 and CD8 T cells in the control of intracellular pathogens such as Leishmania donovani, we wanted to identify vaccines that could raise both CD4 and CD8 T cell responses and determine how to configure immunization strategies to generate the best combined protective T cell response. We examined responses generated against the Leishmania vaccine antigen F3 following its administration in either recombinant form with the Toll-like receptor 4 (TLR4) agonist-containing adjuvant formulation GLA-SE (F3+GLA-SE) or as a gene product delivered in an adenoviral vector (Ad5-F3). Homologous immunization strategies using only F3+GLA-SE or Ad5-F3 preferentially generated either CD4 or CD8 T cells, respectively. In contrast, heterologous strategies generated both antigen-specific CD4 and CD8 T cells. Administration of F3+GLA-SE before Ad5-F3 generated the greatest combined CD4 and CD8 responses. Cytotoxic CD8 T cell responses were highest when Th1 cells were generated prior to their induction by Ad5-F3. Finally, a single immunization with a combination of F3+GLA-SE mixed with Ad5-F3 was found to be sufficient to provide protection against experimental L. donovani infection. Taken together, our data delineate immunization regimens that induce antigen-specific CD4 and CD8 T cell memory responses, and identify a single immunization strategy that could be used to rapidly provide protection against intracellular pathogens in regions where access to health care is limited or sporadic. PMID:27466350

  20. A DNA vaccine encoding foot-and-mouth disease virus B and T-cell epitopes targeted to class II swine leukocyte antigens protects pigs against viral challenge.

    PubMed

    Borrego, Belén; Argilaguet, Jordi M; Pérez-Martín, Eva; Dominguez, Javier; Pérez-Filgueira, Mariano; Escribano, José M; Sobrino, Francisco; Rodriguez, Fernando

    2011-11-01

    Development of efficient and safer vaccines against foot-and-mouth disease virus (FMDV) is a must. Previous results obtained in our laboratory have demonstrated that DNA vaccines encoding B and T cell epitopes from type C FMDV, efficiently controlled virus replication in mice, while they did not protect against FMDV challenge in pigs, one of the FMDV natural hosts. The main finding of this work is the ability to improve the protection afforded in swine using a new DNA-vaccine prototype (pCMV-APCH1BTT), encoding FMDV B and T-cell epitopes fused to the single-chain variable fragment of the 1F12 mouse monoclonal antibody that recognizes Class-II Swine Leukocyte antigens. Half of the DNA-immunized pigs were fully protected upon viral challenge, while the remaining animals were partially protected, showing a delayed, shorter and milder disease than control pigs. Full protection in a given vaccinated-pig correlated with the induction of specific IFNγ-secreting T-cells, detectable prior to FMDV-challenge, together with a rapid development of neutralizing antibodies after viral challenge, pointing towards the relevance that both arms of the immune response can play in protection. Our results open new avenues for developing future FMDV subunit vaccines.

  1. Affinity Maturation of an Anti-V Antigen IgG Expressed In Situ Via Adenovirus Gene Delivery Confers Enhanced Protection Against Yersinia pestis Challenge

    PubMed Central

    Van Blarcom, Thomas J.; Sofer-Podesta, Carolina; Ang, John; Boyer, Julie L.; Crystal, Ronald G.; Georgiou, George

    2013-01-01

    Genetic transfer of neutralizing antibodies has been shown to confer strong and persistent protection against bacterial and viral infectious agents. While it is well established that for many exogenous neutralizing antibodies increased antigen affinity correlates with protection, the effect of antigen affinity on antibodies produced in situ following adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal antibody 2C12.4 recognizes the Yersinia pestis Type III secretion apparatus protein LcrV (V antigen) and confers protection in mice when administered as an IgG intraperitoneally or, following genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad) 1. 2C12.4 was expressed as a scFv fragment in E. coli and was shown to display a KD=3.5 nM by surface plasmon resonance (SPR) analysis. The 2C12.4 scFv was subjected to random mutagenesis and variants with increased affinity were isolated by flow cytometry using the Anchored Periplasmic Expression (APEx) bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower KD values (H8, KD=100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdαV giving rise to AdαV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen antibodies 3 days post-immunization with 109, 1010 or 1011 particle units. Following intranasal challenge with 363 LD50Y. pestis CO92, 54% of the mice immunized with 1010 pu of AdαV.H8 survived at the 14 day end point compared to only 15% survivors for the group immunized with AdαV expressing the lower affinity 2C12.4 (P<0.04, AdαV versus AdαV.H8). These results indicate that affinity maturation of a neutralizing antibody delivered by genetic transfer may confer increased protection not only for Y. pestis challenge but possibly for other pathogens. PMID:20393511

  2. Vaccination-induced protection of lambs against the parasitic nematode Haemonchus contortus correlates with high IgG antibody responses to the LDNF glycan antigen.

    PubMed

    Vervelde, Lonneke; Bakker, Nicole; Kooyman, Frans N J; Cornelissen, Albert W C A; Bank, Christine M C; Nyame, A Kwame; Cummings, Richard D; van Die, Irma

    2003-11-01

    Lambs respond to vaccination against bacteria and viruses but have a poor immunological response to nematodes. Here we report that they are protected against the parasitic nematode Haemonchus contortus after vaccination with excretory/secretory (ES) glycoproteins using Alhydrogel as an adjuvant. Lambs immunized with ES in Alhydrogel and challenged with 300 L3 larvae/kg body weight had a reduction in cumulative egg output of 89% and an increased percentage protection of 54% compared with the adjuvant control group. Compared to the adjuvant dimethyl dioctadecyl ammonium bromide, Alhydrogel induced earlier onset and significantly higher ES- specific IgG, IgA, and IgE antibody responses. In all vaccinated groups a substantial proportion of the antibody response was directed against glycan epitopes, irrespective of the adjuvant used. In lambs vaccinated with ES in Alhydrogel but not in any other group a significant increase was found in antibody levels against the GalNAcbeta1,4 (Fucalpha1,3)GlcNAc (fucosylated LacdiNAc, LDNF) antigen, a carbohydrate antigen that is also involved in the host defense against the human parasite Schistosoma mansoni. In lambs the LDNF-specific response increased from the first immunization onward and was significantly higher in protected lambs. In addition, an isotype switch from LDNF-specific IgM to IgG was induced that correlated with protection. These data demonstrate that hyporesponsiveness of lambs to H. contortus can be overcome by vaccination with ES glycoproteins in a strong T-helper 2 type response-inducing aluminum adjuvant. This combination generated high and specific antiglycan antibody responses that may contribute to the vaccination-induced protection.

  3. Immunization with the MipA, Skp, or ETEC_2479 Antigens Confers Protection against Enterotoxigenic E. coli Strains Expressing Different Colonization Factors in a Mouse Pulmonary Challenge Model

    PubMed Central

    Hays, Michael P.; Kumar, Amit; Martinez-Becerra, Francisco J.; Hardwidge, Philip R.

    2016-01-01

    Achieving cross-protective efficacy against multiple bacterial strains or serotypes is an important goal of vaccine design. Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease in underdeveloped nations. We have been interested in identifying and characterizing ETEC antigens that generate protective immune responses independent of ETEC colonization factor (CF) expression. Our previous studies used proteomics to identify the ETEC MipA, Skp, and ETEC_2479 proteins as effective in protecting mice from homologous challenge with ETEC H10407 using a pulmonary inoculation model. This model permits analysis of mouse survival, bacterial clearance, and the production of secretory IgA (sIgA) and has been employed previously for studies of enteric pathogens for which robust oral challenge models do not exist. MipA belongs to a family of proteins involved in remodeling peptidoglycan. Skp rescues misdirected outer membrane proteins. ETEC_2479 is predicted to function as an outer membrane porin. These proteins are conserved in pathogenic ETEC strains as well as in commensal Proteobacteria. Antibodies produced against the ETEC MipA, Skp, and ETEC_2479 proteins also reduced the adherence of multiple ETEC strains differing in CF type to intestinal epithelial cells. Here we characterized the ability of 10 heterologous ETEC strains that differ in CF type to cause clinical signs of illness in mice after pulmonary challenge. ETEC strains C350C1A, E24377A, E7476A, WS2173A, and PE360 caused variable degrees of lethality in this mouse model, while ETEC strains B7A, WS6866B, 2230, ARG-2, and 8786 did not. Subsequent challenge experiments in which mice were first vaccinated intranasally with MipA, Skp, or ETEC_2479, when combined with cholera toxin, showed both that each antigen was protective and that protection was strongly correlated with fecal IgA concentrations. We conclude that the MipA, Skp, or ETEC_2479 antigens generate protection in the mouse pulmonary

  4. Effect of emergency FMD vaccine antigen payload on protection, sub-clinical infection and persistence following direct contact challenge of cattle.

    PubMed

    Cox, S J; Voyce, C; Parida, S; Reid, S M; Hamblin, P A; Hutchings, G; Paton, D J; Barnett, P V

    2006-04-12

    Previous work, in sheep vaccinated with emergency foot-and-mouth disease (FMD) vaccine, indicated the benefit of increasing the antigen payload in inhibiting local virus replication and consequently persistence following an indirect aerosol challenge with a virus homologous to the vaccine strain. The work presented here investigates this possibility further using cattle and a more severe semi-heterologous direct contact challenge. The quantitative dynamics of virus replication and excretion in both vaccinated and non-vaccinated cattle following challenge are examined. Two experiments were carried out each involving 20 vaccinated and 5 non-vaccinated cattle. An O(1) Manisa vaccine (18 PD(50)) was used for the first, previously reported experiment [Cox SJ, Voyce C, Parida S, Reid SM, Hamblin PA, Paton DJ, et al. Protection against direct contact challenge following emergency FMD vaccination of cattle and the effect on virus excretion from the oropharynx. Vaccine 2005;23:1106-13]. The same vaccine was used for the second experiment described in this paper except the antigen payload was increased 10-fold per bovine dose, resulting in significantly higher FMD virus neutralising antibody titres prior to challenge. Twenty-one days post-vaccination the cattle received a 5-day direct contact challenge with FMD virus from five further non-vaccinated cattle infected 24h earlier with O UKG 34/2001. All vaccinated cattle regardless of antigen payload were protected against clinical disease. Sub-clinical oropharyngeal infection was detected in animals from both experiments but the level of virus replication shortly after direct contact challenge was significantly reduced in vaccinated animals. Cattle immunised with the 10-fold antigen payload cleared the virus more readily and consequently at 28 days post-challenge fewer animals were persistently infected compared to the single strength vaccine. Following a severe challenge, the results from both experiments show that use of

  5. Malaria protection in β2-microglobulin-deficient mice lacking major histocompatibility complex class I antigens: essential role of innate immunity, including γδT cells

    PubMed Central

    Taniguchi, Tomoyo; Tachikawa, Saoko; Kanda, Yasuhiro; Kawamura, Toshihiko; Tomiyama-Miyaji, Chikako; Li, Changchun; Watanabe, Hisami; Sekikawa, Hiroho; Abo, Toru

    2007-01-01

    It is still controversial whether malaria protection is mediated by conventional immunity associated with T and B cells or by innate immunity associated with extrathymic T cells and autoantibody-producing B cells. Given this situation, it is important to examine the mechanism of malaria protection in β2-microglobulin-deficient (β2m(–/–)) mice. These mice lack major histocompatibility complex class I and CD1d antigens, which results in the absence of CD8+ T cells and natural killer T (NKT) cells. When C57BL/6 and β2m(–/–) mice were injected with parasitized (Plasmodium yoelii 17XNL) erythrocytes, both survived from the infection and showed a similar level of parasitaemia. The major expanding T cells were NK1.1– αβΤ-cell receptorint cells in both mice. The difference was a compensatory expansion of NK and γδT cells in β2m(–/–) mice, and an elimination experiment showed that these lymphocytes were critical for protection in these mice. These results suggest that malaria protection might be events of the innate immunity associated with multiple subsets with autoreactivity. CD8+ T and NKT cells may be partially related to this protection. PMID:17916163

  6. A Novel Method of Safely Measuring Influenza Virus Aerosol Using Antigen-Capture Enzyme-Linked Immunosorbent Assay for the Performance Evaluation of Protective Clothing Materials.

    PubMed

    Shimasaki, Noriko; Nojima, Yasuhiro; Okaue, Akira; Takahashi, Hitoshi; Kageyama, Tsutomu; Hamamoto, Itsuki; Shinohara, Katsuaki

    2016-01-01

    Currently, threats caused by pathogens are serious public health problems worldwide. Protective clothing is essential when one is treating infected patients or dealing with unknown pathogens. Therefore, it is necessary to evaluate the performance of protective clothing against pathogens. In Japan, some methods for evaluating the performance of protective clothing have been established in the Japanese Industrial Standards (JIS). However, a test method against virus aerosols has not been established. Because there is a risk of infection from a live virus during the test, it is necessary to devise a safe method for the virus-aerosol-based test. Here, we propose a new method of safely measuring virus aerosols for the performance evaluation of protective clothing materials. To ensure safety, an inactivated virus was used. As a model virus, the influenza virus was selected owing to the proper small diameter of the virus particles. To quantitatively measure the particle-amount of the inactivated influenza virus, we developed an antigen-capture enzyme-linked immunosorbent assay (ELISA) targeting the M1 protein. Furthermore, we evaluated two materials using our method. Significant differences in the protection performance against the virus aerosol were observed between different sample materials, thereby confirming the applicability of our new method for performance evaluation.

  7. Regulated delayed synthesis of lipopolysaccharide and enterobacterial common antigen of Salmonella Typhimurium enhances immunogenicity and cross-protective efficacy against heterologous Salmonella challenge

    PubMed Central

    Huang, Chun; Liu, Qing; Luo, Yali; Li, Pei; Liu, Qiong; Kong, Qingke

    2016-01-01

    Lipopolysaccharide (LPS) O-antigen and enterobacterial common antigen (ECA) are two major polysaccharide structures on the surface of Salmonella enterica serovar Typhimurium. Previous studies have demonstrated that regulated truncation of LPS enhances the cross-reaction against conserved outer membrane proteins (OMPs) from enteric bacteria. We speculate that the regulation of both O-antigen and ECA may enhance the induction of immune responses against conserved OMPs from enteric bacteria. In this work we targeted rfbB and rffG genes which encode dTDP-glucose 4,6-dehydratases and share the same function in regulating O-antigen and ECA synthesis. We constructed a mutant, S496 (ΔrfbB6 ΔrffG7 ΔpagL73::TT araC PBAD rfbB-3), in which rfbB gene expression was dependent on exogenously supplied arabinose during in vitro growth and achieved the simultaneous tight regulation of both LPS and ECA synthesis, as demonstrated by the LPS profile and Western blotting using antisera against LPS and ECA. When administered orally, S. Typhimurium S496 was completely attenuated for virulence but still retained the capacity to colonize and disseminate in mice. In addition, we found that oral immunization with S496 resulted in increased immune responses against OMPs from enteric bacteria and enhanced survival compared with immunization with S492 possessing ΔrfbB6 ΔrffG8 mutations when challenged with lethal doses of Salmonella Choleraesuis or Salmonella Enteritidis. These results indicate that S. Typhimurium arabinose-regulated rfbB strain S496 is a good vaccine candidate, conferring cross-protection against lethal challenge with heterologous Salmonella. PMID:27423383

  8. Bicistronic DNA vaccines simultaneously encoding HIV, HSV and HPV antigens promote CD8⁺ T cell responses and protective immunity.

    PubMed

    Santana, Vinicius C; Diniz, Mariana O; Cariri, Francisco A M O; Ventura, Armando M; Cunha-Neto, Edécio; Almeida, Rafael R; Campos, Marco A; Lima, Graciela K; Ferreira, Luís C S

    2013-01-01

    Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⁺ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⁺ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.

  9. Etanercept prevents airway hyperresponsiveness by protecting neuronal M2 muscarinic receptors in antigen-challenged guinea pigs

    PubMed Central

    Nie, Zhenying; Jacoby, David B; Fryer, Allison D

    2009-01-01

    Background and purpose Increased tumour necrosis factor-α (TNF-α) is associated with airway hyperreactivity in antigen-challenged animals. In human asthmatics, TNF-α is increased and blocking it prevents airway hyperreactivity in some asthmatic patients. However, the mechanisms by which TNF-α mediates hyperreactivity are unknown. Airway hyperreactivity can be caused by dysfunction of neuronal M2 muscarinic receptors that normally limit acetylcholine release from parasympathetic nerves. Here we test whether blocking TNF-α receptors with etanercept prevents M2 receptor dysfunction and airway hyperreactivity in antigen-challenged guinea pigs. Experimental approach Ovalbumin-sensitized guinea pigs were challenged by inhalation of antigen. Some animals received etanercept (3 mg kg−1 i.p.) 3 h before challenge. 24 h after challenge, airway hyperreactivity and M2 receptor function were tested. Inflammatory cells in bronchoalveolar lavage, blood and lung were counted. TNF-α and its receptors were detected by real-time RT-PCR and immunocytochemistry in parasympathetic nerves from humans and guinea pigs and in human neuroblastoma cells. Key results Antigen-challenged animals were hyperreactive to vagal stimulation and neuronal M2 receptors were dysfunctional. Both M2 receptor dysfunction and airway hyperreactivity were prevented by etanercept. Etanercept reduced eosinophils around airway nerves, and in blood, bronchoalveolar lavage and airway smooth muscle. Also, TNF-α decreased M2 receptor mRNA in human and guinea pig parasympathetic neurons. Conclusions and implications Tumour necrosis factor-α may contribute to M2 receptor dysfunction and airway hyperreactivity directly by decreasing receptor expression and indirectly by promoting recruitment of eosinophils, containing major basic protein, an M2 antagonist. This suggests that etanercept may be beneficial in treatment of allergic asthma. PMID:19134001

  10. Reflectance measurement using digital camera and a protecting dome with built in light source.

    PubMed

    Välisuo, Petri; Harju, Toni; Alander, Jarmo

    2011-08-01

    The reflectance of the skin reveals the chemical and physical changes of the skin as well as many metabolic changes. The reflectance measurement is an important method for medical diagnosis, follow-up and screening. This article concentrates on designing and validating an imaging system, based on a digital camera. The proposed system can measure the reflectance of the skin with high spatial and currently four channel spectral resolution, in the range of 450 nm to 980 nm. The accuracy of the system is determined by imaging a colour checker board and comparing the obtained values with both given values and spectrometer measurements. The diffuse interreflections of both, the integrating sphere and the lighting dome of the imaging system, is compensated with a correction factor. The accuracy of the proposed system is only slightly weaker than the spectrometer. The imaging system characteristics are independent of the camera characteristics.

