Science.gov

Sample records for rhabdovirus dna-vaccine depends

  1. The immunogenicity of viral haemorragic septicaemia rhabdovirus (VHSV) DNA vaccines can depend on plasmid regulatory sequences.

    PubMed

    Chico, V; Ortega-Villaizan, M; Falco, A; Tafalla, C; Perez, L; Coll, J M; Estepa, A

    2009-03-18

    A plasmid DNA encoding the viral hemorrhagic septicaemia virus (VHSV)-G glycoprotein under the control of 5' sequences (enhancer/promoter sequence plus both non-coding 1st exon and 1st intron sequences) from carp beta-actin gene (pAE6-G(VHSV)) was compared to the vaccine plasmid usually described the gene expression is regulated by the human cytomegalovirus (CMV) immediate-early promoter (pMCV1.4-G(VHSV)). We observed that these two plasmids produced a markedly different profile in the level and time of expression of the encoded-antigen, and this may have a direct effect upon the intensity and suitability of the in vivo immune response. Thus, fish genetic immunisation assays were carried out to study the immune response of both plasmids. A significantly enhanced specific-antibody response against the viral glycoprotein was found in the fish immunised with pAE6-G(VHSV). However, the protective efficacy against VHSV challenge conferred by both plasmids was similar. Later analysis of the transcription profile of a set of representative immune-related genes in the DNA immunized fish suggested that depending on the plasmid-related regulatory sequences controlling its expression, the plasmid might activate distinct patterns of the immune system. All together, the results from this study mainly point out that the selection of a determinate encoded-antigen/vector combination for genetic immunisation is of extraordinary importance in designing optimised DNA vaccines that, when required for inducing protective immune response, could elicit responses biased to antigen-specific antibodies or cytotoxic T cells generation.

  2. The antiviral defense mechanisms in mandarin fish induced by DNA vaccination against a rhabdovirus.

    PubMed

    Chen, Zhong-Yuan; Lei, Xiao-Ying; Zhang, Qi-Ya

    2012-06-15

    Plasmid DNAs containing Siniperca chuatsi rhabdovirus (SCRV) glycoprotein gene (pcDNA-G) and nucleoprotein gene (pcDNA-N) were constructed, and used to determine the antiviral immune response elicited by DNA vaccination in mandarin fish. In vitro and in vivo expression of the plasmid constructs was confirmed in transfected cells and muscle tissues of vaccinated fish by Western blot, indirect immunofluorescence or RT-PCR analysis. Fish injected with pcDNA-G exhibited protective effect against SCRV challenge with a relative percent survival (RPS) of 77.5%, but no significant protection (RPS of 2.5%) was observed in fish vaccinated with pcDNA-N. Immunohistochemical analysis showed that vaccination with pcDNA-G decreased histological lesions and suppressed the virus replication in fish target organs, e.g. kidney, liver, spleen, gill and heart. Transcriptional analysis further revealed that the expression levels of type I IFN system genes including interferon regulation factor-7 (IRF-7) gene, myxovirus resistance (Mx) gene and virus inhibitory protein (Viperin) gene were strongly up-regulated after injection with pcDNA-G, whereas the level of transcription of immunoglobulin M (IgM) gene did not show a statistically significant change. These results reveal that type I IFN antiviral immune response is rapidly triggered by the plasmid DNA containing rhabdovirus glycoprotein gene in fish, which offers an explanation of molecular mechanisms for DNA vaccination inducing mandarin fish resist to SCRV disease. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. DNA vaccines as a tool for analysing the protective immune response against rhabdoviruses in rainbow trout.

    PubMed

    Lorenzen, N; Lorenzen, E; Einer-Jensen, K; LaPatra, S E

    2002-05-01

    DNA vaccines based on the glycoprotein genes of the salmonid rhabdoviruses VHSV and IHNV have been demonstrated to be very efficient in inducing a protective immune response against the respective diseases in rainbow trout. Nanogram doses of plasmid DNA delivered by intramuscular injection are sufficient to induce high levels of immunity in fingerling-size fish, whereas larger fish require more vaccine for protection. The protection is long lasting and, more surprisingly, is partly established already 4 days post vaccination. The early protection involves cross-protective anti-viral defence mechanisms, while the long duration immunity is highly specific. The nature of these immune response mechanisms is discussed and it is suggested that the efficacy of the vaccines is related to their ability to activate the innate immune system as it is activated by live virus.

  4. Dual DNA vaccination of rainbow trout (Oncorhynchus mykiss) against two different rhabdoviruses, VHSV and IHNV, induces specific divalent protection.

    PubMed

    Einer-Jensen, Katja; Delgado, Lourdes; Lorenzen, Ellen; Bovo, Giuseppe; Evensen, Øystein; Lapatra, Scott; Lorenzen, Niels

    2009-02-18

    DNA vaccines encoding the glycoprotein genes of the salmonid rhabdoviruses VHSV and IHNV are very efficient in eliciting protective immune responses against their respective diseases in rainbow trout (Oncorhynchus mykiss). The early anti-viral response (EAVR) provides protection by 4 days post vaccination and is non-specific and transient while the specific anti-viral response (SAVR) is long lasting and highly specific. Since both VHSV and IHNV are endemic in rainbow trout in several geographical regions of Europe and Atlantic salmon (Salmo salar) on the Pacific coast of North America, co-vaccination against the two diseases would be a preferable option. In the present study we demonstrated that a single injection of mixed DNA vaccines induced long-lasting protection against both individual and a simultaneous virus challenge 80 days post vaccination. Transfected muscle cells at the injection site expressed both G proteins. This study confirms the applied potential of using a combined DNA vaccination for protection of fish against two different rhabdoviral diseases.

  5. An active DNA vaccine against infectious pancreatic necrosis virus (IPNV) with a different mode of action than fish rhabdovirus DNA vaccines.

    PubMed

    Cuesta, A; Chaves-Pozo, E; de Las Heras, A I; Saint-Jean, S Rodríguez; Pérez-Prieto, S; Tafalla, C

    2010-04-26

    Although there are some commercial vaccines available against infectious pancreatic necrosis virus (IPNV), the disease still continues to be a major problem for aquaculture development worldwide. In the current work, we constructed a DNA vaccine against IPNV (pIPNV-PP) by cloning the long open reading frame of the polyprotein encoded by the viral RNA segment A. In vitro, the vaccine is properly translated giving the functional IPNV polyprotein since preVP2, VP2 and VP3 proteins were detected because of the VP4-protease cleavage. EPC cells transfected with the vaccine plasmid expressed the viral proteins and induced the expression of type I interferon (IFN)-induced Mx genes. Furthermore, IPNV synthesized proteins seemed to assemble in virus-like particles as evidenced by electron microscopy. In vivo, rainbow trout specimens were intramuscularly injected with the vaccine and expression of immune-relevant genes, the presence of neutralizing antibodies and effect on viral load was determined. The pIPNV-PP vaccine was expressed at the injection site and up-regulated MHC Ialpha, MHC IIalpha, type-I interferon (IFN), Mx, CD4 and CD8alpha gene expression in the muscle, head kidney or spleen, although to a much lower extent than the up-regulations observed in response to an effective DNA vaccine against viral hemorrhagic septicaemia virus (VHSV). However, the IPNV vaccine was also very effective in terms of acquired immunity since it elicited neutralizing antibodies (in 6 out of 8 trout fingerlings) and decreased 665-fold the viral load after IPNV infection. The effectiveness of this new IPNV DNA vaccine and its possible mechanism of action are discussed and compared to other viral vaccines.

  6. Biotechnology and DNA vaccines for aquatic animals

    USGS Publications Warehouse

    Kurath, G.

    2008-01-01

    Biotechnology has been used extensively in the development of vaccines for aquaculture. Modern molecular methods such as polymerase chain reaction (PCR), cloning and microarray analysis have facilitated antigen discovery, construction of novel candidate vaccines, and assessments of vaccine efficacy, mode of action, and host response. This review focuses on DNA vaccines for finfish to illustrate biotechnology applications in this field. Although DNA vaccines for fish rhabdoviruses continue to show the highest efficacy, DNA vaccines for several other viral and bacterial fish pathogens have now been proven to provide significant protection against pathogen challenge. Studies of the fish rhabdovirus DNA vaccines have elucidated factors that affect DNA vaccine efficacy as well as the nature of the fish innate and adaptive immune responses to DNA vaccines. As tools for managing aquatic animal disease emergencies, DNA vaccines have advantages in speed, flexibility, and safety, and one fish DNA vaccine has been licensed.

  7. Biotechnology and DNA vaccines for aquatic animals.

    PubMed

    Kurath, G

    2008-04-01

    Biotechnology has been used extensively in the development of vaccines for aquaculture. Modern molecular methods such as polymerase chain reaction (PCR), cloning and microarray analysis have facilitated antigen discovery, construction of novel candidate vaccines, and assessments of vaccine efficacy, mode of action, and host response. This review focuses on DNA vaccines for finfish to illustrate biotechnology applications in this field. Although DNA vaccines for fish rhabdoviruses continue to show the highest efficacy, DNA vaccines for several other viral and bacterial fish pathogens have now been proven to provide significant protection against pathogen challenge. Studies of the fish rhabdovirus DNA vaccines have elucidated factors that affect DNA vaccine efficacy as well as the nature of the fish innate and adaptive immune responses to DNA vaccines. As tools for managing aquatic animal disease emergencies, DNA vaccines have advantages in speed, flexibility, and safety, and one fish DNA vaccine has been licensed.

  8. Protection of rainbow trout against infectious hematopoietic necrosis virus four days after specific or semi-specific DNA vaccination

    USGS Publications Warehouse

    LaPatra, S.E.; Corbeil, S.; Jones, G.R.; Shewmaker, W.D.; Lorenzen, N.; Anderson, E.D.; Kurath, G.

    2001-01-01

    A DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was shown to provide significant protection as soon as 4 d after intramuscular vaccination in 2 g rainbow trout (Oncorhynchus mykiss) held at 15??C. Nearly complete protection was also observed at later time points (7, 14, and 28 d) using a standardized waterborne challenge model. In a test of the specificity of this early protection, immunization of rainbow trout with a DNA vaccine against another fish rhabdovirus, viral hemorrhagic septicemia virus, provided a significant level of cross-protection against IHNV challenge for a transient period of time, whereas a rabies virus DNA vaccine was not protective. This indication of distinct early and late protective mechanisms was not dependent on DNA vaccine doses from 0.1 to 2.5 ??g. ?? 2001 Elsevier Science Ltd.

  9. Growth and performance of Atlantic salmon, Salmo salar L., following administration of a rhabdovirus DNA vaccine alone or concurrently with an oil-adjuvanted, polyvalent vaccine.

    PubMed

    Skinner, L A; Schulte, P M; LaPatra, S E; Balfry, S K; McKinley, R S

    2008-09-01

    This research demonstrates for the first time an absence of growth-related side effects in Atlantic salmon, Salmo salar L., following the injection of a DNA vaccine alone or concurrently with a commercially available, polyvalent, oil-adjuvanted vaccine. Using weight and specific growth rate measurements, individually tagged Atlantic salmon were monitored for 2028 degree days (dd) post-vaccination. During this time, DNA-vaccinated fish did not differ in weight, length, condition factor or specific growth rate compared to unvaccinated control fish. While differences in weight were observed between unvaccinated control and concurrently vaccinated fish, there were no significant differences in weight, length, condition factor or specific growth rate between concurrently vaccinated fish and adjuvant-vaccinated fish, suggesting that only adjuvant vaccination affected growth. To further determine if concurrent injection of a DNA vaccine and a polyvalent, oil-adjuvanted vaccine had a physiological impact on the Atlantic salmon, swimming performance tests were performed at 106 dd post-vaccination with U(crit,1), U(crit,2), the U(crit) recovery ratio (RR) and the normalized RR being similar to values obtained from unvaccinated control fish. In summary, this study shows that concurrent injection of a DNA vaccine and a polyvalent, oil-adjuvanted vaccine does not negatively influence the growth or swimming performance of Atlantic salmon compared to adjuvant vaccination alone.

  10. DNA vaccines

    NASA Astrophysics Data System (ADS)

    Gregersen, Jens-Peter

    2001-12-01

    Immunization by genes encoding immunogens, rather than with the immunogen itself, has opened up new possibilities for vaccine research and development and offers chances for new applications and indications for future vaccines. The underlying mechanisms of antigen processing, immune presentation and regulation of immune responses raise high expectations for new and more effective prophylactic or therapeutic vaccines, particularly for vaccines against chronic or persistent infectious diseases and tumors. Our current knowledge and experience of DNA vaccination is summarized and critically reviewed with particular attention to basic immunological mechanisms, the construction of plasmids, screening for protective immunogens to be encoded by these plasmids, modes of application, pharmacokinetics, safety and immunotoxicological aspects. DNA vaccines have the potential to accelerate the research phase of new vaccines and to improve the chances of success, since finding new immunogens with the desired properties is at least technically less demanding than for conventional vaccines. However, on the way to innovative vaccine products, several hurdles have to be overcome. The efficacy of DNA vaccines in humans appears to be much less than indicated by early studies in mice. Open questions remain concerning the persistence and distribution of inoculated plasmid DNA in vivo, its potential to express antigens inappropriately, or the potentially deleterious ability to insert genes into the host cell's genome. Furthermore, the possibility of inducing immunotolerance or autoimmune diseases also needs to be investigated more thoroughly, in order to arrive at a well-founded consensus, which justifies the widespread application of DNA vaccines in a healthy population.

  11. DNA vaccines for aquacultured fish.

    PubMed

    Lorenzen, N; LaPatra, S E

    2005-04-01

    Deoxyribonucleic acid (DNA) vaccination is based on the administration of the gene encoding the vaccine antigen, rather than the antigen itself. Subsequent expression of the antigen by cells in the vaccinated hosts triggers the host immune system. Among the many experimental DNA vaccines tested in various animal species as well as in humans, the vaccines against rhabdovirus diseases in fish have given some of the most promising results. A single intramuscular (IM) injection of microgram amounts of DNA induces rapid and long-lasting protection in farmed salmonids against economically important viruses such as infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV). DNA vaccines against other types of fish pathogens, however, have so far had limited success. The most efficient delivery route at present is IM injection, and suitable delivery strategies for mass vaccination of small fish have yet to be developed. In terms of safety, no adverse effects in the vaccinated fish have been observed to date. As DNA vaccination is a relatively new technology, various theoretical and long-term safety issues related to the environment and the consumer remain to be fully addressed, although inherently the risks should not be any greater than with the commercial fish vaccines that are currently used. Present classification systems lack clarity in distinguishing DNA-vaccinated animals from genetically modified organisms (GMOs), which could raise issues in terms of licensing and public acceptance of the technology. The potential benefits of DNA vaccines for farmed fish include improved animal welfare, reduced environmental impacts of aquaculture activities, increased food quality and quantity, and more sustainable production. Testing under commercial production conditions has recently been initiated in Canada and Denmark.

  12. Immunity to fish rhabdoviruses.

    PubMed

    Purcell, Maureen K; Laing, Kerry J; Winton, James R

    2012-01-01

    Members of the family Rhabdoviridae are single-stranded RNA viruses and globally important pathogens of wild and cultured fish and thus relatively well studied in their respective hosts or other model systems. Here, we review the protective immune mechanisms that fish mount in response to rhabdovirus infections. Teleost fish possess the principal components of innate and adaptive immunity found in other vertebrates. Neutralizing antibodies are critical for long-term protection from fish rhabdoviruses, but several studies also indicate a role for cell-mediated immunity. Survival of acute rhabdoviral infection is also dependent on innate immunity, particularly the interferon (IFN) system that is rapidly induced in response to infection. Paradoxically, rhabdoviruses are sensitive to the effects of IFN but virulent rhabdoviruses can continue to replicate owing to the abilities of the matrix (M) protein to mediate host-cell shutoff and the non‑virion (NV) protein to subvert programmed cell death and suppress functional IFN. While many basic features of the fish immune response to rhabdovirus infections are becoming better understood, much less is known about how factors in the environment affect the ecology of rhabdovirus infections in natural populations of aquatic animals.

  13. Immunity to Fish Rhabdoviruses

    PubMed Central

    Purcell, Maureen K.; Laing, Kerry J.; Winton, James R.

    2012-01-01

    Members of the family Rhabdoviridae are single-stranded RNA viruses and globally important pathogens of wild and cultured fish and thus relatively well studied in their respective hosts or other model systems. Here, we review the protective immune mechanisms that fish mount in response to rhabdovirus infections. Teleost fish possess the principal components of innate and adaptive immunity found in other vertebrates. Neutralizing antibodies are critical for long-term protection from fish rhabdoviruses, but several studies also indicate a role for cell-mediated immunity. Survival of acute rhabdoviral infection is also dependent on innate immunity, particularly the interferon (IFN) system that is rapidly induced in response to infection. Paradoxically, rhabdoviruses are sensitive to the effects of IFN but virulent rhabdoviruses can continue to replicate owing to the abilities of the matrix (M) protein to mediate host-cell shutoff and the non‑virion (NV) protein to subvert programmed cell death and suppress functional IFN. While many basic features of the fish immune response to rhabdovirus infections are becoming better understood, much less is known about how factors in the environment affect the ecology of rhabdovirus infections in natural populations of aquatic animals. PMID:22355456

  14. Immunity to fish rhabdoviruses

    USGS Publications Warehouse

    Purcell, Maureen K.; Laing, Kerry J.; Winton, James R.

    2012-01-01

    Members of the family Rhabdoviridae are single-stranded RNA viruses and globally important pathogens of wild and cultured fish and thus relatively well studied in their respective hosts or other model systems. Here, we review the protective immune mechanisms that fish mount in response to rhabdovirus infections. Teleost fish possess the principal components of innate and adaptive immunity found in other vertebrates. Neutralizing antibodies are critical for long-term protection from fish rhabdoviruses, but several studies also indicate a role for cell-mediated immunity. Survival of acute rhabdoviral infection is also dependent on innate immunity, particularly the interferon (IFN) system that is rapidly induced in response to infection. Paradoxically, rhabdoviruses are sensitive to the effects of IFN but virulent rhabdoviruses can continue to replicate owing to the abilities of the matrix (M) protein to mediate host-cell shutoff and the non-virion (NV) protein to subvert programmed cell death and suppress functional IFN. While many basic features of the fish immune response to rhabdovirus infections are becoming better understood, much less is known about how factors in the environment affect the ecology of rhabdovirus infections in natural populations of aquatic animals.

  15. Fish Rhabdoviruses

    USGS Publications Warehouse

    Kurath, G.; Winton, J.

    2008-01-01

    Many important viral pathogens of fish are members of the family Rhabdoviridae. The viruses in this large group cause significant losses in populations of wild fish as well as among fish reared in aquaculture. Fish rhabdoviruses often have a wide host and geographic range, and infect aquatic animals in both freshwater and seawater. The fish rhabdoviruses comprise a diverse collection of isolates that can be placed in one of two quite different groups: isolates that are members of the established genusNovirhabdovirus, and those that are most similar to members of the genus Vesiculovirus. Because the diseases caused by fish rhabdoviruses are important to aquaculture, diagnostic methods for their detection and identification are well established. In addition to regulations designed to reduce the spread of fish viruses, a significant body of research has addressed methods for the control or prevention of diseases caused by fish rhabdoviruses, including vaccination. The number of reported fish rhabdoviruses continues to grow as a result of the expansion of aquaculture, the increase in global trade, the development of improved diagnostic methods, and heightened surveillance activities. Fish rhabdoviruses serve as useful components of model systems to study vertebrate virus disease, epidemiology, and immunology.

  16. The dose-dependent effect on protection and humoral response to a DNA vaccine against Infectious Hematopoietic Necrosis (IHN) virus in subyearling rainbow trout

    USGS Publications Warehouse

    LaPatra, Scott E.; Corbeil, Serge; Jones, Gerald R.; Shewmaker, William D.; Kurath, Gael

    2000-01-01

    A dose–response study that used the DNA vaccine pIHNw-G against infectious hematopoietic necrosis virus (IHNV) showed that complete and highly significant (P < 0.001) protection against a virus injection challenge can be attained in subyearling rainbow trout Oncorhynchus mykiss (145–160 g, 8- to 10-months-old) 6 weeks after a single intramuscular injection with doses as low as 1 μg. Complete protection was also reproducibly demonstrated at higher vaccine doses; however, no protection was observed with a 0.1-μg dose. Virus-neutralizing antibody titers were detected in fish that had been vaccinated with different doses of the DNA vaccine and then sham-infected; there appeared to be a dose-dependent effect, with higher titers obtained with higher doses of vaccine. The DNA-vaccinated animals that survived virus challenge had significantly (P < 0.05) higher neutralizing antibody titers than sham-infected, DNA-vaccinated control fish. Additionally, the titers detected in the IHN survivors exhibited a significant (P < 0.05) dose-dependent effect, with the highest titers being present in fish that received the highest vaccine doses.

  17. Overview of recent DNA vaccine development for fish

    USGS Publications Warehouse

    Kurath, G.; ,

    2005-01-01

    Since the first description of DNA vaccines for fish in 1996, numerous studies of genetic immunisation against the rhabdovirus pathogens infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV) have established their potential as both highly efficacious biologicals and useful basic research tools. Single small doses of rhabdovirus DNA constructs provide extremely strong protection against severe viral challenge under a variety of conditions. DNA vaccines for several other important fish viruses, bacteria, and parasites are under investigation, but they have not yet shown high efficacy. Therefore, current research is focussed on mechanistic studies to understand the basis of protection, and on improvement of the nucleic acid vaccine applications against a wider range of fish pathogens.

  18. DNA Vaccination in Chickens.

    PubMed

    Gupta, Shishir Kumar; Dey, Sohini; Chellappa, Madhan Mohan

    2016-01-01

    Robust and sustainable development of poultry industry requires prevention of deadly infectious diseases. Vigorous vaccination of the birds is a routine practice; however, the live and inactivated vaccines that are used have inherent disadvantages. New-generation vaccines such as DNA vaccines offer several advantages over conventional vaccines. DNA vaccines, which encode an antigen of interest or multiple antigens in the target host, are stable, easy to produce and administer, do not require cold chain maintenance, and are not affected by the maternal antibodies. In addition, DNA vaccines can also be administered in ovo, and thus, mass vaccination and early induction of immune response can effectively be achieved. In this chapter, we focus on the development of DNA vaccines against important infectious viral as well as parasitic diseases of poultry.

  19. DNA vaccines against tuberculosis.

    PubMed

    Bruffaerts, Nicolas; Huygen, Kris; Romano, Marta

    2014-12-01

    Tuberculosis (TB) remains a major health problem and novel vaccination regimens are urgently needed. DNA vaccines against TB have been tested in various preclinical models and strategies have been developed to increase their immunogenicity in large animal species. DNA vaccines are able to induce a wide variety of immune responses, including CD8(+) T-cell-mediated cytolytic and IFN-γ responses. DNA vaccination may be valuable in heterologous prime-boost strategies with the currently used bacillus Calmette-Guérin (BCG) vaccine. This approach could broaden the antigenic repertoire of BCG and enhance its weak induction of MHC class I-restricted immune responses. DNA vaccines offer a number of advantages over certain other types of vaccines, such as the induction of robust MHC class I-restricted cytotoxic T lymphocyte (CTL), their generic manufacturing platform and their relatively low manufacturing costs. Because of their strong potential for inducing memory responses, DNA vaccines are particularly suited for priming immune responses. Furthermore, DNA vaccine technology may help antigen discovery by facilitating screening of candidate vaccines. Co-administration of BCG with plasmid DNA coding for immunodominant, subdominant and phase-specific antigens, poorly expressed by BCG, may lead to the development of improved TB vaccines.

  20. DNA Vaccination Techniques.

    PubMed

    Fissolo, Nicolás; Montalban, Xavier; Comabella, Manuel

    2016-01-01

    Multiple sclerosis (MS) is the most common inflammatory, demyelinating, and neurodegenerative disorder of the central nervous system (CNS) in humans. Although the etiology of MS remains unknown, several lines of evidence support the notion that autoimmunity against components of the myelin sheath plays a major role in susceptibility to and development of the disease. At present, there are no approved MS therapies aimed specifically toward downregulating antigen-specific autoreactive immune cells. One antigen-specific approach that appears promising for the treatment of MS is DNA vaccination. This technique has demonstrated efficacy in clinical trials while maintaining safety.Here, we describe the generation of DNA vaccines containing immunologically relevant antigens of MS. Moreover, we present a detailed protocol for the prophylactic and therapeutic administration of DNA vaccines via intramuscular injection targeting on the development of experimental autoimmune encephalomyelitis (EAE), an animal model resembling MS.

  1. Initial sequence characterization of the rhabdoviruses of squamate reptiles, including a novel rhabdovirus from a caiman lizard (Dracaena guianensis).

    PubMed

    Wellehan, James F X; Pessier, Allan P; Archer, Linda L; Childress, April L; Jacobson, Elliott R; Tesh, Robert B

    2012-08-17

    Rhabdoviruses infect a variety of hosts, including non-avian reptiles. Consensus PCR techniques were used to obtain partial RNA-dependent RNA polymerase gene sequence from five rhabdoviruses of South American lizards; Marco, Chaco, Timbo, Sena Madureira, and a rhabdovirus from a caiman lizard (Dracaena guianensis). The caiman lizard rhabdovirus formed inclusions in erythrocytes, which may be a route for infecting hematophagous insects. This is the first information on behavior of a rhabdovirus in squamates. We also obtained sequence from two rhabdoviruses of Australian lizards, confirming previous Charleville virus sequence and finding that, unlike a previous sequence report but in agreement with serologic reports, Almpiwar virus is clearly distinct from Charleville virus. Bayesian and maximum likelihood phylogenetic analysis revealed that most known rhabdoviruses of squamates cluster in the Almpiwar subgroup. The exception is Marco virus, which is found in the Hart Park group. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Virus-Like Particle Secretion and Genotype-Dependent Immunogenicity of Dengue Virus Serotype 2 DNA Vaccine

    PubMed Central

    Galula, Jedhan U.; Shen, Wen-Fan; Chuang, Shih-Te

    2014-01-01

    ABSTRACT Dengue virus (DENV), composed of four distinct serotypes, is the most important and rapidly emerging arthropod-borne pathogen and imposes substantial economic and public health burdens. We constructed candidate vaccines containing the DNA of five of the genotypes of dengue virus serotype 2 (DENV-2) and evaluated the immunogenicity, the neutralizing (Nt) activity of the elicited antibodies, and the protective efficacy elicited in mice immunized with the vaccine candidates. We observed a significant correlation between the level of in vitro virus-like particle secretion, the elicited antibody response, and the protective efficacy of the vaccines containing the DNA of the different DENV genotypes in immunized mice. However, higher total IgG antibody levels did not always translate into higher Nt antibodies against homologous and heterologous viruses. We also found that, in contrast to previous reports, more than 50% of total IgG targeted ectodomain III (EDIII) of the E protein, and a substantial fraction of this population was interdomain highly neutralizing flavivirus subgroup-cross-reactive antibodies, such as monoclonal antibody 1B7-5. In addition, the lack of a critical epitope(s) in the Sylvatic genotype virus recognized by interdomain antibodies could be the major cause of the poor protection of mice vaccinated with the Asian 1 genotype vaccine (pVD2-Asian 1) from lethal challenge with virus of the Sylvatic genotype. In conclusion, although the pVD2-Asian 1 vaccine was immunogenic, elicited sufficient titers of Nt antibodies against all DENV-2 genotypes, and provided 100% protection against challenge with virus of the homologous Asian 1 genotype and virus of the heterologous Cosmopolitan genotype, it is critical to monitor the potential emergence of Sylvatic genotype viruses, since vaccine candidates under development may not protect vaccinated humans from these viruses. IMPORTANCE Five genotype-specific dengue virus serotype 2 (DENV-2) DNA vaccine

  3. DNA vaccines: a review.

    PubMed

    Lewis, P J; Babiuk, L A

    1999-01-01

    Therapeutic and prophylactic DNA vaccine clinical trials for a variety of pathogens and cancers are underway (Chattergoon et al., 1997; Taubes, 1997). The speed with which initiation of these trials occurred is no less than astounding; clinical trials for a human immunodeficiency virus (HIV) gp160 DNA-based vaccine were underway within 36 months of the first description of "genetic immunization" (Tang et al., 1992) and within 24 months of publication of the first article describing intramuscular delivery of a DNA vaccine (Ulmer et al., 1993). Despite the relative fervor with which clinical trials have progressed, it can be safely stated that DNA-based vaccines will not be an immunological "silver bullet." In this regard, it was satisfying to see a publication entitled "DNA Vaccines--A Modern Gimmick or a Boon to Vaccinology?" (Manickan et al., 1997b). There is no doubt that this technology is well beyond the phenomenology phase of study. Research niches and models have been established and will allow the truly difficult questions of mechanism and application to target species to be studied. These two aspects of future studies are intricately interwoven and will ultimately determine the necessity for mechanistic understanding and the evolution of target species studies. The basic science of DNA vaccines has yet to be clearly defined and will ultimately determine the success or failure of this technology to find a place in the immunological arsenal against disease. In a commentary on a published study describing DNA vaccine-mediated protection against heterologous challenge with HIV-1 in chimpanzees, Ronald Kennedy (1997) states, "As someone who has been in the trenches of AIDS vaccine research for over a decade and who, together with collaborators, has attempted a number of different vaccine approaches that have not panned out, I have a relatively pessimistic view of new AIDS vaccine approaches." Kennedy then goes on to summarize a DNA-based multigene vaccine

  4. Therapeutic HPV DNA vaccines

    PubMed Central

    Lin, Ken; Roosinovich, Elena; Ma, Barbara; Hung, Chien-Fu

    2010-01-01

    It is now well established that most cervical cancers are causally associated with HPV infection. This realization has led to efforts to control HPV-associated malignancy through prevention or treatment of HPV infection. Currently, commercially available HPV vaccines are not designed to control established HPV infection and associated premalignant and malignant lesions. To treat and eradicate pre-existing HPV infections and associated lesions which remain prevalent in the U.S. and worldwide, effective therapeutic HPV vaccines are needed. DNA vaccination has emerged as a particularly promising form of therapeutic HPV vaccines due to its safety, stability and ability to induce antigen-specific immunity. This review focuses on improving the potency of therapeutic HPV vaccines through modification of dendritic cells (DCs) by [1] increasing the number of antigen-expressing/antigen-loaded DCs, [2] improving HPV antigen expression, processing and presentation in DCs, and [3] enhancing DC and T cell interaction. Continued improvement in therapeutic HPV DNA vaccines may ultimately lead to an effective DNA vaccine for the treatment of HPV-associated malignancies. PMID:20066511

  5. An oral DNA vaccine against infectious haematopoietic necrosis virus (IHNV) encapsulated in alginate microspheres induces dose-dependent immune responses and significant protection in rainbow trout (Oncorrhynchus mykiss).

    PubMed

    Ballesteros, Natalia A; Alonso, Marta; Saint-Jean, Sylvia Rodríguez; Perez-Prieto, Sara I

    2015-08-01

    Administered by intramuscular injection, a DNA vaccine (pIRF1A-G) containing the promoter regions upstream of the rainbow trout interferon regulatory factor 1A gene (IRF1A) driven the expression of the infectious hematopoietic necrosis virus (IHNV) glycoprotein (G) elicited protective immune responses in rainbow trout (Oncorhynchus mykiss). However, less laborious and cost-effective routes of DNA vaccine delivery are required to vaccinate large numbers of susceptible farmed fish. In this study, the pIRF1A-G vaccine was encapsulated into alginate microspheres and orally administered to rainbow trout. At 1, 3, 5, and 7 d post-vaccination, IHNV G transcripts were detected by quantitative real-time PCR in gills, spleen, kidney and intestinal tissues of vaccinated fish. This result suggested that the encapsulation of pIRF1A-G in alginate microparticles protected the DNA vaccine from degradation in the fish stomach and ensured vaccine early delivery to the hindgut, vaccine passage through the intestinal mucosa and its distribution thought internal and external organs of vaccinated fish. We also observed that the oral route required approximately 20-fold more plasmid DNA than the injection route to induce the expression of significant levels of IHNV G transcripts in kidney and spleen of vaccinated fish. Despite this limitation, increased IFN-1, TLR-7 and IgM gene expression was detected by qRT-PCR in kidney of vaccinated fish when a 10 μg dose of the oral pIRF1A-G vaccine was administered. In contrast, significant Mx-1, Vig-1, Vig-2, TLR-3 and TLR-8 gene expression was only detected when higher doses of pIRF1A-G (50 and 100 μg) were orally administered. The pIRF1A-G vaccine also induced the expression of several markers of the adaptive immune response (CD4, CD8, IgM and IgT) in kidney and spleen of immunized fish in a dose-dependent manner. When vaccinated fish were challenged by immersion with live IHNV, evidence of a dose-response effect of the oral vaccine could also

  6. Development of dengue DNA vaccines.

    PubMed

    Danko, Janine R; Beckett, Charmagne G; Porter, Kevin R

    2011-09-23

    Vaccination with plasmid DNA against infectious pathogens including dengue is an active area of investigation. By design, DNA vaccines are able to elicit both antibody responses and cellular immune responses capable of mediating long-term protection. Great technical improvements have been made in dengue DNA vaccine constructs and trials are underway to study these in the clinic. The scope of this review is to highlight the rich history of this vaccine platform and the work in dengue DNA vaccines accomplished by scientists at the Naval Medical Research Center. This work resulted in the only dengue DNA vaccine tested in a clinical trial to date. Additional advancements paving the road ahead in dengue DNA vaccine development are also discussed.

  7. DNA vaccines in veterinary use.

    PubMed

    Redding, Laurel; Weiner, David B

    2009-09-01

    DNA vaccines represent a new frontier in vaccine technology. One important application of this technology is in the veterinary arena. DNA vaccines have already gained a foothold in certain fields of veterinary medicine. However, several important questions must be addressed when developing DNA vaccines for animals, including whether or not the vaccine is efficacious and cost effective compared with currently available options. Another important question to consider is how to apply this developing technology in a wide range of different situations, from the domestic pet to individual fish in fisheries with several thousand animals, to wildlife programs for disease control. In some cases, DNA vaccines represent an interesting option for vaccination, while in others, currently available options are sufficient. This review will examine a number of diseases of veterinary importance and the progress being made in DNA vaccine technology relevant to these diseases, and we compare these with the conventional treatment options available.

  8. DNA vaccine for cancer immunotherapy

    PubMed Central

    Yang, Benjamin; Jeang, Jessica; Yang, Andrew; Wu, T C; Hung, Chien-Fu

    2014-01-01

    DNA vaccination has emerged as an attractive immunotherapeutic approach against cancer due to its simplicity, stability, and safety. Results from numerous clinical trials have demonstrated that DNA vaccines are well tolerated by patients and do not trigger major adverse effects. DNA vaccines are also very cost effective and can be administered repeatedly for long-term protection. Despite all the practical advantages, DNA vaccines face challenges in inducing potent antigen specific cellular immune responses as a result of immune tolerance against endogenous self-antigens in tumors. Strategies to enhance immunogenicity of DNA vaccines against self-antigens have been investigated including encoding of xenogeneic versions of antigens, fusion of antigens to molecules that activate T cells or trigger associative recognition, priming with DNA vectors followed by boosting with viral vector, and utilization of immunomodulatory molecules. This review will focus on discussing strategies that circumvent immune tolerance and provide updates on findings from recent clinical trials. PMID:25625927

  9. DNA vaccines in veterinary use

    PubMed Central

    Redding, Laurel; Werner, David B

    2015-01-01

    DNA vaccines represent a new frontier in vaccine technology. One important application of this technology is in the veterinary arena. DNA vaccines have already gained a foothold in certain fields of veterinary medicine. However, several important questions must be addressed when developing DNA vaccines for animals, including whether or not the vaccine is efficacious and cost effective compared with currently available options. Another important question to consider is how to apply this developing technology in a wide range of different situations, from the domestic pet to individual fish in fisheries with several thousand animals, to wildlife programs for disease control. In some cases, DNA vaccines represent an interesting option for vaccination, while in others, currently available options are sufficient. This review will examine a number of diseases of veterinary importance and the progress being made in DNA vaccine technology relevant to these diseases, and we compare these with the conventional treatment options available. PMID:19722897

  10. DNA vaccine for cancer immunotherapy.

    PubMed

    Yang, Benjamin; Jeang, Jessica; Yang, Andrew; Wu, T C; Hung, Chien-Fu

    2014-01-01

    DNA vaccination has emerged as an attractive immunotherapeutic approach against cancer due to its simplicity, stability, and safety. Results from numerous clinical trials have demonstrated that DNA vaccines are well tolerated by patients and do not trigger major adverse effects. DNA vaccines are also very cost effective and can be administered repeatedly for long-term protection. Despite all the practical advantages, DNA vaccines face challenges in inducing potent antigen specific cellular immune responses as a result of immune tolerance against endogenous self-antigens in tumors. Strategies to enhance immunogenicity of DNA vaccines against self-antigens have been investigated including encoding of xenogeneic versions of antigens, fusion of antigens to molecules that activate T cells or trigger associative recognition, priming with DNA vectors followed by boosting with viral vector, and utilization of immunomodulatory molecules. This review will focus on discussing strategies that circumvent immune tolerance and provide updates on findings from recent clinical trials.

  11. DNA vaccines: roles against diseases

    PubMed Central

    Khan, Kishwar Hayat

    2013-01-01

    Vaccination is the most successful application of immunological principles to human health. Vaccine efficacy needs to be reviewed from time to time and its safety is an overriding consideration. DNA vaccines offer simple yet effective means of inducing broad-based immunity. These vaccines work by allowing the expression of the microbial antigen inside host cells that take up the plasmid. These vaccines function by generating the desired antigen inside the cells, with the advantage that this may facilitate presentation through the major histocompatibility complex. This review article is based on a literature survey and it describes the working and designing strategies of DNA vaccines. Advantages and disadvantages for this type of vaccines have also been explained, together with applications of DNA vaccines. DNA vaccines against cancer, tuberculosis, Edwardsiella tarda, HIV, anthrax, influenza, malaria, dengue, typhoid and other diseases were explored. PMID:24432284

  12. Introduction of translation stop condons into the viral glycoprotein gene in a fish DNA vaccine eliminates induction of protective immunity

    USGS Publications Warehouse

    Garver, Kyle A.; Conway, Carla M.; Kurath, Gael

    2006-01-01

    A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was mutated to introduce two stop codons to prevent glycoprotein translation while maintaining the plasmid DNA integrity and RNA transcription ability. The mutated plasmid vaccine, denoted pIHNw-G2stop, when injected intramuscularly into fish at high doses, lacked detectable glycoprotein expression in the injection site muscle, and did not provide protection against lethal virus challenge 7 days post-vaccination. These results suggest that the G-protein itself is required to stimulate the early protective antiviral response observed after vaccination with the nonmutated parental DNA vaccine.

  13. Introduction of translation stop codons into the viral glycoprotein gene in a fish DNA vaccine eliminates induction of protective immunity

    USGS Publications Warehouse

    Garver, K.A.; Conway, C.M.; Kurath, G.

    2006-01-01

    A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was mutated to introduce two stop codons to prevent glycoprotein translation while maintaining the plasmid DNA integrity and RNA transcription ability. The mutated plasmid vaccine, denoted pIHNw-G2stop, when injected intramuscularly into fish at high doses, lacked detectable glycoprotein expression in the injection site muscle, and did not provide protection against lethal virus challenge 7 days post-vaccination. These results suggest that the G-protein itself is required to stimulate the early protective antiviral response observed after vaccination with the nonmutated parental DNA vaccine. ?? Springer Science+Business Media, Inc. 2006.

  14. A cGAS-Independent STING/IRF7 Pathway Mediates the Immunogenicity of DNA Vaccines.

    PubMed

    Suschak, John J; Wang, Shixia; Fitzgerald, Katherine A; Lu, Shan

    2016-01-01

    It has been known since the discovery of DNA vaccines >20 y ago that DNA vaccines can function as adjuvants. Our recent study reported the involvement of Aim2 as the sensor of DNA vaccines in eliciting Ag-specific Ab responses. Our findings indicated the presence of previously unrecognized innate immune response pathways in addition to the TLR9 pathway, which is mainly activated by the CpG motifs of DNA vaccines. Our data further demonstrated the requirement of type I IFN in DNA vaccine-induced immune responses via the Aim2 pathway, but the exact downstream molecular mechanism was not characterized. In the present study, we investigated the roles of the putative DNA sensor cyclic GMP-AMP synthase (cGas), as well as the downstream IFN regulatory factors (IRF) 3 and 7 in type I IFN induction and Ag-specific immune responses elicited by DNA vaccination. Our results showed that DNA vaccine-induced, Irf7-dependent signaling, as part of the Sting pathway, was critical for generation of both innate cytokine signaling and Ag-specific B and T cell responses. In contrast, Irf3 was not as critical as expected in this pathway and, more surprisingly, immune responses elicited by DNA vaccines were not cGas-dependent in vivo. Data from this study provide more details on the innate immune mechanisms involved in DNA vaccination and further enrich our understanding on the potential utility of DNA vaccines in generating Ag-specific immune responses.

  15. Current progress of DNA vaccine studies in humans.

    PubMed

    Lu, Shan; Wang, Shixia; Grimes-Serrano, Jill M

    2008-03-01

    Despite remarkable progress in the field of DNA vaccine research since its discovery in the early 1990 s, the formal acceptance of this novel technology as a new modality of human vaccines depends on the successful demonstration of its safety and efficacy in advanced clinical trials. Although clinical trials conducted so far have provided overwhelming evidence that DNA vaccines are well tolerated and have an excellent safety profile, the early designs of DNA vaccines failed to demonstrate sufficient immunogenicity in humans. However, studies conducted over the last few years have led to promising results, particularly when DNA vaccines were used in combination with other forms of vaccines. Here, we provide a review of the data from reported DNA vaccine clinical studies with an emphasis on the ability of DNA vaccines to elicit antigen-specific, cell-mediated and antibody responses in humans. The majority of these trials are designed to test candidate vaccines against several major human pathogens and the remaining studies tested the immunogenicity of therapeutic vaccines against cancer.

  16. DNA vaccines expressing the duck hepatitis B virus surface proteins lead to reduced numbers of infected hepatocytes and protect ducks against the development of chronic infection in a virus dose-dependent manner.

    PubMed

    Miller, Darren S; Kotlarski, Ieva; Jilbert, Allison R

    2006-07-20

    We tested the efficacy of DNA vaccines expressing the duck hepatitis B virus (DHBV) pre-surface (pre-S/S) and surface (S) proteins in modifying the outcome of infection in 14-day-old ducks. In two experiments, Pekin Aylesbury ducks were vaccinated on days 4 and 14 of age with plasmid DNA vaccines expressing either the DHBV pre-S/S or S proteins, or the control plasmid vector, pcDNA1.1Amp. All ducks were then challenged intravenously on day 14 of age with 5 x 10(7) or 5 x 10(8) DHBV genomes. Levels of initial DHBV infection were assessed using liver biopsy tissue collected at day 4 post-challenge (p.c.) followed and immunostained for DHBV surface antigen to determine the percentage of infected hepatocytes. All vector vaccinated ducks challenged with 5 x 10(7) and 5 x 10(8) DHBV genomes had an average of 3.21% and 20.1% of DHBV-positive hepatocytes respectively at day 4 p.c. and 16 out of 16 ducks developed chronic DHBV infection. In contrast, pre-S/S and S vaccinated ducks challenged with 5 x 10(7) DHBV genomes had reduced levels of initial infection with an average of 1.38% and 1.93% of DHBV-positive hepatocytes at day 4 p.c. respectively and 10 of 18 ducks were protected against chronic infection. The pre-S/S and the S DNA vaccinated ducks challenged with 5 x 10(8) DHBV genomes had an average of 31.5% and 9.2% of DHBV-positive hepatocytes on day 4 p.c. respectively and only 4 of the 18 vaccinated ducks were protected against chronic infection. There was no statistically significant difference in the efficacy of the DHBV pre-S/S or S DNA vaccines. In conclusion, vaccination of young ducks with DNA vaccines expressing the DHBV pre-S/S and S proteins induced rapid immune responses that reduced the extent of initial DHBV infection in the liver and prevented the development of chronic infection in a virus dose-dependent manner.

  17. Rhabdovirus accessory genes.

    PubMed

    Walker, Peter J; Dietzgen, Ralf G; Joubert, D Albert; Blasdell, Kim R

    2011-12-01

    The Rhabdoviridae is one of the most ecologically diverse families of RNA viruses with members infecting a wide range of organisms including placental mammals, marsupials, birds, reptiles, fish, insects and plants. The availability of complete nucleotide sequences for an increasing number of rhabdoviruses has revealed that their ecological diversity is reflected in the diversity and complexity of their genomes. The five canonical rhabdovirus structural protein genes (N, P, M, G and L) that are shared by all rhabdoviruses are overprinted, overlapped and interspersed with a multitude of novel and diverse accessory genes. Although not essential for replication in cell culture, several of these genes have been shown to have roles associated with pathogenesis and apoptosis in animals, and cell-to-cell movement in plants. Others appear to be secreted or have the characteristics of membrane-anchored glycoproteins or viroporins. However, most encode proteins of unknown function that are unrelated to any other known proteins. Understanding the roles of these accessory genes and the strategies by which rhabdoviruses use them to engage, divert and re-direct cellular processes will not only present opportunities to develop new anti-viral therapies but may also reveal aspects of cellar function that have broader significance in biology, agriculture and medicine. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

  18. DNA Vaccines for Prostate Cancer

    PubMed Central

    McNeel, Douglas G.; Becker, Jordan T.; Johnson, Laura E.; Olson, Brian M.

    2013-01-01

    Delivery of plasmid DNA encoding an antigen of interest has been demonstrated to be an effective means of immunization, capable of eliciting antigen-specific T cells. Plasmid DNA vaccines offer advantages over other anti-tumor vaccine approaches in terms of simplicity, manufacturing, and possibly safety. The primary disadvantage is their poor transfection efficiency and subsequent lower immunogenicity relative to other genetic vaccine approaches. However, multiple preclinical models demonstrate anti-tumor efficacy, and many efforts are underway to improve the immunogenicity and anti-tumor effect of these vaccines. Clinical trials using DNA vaccines as treatments for prostate cancer have begun, and to date have demonstrated safety and immunological effect. This review will focus on DNA vaccines as a specific means of antigen delivery, advantages and disadvantages of this type of immunization, previous experience in preclinical models and human trials specifically conducted for the treatment of prostate cancer, and future directions for the application of DNA vaccines to prostate cancer immunotherapy. PMID:24587772

  19. Can VHS Virus Bypass the Protective Immunity Induced by DNA Vaccination in Rainbow Trout?

    PubMed

    Sepúlveda, Dagoberto; Lorenzen, Niels

    2016-01-01

    DNA vaccines encoding viral glycoproteins have been very successful for induction of protective immunity against diseases caused by rhabdoviruses in cultured fish species. However, the vaccine concept is based on a single viral gene and since RNA viruses are known to possess high variability and adaptation capacity, this work aimed at evaluating whether viral haemorrhagic septicaemia virus (VHSV), an RNA virus and member of Rhabdoviridae family, was able to evade the protective immune response induced by the DNA vaccination of rainbow trout. The experiments comprised repeated passages of a highly pathogenic VHSV isolate in a fish cell line in the presence of neutralizing fish serum (in vitro approach), and in rainbow trout immunized with the VHS DNA vaccine (in vivo approach). For the in vitro approach, the virus collected from the last passage (passaged virus) was as sensitive as the parental virus to serum neutralization, suggesting that the passaging did not promote the selection of virus populations able to bypass the neutralization by serum antibodies. Also, in the in vivo approach, where virus was passaged several times in vaccinated fish, no increased virulence nor increased persistence in vaccinated fish was observed in comparison with the parental virus. However, some of the vaccinated fish did get infected and could transmit the infection to naïve cohabitant fish. The results demonstrated that the DNA vaccine induced a robust protection, but also that the immunity was non-sterile. It is consequently important not to consider vaccinated fish as virus free in veterinary terms.

  20. Can VHS Virus Bypass the Protective Immunity Induced by DNA Vaccination in Rainbow Trout?

    PubMed Central

    Sepúlveda, Dagoberto; Lorenzen, Niels

    2016-01-01

    DNA vaccines encoding viral glycoproteins have been very successful for induction of protective immunity against diseases caused by rhabdoviruses in cultured fish species. However, the vaccine concept is based on a single viral gene and since RNA viruses are known to possess high variability and adaptation capacity, this work aimed at evaluating whether viral haemorrhagic septicaemia virus (VHSV), an RNA virus and member of Rhabdoviridae family, was able to evade the protective immune response induced by the DNA vaccination of rainbow trout. The experiments comprised repeated passages of a highly pathogenic VHSV isolate in a fish cell line in the presence of neutralizing fish serum (in vitro approach), and in rainbow trout immunized with the VHS DNA vaccine (in vivo approach). For the in vitro approach, the virus collected from the last passage (passaged virus) was as sensitive as the parental virus to serum neutralization, suggesting that the passaging did not promote the selection of virus populations able to bypass the neutralization by serum antibodies. Also, in the in vivo approach, where virus was passaged several times in vaccinated fish, no increased virulence nor increased persistence in vaccinated fish was observed in comparison with the parental virus. However, some of the vaccinated fish did get infected and could transmit the infection to naïve cohabitant fish. The results demonstrated that the DNA vaccine induced a robust protection, but also that the immunity was non-sterile. It is consequently important not to consider vaccinated fish as virus free in veterinary terms. PMID:27054895

  1. Dose-Dependent Protection against or Exacerbation of Disease by a Polylactide Glycolide Microparticle-Adsorbed, Alphavirus-Based Measles Virus DNA Vaccine in Rhesus Macaques▿

    PubMed Central

    Pan, Chien-Hsiung; Nair, Nitya; Adams, Robert J.; Zink, M. Christine; Lee, Eun-Young; Polack, Fernando P.; Singh, Manmohan; O'Hagan, Derek T.; Griffin, Diane E.

    2008-01-01

    Measles remains an important cause of vaccine-preventable child mortality. Development of a low-cost, heat-stable vaccine for infants under the age of 6 months could improve measles control by facilitating delivery at the time of other vaccines and by closing a window of susceptibility prior to immunization at 9 months of age. DNA vaccines hold promise for development, but achieving protective levels of antibody has been difficult and there is an incomplete understanding of protective immunity. In the current study, we evaluated the use of a layered alphavirus DNA/RNA vector encoding measles virus H (SINCP-H) adsorbed onto polylactide glycolide (PLG) microparticles. In mice, antibody and T-cell responses to PLG-formulated DNA were substantially improved compared to those to naked DNA. Rhesus macaques received two doses of PLG/SINCP-H delivered either intramuscularly (0.5 mg) or intradermally (0.5 or 0.1 mg). Antibody and T-cell responses were induced but not sustained. On challenge, the intramuscularly vaccinated monkeys did not develop rashes and had lower viremias than vector-treated control monkeys. Monkeys vaccinated with the same dose intradermally developed rashes and viremia. Monkeys vaccinated intradermally with the low dose developed more severe rashes, with histopathologic evidence of syncytia and intense dermal and epidermal inflammation, eosinophilia, and higher viremia compared to vector-treated control monkeys. Protection after challenge correlated with gamma interferon-producing T cells and with early production of high-avidity antibody that bound wild-type H protein. We conclude that PLG/SINCP-H is most efficacious when delivered intramuscularly but does not provide an advantage over standard DNA vaccines for protection against measles. PMID:18287579

  2. Molecular mechanisms for enhanced DNA vaccine immunogenicity

    PubMed Central

    Li, Lei; Petrovsky, Nikolai

    2016-01-01

    Summary In the two decades since their initial discovery, DNA vaccines technologies have come a long way. Unfortunately, when applied to human subjects inadequate immunogenicity is still the biggest challenge for practical DNA vaccine use. Many different strategies have been tested in preclinical models to address this problem, including novel plasmid vectors and codon optimization to enhance antigen expression, new gene transfection systems or electroporation to increase delivery efficiency, protein or live virus vector boosting regimens to maximise immune stimulation, and formulation of DNA vaccines with traditional or molecular adjuvants. Better understanding of the mechanisms of action of DNA vaccines has also enabled better use of the intrinsic host response to DNA to improve vaccine immunogenicity. This review summarizes recent advances in DNA vaccine technologies and related intracellular events and how these might impact on future directions of DNA vaccine development. PMID:26707950

  3. Molecular mechanisms for enhanced DNA vaccine immunogenicity.

    PubMed

    Li, Lei; Petrovsky, Nikolai

    2016-01-01

    In the two decades since their initial discovery, DNA vaccines technologies have come a long way. Unfortunately, when applied to human subjects inadequate immunogenicity is still the biggest challenge for practical DNA vaccine use. Many different strategies have been tested in preclinical models to address this problem, including novel plasmid vectors and codon optimization to enhance antigen expression, new gene transfection systems or electroporation to increase delivery efficiency, protein or live virus vector boosting regimens to maximise immune stimulation, and formulation of DNA vaccines with traditional or molecular adjuvants. Better understanding of the mechanisms of action of DNA vaccines has also enabled better use of the intrinsic host response to DNA to improve vaccine immunogenicity. This review summarizes recent advances in DNA vaccine technologies and related intracellular events and how these might impact on future directions of DNA vaccine development.

  4. Molecular and Cellular Aspects of Rhabdovirus Entry

    PubMed Central

    Albertini, Aurélie A. V.; Baquero, Eduard; Ferlin, Anna; Gaudin, Yves

    2012-01-01

    Rhabdoviruses enter the cell via the endocytic pathway and subsequently fuse with a cellular membrane within the acidic environment of the endosome. Both receptor recognition and membrane fusion are mediated by a single transmembrane viral glycoprotein (G). Fusion is triggered via a low-pH induced structural rearrangement. G is an atypical fusion protein as there is a pH-dependent equilibrium between its pre- and post-fusion conformations. The elucidation of the atomic structures of these two conformations for the vesicular stomatitis virus (VSV) G has revealed that it is different from the previously characterized class I and class II fusion proteins. In this review, the pre- and post-fusion VSV G structures are presented in detail demonstrating that G combines the features of the class I and class II fusion proteins. In addition to these similarities, these G structures also reveal some particularities that expand our understanding of the working of fusion machineries. Combined with data from recent studies that revealed the cellular aspects of the initial stages of rhabdovirus infection, all these data give an integrated view of the entry pathway of rhabdoviruses into their host cell. PMID:22355455

  5. Therapeutic targeting of liver cancer with a recombinant DNA vaccine containing the hemagglutinin-neuraminidase gene of Newcastle disease virus via apoptotic-dependent pathways

    PubMed Central

    Chen, Li-Gang; Liu, Yuan-Sheng; Zheng, Tang-Hui; Chen, Xu; Li, Ping; Xiao, Chuan-Xing; Ren, Jian-Lin

    2016-01-01

    A total of ~38.6 million mortalities occur due to liver cancer annually, worldwide. Although a variety of therapeutic methods are available, the efficacy of treatment at present is extremely limited due to an increased risk of malignancy and inherently poor prognosis of liver cancer. Gene therapy is considered a promising option, and has shown notable potential for the comprehensive therapy of liver cancer, in keeping with advances that have been made in the development of cancer molecular biology. The present study aimed to investigate the synergistic effects of the abilities of the hemagglutinin neuraminidase protein of Newcastle disease virus (NDV), the pro-apoptotic factor apoptin from chicken anaemia virus, and the interferon-γ inducer interleukin-18 (IL-18) in antagonizing liver cancer. Therefore, a recombinant DNA plasmid expressing the three exogenous genes, VP3, IL-18 and hemagglutinin neuraminidase (HN), was constructed. Flow cytometry, acridine orange/ethidium bromide staining and analysis of caspase-3 activity were performed in H22 cell lines transfected with the recombinant DNA plasmid. In addition, 6-week-old C57BL/6 mice were used to establish a H22 hepatoma-bearing mouse model. Mice tumor tissue was analyzed by immunohistochemistry and scanning electron microscopy. The results of the present study revealed that the recombinant DNA vaccine containing the VP3, IL-18 and HN genes inhibited cell proliferation and induced autophagy via the mitochondrial pathway in vivo and in vitro. PMID:27900002

  6. Therapeutic targeting of liver cancer with a recombinant DNA vaccine containing the hemagglutinin-neuraminidase gene of Newcastle disease virus via apoptotic-dependent pathways.

    PubMed

    Chen, Li-Gang; Liu, Yuan-Sheng; Zheng, Tang-Hui; Chen, Xu; Li, Ping; Xiao, Chuan-Xing; Ren, Jian-Lin

    2016-11-01

    A total of ~38.6 million mortalities occur due to liver cancer annually, worldwide. Although a variety of therapeutic methods are available, the efficacy of treatment at present is extremely limited due to an increased risk of malignancy and inherently poor prognosis of liver cancer. Gene therapy is considered a promising option, and has shown notable potential for the comprehensive therapy of liver cancer, in keeping with advances that have been made in the development of cancer molecular biology. The present study aimed to investigate the synergistic effects of the abilities of the hemagglutinin neuraminidase protein of Newcastle disease virus (NDV), the pro-apoptotic factor apoptin from chicken anaemia virus, and the interferon-γ inducer interleukin-18 (IL-18) in antagonizing liver cancer. Therefore, a recombinant DNA plasmid expressing the three exogenous genes, VP3, IL-18 and hemagglutinin neuraminidase (HN), was constructed. Flow cytometry, acridine orange/ethidium bromide staining and analysis of caspase-3 activity were performed in H22 cell lines transfected with the recombinant DNA plasmid. In addition, 6-week-old C57BL/6 mice were used to establish a H22 hepatoma-bearing mouse model. Mice tumor tissue was analyzed by immunohistochemistry and scanning electron microscopy. The results of the present study revealed that the recombinant DNA vaccine containing the VP3, IL-18 and HN genes inhibited cell proliferation and induced autophagy via the mitochondrial pathway in vivo and in vitro.

  7. Transcriptional IL-15-Directed in vivo DC Targeting DNA Vaccine

    PubMed Central

    Tian, S; Liu, Z; Donahue, C; Noh, HS; Falo, LD; You, Z

    2009-01-01

    DC engineered in vitro by DNA encoding OVAhsp70 and IL-15 up-regulated their expressions of CD80, CD86, CCR7 and IL-15Rα and promoted their productions of IL-6, IL-12 and TNF-α. Transcriptional IL-15-directed in vivo DC targeting DNA vaccine encoding OVAhsp70 elicited long-lasting Th1 and CTL responses and anti-B16OVA activity. CD8 T cell-mediated primary tumor protection was abrogated by DC or CD4 T cell depletion during the induction phase of immune responses. However, CD4 T cell depletion during immunization did not impair CD8 T cell-dependent long-lasting tumor protection. Furthermore, in vivo DC-derived IL-15 exerted the enhancements of cellular and humoral immune responses and antitumor immunity elicited by OVAhsp70 DNA vaccine. Importantly, the potency of this novel DNA vaccine strategy was proven using a self/tumor Ag (TRP2) in a clinically relevant B16 melanoma model. These findings have implications for developing next generation DNA vaccines against cancers and infectious diseases in both healthy and CD4 deficient individuals. PMID:19727134

  8. Chemical adjuvants for plasmid DNA vaccines.

    PubMed

    Greenland, John R; Letvin, Norman L

    2007-05-10

    Plasmid DNA vaccines are a promising modality for immunization against a variety of human pathogens. Immunization via multiple routes with plasmid DNA can elicit potent cellular immune responses, and these immunogens can be administered repeatedly without inducing anti-vector immunity. Nonetheless, the immunogenicity of plasmid DNA vaccines has been limited by problems associated with delivery. A number of adjuvants have been designed to improve plasmid DNA immunogenicity, either by directly stimulating the immune system or by enhancing plasmid DNA expression. Chemical adjuvants for enhancing plasmid DNA expression include liposomes, polymers, and microparticles, all of which have shown promise for enhancing the expression and immunogenicity of plasmid DNA vaccines in animal models. Micro- and nanoparticles have not been shown to enhance immune responses to plasmid DNA vaccines. However, formulation of plasmid DNA with some non-particulate polymeric adjuvants has led to a statistically significant enhancement of immune responses. Further development of these technologies will significantly improve the utility of plasmid DNA vaccination.

  9. DNA Vaccines: Developing New Strategies against Cancer

    PubMed Central

    Fioretti, Daniela; Iurescia, Sandra; Fazio, Vito Michele; Rinaldi, Monica

    2010-01-01

    Due to their rapid and widespread development, DNA vaccines have entered into a variety of human clinical trials for vaccines against various diseases including cancer. Evidence that DNA vaccines are well tolerated and have an excellent safety profile proved to be of advantage as many clinical trials combines the first phase with the second, saving both time and money. It is clear from the results obtained in clinical trials that such DNA vaccines require much improvement in antigen expression and delivery methods to make them sufficiently effective in the clinic. Similarly, it is clear that additional strategies are required to activate effective immunity against poorly immunogenic tumor antigens. Engineering vaccine design for manipulating antigen presentation and processing pathways is one of the most important aspects that can be easily handled in the DNA vaccine technology. Several approaches have been investigated including DNA vaccine engineering, co-delivery of immunomodulatory molecules, safe routes of administration, prime-boost regimen and strategies to break the immunosuppressive networks mechanisms adopted by malignant cells to prevent immune cell function. Combined or single strategies to enhance the efficacy and immunogenicity of DNA vaccines are applied in completed and ongoing clinical trials, where the safety and tolerability of the DNA platform are substantiated. In this review on DNA vaccines, salient aspects on this topic going from basic research to the clinic are evaluated. Some representative DNA cancer vaccine studies are also discussed. PMID:20368780

  10. DNA vaccines: developing new strategies against cancer.

    PubMed

    Fioretti, Daniela; Iurescia, Sandra; Fazio, Vito Michele; Rinaldi, Monica

    2010-01-01

    Due to their rapid and widespread development, DNA vaccines have entered into a variety of human clinical trials for vaccines against various diseases including cancer. Evidence that DNA vaccines are well tolerated and have an excellent safety profile proved to be of advantage as many clinical trials combines the first phase with the second, saving both time and money. It is clear from the results obtained in clinical trials that such DNA vaccines require much improvement in antigen expression and delivery methods to make them sufficiently effective in the clinic. Similarly, it is clear that additional strategies are required to activate effective immunity against poorly immunogenic tumor antigens. Engineering vaccine design for manipulating antigen presentation and processing pathways is one of the most important aspects that can be easily handled in the DNA vaccine technology. Several approaches have been investigated including DNA vaccine engineering, co-delivery of immunomodulatory molecules, safe routes of administration, prime-boost regimen and strategies to break the immunosuppressive networks mechanisms adopted by malignant cells to prevent immune cell function. Combined or single strategies to enhance the efficacy and immunogenicity of DNA vaccines are applied in completed and ongoing clinical trials, where the safety and tolerability of the DNA platform are substantiated. In this review on DNA vaccines, salient aspects on this topic going from basic research to the clinic are evaluated. Some representative DNA cancer vaccine studies are also discussed.

  11. DNA vaccination against oncoantigens: A promise.

    PubMed

    Iezzi, Manuela; Quaglino, Elena; Amici, Augusto; Lollini, Pier-Luigi; Forni, Guido; Cavallo, Federica

    2012-05-01

    The emerging evidence that DNA vaccines elicit a protective immune response in rodents, dogs and cancer patients, coupled with the US Food and Drug Administration (FDA) approval of an initial DNA vaccine to treat canine tumors is beginning to close the gap between the optimistic experimental data and their difficult application in a clinical setting. Here we review a series of conceptual and biotechnological advances that are working together to make DNA vaccines targeting molecules that play important roles during cancer progression (oncoantigens) a promise with near-term clinical impact.

  12. Efficacy of an infectious hematopoietic necrosis (IHN) virus DNA vaccine in Chinook Oncorhynchus tshawytscha and sockeye O. nerka salmon.

    PubMed

    Garver, Kyle A; LaPatra, Scott E; Kurath, Gael

    2005-04-06

    The level of protective immunity was determined for Chinook Oncorhynchus tshawytscha and sockeye/kokanee salmon (anadromous and landlocked) O. nerka following intramuscular vaccination with a DNA vaccine against the aquatic rhabdovirus, infectious hematopoietic necrosis virus (IHNV). A DNA vaccine containing the glycoprotein gene of IHNV protected Chinook and sockeye/kokanee salmon against waterborne or injection challenge with IHNV, and relative percent survival (RPS) values of 23 to 86% were obtained under a variety of lethal challenge conditions. Although this is significant protection, it is less than RPS values obtained in previous studies with rainbow trout (O. mykiss). In addition to the variability in the severity of the challenge and inherent host susceptibility differences, it appears that use of a cross-genogroup challenge virus strain may lead to reduced efficacy of the DNA vaccine. Neutralizing antibody titers were detected in both Chinook and sockeye that had been vaccinated with 1.0 and 0.1 pg doses of the DNA vaccine, and vaccinated fish responded to viral challenges with higher antibody titers than mock-vaccinated control fish.

  13. Nanogram quantities of a DNA vaccine protect rainbow trout fry against heterologous strains of infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Corbeil, S.; LaPatra, S.E.; Anderson, E.D.; Kurath, G.

    2000-01-01

    The efficacy of a DNA vaccine containing the glycoprotein gene of infectious hematopoietic necrosis virus (IHNV), a rhabdovirus affecting trout and salmon, was investigated. The minimal dose of vaccine required, the protection against heterologous strains, and the titers of neutralizing antibodies produced were used to evaluate the potential of the vaccine as a control pharmaceutical. Results indicated that a single dose of as little as 1–10 ng of vaccine protected rainbow trout fry against waterborne challenge by IHNV. An optimal dose of 100 ng per fish was selected to assure strong protection under various conditions. Neutralizing antibody titers were detected in fish vaccinated with concentrations of DNA ranging from 5 to 0.01 μg. Furthermore, the DNA vaccine protected fish against a broad range of viral strains from different geographic locations, including isolates from France and Japan, suggesting that the vaccine could be used worldwide. A single dose of this DNA vaccine induced protection in fish at a lower dose than is usually reported in mammalian DNA vaccine studies.

  14. Efficacy of an infectious hematopoietic necrosis (IHN) virus DNA vaccine in Chinook Oncorhynchus tshawytscha and sockeye O. nerka salmon

    USGS Publications Warehouse

    Garver, K.A.; LaPatra, S.E.; Kurath, G.

    2005-01-01

    The level of protective immunity was determined for Chinook Oncorhynchus tshawytscha and sockeye/kokanee salmon (anadromous and landlocked) O. nerka following intramuscular vaccination with a DNA vaccine against the aquatic rhabdovirus, infectious hematopoietic necrosis virus (IHNV). A DNA vaccine containing the glycoprotein gene of IHNV protected Chinook and sockeye/kokanee salmon against waterborne or injection challenge with IHNV, and relative percent survival (RPS) values of 23 to 86% were obtained under a variety of lethal challenge conditions. Although this is significant protection, it is less than RPS values obtained in previous studies with rainbow trout (O. mykiss). In addition to the variability in the severity of the challenge and inherent host susceptibility differences, it appears that use of a cross-genogroup challenge virus strain may lead to reduced efficacy of the DNA vaccine. Neutralizing antibody titers were detected in both Chinook and sockeye that had been vaccinated with 1.0 and 0.1 ??g doses of the DNA vaccine, and vaccinated fish responded to viral challenges with higher antibody titers than mock-vaccinated control fish. ?? Inter-Research 2005.

  15. Transcriptome profiles associated to VHSV infection or DNA vaccination in turbot (Scophthalmus maximus).

    PubMed

    Pereiro, Patricia; Dios, Sonia; Boltaña, Sebastián; Coll, Julio; Estepa, Amparo; Mackenzie, Simon; Novoa, Beatriz; Figueras, Antonio

    2014-01-01

    DNA vaccines encoding the viral G glycoprotein show the most successful protection capability against fish rhabdoviruses. Nowadays, the molecular mechanisms underlying the protective response remain still poorly understood. With the aim of shedding light on the protection conferred by the DNA vaccines based in the G glycoprotein of viral haemorrhagic septicaemia virus (VHSV) in turbot (Scophthalmus maximus) we have used a specific microarray highly enriched in antiviral sequences to carry out the transcriptomic study associated to VHSV DNA vaccination/infection. The differential gene expression pattern in response to empty plasmid (pMCV1.4) and DNA vaccine (pMCV1.4-G860) intramuscular administration with regard to non-stimulated turbot was analyzed in head kidney at 8, 24 and 72 hours post-vaccination. Moreover, the effect of VHSV infection one month after immunization was also analyzed in vaccinated and non-vaccinated fish at the same time points. Genes implicated in the Toll-like receptor signalling pathway, IFN inducible/regulatory proteins, numerous sequences implicated in apoptosis and cytotoxic pathways, MHC class I antigens, as well as complement and coagulation cascades among others were analyzed in the different experimental groups. Fish receiving the pMCV1.4-G860 vaccine showed transcriptomic patterns very different to the ones observed in pMCV1.4-injected turbot after 72 h. On the other hand, VHSV challenge in vaccinated and non-vaccinated turbot induced a highly different response at the transcriptome level, indicating a very relevant role of the acquired immunity in vaccinated fish able to alter the typical innate immune response profile observed in non-vaccinated individuals. This exhaustive transcriptome study will serve as a complete overview for a better understanding of the crosstalk between the innate and adaptive immune response in fish after viral infection/vaccination. Moreover, it provides interesting clues about molecules with a potential

  16. DNA Vaccines: Regulatory Considerations and Safety Aspects.

    PubMed

    Myhr, Anne Ingeborg

    2017-01-01

    DNA vaccines have great potential as preventive or therapeutic vaccines against viral, bacterial, or parasitic diseases as well as cancer, and may also be used as gene therapy products. Although many human and veterinary DNA vaccines have been investigated in laboratory trials, only four of these have been approved for commercial use. In this paper an overview of the regulatory requirements for the development of DNA vaccines is given. The regulatory process in EU and USA is described. A discussion concerning the relevance of national regulations on gene technology is included. In addition the main safety concerns associated with DNA vaccines, relating to unwanted side effects in the vaccinated mammal or fish, are presented. Finally, the need for greater openness regarding the assessment information is discussed.

  17. Increasing versatility of the DNA vaccines through modification of the subcellular location of plasmid-encoded antigen expression in the in vivo transfected cells.

    PubMed

    Martinez-Lopez, Alicia; García-Valtanen, Pablo; Ortega-Villaizan, María Del Mar; Chico, Verónica; Medina-Gali, Regla María; Perez, Luis; Coll, Julio; Estepa, Amparo

    2013-01-01

    The route of administration of DNA vaccines can play a key role in the magnitude and quality of the immune response triggered after their administration. DNA vaccines containing the gene of the membrane-anchored glycoprotein (gpG) of the fish rhabdoviruses infectious haematopoietic necrosis virus (IHNV) or viral haematopoietic septicaemia virus (VHSV), perhaps the most effective DNA vaccines generated so far, confer maximum protection when injected intramuscularly in contrast to their low efficacy when injected intraperitoneally. In this work, taking as a model the DNA vaccine against VHSV, we focused on developing a more versatile DNA vaccine capable of inducing protective immunity regardless of the administration route used. For that, we designed two alternative constructs to gpG₁₋₅₀₇ (the wild type membrane-anchored gpG of VHSV) encoding either a soluble (gpG₁₋₄₆₂) or a secreted soluble (gpG(LmPle20-462)) form of the VHSV-gpG. In vivo immunisation/challenge assays showed that only gpG(LmPle20-462) (the secreted soluble form) conferred protective immunity against VHSV lethal challenge via both intramuscular and intraperitoneal injection, being this the first description of a fish viral DNA vaccine that confers protection when administered intraperitoneally. Moreover, this new DNA vaccine construct also conferred protection when administered in the presence of an oil adjuvant suggesting that DNA vaccines against rhabdoviruses could be included in the formulation of current multicomponent-intaperitoneally injectable fish vaccines formulated with an oil adjuvant. On the other hand, a strong recruitment of membrane immunoglobulin expressing B cells, mainly membrane IgT, as well as t-bet expressing T cells, at early times post-immunisation, was specifically observed in the fish immunised with the secreted soluble form of the VHSV-gpG protein; this may indicate that the subcellular location of plasmid-encoded antigen expression in the in vivo

  18. Budding of PPxY-containing rhabdoviruses is not dependent on host proteins TGS101 and VPS4A.

    PubMed

    Irie, Takashi; Licata, Jillian M; McGettigan, James P; Schnell, Matthias J; Harty, Ronald N

    2004-03-01

    Viral matrix proteins of several enveloped RNA viruses play important roles in virus assembly and budding and are by themselves able to bud from the cell surface in the form of lipid-enveloped, virus-like particles (VLPs). Three motifs (PT/SAP, PPxY, and YxxL) have been identified as late budding domains (L-domains) responsible for efficient budding. L-domains can functionally interact with cellular proteins involved in vacuolar sorting (VPS4A and TSG101) and endocytic pathways (Nedd4), suggesting involvement of these pathways in virus budding. Ebola virus VP40 has overlapping PTAP and PPEY motifs, which can functionally interact with TSG101 and Nedd4, respectively. As for vesicular stomatitis virus (VSV), a PPPY motif within M protein can interact with Nedd4. In addition, M protein has a PSAP sequence downstream of the PPPY motif, but the function of PSAP in budding is not clear. In this study, we compared L-domain functions between Ebola virus and VSV by constructing a chimeric M protein (M40), in which the PPPY motif of VSV M is replaced by the L domains of VP40. The budding efficiency of M40 was 10-fold higher than that of wild-type (wt) M protein. Overexpression of a dominant negative mutant of VPS4A or depletion of cellular TSG101 reduced the budding of only M40-containing VLPs but not that of wt M VLPs or live VSV. These findings suggest that the PSAP motif of M protein is not critical for budding and that there are fundamental differences between PTAP-containing viruses (Ebola virus and human immunodeficiency virus type 1) and PPPY-containing viruses (VSV and rabies virus) regarding their dependence on specific host factors for efficient budding.

  19. c-DNA vaccination against parasitic infections: advantages and disadvantages.

    PubMed

    Kofta, W; Wedrychowicz, H

    2001-09-12

    Recently developed technology for DNA vaccination appears to offer the good prospect for the development of a multivalent vaccines that will effectively activate both the humoral and cell mediated mechanisms of the immune system. Currently, DNA vaccination against such important parasitic diseases like malaria, leishmaniosis, toxoplasmosis, cryptosporidiosis, schistosomosis, fasciolosis offers several new opportunities. However, the outcome of vaccination depends very much on vaccine formulations, dose and route of vaccine delivery, and the species and even strain of the vaccinated host. To overcome these problems much research is still needed, specifically focused on cloning and testing of new c-DNA sequences in the following: genome projects: different ways of delivery: design of vectors containing appropriate immunostimulatory sequences and very detailed studies on safety.

  20. DNA vaccines encoding viral glycoproteins induce nonspecific immunity and Mx protein synthesis in fish.

    PubMed

    Kim, C H; Johnson, M C; Drennan, J D; Simon, B E; Thomann, E; Leong, J A

    2000-08-01

    Protective immunity by vaccination with plasmid DNA encoding a viral glycoprotein (G) has long been assumed to result from the induction of a specific immune response. We report here that the initial protection may be due to the induction of alpha/beta interferon, with long-term protection due to a specific response to the encoded viral G. DNA vaccines encoding the Gs of three serologically unrelated fish rhabdoviruses were used to vaccinate rainbow trout against a lethal challenge with infectious hematopoietic necrosis virus (IHNV). All three vaccines, each encoding the G gene of either IHNV (IHNV-G), snakehead rhabdovirus (SHRV) (SHRV-G), or spring viremia of carp virus (SVCV) (SVCV-G), elicited protective immunity against IHNV. Vaccinated fish were challenged at 30 or 70 days postvaccination with lethal doses of IHNV. At 30 days postvaccination, only 5% of fish that had received any of the G vaccines died, whereas more than 50% of the control fish succumbed to virus challenge. When fish were vaccinated and challenged at 70 days postvaccination, only 12% of the IHNV-G-vaccinated fish died compared to 68% for the SHRV-G- and 76% for the SVCV-G-vaccinated fish. Assays for trout Mx protein, an indicator of alpha/beta interferon induction, showed that only fish vaccinated with a G-containing plasmid produced high levels of Mx protein in the kidneys and liver. Interestingly, at day 7 after virus challenge, all of the fish vaccinated with the IHNV-G plasmid were negative for Mx, but the SHRV-G- and SVCV-G-vaccinated fish still showed detectable levels of Mx. These results suggest that DNA vaccines in fish induce an early, nonspecific antiviral protection mediated by an alpha/beta interferon and, later, a specific immune response.

  1. DNA vaccines: a simple DNA sensing matter?

    PubMed

    Coban, Cevayir; Kobiyama, Kouji; Jounai, Nao; Tozuka, Miyuki; Ishii, Ken J

    2013-10-01

    Since the introduction of DNA vaccines two decades ago, this attractive strategy has been hampered by its low immunogenicity in humans. Studies conducted to improve the immunogenicity of DNA vaccines have shown that understanding the mechanism of action of DNA vaccines might be the key to successfully improving their immunogenicity. Our current understanding is that DNA vaccines induce innate and adaptive immune responses in two ways: (1) encoded protein (or polypeptide) antigen(s) by the DNA plasmid can be expressed in stromal cells (i.e., muscle cells) as well as DCs, where these antigens are processed and presented to naïve CD4 or CD8 T cells either by direct or cross presentation, respectively; and (2) the transfected DNA plasmid itself may bind to an un-identified cytosolic DNA sensor and activate the TBK1-STING pathway and the production of type I interferons (IFNs) which function as an adjuvant. Recent studies investigating double-stranded cytosolic DNA sensor(s) have highlighted new mechanisms in which cytosolic DNA may release secondary metabolites, which are in turn recognized by a novel DNA sensing machinery. Here, we discuss these new metabolites and the possibilities of translating this knowledge into improved immunogenicity for DNA vaccines.

  2. DNA vaccines for targeting bacterial infections

    PubMed Central

    Ingolotti, Mariana; Kawalekar, Omkar; Shedlock, Devon J; Muthumani, Karuppiah; Weiner, David B

    2010-01-01

    DNA vaccination has been of great interest since its discovery in the 1990s due to its ability to elicit both humoral and cellular immune responses. DNA vaccines consist of a DNA plasmid containing a transgene that encodes the sequence of a target protein from a pathogen under the control of a eukaryotic promoter. This revolutionary technology has proven to be effective in animal models and four DNA vaccine products have recently been approved for veterinary use. Although few DNA vaccines against bacterial infections have been tested, the results are encouraging. Because of their versatility, safety and simplicity a wider range of organisms can be targeted by these vaccines, which shows their potential advantages to public health. This article describes the mechanism of action of DNA vaccines and their potential use for targeting bacterial infections. In addition, it provides an updated summary of the methods used to enhance immunogenicity from codon optimization and adjuvants to delivery techniques including electroporation and use of nanoparticles. PMID:20624048

  3. Antiparasitic DNA vaccines in 21st century.

    PubMed

    Wedrychowicz, Halina

    2015-06-01

    Demands for effective vaccines to control parasitic diseases of humans and livestock have been recently exacerbated by the development of resistance of most pathogenic parasites to anti-parasitic drugs. Novel genomic and proteomic technologies have provided opportunities for the discovery and improvement of DNA vaccines which are relatively easy as well as cheap to fabricate and stable at room temperatures. However, their main limitation is rather poor immunogenicity, which makes it necessary to couple the antigens with adjuvant molecules. This paper review recent advances in the development of DNA vaccines to some pathogenic protozoa and helminths. Numerous studies were conducted over the past 14 years of 21st century, employing various administration techniques, adjuvants and new immunogenic antigens to increase efficacy of DNA vaccines. Unfortunately, the results have not been rewarding. Further research is necessary using more extensive combinations of antigens; alternate delivery systems and more efficient adjuvants based on knowledge of the immunomodulatory capacities of parasitic protozoa and helminths.