  11. Antigenicity and Protective Efficacy of a Leishmania Amastigote-specific Protein, Member of the Super-oxygenase Family, against Visceral Leishmaniasis

    PubMed Central

    Martins, Vivian T.; Chávez-Fumagalli, Miguel A.; Costa, Lourena E.; Martins, Adriana M. C. C.; Lage, Paula S.; Lage, Daniela P.; Duarte, Mariana C.; Valadares, Diogo G.; Magalhães, Rubens D. M.; Ribeiro, Tatiana G.; Nagem, Ronaldo A. P.; DaRocha, Wanderson D.; Régis, Wiliam C. B.

    2013-01-01

    Background The present study aimed to evaluate a hypothetical Leishmania amastigote-specific protein (LiHyp1), previously identified by an immunoproteomic approach performed in Leishmania infantum, which showed homology to the super-oxygenase gene family, attempting to select a new candidate antigen for specific serodiagnosis, as well as to compose a vaccine against VL. Methodology/Principal Findings The LiHyp1 DNA sequence was cloned; the recombinant protein (rLiHyp1) was purified and evaluated for its antigenicity and immunogenicity. The rLiHyp1 protein was recognized by antibodies from sera of asymptomatic and symptomatic animals with canine visceral leishmaniasis (CVL), but presented no cross-reactivity with sera of dogs vaccinated with Leish-Tec, a Brazilian commercial vaccine; with Chagas' disease or healthy animals. In addition, the immunogenicity and protective efficacy of rLiHyp1 plus saponin was evaluated in BALB/c mice challenged subcutaneously with virulent L. infantum promastigotes. rLiHyp1 plus saponin vaccinated mice showed a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with the recombinant protein. Immunized and infected mice, as compared to the control groups (saline and saponin), showed significant reductions in the number of parasites found in the liver, spleen, bone marrow, and in the paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, produced mainly by CD4 T cells. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 response could also be observed. Conclusions/Significance The present study showed that this Leishmania oxygenase amastigote-specific protein can be used for a more sensitive and specific serodiagnosis of asymptomatic and symptomatic CVL and, when combined with a Th1-type adjuvant, can also be employ as a candidate antigen to develop vaccines against VL. PMID:23573301

  12. Antigenicity and protective efficacy of a Leishmania amastigote-specific protein, member of the super-oxygenase family, against visceral leishmaniasis.

    PubMed

    Martins, Vivian T; Chávez-Fumagalli, Miguel A; Costa, Lourena E; Canavaci, Adriana M C; Martins, Adriana M C C; Lage, Paula S; Lage, Daniela P; Duarte, Mariana C; Valadares, Diogo G; Magalhães, Rubens D M; Ribeiro, Tatiana G; Nagem, Ronaldo A P; Darocha, Wanderson D; Régis, Wiliam C B; Soto, Manuel; Coelho, Eduardo A F; Fernandes, Ana Paula; Tavares, Carlos A P

    2013-01-01

    The present study aimed to evaluate a hypothetical Leishmania amastigote-specific protein (LiHyp1), previously identified by an immunoproteomic approach performed in Leishmania infantum, which showed homology to the super-oxygenase gene family, attempting to select a new candidate antigen for specific serodiagnosis, as well as to compose a vaccine against VL. The LiHyp1 DNA sequence was cloned; the recombinant protein (rLiHyp1) was purified and evaluated for its antigenicity and immunogenicity. The rLiHyp1 protein was recognized by antibodies from sera of asymptomatic and symptomatic animals with canine visceral leishmaniasis (CVL), but presented no cross-reactivity with sera of dogs vaccinated with Leish-Tec, a Brazilian commercial vaccine; with Chagas' disease or healthy animals. In addition, the immunogenicity and protective efficacy of rLiHyp1 plus saponin was evaluated in BALB/c mice challenged subcutaneously with virulent L. infantum promastigotes. rLiHyp1 plus saponin vaccinated mice showed a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with the recombinant protein. Immunized and infected mice, as compared to the control groups (saline and saponin), showed significant reductions in the number of parasites found in the liver, spleen, bone marrow, and in the paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, produced mainly by CD4 T cells. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 response could also be observed. The present study showed that this Leishmania oxygenase amastigote-specific protein can be used for a more sensitive and specific serodiagnosis of asymptomatic and symptomatic CVL and, when combined with a Th1-type adjuvant, can also be employ as a candidate antigen to develop vaccines against VL.

  13. Towards the development of an oral vaccine against porcine cysticercosis: expression of the protective HP6/TSOL18 antigen in transgenic carrots cells.

    PubMed

    Monreal-Escalante, Elizabeth; Govea-Alonso, Dania O; Hernández, Marisela; Cervantes, Jacquelynne; Salazar-González, Jorge A; Romero-Maldonado, Andrea; Rosas, Gabriela; Garate, Teresa; Fragoso, Gladis; Sciutto, Edda; Rosales-Mendoza, Sergio

    2016-03-01

    The Taenia solium HP6/TSOL18 antigen was produced in carrot cells, yielding an immunogenic protein that induced significant protection in an experimental murine model against T. crassiceps cysticercosis when orally administered. This result supports the potential of HP6/TSOL18-carrot as a low-cost anti-cysticercosis vaccine candidate. Cysticercosis is a zoonosis caused by Taenia solium that can be prevented by interrupting the parasite life cycle through pig vaccination. Several injectable vaccine candidates have been reported, but the logistic difficulties and costs for its application limited its use in nationwide control programs. Oral plant-based vaccines can deal with this limitation, because of their easy administration and low cost. A stable expression of the HP6/TSOL18 anti-T. solium cysticercosis protective antigen in carrot calli transformed with an optimized transgene is herein reported. An antigen accumulation up to 14 µg g(-1) of dry-weight biomass was achieved in the generated carrot lines. Mouse immunization with one of the transformed calli induced both specific IgG and IgA anti-HP6/TSOL18 antibodies. A statistically significant reduction in the expected number of T. crassiceps cysticerci was observed in mice orally immunized with carrot-made HP6/TSOL18, in a similar extent to that obtained by subcutaneous immunization with recombinant HP6/TSOL18 protein. In this study, a new oral plant-made version of the HP6/TSOL18 anti-cysticercosis vaccine is reported. The vaccine candidate should be further tested against porcine cysticercosis.

  14. Induction of protection against leishmaniasis in susceptible BALB/c mice using simple DOTAP cationic nanoliposomes containing soluble Leishmania antigen (SLA).

    PubMed

    Firouzmand, Hengameh; Badiee, Ali; Khamesipour, Ali; Heravi Shargh, Vahid; Alavizadeh, Seyedeh Hoda; Abbasi, Azam; Jaafari, Mahmoud Reza

    2013-12-01

    A suitable adjuvant and delivery system are needed to develop an effective vaccine against leishmaniasis. To induce a Th1 type of response and protection in BALB/c mice against Leishmania major infection, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) nanoliposomes bearing an intrinsic adjuvanticity, were used as an antigen delivery system and immunoadjuvant for soluble Leishmania antigens (SLA). DOTAP liposomes containing different concentrations of SLA were prepared by using lipid film method followed by sonication. The prepared vesicles showed a diameter of about 100nm, a positive zeta potential and approximately 70% encapsulation efficiency of SLA. BALB/c mice were immunized subcutaneously (SC), three times in a 3-week interval with different concentrations of liposomal SLA (12.5, 25, and 50μg of SLA/50μl/mice), free SLA and as well as free liposome. The group of mice received 50μg of SLA in DOTAP-nanoliposomes showed a significantly (p<0.001) smaller footpad swelling and the lowest spleen and footpad parasite burden after the challenge. This group also showed the highest IFN-γ production compared to the other groups, lower IL-4 level and higher IgG2a antibody titer. Taken together, the results indicated that simple DOTAP nanoliposome containing 1μg/μl SLA are appropriate delivery systems to induce a Th1 type of immune response and protection against L. major infection in BALB/c mice.

  15. Novel 6xHis tagged foot-and-mouth disease virus vaccine bound to nanolipoprotein adjuvant via metal ions provides antigenic distinction and effective protective immunity

    SciTech Connect

    Rai, Devendra K.; Segundo, Fayna Diaz-San; Schafer, Elizabeth; Burrage, Thomas G.; Rodriguez, Luis L.; Santos, Teresa de los; Hoeprich, Paul D.; Rieder, Elizabeth

    2016-08-15

    Here, we engineered two FMD viruses with histidine residues inserted into or fused to the FMDV capsid. Both 6xHis viruses exhibited growth kinetics, plaque morphologies and antigenic characteristics similar to wild-type virus. The 6xHis tag allowed one-step purification of the mutant virions by Co{sup 2+} affinity columns. Electron microscopy and biochemical assays showed that the 6xHis FMDVs readily assembled into antigen: adjuvant complexes in solution, by conjugating with Ni{sup 2+}-chelated nanolipoprotein and monophosphoryl lipid A adjuvant (MPLA:NiNLP). Animals Immunized with the inactivated 6xHis-FMDV:MPLA:NiNLP vaccine acquired enhanced protective immunity against FMDV challenge compared to virions alone. Induction of anti-6xHis and anti-FMDV neutralizing antibodies in the immunized animals could be exploited in the differentiation of vaccinated from infected animals needed for the improvement of FMD control measures. The novel marker vaccine/nanolipid technology described here has broad applications for the development of distinctive and effective immune responses to other pathogens of importance. - Highlights: • 6xHis-tags in A{sub 24} FMDV enable purification and biding to adjuvants via metal ions. • 6xHis A{sub 24} FMDV:MPLA:NiNLP vaccine enhanced protective immunity against FMDV. • Surface exposed capsid tags allow distinction of infected from vaccinated animals.

  16. Use of flagellin and cholera toxin as adjuvants in intranasal vaccination of mice to enhance protective immune responses against uropathogenic Escherichia coli antigens.

    PubMed

    Asadi Karam, Mohammad Reza; Habibi, Mehri; Bouzari, Saeid

    2016-09-01

    Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most common infections in human. Antibiotics are common therapy for UTIs, but increase in antibiotic resistance will complicate future treatment of the infections, making the development of an efficacious UTI vaccine more urgent. In this study, we have evaluated intranasally the efficacy of FliC and FimH antigens of UPEC in different vaccine formulations with and without cholera toxin (CT) adjuvant. Immunization of mice with FliC in fusion form or admixed with FimH elicited higher levels of serum, mucosal and cell-mediated responses than FimH alone. Furthermore, the use of CT in synergism with FliC resulted in the stimulation of a mixed Th1 and Th2 responses against FimH and FliC as antigen and maintained the antibody responses for at least 24 weeks following the last vaccine dose. Of the vaccine preparations, Fusion, Fusion + CT, and FimH admixed with FliC and CT showed the best protection against UPEC. These data indicated that intranasal administration of a FliC and CT adjuvant-based vaccine has the potential to provide protective responses against UPEC strains.

  17. Protection against murine intestinal amoebiasis induced by oral immunization with the 29 kDa antigen of Entamoeba histolytica and cholera toxin.

    PubMed

    Carrero, J C; Contreras-Rojas, A; Sánchez-Hernández, B; Petrosyan, P; Bobes, R J; Ortiz-Ortiz, L; Laclette, J P

    2010-11-01

    Entamoeba histolytica antigens recognized by salivary IgA from infected patients include the 29 kDa antigen (Eh29), an alkyl hydroperoxide reductase. Here, we investigate the potential of recombinant Eh29 and an Eh29-cholera toxin subunit B (CTxB) fusion protein to confer protection against intestinal amoebiasis after oral immunization. The purified Eh29-CTxB fusion retained the critical ability to bind ganglioside GM(1), as determined by ELISA. Oral immunization of C3H/HeJ mice with Eh29 administered in combination with a subclinical dose of whole cholera toxin, but not as an Eh29-CTxB fusion, induced elevated levels of intestinal IgA and serum IgG anti-Eh29 antibodies that inhibited trophozoites adherence to MDCK cell monolayers. The 80% of immunized mice seen to develop IgA and IgG immune responses showed no evidence of infection in tissue sections harvested following intracecal challenge with virulent E. histolytica trophozoites. These results suggest that Eh29 is capable of inducing protective anti-amoebic immune responses in mice following oral immunization and could be used in the development of oral vaccines against amoebiasis. (c) 2010 Elsevier Inc. All rights reserved.

  18. Hepatitis B virus core antigen as a carrier for Chlamydia trachomatis MOMP multi-epitope peptide enhances protection against genital chlamydial infection.

    PubMed

    Jiang, Pengfei; Du, Wangqi; Xiong, Yirong; Lv, Yan; Feng, Juan; Zhu, Shanli; Xue, Xiangyang; Chen, Shao; Zhang, Lifang

    2015-12-22

    Chlamydia trachomatis (Ct) is the leading cause of sexually transmitted diseases worldwide. There is no safe and effective vaccine to control the spread of Ct. In development of Ct vaccine, selection of appropriate candidate antigens and an effective delivery system may be the main challenges. Multi-epitope of major outer membrane protein (MOMPm) is the most suitable candidate for a Ct vaccine, while hepatitis B virus core antigen (HBcAg) has unique advantages as vaccine delivery system. Therefore, in this study, we evaluated the immunogenicity and protective immune response of a novel candidate vaccine in a murine model of chlamydial genital infection. This candidate vaccine comprises MOMPm peptide delivered with HBcAg. Our results of Ct-specific serum IgG and secretory IgA assay, cytokine assay, and cytotoxic T-lymphocyte assay revealed that immunogenicity of the candidate vaccine was much better than that of the corresponding synthetic MOMPm peptide. Furthermore, the protective effect of the candidate vaccine was also shown much better than that of the synthetic peptide by calculating the isolation of Chlamydia from vaginal swabs and histopathological analysis. Taken together, our results indicate that HBcAg carrying Ct MOMPm could be an effective immune prophylactic for chlamydial infection.

  19. Signaling when (and when not) to be cautious and self-protective: impulsive and reflective trust in close relationships.

    PubMed

    Murray, Sandra L; Pinkus, Rebecca T; Holmes, John G; Harris, Brianna; Gomillion, Sarah; Aloni, Maya; Derrick, Jaye L; Leder, Sadie

    2011-09-01

    A dual process model is proposed to explain how automatic evaluative associations to the partner (i.e., impulsive trust) and deliberative expectations of partner caring (i.e., reflective trust) interact to govern self-protection in romantic relationships. Experimental and correlational studies of dating and marital relationships supported the model. Subliminally conditioning more positive evaluative associations to the partner increased confidence in the partner's caring, suggesting that trust has an impulsive basis. Being high on impulsive trust (i.e., more positive evaluative associations to the partner on the Implicit Association Test; Zayas & Shoda, 2005) also reduced the automatic inclination to distance in response to doubts about the partner's trustworthiness. It similarly reduced self-protective behavioral reactions to these reflective trust concerns. The studies further revealed that the effects of impulsive trust depend on working memory capacity: Being high on impulsive trust inoculated against reflective trust concerns for people low on working memory capacity. PsycINFO Database Record (c) 2011 APA, all rights reserved.

  20. Protection of rabbits against challenge with rabbit papillomaviruses by immunization with the N terminus of human papillomavirus type 16 minor capsid antigen L2.

    PubMed

    Gambhira, Ratish; Jagu, Subhashini; Karanam, Balasubramanyam; Gravitt, Patti E; Culp, Timothy D; Christensen, Neil D; Roden, Richard B S

    2007-11-01

    Current L1 virus-like particle (VLP) vaccines provide type-restricted protection against a small subset of the human papillomavirus (HPV) genotypes associated with cervical cancer, necessitating continued cytologic screening of vaccinees. Cervical cancer is most problematic in countries that lack the resources for screening or highly multivalent HPV VLP vaccines, suggesting the need for a low-cost, broadly protective vaccinogen. Here, N-terminal L2 polypeptides comprising residues 1 to 88 or 11 to 200 derived from HPV16, bovine papillomavirus type 1 (BPV1), or cottontail rabbit papillomavirus (CRPV) were produced in bacteria. Rabbits were immunized with these N-terminal L2 polypeptides and concurrently challenged with CRPV and rabbit oral papillomavirus (ROPV). Vaccination with either N-terminal L2 polypeptides of CRPV effectively protected rabbits from CRPV challenge but not from papillomas induced by cutaneous challenge with CRPV genomic DNA. Furthermore, papillomas induced by CRPV genomic DNA deficient for L2 expression grew at the same rate as those induced by wild-type CRPV genomic DNA, further suggesting that the L2 polypeptide vaccines lack therapeutic activity. Neutralizing serum antibody titers of >15 correlated with protection (P < 0.001), a finding consistent with neutralizing antibody-mediated protection. Surprisingly, a remarkable degree of protection against heterologous papillomavirus types was observed after vaccination with N-terminal L2 polypeptides. Notably, vaccination with HPV16 L2 11-200 protected against cutaneous and mucosal challenge with CRPV and ROPV, respectively, papillomaviruses that are evolutionarily divergent from HPV16. Further, vaccination with HPV16 L2 11-200 generates broadly cross-neutralizing serum antibody, suggesting the potential of L2 as a second-generation preventive HPV vaccine antigen.

  1. Eimeria maxima recombinant Gam82 gametocyte antigen vaccine protects against coccidiosis and augments humoral and cell-mediated immunity

    USDA-ARS?s Scientific Manuscript database

    Intestinal infection with Eimeria, the etiologic agent of avian coccidiosis, stimulates protective immunity to subsequent colonization by the homologous parasite, whilst cross-protection against heterologous species is poor. As a first step toward the development of a broad specificity Eimeria vacci...

  2. Protection Against Aerosolized Yersinia pestis Challenge Following Homologous and Heterologous Prime-Boost With Recombinant Plague Antigens

    DTIC Science & Technology

    2005-08-01

    INFECTION AND IMMUNITY , Aug. 2005, p. 5256–5261 Vol. 73, No. 8 0019-9567/05/$08.000 doi:10.1128/IAI.73.8.5256–5261.2005 Copyright © 2005, American...and heterologous prime-boost with recombinant plague antigens, Infection and Immunity 73:5256 - 5261 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM... infection following aerosol challenge with Y. pestis with a median survival time (MST) of 72 h. By con- trast, 9/10 positive-control animals immunized

  3. Vaccination of sheep against haemonchosis with H11, a gut membrane-derived protective antigen from the adult parasite: prevention of the periparturient rise and colostral transfer of protective immunity.

    PubMed

    Andrews, S J; Hole, N J; Munn, E A; Rolph, T P

    1995-07-01

    Pregnant ewes were immunised with a fraction highly enriched in the membrane glycoprotein antigen H11, isolated from the intestinal brush border of adult Haemonchus contortus. Immunity induced by immunisation was able to abolish almost completely (98-99%) the worm egg output from pregnant ewes challenged with ca. 10,000 infective larvae of H. contortus during the last trimester. Furthermore, lambs born and reared on vaccinated ewes had substantial antibody levels to H11 derived from maternal transfer. This antibody conferred moderate protection against a bolus challenge of ca. 3000 infective larvae of H. contortus in 5-week-old lambs.

  4. FUV Reflectance of Recently Prepared Al Protected with AlF3: COR Program Technology Development

    NASA Technical Reports Server (NTRS)

    Quijada, Manuel

    2016-01-01

    Astronomical observations in the Far Ultraviolet (FUV) spectral region are some of the more challenging due to the very distant and faint objects that are typically searched for in cosmic origin studies such as origin of large scale structure, the formation, evolution, and age of galaxies and the origin of stellar and planetary systems. These challenges are driving the need to improve the performance of optical coatings over a wide spectral range that would increase reflectance in mirrors and reduced absorption in dielectric filters used in optical telescope for FUV observations. This paper will present recent advances in reflectance performance for Al+AlF3 mirrors optimized for Lyman-alpha wavelength by performing the deposition of the AlF3 overcoat at elevated substrate temperatures.

  5. A Certified Health Physicist’s Reflections on a 40-Year Career in Radiation Protection

    PubMed Central

    2016-01-01

    This is a reflection from a certified health physicist regarding his becoming aware, during his 40-year career, that the linear no-threshold (LNT) model and the associated As Low As Reasonably Achievable concept have no scientific basis and make no positive contribution to radiation safety. They should be replaced by an alternative, scientifically based model that includes a threshold, below which there is no harm, and recognition of hormesis and the adaptive response, which reflect the benefits of low-dose and low-dose-rate radiation exposure. Continued use of the unscientific LNT model is not conservative, as most regulators complacently claim but actually harmful. Examples of these harmful impacts in the areas of nuclear power, nuclear medicine, and environmental management are included. PMID:27867322

  6. A Certified Health Physicist's Reflections on a 40-Year Career in Radiation Protection.

    PubMed

    Miller, Mark L

    2016-01-01

    This is a reflection from a certified health physicist regarding his becoming aware, during his 40-year career, that the linear no-threshold (LNT) model and the associated As Low As Reasonably Achievable concept have no scientific basis and make no positive contribution to radiation safety. They should be replaced by an alternative, scientifically based model that includes a threshold, below which there is no harm, and recognition of hormesis and the adaptive response, which reflect the benefits of low-dose and low-dose-rate radiation exposure. Continued use of the unscientific LNT model is not conservative, as most regulators complacently claim but actually harmful. Examples of these harmful impacts in the areas of nuclear power, nuclear medicine, and environmental management are included.

  7. Thin film transistors on plastic substrates with reflective coatings for radiation protection

    DOEpatents

    Wolfe, Jesse D.; Theiss, Steven D.; Carey, Paul G.; Smith, Patrick M.; Wickboldt, Paul

    2003-11-04

    Fabrication of silicon thin film transistors (TFT) on low-temperature plastic substrates using a reflective coating so that inexpensive plastic substrates may be used in place of standard glass, quartz, and silicon wafer-based substrates. The TFT can be used in large area low cost electronics, such as flat panel displays and portable electronics such as video cameras, personal digital assistants, and cell phones.

  8. Thin film transistors on plastic substrates with reflective coatings for radiation protection

    DOEpatents

    Wolfe, Jesse D.; Theiss, Steven D.; Carey, Paul G.; Smith, Patrick M.; Wickbold, Paul

    2006-09-26

    Fabrication of silicon thin film transistors (TFT) on low-temperature plastic substrates using a reflective coating so that inexpensive plastic substrates may be used in place of standard glass, quartz, and silicon wafer-based substrates. The TFT can be used in large area low cost electronics, such as flat panel displays and portable electronics such as video cameras, personal digital assistants, and cell phones.

  9. Human leukocyte antigen (HLA)-B, DRB1, and DQB1 allotypes associated with disease and protection of trachoma endemic villagers.

    PubMed

    Abbas, Muneer; Bobo, Linda D; Hsieh, Yu-Hsiang; Berka, Noureddine; Dunston, Georgia; Bonney, George E; Apprey, Victor; Quinn, Thomas C; West, Sheila K

    2009-04-01

    Trachoma remains the leading preventable infectious cause of blindness in developing countries. Human leukocyte antigen (HLA) associations with ocular disease severity and persistent Chlamydia trachomatis infection of Tanzanians living in trachoma-endemic villages were examined to determine possible protective candidate allotypes for vaccine development. Buccal swab scrapes were taken from subjects in the Trichiasis Study Group (TSG), which studied females only, and the Family Trachoma Study (FTS), which compared persistently infected probands who had severe disease with disease-free siblings and parents. DNA was purified for polymerase chain reaction sequence-specific oligonucleotide identification of HLA-DRB1, DQB1, and B allotypes. Infection was detected from conjunctival scrapes using a C. trachomatis-specific PCR-enzyme immunoassay for the MOMP-1 gene. In the TSG, DR*B11 (odds ratio [OR], 0.48; 95% confidence interval [CI], 0.26-0.90; P=0.02) was significantly associated with lack of trichiasis, whereas HLA-B*07 (OR, 3.26; 95% CI, 1.42-7.49; P=0.004) and HLA-B*08 (OR, 5.12; 95% CI, 1.74-15.05; P=0.001) were associated with trichiasis. In addition, HLA-B*14 was significantly associated with inflammatory trachoma + follicular trachoma (OR, 3.76; 95% CI, 1.70-8.33; P=0.04). There were no significant allele frequencies for the FTS. The data suggest that HLA-DRB*11 may offer protection from trichiasis in trachoma hyperendemic villages. Complete allotype identification and designation of its respective protective CD4(+) T-cell antigens could provide a testable candidate vaccine for blindness prevention. Additionally, buccal swab DNA was sufficiently stable when acquired under harsh field conditions and stored long term in the freezer for low-resolution HLA typing.

  10. Protective Effect of Human Leukocyte Antigen B27 in Hepatitis C Virus Infection Requires the Presence of a Genotype-Specific Immunodominant CD8+ T-Cell Epitope

    PubMed Central

    Kersting, Nadine; Fitzmaurice, Karen; Oniangue-Ndza, Cesar; Kemper, Michael N.; Humphreys, Isla; McKiernan, Susan; Kelleher, Dermot; Lohmann, Volker; Bowness, Paul; Huzly, Daniela; Rosen, Hugo R.; Kim, Arthur Y.; Lauer, Georg M.; Allen, Todd M.; Barnes, Eleanor; Roggendorf, Michael; Blum, Hubert E.; Thimme, Robert

    2015-01-01

    Human leukocyte antigen B27 (HLA-B27) is associated with protection in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. This protective role is linked to single immunodominant HLA-B27-restricted CD8+ T-cell epitopes in both infections. In order to define the relative contribution of a specific HLA-B27-restricted epitope to the natural course of HCV infection, we compared the biological impact of the highly conserved HCV genotype 1 epitope, for which the protective role has been described, with the corresponding region in genotype 3 that differs in its sequence by three amino acid residues. The genotype 3a peptide was not recognized by CD8+ T cells specific for the genotype 1 peptide. Furthermore, patients with acute or chronic infection with HCV genotype 3a did not mount T-cell responses to this epitope region, and their autologous viral sequences showed no evidence of T-cell pressure. Finally, we found a significantly higher frequency of HLA-B27 positivity in patients with chronic HCV genotype 3a infection compared to genotype 1 infection, indicating that there is no protection by HLA-B27 in HCV genotype 3 infection. Conclusion Our data indicate that the protective effect of HLA-B27 is limited to HCV genotype 1 infection and does not expand to other genotypes such as genotype 3a. This can most likely be explained by intergenotype sequence diversity leading to the loss of the immunodominant HLA-B27 epitope in viral strains other than genotype 1. Our results underline the central role of a single HLA-B27-restricted epitope-specific CD8+ T-cell response in mediating protection in HCV genotype 1 infection. PMID:20034048

  11. A recombinant 63-kDa form of Bacillus anthracis protective antigen produced in the yeast Saccharomyces cerevisiae provides protection in rabbit and primate inhalational challenge models of anthrax infection.

    PubMed

    Hepler, Robert W; Kelly, Rosemarie; McNeely, Tessie B; Fan, Hongxia; Losada, Maria C; George, Hugh A; Woods, Andrea; Cope, Leslie D; Bansal, Alka; Cook, James C; Zang, Gina; Cohen, Steven L; Wei, Xiaorong; Keller, Paul M; Leffel, Elizabeth; Joyce, Joseph G; Pitt, Louise; Schultz, Loren D; Jansen, Kathrin U; Kurtz, Myra

    2006-03-06

    Infection by Bacillus anthracis is preventable by prophylactic vaccination with several naturally derived and recombinant vaccine preparations. Existing data suggests that protection is mediated by antibodies directed against the protective antigen (PA) component of the anthrax toxin complex. PA is an 83-kDa protein cleaved in vivo to yield a biologically active 63-kDa protein. In an effort to evaluate the potential of yeast as an expression system for the production of recombinant PA, and to determine if the yeast-purified rPA63 can protect from a lethal inhalational challenge, the sequence of the 63-kDa form of PA was codon-optimized and expressed in the yeast Saccharomyces cerevisiae. Highly purified rPA63 isolated from Saccharomyces under denaturing conditions demonstrated reduced biological activity in a macrophage-killing assay compared to non-denatured rPA83 purified from Escherichia coli. Rabbits and non-human primates (NHP) immunized with rPA63 and later challenged with a lethal dose of B. anthracis spores were generally protected from infection. These results indicate that epitopes present in the 63-kDa from of PA can protect rabbits and non-human primates from a lethal spore challenge, and further suggest that a fully functional rPA63 is not required in order to provide these epitopes.

  12. Immunostimulatory complexes containing Eimeria tenella antigens and low toxicity plant saponins induce antibody response and provide protection from challenge in broiler chickens

    USDA-ARS?s Scientific Manuscript database

    Immunostimulating complexes (ISCOMs) are unique multimolecular structures formed by encapsulating antigens, lipids and triterpene saponins and are one of the most successful antigen delivery systems for microbial antigens. In the current study, both the route of administration and the antigen conce...

  13. Natural antibody response to Plasmodium falciparum merozoite antigens MSP5, MSP9 and EBA175 is associated to clinical protection in the Brazilian Amazon

    PubMed Central

    2013-01-01

    Background Antibodies have an essential role in the acquired immune response against blood stage P. falciparum infection. Although several antigens have been identified as important antibody targets, it is still elusive which antigens have to be recognized for clinical protection. Herein, we analyzed antibodies from plasmas from symptomatic or asymptomatic individuals living in the same geographic area in the Western Amazon, measuring their recognition of multiple merozoite antigens. Methods Specific fragments of genes encoding merozoite proteins AMA1 and members of MSP and EBL families from circulating P. falciparum field isolates present in asymptomatic and symptomatic patients were amplified by PCR. After cloning and expression of different versions of the antigens as recombinant GST-fusion peptides, we tested the reactivity of patients’ plasmas by ELISA and the presence of IgG subclasses in the most reactive plasmas. Results 11 out of 24 recombinant antigens were recognized by plasmas from either symptomatic or asymptomatic infections. Antibodies to MSP9 (X2DF=1 = 9.26/p = 0.0047) and MSP5 (X2DF=1 = 8.29/p = 0.0069) were more prevalent in asymptomatic individuals whereas the opposite was observed for MSP1 block 2-MAD20 (X2DF=1 = 6.41/p = 0.0206, Fisher’s exact test). Plasmas from asymptomatic individuals reacted more intensely against MSP4 (U = 210.5, p < 0.03), MSP5 (U = 212, p < 0.004), MSP9 (U = 189.5, p < 0.002) and EBA175 (U = 197, p < 0.014, Mann-Whitney’s U test). IgG1 and IgG3 were predominant for all antigens, but some patients also presented with IgG2 and IgG4. The recognition of MSP5 (OR = 0.112, IC95% = 0.021-0.585) and MSP9 (OR = 0.125, IC95% = 0.030-0.529, cross tab analysis) predicted 8.9 and 8 times less chances, respectively, to present symptoms. Higher antibody levels against MSP5 and EBA175 were associated by odds ratios of 9.4 (IC95% = 1.29-69.25) and 5.7 (IC95

  14. Cloning, expression and purification of binding domains of lethal factor and protective antigen of Bacillus anthracis in Escherichia coli and evaluation of their related murine antibody.

    PubMed

    Rezaee, Mehdi; Honari, Hossein; Kooshk, Mohammad Reza Ashrafi

    2014-01-01

    Anthrax is common disease between human and animals caused by Bacillus anthracis. The cell binding domain of protective antigen (PAD4) and the binding domain of lethal factor (LFD1) have high immunogenicity potential and always were considered as a vaccine candidate against anthrax. The aims of this study are cloning and expressing of PAD4 and LFD1 in Escherichia coli, purification of the recombinant proteins and determination of their immunogenicity through evaluating of the relative produced polyclonal antibodies in mice. PAD4 and LFD1 genes were cloned in pET28a(+) vector and expressed in E. coli Bl21(DE3)PlysS. Expression and purification of the two recombinant proteins were confirmed by SDS-PAGE and Western blotting techniques. The PAD4 and LFD1 were purified using Ni(+)-NTA affinity chromatography (95-98 %), yielding 37.5 and 45 mg/l of culture, respectively. The antigens were injected three times into mice and production of relative antibodies was evaluated by ELISA test. The results showed that both PAD4 and LFD1 are immunogenic, but LFD1 has higher potential to stimulate Murine immune system. With regard to the high level of LFD1 and PAD4 expression and also significant increment in produced polyclonal antibodies, these recombinant proteins can be considered as a recombinant vaccine candidate against anthrax.

  15. The Leishmania HSP20 Is Antigenic during Natural Infections, but, as DNA Vaccine, It does not Protect BALB/c Mice against Experimental L. amazonensis Infection

    PubMed Central

    Montalvo-Álvarez, Ana M.; Folgueira, Cristina; Carrión, Javier; Monzote-Fidalgo, Lianet; Cañavate, Carmen; Requena, Jose M.

    2008-01-01

    Protozoa of the genus Leishmania are causative agents of leishmaniasis, an important health problem in both human and veterinary medicine. Here, we describe a new heat shock protein (HSP) in Leishmania, belonging to the small HSP (sHSP) family in kinetoplastids. The protein is highly conserved in different Leishmania species, showing instead significant divergence with sHSP's from other organisms. The humoral response elicited against this protein during Leishmania infection has been investigated in natural infected humans and dogs, and in experimentally infected hamsters. Leishmania HSP20 is a prominent antigen for canine hosts; on the contrary, the protein seems to be a poor antigen for human immune system. Time-course analysis of appearance of anti-HSP20 antibodies in golden hamsters indicated that these antibodies are produced at late stages of the infection, when clinical symptoms of disease are patent. Finally, the protective efficacy of HSP20 was assessed in mice using a DNA vaccine approach prior to challenge with Leishmania amazonensis. PMID:18401455

  16. Induction of protective immunity in chickens immunized with plant-made chimeric Bamboo mosaic virus particles expressing very virulent Infectious bursal disease virus antigen.

    PubMed

    Chen, Tsung-Hsien; Chen, Ten-Hong; Hu, Chung-Chi; Liao, Jia-Teh; Lee, Chin-Wei; Liao, Jiunn-Wang; Lin, Maw-Yeong; Liu, Hung-Jen; Wang, Min-Ying; Lin, Na-Sheng; Hsu, Yau-Heiu

    2012-06-01

    Very virulent Infectious bursal disease virus (vvIBDV) causes a highly contagious disease in young chickens and leads to significant economic loss in the poultry industry. Effective new vaccines are urgently needed. Autonomously replicating plant virus-based vector provides attractive means for producing chimeric virus particles (CVPs) in plants that can be developed into vaccines. In this study, we demonstrate the potential for vaccine development of Bamboo mosaic virus (BaMV) epitope-presentation system, where the antigen from vvIBDV VP2 was fused to the N-terminus of BaMV coat protein. Accordingly, an infections plasmid, pBIBD2, was constructed. Inoculation of the recombinant BaMV clone pBIBD2 enabled the generation of chimeric virus, BIBD2, and stable expression of IBDV VP2 antigen on its coat protein. After intramuscular immunization with BIBD2 CVPs, chickens produced antibodies against IBDV and were protected from vvIBDV (V263/TW strain) challenges. These results corroborate the feasibility of BaMV-based CVP platform in plants for the development and production of vaccines against IBDV. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Protein antigens increase the protective efficacy of a capsule-based vaccine against Staphylococcus aureus in a rat model of osteomyelitis.

    PubMed

    Lattar, Santiago M; Noto Llana, Mariángeles; Denoël, Philippe; Germain, Sophie; Buzzola, Fernanda R; Lee, Jean C; Sordelli, Daniel O

    2014-01-01

    Staphylococcus aureus is an invasive bacterial pathogen, and antibiotic resistance has impeded adequate control of infections caused by this microbe. Moreover, efforts to prevent human infections with single-component S. aureus vaccines have failed. In this study, we evaluated the protective efficacy in rats of vaccines containing both S. aureus capsular polysaccharides (CPs) and proteins. The serotypes 5 CP (CP5) and 8 CP (CP8) were conjugated to tetanus toxoid and administered to rats alone or together with domain A of clumping factor A (ClfA) or genetically detoxified alpha-toxin (dHla). The vaccines were delivered according to a preventive or a therapeutic regimen, and their protective efficacy was evaluated in a rat model of osteomyelitis. Addition of dHla (but not ClfA) to the CP5 or CP8 vaccine induced reductions in bacterial load and bone morphological changes compared with immunization with either conjugate vaccine alone. Both the prophylactic and therapeutic regimens were protective. Immunization with dHla together with a pneumococcal conjugate vaccine used as a control did not reduce staphylococcal osteomyelitis. The emergence of unencapsulated or small-colony variants during infection was negligible and similar for all of the vaccine groups. In conclusion, addition of dHla to a CP5 or CP8 conjugate vaccine enhanced its efficacy against S. aureus osteomyelitis, indicating that the inclusion of multiple antigens will likely enhance the efficacy of vaccines against both chronic and acute forms of staphylococcal disease.