  4. Fish rhabdoviruses: molecular epidemiology and evolution.

    PubMed

    Hoffmann, B; Beer, M; Schütze, H; Mettenleiter, T C

    2005-01-01

    Rhabdoviruses may cause serious diseases in wild and farmed fish. Within the Rhabdoviridae six genera have been established: Ephemerovirus, Cytorhabdovirus, Nucleorhabdovirus, Lyssavirus, Vesiculovirus, and Novirhabdovirus. Viruses that infect fish are official or tentative members of the genera Vesiculovirus and Novirhabdovirus, or are listed as unassigned rhabdoviruses. In this report, we summarize and discuss published and our own unpublished data on the molecular epidemiology and phylogeography of fish rhabdoviruses including intrapopulational differences and subgrouping of fish rhabdoviruses, in particular the species spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV).

  5. Novel approaches to tuberculosis prevention: DNA vaccines.

    PubMed

    Rivas-Santiago, Bruno; Cervantes-Villagrana, Alberto R

    2014-03-01

    It is estimated that there are approximately eight million new cases of active tuberculosis (TB) worldwide annually. There is only 1 vaccine available for prevention: bacillus Calmette-Guérin (BCG). This has variable efficacy and is only protective for certain extrapulmonary TB cases in children, therefore new strategies for the creation of novel vaccines have emerged. One of the promising approaches is the DNA vaccine, used as a direct vaccination or as a prime-boost vaccine. This review describes the experimental data obtained during the design of DNA vaccines for TB.

  6. Fish DNA vaccine against infectious hematopoietic necrosis virus: efficacy of various routes of immunization

    USGS Publications Warehouse

    Corbeil, Serge; Kurath, Gael; LaPatra, Scott E.

    2000-01-01

    The DNA vaccine, pIHNVw-G, contains the gene for the glycoprotein (G) of the rhabdovirus infectious hematopoietic necrosis virus (IHNV), a major pathogen of salmon and trout. The relative efficacy of various routes of immunisation with pIHNVw-G was evaluated using 1.8 g rainbow trout fry vaccinated via intramuscular injection, scarification of the skin, intraperitoneal injection, intrabuccal administration, cutaneous particle bombardment using a gene gun, or immersion in water containing DNA vaccine-coated beads. Twenty-seven days after vaccination neutralising antibody titres were determined, and 2 days later groups of vaccinated and control unvaccinated fish were subjected to an IHNV immersion challenge. Results of the virus challenge showed that the intramuscular injection and the gene gun immunisation induced protective immunity in fry, while intraperitoneal injection provided partial protection. Neutralising antibodies were not detected in sera of vaccinated fish regardless of the route of immunisation used, suggesting that cell mediated immunity may be at least partially responsible for the observed protection.

  7. [Progress of research on DNA vaccines against parasitosis].

    PubMed

    Qi, Wen-Juan; Fang, Qiang

    2011-06-01

    One of the effective prevention and treatment strategies to parasitosis is to develop safe and effective vaccines. The DNA vaccine is a new kind of vaccine developed in last 10 years. In recent years, many advances in DNA vaccines against parasitosis have been made. This article reviews the advances in the mechanism, construction, optimization, adjuvants and delivery ways of DNA vaccines and the advances in the study of DNA vaccines against some parasitosis including malaria, schistosomiasis, cysticercosis and toxoplasmosis in recent years.

  8. Protective immunity and lack of histopathological damage two years after DNA vaccination against infectious hematopoietic necrosis virus in trout

    USGS Publications Warehouse

    Kurath, Gael; Garver, Kyle A.; Corbeil, Serge; Elliott, Diane G.; Anderson, Eric D.; LaPatra, Scott E.

    2006-01-01

    The DNA vaccine pIHNw-G encodes the glycoprotein of the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV). Vaccine performance in rainbow trout was measured 3, 6, 13, 24, and 25 months after vaccination. At three months all fish vaccinated with 0.1 μg pIHNw-G had detectable neutralizing antibody (NAb) and they were completely protected from lethal IHNV challenge with a relative percent survival (RPS) of 100% compared to control fish. Viral challenges at 6, 13, 24, and 25 months post-vaccination showed protection with RPS values of 47–69%, while NAb seroprevalence declined to undetectable levels. Passive transfer experiments with sera from fish after two years post-vaccination were inconsistent but significant protection was observed in some cases. The long-term duration of protection observed here defined a third temporal phase in the immune response to IHNV DNA vaccination, characterized by reduced but significant levels of protection, and decline or absence of detectable NAb titers. Examination of multiple tissues showed an absence of detectable long-term histopathological damage due to DNA vaccination.

  9. Polymer multilayer tattooing for enhanced DNA vaccination

    NASA Astrophysics Data System (ADS)

    Demuth, Peter C.; Min, Younjin; Huang, Bonnie; Kramer, Joshua A.; Miller, Andrew D.; Barouch, Dan H.; Hammond, Paula T.; Irvine, Darrell J.

    2013-04-01

    DNA vaccines have many potential benefits but have failed to generate robust immune responses in humans. Recently, methods such as in vivo electroporation have demonstrated improved performance, but an optimal strategy for safe, reproducible, and pain-free DNA vaccination remains elusive. Here we report an approach for rapid implantation of vaccine-loaded polymer films carrying DNA, immune-stimulatory RNA, and biodegradable polycations into the immune-cell-rich epidermis, using microneedles coated with releasable polyelectrolyte multilayers. Films transferred into the skin following brief microneedle application promoted local transfection and controlled the persistence of DNA and adjuvants in the skin from days to weeks, with kinetics determined by the film composition. These ‘multilayer tattoo’ DNA vaccines induced immune responses against a model HIV antigen comparable to electroporation in mice, enhanced memory T-cell generation, and elicited 140-fold higher gene expression in non-human primate skin than intradermal DNA injection, indicating the potential of this strategy for enhancing DNA vaccination.

  10. Tolerizing DNA vaccines for autoimmune arthritis.

    PubMed

    Ho, Peggy P; Higgins, John P; Kidd, Brian A; Tomooka, Beren; Digennaro, Carla; Lee, Lowen Y; de Vegvar, Henry E Neuman; Steinman, Lawrence; Robinson, William H

    2006-12-01

    Current therapies for rheumatoid arthritis (RA) and other autoimmune diseases non-specifically suppress immune function, and there is great need for fundamental approaches such as antigen-specific tolerizing therapy. In this paper we describe development of antigen-specific tolerizing DNA vaccines to treat collagen-induced arthritis (CIA) in mice, and use of protein microarrays to monitor response to therapy and to identify potential additional autoimmune targets for next generation vaccines. We demonstrate that tolerizing DNA vaccines encoding type II collagen (CII) reduced the incidence and severity of CIA. Atorvastatin, a statin drug found to reduce the severity of autoimmunity, potentiated the effect of DNA vaccines encoding CII. Analysis of cytokines produced by collagen-reactive T cells derived from mice receiving tolerizing DNA encoding CII, as compared to control vaccines, revealed reduced production of the pro-inflammatory cytokines IFN-gamma and TNF-alpha. Arthritis microarray analysis demonstrated reduced spreading of autoantibody responses in mice treated with DNA encoding CII. The development of tolerizing DNA vaccines, and the use of antibody profiling to guide design of and to monitor therapeutic responses to such vaccines, represents a promising approach for the treatment of RA and other autoimmune diseases.

  11. Application of DNA vaccine technology to aquaculture.

    PubMed

    Heppell, J; Davis, H L

    2000-09-15

    The aquaculture industry needs to augment its global production and efficiency to meet the increasing consumer needs for fish and shellfish products. Unfortunately, infectious diseases have been a major impediment to the development and profitability of fish farms. While vaccines offer the most efficient way to control infectious pathogens, current products have only been successful against some diseases. These are mostly bacterial, and there are still several important diseases, mainly of viral and parasitic origin, for which no prophylactic treatment exists. DNA vaccines, compared to traditional antigen vaccines, have several practical and immunological advantages that make them very attractive for the aquaculture industry. The early success of DNA vaccines in animal models was very encouraging, but fish are unique in many aspects, and findings with other classes of vertebrate, namely mammals and birds, do not necessarily apply to aquatic animals. However, more recent studies with reporter genes showed that fish cells efficiently express foreign proteins encoded by eukaryotic expression vectors. A piscine-specific backbone vector might eventually improve immune responses to DNA vaccines, but there is already strong direct evidence for the induction of protective immunity with currently available plasmids. Immune responses to plasmid DNA injected intramuscularly (IM) into fish are characterized by the production of antibodies, which have been shown to be neutralizing in two different viral disease models. There is also indirect evidence suggesting the induction of cell-mediated immunity. Despite this evidence, immune responses to DNA vaccines have only been poorly characterized in fish because of the limited knowledge of the piscine immune system, and the small number of studies on the subject. Apart from optimizing the efficiency of DNA vaccines, other important issues, such as safety and production cost will be determinants for the potential application of this

  12. Micro- and nanoparticulates for DNA vaccine delivery.

    PubMed

    Farris, Eric; Brown, Deborah M; Ramer-Tait, Amanda E; Pannier, Angela K

    2016-05-01

    DNA vaccination has emerged as a promising alternative to traditional protein-based vaccines for the induction of protective immune responses. DNA vaccines offer several advantages over traditional vaccines, including increased stability, rapid and inexpensive production, and flexibility to produce vaccines for a wide variety of infectious diseases. However, the immunogenicity of DNA vaccines delivered as naked plasmid DNA is often weak due to degradation of the DNA by nucleases and inefficient delivery to immune cells. Therefore, biomaterial-based delivery systems based on micro- and nanoparticles that encapsulate plasmid DNA represent the most promising strategy for DNA vaccine delivery. Microparticulate delivery systems allow for passive targeting to antigen presenting cells through size exclusion and can allow for sustained presentation of DNA to cells through degradation and release of encapsulated vaccines. In contrast, nanoparticle encapsulation leads to increased internalization, overall greater transfection efficiency, and the ability to increase uptake across mucosal surfaces. Moreover, selection of the appropriate biomaterial can lead to increased immune stimulation and activation through triggering innate immune response receptors and target DNA to professional antigen presenting cells. Finally, the selection of materials with the appropriate properties to achieve efficient delivery through administration routes conducive to high patient compliance and capable of generating systemic and local (i.e. mucosal) immunity can lead to more effective humoral and cellular protective immune responses. In this review, we discuss the development of novel biomaterial-based delivery systems to enhance the delivery of DNA vaccines through various routes of administration and their implications for generating immune responses. © 2016 by the Society for Experimental Biology and Medicine.

  13. Micro- and nanoparticulates for DNA vaccine delivery

    PubMed Central

    Farris, Eric; Brown, Deborah M; Ramer-Tait, Amanda E

    2016-01-01

    DNA vaccination has emerged as a promising alternative to traditional protein-based vaccines for the induction of protective immune responses. DNA vaccines offer several advantages over traditional vaccines, including increased stability, rapid and inexpensive production, and flexibility to produce vaccines for a wide variety of infectious diseases. However, the immunogenicity of DNA vaccines delivered as naked plasmid DNA is often weak due to degradation of the DNA by nucleases and inefficient delivery to immune cells. Therefore, biomaterial-based delivery systems based on micro- and nanoparticles that encapsulate plasmid DNA represent the most promising strategy for DNA vaccine delivery. Microparticulate delivery systems allow for passive targeting to antigen presenting cells through size exclusion and can allow for sustained presentation of DNA to cells through degradation and release of encapsulated vaccines. In contrast, nanoparticle encapsulation leads to increased internalization, overall greater transfection efficiency, and the ability to increase uptake across mucosal surfaces. Moreover, selection of the appropriate biomaterial can lead to increased immune stimulation and activation through triggering innate immune response receptors and target DNA to professional antigen presenting cells. Finally, the selection of materials with the appropriate properties to achieve efficient delivery through administration routes conducive to high patient compliance and capable of generating systemic and local (i.e. mucosal) immunity can lead to more effective humoral and cellular protective immune responses. In this review, we discuss the development of novel biomaterial-based delivery systems to enhance the delivery of DNA vaccines through various routes of administration and their implications for generating immune responses. PMID:27048557

  14. M cell-targeted DNA vaccination

    NASA Astrophysics Data System (ADS)

    Wu, Yunpeng; Wang, Xinhai; Csencsits, Keri L.; Haddad, Asmahan; Walters, Nancy; Pascual, David W.

    2001-07-01

    DNA immunization, although attractive, is poor for inducing mucosal immunity, thus limiting its protective value against most infectious agents. To surmount this shortcoming, we devised a method for mucosal transgene vaccination by using an M cell ligand to direct the DNA vaccine to mucosal inductive tissues and the respiratory epithelium. This ligand, reovirus protein 1, when conjugated to polylysine (PL), can bind the apical surface of M cells from nasal-associated lymphoid tissues. Intranasal immunizations with protein 1-PL-DNA complexes produced antigen-specific serum IgG and prolonged mucosal IgA, as well as enhanced cell-mediated immunity, made evident by elevated pulmonary cytotoxic T lymphocyte responses. Therefore, targeted transgene vaccination represents an approach for enabling DNA vaccination of the mucosa.

  15. Transcriptome Profiles Associated to VHSV Infection or DNA Vaccination in Turbot (Scophthalmus maximus)

    PubMed Central

    Pereiro, Patricia; Dios, Sonia; Boltaña, Sebastián; Coll, Julio; Estepa, Amparo; Mackenzie, Simon; Novoa, Beatriz; Figueras, Antonio

    2014-01-01

    DNA vaccines encoding the viral G glycoprotein show the most successful protection capability against fish rhabdoviruses. Nowadays, the molecular mechanisms underlying the protective response remain still poorly understood. With the aim of shedding light on the protection conferred by the DNA vaccines based in the G glycoprotein of viral haemorrhagic septicaemia virus (VHSV) in turbot (Scophthalmus maximus) we have used a specific microarray highly enriched in antiviral sequences to carry out the transcriptomic study associated to VHSV DNA vaccination/infection. The differential gene expression pattern in response to empty plasmid (pMCV1.4) and DNA vaccine (pMCV1.4-G860) intramuscular administration with regard to non-stimulated turbot was analyzed in head kidney at 8, 24 and 72 hours post-vaccination. Moreover, the effect of VHSV infection one month after immunization was also analyzed in vaccinated and non-vaccinated fish at the same time points. Genes implicated in the Toll-like receptor signalling pathway, IFN inducible/regulatory proteins, numerous sequences implicated in apoptosis and cytotoxic pathways, MHC class I antigens, as well as complement and coagulation cascades among others were analyzed in the different experimental groups. Fish receiving the pMCV1.4-G860 vaccine showed transcriptomic patterns very different to the ones observed in pMCV1.4-injected turbot after 72 h. On the other hand, VHSV challenge in vaccinated and non-vaccinated turbot induced a highly different response at the transcriptome level, indicating a very relevant role of the acquired immunity in vaccinated fish able to alter the typical innate immune response profile observed in non-vaccinated individuals. This exhaustive transcriptome study will serve as a complete overview for a better understanding of the crosstalk between the innate and adaptive immune response in fish after viral infection/vaccination. Moreover, it provides interesting clues about molecules with a potential

  16. The Web-Based DNA Vaccine Database DNAVaxDB and Its Usage for Rational DNA Vaccine Design.

    PubMed

    Racz, Rebecca; He, Yongqun

    2016-01-01

    A DNA vaccine is a vaccine that uses a mammalian expression vector to express one or more protein antigens and is administered in vivo to induce an adaptive immune response. Since the 1990s, a significant amount of research has been performed on DNA vaccines and the mechanisms behind them. To meet the needs of the DNA vaccine research community, we created DNAVaxDB ( http://www.violinet.org/dnavaxdb ), the first Web-based database and analysis resource of experimentally verified DNA vaccines. All the data in DNAVaxDB, which includes plasmids, antigens, vaccines, and sources, is manually curated and experimentally verified. This chapter goes over the detail of DNAVaxDB system and shows how the DNA vaccine database, combined with the Vaxign vaccine design tool, can be used for rational design of a DNA vaccine against a pathogen, such as Mycobacterium bovis.

  17. Enhancement of glycoprotein-based DNA vaccine for viral hemorrhagic septicemia virus (VHSV) via addition of the molecular adjuvant, DDX41.

    PubMed

    Lazarte, Jassy Mary S; Kim, Young Rim; Lee, Jung Seok; Im, Se Pyeong; Kim, Si Won; Jung, Jae Wook; Kim, Jaesung; Lee, Woo Jai; Jung, Tae Sung

    2017-03-01

    The use of molecular adjuvants to improve the immunogenicity of DNA vaccines has been thoroughly studied in recent years. Glycoprotein (G)-based DNA vaccines had been proven to be effective in combating infection against Rhabdovirus (especially infectious hematopoietic necrosis virus, IHNV) in salmonids. DDX41 is a helicase known to induce antiviral and inflammatory responses by inducing a type I IFN innate immune response. To gain more information regarding G-based DNA vaccines in olive flounder (Paralicthys olivaceus), we tried to develop a more efficient G-based DNA vaccine by adding a molecular adjuvant, DDX41. We designed a DNA vaccine in which the VHSV glycoprotein (G-protein) and DDX41 were driven by the EF-1α and CMV promoters, respectively. Olive flounders were intramuscularly immunized with 1 μg of plasmids encoding the G-based DNA vaccine alone (pEF-G), the molecular adjuvant alone (pEF-D), or the vaccine-adjuvant construct (pEF-GD). At two different time points, 15 and 30 days later, the fish were intraperitoneally infected with VHSV (100 μL; 1 × 10(6) TCID50/mL). Our assays revealed that the plasmid constructs showed up-regulated expression of IFN-1 and its associated genes at day 3 post-vaccination in both kidney and spleen samples. Specifically, pEF-GD showed statistically higher expression of immune response genes than pEF-G and pEF-D treated group (p < 0.05/p < 0.001). After VHSV challenge, the fish group treated with pEF-GD showed higher survival rate than the pEF-G treated group, though difference was not statistically significant in the 15 dpv challenged group however in the 30 dpv challenged group, the difference was statistically significant (p < 0.05). Together, these results clearly demonstrate that DDX41 is an effective adjuvant for the G-based DNA vaccine in olive flounder. Our novel findings could facilitate the development of more effective DNA vaccines for the aquaculture industry.

  18. Isolation and characterization of Scophthalmus maximus rhabdovirus.

    PubMed

    Zhang, Qi-Ya; Tao, Jian-Jun; Gui, Lang; Zhou, Guang-Zhou; Ruan, Hong-Mei; Li, Zhen-Qiu; Gui, Jian-Fang

    2007-02-28

    A rhabdovirus associated with a lethal hemorrhagic disease in cultured turbot Scophthalmus maximus Linnaeus was isolated. The virus induced typical cytopathogenic effects (CPE) in 9 of 15 fish cell lines examined and was then propagated and isolated from infected carp leucocyte cells (CLC). Electron microscopy observations revealed that the negatively stained virions had a typical bullet-shaped morphology with one rounded end and one flat base end. The bullet-shaped morphology was more obvious and clear in ultrathin sections of infected cells. Experimental infections also indicated that the S. maximus rhabdovirus (SMRV) was not only a viral pathogen for cultured turbot, but also had the ability to infect other fish species, such as freshwater grass carp. A partial nucleotide sequence of the SMRV polymerase gene was determined by RT-PCR using 2 pairs of degenerate primers designed according to the conserved sequences of rhabdovirus polymerase genes. Homology analysis, amino acid sequence alignment, and phylogenetic relationship analysis of the partial SMRV polymerase sequence indicated that SMRV was genetically distinct from other rhabdoviruses. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified SMRV revealed 5 major structural proteins, and their molecular masses were estimated to be about 250, 58, 47, 42, and 28 kDa. Significant serological reactivity differences were also observed between SMRV and its nearest neighbor, spring viremia of carp virus (SVCV). The data suggest that SMRV is likely a novel fish rhabdovirus, although it is closely related to rhabdoviruses in the genus Vesiculovirus.

  19. [Plant rhabdoviruses with bipartite genomes].

    PubMed

    Kondo, Hideki

    2013-01-01

    Members of the family Rhabdoviridae (order Mononegavirales) have a broad range of hosts, including humans, livestock, fish, plants, and invertebrates. They have a nonsegmented negative-sense RNA as the genome. Orchid fleck virus (OFV) is distributed world-wide on several orchid plants and transmitted by the false spider mite, Brevipalpus californicus. Based on its virions morphology and cytopathic effects in the infected cells, OFV was tentatively placed as unassigned plant rhabdoviruses in the sixth ICTV Report. However, the molecular studies reveled that OFV has a unique two-segmented negative-sense RNA genome that resembles monopartite genomes of plant nucleorhabdoviruses. In this review, we describe the current knowledge on the genome structure and gene expression strategy of OFV, the possible mechanism of nuclear viroplasm formation, and the taxonomical consideration of the virus as well.

  20. DNA vaccines: ready for prime time?

    PubMed Central

    Kutzler, Michele A.; Weiner, David B.

    2015-01-01

    Since the discovery, over a decade and a half ago, that genetically engineered DNA can be delivered in vaccine form and elicit an immune response, there has been much progress in understanding the basic biology of this platform. A large amount of data has been generated in preclinical model systems, and more sustained cellular responses and more consistent antibody responses are being observed in the clinic. Four DNA vaccine products have recently been approved, all in the area of veterinary medicine. These results suggest a productive future for this technology as more optimized constructs, better trial designs and improved platforms are being brought into the clinic. PMID:18781156

  1. Influenza Plasmid DNA Vaccines: Progress and Prospects.

    PubMed

    Bicho, Diana; Queiroz, João António; Tomaz, Cândida Teixeira

    2015-01-01

    Current influenza vaccines have long been used to fight flu infectious; however, recent advances highlight the importance of produce new alternatives. Even though traditional influenza vaccines are safe and usually effective, they need to be uploaded every year to anticipate circulating flu viruses. This limitation together with the use of embryonated chicken eggs as the substrate for vaccine production, is time-consuming and could involve potential biohazards in growth of new virus strains. Plasmid DNA produced by prokaryote microorganisms and encoding foreign proteins had emerged as a promising therapeutic tool. This technology allows the expression of a gene of interest by eukaryotic cells in order to induce protective immune responses against the pathogen of interest. In this review, we discuss the strategies to choose the best DNA vaccine to be applied in the treatment and prevention of influenza. Specifically, we give an update of influenza DNA vaccines developments, all involved techniques, their main characteristics, applicability and technical features to obtain the best option against influenza infections.

  2. DNA vaccines against leptospirosis: A literature review.

    PubMed

    Silveira, Marcelle Moura; Oliveira, Thaís Larré; Schuch, Rodrigo Andrade; McBride, Alan John Alexander; Dellagostin, Odir Antônio; Hartwig, Daiane Drawanz

    2017-10-09

    Leptospirosis is an infectious disease caused by pathogenic Leptospira species. The vaccines that are currently available for leptospirosis are composed of whole-cell preparations and suffer from limitations such as low efficacy, multiple side-effects, poor immunological memory and lack of cross-protection against different serovars of Leptospira spp. In light of the global prevalence of this disease, the development of a more effective vaccine against leptospirosis is of paramount importance. Genetic immunization is a promising alternative to conventional vaccine development. In the last 25years, several novel strategies have been developed for increasing the efficacy of DNA vaccines. Examples of such strategies include the introduction of novel plasmid vectors, adjuvants, alternate delivery routes, and prime-boost regimens. Herein we discuss the latest and most promising advances that have been made in developing DNA vaccines against leptospirosis. We also deliberate over the future directions that must be undertaken in order to improve results in this field. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Licensed DNA Vaccines against Infectious Hematopoietic Necrosis Virus (IHNV).

    PubMed

    Alonso, Marta; Leong, Jo-Ann C

    2013-04-01

    This article reviews some of the recent patents on DNA vaccines against fish viruses, in particular against the novirhabdovirus infectious hematopoitic necrosis virus (IHNV). Although very effective in protecting fish against IHNV, only one DNA vaccine has been approved to date for use in Canada. In Europe and in US, its commercialization is restricted due to safety concerns.

  4. DNA vaccination as a treatment for chronic kidney disease.

    PubMed

    Wang, Yuan Min; Alexander, Stephen I

    2014-01-01

    Chronic kidney disease is one of the major health problems worldwide. DNA vaccination delivers plasmid DNA encoding the target gene to induce both humoral and cellular immune responses. Here, we describe the methods of CD40 DNA vaccine enhanced by dendritic cell (DC) targeting on the development of Heymann nephritis (HN), a rat model of human membranous nephropathy.

  5. 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.

  6. DNA vaccines for emerging infectious diseases: what if?

    PubMed Central

    Whalen, R. G.

    1996-01-01

    A novel and powerful method for vaccine research, colloquially known as DNA vaccines, involves the deliberate introduction into tissues of a DNA plasmid carrying an antigen-coding gene that transfects cells in vivo and results in an immune response. DNA vaccines have several distinct advantages, which include ease of manipulation, use of a generic technology, simplicity of manufacture, and chemical and biological stability. In addition, DNA vaccines are a great leveler among re-searchers around the world because they provide unprecedented ease of experi-mentation. To facilitate diffusion of information, an Internet site has been established called THE DNA VACCINE WEB (URL:http://www.genweb.com/dnavax/dnavax.html). In this review, a brief survey is undertaken of the experimental models and preclinical work on DNA vaccines to contribute to a greater awareness of the possibilities for emerging infectious diseases. PMID:8903226

  7. DNA vaccines: an historical perspective and view to the future.

    PubMed

    Liu, Margaret A

    2011-01-01

    This review provides a detailed look at the attributes and immunologic mechanisms of plasmid DNA vaccines and their utility as laboratory tools as well as potential human vaccines. The immunogenicity and efficacy of DNA vaccines in a variety of preclinical models is used to illustrate how they differ from traditional vaccines in novel ways due to the in situ antigen production and the ease with which they are constructed. The ability to make new DNA vaccines without needing to handle a virulent pathogen or to adapt the pathogen for manufacturing purposes demonstrates the potential value of this vaccine technology for use against emerging and epidemic pathogens. Similarly, personalized anti-tumor DNA vaccines can also readily be made from a biopsy. Because DNA vaccines bias the T-helper (Th) cell response to a Th1 phenotype, DNA vaccines are also under development for vaccines against allergy and autoimmune diseases. The licensure of four animal health products, including two prophylactic vaccines against infectious diseases, one immunotherapy for cancer, and one gene therapy delivery of a hormone for a food animal, provides evidence of the efficacy of DNA vaccines in multiple species including horses and pigs. The size of these target animals provides evidence that the somewhat disappointing immunogenicity of DNA vaccines in a number of human clinical trials is not due simply to the larger mass of humans compared with most laboratory animals. The insights gained from the mechanisms of protection in the animal vaccines, the advances in the delivery and expression technologies for increasing the potency of DNA vaccines, and encouragingly potent human immune responses in certain clinical trials, provide insights for future efforts to develop DNA vaccines into a broadly useful vaccine and immunotherapy platform with applications for human and animal health.

  8. Evaluation of the protective immunogencity of the N, P, M, NV and G proteins of infectious hematopoietic necrosis virus in rainbow trout Oncorhynchus mykiss using DNA vaccines

    USGS Publications Warehouse

    Corbeil, S.; LaPatra, S.E.; Anderson, E.D.; Jones, J.; Vincent, B.; Hsu, Ya Li; Kurath, G.

    1999-01-01

    The protective immunogenicity of the nucleoprotein (N), phosphoprotein (P), matrix protein (M), non-virion protein (NV) and glycoprotein (G) of the rhabdovirus infectious hematopoietic necrosis virus (IHNV) was assessed in rainbow trout using DNA vaccine technology. DNA vaccines were produced by amplifying and cloning the viral genes in the plasmid pCDNA 3.1. The protective immunity elicited by each vaccine was evaluated through survival of immunized fry after challenge with live virus. Neutralizing antibody titers were also determined in vaccinated rainbow troutOncorhynchus mykiss fry (mean weight 2 g) and 150 g sockeye salmon Oncorhynchus nerka. The serum from the 150 g fish was also used in passive immunization studies with naïve fry. Our results showed that neither the internal structural proteins (N, P and M) nor the NV protein of IHNV induced protective immunity in fry or neutralizing antibodies in fry and 150 g fish when expressed by a DNA vaccine construct. The G protein, however, did confer significant protection in fry up to 80 d post-immunization and induced protective neutralizing antibodies. We are currently investigating the role of different arms of the fish immune system that contribute to the high level of protection against IHNV seen in vaccinated fish.

  9. Induction of protective immunity against Eimeria tenella, Eimeria necatrix, Eimeria maxima and Eimeria acervulina infections using multivalent epitope DNA vaccines.

    PubMed

    Song, Xiaokai; Ren, Zhe; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui

    2015-06-04

    Avian coccidiosis is mostly caused by mixed infection of several Eimeria species under natural conditions and immunity to avian coccidiosis is largely dependent on T-cell immune response. In this study, 14 T-cell epitope fragments from eight antigens of Eimeria tenella (E. tenella), Eimeria necatrix (E. necatrix), Eimeria maxima (E. maxima) and Eimeria acervulina (E. acervulina) were ligated with pVAX1 producing 14 monovalent DNA vaccines, respectively. Protective immunity of the monovalent DNA vaccines was assessed by in vivo challenge experiments and then four most protective fragments of each species were chosen to construct multivalent epitope DNA vaccines with or without chicken IL-2 as genetic adjuvant. Protective efficacies of the epitope DNA vaccines on chickens against E. tenella, E. necatrix, E. maxima and E. acervulina were evaluated. The results showed that the constructed multivalent epitope DNA vaccines significantly increased body weight gain, alleviated enteric lesions and reduced oocyst output of the infected birds. Especially, the multivalent epitope DNA vaccines of pVAX1-NA4-1-TA4-1-LDH-2-EMCDPK-1 and pVAX1-NA4-1-TA4-1-LDH-2-EMCDPK-1-IL-2 not only significantly increased body weight gain, alleviated enteric lesions and reduced oocyst output of the infected birds, but also resulted in anti-coccidial index (ACI) more than 170 against E. tenella, E. necatrix, E. maxima and E. acervulina, which indicated they could induce protective immunity against E. tenella, E. necatrix, E. maxima and E. acervulina. Our findings suggest the constructed multivalent epitope DNA vaccines are the potential candidate multivalent vaccines against mixed infection of Eimeria.

  10. DNA vaccines against cancer come of age.

    PubMed

    Stevenson, Freda K; Ottensmeier, Christian H; Rice, Jason

    2010-04-01

    Genetic technology allows construction of DNA vaccines encoding selected tumor antigens together with molecules to direct and amplify the desired effector pathways. Their enormous promise has been marred by a problem of scaling up to human subjects. This is now largely overcome by electroporation, which increases both antigen expression and the inflammatory milieu. While the principles of vaccine design can be developed in mouse models, the real operative test is in the clinic, using patients in temporary remission. Monitoring of induced immunity, although commonly limited to blood, is providing objective qualitative and quantitative data on T-cell and antibody responses. Prolongation of remission is the goal and an activated immune system should achieve this.

  11. Strategies and hurdles using DNA vaccines to fish

    PubMed Central

    2014-01-01

    DNA vaccinations against fish viral diseases as IHNV at commercial level in Canada against VHSV at experimental level are both success stories. DNA vaccination strategies against many other viral diseases have, however, not yet yielded sufficient results in terms of protection. There is an obvious need to combat many other viral diseases within aquaculture where inactivated vaccines fail. There are many explanations to why DNA vaccine strategies against other viral diseases fail to induce protective immune responses in fish. These obstacles include: 1) too low immunogenicity of the transgene, 2) too low expression of the transgene that is supposed to induce protection, 3) suboptimal immune responses, and 4) too high degradation rate of the delivered plasmid DNA. There are also uncertainties with regard distribution and degradation of DNA vaccines that may have implications for safety and regulatory requirements that need to be clarified. By combining plasmid DNA with different kind of adjuvants one can increase the immunogenicity of the transgene antigen – and perhaps increase the vaccine efficacy. By using molecular adjuvants with or without in combination with targeting assemblies one may expect different responses compared with naked DNA. This includes targeting of DNA vaccines to antigen presenting cells as a central factor in improving their potencies and efficacies by means of encapsulating the DNA vaccine in certain carriers systems that may increase transgene and MHC expression. This review will focus on DNA vaccine delivery, by the use of biodegradable PLGA particles as vehicles for plasmid DNA mainly in fish. PMID:24552235

  12. DNA vaccination of poultry: The current status in 2015.

    PubMed

    Meunier, Marine; Chemaly, Marianne; Dory, Daniel

    2016-01-04

    DNA vaccination is a promising alternative strategy for developing new human and animal vaccines. The massive efforts made these past 25 years to increase the immunizing potential of this kind of vaccine are still ongoing. A relatively small number of studies concerning poultry have been published. Even though there is a need for new poultry vaccines, five parameters must nevertheless be taken into account for their development: the vaccine has to be very effective, safe, inexpensive, suitable for mass vaccination and able to induce immune responses in the presence of maternal antibodies (when appropriate). DNA vaccination should meet these requirements. This review describes studies in this field performed exclusively on birds (chickens, ducks and turkeys). No evaluations of avian DNA vaccine efficacy performed on mice as preliminary tests have been taken into consideration. The review first describes the state of the art for DNA vaccination in poultry: pathogens targeted, plasmids used and different routes of vaccine administration. Second, it presents strategies designed to improve DNA vaccine efficacy: influence of the route of administration, plasmid dose and age of birds on their first inoculation; increasing plasmid uptake by host cells; addition of immunomodulators; optimization of plasmid backbones and codon usage; association of vaccine antigens and finally, heterologous prime-boost regimens. The final part will indicate additional properties of DNA vaccines in poultry: fate of the plasmids upon inoculation, immunological considerations and the use of DNA vaccines for purposes other than preventing infectious diseases.

  13. Immunogenicity of an HPV-16 L2 DNA vaccine

    PubMed Central

    Hitzeroth, Inga I.; Passmore, Jo-Ann S.; Shephard, Enid; Stewart, Debbie; Müller, Martin; Williamson, Anna-Lise; Rybicki, Edward P.; Kast, W. Martin

    2009-01-01

    The ability to elicit cross-neutralizing antibodies makes human papillomavirus (HPV) L2 capsid protein a possible HPV vaccine. We examined and compared the humoral response of mice immunised with a HPV-16 L2 DNA vaccine or with HPV-16 L2 protein. The L2 DNA vaccine elicited a non-neutralising antibody response unlike the L2 protein. L2 DNA vaccination suppressed the growth of L2-expressing C3 tumor cells, which is a T cell mediated effect, demonstrating that the lack of non-neutralizing antibody induction by L2 DNA was not caused by lack of T cell immunogenicity of the construct. PMID:19559114

  14. In vitro capping and transcription of rhabdoviruses.

    PubMed

    Ogino, Tomoaki

    2013-02-01

    The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus (VSV), a prototypic nonsegmented negative strand (NNS) RNA virus classified into the Rhabdoviridae family, has been used to investigate the fundamental molecular mechanisms of NNS RNA viral mRNA synthesis and processing. In vitro studies on mRNA cap formation with the VSV L protein eventually led to the discovery of the unconventional mRNA capping pathway catalyzed by the guanosine 5'-triphosphatase and RNA:GDP polyribonucleotidyltransferase (PRNTase) activities. The PRNTase activity is a novel enzymatic activity, which transfers 5'-monophosphorylated (p-) RNA from 5'-triphosphorylated (ppp-) RNA to GDP to form 5'-capped RNA (GpppRNA) in a viral mRNA-start sequence-dependent manner. This unconventional capping (pRNA transfer) reaction with PRNTase can be experimentally distinguished from the conventional capping (GMP transfer) reaction with eukaryotic GTP:RNA guanylyltransferase (GTase) on the basis of the following differences in their substrate specificity for the cap formation: PRNTase uses GDP and pppRNA, but not ppRNA, whereas GTase employs GTP, but not GDP, and ppRNA. The pRNA transfer reaction with PRNTase proceeds through a covalent enzyme-pRNA intermediate with a phosphoamide bond. Hence, to prove the PRNTase activity, it is necessary to demonstrate the following consecutive steps separately: (1) the enzyme forms a covalent enzyme-pRNA intermediate, and (2) the intermediate transfers pRNA to GDP. This article describes the methods for in vitro transcription and capping with the recombinant VSV L protein, which permit detailed characterization of its enzymatic reactions and mapping of active sites of its enzymatic domains. It is expected that these systems are adaptable to rhabdoviruses and, by extension, other NNS RNA viruses belonging to different families.

  15. Immunogenicity of a multi-epitope DNA vaccine against hantavirus.

    PubMed

    Zhao, Chen; Sun, Ying; Zhao, Yujie; Wang, Si; Yu, Tongtong; Du, Feng; Yang, X Frank; Luo, Enjie

    2012-02-01

    Hemorrhagic fever with renal syndrome (HFRS) is a severe epidemic disease caused by hantaviruses including Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV) and Puumala virus. Three of the four HFRS hantaviruses, HTNV, SEOV, and PUUV are found in China. Currently, there is no effective strategy available to reduce infection risk. In this study, we constructed a multi-epitope chimeric DNA vaccine that encodes expressing 25 glycoprotein epitopes from SEOV, HTNV and PUUV (designated as SHP chimeric gene). Vaccination of BALb/c mice with SHP multi-epitope chimeric DNA vaccine led to a dramatic augmentation of humoral and cellular responses. The SHP vaccine DNA was detected in many organs but not for more than 60 d. There was no risk of mutation due to integration. Thus, the SHP multi-epitope chimeric DNA vaccine is a potential effective and safe DNA vaccine against infection by SEOV, HTNV, and PUUV.

  16. Assessment of a DNA Vaccine Encoding Burkholderia pseudomallei Bacterioferritin

    DTIC Science & Technology

    2007-08-01

    bacterioferritin gene from Brucella abortus, when delivered to mice as a DNA vaccine, evokes a potent Th1 immune response, including strong IFN-γ...blocking buffer containing goat anti-mouse IgG alkaline phosphatase conjugate (Sigma) at a dilution of 1:30000 for 1hr at room temperature. Following...Walravens, and J. J. Letesson. 2001. Induction of immune response in BALB/c mice with a DNA vaccine encoding bacterioferritin or P39 of Brucella

  17. Smallpox DNA Vaccine Protects Nonhuman Primates Against Lethal Monkeypox

    DTIC Science & Technology

    2004-05-01

    PCR. Monkeys were challenged with MPOV on day 0. †, fatality . VOL. 78, 2004 SMALLPOX DNA VACCINE AGAINST LETHAL MONKEYPOX 4441 FIG. 5. Antibody...01 MAY 2004 2. REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Smallpox DNA vaccine protects nonhuman primates against lethal ...vaccinia virus genes (L1R, A27L, A33R, and B5R) were protected from severe disease after an otherwise lethal challenge with monkeypox virus. Animals

  18. Present and future DNA vaccines for chronic hepatitis B treatment.

    PubMed

    Cova, Lucyna

    2017-02-01

    With at least 240 million hepatitis B virus (HBV) carriers worldwide, being at a high risk of cirrhosis and hepatocellular carcinoma (HCC), chronic hepatitis B remains a major public health issue. Because current antiviral treatments are only virostatic, there is an urgent need for the development of innovative anti-HBV strategies leading to the functional cure. In this context, DNA-based vaccines appear as a promising approach. Area covered: In this review, the authors summarize the pertinent features of DNA vaccines for chronic hepatitis B therapy. They review several technologies that improve DNA vaccines efficacy evaluated in animal models of hepadnaviral infection. They also discuss the clinical trials of therapeutic DNA vaccination initiated in HBV-carrier patients. Expert opinion: Preclinical studies in HBV transgenic mice, DHBV-carrier ducks and WHV-infected woodchucks, have clearly demonstrated a benefit of DNA vaccine-based combination therapies for chronic hepatitis B treatment. However, the results of clinical trials conducted in HBV patients were rather disappointing and frustrating, as DNA-vaccines have not shown the same efficacy in patients as in animal models. We are convinced that the design of innovative clinical trials based on strategies able to increase DNA vaccine immunogenicity will allow to advance in this challenging field.