  18. Vaccination with a novel recombinant Leishmania antigen plus MPL provides partial protection against L. donovani challenge in experimental model of visceral leishmaniasis.

    PubMed

    Bhardwaj, Suvercha; Vasishta, R K; Arora, Sunil K

    2009-01-01

    The acquisition of immunity following subclinical or resolved infection with the intracellular parasite Leishmania donovani suggests that vaccination could prevent visceral leishmaniasis. The characteristics and in vitro stimulating capability of the recombinant proteins expressed by previously identified clones on the basis of their capacity to stimulate an indigenously established Leishmania-specific cell line leading to high level of IFN-gamma suggested these to be potential candidates for immunoprophylaxis against leishmaniasis. In this study, we investigated the protective efficacy of purified recombinant proteins from two of the identified cDNA clones along with the adjuvant MPL, in a hamster model of experimental leishmaniasis. We demonstrate here that the immunization of animals with one of the recombinant proteins (rF14) having 97% similarity to C1 clone of L. chagasi ribosomal protein gene P0 (rLiP0) along with MPL provided partial protection against the virulent challenge of L. donovani. The absence of antigen-specific lymphoproliferative responses in these immunized animals may be responsible for the lack of complete and long-lasting protection.

  19. Protein Antigens Increase the Protective Efficacy of a Capsule-Based Vaccine against Staphylococcus aureus in a Rat Model of Osteomyelitis

    PubMed Central

    Lattar, Santiago M.; Noto Llana, Mariángeles; Denoël, Philippe; Germain, Sophie; Buzzola, Fernanda R.; Lee, Jean C.

    2014-01-01

    Staphylococcus aureus is an invasive bacterial pathogen, and antibiotic resistance has impeded adequate control of infections caused by this microbe. Moreover, efforts to prevent human infections with single-component S. aureus vaccines have failed. In this study, we evaluated the protective efficacy in rats of vaccines containing both S. aureus capsular polysaccharides (CPs) and proteins. The serotypes 5 CP (CP5) and 8 CP (CP8) were conjugated to tetanus toxoid and administered to rats alone or together with domain A of clumping factor A (ClfA) or genetically detoxified alpha-toxin (dHla). The vaccines were delivered according to a preventive or a therapeutic regimen, and their protective efficacy was evaluated in a rat model of osteomyelitis. Addition of dHla (but not ClfA) to the CP5 or CP8 vaccine induced reductions in bacterial load and bone morphological changes compared with immunization with either conjugate vaccine alone. Both the prophylactic and therapeutic regimens were protective. Immunization with dHla together with a pneumococcal conjugate vaccine used as a control did not reduce staphylococcal osteomyelitis. The emergence of unencapsulated or small-colony variants during infection was negligible and similar for all of the vaccine groups. In conclusion, addition of dHla to a CP5 or CP8 conjugate vaccine enhanced its efficacy against S. aureus osteomyelitis, indicating that the inclusion of multiple antigens will likely enhance the efficacy of vaccines against both chronic and acute forms of staphylococcal disease. PMID:24126523

  20. Antigen-specific B-1a antibodies induced by Francisella tularensis LPS provide long-term protection against F. tularensis LVS challenge

    PubMed Central

    Cole, Leah E.; Yang, Yang; Elkins, Karen L.; Fernandez, Ellen T.; Qureshi, Nilofer; Shlomchik, Mark J.; Herzenberg, Leonard A.; Herzenberg, Leonore A.; Vogel, Stefanie N.

    2009-01-01

    Francisella tularensis (Ft), a Gram-negative intracellular bacterium, is the etiologic agent of tularemia. Infection of mice with <10 Ft Live Vaccine Strain (Ft LVS) organisms i.p. causes a lethal infection that resembles human tularemia. Here, we show that immunization with as little as 0.1 ng Ft LVS lipopolysaccharide (Ft-LPS), but not Ft lipid A, generates a rapid antibody response that protects wild-type (WT) mice against lethal Ft LVS challenge. Protection is not induced in Ft-LPS-immunized B cell-deficient mice (μMT or JhD), male xid mice, or Ig transgenic mice that produce a single IgH (not reactive with Ft-LPS). Focusing on the cellular mechanisms that underlie this protective response, we show that Ft-LPS specifically stimulates proliferation of B-1a lymphocytes that bind fluorochrome-labeled Ft-LPS and the differentiation of these cells to plasma cells that secrete antibodies specific for Ft-LPS. This exclusively B-1a antibody response is equivalent in WT, T-deficient (TCRαβ−/−, TCRγδ−/−), and Toll-like receptor 4 (TLR4)-deficient (TLR4−/−) mice and thus is not dependent on T cells or typical inflammatory processes. Serum antibody levels peak ≈5 days after Ft-LPS immunization and persist at low levels for months. Thus, immunization with Ft-LPS activates a rare population of antigen-specific B-1a cells to produce a persistent T-independent antibody response that provides long-term protection against lethal Ft LVS infection. These data support the possibility of creating effective, minimally invasive vaccines that can provide effective protection against pathogen invasion. PMID:19251656

  1. A Plasmodium vivax plasmid DNA- and adenovirus-vectored malaria vaccine encoding blood stage antigens AMA1 and MSP142 in a prime/boost heterologous immunization regimen partially protects Aotus monkeys against blood stage challenge.

    PubMed

    Obaldia, Nicanor; Stockelman, Michael G; Otero, William; Cockrill, Jennifer A; Ganeshan, Harini; Abot, Esteban N; Zhang, Jianfeng; Limbach, Keith; Charoenvit, Yupin; Doolan, Denise L; Tang, De-Chu C; Richie, Thomas L

    2017-02-08

    Malaria is caused by parasites of the genus Plasmodium that are transmitted to humans by the bites of Anopheles mosquitoes. After the elimination of P. falciparum it is predicted that Plasmodium vivax will remain an important cause of morbidity and mortality outside of Africa, stressing the importance of developing a vaccine against malaria. In this study we assess the immunogenicity and protective efficacy of two P. vivax antigens, AMA1 and MSP142 in a recombinant DNA plasmid prime/adenoviral vector (Ad) boost regimen in Aotus monkeys. Groups of 4 to 5 monkeys were immunized with DNA alone, Ad alone, prime/boost regimens of each antigen, prime/boost with both antigens, and empty vector controls, and then subjected to blood stage challenge. The heterologous immunization regimen with the antigen pair was more protective than either antigen alone or both antigens delivered with a single vaccine platform, based on their ability to induced the longest pre-patent period and time to peak parasitemia; the lowest peak and mean parasitemia; the smallest area under the parasitemia curve and the highest self-cured rate. Overall, pre-challenge MSP1 antibody titers strongly correlated with decreased parasite burden. Nevertheless, a significant proportion of immunized animals developed anemia. In conclusion, P. vivax plasmid DNA/Ad5 vaccine encoding blood stage parasite antigens AMA1 and MSP142 in a heterologous prime/boost immunization regimen, provided significant protection against blood-stage challenge in Aotus monkeys, indicating the suitability of these antigens and regimen for further development.

  2. Protective Oral Vaccination against Infectious bursal disease virus Using the Major Viral Antigenic Protein VP2 Produced in Pichia pastoris

    PubMed Central

    Taghavian, Omid; Spiegel, Holger; Hauck, Rüdiger; Hafez, Hafez M.; Fischer, Rainer; Schillberg, Stefan

    2013-01-01

    Infectious bursal disease virus (IBDV) causes economically important immunosuppressive disease in young chickens. The self-assembling capsid protein (VP2) from IBDV strain IR01 was expressed in Pichia pastoris resulting in the formation of homomeric, 23-nm infectious bursal disease subviral particles (IBD-SVPs) with a yield of 76 mg/l before and 38 mg/l after purification. Anti-IBDV antibodies were detected in chickens injected with purified IBD-SVPs or fed with either purified IBD-SVPs or inactivated P. pastoris cells containing IBD-VP2 (cell-encapsulated). Challenge studies using the heterologous classical IBDV strain (MB3) showed that intramuscular vaccination with 20 µg purified IBD-SVPs conferred full protection, achieved complete virus clearance and prevented bursal damage and atrophy, compared with only 40% protection, 0–10% virus clearance accompanied by severe atrophy and substantial bursal damage in mock-vaccinated and challenge controls. The commercial IBDV vaccine also conferred full protection and achieved complete virus clearance, albeit with partial bursal atrophy. Oral administration of 500 µg purified IBD-SVPs with and without adjuvant conferred 100% protection but achieved only 60% virus clearance with adjuvant and none without it. Moderate bursal damage was observed in both cases but the inclusion of adjuvant resulted in bursal atrophy similar to that observed with live-attenuated vaccine and parenteral administration of 20 µg purified IBD-SVPs. The oral administration of 250 mg P. pastoris cells containing IBD-VP2 resulted in 100% protection with adjuvant and 60% without, accompanied by moderate bursal damage and atrophy in both groups, whereas 25 mg P. pastoris cells containing IBD-VP2 resulted in 90–100% protection with moderate bursal lesions and severe atrophy. Finally, the oral delivery of 50 µg purified IBD-SVPs achieved 40–60% protection with severe bursal lesions and atrophy. Both oral and parenteral administration of yeast

  3. Utility of a Novel Reflective Marker Visualized by Flash Photography for Assessment of Personnel Contamination During Removal of Personal Protective Equipment.

    PubMed

    Tomas, Myreen E; Cadnum, Jennifer L; Mana, Thriveen S C; Jencson, Annette L; Koganti, Sreelatha; Alhmidi, Heba; Kundrapu, Sirisha; Sunkesula, Venkata C K; Donskey, Curtis J

    2016-06-01

    In an experimental study, the frequency of contamination of healthcare personnel during removal of contaminated personal protective equipment (PPE) was similar for bacteriophage MS2 and a novel reflective marker visualized using flash photography. The reflective marker could be a useful tool to visualize and document personnel contamination during PPE removal. Infect Control Hosp Epidemiol 2016;37:711-713.

  4. A Multi-Antigenic Adenoviral-Vectored Vaccine Improves BCG-Induced Protection of Goats against Pulmonary Tuberculosis Infection and Prevents Disease Progression

    PubMed Central

    Pérez de Val, Bernat; Vidal, Enric; Villarreal-Ramos, Bernardo; Gilbert, Sarah C.; Andaluz, Anna; Moll, Xavier; Martín, Maite; Nofrarías, Miquel; McShane, Helen; Vordermeier, H. Martin; Domingo, Mariano

    2013-01-01

    The “One world, one health” initiative emphasizes the need for new strategies to control human and animal tuberculosis (TB) based on their shared interface. A good example would be the development of novel universal vaccines against Mycobacterium tuberculosis complex (MTBC) infection. This study uses the goat model, a natural TB host, to assess the protective effectiveness of a new vaccine candidate in combination with Bacillus Calmette-Guerin (BCG) vaccine. Thirty-three goat kids were divided in three groups: Group 1) vaccinated with BCG (week 0), Group 2) vaccinated with BCG and boosted 8 weeks later with a recombinant adenovirus expressing the MTBC antigens Ag85A, TB10.4, TB9.8 and Acr2 (AdTBF), and Group 3) unvaccinated controls. Later on, an endobronchial challenge with a low dose of M. caprae was performed (week 15). After necropsy (week 28), the pulmonary gross pathology was quantified using high resolution Computed Tomography. Small granulomatous pulmonary lesions (< 0.5 cm diameter) were also evaluated through a comprehensive qualitative histopathological analysis. M. caprae CFU were counted from pulmonary lymph nodes. The AdTBF improved the effects of BCG reducing gross lesion volume and bacterial load, as well as increasing weight gain. The number of Ag85A-specific gamma interferon-producing memory T-cells was identified as a predictor of vaccine efficacy. Specific cellular and humoral responses were measured throughout the 13-week post-challenge period, and correlated with the severity of lesions. Unvaccinated goats exhibited the typical pathological features of active TB in humans and domestic ruminants, while vaccinated goats showed only very small lesions. The data presented in this study indicate that multi-antigenic adenoviral vectored vaccines boosts protection conferred by vaccination with BCG. PMID:24278420

  5. Antibody to the Filarial Antigen Wb123 Reflects Reduced Transmission and Decreased Exposure in Children Born following Single Mass Drug Administration (MDA)

    PubMed Central

    Steel, Cathy; Kubofcik, Joseph; Ottesen, Eric A.; Nutman, Thomas B.

    2012-01-01

    Background Antibody (Ab) to the Wuchereria bancrofti (Wb) infective larval (L3) antigen Wb123, using a Luciferase Immunoprecipitation System (LIPS) assay, has been shown to be a species-specific, early marker of infection developed for potential use as a surveillance tool following transmission interruption post mass drug administration. To examine its usefulness in a single filarial-endemic island assessed at two time points with markedly different levels of transmission, Ab to Wb123 was measured in sera collected from subjects from Mauke, Cook Islands in 1975 (no previous treatment) and 1992 (5 years after a one time island-wide treatment with diethylcarbamazine [DEC]). Findings Between 1975 and 1992, Wb transmission decreased dramatically as evidenced by reduced prevalences of microfilariae (31% vs. 5%) and circulating Ag (CAg, 49% vs. 16%). Age specific prevalence analysis showed a dramatic reduction in Wb123 Ab positivity from 54% (25/46) in 1975 to 8% (3/38) in 1992 in children 1–5 years (p<0.0001), reflecting the effects of single-dose treatment five years earlier. By 1992, Wb123 Ab prevalence in children 6–10 years had fallen from 75% (42/56) in 1975 to 42% (33/79) consistent with a lower cumulative transmission potential. In the whole population, Wb123 seropositivity decreased from 86% to 60% between 1975 and 1992. In CAg+ subjects the levels of Wb123 Ab were indistinguishable between the 2 time points but differed in those who were CAg− (p<0.0001). In paired sample analysis, individuals who were CAg+ in 1975 but became CAg− in 1992 had significantly lower Ab levels in 1992 (p<0.0001), with 9/40 (23%) becoming seronegative for Wb123. Conclusions The relationship between reduction in Wb123 Ab prevalence and the reduction of transmission, seen most clearly in young children, strongly advocates for the continuing assessment and rapid development of Wb123 as a surveillance tool to detect potential transmission of bancroftian filariasis in treated

  6. Antibody to the filarial antigen Wb123 reflects reduced transmission and decreased exposure in children born following single mass drug administration (MDA).

    PubMed

    Steel, Cathy; Kubofcik, Joseph; Ottesen, Eric A; Nutman, Thomas B

    2012-01-01

    Antibody (Ab) to the Wuchereria bancrofti (Wb) infective larval (L3) antigen Wb123, using a Luciferase Immunoprecipitation System (LIPS) assay, has been shown to be a species-specific, early marker of infection developed for potential use as a surveillance tool following transmission interruption post mass drug administration. To examine its usefulness in a single filarial-endemic island assessed at two time points with markedly different levels of transmission, Ab to Wb123 was measured in sera collected from subjects from Mauke, Cook Islands in 1975 (no previous treatment) and 1992 (5 years after a one time island-wide treatment with diethylcarbamazine [DEC]). Between 1975 and 1992, Wb transmission decreased dramatically as evidenced by reduced prevalences of microfilariae (31% vs. 5%) and circulating Ag (CAg, 49% vs. 16%). Age specific prevalence analysis showed a dramatic reduction in Wb123 Ab positivity from 54% (25/46) in 1975 to 8% (3/38) in 1992 in children 1-5 years (p<0.0001), reflecting the effects of single-dose treatment five years earlier. By 1992, Wb123 Ab prevalence in children 6-10 years had fallen from 75% (42/56) in 1975 to 42% (33/79) consistent with a lower cumulative transmission potential. In the whole population, Wb123 seropositivity decreased from 86% to 60% between 1975 and 1992. In CAg+ subjects the levels of Wb123 Ab were indistinguishable between the 2 time points but differed in those who were CAg- (p<0.0001). In paired sample analysis, individuals who were CAg+ in 1975 but became CAg- in 1992 had significantly lower Ab levels in 1992 (p<0.0001), with 9/40 (23%) becoming seronegative for Wb123. The relationship between reduction in Wb123 Ab prevalence and the reduction of transmission, seen most clearly in young children, strongly advocates for the continuing assessment and rapid development of Wb123 as a surveillance tool to detect potential transmission of bancroftian filariasis in treated endemic areas.

  7. Antigenic sites on the HN domain of botulinum neurotoxin A stimulate protective antibody responses against active toxin.

    PubMed

    Ayyar, B Vijayalakshmi; Tajhya, Rajeev B; Beeton, Christine; Atassi, M Zouhair

    2015-10-28

    Botulinum neurotoxins (BoNTs) are the most toxic substances known. BoNT intoxicates cells in a highly programmed fashion initiated by binding to the cell surface, internalization and enzymatic cleavage of substrate, thus, inhibiting synaptic exocytosis. Over the past two decades, immunological significance of BoNT/A C-terminal heavy chain (HC) and light chain (LC) domains were investigated extensively leading to important findings. In the current work, we explored the significance of BoNT/A heavy chain N-terminal (HN) region as a vaccine candidate. Mice were immunized with recombinant HN519-845 generating antibodies (Abs) that were found to be protective against lethal dose of BoNT/A. Immuno-dominant regions of HN519-845 were identified and individually investigated for antibody response along with synthetic peptides within those regions, using in vivo protection assays against BoNT/A. Results were confirmed by patch-clamp analysis where anti-HN antibodies were studied for the ability to block toxin-induced channel formation. This data strongly indicated that HN519-593 is an important region in generating protective antibodies and should be valuable in a vaccine design. These results are the first to describe and dissect the protective activity of the BoNT/A HN domain.

  8. A reflection about the social and technological aspects in flood risk management - the case of the Italian Civil Protection

    NASA Astrophysics Data System (ADS)

    Llasat, M. Del Carmen; Siccardi, F.

    2010-01-01

    The right of a person to be protected from natural hazards is a characteristic of the social and economical development of the society. This paper is a contribution to the reflection about the role of Civil Protection organizations in a modern society. The paper is based in the inaugural conference made by the authors on the 9th Plinius Conference on Mediterranean Storms. Two major issues are considered. The first one is sociological; the Civil Protection organizations and the responsible administration of the land use planning should be perceived as reliable as possible, in order to get consensus on the restrictions they pose, temporary or definitely, on the individual free use of the territory as well as in the entire warning system. The second one is technological: in order to be reliable they have to issue timely alert and warning to the population at large, but such alarms should be as "true" as possible. With this aim, the paper summarizes the historical evolution of the risk assessment, starting from the original concept of "hazard", introducing the concepts of "scenario of event" and "scenario of risk" and ending with a discussion about the uncertainties and limits of the most advanced and efficient tools to predict, to forecast and to observe the ground effects affecting people and their properties. The discussion is centred in the case of heavy rains and flood events in the North-West of Mediterranean Region.

  9. HSP70 Domain II of Mycobacterium tuberculosis Modulates Immune Response and Protective Potential of F1 and LcrV Antigens of Yersinia pestis in a Mouse Model

    PubMed Central

    Batra, Lalit; Verma, Shailendra K.; Nagar, Durgesh P.; Saxena, Nandita; Pathak, Prachi; Pant, Satish C.; Tuteja, Urmil

    2014-01-01

    No ideal vaccine exists to control plague, a deadly dangerous disease caused by Yersinia pestis. In this context, we cloned, expressed and purified recombinant F1, LcrV antigens of Y. pestis and heat shock protein70 (HSP70) domain II of M. tuberculosis in E. coli. To evaluate the protective potential of each purified protein alone or in combination, Balb/C mice were immunized. Humoral and cell mediated immune responses were evaluated. Immunized animals were challenged with 100 LD50 of Y. pestis via intra-peritoneal route. Vaccine candidates i.e., F1 and LcrV generated highly significant titres of anti-F1 and anti-LcrV IgG antibodies. A significant difference was noticed in the expression level of IL-2, IFN-γ and TNF-α in splenocytes of immunized animals. Significantly increased percentages of CD4+ and CD8+ T cells producing IFN-γ in spleen of vaccinated animals were observed in comparison to control group by flow cytometric analysis. We investigated whether the F1, LcrV and HSP70(II) antigens alone or in combination can effectively protect immunized animals from any histopathological changes. Signs of histopathological lesions noticed in lung, liver, kidney and spleen of immunized animals on 3rd day post challenge whereas no lesions in animals that survived to day 20 post-infection were observed. Immunohistochemistry showed bacteria in lung, liver, spleen and kidney on 3rd day post-infection whereas no bacteria was observed on day 20 post-infection in surviving animals in LcrV, LcrV+HSP70(II), F1+LcrV, and F1+LcrV+HSP70(II) vaccinated groups. A significant difference was observed in the expression of IL-2, IFN-γ, TNF-α, and CD4+/CD8+ T cells secreting IFN-γ in the F1+LcrV+HSP70(II) vaccinated group in comparison to the F1+LcrV vaccinated group. Three combinations that included LcrV+HSP70(II), F1+LcrV or F1+LcrV+HSP70(II) provided 100% protection, whereas LcrV alone provided only 75% protection. These findings suggest that HSP70(II) of M. tuberculosis can be a

  10. Immune response and protection provided by live tachyzoites and native antigens from the NC-6 Argentina strain of Neospora caninum in pregnant heifers.

    PubMed

    Hecker, Y P; Moore, D P; Quattrocchi, V; Regidor-Cerrillo, J; Verna, A; Leunda, M R; Morrell, E; Ortega-Mora, L M; Zamorano, P; Venturini, M C; Campero, C M

    2013-11-08

    The aim of the present study was to compare the immunogenicity and protective efficacy of live tachyzoites and native antigen extract obtained from the NC-6 Argentina strain against vertical transmission of Neospora caninum, following experimental challenge in pregnant heifers with the NC-1 strain. Sixteen pregnant heifers were divided in 4 groups of 4 animals, each receiving different inoculation before mating: group A animals were intravenously (iv) inoculated with 6.25×10(7) live tachyzoites of the NC-6 strain, group B heifers were inoculated twice subcutaneously (sc) with N. caninum native antigen extract formulated with ISCOMs, group C heifers were sc injected with sterile phosphate-buffered saline (PBS) and group D heifers received sc ISCOM-matrix (ISCOMs without antigen). All groups were iv challenged with the NC-1 strain at 70 days of gestation. Serum and heparinized blood samples were collected eight times on weeks 0, 2, 3, 5, 9, 13, 16 and 17 post-inoculation. Dams were slaughtered at the 17th week of experiment (104 days of pregnancy) and placental and fetal tissue samples were collected. Specific antibody responses in heifers were tested by indirect enzyme linked immunosorbent assay (iELISA). The cellular immune response in dams was assessed by quantifying IFN-γ production and the percentages of T-cells (CD4(+), CD8(+) and γδ(+)) and monocytes in peripheral blood mononuclear cells (PBMC). Fetal fluids and tissue samples were tested using the indirect fluorescence antibody test, western blot, histopathology, immunohistochemistry and nested-PCR. A significant increase in N. caninum antibody response was detected in heifers of groups A and B from week 3 after inoculation (P<0.001). IFN-γ production was similar in groups A and B at week 13 (P>0.05). All fetuses were viable at necropsy. Specific IgG against N. caninum was detected in 1/4 fetal fluids recovered from groups A, C and D heifers and 3/4 fetal fluids from group B. Transplacental transmission

  11. A method to identify protein antigens of Dermanyssus gallinae for the protection of birds from poultry mites.

    PubMed

    Makert, Gustavo R; Vorbrüggen, Susanne; Krautwald-Junghanns, Maria-Elisabeth; Voss, Matthias; Sohn, Kai; Buschmann, Tilo; Ulbert, Sebastian

    2016-07-01

    The poultry red mite (PRM) Dermanyssus gallinae causes high economic losses and is among the most important parasites in poultry farming worldwide. Different chemical, physical, and biological strategies try to control the expansion of PRM. However, effective solutions to this problem still have to be found. Here, we present a method for the development of an immunological control strategy, based on the identification of mite protein antigens which elicit antibodies with anti-mite activity in the immunized chicken. Hens were immunized with different PRM protein extracts formulated with two different adjuvants, and IgY-antibodies were isolated from the eggs. A PRM in vitro feeding assay which used chicken blood spiked with these IgY-preparations was used to detect antibodies which caused PRM mortality. In vitro feeding of mites with IgY isolated from hens immunized with PRM extract formulated with one of the adjuvants showed a statistically significant increase in the mortality as compared to control mites. After the separation of total PRM extracts in two-dimensional gels, several protein spots were recognized by such IgY preparations. Ten protein spots were subjected to mass spectrometry (MS/MS) for the identification of the corresponding proteins. Complete protein sequences were deduced from genomic and transcriptomic assemblies derived from high throughput sequencing of total PRM DNA and RNA. The results may contribute to the development of an immunological control strategy of D. gallinae.

  12. Diet pills and the cataract outbreak of 1935: reflections on the evolution of consumer protection legislation.

    PubMed

    Margo, Curtis E; Harman, Lynn E

    2014-01-01

    An outbreak of cataracts in 1935 caused by dinitrophenol (DNP), the active ingredient of popular diet pills, highlighted the inability of the U.S. Food and Drug Administration (FDA) to prevent harmful drugs from entering the marketplace. Just two years earlier, the FDA used horrific images of ocular surface injury caused by cosmetics at the World's Fair in Chicago to garner public support for legislative reform. The FDA had to walk a fine line between a public awareness campaign and lobbying Congress while lawmakers debated the need for consumer protection. The cataract outbreak of 1935 was conspicuous in the medical literature during the height of New Deal legislation, but questions persist as to how much it affected passage of the proposed Food, Drug, and Cosmetic Act (of 1938). The legislation languished in committee for years. The cataract outbreak probably had little impact on the eventual outcome, but medical opinion concerning the safety of DNP may have contributed to the voluntary withdrawal of the diet drug from the market. We review the DNP cataract outbreak and examine it in context of the challenges facing regulatory reform at that time.

  13. Cooperation between CD4+ T Cells and Humoral Immunity Is Critical for Protection against Dengue Using a DNA Vaccine Based on the NS1 Antigen

    PubMed Central

    Gonçalves, Antônio J. S.; Oliveira, Edson R. A.; Costa, Simone M.; Paes, Marciano V.; Silva, Juliana F. A.; Azevedo, Adriana S.; Mantuano-Barradas, Marcio; Nogueira, Ana Cristina M. A.; Almeida, Cecília J.; Alves, Ada M. B.

    2015-01-01

    Dengue virus (DENV) is spread through most tropical and subtropical areas of the world and represents a serious public health problem. At present, the control of dengue disease is mainly hampered by the absence of antivirals or a vaccine, which results in an estimated half worldwide population at risk of infection. The immune response against DENV is not yet fully understood and a better knowledge of it is now recognized as one of the main challenge for vaccine development. In previous studies, we reported that a DNA vaccine containing the signal peptide sequence from the human tissue plasminogen activator (t-PA) fused to the DENV2 NS1 gene (pcTPANS1) induced protection against dengue in mice. In the present work, we aimed to elucidate the contribution of cellular and humoral responses elicited by this vaccine candidate for protective immunity. We observed that pcTPANS1 exerts a robust protection against dengue, inducing considerable levels of anti-NS1 antibodies and T cell responses. Passive immunization with anti-NS1 antibodies conferred partial protection in mice infected with low virus load (4 LD50), which was abrogated with the increase of viral dose (40 LD50). The pcTPANS1 also induced activation of CD4+ and CD8+ T cells. We detected production of IFN-γ and a cytotoxic activity by CD8+ T lymphocytes induced by this vaccine, although its contribution in the protection was not so evident when compared to CD4+ cells. Depletion of CD4+ cells in immunized mice completely abolished protection. Furthermore, transfer experiments revealed that animals receiving CD4+ T cells combined with anti-NS1 antiserum, both obtained from vaccinated mice, survived virus infection with survival rates not significantly different from pcTPANS1-immunized animals. Taken together, results showed that the protective immune response induced by the expression of NS1 antigen mediated by the pcTPANS1 requires a cooperation between CD4+ T cells and the humoral immunity. PMID:26650916

  14. Cooperation between CD4+ T Cells and Humoral Immunity Is Critical for Protection against Dengue Using a DNA Vaccine Based on the NS1 Antigen.

    PubMed

    Gonçalves, Antônio J S; Oliveira, Edson R A; Costa, Simone M; Paes, Marciano V; Silva, Juliana F A; Azevedo, Adriana S; Mantuano-Barradas, Marcio; Nogueira, Ana Cristina M A; Almeida, Cecília J; Alves, Ada M B

    2015-12-01

    Dengue virus (DENV) is spread through most tropical and subtropical areas of the world and represents a serious public health problem. At present, the control of dengue disease is mainly hampered by the absence of antivirals or a vaccine, which results in an estimated half worldwide population at risk of infection. The immune response against DENV is not yet fully understood and a better knowledge of it is now recognized as one of the main challenge for vaccine development. In previous studies, we reported that a DNA vaccine containing the signal peptide sequence from the human tissue plasminogen activator (t-PA) fused to the DENV2 NS1 gene (pcTPANS1) induced protection against dengue in mice. In the present work, we aimed to elucidate the contribution of cellular and humoral responses elicited by this vaccine candidate for protective immunity. We observed that pcTPANS1 exerts a robust protection against dengue, inducing considerable levels of anti-NS1 antibodies and T cell responses. Passive immunization with anti-NS1 antibodies conferred partial protection in mice infected with low virus load (4 LD50), which was abrogated with the increase of viral dose (40 LD50). The pcTPANS1 also induced activation of CD4+ and CD8+ T cells. We detected production of IFN-γ and a cytotoxic activity by CD8+ T lymphocytes induced by this vaccine, although its contribution in the protection was not so evident when compared to CD4+ cells. Depletion of CD4+ cells in immunized mice completely abolished protection. Furthermore, transfer experiments revealed that animals receiving CD4+ T cells combined with anti-NS1 antiserum, both obtained from vaccinated mice, survived virus infection with survival rates not significantly different from pcTPANS1-immunized animals. Taken together, results showed that the protective immune response induced by the expression of NS1 antigen mediated by the pcTPANS1 requires a cooperation between CD4+ T cells and the humoral immunity.

  15. Whole recombinant Pichia pastoris expressing HPV16 L1 antigen is superior in inducing protection against tumor growth as compared to killed transgenic Leishmania

    PubMed Central

    Bolhassani, Azam; Muller, Martin; Roohvand, Farzin; Motevalli, Fatemeh; Agi, Elnaz; Shokri, Mehdi; Rad, Mahdieh Motamedi; Hosseinzadeh, Sahar

    2015-01-01

    The development of an efficient vaccine against high-risk HPV types can reduce the incidence rates of cervical cancer by generating anti-tumor protective responses. Traditionally, the majority of prophylactic viral vaccines are composed of live, attenuated or inactivated viruses. Among them, the design of an effective and low-cost vaccine is critical. Inactivated vaccines especially heat-killed yeast cells have emerged as a promising approach for generating antigen-specific immunotherapy. Recent studies have indicated that yeast cell wall components possess adjuvant activities. Moreover, a non-pathogenic protozoan, Leishmania tarentolae (L.tar) has attracted a great attention as a live candidate vaccine. In current study, immunological and protective efficacy of whole recombinant killed Pichia pastoris and Leishmania tarentolae expressing HPV16 L1 capsid protein was evaluated in tumor mice model. We found that Pichia-L1, L.tar-L1 and Gardasil groups increase the IgG2a/IgG1 ratio, indicating a relative preference for the induction of Th1 immune responses. Furthermore, subcutaneous injection of killed Pichia-L1 generated the significant L1-specific IFN-γ immune response as well as the best protective effects in vaccinated mice as compared to killed L.tar-L1, killed Pichia pastoris, killed L.tar and PBS groups. Indeed, whole recombinant Leishmania tarentolae could not protect mice against C3 tumor mice model. These data suggest that Pichia-L1 may be a candidate for the control of HPV infections. PMID:25668661

  16. CXCL10 Is Critical for the Generation of Protective CD8 T Cell Response Induced by Antigen Pulsed CpG-ODN Activated Dendritic Cells

    PubMed Central

    Majumder, Saikat; Bhattacharjee, Surajit; Paul Chowdhury, Bidisha; Majumdar, Subrata

    2012-01-01

    The visceral form of leishmaniasis is the most severe form of the disease and of particular concern due to the emerging problem of HIV/visceral leishmaniasis (VL) co-infection in the tropics. Till date miltefosine, amphotericin B and pentavalent antimony compounds remain the main treatment regimens for leishmaniasis. However, because of severe side effects, there is an urgent need for alternative improved therapies to combat this dreaded disease. In the present study, we have used the murine model of leishmaniasis to evaluate the potential role played by soluble leishmanial antigen (SLA) pulsed-CpG-ODN stimulated dendritic cells (SLA-CpG-DCs) in restricting the intracellular leishmanial growth. We found that mice vaccinated with a single dose of SLA-pulsed DC stimulated by CpG-ODN were protected against a subsequent leishmanial challenge and had a dramatic reduction in parasite burden along with the generation of parasite specific cytotoxic T lymphocytes. Moreover, we demonstrate that the induction of protective immunity conferred by SLA-CpG-DCs depends entirely on the CXC chemokine IFN-γ-inducible protein 10 (CXCL10; IP-10). CXCL10 is directly involved in the generation of a parasite specific CD8+ T cell-mediated immune response. We observed significant reduction of CD8+ T cells in mice depleted of CXCL10 suggesting a direct role of CXCL10 in the generation of CD8+ T cells in SLA-CpG-DCs vaccinated mice. CXCL10 also contributed towards the generation of perforin and granzyme B, two important cytolytic mediators of CD8+ T cells, following SLA-CpG-DCs vaccination. Together, these findings strongly demonstrate that CXCL10 is critical for rendering a protective cellular immunity during SLA-CpG-DC vaccination that confers protection against Leishmania donovani infection. PMID:23144947

  17. Functional and Antigen-Specific Serum Antibody Levels as Correlates of Protection against Shigellosis in a Controlled Human Challenge Study

    PubMed Central

    Shimanovich, Avital A.; Buskirk, Amanda D.; Heine, Shannon J.; Blackwelder, William C.; Wahid, Rezwanul; Kotloff, Karen L.

    2016-01-01

    ABSTRACT Shigella is an important cause of diarrheal disease in young children living in developing countries. No approved vaccines are available, and the development of vaccine candidates has been hindered by the lack of firm immunological correlates of protection, among other reasons. To address this gap in knowledge, we established quantitative assays to measure Shigella-specific serum bactericidal antibody (SBA) and opsonophagocytic killing antibody (OPKA) activities and investigated their potential association with protection against disease in humans. SBA, OPKA, and Ipa-, VirG (IscA)-, and Shigella flexneri 2a lipopolysaccharide-specific serum IgG titers were determined in adult volunteers who received Shigella vaccine candidate EcSf2a-2 and in unvaccinated controls, all of whom were challenged with virulent Shigella flexneri 2a. Prechallenge antibody titers were compared with disease severity after challenge. SBA and OPKA, as well as IpaB- and VirG-specific IgG, significantly correlated with reduced illness. SBA and OPKA assays were also used to evaluate the immunogenicity of leading live attenuated vaccine candidates Shigella CVD 1204 and CVD 1208S in humans. A single oral immunization with CVD 1204 or CVD 1208S resulted in SBA seroconversion rates of 71% and 47% and OPKA seroconversion rates of 57% and 35%, respectively. Higher functional antibody responses were induced by CVD 1204, which is consistent with its lower attenuation. This is the first demonstration of SBA, OPKA, and IpaB- and VirG-specific IgG levels as potential serological correlates of protection against shigellosis in humans. These results warrant further studies to establish their capacity to predict protective immunity and vaccine efficacy. PMID:27927680

  18. Protective response to Leishmania major in BALB/c mice requires antigen processing in the absence of DM.

    PubMed

    Kamala, Tirumalai; Nanda, Navreet K

    2009-04-15

    Protection from the parasite Leishmania major is mediated by CD4 T cells. BALB/c mice are susceptible to L. major and show a nonprotective immunodominant CD4 T cell response to Leishmania homolog of activated receptor for c-kinase (LACK) 158-173. Host genes that underlie BALB/c susceptibility to L. major infections are poorly defined. DM, a nonclassical MHC class II molecule, due to its peptide editing properties has been shown to 1) edit the repertoire of peptides displayed by APC, and 2) focus the display of epitopes by APC to the immunodominant ones. We tested the hypothesis that deficiency of DM, by causing presentation of a different array of epitopes by infected APC than that presented by DM-sufficient APC, may change the course of L. major infection in the susceptible BALB/c mice. We show herein that unlike their susceptible wild-type counterparts, BALB/c mice deficient in DM are protected from infections with L. major. Furthermore, DM-deficient mice fail to display the immunodominant LACK 158-173 on infected APC. In its place, infected DM(-/-) hosts show elicitation of CD4 T cells specific for newer epitopes not presented by wild-type L. major-infected APC. Protection of BALB/c DM(-/-) mice is dependent on IFN-gamma. DM is thus a host susceptibility gene in BALB/c mice, and Ag processing in the absence of DM results in elicitation of a protective T cell response against L. major infections. This report suggests a novel mechanism to trigger host resistance against pathogens.