  19. Optimization of DNA vaccination against cutaneous leishmaniasis.

    PubMed

    Méndez, Susana; Belkaid, Yasmine; Seder, Robert A; Sacks, David

    2002-11-01

    The present studies were designed to examine the requirements of dose, route of inoculation and constituent antigens for the maintenance of complete and long lasting protection against cutaneous leishmaniasis due to Leishmania major conferred by a cocktail DNA vaccine encoding the Leishmania antigens LACK, LmST11 and TSA. Vaccination of C57Bl/6 mice with LACK DNA alone resulted in partial protection, whereas the combination of LmST11 and TSA provided stronger, though still incomplete protection compared to the combination of all three Ag DNAs. When intradermal (i.d), intramuscular (i.m.), and subcutaneous (s.c.) vaccination routes were compared, i.d. immunization reduced by five-fold the dose necessary to maintain complete protection. In vivo depletion of CD4+ or CD8+ T cells provided direct evidence that both populations are necessary to mediate complete protection. These results establish intradermal vaccination using DNA encoding multiple Leishmania antigens as a way to optimize priming of CD4+ and CD8+ T cells necessary for potent and durable protection against cutaneous leishmaniasis.

  20. Optimization of a DNA vaccine against SARS.

    PubMed

    Zakhartchouk, Alexander N; Viswanathan, Sathiyanarayanan; Moshynskyy, Igor; Petric, Martin; Babiuk, Lorne A

    2007-10-01

    Severe acute respiratory syndrome coronavirus (SARS-CoV) first appeared in Southern China in November 2002, and then quickly spread to 33 countries on five continents along international air travel routes. Although the SARS epidemic has been contained, there is a clear need for a safe and effective vaccine should an outbreak of a SARS-CoV infection reappear in human population. In this study, we tested four DNA-vaccine constructs: (1) pLL70, containing cDNA for the SARS-CoV spike (S) gene; (2) pcDNA-SS, containing codon-optimized S gene for SARS-CoV S protein (residues 12-1255) fused with a leader sequence derived from the human CD5 gene; (3) pcDNA-St, containing the gene encoding the N-portion of the codon-optimized S gene (residues 12-532) with the CD5 leader sequence; (4) pcDNA-St-VP22C, containing the gene encoding the N-portion of the codon-optimized S protein with the CD5 leader sequence fused with the C-terminal 138 amino acids of the bovine herpesvirus-1 (BHV-1) major tegument protein VP22. Each of these plasmids was intradermally administered to C57BL/6 mice in three separate immunizations. Analysis of humoral and cellular immune responses in immunized mice demonstrated that pcDNA-SS and pcDNA-St-VP22C are the most immunogenic SARS vaccine candidates.

  1. DNA vaccination strategies against infectious diseases.

    PubMed

    Watts, A M; Kennedy, R C

    1999-08-01

    DNA immunisation represents a novel approach to vaccine and immunotherapeutic development. Injection of plasmid DNA encoding a foreign gene of interest can result in the subsequent expression of the foreign gene products and the induction of an immune response within a host. This is relevant to prophylactic and therapeutic vaccination strategies when the foreign gene represents a protective epitope from a pathogen. The recent demonstration by a number of laboratories that these immune responses evoke protective immunity against some infectious diseases and cancers provides support for the use of this approach. In this article, we attempt to present an informative and unbiased representation of the field of DNA immunisation. The focus is on studies that impart information on the development of vaccination strategies against a number of human and animal pathogens. Investigations that describe the mechanism(s) of protective immunity induced by DNA immunisation highlight the advantages and disadvantages of this approach to developing vaccines within a given system. A variety of systems in which DNA vaccination has resulted in the induction of protective immunity, as well as the correlates associated with these protective immune responses, will be described. Particular attention will focus on systems involving parasitic diseases. Finally, the potential of DNA immunisation is discussed as it relates to veterinary medicine and its role as a possible vaccine strategy against animal coccidioses.

  2. The degree of apoptosis as an immunostimulant for a DNA vaccine against HIV-1 infection.

    PubMed

    Kojima, Yoshitsugu; Jounai, Nao; Takeshita, Fumihiko; Nakazawa, Masatoshi; Okuda, Kentaro; Watabe, Setsuko; Xin, Ke-Qin; Okuda, Kenji

    2007-01-05

    To regulate the expression of the apoptotic gene, we constructed bicistronic DNA vaccines that encode for HIV env and caspase-3 mutant (casp 3m) that are expressed via the encephalomyocarditis virus internal ribosomal entry site (IRES) or cytomegalovirus (CMV) promoter-dependent translations. While IRES-casp 3m induced weak apoptosis and caused little reduction in antigen expression, CMV-casp 3m elicited strong apoptosis and led to a marked decrease in the antigen expression. Therefore, IRES-casp 3m augmented HIV-specific immune responses, and IRES-casp 3m induced significant protection against the vaccinia-HIV chimeric virus. These results suggest that the appropriate level of apoptosis is important for DNA vaccine development.

  3. Distribution and variation of NV genes in fish rhabdoviruses.

    PubMed

    Kurath, G; Higman, K H; Björklund, H V

    1997-01-01

    The fish rhabdovirus infectious haematopoietic necrosis virus (IHNV) contains a non-virion (NV) gene between the glycoprotein (G) and polymerase (L) genes on its RNA genome. The present study investigated three other fish rhabdovirus genomes and found that the NV gene of hirame rhabdovirus is closely related to the NV of IHNV, whereas the viral haemorrhagic septicemia NV gene showed evidence of significant divergence. Most importantly, spring viraemia of carp virus, the only vesiculovirus-like fish rhabdovirus examined, did not have an NV gene at its genomic RNA G-L junction. These results suggest that the presence of an NV gene is characteristic of the unassigned fish rhabdovirus subgroup previously classified as lyssaviruses, and that the NV gene is not essential for replication in fish cells per se, since it is absent in a vesiculovirus-like fish rhabdovirus.

  4. Distribution and variation of NV genes in fish rhabdoviruses

    USGS Publications Warehouse

    Kurath, G.; Higman, K.H.; Bjorklund, H.V.

    1997-01-01

    The fish rhabdovirus infectious haematopoietic necrosis virus (IHNV) contains a non-virion (NV) gene between the glycoprotein (G) and polymerase (L) genes on its RNA genome. The present study investigated three other fish rhabdovirus genomes and found that the NV gene of hirame rhabdovirus is closely related to the NV of IHNV, whereas the viral haemorrhagic septicemia NV gene showed evidence of significant divergence. Most importantly, spring viraemia of carp virus, the only vesiculovirus-like fish rhabdovirus examined, did not have an NV gene at its genomic RNA G-L junction. These results suggest that the presence of an NV gene is characteristic of the unassigned fish rhabdovirus subgroup previously classified as lyssaviruses, and that the NV gene is not essential for replication in fish cells per se, since it is absent in a vesiculovirus-like fish rhabdovirus.

  5. Identification of Genetically Modified Maraba Virus as an Oncolytic Rhabdovirus

    PubMed Central

    Brun, Jan; McManus, Dan; Lefebvre, Charles; Hu, Kang; Falls, Theresa; Atkins, Harold; Bell, John C; McCart, J. Andrea; Mahoney, Douglas; Stojdl, David F

    2010-01-01

    To expand our current array of safe and potent oncolytic viruses, we screened a variety of wild-type (WT) rhabdoviruses against a panel of tumor cell lines. Our screen identified a number of viruses with varying degrees of killing activity. Maraba virus was the most potent of these strains. We built a recombinant system for the Maraba virus platform, engineered a series of attenuating mutations to expand its therapeutic index, and tested their potency in vitro and in vivo. A double mutant (MG1) strain containing both G protein (Q242R) and M protein (L123W) mutations attenuated Maraba virus in normal diploid cell lines, yet appeared to be hypervirulent in cancer cells. This selective attenuation was mediated through interferon (IFN)-dependent and -independent mechanisms. Finally, the Maraba MG1 strain had a 100-fold greater maximum tolerable dose (MTD) than WT Maraba in vivo and resulted in durable cures when systemically administered in syngeneic and xenograft models. In summary, we report a potent new oncolytic rhabdovirus platform with unique tumor-selective attenuating mutations. PMID:20551913

  6. Internalization and fusion mechanism of vesicular stomatitis virus and related rhabdoviruses

    PubMed Central

    Sun, Xiangjie; Roth, Shoshannah L; Bialecki, Michele A; Whittaker, Gary R

    2013-01-01

    Members of the Rhabdoviridae infect a wide variety of animals and plants, and are the causative agents of many important diseases. Rhabdoviruses enter host cells following internalization into endosomes, with the glycoprotein (G protein) mediating both receptor binding to host cells and fusion with the cellular membrane. The recently solved crystal structure of vesicular stomatitis virus G has allowed considerable insight into the mechanism of rhabdovirus entry, in particular the low pH-dependent conformational changes that lead to fusion activation. Rhabdovirus entry shows several distinct features compared with other enveloped viruses; first, the entry process appears to consist of two distinct fusion events, initial fusion into vesicles within endosomes followed by back-fusion into the cytosol; second, the conformational changes in the G protein that lead to fusion activation are reversible; and third, the G protein is structurally distinct from other viral fusion proteins and is not proteolytically cleaved. The internalization and fusion mechanisms of rhabdoviruses are discussed in this article, with a focus on viral systems where the G protein has been studied extensively: vesicular stomatitis virus and rabies virus, as well as viral hemorrhagic septicemia virus. PMID:23516023

  7. Targeting DNA Vaccines to Myeloid Cells Using a Small Peptide

    PubMed Central

    Ye, Chunting; Choi, Jang Gi; Abraham, Sojan; Shankar, Premlata; Manjunath, N.

    2014-01-01

    Targeting DNA vaccines to dendritic cells (DCs) greatly enhances immunity. Although several approaches have been used to target protein antigens to DCs, currently there is no method that targets DNA vaccines directly to DCs. Here, we show that a small peptide derived from the rabies virus glycoprotein, fused to protamine residues (RVG-P) can target DNA to myeloid cells, including DCs, that results in enhanced humoral and T-cell responses. DCs targeted with a DNA vaccine encoding the immunodominant vaccinia B8R gene via RVG-P were able to restimulate vaccinia-specific memory T cells in vitro. Importantly, a single i.v. injection of B8R gene bound to RVG-P was able prime a vaccinia-specific T-cell response that was able to rapidly clear a subsequent vaccinia challenge in mice. Moreover, delivery of DNA in DCs was enough to induce DC maturation and efficient antigen presentation without the need for adjuvants. Finally, immunization of mice with a DNA-vaccine encoding West Nile virus (WNV) prM and E proteins via RVG-P elicited high titers of WN neutralizing antibodies that protected mice from lethal WNV challenge. Thus, RVG-P provides a reagent to target DNA vaccines to myeloid cells and elicit robust T-cell and humoral immune responses. PMID:25270431

  8. Targeting DNA vaccines to myeloid cells using a small peptide.

    PubMed

    Ye, Chunting; Choi, Jang Gi; Abraham, Sojan; Shankar, Premlata; Manjunath, N

    2015-01-01

    Targeting DNA vaccines to dendritic cells (DCs) greatly enhances immunity. Although several approaches have been used to target protein Ags to DCs, currently there is no method that targets DNA vaccines directly to DCs. Here, we show that a small peptide derived from the rabies virus glycoprotein fused to protamine residues (RVG-P) can target DNA to myeloid cells, including DCs, which results in enhanced humoral and T-cell responses. DCs targeted with a DNA vaccine encoding the immunodominant vaccinia B8R gene via RVG-P were able to restimulate vaccinia-specific memory T cells in vitro. Importantly, a single i.v. injection of B8R gene bound to RVG-P was able to prime a vaccinia-specific T-cell response that was able to rapidly clear a subsequent vaccinia challenge in mice. Moreover, delivery of DNA in DCs was enough to induce DC maturation and efficient Ag presentation without the need for adjuvants. Finally, immunization of mice with a DNA-vaccine encoding West Nile virus (WNV) prM and E proteins via RVG-P elicited high titers of WNV-neutralizing Abs that protected mice from lethal WNV challenge. Thus, RVG-P provides a reagent to target DNA vaccines to myeloid cells and elicit robust T-cell and humoral immune responses.

  9. VHSV G glycoprotein major determinants implicated in triggering the host type I IFN antiviral response as DNA vaccine molecular adjuvants.

    PubMed

    Martinez-Lopez, A; Garcia-Valtanen, P; Ortega-Villaizan, M; Chico, V; Gomez-Casado, E; Coll, J M; Estepa, A

    2014-10-14

    We have recently identified the two major determinants of the glycoprotein G of the viral hemorrhagic septicaemia rhabdovirus (gpGVHSV), peptides p31 and p33 implicated in triggering the host type I IFN antiviral response associated to these rhabdoviral antigens. With the aim to investigate the properties of these viral glycoprotein regions as DNA molecular adjuvants, their corresponding cDNA sequences were cloned into a plasmid (pMCV1.4) flanked by the signal peptide and transmembrane sequences of gpGVHSV. In addition, a plasmid construct encoding both sequences p31 and p33 (pMCV1.4-p31+p33) was also designed. In vitro transitory cell transfection assays showed that these VHSV gpG regions were able to induce the expression of type I IFN stimulated genes as well as to confer resistance to the infection with a different fish rhabdovirus, the spring viremia of carp virus (SVCV). In vivo, zebrafish intramuscular injection of only 1μg of the construct pMCV1.4-p31+p33 conferred fish protection against SVCV lethal challenge up to 45 days post-immunization. Moreover, pMCV1.4-p31+p33 construct was assayed for molecular adjuvantcity's for a DNA vaccine against SVCV based in the surface antigen of this virus (pAE6-GSVCV). The results showed that the co-injection of the SVCV DNA vaccine and the molecular adjuvant allowed (i) a ten-fold reduction in the dose of pAE6-Gsvcv without compromising its efficacy (ii) an increase in the duration of protection, and (iii) an increase in the survival rate. To our knowledge, this is the first report in which specific IFN-inducing regions from a viral gpG are used to design more-efficient and cost-effective viral vaccines, as well as to improve our knowledge on how to stimulate the innate immune system.

  10. Discovery of novel rhabdoviruses in the blood of healthy individuals from West Africa.

    PubMed

    Stremlau, Matthew H; Andersen, Kristian G; Folarin, Onikepe A; Grove, Jessica N; Odia, Ikponmwonsa; Ehiane, Philomena E; Omoniwa, Omowunmi; Omoregie, Omigie; Jiang, Pan-Pan; Yozwiak, Nathan L; Matranga, Christian B; Yang, Xiao; Gire, Stephen K; Winnicki, Sarah; Tariyal, Ridhi; Schaffner, Stephen F; Okokhere, Peter O; Okogbenin, Sylvanus; Akpede, George O; Asogun, Danny A; Agbonlahor, Dennis E; Walker, Peter J; Tesh, Robert B; Levin, Joshua Z; Garry, Robert F; Sabeti, Pardis C; Happi, Christian T

    2015-03-01

    Next-generation sequencing (NGS) has the potential to transform the discovery of viruses causing unexplained acute febrile illness (UAFI) because it does not depend on culturing the pathogen or a priori knowledge of the pathogen's nucleic acid sequence. More generally, it has the potential to elucidate the complete human virome, including viruses that cause no overt symptoms of disease, but may have unrecognized immunological or developmental consequences. We have used NGS to identify RNA viruses in the blood of 195 patients with UAFI and compared them with those found in 328 apparently healthy (i.e., no overt signs of illness) control individuals, all from communities in southeastern Nigeria. Among UAFI patients, we identified the presence of nucleic acids from several well-characterized pathogenic viruses, such as HIV-1, hepatitis, and Lassa virus. In our cohort of healthy individuals, however, we detected the nucleic acids of two novel rhabdoviruses. These viruses, which we call Ekpoma virus-1 (EKV-1) and Ekpoma virus-2 (EKV-2), are highly divergent, with little identity to each other or other known viruses. The most closely related rhabdoviruses are members of the genus Tibrovirus and Bas-Congo virus (BASV), which was recently identified in an individual with symptoms resembling hemorrhagic fever. Furthermore, by conducting a serosurvey of our study cohort, we find evidence for remarkably high exposure rates to the identified rhabdoviruses. The recent discoveries of novel rhabdoviruses by multiple research groups suggest that human infection with rhabdoviruses might be common. While the prevalence and clinical significance of these viruses are currently unknown, these viruses could have previously unrecognized impacts on human health; further research to understand the immunological and developmental impact of these viruses should be explored. More generally, the identification of similar novel viruses in individuals with and without overt symptoms of disease

  11. DNA vaccines: a rational design against parasitic diseases.

    PubMed

    Carvalho, Joana A; Rodgers, Jean; Atouguia, Jorge; Prazeres, Duarte M F; Monteiro, Gabriel A

    2010-02-01

    Parasitic diseases are one of the most devastating causes of morbidity and mortality worldwide. Although immunization against these infections would be an ideal solution, the development of effective vaccines has been hampered by specific challenges posed by parasitic pathogens. Plasmid-based DNA vaccines may prove to be promising immunization tools in this area because vectors can be designed to integrate several antigens from different stages of the parasite life cycle or different subspecies; vaccines, formulations and immunization protocols can be tuned to match the immune response that offers protective immunity; and DNA vaccination is an affordable platform for developing countries. Partial and full protective immunity have been reported following DNA vaccination against the most significant parasitic diseases in the world.

  12. Vector Design for Improved DNA Vaccine Efficacy, Safety and Production

    PubMed Central

    Williams, James A.

    2013-01-01

    DNA vaccination is a disruptive technology that offers the promise of a new rapidly deployed vaccination platform to treat human and animal disease with gene-based materials. Innovations such as electroporation, needle free jet delivery and lipid-based carriers increase transgene expression and immunogenicity through more effective gene delivery. This review summarizes complementary vector design innovations that, when combined with leading delivery platforms, further enhance DNA vaccine performance. These next generation vectors also address potential safety issues such as antibiotic selection, and increase plasmid manufacturing quality and yield in exemplary fermentation production processes. Application of optimized constructs in combination with improved delivery platforms tangibly improves the prospect of successful application of DNA vaccination as prophylactic vaccines for diverse human infectious disease targets or as therapeutic vaccines for cancer and allergy. PMID:26344110

  13. DAI (DLM-1/ZBP1) as a genetic adjuvant for DNA vaccines that promotes effective antitumor CTL immunity.

    PubMed

    Lladser, Alvaro; Mougiakakos, Dimitrios; Tufvesson, Helena; Ligtenberg, Maarten A; Quest, Andrew Fg; Kiessling, Rolf; Ljungberg, Karl

    2011-03-01

    DNA vaccination is an attractive approach to induce antigen-specific cytotoxic CD8(+) T lymphocytes (CTLs), which can mediate protective antitumor immunity. The potency of DNA vaccines encoding weakly immunogenic tumor-associated antigens (TAAs) can be enhanced by codelivering gene-encoded adjuvants. Pattern recognition receptors (PRRs) that sense intracellular DNA could potentially be used to harness intrinsic immune-stimulating properties of plasmid DNA vaccines. Consequently, the cytosolic DNA sensor, DNA-dependent activator of interferon (IFN) regulatory factors (DAI), was used as a genetic adjuvant. In vivo electroporation (EP) of mice with a DAI-encoding plasmid (pDAI) promoted transcription of genes encoding type I IFNs, proinflammatory cytokines, and costimulatory molecules. Coimmunization with pDAI and antigen-encoding plasmids enhanced in vivo antigen-specific proliferation, and induction of effector and memory CTLs. Moreover, codelivery of pDAI effectively promoted CTL and CD4(+) Th1 responses to the TAA survivin. The DAI-enhanced CTL induction required nuclear factor κB (NF-κB) activation and type I IFN signaling, but did not involve the IFN regulatory factor 3 (IRF3). Codelivery of pDAI also increased CTL responses to the melanoma-associated antigen tyrosinase-related protein-2 (TRP2), enhanced tumor rejection and conferred long-term protection against B16 melanoma challenge. This study constitutes "proof-of-principle" validating the use of intracellular PRRs as genetic adjuvants to enhance DNA vaccine potency.

  14. Characterization of the complete genome sequence of pike fry rhabdovirus.

    PubMed

    Chen, Hong-Lian; Liu, Hong; Liu, Zong-Xiao; He, Jun-Qiang; Gao, Long-Ying; Shi, Xiu-Jie; Jiang, Yu-Lin

    2009-01-01

    The complete genome sequence of pike fry rhabdovirus (PFRV), consisting of 11,097 nucleotides, was determined. The genome contains five genes, encoding the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L) protein in the order 3'-N-P-M-G-L-5'. 3' leader- and 5' trailer-sequences in the PFRV genome show inverse complementarity. The PFRV proteins share the highest homology to the proteins of spring viremia of carp virus (SVCV), ranging from 55.3 to 91.4%. Phylogenetic analysis of the five proteins showed that PFRV clusters with SVCV and is closely related to the mammalian vesiculoviruses, 903/87, STRV and SCRV.

  15. Transcription of immune genes upon challenge with viral hemorrhagic septicemia virus (VHSV) in DNA vaccinated rainbow trout (Oncorhynchus mykiss).

    PubMed

    Cuesta, A; Tafalla, C

    2009-01-07

    Even though DNA vaccination has proven as one of the most effective methods in controlling fish rhabdoviruses, the immune mechanisms responsible for protection are still unknown. Many studies have focused on studying which cytokines and immune genes are triggered in response to the vaccine at different times post-vaccination. However, to elucidate the mechanism(s) responsible for protection, to our understanding it is also of great relevance to study the immune response to the virus in fish that have been previously vaccinated and compare it to the effects that the virus might have on non-vaccinated fish. This type of study has never been performed to date in fish. Thus, in the current work, we vaccinated rainbow trout (Oncorhynchus mykiss) with a DNA vaccine against viral hemorrhagic septicemia virus (VHSV), and 30 days post-vaccination we challenged the fish with a virulent VHSV. It was then, that we studied the immune response to the virus at very early times post-infection in fish, in order to compare the effects of VHSV on vaccinated or non-vaccinated trout. We studied the levels of expression of interleukin 1beta (IL-1beta), major histocompatibility complex (MHC) class Ialpha and IIalpha genes, immunoglobulin M (IgM), CD8alpha, type I interferon (IFN), Mx, IFN-gamma and natural killer enhancing factor (NKEF) in head kidney, spleen and blood. When we compared the effect that VHSV had on vaccinated fish to the effect that the virus produced in fish vaccinated with the empty plasmid, the genes that were significantly up-regulated were IL-1beta and MHC IIalpha in the spleen at day 1 post-infection, MHC Ialpha in all organs at day 1 post-infection, and IFN and Mx in the spleen and blood at days 1 and 3 post-infection, respectively. Genes that correlate with an increased specific immune response were not significantly increased in response to VHSV in these vaccinated animals. The results suggest that DNA vaccination induces a memory state in fish that, on the

  16. Using Plasmids as DNA Vaccines for Infectious Diseases.

    PubMed

    Tregoning, John S; Kinnear, Ekaterina

    2014-12-01

    DNA plasmids can be used to induce a protective (or therapeutic) immune response by delivering genes encoding vaccine antigens. That naked DNA (without the refinement of coat proteins or host evasion systems) can cross from outside the cell into the nucleus and be expressed is particularly remarkable given the sophistication of the immune system in preventing infection by pathogens. As a result of the ease, low cost, and speed of custom gene synthesis, DNA vaccines dangle a tantalizing prospect of the next wave of vaccine technology, promising individual designer vaccines for cancer or mass vaccines with a rapid response time to emerging pandemics. There is considerable enthusiasm for the use of DNA vaccination as an approach, but this enthusiasm should be tempered by the successive failures in clinical trials to induce a potent immune response. The technology is evolving with the development of improved delivery systems that increase expression levels, particularly electroporation and the incorporation of genetically encoded adjuvants. This review will introduce some key concepts in the use of DNA plasmids as vaccines, including how the DNA enters the cell and is expressed, how it induces an immune response, and a summary of clinical trials with DNA vaccines. The review also explores the advances being made in vector design, delivery, formulation, and adjuvants to try to realize the promise of this technology for new vaccines. If the immunogenicity and expression barriers can be cracked, then DNA vaccines may offer a step change in mass vaccination.

  17. Hepatitis E virus DNA vaccine elicits immunologic memory in mice.

    PubMed

    He, J; Hayes, C G; Binn, L N; Seriwatana, J; Vaughn, D W; Kuschner, R A; Innis, B L

    2001-01-01

    Injection of an expression vector pJHEV containing hepatitis E virus (HEV) structural protein open reading frame 2 gene generates a strong antibody response in BALB/c mice that can bind to and agglutinate HEV. In this study, we tested for immunologic memory in immunized mice whose current levels of IgG to HEV were low or undetectable despite 3 doses of HEV DNA vaccine 18 months earlier. Mice previously vaccinated with vector alone were controls. All mice were administered a dose of HEV DNA vaccine to simulate an infectious challenge with HEV. The endpoint was IgG to HEV determined by ELISA. Ten days after the vaccine dose, 5 of 9 mice previously immunized with HEV DNA vaccine had a slight increase in IgG to HEV. By 40 days after the vaccine dose, the level of IgG to HEV had increased dramatically in all 9 mice (108-fold increase in geometric mean titer). In contrast, no control mice became seropositive. These results indicate that mice vaccinated with 3 doses of HEV DNA vaccine retain immunologic memory. In response to a small antigenic challenge delivered as DNA, possibly less than delivered by a human infective dose of virus, mice with memory were able to generate high levels of antibody in less time than the usual incubation period of hepatitis E. We speculate that this type of response could protect a human from overt disease.

  18. Bacillus subtilis spores as adjuvants for DNA vaccines.

    PubMed

    Aps, Luana R M M; Diniz, Mariana O; Porchia, Bruna F M M; Sales, Natiely S; Moreno, Ana Carolina R; Ferreira, Luís C S

    2015-05-11

    Recently, Bacillus subtilis spores were shown to be endowed with strong adjuvant capacity when co-administered with purified antigenic proteins. In the present study we assessed whether spores possess adjuvant properties when combined with DNA vaccines. We showed that B. subtilis spores promoted the activation of dendritic cells in vitro and induced migration of pro-inflammatory cells after parenteral administration to mice. Likewise, co-administration of spores with a DNA vaccine encoding the human papillomavirus type 16 (HPV-16) E7 protein enhanced the activation of antigen-specific CD8(+) T cell responses in vivo. Mice immunized with the DNA vaccine admixed with spores presented a protective immunity increase to previously implanted tumor cells, capable of expressing HPV-16 oncoproteins. Finally, we observed that the adjuvant effect can vary accordingly to the number of co-administered spores which may be ascribed with the ability to induce. Collectively, the present results demonstrate for the first time that B. subtilis spores can also confer adjuvant effects to DNA vaccines.

  19. Preclinical and clinical development of DNA vaccines for prostate cancer.

    PubMed

    Colluru, V T; Johnson, Laura E; Olson, Brian M; McNeel, Douglas G

    2016-04-01

    Prostate cancer is the most commonly diagnosed cancer in the United States. It is also the second leading cause of cancer-related death in men, making it one of the largest public health concerns today. Prostate cancer is an ideal disease for immunotherapies because of the generally slow progression, the dispensability of the target organ in the patient population, and the availability of several tissue-specific antigens. As such, several therapeutic vaccines have entered clinical trials, with one autologous cellular vaccine (sipuleucel-T) recently gaining Food and Drug Administration approval after demonstrating overall survival benefit in randomized phase III clinical trials. DNA-based vaccines are safe, economical, alternative "off-the-shelf" approaches that have undergone extensive evaluation in preclinical models. In fact, the first vaccine approved in the United States for the treatment of cancer was a DNA vaccine for canine melanoma. Several prostate cancer-specific DNA vaccines have been developed in the last decade and have shown promising results in early phase clinical trials. This review summarizes anticancer human DNA vaccine trials, with a focus on those conducted for prostate cancer. We conclude with an outline of special considerations important for the development and successful translation of DNA vaccines from the laboratory to the clinic.

  20. Analysis of DNA-vaccinated fish reveals viral antigen in muscle, kidney, and thymus, and transient histopathologic changes

    USGS Publications Warehouse

    Garver, K.A.; Conway, C.M.; Elliott, D.G.; Kurath, G.

    2005-01-01

    A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was used in a systematic study to analyze vaccine tissue distribution, persistence, expression patterns, and histopathologic effects. Vaccine plasmid pIHNw-G, containing the gene for the viral glycoprotein, was detected immediately after intramuscular injection in all tissues analyzed, including blood, but at later time points was found primarily in muscle tissue, where it persisted to 90 days. Glycoprotein expression was detected in muscle, kidney, and thymus tissues, with levels peaking at 14 days and becoming undetectable by 28 days. Histologic examination revealed no vaccine-specific pathologic changes at the standard effective dose of 0.1 ??g DNA per fish, but at a high dose of 50 ??g an increased inflammatory response was evident. Transient damage associated with needle injection was localized in muscle tissue, but by 90 days after vaccination no damage was detected in any tissue, indicating the vaccine to be safe and well tolerated. ?? Springer Science+Business Media, Inc. 2005.

  1. Induction of antigen-positive cell death by the expression of perforin, but not DTa, from a DNA vaccine enhances the immune response.

    PubMed

    Gargett, Tessa; Grubor-Bauk, Branka; Garrod, Tamsin J; Yu, Wenbo; Miller, Darren; Major, Lee; Wesselingh, Steve; Suhrbier, Andreas; Gowans, Eric J

    2014-04-01

    The failure of traditional protein-based vaccines to prevent infection by viruses such as HIV or hepatitis C highlights the need for novel vaccine strategies. DNA vaccines have shown promise in small animal models, and are effective at generating anti-viral T cell-mediated immune responses; however, they have proved to be poorly immunogenic in clinical trials. We propose that the induction of necrosis will enhance the immune response to vaccine antigens encoded by DNA vaccines, as necrotic cells are known to release a range of intracellular factors that lead to dendritic cell (DC) activation and enhanced cross-presentation of antigen. Here we provide evidence that induction of cell death in DNA vaccine-targeted cells provides an adjuvant effect following intradermal vaccination of mice; however, this enhancement of the immune response is dependent on both the mechanism and timing of cell death after antigen expression. We report that a DNA vaccine encoding the cytolytic protein, perforin, resulted in DC activation, enhanced broad and multifunctional CD8 T-cell responses to the HIV-1 antigen GAG and reduced viral load following challenge with a chimeric virus, EcoHIV, compared with the canonical GAG DNA vaccine. This effect was not observed for a DNA vaccine encoding an apoptosis-inducing toxin, DTa, or when the level of perforin expression was increased to induce cell death sooner after vaccination. Thus, inducing lytic cell death following a threshold level of expression of a viral antigen can improve the immunogenicity of DNA vaccines, whereas apoptotic cell death has an inhibitory effect on the immune response.

  2. Development of a DNA vaccine targeting Merkel cell polyomavirus.

    PubMed

    Zeng, Qi; Gomez, Bianca P; Viscidi, Raphael P; Peng, Shiwen; He, Liangmei; Ma, Barbara; Wu, T-C; Hung, Chien-Fu

    2012-02-08

    Merkel cell carcinoma (MCC) is a rare but devastating skin disease that is increasing in incidence within the United States. The poor prognosis of MCC patients and limited understanding of MCC pathogenesis warrants innovative treatments to control MCC. Several lines of evidence have pointed to Merkel cell polyomavirus (MCPyV) as the etiological agent of MCC. In particular, the amino terminus of MCPyV large T antigen (LT) (aa1-258) is expressed in all MCPyV-positive tumors and plays an important role in MCC oncogenesis, rendering it an ideal therapeutic target for vaccination. In the current study, we developed a DNA vaccine encoding MCPyV LT aa1-258 (pcDNA3-LT). Within our pcDNA3-LT DNA vaccine, we identified that MCPyV LT aa136-160 likely contains an LT-specific CD4+ T helper epitope. We have also created an LT-expressing B16/LT tumor model using B16, a murine melanoma cell line, to characterize the potency of our DNA vaccine. Using this tumorigenic B16/LT tumor model, we found that pcDNA3-LT DNA vaccine generates antitumor effects mainly mediated by CD4+ T cells against B16/LT tumors in vaccinated C57BL/6 mice. Thus, immunotherapy using pcDNA3-LT DNA vaccine may represent a promising approach for the control of MCPyV-associated lesions. The B16/LT tumor model further serves as a useful model for testing various vaccine strategies against MCC.

  3. Zahedan rhabdovirus, a novel virus detected in ticks from Iran.

    PubMed

    Dilcher, Meik; Faye, Oumar; Faye, Ousmane; Weber, Franziska; Koch, Andrea; Sadegh, Chinikar; Weidmann, Manfred; Sall, Amadou Alpha

    2015-11-05

    Rhabdoviridae infect a wide range of vertebrates, invertebrates and plants. Their transmission can occur via various arthropod vectors. In recent years, a number of novel rhabdoviruses have been identified from various animal species, but so far only few tick-transmitted rhabdoviruses have been described. We isolated a novel rhabdovirus, provisionally named Zahedan rhabdovirus (ZARV), from Hyalomma anatolicum anatolicum ticks collected in Iran. The full-length genome was determined using 454 next-generation sequencing and the phylogenetic relationship to other rhabdoviruses was analyzed. Inoculation experiments in mammalian Vero cells and mice were conducted and a specific PCR assay was developed. The complete genome of ZARV has a size of 11,230 nucleotides (nt) with the typical genomic organization of Rhabdoviridae. Phylogenetic analysis confirms that ZARV is closely related to Moussa virus (MOUV) from West Africa and Long Island tick rhabdovirus (LITRV) from the U.S., all forming a new monophyletic clade, provisionally designated Zamolirhabdovirus, within the Dimarhabdovirus supergroup. The glycoprotein (G) contains 12 conserved cysteins which are specific for animal rhabdoviruses infecting fish and mammals. In addition, ZARV is able to infect mammalian Vero cells and is lethal for mice when inoculated intracerebrally or subcutaneously. The developed PCR assay can be used to specifically detect ZARV. The novel tick-transmitted rhabdovirus ZARV is closely related to MOUV and LITRV. All three viruses seem to form a new monophyletic clade. ZARV might be pathogenic for mammals, since it can infect Vero cells, is lethal for mice and its glycoprotein contains 12 conserved cysteins only found in animal rhabdoviruses. The mammalian host of ZARV remains to be identified.

  4. DNA vaccination by electroporation and boosting with recombinant proteins enhances the efficacy of DNA vaccines for Schistosomiasis japonica.

    PubMed

    Dai, Yang; Zhu, Yinchang; Harn, Donald A; Wang, Xiaoting; Tang, Jianxia; Zhao, Song; Lu, Fei; Guan, Xiaohong

    2009-12-01

    Schistosomiasis japonica is an endemic, zoonotic disease of major public health importance in China. Control programs combining chemotherapy and snail killing have not been able to block transmission of infection in lakes and marsh regions. Vaccination is needed as a complementary approach to the ongoing control programs. In the present study, we wanted to determine if the efficacies of DNA vaccines encoding the 23-kDa tetraspanin membrane protein (SjC23), triose phosphate isomerase (SjCTPI), and sixfold-repeated genes of the complementarity determining region 3 (CDR3) in the H chain of NP30 could be enhanced by boosting via electroporation in vivo and/or with cocktail protein vaccines. Mice vaccinated with cocktail DNA vaccines showed a significant worm reduction of 32.88% (P < 0.01) and egg reduction of 36.20% (P < 0.01). Vaccine efficacy was enhanced when animals were boosted with cocktail protein vaccines; adult worm and liver egg burdens were reduced 45.35% and 48.54%, respectively. Nearly identical results were obtained in mice boosted by electroporation in vivo, with adult worm and egg burdens reduced by 45.00% and 50.88%, respectively. The addition of a protein vaccine boost to this regimen further elevated efficacy to approximately 60% for adult worm burden and greater than 60% for liver egg reduction. The levels of interleukin-2, gamma interferon, and the ratios of immunoglobulin G2a (IgG2a)/IgG1 clearly showed that cocktail DNA vaccines induced CD4(+) Th1-type responses. Boosting via either electroporation or with recombinant proteins significantly increased associated immune responses over those seen in mice vaccinated solely with DNA vaccines. Thus, schistosome DNA vaccine efficacy was significantly enhanced via boosting by electroporation in vivo and/or cocktail protein vaccines.

  5. Variable effects of the co-administration of a GM-CSF-expressing plasmid on the immune response to flavivirus DNA vaccines in mice.

    PubMed

    Chen, Hui; Gao, Na; Wu, Jiangman; Zheng, Xiaoyan; Li, Jieqiong; Fan, Dongying; An, Jing

    2014-11-01

    As a cytokine adjuvant, granulocyte-macrophage colony-stimulating factor (GM-CSF) has been demonstrated to play central roles in the enhancement of the immune response and protection elicited by experimental vaccines. However, in our previous work, the co-administration of GM-CSF produced untoward effects on the immune response induced by a Japanese encephalitis virus DNA vaccine candidate. This study aimed to elucidate the adjuvant roles of GM-CSF in several Flaviviridae virus DNA vaccine candidates. Our results showed that the effects of GM-CSF were diverse: co-inoculated GM-CSF caused significant suppression to the dengue virus type 1 and type 2 prM-E DNA vaccinations and influenced protective efficiency against virus challenge. In contrast, GM-CSF showed little effect or an enhancement on the immune response elicited by hepatitis C virus C or E1 DNA vaccine candidates. Notably, these effects of GM-CSF were highly durable. Our results suggested that the adjuvant roles of the GM-CSF plasmid were complex and diverse, ranging from enhancement to suppression, depending on the immunogen of Flaviviridae virus DNA vaccine candidates. Therefore, the application of GM-CSF as a vaccine adjuvant or a therapeutic agent should be evaluated carefully.

  6. The glycoprotein genes and gene junctions of the fish rhabdoviruses spring viremia of carp virus and hirame rhabdovirus: analysis of relationships with other rhabdoviruses.

    PubMed

    Björklund, H V; Higman, K H; Kurath, G

    1996-06-01

    The nucleotide sequences of the glycoprotein genes and all of the internal gene junctions of the fish pathogenic rhabdoviruses spring viremia of carp virus (SVCV) and hirame rhabdovirus (HIRRV) have been determined from cDNA clones generated from viral genomic RNA. The SVCV glycoprotein gene sequence is 1588 nucleotides (nt) long and encodes a 509 amino acid (aa) protein. The HIRRV glycoprotein gene sequence comprises 1612 nt, coding for a 508 aa protein. In sequence comparisons of 15 rhabdovirus glycoproteins, the SVCV glycoprotein gene showed the highest amino acid sequence identity (31.2-33.2%) with vesicular stomatitis New Jersey virus (VSNJV), Chandipura virus (CHPV) and vesicular stomatitis Indiana virus (VSIV). The HIRRV glycoprotein gene showed a very high amino acid sequence identity (74.3%) with the glycoprotein gene of another fish pathogenic rhabdovirus, infectious hematopoietic necrosis virus (IHNV), but no significant similarity with glycoproteins of VSIV or rabies virus (RABV). In phylogenetic analyses SVCV was grouped consistently with VSIV, VSNJV and CHPV in the Vesiculovirus genus of Rhabdoviridae. The fish rhabdoviruses HIRRV, IHNV and viral hemorrhagic septicemia virus (VHSV) showed close relationships with each other, but only very distant relationships with mammalian rhabdoviruses. The gene junctions are highly conserved between SVCV and VSIV, well conserved between IHNV and HIRRV, but not conserved between HIRRV/IHNV and RABV. Based on the combined results we suggest that the fish lyssa-type rhabdoviruses HIRRV, IHNV and VHSV may be grouped in their own genus within the family Rhabdoviridae. Aquarhabdovirus has been proposed for the name of this new genus.

  7. The glycoprotein genes and gene junctions of the fish rhabdoviruses spring viremia of carp virus and hirame rhabdovirus: Analysis of relationships with other rhabdoviruses

    USGS Publications Warehouse

    Bjorklund, H.V.; Higman, K.H.; Kurath, G.