  19. Multiple Asparagine Deamidation of Bacillus anthracis Protective Antigen Causes Charge Isoforms Whose Complexity Correlates with Reduced Biological Activity

    DTIC Science & Technology

    2007-01-01

    Report Documentation Page Form ApprovedOMB No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour...subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. 1 . REPORT DATE 1 ...FEB 2007 2. REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Multiple asparagine deamidation of Bacillus anthracis protective

  20. Manipulation of the immune evasive properties of circulating cathodic antigen induces protective immunity against Schistosomiasis mansoni in C57BL/6 mice.

    PubMed

    Abdeen, Sherif H

    2010-01-01

    Schistosome circulating cathodic antigen (CCA) has been hypothesized to take part in immune evasion mechanisms that protect against host's immunity. To dissect its immunogenicity, lymphoproliferative responses of splenocytes were assessed in C57BL/6 mice immunized with recombinant plasmid constructs expressing full-length and truncated fragments of CCA cDNA, before and after challenge infection with S. mansoni cercariae. Prior to challenge, splenocytes of immunized mice showed low responses to phytohaemagglutinin (PHA) and high responses to native CCA, compared to controls. After challenge, PHA-responses increased on day 3 through day 7 and subsequently declined. On the other hand, CCA-induced responses increased on day 3 post-challenge then declined in mice immunized with CCA fragments lacking the N-terminus (-N CCA). Whereas, mice immunized with full-length or CCA fragment lacking the C-terminus (-C CCA) showed a delayed increase of CCA-induced responses that maximize on day 25. Interestingly, animals immunized with -N CCA showed a significant reduction in worm burden between 42-51%, while, mice immunized with full-length or -C CCA showed lower protection levels of about 15 and 37%, respectively. These findings suggest that CCA may contain immunosuppressive epitopes on the N-terminus. Abrogation of these epitopes could disrupt the immune evasion mechanism orchestrated by CCA, which could aid the development of an alternative vaccination approach.

  1. Optimal attenuation of a PR8-derived mouse pathogenic H5N1 recombinant virus for testing antigenicity and protective efficacy in mice.

    PubMed

    Kim, Il-Hwan; Kwon, Hyuk-Joon; Park, Jae-Keun; Song, Chang-Seon; Kim, Jae-Hong

    2015-11-17

    The PR8-based reverse genetics vector system is widely used to generate commercial vaccine strains, but the pathogenicity of PR8-derived recombinant viruses in mice hinders further immunological studies. In the present study, we generated PR8-derived H5N1 recombinant viruses, in which haemagglutinin (HA) and neuraminidase (NA) originated from a mouse-pathogenic H5N1 low pathogenic avian influenza virus (LPAIV), and the non-structural proteins (NS) and polymerase basic protein 2 (PB2) originated from different H9N2 LPAIVs. In contrast to the control H5N1 recombinant virus, harboring six internal genes from PR8, the NS and PB2 recombinant viruses did not cause body weight loss in mice. However, the NS recombinant virus replicated in the lungs of mice. It was more immunogenic than the PB2 recombinant virus to protect efficiently against a lethal challenge of a H5N1 highly pathogenic AIV with 89 and 88% amino acid identity in HA and NA, respectively. Therefore, the NS gene may be useful for generating nonpathogenic and immunogenic PR8-derived recombinant viruses for studies of antigenicity and protective efficacy in mice.

  2. A CpG-Ficoll Nanoparticle Adjuvant for Anthrax Protective Antigen Enhances Immunogenicity and Provides Single-Immunization Protection against Inhaled Anthrax in Monkeys.

    PubMed

    Kachura, Melissa A; Hickle, Colin; Kell, Sariah A; Sathe, Atul; Calacsan, Carlo; Kiwan, Radwan; Hall, Brian; Milley, Robert; Ott, Gary; Coffman, Robert L; Kanzler, Holger; Campbell, John D

    2016-01-01

    Nanoparticulate delivery systems for vaccine adjuvants, designed to enhance targeting of secondary lymphoid organs and activation of APCs, have shown substantial promise for enhanced immunopotentiation. We investigated the adjuvant activity of synthetic oligonucleotides containing CpG-rich motifs linked to the sucrose polymer Ficoll, forming soluble 50-nm particles (DV230-Ficoll), each containing >100 molecules of the TLR9 ligand, DV230. DV230-Ficoll was evaluated as an adjuvant for a candidate vaccine for anthrax using recombinant protective Ag (rPA) from Bacillus anthracis. A single immunization with rPA plus DV230-Ficoll induced 10-fold higher titers of toxin-neutralizing Abs in cynomolgus monkeys at 2 wk compared with animals immunized with equivalent amounts of monomeric DV230. Monkeys immunized either once or twice with rPA plus DV230-Ficoll were completely protected from challenge with 200 LD50 aerosolized anthrax spores. In mice, DV230-Ficoll was more potent than DV230 for the induction of innate immune responses at the injection site and draining lymph nodes. DV230-Ficoll was preferentially colocalized with rPA in key APC populations and induced greater maturation marker expression (CD69 and CD86) on these cells and stronger germinal center B and T cell responses, relative to DV230. DV230-Ficoll was also preferentially retained at the injection site and draining lymph nodes and produced fewer systemic inflammatory responses. These findings support the development of DV230-Ficoll as an adjuvant platform, particularly for vaccines such as for anthrax, for which rapid induction of protective immunity and memory with a single injection is very important. Copyright © 2015 by The American Association of Immunologists, Inc.

  3. Oral vaccination with salmonella simultaneously expressing Yersinia pestis F1 and V antigens protects against bubonic and pneumonic plague.

    PubMed

    Yang, Xinghong; Hinnebusch, B Joseph; Trunkle, Theresa; Bosio, Catharine M; Suo, Zhiyong; Tighe, Mike; Harmsen, Ann; Becker, Todd; Crist, Kathryn; Walters, Nancy; Avci, Recep; Pascual, David W

    2007-01-15

    The gut provides a large area for immunization enabling the development of mucosal and systemic Ab responses. To test whether the protective Ags to Yersinia pestis can be orally delivered, the Y. pestis caf1 operon, encoding the F1-Ag and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vectors. F1-Ag expression was controlled under a promoter from the caf1 operon; two different promoters (P), PtetA in pV3, PphoP in pV4, as well as a chimera of the two in pV55 were tested. F1-Ag was amply expressed; the chimera in the pV55 showed the best V-Ag expression. Oral immunization with Salmonella-F1 elicited elevated secretory (S)-IgA and serum IgG titers, and Salmonella-V-Ag(pV55) elicited much greater S-IgA and serum IgG Ab titers than Salmonella-V-Ag(pV3) or Salmonella-V-Ag(pV4). Hence, a new Salmonella vaccine, Salmonella-(F1+V)Ags, made with a single plasmid containing the caf1 operon and the chimeric promoter for V-Ag allowed the simultaneous expression of F1 capsule and V-Ag. Salmonella-(F1+V)Ags elicited elevated Ab titers similar to their monotypic derivatives. For bubonic plague, mice dosed with Salmonella-(F1+V)Ags and Salmonella-F1-Ag showed similar efficacy (>83% survival) against approximately 1000 LD(50) Y. pestis. For pneumonic plague, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1+V)Ags protected against 100 LD(50) Y. pestis. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect both systemic and mucosal immune protection against Y. pestis.

  4. The Use of Directional Reflectance Measurement for in vivo Assessment of Protective Properties of Cosmetics in the Infrared Radiation Range.

    PubMed

    Wilczyński, Sławomir; Deda, Anna; Koprowski, Robert; Banyś, Anna; Błońska-Fajfrowska, Barbara

    2017-10-01

    Photoprotection of skin is now focused on UV radiation. The aim of this study was to evaluate in vivo cosmetic products in terms of protection against infrared radiation (IR) and propose a methodology for conducting such measurements. The directional reflectance (DR) of 12 UV filters, six care creams and two preparations containing fumed silica applied on the forearm of 36 volunteers was examined in six spectral bands for two angles of incidence. SOC-410 Directional Hemispherical Reflectometer was used to measure DR. There is very little change in DR for all spectral bands for both incident angles for both UV filters, care creams and preparations containing fumed silica. For example, for 15% of fumed silica in glycerin for the spectral band of 0.9-1.1 μm and the incident angle of 20°, skin DR prior to application was 0.543; 5 min after application was 0.533 and 30 min after application was 0.559. Both UV filters, care creams and fumed silica do not protect skin against IR. The proposed method of in vivo measurements is superior to in vitro studies which have been conducted so far because it takes into account both the refractive index at the tissue/air interface and the absorption of IR by adipose tissue. © 2017 The American Society of Photobiology.

  5. Use of Site-Directed Mutagenesis To Model the Effects of Spontaneous Deamidation on the Immunogenicity of Bacillus anthracis Protective Antigen

    PubMed Central

    Verma, Anita; McNichol, Beth; Domínguez-Castillo, Rocío I.; Amador-Molina, Juan C.; Arciniega, Juan L.; Reiter, Karine; Meade, Bruce D.; Ngundi, Miriam M.; Stibitz, Scott

    2013-01-01

    Long-term stability is a desired characteristic of vaccines, especially anthrax vaccines, which must be stockpiled for large-scale use in an emergency situation; however, spontaneous deamidation of purified vaccine antigens has the potential to adversely affect vaccine immunogenicity over time. In order to explore whether spontaneous deamidation of recombinant protective antigen (rPA)—the major component of new-generation anthrax vaccines—affects vaccine immunogenicity, we created a “genetically deamidated” form of rPA using site-directed mutagenesis to replace six deamidation-prone asparagine residues, at positions 408, 466, 537, 601, 713, and 719, with either aspartate, glutamine, or alanine residues. We found that the structure of the six-Asp mutant rPA was not significantly altered relative to that of the wild-type protein as assessed by circular dichroism (CD) spectroscopy and biological activity. In contrast, immunogenicity of aluminum-adjuvanted six-Asp mutant rPA, as measured by induction of toxin-neutralizing antibodies, was significantly lower than that of the corresponding wild-type rPA vaccine formulation. The six-Gln and six-Ala mutants also exhibited lower immunogenicity than the wild type. While the wild-type rPA vaccine formulation exhibited a high level of immunogenicity initially, its immunogenicity declined significantly upon storage at 25°C for 4 weeks. In contrast, the immunogenicity of the six-Asp mutant rPA vaccine formulation was low initially but did not change significantly upon storage. Taken together, results from this study suggest that spontaneous deamidation of asparagine residues predicted to occur during storage of rPA vaccines would adversely affect vaccine immunogenicity and therefore the storage life of vaccines. PMID:23115046

  6. Nonclassical MHC Ib-restricted CD8+ T Cells Recognize Mycobacterium tuberculosis-Derived Protein Antigens and Contribute to Protection Against Infection.

    PubMed

    Shang, Shaobin; Siddiqui, Sarah; Bian, Yao; Zhao, Jie; Wang, Chyung-Ru

    2016-06-01

    MHC Ib-restricted CD8+ T cells have been implicated in host defense against Mycobacterium tuberculosis (Mtb) infection. However, the relative contribution of various MHC Ib-restricted T cell populations to anti-mycobacterial immunity remains elusive. In this study, we used mice that lack MHC Ia (Kb-/-Db-/-), MHC Ia/H2-M3 (Kb-/-Db-/-M3-/-), or β2m (β2m-/-) to study the role of M3-restricted and other MHC Ib-restricted T cells in immunity against Mtb. Unlike their dominant role in Listeria infection, we found that M3-restricted CD8+ T cells only represented a small proportion of the CD8+ T cells responding to Mtb infection. Non-M3, MHC Ib-restricted CD8+ T cells expanded preferentially in the lungs of Mtb-infected Kb-/-Db-/-M3-/- mice, exhibited polyfunctional capacities and conferred protection against Mtb. These MHC Ib-restricted CD8+ T cells recognized several Mtb-derived protein antigens at a higher frequency than MHC Ia-restricted CD8+ T cells. The presentation of Mtb antigens to MHC Ib-restricted CD8+ T cells was mostly β2m-dependent but TAP-independent. Interestingly, a large proportion of Mtb-specific MHC Ib-restricted CD8+ T cells in Kb-/-Db-/-M3-/- mice were Qa-2-restricted while no considerable numbers of MR1 or CD1-restricted Mtb-specific CD8+ T cells were detected. Our findings indicate that nonclassical CD8+ T cells other than the known M3, CD1, and MR1-restricted CD8+ T cells contribute to host immune responses against Mtb infection. Targeting these MHC Ib-restricted CD8+ T cells would facilitate the design of better Mtb vaccines with broader coverage across MHC haplotypes due to the limited polymorphism of MHC class Ib molecules.

  7. A novel homogeneous immunoassay for anthrax detection based on the AlphaLISA method: detection of B. anthracis spores and protective antigen (PA) in complex samples.

    PubMed

    Mechaly, Adva; Cohen, Noam; Weiss, Shay; Zahavy, Eran

    2013-05-01

    Amplified Luminescent Proximity Homogeneous Assay (AlphaLISA) technology is an energy-transfer-based assay, utilizing singlet oxygen as an energy donor to a fluorescent acceptor. The long singlet oxygen migration distance allows the energy transfer mechanism to go up to ~200 nm, facilitating flexible and sensitive homogeneous immunoassays. While soluble protein detection using AlphaLISA was previously described, the detection of particles such as bacteria and viruses was not reported. In this work, we show for the first time the implementation of the AlphaLISA technology for the detection of a particulate antigen, i.e., Bacillus anthracis spores. Here, we show that an efficient particle immunoassay requires a high acceptor-to-donor ratio (>4:1). The results suggested that the high acceptor/donor ratio is required to avoid donor aggregation ("islands") on the spore surface, hence facilitating donor/acceptor interaction. The developed assay enabled the detection of 10(6) spores/mL spiked in PBS. We also demonstrate the development of a highly sensitive AlphaLISA assay for the detection of the main toxin component of anthrax, protective antigen (PA). The assay enabled the detection of 10 and 100 pg/mL PA in buffer and spiked naïve rabbit sera, respectively, and was successfully implemented in sera of anthrax-infected rabbits. To summarize, this study demonstrates that AlphaLISA enables detection of anthrax spores and toxin, utilizing short homogeneous assays. Moreover, it is shown for the first time that this technology facilitates the detection of particulate entities and might be suitable for the detection of other bacteria or viruses.

  8. BoHV-4-Based Vector Single Heterologous Antigen Delivery Protects STAT1(-/-) Mice from Monkeypoxvirus Lethal Challenge

    PubMed Central

    Crump, Ryan W.; Doronin, Konstantin; Hembrador, Edguardo; Pompilio, Daniela; Tebaldi, Giulia; Estep, Ryan D.; Wong, Scott W.; Buller, Mark R.; Donofrio, Gaetano

    2015-01-01

    Monkeypox virus (MPXV) is the etiological agent of human (MPX). It is an emerging orthopoxvirus zoonosis in the tropical rain forest of Africa and is endemic in the Congo-basin and sporadic in West Africa; it remains a tropical neglected disease of persons in impoverished rural areas. Interaction of the human population with wildlife increases human infection with MPX virus (MPXV), and infection from human to human is possible. Smallpox vaccination provides good cross-protection against MPX; however, the vaccination campaign ended in Africa in 1980, meaning that a large proportion of the population is currently unprotected against MPXV infection. Disease control hinges on deterring zoonotic exposure to the virus and, barring that, interrupting person-to-person spread. However, there are no FDA-approved therapies against MPX, and current vaccines are limited due to safety concerns. For this reason, new studies on pathogenesis, prophylaxis and therapeutics are still of great interest, not only for the scientific community but also for the governments concerned that MPXV could be used as a bioterror agent. In the present study, a new vaccination strategy approach based on three recombinant bovine herpesvirus 4 (BoHV-4) vectors, each expressing different MPXV glycoproteins, A29L, M1R and B6R were investigated in terms of protection from a lethal MPXV challenge in STAT1 knockout mice. BoHV-4-A-CMV-A29LgD106ΔTK, BoHV-4-A-EF1α-M1RgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK were successfully constructed by recombineering, and their capacity to express their transgene was demonstrated. A small challenge study was performed, and all three recombinant BoHV-4 appeared safe (no weight-loss or obvious adverse events) following intraperitoneal administration. Further, BoHV-4-A-EF1α-M1RgD106ΔTK alone or in combination with BoHV-4-A-CMV-A29LgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK, was shown to be able to protect, 100% alone and 80% in combination, STAT1(-/-) mice against

  9. Bordetella pertussis filamentous hemagglutinin: evaluation as a protective antigen and colonization factor in a mouse respiratory infection model.

    PubMed Central

    Kimura, A; Mountzouros, K T; Relman, D A; Falkow, S; Cowell, J L

    1990-01-01

    Filamentous hemagglutinin (FHA) is a cell surface protein of Bordetella pertussis which functions as an adhesin for this organism. It is a component of many new acellular pertussis vaccines. The proposed role of FHA in immunity to pertussis is based on animal studies which have produced some conflicting results. To clarify this situation, we reexamined the protective activity of FHA in an adult mouse respiratory infection model. Four-week-old BALB/c mice were immunized with one or two doses of 4 or 8 micrograms of FHA and then aerosol challenged with B. pertussis Tohama I. In control mice receiving tetanus toxoid, the CFU in the lungs increased from 10(5) immediately following infection to greater than 10(6) by days 5 and 9 after challenge. Mice immunized with FHA by the intraperitoneal or intramuscular route had significantly reduced bacterial colonization in the lungs. A decrease in colonization of the trachea was also observed in FHA-immunized mice. Evaluation of antibody responses in these mice revealed high titers of immunoglobulin G (IgG) and IgM to FHA in sera and of IgG to FHA in lung lavage fluids. No IgA to FHA was detected. BALB/c mice were also passively immunized intravenously with either goat or rat antibodies to FHA and then aerosol challenged 24 h later, when anti-FHA antibodies were detected in the respiratory tract. Lung and tracheal colonization was markedly reduced in mice immunized with FHA-specific antibodies compared with those receiving control antibodies. In additional studies, the role of FHA in the colonization of the mouse respiratory tract was evaluated by using strain BP101, an FHA mutant of B. pertussis. FHA was important in the initial colonization of the mouse trachea, but was not required for colonization of the trachea later in the infection. FHA was not a factor in colonization of the lungs. Collectively, these experiments demonstrate (i) that systemic immunization with FHA can provide significant protection against B. pertussis

  10. Chimeric Antigen Receptor-Engineered NK-92 Cells: An Off-the-Shelf Cellular Therapeutic for Targeted Elimination of Cancer Cells and Induction of Protective Antitumor Immunity.