    1996-01-01

    The nucleotide sequences of the glycoprotein genes and all of the internal gene junctions of the fish pathogenic rhabdoviruses spring viremia of carp virus (SVCV) and hirame rhabdovirus (HIRRV) have been determined from cDNA clones generated from viral genomic RNA. The SVCV glycoprotein gene sequence is 1588 nucleotides (nt) long and encodes a 509 amino acid (aa) protein. The HIRRV glycoprotein gene sequence comprises 1612 nt, coding for a 508 aa protein. In sequence comparisons of 15 rhabdovirus glycoproteins, the SVCV glycoprotein gene showed the highest amino acid sequence identity (31.2–33.2%) with vesicular stomatitis New Jersey virus (VSNJV), Chandipura virus (CHPV) and vesicular stomatitis Indiana virus (VSIV). The HIRRV glycoprotein gene showed a very high amino acid sequence identity (74.3%) with the glycoprotein gene of another fish pathogenic rhabdovirus, infectious hematopoietic necrosis virus (IHNV), but no significant similarity with glycoproteins of VSIV or rabies virus (RABV). In phylogenetic analyses SVCV was grouped consistently with VSIV, VSNJV and CHPV in the Vesiculovirus genus of Rhabdoviridae. The fish rhabdoviruses HIRRV, IHNV and viral hemorrhagic septicemia virus (VHSV) showed close relationships with each other, but only very distant relationships with mammalian rhabdoviruses. The gene junctions are highly conserved between SVCV and VSIV, well conserved between IHNV and HIRRV, but not conserved between HIRRV/IHNV and RABV. Based on the combined results we suggest that the fish lyssa-type rhabdoviruses HIRRV, IHNV and VHSV may be grouped in their own genus within the family Rhabdoviridae. Aquarhabdovirus has been proposed for the name of this new genus.

  8. The glycoprotein genes and gene junctions of the fish rhabdoviruses spring viremia of carp virus and hirame rhabdovirus: Analysis of relationships with other rhabdoviruses

    USGS Publications Warehouse

    Bjorklund, H.V.; Higman, K.H.; Kurath, G.

    1996-01-01

    The nucleotide sequences of the glycoprotein genes and all of the internal gene junctions of the fish pathogenic rhabdoviruses spring viremia of carp virus (SVCV) and hirame rhabdovirus (HIRRV) have been determined from cDNA clones generated from viral genomic RNA. The SVCV glycoprotein gene sequence is 1588 nucleotides (nt) long and encodes a 509 amino acid (aa) protein. The HIRRV glycoprotein gene sequence comprises 1612 nt, coding for a 508 aa protein. In sequence comparisons of 15 rhabdovirus glycoproteins, the SVCV glycoprotein gene showed the highest amino acid sequence identity (31.2-33.2%) with vesicular stomatitis New Jersey virus (VSNJV), Chandipura virus (CHPV) and vesicular stomatitis Indiana virus (VSIV). The HIRRV glycoprotein gene showed a very high amino acid sequence identity (74.3%) with the glycoprotein gene of another fish pathogenic rhabdovirus, infectious hematopoietic necrosis virus (IHNV), but no significant similarity with glycoproteins of VSIV or rabies virus (RABV). In phylogenetic analyses SVCV was grouped consistently with VSIV, VSNJV and CHPV in the Vesiculovirus genus of Rhabdoviridae. The fish rhabdoviruses HIRRV, IHNV and viral hemorrhagic septicemia virus (VHSV) showed close relationships with each other, but only very distant relationships with mammalian rhabdoviruses. The gene junctions are highly conserved between SVCV and VSIV, well conserved between IHNV and HIRRV, but not conserved between HIRRV/IHNV and RABV. Based on the combined results we suggest that the fish lyssa-type rhabdoviruses HIRRV, IHNV and VHSV may be grouped in their own genus within the family Rhabdoviridae. Aquarhabdovirus has been proposed for the name of this new genus.

  9. Reverse genetics on fish rhabdoviruses: tools to study the pathogenesis of fish rhabdoviruses.

    PubMed

    Brémont, M

    2005-01-01

    Rhabdoviruses, mainly in rainbow trout, are among the most devastating viruses for worldwide aquaculture. To date no effective treatments to fight against these viruses are available. During the past years, several approaches to develop efficient vaccines have been undertaken such as the use of immunogenic recombinant viral proteins, naked DNA or inactivated viruses. However, although these vaccines have been proven to be very effective on a small scale, they have never been used in the field because the vaccines would have to be injected into thousands of yearling trouts. The only alternative to injection consists of the development of attenuated live vaccines that can be administrated to trouts by bath immersion. Reverse genetics on trout rhabdoviruses offer the possibility of recovering a series of live recombinant viruses in which the viral genome has been irreversibly modified to generate cost-effective live, safe vaccines.

  10. Genomic sequence of mandarin fish rhabdovirus with an unusual small non-transcriptional ORF.

    PubMed

    Tao, Jian-Jun; Zhou, Guang-Zhou; Gui, Jian-Fang; Zhang, Qi-Ya

    2008-03-01

    The complete genome of mandarin fish Siniperca chuatsi rhabdovirus (SCRV) was cloned and sequenced. It comprises 11,545 nucleotides and contains five genes encoding the nucleoprotein N, the phosphoprotein P, the matrix protein M, the glycoprotein G, and the RNA-dependent RNA polymerase protein L. At the 3' and 5' termini of SCRV genome, leader and trailer sequences show inverse complementarity. The N, P, M and G proteins share the highest sequence identities (ranging from 14.8 to 41.5%) with the respective proteins of rhabdovirus 903/87, the L protein has the highest identity with those of vesiculoviruses, especially with Chandipura virus (44.7%). Phylogenetic analysis of L proteins showed that SCRV clustered with spring vireamia of carp virus (SVCV) and was most closely related to viruses in the genus Vesiculovirus. In addition, an overlapping open reading frame (ORF) predicted to encode a protein similar to vesicular stomatitis virus C protein is present within the P gene of SCRV. Furthermore, an unoverlapping small ORF downstream of M ORF within M gene is predicted (tentatively called orf4). Therefore, the genomic organization of SCRV can be proposed as 3' leader-N-P/C-M-(orf4)-G-L-trailer 5'. Orf4 transcription or translation products could not be detected by northern or Western blot, respectively, though one similar mRNA band to M mRNA was found. This is the first report on one small unoverlapping ORF in M gene of a fish rhabdovirus.

  11. Current trends in separation of plasmid DNA vaccines: a review.

    PubMed

    Ghanem, Ashraf; Healey, Robert; Adly, Frady G

    2013-01-14

    Plasmid DNA (pDNA)-based vaccines offer more rapid avenues for development and production if compared to those of conventional virus-based vaccines. They do not rely on time- or labour-intensive cell culture processes and allow greater flexibility in shipping and storage. Stimulating antibodies and cell-mediated components of the immune system are considered as some of the major advantages associated with the use of pDNA vaccines. This review summarizes the current trends in the purification of pDNA vaccines for practical and analytical applications. Special attention is paid to chromatographic techniques aimed at reducing the steps of final purification, post primary isolation and intermediate recovery, in order to reduce the number of steps necessary to reach a purified end product from the crude plasmid.

  12. Systems Vaccinology Applied to DNA Vaccines: Perspective and Challenges.

    PubMed

    Lever, Melissa; Silveira, Eduardo L; Nakaya, Helder I

    2017-01-01

    DNA vaccination represents a new milestone in our technological efforts to avoid infectious diseases. Although this method of vaccination has had success in providing protection in animals, these vaccines suffer from low immunogenicity in humans. Questions remain over the molecular mechanism of DNA vaccination, the best ways in which to safely increase vaccine reactogenecity, and what biomarkers can be used as correlates of protection. Systems vaccinology, which utilizes modern experimental and computational approaches to provide an integrated view of the vaccination process, offers the potential to answer these questions. In this review we discuss the current tools utilized in systems vaccinology, the ways in which they have and can be applied to DNA vaccinology, and challenges faced in the field.

  13. Enhancing the Immunogenicity of a Tetravalent Dengue DNA Vaccine

    DTIC Science & Technology

    2016-08-01

    AWARD NUMBER: W81XWH-15-2-0029 TITLE: Enhancing the Immunogenicity of a Tetravalent Dengue DNA Vaccine PRINCIPAL INVESTIGATOR: Maya...TITLE AND SUBTITLE Enhancing the Immunogenicity of a Tetravalent Dengue DNA 5a. CONTRACT NUMBER Vaccine 5b. GRANT NUMBER 5c. PROGRAM ELEMENT...personnel is priority research area for the US DoD. Phase 1 clinical trials demonstrated that the Naval Medical Research Center’s DNA based dengue vaccine

  14. Molecular characterization of the glycoproteins from two warm water rhabdoviruses: snakehead rhabdovirus (SHRV) and rhabdovirus of penaeid shrimp (RPS)/spring viremia of carp virus (SVCV).

    PubMed

    Johnson, M C; Maxwell, J M; Loh, P C; Leong, J A

    1999-11-01

    We have determined the complete coding sequences for the glycoprotein (G) genes from two rhabdoviruses that infect warm water aquatic animals, the snakehead rhabdovirus (SHRV) and rhabdovirus of penaeid shrimp (RPS). Surprisingly, the G nucleotide sequence from RPS, a virus which has been isolated from diseased shrimp in Hawaii on numerous occasions, was over 99% identical to the G nucleotide sequence from spring viremia of carp virus (SVCV), a fish virus from Europe and Asia. This is the first report of SVCV isolation outside of Europe and Asia, and it is also the first report of SVCV infecting a non-vertebrate species. The G gene from SHRV was most closely related to the G genes from the three Novirhabdoviruses, viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and hirame rhabdovirus (HIRRV), with 47, 37, and 36% amino acid identity, respectively. In addition, a phylogenetic analysis using the amino acid sequence from rhabdovirus G genes indicated that SHRV should be classified within the Novirhabdovirus genus. Finally, the SHRV-G gene was successfully expressed in mammalian cells under the control of the cytomegalovirus (CMV) promoter, establishing that it can potentially be used in the production of pseudotyped retroviruses designed to infect fish.

  15. Enhancement of HIV-1 DNA vaccine immunogenicity by BCG-PSN, a novel adjuvant.

    PubMed

    Sun, Jing; Hou, Jue; Li, Dingfeng; Liu, Yong; Hu, Ningzhu; Hao, Yanling; Fu, Jingjing; Hu, Yunzhang; Shao, Yiming

    2013-01-07

    Although the importance of DNA vaccines, especially as a priming immunization has been well established in numerous HIV vaccine studies, the immunogenictiy of DNA vaccines is generally moderate. Novel adjuvant is in urgent need for improving the immunogenicity of DNA vaccine. Polysaccharide and nucleic acid fraction extracted by hot phenol method from Mycobacterium bovis bacillus Calmette-Guérin, known as BCG-PSN, is a widely used immunomodulatory product in China clinical practice. In this study, we evaluated whether the BCG-PSN could serve as a novel adjuvant of DNA vaccine to trigger better cellular and humoral immune responses against the HIV-1 Env antigen in Balb/C mouse model. The BCG-PSN was mixed with 10 μg or 100 μg of pDRVI1.0gp145 (HIV-1 CN54 gp145 gene) DNA vaccine and intramuscularly immunized two or three times. We found that BCG-PSN could significantly improve the immunogenicity of DNA vaccine when co-administered with DNA vaccine. Further, at the same vaccination schedule, BCG-PSN co-immunization with 10 μg DNA vaccine could elicit cellular and humoral immune responses which were comparable to that induced by 100 μg DNA vaccine alone. Moreover, our results demonstrate that BCG-PSN can activate TLR signaling pathways and induce Th1-type cytokines secretion. These findings suggest that BCG-PSN can serve as a novel and effective adjuvant for DNA vaccination.

  16. Tailoring DNA Vaccines: Designing Strategies Against HER2-Positive Cancers

    PubMed Central

    Marchini, Cristina; Kalogris, Cristina; Garulli, Chiara; Pietrella, Lucia; Gabrielli, Federico; Curcio, Claudia; Quaglino, Elena; Cavallo, Federica; Amici, Augusto

    2013-01-01

    The crucial role of HER2 in epithelial transformation and its selective overexpression on cancer tissues makes it an ideal target for cancer immunotherapies such as passive immunotherapy with Trastuzumab. There are, however, a number of concerns regarding the use of monoclonal antibodies which include resistance, repeated treatments, considerable costs, and side effects that make active immunotherapies against HER2 desirable alternative approaches. The efficacy of anti-HER2 DNA vaccination has been widely demonstrated in transgenic cancer-prone mice, which recapitulate several features of human breast cancers. Nonetheless, the rational design of a cancer vaccine able to trigger a long-lasting immunity, and thus prevent tumor recurrence in patients, would require the understanding of how tolerance and immunosuppression regulate antitumor immune responses and, at the same time, the identification of the most immunogenic portions of the target protein. We herein retrace the findings that led to our most promising DNA vaccines that, by encoding human/rat chimeric forms of HER2, are able to circumvent peripheral tolerance. Preclinical data obtained with these chimeric DNA vaccines have provided the rationale for their use in an ongoing Phase I clinical trial (EudraCT 2011-001104-34). PMID:23675574

  17. Prophylactic and therapeutic DNA vaccines against Chagas disease.

    PubMed

    Arce-Fonseca, Minerva; Rios-Castro, Martha; Carrillo-Sánchez, Silvia del Carmen; Martínez-Cruz, Mariana; Rodríguez-Morales, Olivia

    2015-02-24

    Chagas disease is a zoonosis caused by Trypanosoma cruzi in which the most affected organ is the heart. Conventional chemotherapy has a very low effectiveness; despite recent efforts, there is currently no better or more effective treatment available. DNA vaccines provide a new alternative for both prevention and treatment of a variety of infectious disorders, including Chagas disease. Recombinant DNA technology has allowed some vaccines to be developed using recombinant proteins or virus-like particles capable of inducing both a humoral and cellular specific immune response. This type of immunization has been successfully used in preclinical studies and there are diverse models for viral, bacterial and/or parasitic diseases, allergies, tumors and other diseases. Therefore, several research groups have been given the task of designing a DNA vaccine against experimental infection with T. cruzi. In this review we explain what DNA vaccines are and the most recent studies that have been done to develop them with prophylactic or therapeutic purposes against Chagas disease.

  18. Lipopolysaccharide contamination in intradermal DNA vaccination: toxic impurity or adjuvant?

    PubMed

    van den Berg, Joost H; Quaak, Susanne G L; Beijnen, Jos H; Hennink, Wim E; Storm, Gert; Schumacher, Ton N; Haanen, John B A G; Nuijen, Bastiaan

    2010-05-05

    Lipopolysaccharides (LPS) are known both as potential adjuvants for vaccines and as toxic impurity in pharmaceutical preparations. The aim of this study was to assess the role of LPS in intradermal DNA vaccination administered by DNA tattooing. Mice were vaccinated with a model DNA vaccine (Luc-NP) with an increasing content of residual LPS. The effect of LPS on systemic toxicity, antigen expression and cellular immunity was studied. The presence of LPS in the DNA vaccine neither induced systemic toxicity (as reflected by IL-6 concentration in serum), nor influenced antigen expression (measured by intravital imaging). Higher LPS contents however, appeared to be associated with an elevated cytotoxic T-lymphocyte (CTL) response but without reaching statistical significance. Interestingly, the DNA tattoo procedure by itself was shown to induce a serum cytokine response that was at least as potent as that induced by parenteral LPS administration. LPS does not show toxicity in mice vaccinated by DNA tattooing at dose levels well above those encountered in GMP-grade DNA preparations. Thus, residual LPS levels in the pharmaceutical range are not expected to adversely affect clinical outcome of vaccination trials and may in fact have some beneficial adjuvant effect. The observed pro-inflammatory effects of DNA tattoo may help explain the high immunogenicity of this procedure. Copyright 2009 Elsevier B.V. All rights reserved.

  19. Delivery methods to increase cellular uptake and immunogenicity of DNA vaccines.

    PubMed

    Jorritsma, S H T; Gowans, E J; Grubor-Bauk, B; Wijesundara, D K

    2016-11-04

    DNA vaccines are ideal candidates for global vaccination purposes because they are inexpensive and easy to manufacture on a large scale such that even people living in low-income countries can benefit from vaccination. However, the potential of DNA vaccines has not been realized owing mainly to the poor cellular uptake of DNA in vivo resulting in the poor immunogenicity of DNA vaccines. In this review, we discuss the benefits and shortcomings of several promising and innovative non-biological methods of DNA delivery that can be used to increase cellular delivery and efficacy of DNA vaccines.

  20. The enhanced immune response of hepatitis B virus DNA vaccine using SiO2@LDH nanoparticles as an adjuvant.

    PubMed

    Wang, Jin; Zhu, Rongrong; Gao, Bo; Wu, Bin; Li, Kun; Sun, Xiaoyu; Liu, Hui; Wang, Shilong

    2014-01-01

    Various approaches have been used to improve systemic immune response to infectious disease or virus, and DNA vaccination has been demonstrated to be one of these effective ways to elicit protective immunity against pathogens. Our previous studies showed that layered double hydroxides (LDH) nanoparticles could be efficiently taken up by the MDDCs and had an adjuvant activity for DC maturation. To further enhance the immune adjuvant activity of LDH, core-shell structure SiO2@LDH nanoparticles were synthesized with an average diameter of about 210 nm. And its high transfection efficiency in vitro was demonstrated by using GFP expression plasmid as model DNA. Exposing SiO2@LDH nanoparticles to macrophages caused a higher dose-dependent expression of IFN-γ, IL-6, CD86 and MHC II, compared with SiO2 and LDH respectively. Furthermore, in vivo immunization of BALB/c mice indicated that, DNA vaccine loaded-SiO2@LDH nanoparticles not only induced much higher serum antibody response than naked DNA vaccine and plain nanoparticles, but also obviously promoted T-cell proliferation and skewed T helper to Th1 polarization. Additionally, it was proved that the caveolae-mediated uptake of SiO2@LDH nanoparticles by macrophage lead to macrophages activation via NF-κB signaling pathway. Our results indicate that SiO2@LDH nanoparticles could serve as a potential non-viral gene delivery system. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Immunogenicity of candidate chimeric DNA vaccine against tuberculosis and leishmaniasis.

    PubMed

    Dey, Ayan; Kumar, Umesh; Sharma, Pawan; Singh, Sarman

    2009-08-13

    Mycobacterium tuberculosis and Leishmania donovani are important intracellular pathogens, especially in Indian context. In India and other South East Asian countries, both these infections are highly endemic and in about 20% cases co-infection of these pathogens is reported. For both these pathogens cell mediated immunity plays most important role. The available treatment of these infections is either prolonged or cumbersome or it is ineffective in controlling the outbreaks and spread. Therefore, potentiation of a common host defense mechanism can be used to prevent both the infections simultaneously. In this study we have developed a novel chimeric DNA vaccine candidate comprising the esat-6 gene of M. tuberculosis and kinesin motor domain gene of L. donovani. After developing this novel chimera, its immunogenicity was studied in mouse model. The immune response was compared with individual constructs of esat-6 and kinesin motor domain. The results showed that immunization with chimeric DNA vaccine construct resulted in stronger IFN-gamma and IL-2 response against kinesin (3012+/-102 and 367.5+/-8.92pg/ml) and ESAT-6 (1334+/-46.5 and 245.1+/-7.72pg/ml) in comparison to the individual vaccine constructs. The reciprocal immune response (IFN-gamma and IL-2) against individual construct was lower (kinesin motor domain: 1788+/-36.48 and 341.8+/-9.801pg/ml and ESAT-6: 867.0+/-47.23 and 170.8+/-4.578pg/ml, respectively). The results also suggest that using the chimeric construct both proteins yielded a reciprocal adjuvant affect over each other as the IFN-gamma production against chimera vaccination is statistically significant (p<0.0001) than individual construct vaccination. From this pilot study we could envisage that the chimeric DNA vaccine construct may offer an attractive strategy in controlling co-infection of leishmaniasis and tuberculosis and have important implication in future vaccine design.

  2. Transposon leads to contamination of clinical pDNA vaccine.

    PubMed

    van der Heijden, I; Gomez-Eerland, R; van den Berg, J H; Oosterhuis, K; Schumacher, T N; Haanen, J B A G; Beijnen, J H; Nuijen, B

    2013-07-11

    We report an unexpected contamination during clinical manufacture of a Human Papilomavirus (HPV) 16 E6 encoding plasmid DNA (pDNA) vaccine, with a transposon originating from the Escherichia coli DH5 host cell genome. During processing, presence of this transposable element, insertion sequence 2 (IS2) in the plasmid vector was not noticed until quality control of the bulk pDNA vaccine when results of restriction digestion, sequencing, and CGE analysis were clearly indicative for the presence of a contaminant. Due to the very low level of contamination, only an insert-specific PCR method was capable of tracing back the presence of the transposon in the source pDNA and master cell bank (MCB). Based on the presence of an uncontrolled contamination with unknown clinical relevance, the product was rejected for clinical use. In order to prevent costly rejection of clinical material, both in-process controls and quality control methods must be sensitive enough to detect such a contamination as early as possible, i.e. preferably during plasmid DNA source generation, MCB production and ultimately during upstream processing. However, as we have shown that contamination early in the process development pipeline (source pDNA, MCB) can be present below limits of detection of generally applied analytical methods, the introduction of "engineered" or transposon-free host cells seems the only 100% effective solution to avoid contamination with movable elements and should be considered when searching for a suitable host cell-vector combination.

  3. Three types of human CpG motifs differentially modulate and augment immunogenicity of nonviral and viral replicon DNA vaccines as built-in adjuvants.

    PubMed

    Yu, Yun-Zhou; Li, Na; Ma, Yao; Wang, Shuang; Yu, Wei-Yuan; Sun, Zhi-Wei

    2013-01-01

    NakedDNA vaccines given by intramuscular injection are efficient in mouse models, but they require improvement for human use. As the immunogenicity of DNA vaccines depends, to a large extent, on the presence of CpG motifs as built-in adjuvants, we addressed this issue by inserting three types of human CpG motifs (A-type, B-type, and C-type) into the backbone of nonviral DNA and viral DNA replicon vectors with distinct immunostimulatory activities on human PBMCs. The adjuvant effects of CpG modifications in DNA vaccines expressing three types of antigens (β-Gal, AHc, or PA4) were then characterized in mice and found to significantly enhance antigen-specific humoral and cell-mediated immune responses. The three types of CpG motifs also differentially affected and modulated immune responses and protective potency against botulinum neurotoxin serotype A and Bacillus anthracis A16R challenge. Taken together, these results demonstrate that insertion of human CpG motifs can differentially modulate the immunogenicity of nonviral DNA vaccines as well as viral DNA replicon vaccines. Our study provides not only a better understanding of the in vivo activities of CpG motif adjuvants but implications for the rational design of such motifs as built-in adjuvants for DNA vectors targeting specific antigens.

  4. Aluminium phosphate potentiates the efficacy of DNA vaccines against Leishmania mexicana.

    PubMed

    Rosado-Vallado, Miguel; Mut-Martin, Mirza; García-Miss, Maria del Rosario; Dumonteil, Eric

    2005-11-16

    DNA vaccines have been able to induce partial protection against infection with Leishmania in mice, but it is still necessary to increase their efficacy. In the present study we evaluated aluminium phosphate as an adjuvant of different formulations and doses of DNA vaccines against L. mexicana in BALB/c mice. Aluminium phosphate had no effect on the humoral response induced by a high dose (100 microg) DNA vaccine, but slightly increased the cellular response and the protection against infection. It also allowed a non-immunoprotective low dose (20 microg) DNA vaccine encoding L. mexicana GP63 and Leishmania donovani NH36 to become protective. Aluminium phosphate may thus be an effective, low cost and safe adjuvant for DNA vaccines, and the vaccine formulation described here may be an excellent candidate for further vaccine development against Leishmania.

  5. Preparation and immunological effectiveness of a swine influenza DNA vaccine encapsulated in chitosan nanoparticles.

    PubMed

    Zhao, Kai; Shi, Xingming; Zhao, Yan; Wei, Haixia; Sun, Qingshen; Huang, Tingting; Zhang, Xiaoyan; Wang, Yunfeng

    2011-11-03

    Preparation conditions of a DNA vaccine against swine influenza encapsulated in chitosan nanoparticles were determined. The nanoparticles were prepared according to a complex coacervation method using chitosan as a biodegradable matrix forming polymer. Under the preparation conditions, chitosan nanoparticles containing the DNA vaccine were produced with good morphology, high encapsulation rate and high stability. Transfection test indicated that the vaccine could be expressed as an antigen in cells, and maintained good bioactivity. In addition, better immune responses of mice immunized with the chitosan nanoparticles containing the DNA vaccine were induced and prolonged release of the plasmid DNA was achieved compared to the DNA vaccine alone. These results laid a foundation for further development of DNA vaccines in nanoparticles before ultimate industrial application.

  6. Efficient vaccine against pandemic influenza: combining DNA vaccination and targeted delivery to MHC class II molecules.

    PubMed

    Grødeland, Gunnveig; Bogen, Bjarne

    2015-06-01

    There are two major limitations to vaccine preparedness in the event of devastating influenza pandemics: the time needed to generate a vaccine and rapid generation of sufficient amounts. DNA vaccination could represent a solution to these problems, but efficacy needs to be enhanced. In a separate line of research, it has been established that targeting of vaccine molecules to antigen-presenting cells enhances immune responses. We have combined the two principles by constructing DNA vaccines that encode bivalent fusion proteins; these target hemagglutinin to MHC class II molecules on antigen-presenting cells. Such DNA vaccines rapidly induce hemagglutinin-specific antibodies and T cell responses in immunized mice. Responses are long-lasting and protect mice against challenge with influenza virus. In a pandemic situation, targeted DNA vaccines could be produced and tested within a month. The novel DNA vaccines could represent a solution to pandemic preparedness in the advent of novel influenza pandemics.

  7. IFN-γ increases efficiency of DNA vaccine in protecting ducks against infection

    PubMed Central

    Long, Jian-Er; Huang, Li-Na; Qin, Zhi-Qiang; Wang, Wen-Yi; Qu, Di

    2005-01-01

    AIM: To detect the effects of DNA vaccines in combination with duck IFN-γ gene on the protection of ducks against duck hepatitis B virus (DHBV) infection. METHODS: DuIFN-γ cDNA was cloned and expressed in COS-7 cells, and the antiviral activity of DuIFN-γ was detected and neutralized by specific antibodies. Ducks were vaccinated with DHBpreS/S DNA alone or co-immunized with plasmid expressing DuIFN-γ. DuIFN-γ mRNA in peripheral blood mononuclear cells (PBMCs) from immunized ducks was detected by semi-quantitative competitive RT-PCR. Anti-DHBpreS was titrated by enzyme-linked immunosorbent assay (ELISA). DHBV DNA in sera and liver was detected by Southern blot hybridization, after ducks were challenged with high doses of DHBV. RESULTS: DuIFN-γ expressed by COS-7 was able to protect duck fibroblasts against vesicular stomatitis virus (VSV) infection in a dose-dependent fashion, and anti-DuIFN-γ antibodies neutralized the antiviral effects. DuIFN-γ in the supernatant also inhibited the release of DHBV DNA from LMH-D2 cells. When ducks were co-immunized with DNA vaccine expressing DHBpreS/S and DuIFN-γ gene as an adjuvant, the level of DuIFN-γ mRNA in PBMCs was higher than that in ducks vaccinated with DHBpreS/S DNA alone. However, the titer of anti-DHBpreS elicited by DHBpreS/S DNA alone was higher than that co-immunized with DuIFN-γ gene and DHBpreS/S DNA. After being challenged with DHBV at high doses, the load of DHBV in sera dropped faster, and the amount of total DNA and cccDNA in the liver decreased more significantly in the group of ducks co-immunized with DuIFN-γ gene and DHBpreS/S DNA than in other groups. CONCLUSION: DHBV preS/S DNA vaccine can protect ducks against DHBV infection, DuIFN-γ gene as an immune adjuvant enhances its efficacy. PMID:16124047

  8. Superparamagnetic nanoparticles for effective delivery of malaria DNA vaccine.

    PubMed

    Al-Deen, Fatin Nawwab; Ho, Jenny; Selomulya, Cordelia; Ma, Charles; Coppel, Ross

    2011-04-05

    Low efficiency is often observed in the delivery of DNA vaccines. The use of superparamagnetic nanoparticles (SPIONs) to deliver genes via magnetofection could improve transfection efficiency and target the vector to its desired locality. Here, magnetofection was used to enhance the delivery of a malaria DNA vaccine encoding Plasmodium yoelii merozoite surface protein MSP1(19) (VR1020-PyMSP1(19)) that plays a critical role in Plasmodium immunity. The plasmid DNA (pDNA) containing membrane associated 19-kDa carboxyl-terminal fragment of merozoite surface protein 1 (PyMSP1(19)) was conjugated with superparamagnetic nanoparticles coated with polyethyleneimine (PEI) polymer, with different molar ratio of PEI nitrogen to DNA phosphate. We reported the effects of SPIONs-PEI complexation pH values on the properties of the resulting particles, including their ability to condense DNA and the gene expression in vitro. By initially lowering the pH value of SPIONs-PEI complexes to 2.0, the size of the complexes decreased since PEI contained a large number of amino groups that became increasingly protonated under acidic condition, with the electrostatic repulsion inducing less aggregation. Further reaggregation was prevented when the pHs of the complexes were increased to 4.0 and 7.0, respectively, before DNA addition. SPIONs/PEI complexes at pH 4.0 showed better binding capability with PyMSP1(19) gene-containing pDNA than those at neutral pH, despite the negligible differences in the size and surface charge of the complexes. This study indicated that the ability to protect DNA molecules due to the structure of the polymer at acidic pH could help improve the transfection efficiency. The transfection efficiency of magnetic nanoparticle as carrier for malaria DNA vaccine in vitro into eukaryotic cells, as indicated via PyMSP1(19) expression, was significantly enhanced under the application of external magnetic field, while the cytotoxicity was comparable to the benchmark nonviral

  9. A DN-mda5 transgenic zebrafish model demonstrates that Mda5 plays an important role in snakehead rhabdovirus resistance.

    PubMed

    Gabor, K A; Charette, J R; Pietraszewski, M J; Wingfield, D J; Shim, J S; Millard, P J; Kim, C H

    2015-08-01

    Melanoma Differentiation-Associated protein 5 (MDA5) is a member of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family, which is a cytosolic pattern recognition receptor that detects viral nucleic acids. Here we show an Mda5-dependent response to rhabdovirus infection in vivo using a dominant-negative mda5 transgenic zebrafish. Dominant-negative mda5 zebrafish embryos displayed an impaired antiviral immune response compared to wild-type counterparts that can be rescued by recombinant full-length Mda5. To our knowledge, we have generated the first dominant-negative mda5 transgenic zebrafish and demonstrated a critical role for Mda5 in the antiviral response to rhabdovirus. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Administration of HPV DNA vaccine via electroporation elicits the strongest CD8+ T cell immune responses compared to intramuscular injection and intradermal gene gun delivery.

    PubMed

    Best, Simon R; Peng, Shiwen; Juang, Chi-Mou; Hung, Chien-Fu; Hannaman, Drew; Saunders, John R; Wu, T-C; Pai, Sara I

    2009-09-04

    DNA vaccines are an attractive approach to eliciting antigen-specific immunity. Intracellular targeting of tumor antigens through its linkage to immunostimulatory molecules such as calreticulin (CRT) can improve antigen processing and presentation through the MHC class I pathway and increase cytotoxic CD8+ T cell production. However, even with these enhancements, the efficacy of such immunotherapeutic strategies is dependent on the identification of an effective route and method of DNA administration. Electroporation and gene gun-mediated particle delivery are leading methods of DNA vaccine delivery that can generate protective and therapeutic levels of immune responses in experimental models. In this study, we perform a head-to-head comparison of three methods of vaccination--conventional intramuscular injection, electroporation-mediated intramuscular delivery, and epidermal gene gun-mediated particle delivery--in the ability to generate antigen-specific cytotoxic CD8+ T cell responses as well as anti-tumor immune responses against an HPV-16 E7 expressing tumor cell line using the pNGVL4a-CRT/E7(detox) DNA vaccine. Vaccination via electroporation generated the highest number of E7-specific cytotoxic CD8+ T cells, which correlated to improved outcomes in the treatment of growing tumors. In addition, we demonstrate that electroporation results in significantly higher levels of circulating protein compared to gene gun or intramuscular vaccination, which likely enhances calreticulin's role as a local tumor anti-angiogenesis agent. We conclude that electroporation is a promising method for delivery of HPV DNA vaccines and should be considered for DNA vaccine delivery in human clinical trials.

  11. Administration of HPV DNA vaccine via electroporation elicits the strongest CD8+ T cell immune responses compared to intramuscular injection and intradermal gene gun delivery

    PubMed Central

    Best, Simon R.; Peng, Shiwen; Juang, Chi-Mou; Hung, Chien-Fu; Hannaman, Drew; Saunders, John R.; Wu, T.-C.; Pai, Sara I.

    2009-01-01

    DNA vaccines are an attractive approach to eliciting antigen-specific immunity. Intracellular targeting of tumor antigens through its linkage to immunostimulatory molecules such as calreticulin (CRT) can improve antigen processing and presentation through the MHC Class I pathway and increase cytotoxic CD8+ T cell production. However, even with these enhancements, the efficacy of such immunotherapeutic strategies is dependent on the identification of an effective route and method of DNA administration. Electroporation and gene gun-mediated particle delivery are leading methods of DNA vaccine delivery that can generate protective and therapeutic levels of immune responses in experimental models. In this study, we perform a head-to-head comparison of three methods of vaccination – conventional intramuscular injection, electroporation mediated intramuscular delivery, and epidermal gene gun-mediated particle delivery - in the ability to generate antigen specific cytotoxic CD8+ T cell responses as well as anti-tumor immune responses against an HPV-16 E7 expressing tumor cell line using the pNGVL4a-CRT/E7(detox) DNA vaccine. Vaccination via electroporation generated the highest number of E7-specific cytotoxic CD8+ T cells, which correlated to improved outcomes in the treatment of growing tumors. In addition, we demonstrate that electroporation results in significantly higher levels of circulating protein compared to gene gun or intramuscular vaccination, which likely enhances calreticulin’s role as a local tumor anti-angiogenesis agent. We conclude that electroporation is a promising method for delivery of HPV DNA vaccines and should be considered for DNA vaccine delivery in human clinical trials. PMID:19622402

  12. DNA vaccination: using the patient's immune system to overcome cancer.

    PubMed

    Eschenburg, Georg; Stermann, Alexander; Preissner, Robert; Meyer, Hellmuth-Alexander; Lode, Holger N

    2010-01-01

    Cancer is one of the most challenging diseases of today. Optimization of standard treatment protocols consisting of the main columns of chemo- and radiotherapy followed or preceded by surgical intervention is often limited by toxic side effects and induction of concomitant malignancies and/or development of resistant mechanisms. This requires the development of therapeutic strategies which are as effective as standard therapies but permit the patients a life without severe negative side effects. Along this line, the development of immunotherapy in general and the innovative concept of DNA vaccination in particular may provide a venue to achieve this goal. Using the patient's own immune system by activation of humoral and cellular immune responses to target the cancer cells has shown first promising results in clinical trials and may allow reduced toxicity standard therapy regimen in the future. The main challenge of this concept is to transfer the plethora of convincing preclinical and early clinical results to an effective treatment of patients.

  13. Rapid development of a DNA vaccine for Zika virus.

    PubMed

    Dowd, Kimberly A; Ko, Sung-Youl; Morabito, Kaitlyn M; Yang, Eun Sung; Pelc, Rebecca S; DeMaso, Christina R; Castilho, Leda R; Abbink, Peter; Boyd, Michael; Nityanandam, Ramya; Gordon, David N; Gallagher, John Robert; Chen, Xuejun; Todd, John-Paul; Tsybovsky, Yaroslav; Harris, Audray; Huang, Yan-Jang S; Higgs, Stephen; Vanlandingham, Dana L; Andersen, Hanne; Lewis, Mark G; De La Barrera, Rafael; Eckels, Kenneth H; Jarman, Richard G; Nason, Martha C; Barouch, Dan H; Roederer, Mario; Kong, Wing-Pui; Mascola, John R; Pierson, Theodore C; Graham, Barney S

    2016-10-14

    Zika virus (ZIKV) was identified as a cause of congenital disease during the explosive outbreak in the Americas and Caribbean that began in 2015. Because of the ongoing fetal risk from endemic disease and travel-related exposures, a vaccine to prevent viremia in women of childbearing age and their partners is imperative. We found that vaccination with DNA expressing the premembrane and envelope proteins of ZIKV was immunogenic in mice and nonhuman primates, and protection against viremia after ZIKV challenge correlated with serum neutralizing activity. These data not only indicate that DNA vaccination could be a successful approach to protect against ZIKV infection, but also suggest a protective threshold of vaccine-induced neutralizing activity that prevents viremia after acute infection. Copyright © 2016, American Association for the Advancement of Science.

  14. RNA splicing in a new rhabdovirus from Culex mosquitoes.

    PubMed

    Kuwata, Ryusei; Isawa, Haruhiko; Hoshino, Keita; Tsuda, Yoshio; Yanase, Tohru; Sasaki, Toshinori; Kobayashi, Mutsuo; Sawabe, Kyoko

    2011-07-01

    Among members of the order Mononegavirales, RNA splicing events have been found only in the family Bornaviridae. Here, we report that a new rhabdovirus isolated from the mosquito Culex tritaeniorhynchus replicates in the nuclei of infected cells and requires RNA splicing for viral mRNA maturation. The virus, designated Culex tritaeniorhynchus rhabdovirus (CTRV), shares a similar genome organization with other rhabdoviruses, except for the presence of a putative intron in the coding region for the L protein. Molecular phylogenetic studies indicated that CTRV belongs to the family Rhabdoviridae, but it is yet to be assigned a genus. Electron microscopic analysis revealed that the CTRV virion is extremely elongated, unlike virions of rhabdoviruses, which are generally bullet shaped. Northern hybridization confirmed that a large transcript (approximately 6,500 nucleotides [nt]) from the CTRV L gene was present in the infected cells. Strand-specific reverse transcription-PCR (RT-PCR) analyses identified the intron-exon boundaries and the 76-nt intron sequence, which contains the typical motif for eukaryotic spliceosomal intron-splice donor/acceptor sites (GU-AG), a predicted branch point, and a polypyrimidine tract. In situ hybridization exhibited that viral RNAs are primarily localized in the nucleus of infected cells, indicating that CTRV replicates in the nucleus and is allowed to utilize the host's nuclear splicing machinery. This is the first report of RNA splicing among the members of the family Rhabdoviridae.

  15. Novel chemotactic-antigen DNA vaccine against cancer.

    PubMed

    Zhang, Shuren; Zhang, Youhui

    2008-04-01

    Dendritic cells play a pivotal role in immune induction. Dendritic cells perform antigen uptake, processing and presentation to T cells only when they are matured and in the functional state. In the development of a vaccine, it is of utmost importance to consider how to make dendritic cells' functions immunologically adequate. In this paper, we report the development of a series of antitumor DNA vaccines with similar structural framework, in which a gene encoding tumor-associated antigenic peptide is ligated upstream to the gene coding secondary lymphoid-tissue chemokine and downstream to the gene encoding the Fc portion of IgG (named chemotactic-antigen DNA vaccine [CADV]). CCR7(+) T, B, natural killer and dendritic cells can be attracted by secondary lymphoid-tissue chemokine, and Fc facilitates antigen uptake via Fc receptors expressed on dendritic cells. In a series of experiments in mice vaccinated by CADV with such tumor-associated antigenic specificities as HPV-16 E7, PSA-PSM-PAP, HER-2/neu, p53 and hTERT, CADV can attract immune cells to the vaccine inoculation site, remarkably inhibit tumor growth and extend survival time in tumor-bearing mice. The antitumor effect is more efficacious than that in mice treated with SLC-Ag or Ag-Fc hybrid gene. Tumor-associated antigenic-specific cytotoxic T lymphocytes can be induced by in vitro experiment in a human system. When combined with measures blocking the negative immune feedback circuits, the therapeutic effect of the vaccine can be further enhanced. Large-scale production of CADV is possible for clinical application.

  16. A comparative evaluation of different DNA vaccine candidates against experimental murine leishmaniasis due to L. major.

    PubMed

    Ahmed, Sami Ben Hadj; Bahloul, Chokri; Robbana, Cyrine; Askri, Souhir; Dellagi, Koussay

    2004-04-16

    Over the past few years, several reports of DNA vaccines against murine cutaneous experimental leishmaniasis came out with promising but sometimes discordant results. The present studies were designed to compare, under similar conditions, the protective effects in the highly susceptible BALB/c mice of DNA vaccine candidates encoding to various Leishmania major antigens. The candidate DNA vaccines encode to the following antigens: LACK, PSA2, Gp63, LeIF and two newly identified p20 and Ribosomal like protein, in addition to different truncated portions of the LACK antigen. The most promising gene was LACK and it is more protective when it is used as a p24 truncated form. Furthermore, the presence of a tandem repeats of immunostimulating sequences (ISS) in the plasmid backbone played an important adjuvant effect in the observed protective effect induced by the DNA vaccine encoding to the LACKp24. Nevertheless, neither of the DNA vaccine candidates was able to mount a full protection in BALB/c mice challenged with a highly virulent L. major strain. Further improvements of the DNA vaccination approach are still needed to design a fully protective vaccine against leishmaniasis. Three directions of investigations are currently explored: DNA vaccines using a cocktail of antigens; Prime/Boost approach; and association of immune modulators with the candidate antigens.

  17. Intranasal DNA Vaccine for Protection against Respiratory Infectious Diseases: The Delivery Perspectives

    PubMed Central

    Xu, Yingying; Yuen, Pak-Wai; Lam, Jenny Ka-Wing

    2014-01-01

    Intranasal delivery of DNA vaccines has become a popular research area recently. It offers some distinguished advantages over parenteral and other routes of vaccine administration. Nasal mucosa as site of vaccine administration can stimulate respiratory mucosal immunity by interacting with the nasopharyngeal-associated lymphoid tissues (NALT). Different kinds of DNA vaccines are investigated to provide protection against respiratory infectious diseases including tuberculosis, coronavirus, influenza and respiratory syncytial virus (RSV) etc. DNA vaccines have several attractive development potential, such as producing cross-protection towards different virus subtypes, enabling the possibility of mass manufacture in a relatively short time and a better safety profile. The biggest obstacle to DNA vaccines is low immunogenicity. One of the approaches to enhance the efficacy of DNA vaccine is to improve DNA delivery efficiency. This review provides insight on the development of intranasal DNA vaccine for respiratory infections, with special attention paid to the strategies to improve the delivery of DNA vaccines using non-viral delivery agents. PMID:25014738

  18. Protective immunity of grass carp immunized with DNA vaccine against Aeromonas hydrophila by using carbon nanotubes as a carrier molecule.

    PubMed

    Liu, Lei; Gong, Yu-Xin; Liu, Guang-Lu; Zhu, Bin; Wang, Gao-Xue

    2016-08-01

    To reduce the economic losses caused by diseases in aquaculture industry, more efficient and economic prophylactic measures should be urgently investigated. In this research, the effects of a novel functionalized single-walled carbon nanotubes (SWCNTs) applied as a delivery vehicle for DNA vaccine administration in juvenile grass carp against Aeromonas hydrophila were studied. Our results showed that SWCNTs loaded with DNA vaccine induced a better protection to juvenile grass carp against A. hydrophila. Moreover, SWCNTs conjugated with DNA vaccine provided significantly protective immunity compared with free DNA vaccine. Thereby, SWCNTs may be considered as a potential efficient DNA vaccine carrier to enhance the immunological activity.

  19. The evolution, diversity, and host associations of rhabdoviruses.

    PubMed

    Longdon, Ben; Murray, Gemma G R; Palmer, William J; Day, Jonathan P; Parker, Darren J; Welch, John J; Obbard, Darren J; Jiggins, Francis M

    2015-01-01

    Metagenomic studies are leading to the discovery of a hidden diversity of RNA viruses. These new viruses are poorly characterized and new approaches are needed predict the host species these viruses pose a risk to. The rhabdoviruses are a diverse family of RNA viruses that includes important pathogens of humans, animals, and plants. We have discovered thirty-two new rhabdoviruses through a combination of our own RNA sequencing of insects and searching public sequence databases. Combining these with previously known sequences we reconstructed the phylogeny of 195 rhabdovirus sequences, and produced the most in depth analysis of the family to date. In most cases we know nothing about the biology of the viruses beyond the host they were identified from, but our dataset provides a powerful phylogenetic approach to predict which are vector-borne viruses and which are specific to vertebrates or arthropods. By reconstructing ancestral and present host states we found that switches between major groups of hosts have occurred rarely during rhabdovirus evolution. This allowed us to propose seventy-six new likely vector-borne vertebrate viruses among viruses identified from vertebrates or biting insects. Based on currently available data, our analysis suggests it is likely there was a single origin of the known plant viruses and arthropod-borne vertebrate viruses, while vertebrate- and arthropod-specific viruses arose at least twice. There are also few transitions between aquatic and terrestrial ecosystems. Viruses also cluster together at a finer scale, with closely related viruses tending to be found in closely related hosts. Our data therefore suggest that throughout their evolution, rhabdoviruses have occasionally jumped between distantly related host species before spreading through related hosts in the same environment. This approach offers a way to predict the most probable biology and key traits of newly discovered viruses.

  20. The evolution, diversity, and host associations of rhabdoviruses

    PubMed Central

    Longdon, Ben; Murray, Gemma G. R.; Palmer, William J.; Day, Jonathan P.; Parker, Darren J; Welch, John J.; Obbard, Darren J.; Jiggins, Francis M.

    2015-01-01

    Metagenomic studies are leading to the discovery of a hidden diversity of RNA viruses. These new viruses are poorly characterized and new approaches are needed predict the host species these viruses pose a risk to. The rhabdoviruses are a diverse family of RNA viruses that includes important pathogens of humans, animals, and plants. We have discovered thirty-two new rhabdoviruses through a combination of our own RNA sequencing of insects and searching public sequence databases. Combining these with previously known sequences we reconstructed the phylogeny of 195 rhabdovirus sequences, and produced the most in depth analysis of the family to date. In most cases we know nothing about the biology of the viruses beyond the host they were identified from, but our dataset provides a powerful phylogenetic approach to predict which are vector-borne viruses and which are specific to vertebrates or arthropods. By reconstructing ancestral and present host states we found that switches between major groups of hosts have occurred rarely during rhabdovirus evolution. This allowed us to propose seventy-six new likely vector-borne vertebrate viruses among viruses identified from vertebrates or biting insects. Based on currently available data, our analysis suggests it is likely there was a single origin of the known plant viruses and arthropod-borne vertebrate viruses, while vertebrate- and arthropod-specific viruses arose at least twice. There are also few transitions between aquatic and terrestrial ecosystems. Viruses also cluster together at a finer scale, with closely related viruses tending to be found in closely related hosts. Our data therefore suggest that throughout their evolution, rhabdoviruses have occasionally jumped between distantly related host species before spreading through related hosts in the same environment. This approach offers a way to predict the most probable biology and key traits of newly discovered viruses. PMID:27774286

  1. Protective DNA vaccination against experimental autoimmune encephalomyelitis is associated with induction of IFNbeta.

    PubMed

    Wefer, Judit; Harris, Robert A; Lobell, Anna

    2004-04-01

    DNA vaccines encoding encephalitogenic peptides protect against subsequent development of rat experimental autoimmune encephalomyelitis (EAE) through unknown mechanisms. We investigated immune cell phenotypes at different time points after DNA vaccination with vaccine encoding myelin oligodendrocyte glycoprotein peptide 91-108 and subsequent induction of EAE. In protected rats, we observed (i) no alterations in antigen-specific Th2 or Th3 responses, (ii) reduced MHC II expression on splenocytes early after EAE induction, (iii) antigen-specific upregulation of IFNbeta upon recall stimulation and (iv) reduced IL-12Rbeta2 on lymphocytes. We suggest that the underlying mechanism of DNA vaccination is associated with immunomodulation exerted by induced IFNbeta.

  2. Immunogenicity of a DNA-launched replicon-based canine parvovirus DNA vaccine expressing VP2 antigen in dogs.

    PubMed

    Dahiya, Shyam S; Saini, Mohini; Kumar, Pankaj; Gupta, Praveen K

    2012-10-01

    A replicon-based DNA vaccine encoding VP2 gene of canine parvovirus (CPV) was developed by cloning CPV-VP2 gene into a replicon-based DNA vaccine vector (pAlpha). The characteristics of a replicon-based DNA vaccine like, self-amplification of transcripts and induction of apoptosis were analyzed in transfected mammalian cells. When the pAlpha-CPV-VP2 was injected intradermal as DNA-launched replicon-based DNA vaccine in dogs, it induced CPV-specific humoral and cell mediated immune responses. The virus neutralization antibody and lymphocyte proliferative responses were higher than conventional CPV DNA vaccine and commercial CPV vaccine. These results indicated that DNA-launched replicon-based CPV DNA vaccine was effective in inducing both CPV-specific humoral and cellular immune responses and can be considered as effective alternative to conventional CPV DNA vaccine and commercial CPV vaccine.

  3. Merida virus, a putative novel rhabdovirus discovered in Culex and Ochlerotatus spp. mosquitoes in the Yucatan Peninsula of Mexico

    PubMed Central

    Charles, Jermilia; Firth, Andrew E.; Loroño-Pino, Maria A.; Garcia-Rejon, Julian E.; Farfan-Ale, Jose A.; Lipkin, W. Ian; Briese, Thomas

    2016-01-01

    Sequences corresponding to a putative, novel rhabdovirus [designated Merida virus (MERDV)] were initially detected in a pool of Culex quinquefasciatus collected in the Yucatan Peninsula of Mexico. The entire genome was sequenced, revealing 11 798 nt and five major ORFs, which encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). The deduced amino acid sequences of the N, G and L proteins have no more than 24, 38 and 43 % identity, respectively, to the corresponding sequences of all other known rhabdoviruses, whereas those of the P and M proteins have no significant identity with any sequences in GenBank and their identity is only suggested based on their genome position. Using specific reverse transcription-PCR assays established from the genome sequence, 27 571 C. quinquefasciatus which had been sorted in 728 pools were screened to assess the prevalence of MERDV in nature and 25 pools were found positive. The minimal infection rate (calculated as the number of positive mosquito pools per 1000 mosquitoes tested) was 0.9, and similar for both females and males. Screening another 140 pools of 5484 mosquitoes belonging to four other genera identified positive pools of Ochlerotatus spp. mosquitoes, indicating that the host range is not restricted to C. quinquefasciatus. Attempts to isolate MERDV in C6/36 and Vero cells were unsuccessful. In summary, we provide evidence that a previously undescribed rhabdovirus occurs in mosquitoes in Mexico. PMID:26868915

  4. Merida virus, a putative novel rhabdovirus discovered in Culex and Ochlerotatus spp. mosquitoes in the Yucatan Peninsula of Mexico.

    PubMed

    Charles, Jermilia; Firth, Andrew E; Loroño-Pino, Maria A; Garcia-Rejon, Julian E; Farfan-Ale, Jose A; Lipkin, W Ian; Blitvich, Bradley J; Briese, Thomas

    2016-04-01

    Sequences corresponding to a putative, novel rhabdovirus [designated Merida virus (MERDV)] were initially detected in a pool of Culex quinquefasciatus collected in the Yucatan Peninsula of Mexico. The entire genome was sequenced, revealing 11 798 nt and five major ORFs, which encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). The deduced amino acid sequences of the N, G and L proteins have no more than 24, 38 and 43 % identity, respectively, to the corresponding sequences of all other known rhabdoviruses, whereas those of the P and M proteins have no significant identity with any sequences in GenBank and their identity is only suggested based on their genome position. Using specific reverse transcription-PCR assays established from the genome sequence, 27 571 C. quinquefasciatus which had been sorted in 728 pools were screened to assess the prevalence of MERDV in nature and 25 pools were found positive. The minimal infection rate (calculated as the number of positive mosquito pools per 1000 mosquitoes tested) was 0.9, and similar for both females and males. Screening another 140 pools of 5484 mosquitoes belonging to four other genera identified positive pools of Ochlerotatus spp. mosquitoes, indicating that the host range is not restricted to C. quinquefasciatus. Attempts to isolate MERDV in C6/36 and Vero cells were unsuccessful. In summary, we provide evidence that a previously undescribed rhabdovirus occurs in mosquitoes in Mexico.

  5. Detection of rhabdovirus viral RNA in oropharyngeal swabs and ectoparasites of Spanish bats.

    PubMed

    Aznar-Lopez, Carolina; Vazquez-Moron, Sonia; Marston, Denise A; Juste, Javier; Ibáñez, Carlos; Berciano, Jose Miguel; Salsamendi, Egoitz; Aihartza, Joxerra; Banyard, Ashley C; McElhinney, Lorraine; Fooks, Anthony R; Echevarria, Juan

    2013-01-01

    Rhabdoviruses infect a variety of hosts, including mammals, birds, reptiles, fish, insects and plants. As bats are the natural host for most members of the genus Lyssavirus, the specificity of the amplification methods used for active surveillance is usually restricted to lyssaviruses. However, the presence of other rhabdoviruses in bats has also been reported. In order to broaden the scope of such methods, a new RT-PCR, able to detect a diverse range of rhabdoviruses, was designed. The method detected 81 of 86 different rhabdoviruses. In total, 1488 oropharyngeal bat swabs and 38 nycteribiid samples were analysed, and 17 unique rhabdovirus-related sequences were detected. Phylogenetic analysis suggested that those sequences detected in bats did not constitute a monophyletic group, even when originating from the same bat species. However, all of the sequences detected in nycteribiids and one sequence obtained from a bat did constitute a monophyletic group with Drosophila melanogaster sigma rhabdovirus.

  6. Genomic characterization and taxonomic position of a rhabdovirus from a hybrid snakehead.

    PubMed

    Zeng, Weiwei; Wang, Qing; Wang, Yingying; Liu, Cun; Liang, Hongru; Fang, Xiang; Wu, Shuqin

    2014-09-01

    A new rhabdovirus, tentatively designated as hybrid snakehead rhabdovirus C1207 (HSHRV-C1207), was first isolated from a moribund hybrid snakehead (Channa maculata×Channa argus) in China. We present the complete genome sequence of HSHRV-C1207 and a comprehensive sequence comparison between HSHRV-C1207 and other rhabdoviruses. Sequence alignment and phylogenetic analysis revealed that HSHRV-C1207 shared the highest degree of homology with Monopterus albus rhabdovirus and Siniperca chuatsi rhabdovirus. All three viruses clustered into a single group that was distinct from the recognized genera in the family Rhabdoviridae. Our analysis suggests that HSHRV-C1207, as well as MARV and SCRV, should be assigned to a new rhabdovirus genus.

  7. Cimetidine synergizes with Praziquantel to enhance the immune response of HBV DNA vaccine via activating cytotoxic CD8(+) T cell.

    PubMed

    Xie, Xiaoping; Geng, Shuang; Liu, Hu; Li, Chaofan; Yang, Yuqin; Wang, Bin

    2014-01-01

    Previously, we have reported that either CIM or PZQ, 2 clinical drugs, could be used to develop as adjuvants on HBV DNA vaccine to elicit both humoral and cellular immune responses. Here, we demonstrate that combinations of CIM and PZQ as adjuvants for a HBV DNA vaccine, could induce much stronger antigen specific CD4(+) and CD8(+) T cell responses compared either with CIM or PZQ alone. The synergistic effects of CIM plus PZQ to HBV DNA vaccine were observed on a higher IgG2a/IgG1 ratio, an increase of HBsAg-specific CD4(+) T cells capable of producing IFN-γ or IL-17A and a robust IFN-γ-, IL-17A-, or TNF-α-producing CD8(+) T cells to HBsAg. Most importantly, the antigen-specific CTL response was also elevated significantly, which is critical for the eradication of hepatitis B virus (HBV) infected cells. Using an HBsAg transgenic mouse model, the expression of HBsAg in the hepatic cells was also significantly reduced after immunized with pCD-S 2 in the presence of 0.5% CIM and 0.25% PZQ. Further investigations demonstrated that the synergistic effects of combination of CIM and PZQ were dependent on enhanced cytotoxic CD8(+) T cells, which was correlated with impaired activities of regulatory T cells. Therefore, combinations of CIM and PZQ have great potential to be used as effective adjuvants on DNA-based vaccinations for the treatment of chronic hepatitis B.

  8. Spread and morphological-structural properties of plant rhabdoviruses and similar pathogens in Basidiomycetes.

    PubMed

    Boyko, A L; Zarytskyi, N N; Demchenko, A A; Spivak, N Ya; Boyko, O A; Orlovska, G M; Polischuk, V P; Yuzvenko, L E; Lazarenko, L N; Babenko, L R

    2014-01-01

    Long-term studies of spread of rhabdoviruses which indicated their harmfulness in different plant species under conditions of environmental factors were first discussed. Their harmfulness to different plant species under environmental conditions was shown. A comparative description of rhabdoviruses with similar pathogens of the mushrooms is carried out. Thus the main focus was on the morphology and structure of the pathogens. These data are extremely important in the study of distribution of the rhabdovirus on crops in different regions.

  9. Efficacy of DNA vaccines expressing the type F botulinum toxin Hc fragment using different promoters.

    PubMed

    Jathoul, Amit P; Holley, Jane L; Garmory, Helen S

    2004-09-28

    DNA vaccines which expressed the Hc fragment of the Clostridium botulinum type F neurotoxin (BoNT/F Hc) fused to a signal peptide downstream of four different eukaryotic promoters were prepared. Subsequently, the immunogenicity of the DNA vaccines and protection afforded in mice against challenge with 10(4) MLD of type F botulinum toxin was evaluated. The DNA vaccine containing the human ubiquitin gene (UbC) promoter induced the highest BoNT/F Hc-specific antibody concentration following two intramuscular immunisations and afforded 90% protection against challenge. The results from this study indicate that the selection of promoter used in DNA vaccination studies may be of importance in designing optimised vaccines.

  10. HIV vaccine update. DNA vaccination: a promising candidate for a vaccine against HIV-1?

    PubMed

    Van Der Ryst, E

    1996-01-01

    According to animal studies, DNA vaccines employ the genes encoding proteins of pathogens or tumors, in contrast to the more conventional vaccine approaches. In addition, DNA vaccinations do not involve infectious agents, proteins are expressed in their natural form resulting to better recognition of viral proteins by the antibodies, and both strong and durable cellular immune responses as well as neutralizing antibodies are induced. Altogether, this makes DNA vaccinations one of the most promising future candidates in the field of HIV vaccines. However, safety of DNA vaccines should be examined before these vaccines can be considered for large-scale clinical trials in humans. The question of a possible induction of anti-DNA antibodies, with the consequent development of autoimmune manifestations is emphasized. Another is the possible integration of DNA with insertional mutagenesis, which could lead to tumor formation and development of immunologic tolerance of antigen production persists.

  11. Infectious bursal disease DNA vaccination conferring protection by delayed appearance and rapid clearance of invading viruses.

    PubMed

    Chen, Yung-Yi; Hsieh, Ming Kun; Tung, Chun-Yu; Wu, Ching Ching; Lin, Tsang Long

    2011-12-01

    The present study was undertaken to determine the kinetics of viral load and immune response in protection against infectious bursal disease virus (IBDV) by DNA vaccination. Chickens were DNA-vaccinated and challenged with IBDV one week after the third vaccination. Tissues were collected at 12 hours postinfection (HPI), 1 day postinfection (DPI), 3, 5, 7 and 10 DPI. The vaccinated chickens had less viral RNA, with delayed appearance and shorter duration in the bursa of Fabricius, spleen, and cecal tonsil than the challenged control chickens. Their ELISA and neutralizing antibody titers were decreased at 12 HPI and significantly lower (P < 0.05) than those in the challenged control chickens at later time points. Their spleen IFNγ expression was up-regulated compared to that in the DNA-vaccinated chickens without IBDV challenge. These results indicate that DNA vaccination confers protection against IBDV challenge by delayed appearance and rapid clearance of the invading viruses.

  12. Oral vaccination with a liposome-encapsulated influenza DNA vaccine protects mice against respiratory challenge infection.

    PubMed

    Liu, Jing; Wu, Jianqi; Wang, Bing; Zeng, Sheng; Qi, Feifei; Lu, Changlong; Kimura, Yoshinobu; Liu, Beixing

    2014-05-01

    It is well accepted that vaccination by oral administration has many advantages over injected parenteral immunization. The present study focuses on whether oral vaccination with a DNA vaccine could induce protective immunity against respiratory challenge infection. The M1 gene of influenza A virus was used to construct DNA vaccine using pcDNA 3.1(+) plasmid, a eukaryotic expression vector. The cationic liposomes were used to deliver the constructed DNA vaccine. In vitro and in vivo expression of M1 gene was observed in the cell line and in the intestine of orally vaccinated C57BL/6 mice, respectively. It became clear that this type of oral DNA vaccination was capable of inducing both humoral and cellular immune responses, together with an augmentation of IFN-γ production. In addition, oral vaccination with liposome-encapsulated DNA vaccine could protect the mice against respiratory challenge infection. These results suggest that gastrointestinal tract, a constituent member of the common mucosal immune system, is a potent candidate applicable as a DNA vaccine route against virus respiratory diseases.

  13. Recent patents on immunoregulatory DNA vaccines for autoimmune diseases and allograft rejection.

    PubMed

    Shabahang, Shahrokh; Li, Alice F; Escher, Alan

    2010-06-01

    The goal of immunoregulatory DNA vaccination is the antigen- and tissue-specific suppression of pathological inflammation that underlies immune-mediated inflammatory disorders like autoimmune diseases and allograft rejection. Recent patents and patent applications have applied immunoregulatory DNA vaccines in rodent model systems and human clinical trials using plasmid DNA coding for autoantigens such as insulin and glutamic acid decarboxylase for type 1 diabetes, myelin-associated proteins for multiple sclerosis, and heat-sock protein 60 for rheumatoid arthritis. In these cases, the objective is to induce a homeostatic-like regulatory immune response to suppress pathological inflammation. In addition, patent applications have disclosed the use of DNA vaccines encoding the pro-inflammatory MIF cytokine and the CD25 IL-2 receptor subunit to interfere with the inflammatory process. Approaches have also been taken to improve DNA vaccination efficacy, including covalent modification of plasmid DNA, engineering secretion of vaccine-encoded antigen, and co-delivery of DNA coding for anti-inflammatory cytokines, a mutant co-stimulatory molecule, a growth factor, or a pro-apoptotic protein. Furthermore, a patent application has disclosed the use of a DNA vaccine previously shown to treat successfully an autoimmune disease to prolong allograft survival. Taken together, these patents and patent applications indicate a promising bench-to-bedside potential for immunoregulatory DNA vaccination applied to autoimmune diseases and allograft rejection.

  14. Immunogenicity and protective efficacy of a vaxfectin-adjuvanted tetravalent dengue DNA vaccine.

    PubMed

    Porter, Kevin R; Ewing, Daniel; Chen, Lan; Wu, Shuenn-Jue; Hayes, Curtis G; Ferrari, Marilyn; Teneza-Mora, Nimfa; Raviprakash, Kanakatte

    2012-01-05

    A prototype dengue-1 DNA vaccine was shown to be safe and immunogenic in a previous Phase 1 clinical trial. Anti-dengue-1 neutralizing antibody responses were detectable only in the group of volunteers receiving the high dose of nonadjuvanted vaccine and the antibody titers were low. Vaxfectin(®), a lipid-based adjuvant, enhances the immunogenicity of DNA vaccines. We conducted a nonhuman primate study to evaluate the effect of Vaxfectin(®) on the immunogenicity of a tetravalent dengue DNA vaccine. Animals were immunized on days 0, 28 and 84, with each immunization consisting of 3mg of Vaxfectin(®)-adjuvanted tetravalent dengue DNA vaccine. The use of Vaxfectin(®) resulted in a significant increase in anti-dengue neutralizing antibody responses against dengue-1, -3 and -4. There was little to no effect on T cell responses as measured by interferon gamma ELISPOT assay. Animals immunized with the Vaxfectin(®)-formulated tetravalent DNA vaccine showed significant protection against live dengue-2 virus challenge compared to control animals (0.75 mean days of viremia vs 3.3 days). Animals vaccinated with nonadjuvanted DNA had a mean 2.0 days of viremia. These results support further evaluation of the Vaxfectin(®)-adjuvanted tetravalent dengue DNA vaccine in a Phase 1 clinical trial.

  15. DNA vaccine against visceral leishmaniasis: a promising approach for prevention and control.

    PubMed

    Kumar, A; Samant, M

    2016-05-01

    The visceral leishmaniasis (VL) caused by Leishmania donovani parasite severely affects large populations in tropical and subtropical regions of the world. The arsenal of drugs available is limited, and resistance is common in clinical field isolates. Therefore, vaccines could be an important alternative for prevention against VL. Recently, some investigators advocated the protective efficacy of DNA vaccines, which induces the T cell-based immunity against VL. The vaccine antigens are selected as conserved in various Leishmania species and provide a viable strategy for DNA vaccine development. Our understanding for DNA vaccine development against VL is not enough and much technological advancement is required. Improved formulations and methods of delivery are required, which increase the uptake of DNA vaccine by cells; optimization of vaccine vectors/encoded antigens to augment and direct the host immune response in VL. Despite the many genes identified as vaccine candidates, the disappointing potency of the DNA vaccines in VL underscores the challenges encountered in the efforts to translate efficacy in preclinical models into clinical realities. This review will provide a brief background of DNA vaccines including the insights gained about the design, strategy, safety issues, varied candidates, progress and challenges that play a role in their ability against VL.

  16. Electroporation of a multivalent DNA vaccine cocktail elicits a protective immune response against anthrax and plague.

    PubMed

    Albrecht, Mark T; Livingston, Brian D; Pesce, John T; Bell, Matt G; Hannaman, Drew; Keane-Myers, Andrea M

    2012-07-06

    Electroporation of DNA vaccines represents a platform technology well positioned for the development of multivalent biodefense vaccines. To evaluate this hypothesis, three vaccine constructs were produced using codon-optimized genes encoding Bacillus anthracis Protective Antigen (PA), and the Yersinia pestis genes LcrV and F1, cloned into pVAX1. A/J mice were immunized on a prime-boost schedule with these constructs using the electroporation-based TriGrid Delivery System. Immunization with the individual pDNA vaccines elicited higher levels of antigen-specific IgG than when used in combination. DNA vaccine effectiveness was proven, the pVAX-PA titers were toxin neutralizing and fully protective against a lethal B. anthracis spore challenge when administered alone or co-formulated with the plague pDNA vaccines. LcrV and F1 pVAX vaccines against plague were synergistic, resulting in 100% survival, but less protective individually and when co-formulated with pVAX-PA. These DNA vaccine responses were Th1/Th2 balanced with high levels of IFN-γ and IL-4 in splenocyte recall assays, contrary to complimentary protein Alum vaccinations displaying a Th2 bias with increased IL-4 and low levels of IFN-γ. These results demonstrate the feasibility of electroporation to deliver and maintain the overall efficacy of an anthrax-plague DNA vaccine cocktail whose individual components have qualitative immunological differences when combined.

  17. Naked DNA vaccination of Atlantic salmon Salmo salar against IHNV.

    PubMed

    Traxler, G S; Anderson, E; LaPatra, S E; Richard, J; Shewmaker, B; Kurath, G

    1999-11-30

    A naked plasmid DNA encoding the glycoprotein (pCMV4-G) of a 1976 isolate of infectious hematopoietic necrosis virus (IHNV) obtained from steelhead Oncorhynchus mykiss was used to vaccinate Atlantic salmon Salmo salar against IHNV. Eight weeks post-vaccination the fish were challenged with a strain of IHNV originally isolated from farmed Atlantic salmon undergoing an epizootic. Fish injected with the glycoprotein-encoding plasmid were significantly (p < 0.05) protected against IHNV by both immersion and cohabitation challenge. Survivors of the first challenges were pooled and re-challenged by immersion 12 wk after the initial challenge. Significant (p < 0.05) protection was observed in all of the previously challenged groups including those receiving the complete vaccine. Fish injected with the glycoprotein-encoding plasmid produced low levels of virus-neutralizing antibodies prior to the first challenge. Neutralizing antibodies increased in all groups after exposure to the IHNV. Passive transfer of pooled sera from pCMV4-G vaccinates and IHN survivors provided relative survivals of 40 to 100% compared to fish injected with sera collected from fish immunized with control vaccines or left unhandled. In this study, DNA vaccination effectively protected Atlantic salmon smolts against challenges with IHNV.

  18. Vaginal DNA vaccination against infectious diseases transmitted through the vagina.

    PubMed

    Kanazawa, Takanori; Takashima, Yuuki; Okada, Hiroaki

    2012-06-01

    There is an urgent need for the development of vaccines against genital virus infections that are transmitted through heterosexual intercourse, including the HIV and HPV. In general, the surface of female genital mucosa, including vaginal mucosa, is the most common site of initiation of these infections. Thus, it is becoming clear that successful vaccines must induce both cellular and humoral immune responses in both the local genital tract and systemically. We believe that a strong vaginal immune response could be obtained by inducing strong gene expression of antigen-coding DNA in the local targeted tissue. In order to improve transfection efficiency in the vagina, it is important that methods allowing breakthrough of the various barriers, such as the epithelial layer, cellular and nuclear membrane, are developed. Therefore, systems providing less invasive and more effective delivery into the subepithelial layer are required. In this review, we will introduce our studies into efficient vaginal DNA vaccination methods, focusing on the effects of the menstrual cycle, utilization of the combination of functional peptides, and use of a needle-free injector.

  19. MG7 mimotope-based DNA vaccination for gastric cancer.

    PubMed

    Zhang, Dexin; Chen, Yu; Fan, Daiming

    2006-04-01

    Gastric cancer is still one of the leading causes of cancer-related death worldwide. Prevention and treatment of gastric cancer through vaccination has been difficult owing to lack of a specific target and poor immunity. A number of vaccination strategies have been used to augment immune responses against gastric cancer and some progress has been made. In a series of studies, the authors have focused on gastric cancer vaccination approaches based on MG7 mimotopes, which are mimicry epitopes selected from phage-displayed oligopeptide libraries with a gastric cancer cell-specific monoclonal antibody, MG7-Ab. Strategies employed in these studies include viral or plasmid vectors in combination with carrier sequence or unmethylated CpG with synthetic peptides in nanoemulsion. The results demonstrated that MG7 mimotopes could effectively and specifically induce both cellular and humoral immune reactions and in vivo antitumor responses. In particular, a four-MG7 mimotope DNA vaccine was found to elicit much stronger antitumor immune responses in mice compared with its single-mimotope counterpart. These encouraging findings might pave the way for the development of novel MG7 antigen-based vaccination approaches for human gastric cancer. The review also discusses other immune-enhancing vaccination strategies for gastric cancer.

  20. Specific Cleavage of the Nucleoprotein of Fish Rhabdovirus.

    PubMed

    Zhou, G-Z; Yi, Y-J; Chen, Z-Y; Zhang, Q-Y

    2015-11-01

    Siniperca chuatsi rhabdovirus (SCRV) is one of myriad rhabdoviruses recorded in fish. Preliminary data show that inhibition of the SCRV nucleoprotein (N) could significantly reduce the progeny virus titers in infected Epithelioma papulosum cyprinid (EPC) cells. Here, the authors propose that cleavage of the viral 47-kDa N protein is caspase-mediated based on caspase inhibition experiments, transient expression in EPC transfection, and analysis of cleavage sites. Cleavage of the SCRV N protein in culture was prevented by a pan-caspase inhibitor, z-VAD-FMK (z-Val-Ala-DL-Asp-fluoromethyl ketone). Subsequently, N was transiently expressed in EPC cells, the results of which indicated that the specific cleavage of N also occurred in the cells transfected with N-GFP plasmid. Several truncated fragments of the N gene were constructed and transiently transfected into EPC cells. Immunoblotting results indicated that D324 and D374 are the cleavage sites of N by caspases. The authors also found that z-VAD-FMK could inhibit the cytopathic effect in SCRV-infected EPC cells but not affect the production of infectious progeny, suggesting that the caspase-mediated cleavage of N protein is not required for in vitro SCRV replication. To the authors' knowledge, this is the first report on the cleavage of rhabdovirus proteins. © The Author(s) 2015.

  1. Fish Rhabdovirus Cell Entry Is Mediated by Fibronectin

    PubMed Central

    Bearzotti, Monique; Delmas, Bernard; Lamoureux, Annie; Loustau, Anne-Marie; Chilmonczyk, Stefan; Bremont, Michel

    1999-01-01

    Three monoclonal antibodies (MAbs) generated against rainbow trout gonad cells (RTG-2) have been selected for their ability to protect cells from the viral hemorrhagic septicemia virus (VHSV) infection, a salmonid rhabdovirus. Protection from infection was restricted to the salmonid-derived cell lines indicating species specificity of the blocking MAbs. Surprisingly, the blocking activity of these MAbs was also effective against other nonantigenically related fish rhabdoviruses. Indirect immunofluorescence and immunoelectron microscopy observations demonstrated that the three MAbs were all directed against an abundant cell plasma membrane component, and immunoprecipitation studies indicated that the target consisted of a heterodimeric complex with molecular masses of 200 and 44 kDa. Biochemical data provided the following evidence that fibronectin is part of this complex and that it could represent the main receptor for fish rhabdoviruses. (i) An antiserum generated against the 200-kDa protein reacted against the recombinant rainbow trout fibronectin expressed in Escherichia coli. (ii) The purified rainbow trout fibronectin was able to bind specifically to VHSV. To our knowledge, this is the first identification of a cellular component acting as a primary receptor for a virus replicating in lower vertebrates and, more interestingly, for viruses belonging to the Rhabdoviridae family. PMID:10438860

  2. Phylogenetic relationships among rhabdoviruses inferred using the L polymerase gene.

    PubMed

    Bourhy, H; Cowley, J A; Larrous, F; Holmes, E C; Walker, P J

    2005-10-01

    RNA viruses of the family Rhabdoviridae include arthropod-borne agents that infect plants, fish and mammals, and also include a variety of non-vector-borne mammalian viruses. Herein is presented a molecular phylogenetic analysis, the largest undertaken to date, of 56 rhabdoviruses, including 20 viruses which are currently unassigned or assigned as tentative species within the Rhabdoviridae. Degenerate primers targeting a region of block III of the L polymerase gene were defined and used for RT-PCR amplification and sequencing. A maximum-likelihood phylogenetic analysis of a 158-residue L polymerase amino acid sequence produced an evolutionary tree containing the six recognized genera of the Rhabdoviridae and also enabled us to identify four more monophyletic groups of currently unclassified rhabdoviruses that we refer to as the 'Hart Park', 'Almpiwar', 'Le Dantec' and 'Tibrogargan' groups. The broad phylogenetic relationships among these groups and genera also indicate that the evolutionary history of rhabdoviruses was strongly influenced by mode of transmission, host species (plant, fish or mammal) and vector (orthopteran, homopteran or dipteran).

  3. Efficacy of a glycoprotein DNA vaccine against viral haemorrhagic septicaemia (VHS) in Pacific herring, Clupea pallasii Valenciennes

    USGS Publications Warehouse

    Hart, L.M.; Lorenzen, Niels; LaPatra, S.E.; Grady, C.A.; Roon, S.E.; O’Reilly, J.; Gregg, J.L.; Hershberger, P.K.

    2012-01-01

    Viral haemorrhagic septicaemia virus (VHSV) and its associated disease state, viral haemorrhagic septicaemia (VHS), is hypothesized to be a proximate factor accounting for the decline and failed recovery of Pacific herring populations in Prince William Sound, AK (Marty et al. 1998, 2003, 2010). Survivors of laboratory-induced VHSV epizootics develop resistance to subsequent viral exposure (Kocan et al. 2001; Hershberger et al. 2007, 2010), which is likely the result of immune system recognition of the viral glycoprotein (G) (Lecocq-Xhonneux et al. 1994), a surface antigen that contains neutralizing epitopes (Lorenzen, Olesen & Jorgensen 1990; Jørgensen et al. 1995) and cell attachment domains (Lecocq-Xhonneux et al. 1994; Estepa & Coll 1996). These properties have proven useful in the development of G-gene-based DNA vaccines for VHSV and a related rhabdovirus, infectious haematopoietic necrosis virus (IHNV) (Anderson et al. 1996; Heppell et al. 1998; Corbeil et al. 1999; Einer-Jensen et al. 2009). Rainbow trout fingerlings, Oncorhynchus mykiss (Walbaum), vaccinated with 1 µg of either the VHS or IHN vaccine are protected from VHS when exposed to virus as early as 4 days (44 degree days) post-vaccination (p.v.) (Lorenzen et al. 2002). At later time points (80 days p.v.; 880 degree days), the level of cross-protection against VHS by IHN vaccination is either completely lost (60 days p.v.; 660 degree days) (3 g rainbow trout; 1 µg vaccine dose) (Lorenzen et al. 2002) or present at intermediate levels (6.5 g rainbow trout; 1 µg vaccine dose) (Einer-Jensen et al. 2009). Comparatively, VHS vaccination remains effective as long as 9 months (2520 degree days) p.v. (100 g rainbow trout; 0.5 µg vaccine dose) (McLauchlan et al. 2003). These results suggest that IHN and VHS vaccination activate a rapid transitory innate immune response against VHSV that is followed by long-term adaptive immunity in VHS-vaccinated trout (Lorenzen et al. 2002).

  4. Construction of Eimeria tenella multi-epitope DNA vaccines and their protective efficacies against experimental infection.

    PubMed

    Song, Xiaokai; Xu, Lixin; Yan, Ruofeng; Huang, Xinmei; Li, Xiangrui

    2015-08-15

    The search for effective vaccines against chicken coccidiosis remains a challenge because of the complex organisms with multiple life cycle stages of Eimeria. Combination of T-cell epitopes from different stages of Eimeria life cycle could be an optimal strategy to overcome the antigen complexity of the parasite. In this study, 4 fragments with concentrated T-cell epitopes from the sporozoite antigen SO7 and the merozoite antigen MZ5-7 of Eimeria tenella were cloned into eukaryotic expression vector pVAX1 in different forms, with or without chicken cytokines IL-2 or IFN-γ genes as genetic adjuvants, to construct multistage, multi-epitope DNA vaccines against Eimeria tenella. Transcription and expression of the multi-epitope DNA vaccines in vivo were detected by reverse transcription-PCR (RT-PCR) and Western blot. On the basis of survival rate, lesion score, body weight gain, oocyst decrease ratio and the anti-coccidial index (ACI), Animal experiments were carried out to evaluate the protective efficacy against Eimeria tenella. Results showed the constructed DNA vaccines were transcribed and translated successfully in vivo. Animal experiment showed that the multi-epitopes DNA vaccines were more effective to stimulate immune response than single fragment. Compared with the DNA vaccines composed with less T-cell epitopes, DNA vaccine pVAX1-m1-m2-s1-s2 containing 4 fragments with concentrated T-epitopes provided the highest ACI of 180.39. DNA vaccines composed of antigens from two developmental stages were more effective than the single-stage ones. Especially DNA vaccine pVAX1-m1-m2-s1-s2 provided the most effective protection with the ACI of 180.39. Furthermore, cytokines IL-2 or IFN-γ could improve the efficacy of the multi-epitope DNA vaccines significantly. Overall, pVAX1-m1-m2-s1-s2-IFN-γ provided the most effective protection with the ACI of 189.92. The multi-epitope DNA vaccines revealed in this study provide new candidates for Eimeria vaccine development.

  5. Cellular and Molecular Interactions of Rhabdoviruses with their Insect and Plant Hosts

    USDA-ARS?s Scientific Manuscript database

    The rhabdoviruses form a large family (Rhabdoviridae) whose host ranges include humans, other vertebrates, invertebrates, and plants. There are about 75 plant-infecting rhabdoviruses described, several of which are economically important pathogens that are persistently transmitted to their plant ho...

  6. Specificity of DNA vaccines against the U and M genogroups of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Peñaranda, Ma Michelle D; Lapatra, Scott E; Kurath, Gael

    2011-07-01

    Infectious hematopoietic necrosis virus (IHNV) is a fish rhabdovirus that causes significant mortality in salmonid species. In North America IHNV has three major genogroups designated U, M, and L. Host-specificity of the M and U genogroups of IHNV has been established both in the field and in experimental challenges, with M isolates being more prevalent and more virulent in rainbow trout (Oncorhynchus mykiss), and U isolates being more prevalent and highly virulent in sockeye salmon (Oncorhynchus nerka). In this study, efficacy of DNA vaccines containing either M (pM) or U (pU) virus glycoprotein genes was investigated during intra- and cross-genogroup challenges in rainbow trout. In virus challenges at 7 days post-vaccination (early antiviral response), both pM and pU were highly protective against either M or U IHNV. In challenges at 28 days post-vaccination (specific antiviral response), both pM and pU were protective against M IHNV but the homologous pM vaccine was significantly more protective than pU in one of two experiments. At this stage both pM and pU induced comparably high protection against U IHNV challenge. Correlates of protection were also investigated by assessing the expression of the interferon-stimulated gene Mx-1 and the production of neutralizing antibodies (NAbs) following pM or pU DNA vaccination. Mx-1 gene expression, measured at 4 and 7 days post-vaccination as an indicator of the host innate immune response, was found to be significantly higher after pM than pU vaccination in some cases. Neutralizing antibody was produced in response to the two vaccines, but antibody titers did not show consistent correlation with protection. The results show that the rainbow trout innate and adaptive immune responses have some ability to distinguish between the U and M genogroup IHNV, but overall the pM and pU vaccines were protective against both homologous and cross-genogroup challenges.