    PubMed

    Zhang, Congcong; Oberoi, Pranav; Oelsner, Sarah; Waldmann, Anja; Lindner, Aline; Tonn, Torsten; Wels, Winfried S

    2017-01-01

    Significant progress has been made in recent years toward realizing the potential of natural killer (NK) cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also the established NK cell line NK-92 is being developed for adoptive immunotherapy, and general safety of infusion of irradiated NK-92 cells has been established in phase I clinical trials with clinical responses observed in some of the cancer patients treated. To enhance their therapeutic utility, NK-92 cells have been modified to express chimeric antigen receptors (CARs) composed of a tumor-specific single chain fragment variable antibody fragment fused via hinge and transmembrane regions to intracellular signaling moieties such as CD3ζ or composite signaling domains containing a costimulatory protein together with CD3ζ. CAR-mediated activation of NK cells then bypasses inhibitory signals and overcomes NK resistance of tumor cells. In contrast to primary NK cells, CAR-engineered NK-92 cell lines suitable for clinical development can be established from molecularly and functionally well-characterized single cell clones following good manufacturing practice-compliant procedures. In preclinical in vitro and in vivo models, potent antitumor activity of NK-92 variants targeted to differentiation antigens expressed by hematologic malignancies, and overexpressed or mutated self-antigens associated with solid tumors has been found, encouraging further development of CAR-engineered NK-92 cells. Importantly, in syngeneic mouse tumor models, induction of endogenous antitumor immunity after treatment with CAR-expressing NK-92 cells has been demonstrated, resulting in cures and long-lasting immunological memory protecting against tumor rechallenge at distant sites. Here, we summarize the current status and future prospects of CAR

  11. Chimeric Antigen Receptor-Engineered NK-92 Cells: An Off-the-Shelf Cellular Therapeutic for Targeted Elimination of Cancer Cells and Induction of Protective Antitumor Immunity

    PubMed Central

    Zhang, Congcong; Oberoi, Pranav; Oelsner, Sarah; Waldmann, Anja; Lindner, Aline; Tonn, Torsten; Wels, Winfried S.

    2017-01-01

    Significant progress has been made in recent years toward realizing the potential of natural killer (NK) cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also the established NK cell line NK-92 is being developed for adoptive immunotherapy, and general safety of infusion of irradiated NK-92 cells has been established in phase I clinical trials with clinical responses observed in some of the cancer patients treated. To enhance their therapeutic utility, NK-92 cells have been modified to express chimeric antigen receptors (CARs) composed of a tumor-specific single chain fragment variable antibody fragment fused via hinge and transmembrane regions to intracellular signaling moieties such as CD3ζ or composite signaling domains containing a costimulatory protein together with CD3ζ. CAR-mediated activation of NK cells then bypasses inhibitory signals and overcomes NK resistance of tumor cells. In contrast to primary NK cells, CAR-engineered NK-92 cell lines suitable for clinical development can be established from molecularly and functionally well-characterized single cell clones following good manufacturing practice-compliant procedures. In preclinical in vitro and in vivo models, potent antitumor activity of NK-92 variants targeted to differentiation antigens expressed by hematologic malignancies, and overexpressed or mutated self-antigens associated with solid tumors has been found, encouraging further development of CAR-engineered NK-92 cells. Importantly, in syngeneic mouse tumor models, induction of endogenous antitumor immunity after treatment with CAR-expressing NK-92 cells has been demonstrated, resulting in cures and long-lasting immunological memory protecting against tumor rechallenge at distant sites. Here, we summarize the current status and future prospects of CAR

  12. Phage displaying peptides mimic schistosoma antigenic epitopes selected by rat natural antibodies and protective immunity induced by their immunization in mice.

    PubMed

    Wang, Min; Yi, Xin-Yuan; Li, Xian-Ping; Zhou, Dong-Ming; Larry, McReynolds; Zeng, Xian-Fang

    2005-05-21

    To obtain the short peptides mimic antigenic epitopes selected by rat natural antibodies to schistosomes, and to explore their immunoprotection against schistosomiasis in mice. Adults worm antigens (AWA) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked transferred immunoblotting methods with normal SD rat sera (NRS). The killing effects on schistosomula with fresh and heat-inactivated sera from SD rats were observed. Then the purified IgG from sera of SD rats was used to biopan a phage random peptide library and 20 randomly selected positive clones were detected by ELISA and 2 of them were sequenced. Sixty female mice were immunized thrice with positive phage clones (0, 2nd), 4th wk). Each mouse was challenged with 40 cercariae, and all mice were killed 42 d after challenge. The worms and the liver eggs were counted. NRS could specifically react to the molecules of 75,000, 47,000, 34,500 and 23,000 of AWA. Sera from SD rats showed that the mortality rate of schistosomula was 76.2%, and when the sera were heat-inactivated in vitro, the mortality rate was decreased to 41.0% after being cultured for 48 h. The specific phages bound to IgG were enriched about 300-folds after three rounds of biopanning. Twenty clones were detected by ELISA, 19 of them bound to the specific IgG of rat sera. Immunization with these epitopes was carried out in mice. Compared with the control groups, the mixture of two mimic peptides could induce 34.9% (P=0.000) worm reduction and 67.6% (P=0.000) total liver egg reduction in mice. Two different mimic peptides could respectively induce 31.0% (P=0.001), 14.5% (P=0.074) worm reduction and 61.2% (P=0.000), 35.7% (P=0.000) total liver egg reduction. The specific antibody could be induced by immunization of the mimic peptides, and the antibody titer in immunized mice reached more than 1:6,400 as detected by ELISA. Specific peptides mimic antigenic molecules can be obtained by biopanning the

  13. Vaccination with Replication Deficient Adenovectors Encoding YF-17D Antigens Induces Long-Lasting Protection from Severe Yellow Fever Virus Infection in Mice.

    PubMed

    Bassi, Maria R; Larsen, Mads A B; Kongsgaard, Michael; Rasmussen, Michael; Buus, Søren; Stryhn, Anette; Thomsen, Allan R; Christensen, Jan P

    2016-02-01

    The live attenuated yellow fever vaccine (YF-17D) has been successfully used for more than 70 years. It is generally considered a safe vaccine, however, recent reports of serious adverse events following vaccination have raised concerns and led to suggestions that even safer YF vaccines should be developed. Replication deficient adenoviruses (Ad) have been widely evaluated as recombinant vectors, particularly in the context of prophylactic vaccination against viral infections in which induction of CD8+ T-cell mediated immunity is crucial, but potent antibody responses may also be elicited using these vectors. In this study, we present two adenobased vectors targeting non-structural and structural YF antigens and characterize their immunological properties. We report that a single immunization with an Ad-vector encoding the non-structural protein 3 from YF-17D could elicit a strong CD8+ T-cell response, which afforded a high degree of protection from subsequent intracranial challenge of vaccinated mice. However, full protection was only observed using a vector encoding the structural proteins from YF-17D. This vector elicited virus-specific CD8+ T cells as well as neutralizing antibodies, and both components were shown to be important for protection thus mimicking the situation recently uncovered in YF-17D vaccinated mice. Considering that Ad-vectors are very safe, easy to produce and highly immunogenic in humans, our data indicate that a replication deficient adenovector-based YF vaccine may represent a safe and efficient alternative to the classical live attenuated YF vaccine and should be further tested.

  14. CD4+ T Cells Recognizing PE/PPE Antigens Directly or via Cross Reactivity Are Protective against Pulmonary Mycobacterium tuberculosis Infection

    PubMed Central

    Sayes, Fadel; Pawlik, Alexandre; Frigui, Wafa; Gröschel, Matthias I.; Crommelynck, Samuel; Fayolle, Catherine; Cia, Felipe; Bancroft, Gregory J.; Bottai, Daria; Leclerc, Claude; Brosch, Roland; Majlessi, Laleh

    2016-01-01

    Mycobacterium tuberculosis (Mtb), possesses at least three type VII secretion systems, ESX-1, -3 and -5 that are actively involved in pathogenesis and host-pathogen interaction. We recently showed that an attenuated Mtb vaccine candidate (Mtb Δppe25-pe19), which lacks the characteristic ESX-5-associated pe/ppe genes, but harbors all other components of the ESX-5 system, induces CD4+ T-cell immune responses against non-esx-5-associated PE/PPE protein homologs. These T cells strongly cross-recognize the missing esx-5-associated PE/PPE proteins. Here, we characterized the fine composition of the functional cross-reactive Th1 effector subsets specific to the shared PE/PPE epitopes in mice immunized with the Mtb Δppe25-pe19 vaccine candidate. We provide evidence that the Mtb Δppe25-pe19 strain, despite its significant attenuation, is comparable to the WT Mtb strain with regard to: (i) its antigenic repertoire related to the different ESX systems, (ii) the induced Th1 effector subset composition, (iii) the differentiation status of the Th1 cells induced, and (iv) its particular features at stimulating the innate immune response. Indeed, we found significant contribution of PE/PPE-specific Th1 effector cells in the protective immunity against pulmonary Mtb infection. These results offer detailed insights into the immune mechanisms underlying the remarkable protective efficacy of the live attenuated Mtb Δppe25-pe19 vaccine candidate, as well as the specific potential of PE/PPE proteins as protective immunogens. PMID:27467705

  15. Acquisition of Functional Antibodies That Block the Binding of Erythrocyte-Binding Antigen 175 and Protection Against Plasmodium falciparum Malaria in Children.

    PubMed

    Irani, Vashti; Ramsland, Paul A; Guy, Andrew J; Siba, Peter M; Mueller, Ivo; Richards, Jack S; Beeson, James G

    2015-10-15

    The targets and mechanisms of human immunity to malaria are poorly understood, which poses a major barrier to malaria vaccine development. Antibodies play a key role in human immunity and may act by inhibiting receptor-binding functions of key merozoite invasion ligands. Antibodies to the major invasion ligand and vaccine candidate, erythrocyte-binding antigen 175 (EBA-175), have been linked with protection, but how these antibodies function has not been established. We developed 2 new assays that quantify the ability of antibodies to inhibit binding of EBA-175 to its erythrocyte receptor, glycophorin A, using either native or recombinant EBA-175. Binding-inhibitory antibodies were evaluated in a longitudinal cohort study of Papua New Guinean children and related to risk of malaria, age, infection status, and markers of parasite exposure. Binding-inhibition assays (BIAs) were reproducible, and the 2 assays had a high level of agreement. Inhibitory antibodies were common among children, acquired in association with markers of increasing parasite exposure, and high in those children with active infection. Inhibitory antibodies correlated with total immunoglobulin G levels to the EBA-175 binding domain (region II). Importantly, binding-inhibitory antibodies were significantly associated with protection from symptomatic malaria when measured using either BIA. Findings suggest that naturally acquired binding-inhibitory antibodies are an important functional mechanism that contributes to protection against malaria and further supports the potential of EBA-175 as a vaccine candidate. Identifying vaccines and approaches that induce potent binding-inhibitory antibodies may be a valuable strategy in the development of highly efficacious malaria vaccines. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. Eimeria tenella heat shock protein 70 enhances protection of recombinant microneme protein MIC2 subunit antigen vaccination against E. tenella challenge.

    PubMed

    Zhang, Lei; Ma, Liping; Liu, Renqiang; Zhang, Yunfei; Zhang, Shouping; Hu, Chunmei; Song, Meng; Cai, Jianping; Wang, Ming

    2012-09-10

    Heat shock proteins have been reported to stimulate the immune system via innate receptors. Our study found that the novel immunopotentiator, Eimeria tenella (E. tenella) heat shock protein 70 (HSP70), enhanced protective immunity elicited by E. tenella antigen microneme protein 2 (EtMIC2) against avian coccidiosis. It demonstrated that the expression of TLR2 and TLR4 were strongly upregulated in EtHSP70 and EtMIC2 plus EtHSP70 stimulated chicken embryo fibroblasts (CEF) compared with untreated controls and EtMIC2 alone. In addition, the same treatment induced high levels of interleukin (IL)-12 and interferon (IFN)-γ that are critical cytokines of innate immunity. In vivo experiments involved using broiler chickens subcutaneously immunized with EtMIC2 alone or EtMIC2 plus EtHSP70 at 7 and 14 days post-hatch, which were then orally challenged with live E. tenella at 7 days following secondary immunization. Body weight gains, cecal lesion scores, fecal oocyst shedding, serum antibody responses against MIC2, and intestinal cytokine transcript levels were assessed as measures of protective immunity. Chickens immunized with EtMIC2 plus EtHSP70 showed increased body weight gains, decreased oocyst shedding, increased serum antibody responses, and high levels of IL-12, IFN-γ, and IL-17 compared with the EtMIC2 only or control groups. Moreover, chickens immunized with EtHSP70 alone showed significantly protective effect against E. tenella infection. In summary, this study provides the first evidence of the immunoenhancing activities of EtHSP70 in poultry.

  17. CD4+ T Cells Recognizing PE/PPE Antigens Directly or via Cross Reactivity Are Protective against Pulmonary Mycobacterium tuberculosis Infection.

    PubMed

    Sayes, Fadel; Pawlik, Alexandre; Frigui, Wafa; Gröschel, Matthias I; Crommelynck, Samuel; Fayolle, Catherine; Cia, Felipe; Bancroft, Gregory J; Bottai, Daria; Leclerc, Claude; Brosch, Roland; Majlessi, Laleh

    2016-07-01

    Mycobacterium tuberculosis (Mtb), possesses at least three type VII secretion systems, ESX-1, -3 and -5 that are actively involved in pathogenesis and host-pathogen interaction. We recently showed that an attenuated Mtb vaccine candidate (Mtb Δppe25-pe19), which lacks the characteristic ESX-5-associated pe/ppe genes, but harbors all other components of the ESX-5 system, induces CD4+ T-cell immune responses against non-esx-5-associated PE/PPE protein homologs. These T cells strongly cross-recognize the missing esx-5-associated PE/PPE proteins. Here, we characterized the fine composition of the functional cross-reactive Th1 effector subsets specific to the shared PE/PPE epitopes in mice immunized with the Mtb Δppe25-pe19 vaccine candidate. We provide evidence that the Mtb Δppe25-pe19 strain, despite its significant attenuation, is comparable to the WT Mtb strain with regard to: (i) its antigenic repertoire related to the different ESX systems, (ii) the induced Th1 effector subset composition, (iii) the differentiation status of the Th1 cells induced, and (iv) its particular features at stimulating the innate immune response. Indeed, we found significant contribution of PE/PPE-specific Th1 effector cells in the protective immunity against pulmonary Mtb infection. These results offer detailed insights into the immune mechanisms underlying the remarkable protective efficacy of the live attenuated Mtb Δppe25-pe19 vaccine candidate, as well as the specific potential of PE/PPE proteins as protective immunogens.

  18. DNA vaccine encoding the Toxoplasma gondii bradyzoite-specific surface antigens SAG2CDX protect BALB/c mice against type II parasite infection.

    PubMed

    Zhang, Min; Zhao, Lingxiao; Song, Jing; Li, Ying; Zhao, Qunli; He, Shenyi; Cong, Hua

    2013-09-23

    The surface antigens SAG2C, SAG2D, and SAG2X, which expressed specifically on bradyzoite stage of Toxoplasma gondii, have been demonstrated to be important for persistence of cyst in the brain. In this study, DNA vaccines expressing SAG2C, SAG2D, and SAG2X of T. gondii were constructed and their protective efficacy were evaluated in BALB/c mice. Mice vaccinated with pVAX1-SAG2C (pSAG2C), pVAX1-2D (pSAG2D) or pVAX1-2X (pSAG2C) showed higher levels of serum IgG antibodies and lymphocyte proliferation response compared to PBS and pVAX1 treated mice (p<0.05). The immune response was characterized by a strong Th1 response and increased cytokine production of IL-2 and IFN-γ. Vaccinated mice displayed significant protection against the challenge with the cyst of T. gondii genotype II strain of PRU (cyst-forming in mouse). A significant reduction in the brain cyst burden was detected in the mice immunized with pSAG2C (72%), pSAG2D (23%), pSAG2X (69%) alone and even more reduction rate, 77%, was achieved in the combination group compared to PBS treated mice. The results implied that immunization with DNA vaccines expressing SAG2C, SAG2D, and SAG2X, and, in particular, a combination of all three DNA plasmids, could effectively protect the mice against T. gondii chronic infection.

  19. Antigenic requirement for Gag in a vaccine that protects against high-dose mucosal challenge with simian immunodeficiency virus.

    PubMed

    Schell, John B; Bahl, Kapil; Folta-Stogniew, Ewa; Rose, Nina; Buonocore, Linda; Marx, Preston A; Gambhira, Ratish; Rose, John K

    2015-02-01

    We reported previously on a vaccine approach that conferred apparent sterilizing immunity to SIVsmE660. The vaccine regimen employed a prime-boost using vectors based on recombinant vesicular stomatitis virus (VSV) and an alphavirus replicon expressing either SIV Gag or SIV Env. In the current study, we tested the ability of vectors expressing only the SIVsmE660 Env protein to protect macaques against the same high-dose mucosal challenge. Animals developed neutralizing antibody levels comparable to or greater than seen in the previous vaccine study. When the vaccinated animals were challenged with the same high-dose of SIVsmE660, all became infected. While average peak viral loads in animals were slightly lower than those of previous controls, the viral set points were not significantly different. These data indicate that Gag, or the combination of Gag and Env are required for the generation of apparent sterilizing immunity to the SIVsmE660 challenge.

  20. Vaccination of Mice with Virulence-Associated Protein G (VapG) Antigen Confers Partial Protection against Rhodococcus equi Infection through Induced Humoral Immunity

    PubMed Central

    Trevisani, Marcel M.; Hanna, Ebert S.; Oliveira, Aline F.; Cardoso, Silvia A.; Roque-Barreira, Maria C.; Soares, Sandro G.