  7. Specificity of DNA vaccines against the U and M genogroups of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss)

    USGS Publications Warehouse

    Penaranda, M.M.D.; LaPatra, S.E.; Kurath, G.

    2011-01-01

    Infectious hematopoietic necrosis virus (IHNV) is a fish rhabdovirus that causes significant mortality in salmonid species. In North America IHNV has three major genogroups designated U, M, and L. Host-specificity of the M and U genogroups of IHNV has been established both in the field and in experimental challenges, with M isolates being more prevalent and more virulent in rainbow trout (Oncorhynchus mykiss), and U isolates being more prevalent and highly virulent in sockeye salmon (Oncorhynchus nerka). In this study, efficacy of DNA vaccines containing either M (pM) or U (pU) virus glycoprotein genes was investigated during intra- and cross-genogroup challenges in rainbow trout. In virus challenges at 7 days post-vaccination (early antiviral response), both pM and pU were highly protective against either M or U IHNV. In challenges at 28 days post-vaccination (specific antiviral response), both pM and pU were protective against M IHNV but the homologous pM vaccine was significantly more protective than pU in one of two experiments. At this stage both pM and pU induced comparably high protection against U IHNV challenge. Correlates of protection were also investigated by assessing the expression of the interferon-stimulated gene Mx-1 and the production of neutralizing antibodies (NAbs) following pM or pU DNA vaccination. Mx-1 gene expression, measured at 4 and 7 days post-vaccination as an indicator of the host innate immune response, was found to be significantly higher after pM than pU vaccination in some cases. Neutralizing antibody was produced in response to the two vaccines, but antibody titers did not show consistent correlation with protection. The results show that the rainbow trout innate and adaptive immune responses have some ability to distinguish between the U and M genogroup IHNV, but overall the pM and pU vaccines were protective against both homologous and cross-genogroup challenges.

  8. MyD88/CD40 Genetic Adjuvant Function in Cutaneous Atypical Antigen-Presenting Cells Contributes to DNA Vaccine Immunogenicity

    PubMed Central

    Slawin, Kevin M.; Levitt, Jonathan M.; Spencer, David M.

    2016-01-01

    Therapeutic DNA-based vaccines aim to prime an adaptive host immune response against tumor-associated antigens, eliminating cancer cells primarily through CD8+ cytotoxic T cell-mediated destruction. To be optimally effective, immunological adjuvants are required for the activation of tumor-specific CD8+ T cells responses by DNA vaccination. Here, we describe enhanced anti-tumor efficacy of an in vivo electroporation-delivered DNA vaccine by inclusion of a genetically encoded chimeric MyD88/CD40 (MC) adjuvant, which integrates both innate and adaptive immune signaling pathways. When incorporated into a DNA vaccine, signaling by the MC adjuvant increased antigen-specific CD8+ T cells and promoted elimination of pre-established tumors. Interestingly, MC-enhanced vaccine efficacy did not require direct-expression of either antigen or adjuvant by local antigen-presenting cells, but rather our data supports a key role for MC function in “atypical” antigen-presenting cells of skin. In particular, MC adjuvant-modified keratinocytes increased inflammatory cytokine secretion, upregulated surface MHC class I, and were able to increase in vitro and in vivo priming of antigen-specific CD8+ T cells. Furthermore, in the absence of critical CD8α+/CD103+ cross-priming dendritic cells, MC was still able to promote immune priming in vivo, albeit at a reduced level. Altogether, our data support a mechanism by which MC signaling activates an inflammatory phenotype in atypical antigen-presenting cells within the cutaneous vaccination site, leading to an enhanced CD8+ T cell response against DNA vaccine-encoded antigens, through both CD8α+/CD103+ dendritic cell-dependent and independent pathways. PMID:27741278

  9. Enhanced nasal mucosal delivery and immunogenicity of anti-caries DNA vaccine through incorporation of anionic liposomes in chitosan/DNA complexes.

    PubMed

    Chen, Liulin; Zhu, Junming; Li, Yuhong; Lu, Jie; Gao, Li; Xu, Huibi; Fan, Mingwen; Yang, Xiangliang

    2013-01-01

    The design of optimized nanoparticles offers a promising strategy to enable DNA vaccines to cross various physiological barriers for eliciting a specific and protective mucosal immunity via intranasal administration. Here, we reported a new designed nanoparticle system through incorporating anionic liposomes (AL) into chitosan/DNA (CS/DNA) complexes. With enhanced cellular uptake, the constructed AL/CS/DNA nanoparticles can deliver the anti-caries DNA vaccine pGJA-P/VAX into nasal mucosa. TEM results showed the AL/CS/DNA had a spherical structure. High DNA loading ability and effective DNA protection against nuclease were proved by gel electrophoresis. The surface charge of the AL/CS/DNA depended strongly on pH environment, enabling the intracellular release of loaded DNA via a pH-mediated manner. In comparison to the traditional CS/DNA system, our new design rendered a higher transfection efficiency and longer residence time of the AL/CS/DNA at nasal mucosal surface. These outstanding features enable the AL/CS/DNA to induce a significantly (p<0.01) higher level of secretory IgA (SIgA) than the CS/DNA in animal study, and a longer-term mucosal immunity. On the other hand, the AL/CS/DNA exhibited minimal cytotoxicity. These results suggest that the developed nanoparticles offer a potential platform for DNA vaccine packaging and delivery for more efficient elicitation of mucosal immunity.

  10. Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Purcell, Maureen K.; Nichols, Krista M.; Winton, James R.; Kurath, Gael; Thorgaard, Gary H.; Wheeler, Paul; Hansen, John D.; Herwig, Russell P.; Park, Linda K.

    2006-01-01

    The DNA vaccine based on the glycoprotein gene of Infectious hematopoietic necrosis virus induces a non-specific anti-viral immune response and long-term specific immunity against IHNV. This study characterized gene expression responses associated with the early anti-viral response. Homozygous rainbow trout were injected intra-muscularly (I.M.) with vector DNA or the IHNV DNA vaccine. Gene expression in muscle tissue (I.M. site) was evaluated using a 16,008 feature salmon cDNA microarray. Eighty different genes were significantly modulated in the vector DNA group while 910 genes were modulated in the IHNV DNA vaccinate group relative to control group. Quantitative reverse-transcriptase PCR was used to examine expression of selected immune genes at the I.M. site and in other secondary tissues. In the localized response (I.M. site), the magnitudes of gene expression changes were much greater in the vaccinate group relative to the vector DNA group for the majority of genes analyzed. At secondary systemic sites (e.g. gill, kidney and spleen), type I IFN-related genes were up-regulated in only the IHNV DNA vaccinated group. The results presented here suggest that the IHNV DNA vaccine induces up-regulation of the type I IFN system across multiple tissues, which is the functional basis of early anti-viral immunity.

  11. Efficacy of Leishmania donovani ribosomal P1 gene as DNA vaccine in experimental visceral leishmaniasis.

    PubMed

    Masih, Shet; Arora, Sunil K; Vasishta, Rakesh K

    2011-09-01

    The acidic ribosomal proteins of the protozoan parasites have been described as prominent antigens during human disease. We present here data showing the molecular cloning and protective efficacy of P1 gene of Leishmania donovani as DNA vaccine. The PCR amplified complete ORF cloned in either pQE or pVAX vector was used either as peptide or DNA vaccine against experimentally induced visceral leishmaniasis in hamsters. The recombinant protein rLdP1 was given along with Freund's adjuvant and the plasmid DNA vaccine, pVAX-P1 was used alone either as single dose or double dose (prime and boost) in different groups of hamsters which were subsequently challenged with a virulent dose of 1×10(7) L. donovani (MHOM/IN/DD8/1968 strain) promastigotes by intra-cardiac route. While the recombinant protein rLdP1 or DNA vaccine pVAX-P1 in single dose format were not found to be protective, DNA vaccine in a prime-boost mode was able to induce protection with reduced mortality, a significant (75.68%) decrease in splenic parasite burden and increased expression of Th1 type cytokines in immunized hamsters. Histopathology of livers and spleens from these animals showed formation of mature granulomas with compact arrangement of lymphocytes and histiocytes, indicating its protective potential as vaccine candidate.

  12. DNA Vaccination in the Skin Using Microneedles Improves Protection Against Influenza

    PubMed Central

    Song, Jae-Min; Kim, Yeu-Chun; O, Eunju; Compans, Richard W; Prausnitz, Mark R; Kang, Sang-Moo

    2012-01-01

    In this study, we tested the hypothesis that DNA vaccination in the skin using microneedles improves protective immunity compared to conventional intramuscular (IM) injection of a plasmid DNA vaccine encoding the influenza hemagglutinin (HA). In vivo fluorescence imaging demonstrated the expression of a reporter gene delivered to the skin using a solid microneedle patch coated with plasmid DNA. Vaccination at a low dose (3 µg HA DNA) using microneedles generated significantly stronger humoral immune responses and better protective responses post-challenge compared to IM vaccination at either low or high (10 µg HA DNA) dose. Vaccination using microneedles at a high (10 µg) dose further generated improved post-challenge protection, as measured by survival, recall antibody-secreting cell responses in spleen and bone marrow, and interferon (IFN)-γ cytokine T-cell responses. This study demonstrates that DNA vaccination in the skin using microneedles induces higher humoral and cellular immune responses as well as improves protective immunity compared to conventional IM injection of HA DNA vaccine. PMID:22508490

  13. Functional evaluation of malaria Pfs25 DNA vaccine by in vivo electroporation in olive baboons.

    PubMed

    Kumar, Rajesh; Nyakundi, Ruth; Kariuki, Thomas; Ozwara, Hastings; Nyamongo, Onkoba; Mlambo, Godfree; Ellefsen, Barry; Hannaman, Drew; Kumar, Nirbhay

    2013-06-28

    Plasmodium falciparum Pfs25 antigen, expressed on the surface of zygotes and ookinetes, is one of the leading targets for the development of a malaria transmission-blocking vaccine (TBV). Our laboratory has been evaluating DNA plasmid based Pfs25 vaccine in mice and non-human primates. Previously, we established that in vivo electroporation (EP) delivery is an effective method to improve the immunogenicity of DNA vaccine encoding Pfs25 in mice. In order to optimize the in vivo EP procedure and test for its efficacy in more clinically relevant larger animal models, we employed in vivo EP to evaluate the immune response and protective efficacy of Pfs25 encoding DNA vaccine in nonhuman primates (olive baboons, Papio anubis). The results showed that at a dose of 2.5mg DNA vaccine, antibody responses were significantly enhanced with EP as compared to without EP resulting in effective transmission blocking efficiency. Similar immunogenicity enhancing effect of EP was also observed with lower doses (0.5mg and 1mg) of DNA plasmids. Further, final boosting with a single dose of recombinant Pfs25 protein resulted in dramatically enhanced antibody titers and significantly increased functional transmission blocking efficiency. Our study suggests priming with DNA vaccine via EP along with protein boost regimen as an effective method to elicit potent immunogenicity of malaria DNA vaccines in nonhuman primates and provides the basis for further evaluation in human volunteers.

  14. Genetic diversity of perch rhabdoviruses isolates based on the nucleoprotein and glycoprotein genes.

    PubMed

    Talbi, Chiraz; Cabon, Joelle; Baud, Marine; Bourjaily, Maya; de Boisséson, Claire; Castric, Jeannette; Bigarré, Laurent

    2011-12-01

    Despite the increasing impact of rhabdoviruses in European percid farming, the diversity of the viral populations is still poorly investigated. To address this issue, we sequenced the partial nucleoprotein (N) and complete glycoprotein (G) genes of nine rhabdoviruses isolated from perch (Perca fluviatilis) between 1999 and 2010, mostly from France, and analyzed six of them by immunofluorescence antibody test (IFAT). Using two rabbit antisera raised against either the reference perch rhabdovirus (PRhV) isolated in 1980 or the perch isolate R6146, two serogroups were distinguished. Meanwhile, based on partial N and complete G gene analysis, perch rhabdoviruses were divided into four genogroups, A-B-D and E, with a maximum of 32.9% divergence (G gene) between isolates. A comparison of the G amino acid sequences of isolates from the two identified serogroups revealed several variable regions that might account for antigenic differences. Comparative analysis of perch isolates with other rhabdoviruses isolated from black bass, pike-perch and pike showed some strong phylogenetic relationships, suggesting cross-host transmission. Similarly, striking genetic similarities were shown between perch rhabdoviruses and isolates from other European countries and various ecological niches, most likely reflecting the circulation of viruses through fish trade as well as putative transfers from marine to freshwater fish. Phylogenetic relationships of the newly characterized viruses were also determined within the family Rhabdoviridae. The analysis revealed a genetic cluster containing only fish viruses, including all rhabdoviruses from perch, as well as siniperca chuatsi rhabdovirus (SCRV) and eel virus X (EVEX). This cluster was distinct from the one represented by spring viraemia of carp vesiculovirus (SVCV), pike fry rhabdovirus (PFRV) and mammalian vesiculoviruses. The new genetic data provided in the present study shed light on the diversity of rhabdoviruses infecting perch in

  15. Non-contact helium-based plasma for delivery of DNA vaccines

    PubMed Central

    Connolly, Richard J.; Chapman, Taryn; Hoff, Andrew M.; Kutzler, Michele A.; Jaroszeski, Mark J.; Ugen, Kenneth E.

    2012-01-01

    Non-viral in vivo administration of plasmid DNA for vaccines and immunotherapeutics has been hampered by inefficient delivery. Methods to enhance delivery such as in vivo electroporation (EP) have demonstrated effectiveness in circumventing this difficulty. However, the contact-dependent nature of EP has resulting side effects in animals and humans. Noncontact delivery methods should, in principle, overcome some of these obstacles. This report describes a helium plasma–based delivery system that enhanced humoral and cellular antigen-specific immune responses in mice against an intradermally administered HIV gp120-expressing plasmid vaccine (pJRFLgp120). The most efficient plasma delivery parameters investigated resulted in the generation of geometric mean antibody-binding titers that were 19-fold higher than plasmid delivery alone. Plasma mediated delivery of pJRFLgp120 also resulted in a 17-fold increase in the number of interferon-gamma spot-forming cells, a measure of CD8+ cytotoxic T cells, compared with non-facilitated plasmid delivery. This is the first report demonstrating the ability of this contact-independent delivery method to enhance antigen-specific immune responses against a protein generated by a DNA vaccine. PMID:22894954

  16. From plasmids to protection: a review of DNA vaccines against infectious diseases.

    PubMed

    Laddy, Dominick J; Weiner, David B

    2006-01-01

    The field of DNA vaccine development began over 16 years ago with the observation that plasmid DNA could be injected into and expressed in vivo and drive adaptive immune responses. Since then, there has been great interest in developing this technology to create a new generation of vaccines with the ability to elicit both humoral and cellular immune responses from an inherently innocuous injection. However, DNA vaccines have yet to proceed past phase I/II clinical trials in humans--primarily due to a desire to induce more potent immune responses. This review will examine how DNA vaccines function to induce an immune response and how this information might be useful in future vaccine design.

  17. Immunogenicity of a multicomponent DNA vaccine against visceral leishmaniasis in dogs.

    PubMed

    Saldarriaga, Omar A; Travi, Bruno L; Park, Won; Perez, Luis E; Melby, Peter C

    2006-03-10

    Vaccination of dogs, the domestic reservoir of Leishmania chagasi, could not only decrease the burden of canine visceral leishmaniasis (VL), but could also indirectly reduce the incidence of human VL. Intramuscular vaccination of foxhounds with a Leishmania multicomponent (10 antigen) DNA vaccine resulted in antigen-induced lymphoproliferative and IFN-gamma (but not IL-4) responses. This response was not augmented by co-administration of canine IL-12 or GM-CSF DNA adjuvants. The multicomponent DNA vaccine also induced a delayed type hypersensitivity (DTH) response to viable L. donovani promastigotes and led to a reduction of parasite burden in an in vitro intracellular infection model, and in the draining lymph node of dogs early after cutaneous challenge. Thus, the multicomponent DNA vaccine was effective in priming dogs for a parasite-specific type 1 cellular immune response, which was able to restrict parasite growth.

  18. DNA vaccination for prostate cancer, from preclinical to clinical trials - where we stand?

    PubMed

    Ahmad, Sarfraz; Sweeney, Paul; Sullivan, Gerald C; Tangney, Mark

    2012-10-09

    Development of various vaccines for prostate cancer (PCa) is becoming an active research area. PCa vaccines are perceived to have less toxicity compared with the available cytotoxic agents. While various immune-based strategies can elicit anti-tumour responses, DNA vaccines present increased efficacy, inducing both humoural and cellular immunity. This immune activation has been proven effective in animal models and initial clinical trials are encouraging. However, to validate the role of DNA vaccination in currently available PCa management paradigms, strong clinical evidence is still lacking. This article provides an overview of the basic principles of DNA vaccines and aims to provide a summary of preclinical and clinical trials outlining the benefits of this immunotherapy in the management of PCa.

  19. Molecularly engineered poly(ortho ester) microspheres for enhanced delivery of DNA vaccines

    NASA Astrophysics Data System (ADS)

    Wang, Chun; Ge, Qing; Ting, David; Nguyen, David; Shen, Hui-Rong; Chen, Jianzhu; Eisen, Herman N.; Heller, Jorge; Langer, Robert; Putnam, David

    2004-03-01

    Genetic vaccination using plasmid DNA presents a unique opportunity for achieving potent immune responses without the potential limitations of many conventional vaccines. Here we report the design of synthetic biodegradable polymers specifically for enhancing DNA vaccine efficacy in vivo. We molecularly engineered poly(ortho ester) microspheres that are non-toxic to cells, protect DNA from degradation, enable uptake by antigen-presenting cells, and release DNA rapidly in response to phagosomal pH. One type of microsphere of poly(ortho esters) that releases DNA vaccines in synchrony with the natural development of adaptive immunity, elicited distinct primary and secondary humoral and cellular immune responses in mice, and suppressed the growth of tumour cells bearing a model antigen. This polymer microparticulate system could, with further study, have implications for advancing the clinical utility of DNA vaccines as well as other nucleic-acid-based therapeutics against viral infections and cancer.

  20. Turning self-destructing Salmonella into a universal DNA vaccine delivery platform.

    PubMed

    Kong, Wei; Brovold, Matthew; Koeneman, Brian A; Clark-Curtiss, Josephine; Curtiss, Roy

    2012-11-20

    We previously developed a biological containment system using recombinant Salmonella Typhimurium strains that are attenuated yet capable of synthesizing protective antigens. The regulated delayed attenuation and programmed self-destructing features designed into these S. Typhimurium strains enable them to efficiently colonize host tissues and allow release of the bacterial cell contents after lysis. To turn such a recombinant attenuated Salmonella vaccine (RASV) strain into a universal DNA vaccine-delivery vehicle, our approach was to genetically modify RASV strains to display a hyperinvasive phenotype to maximize Salmonella host entry and host cell internalization, to enable Salmonella endosomal escape to release a DNA vaccine into the cytosol, and to decrease Salmonella-induced pyroptosis/apoptosis that allows the DNA vaccine time to traffic to the nucleus for efficient synthesis of encoded protective antigens. A DNA vaccine vector that encodes a domain that contributes to the arabinose-regulated lysis phenotype but has a eukaryotic promoter was constructed. The vector was then improved by insertion of multiple DNA nuclear-targeting sequences for efficient nuclear trafficking and gene expression, and by increasing nuclease resistance to protect the plasmid from host degradation. A DNA vaccine encoding influenza WSN virus HA antigen delivered by the RASV strain with the best genetic attributes induced complete protection to mice against a lethal influenza virus challenge. Adoption of these technological improvements will revolutionize means for effective delivery of DNA vaccines to stimulate mucosal, systemic, and cellular protective immunities, and lead to a paradigm shift in cost-effective control and prevention of a diversity of diseases.

  1. Phase 1 Study of Pandemic H1 DNA Vaccine in Healthy Adults

    PubMed Central

    Crank, Michelle C.; Gordon, Ingelise J.; Yamshchikov, Galina V.; Sitar, Sandra; Hu, Zonghui; Enama, Mary E.; Holman, LaSonji A.; Bailer, Robert T.; Pearce, Melissa B.; Koup, Richard A.; Mascola, John R.; Nabel, Gary J.; Tumpey, Terrence M.; Schwartz, Richard M.; Graham, Barney S.; Ledgerwood, Julie E.

    2015-01-01

    Background A novel, swine-origin influenza A (H1N1) virus was detected worldwide in April 2009, and the World Health Organization (WHO) declared a global pandemic that June. DNA vaccine priming improves responses to inactivated influenza vaccines. We describe the rapid production and clinical evaluation of a DNA vaccine encoding the hemagglutinin protein of the 2009 pandemic A/California/04/2009(H1N1) influenza virus, accomplished nearly two months faster than production of A/California/07/2009(H1N1) licensed monovalent inactivated vaccine (MIV). Methods 20 subjects received three H1 DNA vaccinations (4 mg intramuscularly with Biojector) at 4-week intervals. Eighteen subjects received an optional boost when the licensed H1N1 MIV became available. The interval between the third H1 DNA injection and MIV boost was 3–17 weeks. Vaccine safety was assessed by clinical observation, laboratory parameters, and 7-day solicited reactogenicity. Antibody responses were assessed by ELISA, HAI and neutralization assays, and T cell responses by ELISpot and flow cytometry. Results Vaccinations were safe and well-tolerated. As evaluated by HAI, 6/20 developed positive responses at 4 weeks after third DNA injection and 13/18 at 4 weeks after MIV boost. Similar results were detected in neutralization assays. T cell responses were detected after DNA and MIV. The antibody responses were significantly amplified by the MIV boost, however, the boost did not increased T cell responses induced by DNA vaccine. Conclusions H1 DNA vaccine was produced quickly, was well-tolerated, and had modest immunogenicity as a single agent. Other HA DNA prime-MIV boost regimens utilizing one DNA prime vaccination and longer boost intervals have shown significant immunogenicity. Rapid and large-scale production of HA DNA vaccines has the potential to contribute to an efficient response against future influenza pandemics. Trial Registration Clinicaltrials.gov NCT00973895 PMID:25884189

  2. Characterization of GD2 peptide mimotope DNA vaccines effective against spontaneous neuroblastoma metastases.

    PubMed

    Fest, Stefan; Huebener, Nicole; Weixler, Silke; Bleeke, Matthias; Zeng, Yan; Strandsby, Anne; Volkmer-Engert, Rudolf; Landgraf, Christiane; Gaedicke, Gerhard; Riemer, Angelika B; Michalsky, Elke; Jaeger, Ines S; Preissner, Robert; Förster-Wald, Elisabeth; Jensen-Jarolim, Erika; Lode, Holger N

    2006-11-01

    Disialoganglioside GD2 is an established target for immunotherapy in neuroblastoma. We tested the hypothesis that active immunization against the glycolipid GD2 using DNA vaccines encoding for cyclic GD2-mimicking decapeptides (i.e., GD2 mimotopes) is effective against neuroblastoma. For this purpose, two GD2 peptide mimotopes (MA and MD) were selected based on docking experiments to anti-GD2 antibody ch14.18 (binding free energy: -41.23 kJ/mol for MA and -48.06 kJ/mol for MD) and Biacore analysis (K(d) = 12.3 x 10(-5) mol/L for MA and 5.3 x 10(-5) mol/L for MD), showing a higher affinity of MD over MA. These sequences were selected for DNA vaccine design based on pSecTag2-A (pSA) also including a T-cell helper epitope. GD2 mimicry was shown following transfection of CHO-1 cells with pSA-MA and pSA-MD DNA vaccines, with twice-higher signal intensity for cells expressing MD over MA. Finally, these DNA vaccines were tested for induction of tumor protective immunity in a syngeneic neuroblastoma model following oral DNA vaccine delivery with attenuated Salmonella typhimurium (SL 7207). Only mice receiving the DNA vaccines revealed a reduction of spontaneous liver metastases. The highest anti-GD2 humoral immune response and natural killer cell activation was observed in mice immunized with the pSA-MD, a finding consistent with superior calculated binding free energy, dissociation constant, and GD2 mimicry potential for GD2 mimotope MD over MA. In summary, we show that DNA immunization with pSA-MD may provide a useful strategy for active immunization against neuroblastoma.

  3. The Murine Intravaginal HSV-2 Challenge Model for Investigation of DNA Vaccines

    PubMed Central

    Marshak, Joshua O.; Dong, Lichun; Koelle, David M.

    2014-01-01

    DNA vaccines have been licensed in veterinary medicine and have promise for humans. This format is relatively immunogenic in mice and guinea pigs, the two principle HSV-2 animal models, permitting rapid assessment of vectors, antigens, adjuvants, and delivery systems. Limitations include the relatively poor immunogenicity of naked DNA in humans and the profound differences in HSV-2 pathogenesis between host species. Herein, we detail lessons learned over the last few years investigating candidate DNA vaccines in the progesterone-primed female mouse vaginal model of HSV-2 infection as a guide to investigators in the field. PMID:24671693

  4. A DNA vaccine targeting the receptor-binding domain of Clostridium difficile toxin A

    PubMed Central

    Gardiner, David F.; Rosenberg, Talia; Zaharatos, Jerry; Franco, David; Ho, David D

    2009-01-01

    Clostridium difficile is a pathogen with increasing severity for which host antibody responses provide protection from disease. DNA vaccination has several advantages compared to traditional vaccine methods, however no study has examined this platform against C. difficile toxins. A synthetic gene was created encoding the receptor-binding domain (RBD) of C. difficile toxin A, optimized for expression in human cells. Gene expression was examined in vitro. Mice were inoculated then challenged with parenteral toxin A. Vaccination provided high titer antibodies and protected mice from death. This represents the first report of DNA vaccine inducing neutralizing antibodies to Clostridium difficile toxin A. PMID:19464540

  5. A DNA vaccine targeting the receptor-binding domain of Clostridium difficile toxin A.

    PubMed

    Gardiner, David F; Rosenberg, Talia; Zaharatos, Jerry; Franco, David; Ho, David D

    2009-06-02

    Clostridium difficile is a pathogen with increasing severity for which host antibody responses provide protection from disease. DNA vaccination has several advantages compared to traditional vaccine methods, however no study has examined this platform against C. difficile toxins. A synthetic gene was created encoding the receptor-binding domain (RBD) of C. difficile toxin A, optimized for expression in human cells. Gene expression was examined in vitro. Mice were inoculated and then challenged with parenteral toxin A. Vaccination provided high titer antibodies and protected mice from death. This represents the first report of DNA vaccine inducing neutralizing antibodies to C. difficile toxin A.

  6. Attenuated Salmonella Typhimurium delivery of a novel DNA vaccine induces immune responses and provides protection against duck enteritis virus.

    PubMed

    Liu, Xueyan; Liu, Qing; Xiao, Kangpeng; Li, Pei; Liu, Qiong; Zhao, Xinxin; Kong, Qingke

    2016-04-15

    DNA vaccines are widely used to prevent and treat infectious diseases, cancer and autoimmune diseases; however, their relatively low immunogenicity is an obstacle to their use. In this study, we constructed a novel and universal DNA vaccine vector (pSS898) that can be used to build DNA vaccines against duck enteritis virus (DEV) and other viruses that require DNA vaccines to provide protection. This vaccine vector has many advantages, including innate immunogenicity, efficient nuclear trafficking and resistance to attack from nucleases. UL24 and tgB from DEV were chosen as the antigens, and the heat labile enterotoxin B subunit (LTB) from Escherichia coli and the IL-2 gene (DuIL-2) from duck were used as adjuvants for the construction of DNA vaccine plasmids. Ducklings that were orally immunized with S739 (Salmonella Typhimurium Δasd-66 Δcrp-24 Δcya-25) and harboring these DEV DNA vaccines produced strong mucosal and systemic immune responses, and they resisted an otherwise lethal DEV challenge. More importantly, S739 (UL24-LTB) provided 90% protection after a priming-boost immunization. This study shows that our novel and universal DNA vaccine vector can be used efficiently in practical applications and may provide a promising method of orally inoculating ducks with a DEV DNA vaccine delivered by attenuated Salmonella Typhimurium for prevention of DVE.

  7. DNA vaccine elicits an efficient antitumor response by targeting the mutant Kras in a transgenic mouse lung cancer model.

    PubMed

    Weng, T-Y; Yen, M-C; Huang, C-T; Hung, J-J; Chen, Y-L; Chen, W-C; Wang, C-Y; Chang, J-Y; Lai, M-D

    2014-10-01

    Mutant Kras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) is observed in more than 20% of non-small-cell lung cancers; however, no effective Kras target therapy is available at present. The Kras DNA vaccine may represent as a novel immunotherapeutic agent in lung cancer. In this study, we investigated the antitumor efficacy of the Kras DNA vaccine in a genetically engineered inducible mouse lung tumor model driven by Kras(G12D). Lung tumors were induced by doxycycline, and the therapeutic effects of Kras DNA vaccine were evaluated with delivery of Kras(G12D) plasmids. Mutant Kras(G12D) DNA vaccine significantly decreased the tumor nodules. A dominant-negative mutant Kras(G12D)N17, devoid of oncogenic activity, achieved similar therapeutic effects. The T-helper 1 immune response was enhanced in mice treated with Kras DNA vaccine. Splenocytes from mice receiving Kras DNA vaccine presented an antigen-specific response by treatment with peptides of Kras but not Hras or OVA. The number of tumor-infiltrating CD8(+) T cells increased after Kras vaccination. In contrast, Kras DNA vaccine was not effective in the lung tumor in transgenic mice, which was induced by mutant L858R epidermal growth factor receptor. Overall, these results indicate that Kras DNA vaccine produces an effective antitumor response in transgenic mice, and may be useful in treating lung cancer-carrying Ras mutation.

  8. Directed Molecular Evolution Improves the Immunogenicity and Protective Efficacy of a Venezuelan Equine Encephalitis Virus DNA Vaccine

    DTIC Science & Technology

    2009-05-01

    VEEV IA/B challenge. Our results indicate that it is pos- sible to improve the immunogenicity and protective efficacy of alphavirus DNA vaccines using... alphaviruses that ause periodic epizootics in the Americas [1]. These New World lphaviruses cause diseases in humans characterized by fever, eadache...equine encephalitis virus, VEE, alphavirus , DNA vaccine, envelope glycoproteins, directed molecular evolution, efficacy, immunogenicity, laboratory

  9. Characterization of Durham virus, a novel rhabdovirus that encodes both a C and SH protein

    PubMed Central

    Allison, A. B.; Palacios, G.; Rosa, A. Travassos da; Popov, V. L.; Lu, L.; Xiao, S. Y.; DeToy, K.; Briese, T.; Lipkin, W. Ian; Keel, M. K.; Stallknecht, D. E.; Bishop, G. R.; Tesh, R. B.

    2010-01-01

    The family Rhabdoviridae is a diverse group of non-segmented, negative-sense RNA viruses that are distributed worldwide and infect a wide range of hosts including vertebrates, invertebrates, and plants. Of the 114 currently recognized vertebrate rhabdoviruses, relatively few have been well characterized at both the antigenic and genetic level; hence, the phylogenetic relationships between many of the vertebrate rhabdoviruses remain unknown. The present report describes a novel rhabdovirus isolated from the brain of a moribund American coot (Fulica americana) that exhibited neurological signs when found in Durham County, North Carolina, in 2005. Antigenic characterization of the virus revealed that it was serologically unrelated to 68 other known vertebrate rhabdoviruses. Genomic sequencing of the virus indicated that it shared the highest identity to Tupaia rhabdovirus (TUPV), and as only previously observed in TUPV, the genome encoded a putative C protein in an overlapping open reading frame (ORF) of the phosphoprotein gene and a small hydrophobic protein located in a novel ORF between the matrix and glycoprotein genes. Phylogenetic analysis of partial amino acid sequences of the nucleoprotein and polymerase proteins indicated that, in addition to TUPV, the virus was most closely related to avian and small mammal rhabdoviruses from Africa and North America. In this report, we present the morphological, pathological, antigenic, and genetic characterization of the new virus, tentatively named Durham virus (DURV), and discuss its potential evolutionary relationship to other vertebrate rhabdoviruses. PMID:20863863

  10. Immune-Enhancing Effects of Taishan Pinus massoniana Pollen Polysaccharides on DNA Vaccine Expressing Bordetella avium ompA

    PubMed Central

    Zhu, Fujie; Liu, Xiao; Sun, Zhenhong; Yu, Cuilian; Liu, Liping; Yang, Shifa; Li, Bing; Wei, Kai; Zhu, Ruiliang

    2016-01-01

    Bordetella avium is the causative agent of bordetellosis, which remains to be the cause of severe losses in the turkey industry. Given the lack of vaccines that can provide good protection, developing a novel vaccine against B. avium infection is crucial. In this study, we constructed a eukaryotic expression plasmid, which expressed the outer membrane protein A (ompA) of B. avium, to prepare a B. avium recombinant ompA-DNA vaccine. Three concentrations (low, middle, and high) of Taishan Pinus massoniana pollen polysaccharides (TPPPS), a known immunomodulator, were used as adjuvants, and their immune conditioning effects on the developed DNA vaccine were examined. The pure ompA-DNA vaccine, Freund’s incomplete adjuvant ompA-DNA vaccine, and the empty plasmid served as the controls. The chickens in each group were separately inoculated with these vaccines three times at 1, 7, and 14 days old. Dynamic changes in antibody production, cytokine secretion, and lymphocyte count were then determined from 7 to 49 days after the first inoculation. Protective rates of the vaccines were also determined after the third inoculation. Results showed that the pure DNA vaccine obviously induced the production of antibodies, the secretion of cytokines, and the increase in CD4+ and CD8+ T lymphocyte counts in peripheral blood, as well as provided a protective rate of 50% to the B. avium-challenged chickens. The chickens inoculated with the TPPPS adjuvant ompA-DNA vaccine and Freund’s adjuvant ompA-DNA vaccine demonstrated higher levels of immune responses than those inoculated with pure ompA-DNA vaccine, whereas only the ompA-DNA vaccine with 200 mg/mL TPPPS completely protected the chickens against B. avium infection. These findings indicate that the B. avium ompA-DNA vaccine combined with TPPPS is a potentially effective B. avium vaccine. PMID:26870023

  11. Characterization of perch rhabdovirus (PRV) in farmed grayling Thymallus thymallus.

    PubMed

    Gadd, Tuija; Viljamaa-Dirks, Satu; Holopainen, Riikka; Koski, Perttu; Jakava-Viljanen, Miia

    2013-10-11

    Two Finnish fish farms experienced elevated mortality rates in farmed grayling Thymallus thymallus fry during the summer months, most typically in July. The mortalities occurred during several years and were connected with a few neurological disorders and peritonitis. Virological investigation detected an infection with an unknown rhabdovirus. Based on the entire glycoprotein (G) and partial RNA polymerase (L) gene sequences, the virus was classified as a perch rhabdovirus (PRV). Pairwise comparisons of the G and L gene regions of grayling isolates revealed that all isolates were very closely related, with 99 to 100% nucleotide identity, which suggests the same origin of infection. Phylogenetic analysis demonstrated that they were closely related to the strain isolated from perch Perca fluviatilis and sea trout Salmo trutta trutta caught from the Baltic Sea. The entire G gene sequences revealed that all Finnish grayling isolates, and both the perch and sea trout isolates, were most closely related to a PRV isolated in France in 2004. According to the partial L gene sequences, all of the Finnish grayling isolates were most closely related to the Danish isolate DK5533 from pike. The genetic analysis of entire G gene and partial L gene sequences showed that the Finnish brown trout isolate ka907_87 shared only approximately 67 and 78% identity, respectively, with our grayling isolates. The grayling isolates were also analysed by an immunofluorescence antibody test. This is the first report of a PRV causing disease in grayling in Finland.

  12. Smallpox DNA Vaccine Delivered by Novel Skin Electroporation Device Protects Mice Against Intranasal Poxvirus Challenge

    DTIC Science & Technology

    2006-11-27

    involved topical dministration of DNA followed by electroporation [21–23] r subcutaneous injection of DNA followed by electropora - ion [24–26]. Others have...demonstrated improved delivery f DNA vaccines to the skin by micromechanical disruption 27]; however this procedure did not involve electropora - ion

  13. Evaluation of a prototype dengue-1 DNA vaccine in a Phase 1 clinical trial.

    PubMed

    Beckett, Charmagne G; Tjaden, Jeffrey; Burgess, Timothy; Danko, Janine R; Tamminga, Cindy; Simmons, Monika; Wu, Shuenn-Jue; Sun, Peifang; Kochel, Tadeusz; Raviprakash, Kanakatte; Hayes, Curtis G; Porter, Kevin R

    2011-01-29

    Candidate dengue DNA vaccine constructs for each dengue serotype were developed by incorporating pre-membrane and envelope genes into a plasmid vector. A Phase 1 clinical trial was performed using the dengue virus serotype-1 (DENV-1) vaccine construct (D1ME(100)). The study was an open-label, dose-escalation, safety and immunogenicity trial involving 22 healthy flavivirus-naïve adults assigned to one of two groups. Each group received three intramuscular injections (0, 1, and 5 months) of either a high dose (5.0mg, n=12) or a low dose (1.0mg, n=10) DNA vaccine using the needle-free Biojector(®) 2000. The most commonly reported solicited signs and symptoms were local mild pain or tenderness (10/22, 45%), local mild swelling (6/22, 27%), muscle pain (6/22, 27%) and fatigue (6/22, 27%). Five subjects (41.6%) in the high dose group and none in the low dose group developed detectable anti-dengue neutralizing antibodies. T-cell IFN gamma responses were detected in 50% (4/8) and 83.3% (10/12) of subjects in the low and high dose groups, respectively. The safety profile of the DENV-1 DNA vaccine is acceptable at both doses administered in the study. These results demonstrate a favorable reactogenicity and safety profile of the first in human evaluation of a DENV-1 DNA vaccine.

  14. Recent advances in design of immunogenic and effective naked DNA vaccines against cancer.

    PubMed

    Fioretti, Daniela; Iurescia, Sandra; Rinaldi, Monica

    2014-01-01

    A variety of clinical trials for vaccines against cancer have provided evidence that DNA vaccines are well tolerated and have an excellent safety profile. DNA vaccines require much improvement to make them sufficiently effective against cancer in the clinic. Nowadays, it is clear that an increased antigen expression correlates with improved immunogenicity and it is critical to vaccine performance in large animals and humans. Similarly, additional strategies are required to activate effective immunity against poorly immunogenic tumour antigens. This review discusses very recent scientific references focused on the development of sophisticated DNA vaccines against cancer. We report a selection of novel and relevant patents employed to improve their immunogenicity through several strategies such as the use of tissue-specific transcriptional elements, nuclear localisation signalling, codon-optimisation and by targeting antigenic proteins to secretory pathway. Recent patents validating portions or splice variants of tumour antigens as candidates for cancer DNA vaccines with improved specificity, such as mesothelin and hTERT, are also discussed. Lastly, we review novel patents on the use of genetic immunomodulators, such as "universal" T helper epitopes derived from tetanus toxin, E. coli heat labile enterotoxin and vegetable proteins, as well as cytokines, chemokines or costimulatory molecules such as IL-6, IL-15, IL- 21 to amplify immunity against cancer.

  15. DNA vaccines against infectious agents: recent strategies for enhancing immune responses

    USDA-ARS?s Scientific Manuscript database

    There have been widespread efforts to develop plasmid DNA vaccines against animal and human pathogens, and for use as therapies in the treatment of cancers, autoimmune diseases, and allergies. The impetus for this research activity was based on early promising results in laboratory animals that sho...