    2017-01-01

    Rhodococcus equi is a facultative intracellular bacterium causing severe pyogranulomatous pneumonia, ulcerative enterocolitis, and mesenteric lymphadenopathy in foals aged less than 6 months. Less frequently, this pathogen affects various other species, such as pigs, cattle, cats, and even humans. Although rhodococcosis is treated with a combination of antimicrobial agents, resistance is developed in some cases, and thus, antimicrobial susceptibility must be monitored and managed. Considering these limitations of the current therapy and unavailability of a vaccine to prevent the disease, research is particularly focused on the development of an effective vaccine against rhodococcosis. Most vaccines undergoing development utilize the virulence-associated protein (Vap) A antigen, which was identified previously as a key virulence factor of R. equi. Nevertheless, other proteins, such as VapG, present in most virulent R. equi strains, are also encoded by vap genes located on the R. equi bacterial virulence plasmid. In the present study, we evaluated the effect of VapG immunization on the survival of R. equi-challenged mice. We used attenuated Salmonella as a carrier for VapG (Salmonella-vapG+), a procedure previously adopted to develop a VapA-based vaccine. We observed that vaccination with Salmonella-vapG+ induced both an increased IFN-γ, IL-12, and TNF-α production, and a decreased bacterial burden in organs of the R. equi-challenged mice. Nevertheless, Salmonella-vapG+ vaccination protected only 50% of the mice challenged with a lethal dose of R. equi. Interestingly, we observed an increased frequency of B cells in the spleen of Salmonella-vapG+-vaccinated mice and showed that Salmonella-vapG+-vaccinated R. equi-challenged B-cell-knockout mice did not reduce the bacterial burden. Given these results, we discussed the potential role of the humoral immune response induced by Salmonella-vapG+ vaccination in conferring protection against R. equi infection, as well as

  1. Vaccination of Mice with Virulence-Associated Protein G (VapG) Antigen Confers Partial Protection against Rhodococcus equi Infection through Induced Humoral Immunity.

    PubMed

    Trevisani, Marcel M; Hanna, Ebert S; Oliveira, Aline F; Cardoso, Silvia A; Roque-Barreira, Maria C; Soares, Sandro G

    2017-01-01

    Rhodococcus equi is a facultative intracellular bacterium causing severe pyogranulomatous pneumonia, ulcerative enterocolitis, and mesenteric lymphadenopathy in foals aged less than 6 months. Less frequently, this pathogen affects various other species, such as pigs, cattle, cats, and even humans. Although rhodococcosis is treated with a combination of antimicrobial agents, resistance is developed in some cases, and thus, antimicrobial susceptibility must be monitored and managed. Considering these limitations of the current therapy and unavailability of a vaccine to prevent the disease, research is particularly focused on the development of an effective vaccine against rhodococcosis. Most vaccines undergoing development utilize the virulence-associated protein (Vap) A antigen, which was identified previously as a key virulence factor of R. equi. Nevertheless, other proteins, such as VapG, present in most virulent R. equi strains, are also encoded by vap genes located on the R. equi bacterial virulence plasmid. In the present study, we evaluated the effect of VapG immunization on the survival of R. equi-challenged mice. We used attenuated Salmonella as a carrier for VapG (Salmonella-vapG+), a procedure previously adopted to develop a VapA-based vaccine. We observed that vaccination with Salmonella-vapG+ induced both an increased IFN-γ, IL-12, and TNF-α production, and a decreased bacterial burden in organs of the R. equi-challenged mice. Nevertheless, Salmonella-vapG+ vaccination protected only 50% of the mice challenged with a lethal dose of R. equi. Interestingly, we observed an increased frequency of B cells in the spleen of Salmonella-vapG+-vaccinated mice and showed that Salmonella-vapG+-vaccinated R. equi-challenged B-cell-knockout mice did not reduce the bacterial burden. Given these results, we discussed the potential role of the humoral immune response induced by Salmonella-vapG+ vaccination in conferring protection against R. equi infection, as well as

  2. Evaluation of protective immune responses induced by DNA vaccines encoding Toxoplasma gondii surface antigen 1 (SAG1) and 14-3-3 protein in BALB/c mice.

    PubMed

    Meng, Min; He, Shenyi; Zhao, Guanghui; Bai, Yang; Zhou, Huaiyu; Cong, Hua; Lu, Gang; Zhao, Qunli; Zhu, Xing-Quan

    2012-11-26

    Toxoplasmosis, caused by an obligate intracellular protozoan parasite Toxoplasma gondii, has been a serious clinical and veterinary problem. Effective DNA vaccines against T. gondii can prevent and control the spread of toxoplasmosis, which is important for both human health and the farming industry. The T. gondii 14-3-3 protein has been proved to be antigenic and immunogenic and was a potential vaccine candidate against toxoplasmosis. In this study, we evaluated the immune responses induced by recombinant plasmids encoding T. gondii surface antigen 1 (SAG1) and 14-3-3 protein by immunizing BALB/c mice intramuscularly. In the present study, BALB/c mice were randomly divided into five groups, including three experimental groups (pSAG1, p14-3-3 and pSAG1/14-3-3) and two control groups (PBS and pBudCE4.1), and were immunized intramuscularly three times. The levels of IgG antibodies and cytokine production in mouse sera were determined by enzyme-linked immunosorbent assays (ELISA). Two weeks after the last immunization, all mice were challenged intraperitoneally (i.p.) with 1×10(4) tachyzoites of T. gondii and the survival time of mice was observed and recorded every day. Mice vaccinated with pSAG1, p14-3-3 or pSAG1/14-3-3 developed high levels of IgG2a and gamma interferon (IFN-γ) and low levels of interleukin-4 (IL-4) and interleukin-10 (IL-10) compared to control groups (PBS or pBudCE4.1), which suggested a modulated Th1 type immune response (P<0.05). After intraperitoneal challenge with 1×10(4) tachyzoites of T. gondii (RH strain), the survival time of mice in experimental groups was longer than control groups (P<0.05). Mouse immunized with pSAG1/14-3-3 induced a higher level of IgG antibody response and significantly prolonged the survival time when compared with pSAG1 or p14-3-3 (P<0.05). The study suggested that T. gondii 14-3-3 protein can induce effective immune responses in BALB/c mice and was a novel DNA vaccine candidate against toxoplasmosis, and the

  3. A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoite surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulent Plasmodium chabaudi adami DS malaria.

    PubMed

    Rainczuk, A; Scorza, T; Spithill, T W; Smooker, P M

    2004-10-01

    The ultimate malaria vaccine will require the delivery of multiple antigens from different stages of the complex malaria life cycle. In order to efficiently deliver multiple antigens with use of DNA vaccine technology, new antigen delivery systems must be assessed. This study utilized a bicistronic vector construct, containing an internal ribosome entry site, expressing a combination of malarial candidate antigens: merozoite surface protein 4/5 (MSP4/5) (fused to a monocyte chemotactic protein 3 chemoattractant sequence) and apical membrane antigen 1 (AMA-1) (fused to a tissue plasminogen activator secretion signal). Transfection of COS 7 cells with bicistronic plasmids resulted in production and secretion of both AMA-1 and MSP4/5 in vitro. Vaccination of BALB/c mice via intraepidermal gene gun and intramuscular routes against AMA-1 and MSP4/5 resulted in antibody production and significant in vitro proliferation of splenocytes stimulated by both AMA-1 and MSP4/5. Survival of BALB/c mice vaccinated with bicistronic constructs after lethal Plasmodium chabaudi adami DS erythrocytic-stage challenge was variable, although significant increases in survival and reductions in peak parasitemia were observed in several challenge trials when the vaccine was delivered by the intramuscular route. This study using a murine model demonstrates that the delivery of malarial antigens via bicistronic vectors is feasible. Further experimentation with bicistronic delivery systems is required for the optimization and refinement of DNA vaccines to effectively prime protective immune responses against malaria.

  4. Use of the surface proteins GapC and Mig of Streptococcus dysgalactiae as potential protective antigens against bovine mastitis.

    PubMed

    Bolton, Alexandra; Song, Xin-Ming; Willson, Philip; Fontaine, Michael C; Potter, Andrew A; Perez-Casal, Jose

    2004-06-01

    Streptococcus dysgalactiae is a significant pathogen associated with bovine mastitis in lactating and nonlactating dairy cows, causing a severe inflammatory response of the mammary gland, which results in major economic losses to the dairy industry. Two proteins from S. dysgalactiae strain SDG8 were tested for their protective capacity against a homologous bacterial challenge in a dry cow model. The first was a bovine plasmin receptor protein (GapC), which shares 99.4% sequence identity to the plasmin-binding Plr protein of group A streptococci. The second protein product was Mig, a alpha2-M-, IgG-, and IgA-binding protein present on the cell surface of SDG8. We investigated the efficacy of immunization with purified recombinant forms of GapC and Mig by measuring the number of somatic cells and assessing the presence of the challenge strain in mammary secretions following challenge. In this model, we found that, although the number of quarters containing SDG8 was significantly reduced in the GapC- but not in the Mig-immunized animals, the somatic cell counts from teat secretions were significantly decreased in both the GapC and Mig vaccinates.

  5. Intranasal delivery of naked DNA encoding the LACK antigen leads to protective immunity against visceral leishmaniasis in mice.

    PubMed

    Gomes, Daniel Cláudio de Oliveira; Pinto, Eduardo Fonseca; de Melo, Luiz Dione Barbosa; Lima, Wallace Pacienza; Larraga, Vicente; Lopes, Ulisses Gazos; Rossi-Bergmann, Bartira

    2007-03-08

    We previously showed that intranasal (i.n.) vaccination with pCIneo plasmid encoding the leishmanial LACK gene (pCIneo-LACK) induces long-lasting protective immunity against cutaneous leishmaniasis in mice. In this work, we proposed to investigate whether the efficacy of i.n. pCIneo-LACK is extensive to visceral leishmaniasis. BALB/c mice received two i.n. doses of 30 microg pCIneo-LACK prior to intravenous (i.v.) infection with Leishmania chagasi. Vaccinated mice developed significantly lower parasite burden in the liver and spleen than control mice receiving empty pCIneo or saline. The spleen cells of vaccinated mice produced significantly increased IFN-gamma and IL-4 concomitant with decreased IL-10 production during infection. Serum levels of specific IgG were elevated whereas TNF-alpha were decreased as compared with controls. These results show that the practical needle-free i.n. pCIneo-LACK vaccine displays potential broad-spectrum activity against leishmaniasis.

  6. Antigenicity, immunogenicity, and protective efficacy of Plasmodium vivax MSP1 PV200l: a potential malaria vaccine subunit.

    PubMed

    Valderrama-Aguirre, Augusto; Quintero, Gustavo; Gómez, Andrés; Castellanos, Alejandro; Pérez, Yobana; Méndez, Fabián; Arévalo-Herrera, Myriam; Herrera, Sócrates

    2005-11-01

    The merozoite surface protein 1 (MSP-1) is expressed in all Plasmodium species and is considered a major malaria vaccine candidate. We found that MSP-1 from Plasmodium vivax (PvMSP-1) contains a region of significant sequence homology with the 190L subunit vaccine derived from the P. falciparum MSP-1. The fragment, termed Pv200L, was expressed as a recombinant protein in Escherichia coli (rPv200L) and used to asses its immunologic relevance as a vaccine target. A cross-sectional, seroepidemiologic study conducted in Buenaventura, Colombia showed that 52.2% (95% confidence interval [CI] = 39.8-64.3) of individuals previously exposed to P. vivax and 72.8% (95% CI = 61.8-82.1) of P. vivax-infected patients had IgG antibodies to rPv200L. Immunization of BALB/c mice and Aotus monkeys induced IgG antibodies (titer > 10(6)) that cross-reacted with P. vivax parasites. Immunized monkeys displayed partial protection against a challenge with P. vivax blood stages. Our results suggest that Pv200L is a new malaria vaccine subunit and deserves further testing.

  7. Preclinical identification of vaccine induced protective correlates in human leukocyte antigen expressing transgenic mice infected with Coccidioides posadasii.

    PubMed

    Hurtgen, Brady J; Castro-Lopez, Natalia; Jiménez-Alzate, Maria Del Pilar; Cole, Garry T; Hung, Chiung-Yu

    2016-10-17

    There is an emerging interest to develop human vaccines against medically-important fungal pathogens and a need for a preclinical animal model to assess vaccine efficacies and protective correlates. HLA-DR4 (DRB1∗0401 allele) transgenic mice express a human major histocompatibility complex class II (MHC II) receptor in such a way that CD4(+) T-cell response is solely restricted by this human molecule. In this study HLA-DR4 transgenic mice were immunized with a live-attenuated vaccine (ΔT) and challenged by the intranasal route with 50-70 Coccidioides posadasii spores, a potentially lethal dose. The same vaccination regimen offers 100% survival for C57BL/6 mice. Conversely, ΔT-vaccinated HLA-DR4 mice displayed 3 distinct manifestations of Coccidioides infection including 40% fatal acute (FAD), 30% disseminated (DD) and 30% pulmonary disease (PD). The latter 2 groups of mice had reduced loss of body weight and survived to at least 50days postchallenge (dpc). These results suggest that ΔT vaccinated HLA-DR4 mice activated heterogeneous immunity against pulmonary Coccidioides infection. Vaccinated HLA-DR4 mice displayed early expansion of Th1 and Th17 cells and recruitment of inflammatory innate cells into Coccidioides-infected lungs during the first 9dpc. While contraction rates of Th cells and the inflammatory response during 14-35dpc significantly differed among the 3 groups of vaccinated HLA-DR4 mice. The FAD group displayed a sharply reduced Th1 and Th17 response, while overwhelmingly recruiting neutrophils into lungs during 9-14days. The FAD group approached moribund by 14dpc. In contrast, vaccinated HLA-DR4 survivors gradually contracted Th cells and inflammatory response with the greatest rate in the PD group. While vaccinated HLA-DR4 mice are susceptible to Coccidioides infection, they are useful for evaluation of vaccine efficacy and identification of immunological correlates against this mycosis.

  8. Intranasal immunization with the cholera toxin B subunit-pneumococcal surface antigen A fusion protein induces protection against colonization with Streptococcus pneumoniae and has negligible impact on the nasopharyngeal and oral microbiota of mice.

    PubMed

    Pimenta, F C; Miyaji, E N; Arêas, A P M; Oliveira, M L S; de Andrade, A L S S; Ho, P L; Hollingshead, S K; Leite, L C C

    2006-08-01

    One of the candidate proteins for a mucosal vaccine antigen against Streptococcus pneumoniae is PsaA (pneumococcal surface antigen A). Vaccines targeting mucosal immunity may raise concerns as to possible alterations in the normal microbiota, especially in the case of PsaA, which was shown to have homologs with elevated sequence identity in other viridans group streptococci. In this work, we demonstrate that intranasal immunization with a cholera toxin B subunit-PsaA fusion protein is able to protect mice against colonization with S. pneumoniae but does not significantly alter the natural oral or nasopharyngeal microbiota of mice.

  9. A vaccine prepared from a non-pathogenic H5N1 influenza virus strain from the influenza virus library conferred protective immunity to chickens against the challenge with antigenically drifted highly pathogenic avian influenza virus.

    PubMed

    Samad, Rozanah Asmah Abdul; Nomura, Naoki; Tsuda, Yoshimi; Manzoor, Rashid; Kajihara, Masahiro; Tomabechi, Daisuke; Sasaki, Takashi; Kokumai, Norihide; Ohgitani, Toshiaki; Okamatsu, Masatoshi; Takada, Ayato; Sakoda, Yoshihiro; Kida, Hiroshi

    2011-02-01

    Inactivated influenza virus vaccine prepared from a non-pathogenic influenza virus strain A/duck/Hokkaido/Vac-1/2004 (H5N1) from the virus library conferred protective immunity to chickens against the challenge of antigenically drifted highly pathogenic avian influenza virus (HPAIV), A/whooper swan/Hokkaido/1/2008 (H5N1). The efficacy of the vaccine was comparable to that prepared from genetically modified HPAIV strain deltaRRRRK rg-A/ whooper swan/Mongolia/3/2005 (H5N1), which is more antigenically related to the challenge virus strain, in chickens.

  10. Oral immunization with HpaA affords therapeutic protective immunity against H. pylori that is reflected by specific mucosal immune responses.

    PubMed

    Nyström, Johanna; Svennerholm, Ann-Mari

    2007-03-30

    In the present study, we evaluated the capacity of Helicobacter pylori adhesin A (HpaA), a H. pylori specific colonization factor, to induce therapeutic protection against H. pylori infection in mice. We found that oral immunization of H. pylori infected mice with HpaA induced protection, i.e. significant reduction in bacterial load in the stomach. This was even more pronounced when a combination of HpaA and urease was used. The protection was strongly related to specific mucosal CD4+ T cell responses with a Th1 profile as well as to mucosal IgA responses locally in the stomach. These findings suggest that HpaA is a promising vaccine candidate antigen for use in a therapeutic vaccine against H. pylori.

  11. Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague

    PubMed Central

    Goodin, Jeremy L.; Nellis, David F.; Powell, Bradford S.; Vyas, Vinay V.; Enama, Jeffrey T.; Wang, Lena C.; Clark, Patrick K.; Giardina, Steven L.; Adamovicz, Jeffery. J.; Michiel, Dennis F.

    2009-01-01

    The F1-V vaccine antigen, protective against Yersinia pestis, exhibits a strong tendency to multimerize that affects larger-scale manufacture and characterization. In this work, the sole F1-V cysteine was replaced with serine by site-directed mutagenesis for characterization of F1-V non-covalent multimer interactions and protective potency without participation by disulfide-linkages. F1-V and F1-VC424S proteins were over-expressed in Escherichia coli, recovered using mechanical lysis/pH-modulation and purified from urea-solubilized soft inclusion bodies, using successive ion-exchange, ceramic hydroxyapatite, and size-exclusion chromatography. This purification method resulted in up to 2 mg per gram of cell paste of 95% pure, mono-disperse protein having ≤ 0.5 endotoxin units per mg by a kinetic chromogenic limulus amoebocyte lysate reactivity assay. Both F1-V and F1-VC424S were monomeric at pH 10.0 and progressively self-associated as pH conditions decreased to pH 6.0. Solution additives were screened for their ability to inhibit F1-V self-association at pH 6.5. An L-arginine buffer provided the greatest stabilizing effect. Conversion to >500-kDa multimers occurred between pH 6.0 and 5.0. Conditions for efficient F1-V adsorption to the cGMP-compatible Alhydrogel® adjuvant were optimized. Side-by-side evaluation for protective potency against subcuta