  16. GITRL as a genetic adjuvant enhances enterovirus 71 VP1 DNA vaccine immunogenicity.

    PubMed

    Yuan, Jing; Tang, Xinyi; Yin, Kai; Tian, Jie; Rui, Ke; Ma, Jie; Mao, Chaoming; Chen, Jianguo; Lu, Liwei; Xu, Huaxi; Wang, Shengjun

    2015-05-01

    VP1 protein is the immunodominant capsid protein of enterovirus 71 (EV71) which is responsible for large outbreaks of hand, foot and mouth disease. It has been reported that glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and its ligand (GITRL) are involved in modulating both innate and adaptive immune responses. In this study, a DNA vaccine vector encoding EV71 VP1 gene and mGITRL gene (pIRES/VP1/mGITRL) was constructed. And female Balb/c mice were immunized intramuscularly with the DNA vaccine. Compared with the groups immunized with pIRES, pIRES/VP1, pIRES/mGITRL and PBS, the inoculation of pIRES/VP1/mGITRL induced a higher levels of EV71 VP1-specific antibody and specific antibody-forming cells. However, significantly higher levels of CD4(+)Th1, Th2 and CD8(+)IFN-γ(+)T cells were found in the pIRES/VP1/mGITRL group compared with control groups. Our results demonstrate that a novel DNA vaccine, expressing VP1 and mGITRL, could effectively elicit both humoral and cell-mediated immune responses against EV71 VP1 in mice. Thus, the mGITRL may be used as molecular adjuvant for EV71 DNA vaccine.

  17. Electroporation-mediated administration of candidate DNA vaccines against HIV-1.

    PubMed

    Vasan, Sandhya

    2014-01-01

    Vaccines to prevent HIV remain desperately needed, but a number of challenges, including retroviral integration, establishment of anatomic reservoir sites, high sequence diversity, and heavy envelope glycosylation. have precluded development of a highly effective vaccine. DNA vaccines have been utilized as candidate HIV vaccines because of their ability to generate cellular and humoral immune responses, the lack of anti-vector response allowing for repeat administration, and their ability to prime the response to viral-vectored vaccines. Because the HIV epidemic has disproportionately affected the developing world, the favorable thermostability profile and relative ease and low cost of manufacture of DNA vaccines offer additional advantages. In vivo electroporation (EP) has been utilized to improve immune responses to DNA vaccines as candidate HIV-1 vaccines in standalone or prime-boost regimens with both proteins and viral-vectored vaccines in several animal models and, more recently, in human clinical trials. This chapter describes the preclinical and clinical development of candidate DNA vaccines for HIV-1 delivered by EP, including challenges to bringing this technology to the developing world.

  18. Pilot study of p62 DNA vaccine in dogs with mammary tumors.

    PubMed

    Gabai, Vladimir; Venanzi, Franco M; Bagashova, Elena; Rud, Oksana; Mariotti, Francesca; Vullo, Cecilia; Catone, Giuseppe; Sherman, Michael Y; Concetti, Antonio; Chursov, Andrey; Latanova, Anastasia; Shcherbinina, Vita; Shifrin, Victor; Shneider, Alexander

    2014-12-30

    Our previous data demonstrated profound anti-tumor and anti-metastatic effects of p62 (sqstm1) DNA vaccine in rodents with various types of transplantable tumors. Testing anti-cancer medicine in dogs as an intermediary step of translational research program provides two major benefits. First, clinical data collected in target animals is required for FDA/USDA approval as a veterinary anti-cancer drug or vaccine. It is noteworthy that the veterinary community is in need of novel medicine for the prevention and treatment of canine and feline cancers. The second more important benefit of testing anti-cancer vaccines in dogs is that spontaneous tumors in dogs may provide invaluable information for human trials. Here, we evaluated the effect(s) of p62 DNA vaccine on mammary tumors of dogs. We found that p62 DNA vaccine administered i.m. decreased or stabilized growth of locally advanced lesions in absence of its overall toxic effects. The observed antitumor activity was associated with lymphocyte infiltration and tumor encapsulation via fibrotic reaction. This data justifies both human clinical trials and veterinary application of p62 DNA vaccine.

  19. A DNA vaccine targeting angiomotin inhibits angiogenesis and suppresses tumor growth

    NASA Astrophysics Data System (ADS)

    Holmgren, Lars; Ambrosino, Elena; Birot, Olivier; Tullus, Carl; Veitonmäki, Niina; Levchenko, Tetyana; Carlson, Lena-Maria; Musiani, Piero; Iezzi, Manuela; Curcio, Claudia; Forni, Guido; Cavallo, Federica; Kiessling, Rolf

    2006-06-01

    Endogenous angiogenesis inhibitors have shown promise in preclinical trials, but clinical use has been hindered by low half-life in circulation and high production costs. Here, we describe a strategy that targets the angiostatin receptor angiomotin (Amot) by DNA vaccination. The vaccination procedure generated antibodies that detected Amot on the endothelial cell surface. Purified Ig bound to the endothelial cell membrane and inhibited endothelial cell migration. In vivo, DNA vaccination blocked angiogenesis in the matrigel plug assay and prevented growth of transplanted tumors for up to 150 days. We further demonstrate that a combination of DNA vaccines encoding Amot and the extracellular and transmembrane domains of the human EGF receptor 2 (Her-2)/neu oncogene inhibited breast cancer progression and impaired tumor vascularization in Her-2/neu transgenic mice. No toxicity or impairment of normal blood vessels could be detected. This work shows that DNA vaccination targeting Amot may be used to mimic the effect of angiostatin. cancer vaccines | neoplasia | neovascularization | breast cancer | angiostatin

  20. Antigen Targeting to Human HLA Class II Molecules Increases Efficacy of DNA Vaccination

    PubMed Central

    Fredriksen, Agnete Brunsvik; Løset, Geir Åge; Vikse, Elisabeth; Fugger, Lars

    2016-01-01

    It has been difficult to translate promising results from DNA vaccination in mice to larger animals and humans. Previously, DNA vaccines encoding proteins that target Ag to MHC class II (MHC-II) molecules on APCs have been shown to induce rapid, enhanced, and long-lasting Ag-specific Ab titers in mice. In this study, we describe two novel DNA vaccines that as proteins target HLA class II (HLA-II) molecules. These vaccine proteins cross-react with MHC-II molecules in several species of larger mammals. When tested in ferrets and pigs, a single DNA delivery with low doses of the HLA-II–targeted vaccines resulted in rapid and increased Ab responses. Importantly, painless intradermal jet delivery of DNA was as effective as delivery by needle injection followed by electroporation. As an indication that the vaccines could also be useful for human application, HLA-II–targeted vaccine proteins were found to increase human CD4+ T cell responses by a factor of ×103 in vitro. Thus, targeting of Ag to MHC-II molecules may represent an attractive strategy for increasing efficacy of DNA vaccines in larger animals and humans. PMID:27671110

  1. DNA vaccines targeting the encoded antigens to dendritic cells induce potent antitumor immunity in mice.

    PubMed

    Cao, Jun; Jin, Yiqi; Li, Wei; Zhang, Bin; He, Yang; Liu, Hongqiang; Xia, Ning; Wei, Huafeng; Yan, Jian

    2013-08-14

    Although DNA vaccine holds a great potential for cancer immunotherapy, effective long-lasting antitumoral immunity sufficient to induce durable responses in cancer patients remains to be achieved. Considering the pivotal role of dendritic cells (DC) in the antigen processing and presentation, we prepared DC-targeting DNA vaccines by fusing tumor-associated antigen HER2/neu ectodomain to single chain antibody fragment (scFv) from NLDC-145 antibody specific for DC-restricted surface molecule DEC-205 (scFvNLDC-145), and explored its antitumoral efficacy and underlying mechanisms in mouse breast cancer models. In vivo targeting assay demonstrated that scFvNLDC-145 specifically delivered DNA vaccine-encoded antigen to DC. Compared with untargeted HER2/neu DNA vaccines, vaccination with scFvNLDC-145-HER2/neu markedly promoted the HER2/neu-specific cellular and humoral immune responses with long-lasting immune memory, resulting in effective protection against challenge of HER2/neu-positive D2F2/E2 breast tumor while ineffective in parental HER2/neu-negative D2F2 breast tumor. More importantly, in combination with temporary depletion of regulatory T cells (Treg) by low-dose cyclophosphamide, vaccination with scFvNLDC-145-HER2/neu induced the regression of established D2F2/E2 breast tumor and significantly retarded the development of spontaneous mammary carcinomas in transgenic BALB-neuT mice. Our findings demonstrate that DC-targeted DNA vaccines for in vivo direct delivery of tumor antigens to DC could induce potent antigen-specific cellular and humoral immune responses and, if additional combination with systemic Treg depletion, was able to elicit an impressively therapeutic antitumoral activity, providing a rationale for further development of this approach for cancer treatment.

  2. For t 2 DNA vaccine prevents Forcipomyia taiwana (biting midge) allergy in a mouse model.

    PubMed

    Lee, M-F; Song, P-P; Lin, T-M; Chiu, Y-T; Chen, Y-H

    2016-04-01

    Forcipomyia taiwana (biting midge) is the most prevalent allergenic biting insect in Taiwan, and 60% of the exposed subjects develop allergic reactions. Subjects with insect allergy frequently limit their outdoor activities to avoid the annoyingly intense itchy allergic reactions, leading to significant worsening of their quality of life. Allergen-specific immunotherapy is the only known therapy that provides long-term host immune tolerance to the allergen, but is time-consuming and cumbersome. This study tested whether the For t 2 DNA vaccine can prevent allergic symptoms in For t 2-sensitized mice. Two consecutive shots of For t 2 DNA vaccine were given to mice with a 7-day interval before sensitization with recombinant For t 2 proteins, using the two-step sensitization protocol reported previously. The For t 2 DNA vaccine at 50 μg prevented the production of For t 2-specific IgE (P < 0.05), as well as midge allergen-challenge-induced scratch bouts, midge allergen-induced IL-13 and IL-4 production from splenocytes, and inflammatory cell infiltrations in the lesions 48 h after intradermal challenge. This study is the first to demonstrate that DNA vaccine encoding midge allergen is effective in preventing allergic skin inflammation induced by biting midge. Immunotherapy using For t 2 DNA vaccine can protect mice from being sensitized by midge allergen and may be a promising treatment for biting midge allergy in the future. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Chicken HSP70 DNA vaccine inhibits tumor growth in a canine cancer model.

    PubMed

    Yu, Wen-Ying; Chuang, Tien-Fu; Guichard, Cécile; El-Garch, Hanane; Tierny, Dominique; Laio, Albert Taiching; Lin, Ching-Si; Chiou, Kuo-Hao; Tsai, Cheng-Long; Liu, Chen-Hsuan; Li, Wen-Chiuan; Fischer, Laurent; Chu, Rea-Min

    2011-04-18

    Immunization with xenogeneic DNA is a promising cancer treatment to overcome tolerance to self-antigens. Heat shock protein 70 (HSP70) is over-expressed in various kinds of tumors and is believed to be involved in tumor progression. This study tested a xenogeneic chicken HSP70 (chHSP70) DNA vaccine in an experimental canine transmissible venereal tumor (CTVT) model. Three vaccination strategies were compared: the first (PE) was designed to evaluate the prophylactic efficacy of chHSP70 DNA vaccination by delivering the vaccine before tumor inoculation in a prime boost setting, the second (T) was designed to evaluate the therapeutic efficacy of the same prime boost vaccine by vaccinating the dogs after tumor inoculation; the third (PT) was similar to the first strategy (PE), with the exception that the electroporation booster injection was replaced with a transdermal needle-free injection. Tumor growth was notably inhibited only in the PE dogs, in which the vaccination program triggered tumor regression significantly sooner than in control dogs (NT). The CD4(+) subpopulation of tumor-infiltrating lymphocytes and canine HSP70 (caHSP70)-specific IFN-γ-secreting lymphocytes were significantly increased during tumor regression in the PE dogs as compared to control dogs, demonstrating that specific tolerance to caHSP70 has been overcome. In contrast, no benefit of the therapeutic strategy (T) could be noticed and the (PT) strategy only led to partial control of tumor growth. In summary, antitumor prophylactic activity was demonstrated using the chHSP70 DNA vaccine including a boost via electroporation. Our data stressed the importance of DNA electroporation as a booster to get the full benefit of DNA vaccination but also of cancer immunotherapy initiation as early as possible. Xenogeneic chHSP70 DNA vaccination including an electroporation boost is a potential vaccine to HSP70-expressing tumors, although further research is still required to better understand true

  4. DNA vaccination of bison to brucellar antigens elicits elevated antibody and IFN-γ responses.

    PubMed

    Clapp, Beata; Walters, Nancy; Thornburg, Theresa; Hoyt, Teri; Yang, Xinghong; Pascual, David W

    2011-07-01

    Brucella abortus remains a threat to the health and well-being of livestock in states bordering the Greater Yellowstone Area. During the past several years, cohabitation of infected wildlife with cattle has jeopardized the brucellosis-free status of Idaho, USA; Wyoming, USA; and Montana, USA. Current livestock B. abortus vaccines have not proven to be efficacious in bison (Bison bison) or elk (Cervus elaphus nelsoni). One problem with the lack of vaccine efficacy may stem from the failure to understand wildlife immune responses to vaccines. In an attempt to understand their immune responses, bison were vaccinated with eukaryotic DNA expression vectors encoding the Brucella periplasmic protein, bp26, and the chaperone protein, trigger factor (TF). These DNA vaccines have previously been shown to be protective against Brucella infection in mice. Bison were immunized intramuscularly at weeks 0, 2, and 4 with bp26 and TF DNA vaccines plus CpG adjuvant or empty vector (control) plus CpG. Blood samples were collected before vaccination and at 8, 10, and 12 wk after primary vaccination. The results showed that bison immunized with bp26 and TF DNA vaccines developed enhanced antibody, proliferative T cell, and interferon-gamma (IFN-γ) responses upon in vitro restimulation with purified recombinant bp26 or TF antigens, unlike bison immunized with empty vector. Flow cytometric analysis revealed that the percentages of CD4(+) and CD8(+) T lymphocytes from the DNA-vaccinated groups were significantly greater than they were for those bison given empty vector. These data suggest that DNA vaccination of bison may elicit strong cellular immune responses and serve as an alternative for vaccination of bison for brucellosis.

  5. Kolente virus, a rhabdovirus species isolated from ticks and bats in the Republic of Guinea

    PubMed Central

    Ghedin, Elodie; Rogers, Matthew B.; Widen, Steven G.; Guzman, Hilda; Travassos da Rosa, Amelia P. A.; Wood, Thomas G.; Fitch, Adam; Popov, Vsevolod; Holmes, Edward C.; Walker, Peter J.; Tesh, Robert B.

    2013-01-01

    Kolente virus (KOLEV) is a rhabdovirus originally isolated from ticks and a bat in Guinea, West Africa, in 1985. Although tests at the time of isolation suggested that KOLEV is a novel rhabdovirus, it has remained largely uncharacterized. We assembled the complete genome sequence of the prototype strain DakAr K7292, which was found to encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with alternative ORFs (>180 nt) in the P and L genes. Serologically, KOLEV exhibited a weak antigenic relationship with Barur and Fukuoka viruses in the Kern Canyon group. Phylogenetic analysis revealed that KOLEV represents a distinct and divergent lineage that shows no clear relationship to any rhabdovirus except Oita virus, although with limited phylogenetic resolution. In summary, KOLEV represents a novel species in the family Rhabdoviridae. PMID:24062532

  6. DNA vaccines encoding altered peptide ligands for SSX2 enhance epitope-specific CD8+ T-cell immune responses.

    PubMed

    Smith, Heath A; Rekoske, Brian T; McNeel, Douglas G

    2014-03-26

    Plasmid DNA serves as a simple and easily modifiable form of antigen delivery for vaccines. The USDA approval of DNA vaccines for several non-human diseases underscores the potential of this type of antigen delivery method as a cost-effective approach for the treatment or prevention of human diseases, including cancer. However, while DNA vaccines have demonstrated safety and immunological effect in early phase clinical trials, they have not consistently elicited robust anti-tumor responses. Hence many recent efforts have sought to increase the immunological efficacy of DNA vaccines, and we have specifically evaluated several target antigens encoded by DNA vaccine as treatments for human prostate cancer. In particular, we have focused on SSX2 as one potential target antigen, given its frequent expression in metastatic prostate cancer. We have previously identified two peptides, p41-49 and p103-111, as HLA-A2-restricted SSX2-specific epitopes. In the present study we sought to determine whether the efficacy of a DNA vaccine could be enhanced by an altered peptide ligand (APL) strategy wherein modifications were made to anchor residues of these epitopes to enhance or ablate their binding to HLA-A2. A DNA vaccine encoding APL modified to increase epitope binding elicited robust peptide-specific CD8+ T cells producing Th1 cytokines specific for each epitope. Ablation of one epitope in a DNA vaccine did not enhance immune responses to the other epitope. These results demonstrate that APL encoded by a DNA vaccine can be used to elicit increased numbers of antigen-specific T cells specific for multiple epitopes simultaneously, and suggest this could be a general approach to improve the immunogenicity of DNA vaccines encoding tumor antigens.

  7. Stable antigen is most effective for eliciting CD8+ T-cell responses after DNA vaccination and infection with recombinant vaccinia virus in vivo.

    PubMed

    Schliehe, Christopher; Bitzer, Annegret; van den Broek, Maries; Groettrup, Marcus

    2012-09-01

    The induction of strong CD8(+) T-cell responses against infectious diseases and cancer has remained a major challenge. Depending on the source of antigen and the infectious agent, priming of CD8(+) T cells requires direct and/or cross-presentation of antigenic peptides on major histocompatibility complex (MHC) class I molecules by professional antigen-presenting cells (APCs). However, both pathways show distinct preferences concerning antigen stability. Whereas direct presentation was shown to efficiently present peptides derived from rapidly degraded proteins, cross-presentation is dependent on long-lived antigen species. In this report, we analyzed the role of antigen stability on DNA vaccination and recombinant vaccinia virus (VV) infection using altered versions of the same antigen. The long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) can be targeted for degradation by N-terminal fusion to ubiquitin or, as we show here, to the ubiquitin-like modifier FAT10. Direct presentation by cells either transfected with NP-encoding plasmids or infected with recombinant VV in vitro was enhanced in the presence of short-lived antigens. In vivo, however, the highest induction of NP-specific CD8(+) T-cell responses was achieved in the presence of long-lived NP. Our experiments provide evidence that targeting antigens for proteasomal degradation does not improve the immunogenicity of DNA vaccines and recombinant VVs. Rather, it is the long-lived antigen that is superior for the efficient activation of MHC class I-restricted immune responses in vivo. Hence, our results suggest a dominant role for antigen cross-priming in DNA vaccination and recombinant VV infection.

  8. Inhibition of Hirame rhabdovirus growth by RNA aptamers.

    PubMed

    Hwang, S D; Midorikawa, N; Punnarak, P; Kikuchi, Y; Kondo, H; Hirono, I; Aoki, T

    2012-12-01

    RNA aptamers are artificial nucleic acids that specifically bind to a wide variety of targets. They are an effective tool for pharmaceutical research and development of antiviral agents. Here, we describe four Hirame rhabdovirus (HIRRV)-RNA aptamers (H1, H2, H3 and H4) that we obtained from an in vitro process called the systematic evolution of ligands by exponential enrichment (SELEX). The HIRRV-RNA aptamers specifically bind to HIRRV. Hirame natural embryo (HINAE) cells treated with virus and the RNA aptamer showed a decrease in appearance of cytopathic effect when compared with control (treated only with virus). Rhodovulum sulfidophilum was transformed with genes for the RNA aptamers, and the aptamers were detected in the culture medium, indicating that they were secreted from the cells. Thus, the recombinant R. sulfidophilum might be a powerful tool for the prevention of HIRRV in aquaculture.

  9. Characterization of structural proteins of hirame rhabdovirus, HRV

    USGS Publications Warehouse

    Nishizawa, Toyohiko; Yoshimizu, Mamoru; Winton, James; Ahne, Winfried; Kimura, Takahisa

    1991-01-01

    Structural proteins of hirame rhabdovirus (HRV) were analyzed by SDS-polyacrylarnide gel electrophoresis, western blotting, 2-dimensional gel electrophoresis, and Triton X-100 treatment. Purified HRV virions were composed of: polymerase (L), glycoprotein (G), nucleoprotein (N), and 2 matrix proteins (M1 and M2). Based upon their relative mobilities, the estimated molecular weights of the proteins were: L, 156 KDa; G, 68 KDa; N, 46.4 KDa; M1, 26.4 KDa; and M2, 19.9 KDa. The electrophorehc pattern formed by the structural proteins of HRV was clearly different from that formed by pike fry rhabdovirus, spring viremia of carp virus, eel virus of America, and eel virus European X which belong to the Vesiculovirus genus; however, it resembled the pattern formed by structural proteins of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) which are members of the Lyssavirus genus. Among HRV, IHNV, and VHSV, differences were observed in the relative mobilities of the G, N, M1, and M2 proteins. Western blot analysis revealed that the G. N, and M2 proteins of HRV shared antigenic determinants with IHNV and VHSV, but not with any of the 4 fish vesiculoviruses tested. Cross-reactions between the M1 proteins of HRV, IHNV, or VHSV were not detected in this assay. Two-dimensional gel electrophoresis was used to show that HRV differed from IHNV or VHSV in the isoelectric point (PI) of the M1 and M2 proteins. In this system, 2 forms of the M1 protein of HRV and IHNV were observed.These subspecies of M1 had the same relative mobility but different p1 values. Treatment of purified virions with 2% Triton X-100 in Tris buffer containing NaCl removed the G, M1, and M2 proteins of IHNV, but HRV virions were more stable under these conditions.

  10. [Expression and immunity of multi-HIV B'/C subype genes in replicating DNA vaccines].

    PubMed

    Gao, Ying-ying; Deng, Yao; Qi, Xiang-rong; Zhang, Xiang-min; Meng, Xin; Wang, Hui-juan; Tan, Wen-jie; Ruan, Li

    2010-05-01

    To understand the effect of various gene structures of HIV B'/C subtype on the gene expression and immunity in DNA vaccine, replicating DNA vector pSCK2 was used to construct seven DNA vaccines carrying one or more of HIV B'/C subtype genes: gagpol, gp160 and rtn (rev, tat and nef fusion gene). Immunofluorescence staining indicated that Gag, Gp160, Rev, Tat and Nef could be expressed from the seven DNA vaccines. Stronger expression was observed with the gene in single-gene expression plasmid or with the gene located at upper-IRES in double- or multi-gene expression plasmid. ELISA test showed that Gag induced higher antibody response, but the antibody titers stimulated by Gp160, Pol, or RTN were very low. Both Gag single-gene expression plasmid and Gag-RTN double-gene expression plasmid separately inoculating induced stronger antibody response against Gag than Gag-Gp160 double-gene expression plasmid and Gagpol-Gp160-RTN multi-gene expression plasmid or combined inoculation of Gag and Gp160 single-gene expression plasmids did. ELISPOT detection showed that all the seven DNA vaccines could stimulate cellular immune response against Gag, Pol, Gp160, Tat, and Nef, respectively. Gagpol or Gp160 single-gene expression plasmid separately inoculating stimulated the strongest cellular immune response. Tat and Nef expressed in all the plasmids induced similar immune response. These results indicated that HIV B'/C subtype genes gagpol, gp160 and rtn could be efficiently expressed in the replicating DNA vaccine vector, single-gene expression plasmid had the higher gene expression level and induced stronger immune response; combined immunization of Gagpol and Gp160 had dramatically lower immunity than Gagpol or Gp160 separated immunization did. Immunity of RTN had no difference between combined and separated immunizations. Therefore, in case of immunization with DNA vaccines containing different HIV genes, it is necessary to optimize the combined immunization procedure

  11. A cytomegalovirus DNA vaccine induces antibodies that block viral entry into fibroblasts and epithelial cells.

    PubMed

    McVoy, Michael A; Lee, Ronzo; Saccoccio, Frances M; Hartikka, Jukka; Smith, Larry R; Mahajan, Rohit; Wang, Jian Ben; Cui, Xiaohong; Adler, Stuart P

    2015-12-16

    A vaccine to prevent congenital cytomegalovirus (CMV) infections is a national priority. Investigational vaccines have targeted the viral glycoprotein B (gB) as an inducer of neutralizing antibodies and phosphoprotein 65 (pp65) as an inducer of cytotoxic T cells. Antibodies to gB neutralize CMV entry into all cell types but their potency is low compared to antibodies that block epithelial cell entry through targeting the pentameric complex (gH/gL/UL128/UL130/UL131). Hence, more potent overall neutralizing responses may result from a vaccine that combines gB with pentameric complex-derived antigens. To assess the ability of pentameric complex subunits to generate epithelial entry neutralizing antibodies, DNA vaccines encoding UL128, UL130, and/or UL131 were formulated with Vaxfectin(®), an adjuvant that enhances antibody responses to DNA vaccines. Mice were immunized with individual DNA vaccines or with pair-wise or trivalent combinations. Only the UL130 vaccine induced epithelial entry neutralizing antibodies and no synergy was observed from bi- or trivalent combinations. In rabbits the UL130 vaccine again induced epithelial entry neutralizing antibodies while UL128 or UL131 vaccines did not. To evaluate compatibility of the UL130 vaccine with DNA vaccines encoding gB or pp65, mono-, bi-, or trivalent combinations were evaluated. Fibroblast and epithelial entry neutralizing titers did not differ between rabbits immunized with gB alone vs. gB/UL130, gB/pp65, or gB/UL130/pp65 combinations, indicating a lack of antagonism from coadministration of DNA vaccines. Importantly, gB-induced epithelial entry neutralizing titers were substantially higher than activities induced by UL130, and both fibroblast and epithelial entry neutralizing titers induced by gB alone as well as gB/pp65 or gB/UL130/pp65 combinations were comparable to those observed in sera from humans with naturally-acquired CMV infections. These findings support further development of Vaxfectin

  12. Quantitative real-time PCR study on persistence of pDNA vaccine pVax-Hsp60 TM814 in beef muscles.

    PubMed

    Orság, Petr; Kvardová, Veronika; Raska, Milan; Miller, Andrew D; Ledvina, Miroslav; Turánek, Jaroslav

    2008-09-02

    Application of plasmid DNA for immunization of food-producing animals established new standards of food safety. The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects. A quantitative real-time PCR (QRTPCR) methodology was developed to study the biodistribution and persistence of plasmid DNA vaccine pDNAX (pVAX-Hsp60 TM814) in mice and beef cattle. The linear quantification range and the sensitivity of the method was found to be 10 - 10(9) copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively. Persistence of pDNAX in mice muscle tissue was restricted to injection site and the amount of pDNAX showed delivery formulation dependent (naked pDNA, electroporation, cationic liposome complexes) and mouse age-dependent clearance form injection site but pDNAX was still detectable even after 365 days. The QRTPCR analysis of various muscle tissue samples of vaccinated beef bulls performed 242-292 days after the last revaccination proved that residual pDNAX was found only in the injection site. The highest plasmid levels (up to 290 copies per reaction) were detected in the pDNAX:CDAN/DOPE group similarly to mice model. No pDNA was detected in the samples from distant muscles and draining lymph nodes. Quantitative real-time PCR (QRTPCR) assay was developed to assess the residual pDNA vaccine pVAX-Hsp60 TM814 in mice and beef cattle. In beef cattle, ultra low residual level of pDNA vaccine was only found at the injection site. According to rough estimation, consumption of muscles from the injection site represents almost an undetectable intake of pDNA (400 fg/g muscle tissue) for consumers. Residual plasmid in native state will hardly be found at measurable level following further meat processing. This study brings supportive data for animal and food safety and hence for further approval of pDNA vaccine field trials.

  13. Quantitative real-time PCR study on persistence of pDNA vaccine pVax-Hsp60 TM814 in beef muscles

    PubMed Central

    Orság, Petr; Kvardová, Veronika; Raška, Milan; Miller, Andrew D; Ledvina, Miroslav; Turánek, Jaroslav

    2008-01-01

    Background Application of plasmid DNA for immunization of food-producing animals established new standards of food safety. The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects. Methods A quantitative real-time PCR (QRTPCR) methodology was developed to study the biodistribution and persistence of plasmid DNA vaccine pDNAX (pVAX-Hsp60 TM814) in mice and beef cattle. The linear quantification range and the sensitivity of the method was found to be 10 – 109 copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively. Results Persistence of pDNAX in mice muscle tissue was restricted to injection site and the amount of pDNAX showed delivery formulation dependent (naked pDNA, electroporation, cationic liposome complexes) and mouse age-dependent clearance form injection site but pDNAX was still detectable even after 365 days. The QRTPCR analysis of various muscle tissue samples of vaccinated beef bulls performed 242–292 days after the last revaccination proved that residual pDNAX was found only in the injection site. The highest plasmid levels (up to 290 copies per reaction) were detected in the pDNAX:CDAN/DOPE group similarly to mice model. No pDNA was detected in the samples from distant muscles and draining lymph nodes. Conclusion Quantitative real-time PCR (QRTPCR) assay was developed to assess the residual pDNA vaccine pVAX-Hsp60 TM814 in mice and beef cattle. In beef cattle, ultra low residual level of pDNA vaccine was only found at the injection site. According to rough estimation, consumption of muscles from the injection site represents almost an undetectable intake of pDNA (400 fg/g muscle tissue) for consumers. Residual plasmid in native state will hardly be found at measurable level following further meat processing. This study brings supportive data for animal and food safety and hence for further

  14. DNA Vaccines Encoding Antigen Targeted to MHC Class II Induce Influenza-Specific CD8+ T Cell Responses, Enabling Faster Resolution of Influenza Disease

    PubMed Central

    Lambert, Laura; Kinnear, Ekaterina; McDonald, Jacqueline U.; Grodeland, Gunnveig; Bogen, Bjarne; Stubsrud, Elisabeth; Lindeberg, Mona M.; Fredriksen, Agnete Brunsvik; Tregoning, John S.

    2016-01-01

    Current influenza vaccines are effective but imperfect, failing to cover against emerging strains of virus and requiring seasonal administration to protect against new strains. A key step to improving influenza vaccines is to improve our understanding of vaccine-induced protection. While it is clear that antibodies play a protective role, vaccine-induced CD8+ T cells can improve protection. To further explore the role of CD8+ T cells, we used a DNA vaccine that encodes antigen dimerized to an immune cell targeting module. Immunizing CB6F1 mice with the DNA vaccine in a heterologous prime-boost regime with the seasonal protein vaccine improved the resolution of influenza disease compared with protein alone. This improved disease resolution was dependent on CD8+ T cells. However, DNA vaccine regimes that induced CD8+ T cells alone were not protective and did not boost the protection provided by protein. The MHC-targeting module used was an anti-I-Ed single chain antibody specific to the BALB/c strain of mice. To test the role of MHC targeting, we compared the response between BALB/c, C57BL/6 mice, and an F1 cross of the two strains (CB6F1). BALB/c mice were protected, C57BL/6 were not, and the F1 had an intermediate phenotype; showing that the targeting of antigen is important in the response. Based on these findings, and in agreement with other studies using different vaccines, we conclude that, in addition to antibody, inducing a protective CD8 response is important in future influenza vaccines. PMID:27602032

  15. Suppressive Effects on the Immune Response and Protective Immunity to a JEV DNA Vaccine by Co-administration of a GM-CSF-Expressing Plasmid in Mice

    PubMed Central

    Chen, Hui; Gao, Na; Fan, Dongying; Wu, Jiangman; Zhu, Junping; Li, Jieqiong; Wang, Juan; Chen, Yanlei; An, Jing

    2012-01-01

    As a potential cytokine adjuvant of DNA vaccines, granulocyte-macrophage colony–stimulating factor (GM-CSF) has received considerable attention due to its essential role in the recruitment of antigen-presenting cells, differentiation and maturation of dendritic cells. However, in our recent study of a Japanese encephalitis virus (JEV) DNA vaccine, co-inoculation of a GM-CSF plasmid dramatically suppressed the specific IgG response and resulted in decreased protection against JEV challenge. It is known that GM-CSF has been used in clinic to treat neutropenia for repopulating myeloid cells, and as an adjuvant in vaccine studies; it has shown various effects on the immune response. Therefore, in this study, we characterized the suppressive effects on the immune response to a JEV DNA vaccine by the co-administration of the GM-CSF-expressing plasmid and clarified the underlying mechanisms of the suppression in mice. Our results demonstrated that co-immunization with GM-CSF caused a substantial dampening of the vaccine-induced antibody responses. The suppressive effect was dose- and timing-dependent and likely related to the immunogenicity of the antigen. The suppression was associated with the induction of immature dendritic cells and the expansion of regulatory T cells but not myeloid-derived suppressor cells. Collectively, our findings not only provide valuable information for the application of GM-CSF in clinic and using as a vaccine adjuvant but also offer further insight into the understanding of the complex roles of GM-CSF. PMID:22493704

  16. 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.

  17. Adjunctive immunotherapy with α-crystallin based DNA vaccination reduces Tuberculosis chemotherapy period in chronically infected mice.

    PubMed

    Chauhan, Priyanka; Jain, Ruchi; Dey, Bappaditya; Tyagi, Anil K

    2013-01-01

    By employing modified Cornell model, we have evaluated the potential of adjunctive immunotherapy with DNA vaccines to shorten the tuberculosis chemotherapy period and reduce disease reactivation. We demonstrate that α-crystallin based DNA vaccine (DNAacr) significantly reduced the chemotherapy period from 12 weeks to 8 weeks when compared with the chemotherapy alone. Immunotherapy with SodA based DNA vaccine (DNAsod) reduced the pulmonary bacilli only as much as DNAvec. Both DNAacr and DNAsod, although significantly delayed the reactivation in comparison to the chemotherapy alone, this delay was associated with the immunostimulatory sequences present in the vector backbone and was not antigen specific. Both DNA vaccines resulted in the production of significantly higher number of TEM cells than the chemotherapy alone, however, only in the case of DNAsod, this enhancement was significant over the DNAvec treatment. Overall, our findings emphasize the immunotherapeutic potential of DNAacr in shortening the duration of TB chemotherapy.

  18. DNA vaccination protects mice against Zika virus-induced damage to the testes.

    PubMed

    Griffin, Bryan D; Muthumani, Kar; Warner, Bryce M; Majer, Anna; Hagan, Mable; Audet, Jonathan; Stein, Derek R; Ranadheera, Charlene; Racine, Trina; De La Vega, Marc-Antoine; Piret, Jocelyne; Kucas, Stephanie; Tran, Kaylie N; Frost, Kathy L; De Graff, Christine; Soule, Geoff; Scharikow, Leanne; Scott, Jennifer; McTavish, Gordon; Smid, Valerie; Park, Young K; Maslow, Joel N; Sardesai, Niranjan Y; Kim, J Joseph; Yao, Xiao-Jian; Bello, Alexander; Lindsay, Robbin; Boivin, Guy; Booth, Stephanie A; Kobasa, Darwyn; Embury-Hyatt, Carissa; Safronetz, David; Weiner, David B; Kobinger, Gary P

    2017-06-07

    Zika virus (ZIKV) is an emerging pathogen causally associated with serious sequelae in fetuses, inducing fetal microcephaly and other neurodevelopment defects. ZIKV is primarily transmitted by mosquitoes, but can persist in human semen and sperm, and sexual transmission has been documented. Moreover, exposure of type-I interferon knockout mice to ZIKV results in severe damage to the testes, epididymis and sperm. Candidate ZIKV vaccines have shown protective efficacy in preclinical studies carried out in animal models, and several vaccines have entered clinical trials. Here, we report that administration of a synthetic DNA vaccine encoding ZIKV pre-membrane and envelope (prME) completely protects mice against ZIKV-associated damage to the testes and sperm and prevents viral persistence in the testes following challenge with a contemporary strain of ZIKV. These data suggest that DNA vaccination merits further investigation as a potential means to reduce ZIKV persistence in the male reproductive tract.

  19. DNA vaccination protects mice against Zika virus-induced damage to the testes

    PubMed Central

    Griffin, Bryan D.; Muthumani, Kar; Warner, Bryce M.; Majer, Anna; Hagan, Mable; Audet, Jonathan; Stein, Derek R.; Ranadheera, Charlene; Racine, Trina; De La Vega, Marc-Antoine; Piret, Jocelyne; Kucas, Stephanie; Tran, Kaylie N.; Frost, Kathy L.; De Graff, Christine; Soule, Geoff; Scharikow, Leanne; Scott, Jennifer; McTavish, Gordon; Smid, Valerie; Park, Young K.; Maslow, Joel N.; Sardesai, Niranjan Y.; Kim, J. Joseph; Yao, Xiao-jian; Bello, Alexander; Lindsay, Robbin; Boivin, Guy; Booth, Stephanie A.; Kobasa, Darwyn; Embury-Hyatt, Carissa; Safronetz, David; Weiner, David B.; Kobinger, Gary P.

    2017-01-01

    Zika virus (ZIKV) is an emerging pathogen causally associated with serious sequelae in fetuses, inducing fetal microcephaly and other neurodevelopment defects. ZIKV is primarily transmitted by mosquitoes, but can persist in human semen and sperm, and sexual transmission has been documented. Moreover, exposure of type-I interferon knockout mice to ZIKV results in severe damage to the testes, epididymis and sperm. Candidate ZIKV vaccines have shown protective efficacy in preclinical studies carried out in animal models, and several vaccines have entered clinical trials. Here, we report that administration of a synthetic DNA vaccine encoding ZIKV pre-membrane and envelope (prME) completely protects mice against ZIKV-associated damage to the testes and sperm and prevents viral persistence in the testes following challenge with a contemporary strain of ZIKV. These data suggest that DNA vaccination merits further investigation as a potential means to reduce ZIKV persistence in the male reproductive tract. PMID:28589934

  20. Improvement influenza HA2 DNA vaccine cellular and humoral immune responses with Mx bio adjuvant.

    PubMed

    Soleimani, Sina; Shahsavandi, Shahla; Maddadgar, Omid

    2017-03-01

    Immunization with DNA vaccines as a novel alternative to conventional vaccination strategy requires adjuvant for improving vaccine efficacy. The conserved immunogenic HA2 subunit, which harbors neutralizing epitopes is a promising vaccine candidate against influenza viruses. In this study, for the first time we explore the idea of using host interferon inducible Mx protein to increase the immunogenicity of HA2 H9N2 influenza DNA vaccine. The potency and safety of the Mx adjuvanted-HA2 vaccine was evaluated in BALB/c mice by different prime-boost strategies. To assess the effect of the vaccination on the virus clearance rate, mice were challenged with homologous influenza virus. Administration of the adjuvanted vaccine and boosting with the same regimen could effectively enhance both humoral and cellular immune responses in treated mice. These data demonstrated that Mx as host defense peptide can be potentiated for improving influenza vaccine efficacy.

  1. Approaches towards the development of a vaccine against tuberculosis: recombinant BCG and DNA vaccine.

    PubMed

    Nor, Norazmi Mohd; Musa, Mustaffa

    2004-01-01

    The last few years have witnessed intense research on vaccine development against tuberculosis. This has been driven by the upsurge of tuberculosis cases globally, especially those caused by multi-drug-resistant Mycobacterium tuberculosis strains. Various vaccine strategies are currently being developed which can be broadly divided into the so-called living and non-living vaccines. Examples are attenuated members of the M. tuberculosis complex, recombinant mycobacteria, subunit proteins and DNA vaccines. Given current developments, we anticipate that recombinant BCG and DNA vaccines are the most promising. Multiple epitopes of M. tuberculosis may need to be cloned in a vaccine construct for the desired efficacy to be achieved. The technique of assembly polymerase chain reaction could facilitate such a cloning procedure.

  2. A rapid and potent DNA vaccination strategy defined by in vivo monitoring of antigen expression.

    PubMed

    Bins, Adriaan D; Jorritsma, Annelies; Wolkers, Monika C; Hung, Chien-Fu; Wu, T-C; Schumacher, Ton N M; Haanen, John B A G

    2005-08-01

    Induction of immunity after DNA vaccination is generally considered a slow process. Here we show that DNA delivery to the skin results in a highly transient pulse of antigen expression. Based on this information, we developed a new rapid and potent intradermal DNA vaccination method. By short-interval intradermal DNA delivery, robust T-cell responses, of a magnitude sufficient to reject established subcutaneous tumors, are generated within 12 d. Moreover, this vaccination strategy confers protecting humoral immunity against influenza A infection within 2 weeks after the start of vaccination. The strength and speed of this newly developed strategy will be beneficial in situations in which immunity is required in the shortest possible time.

  3. Design and Construction of Shrimp Antiviral DNA Vaccines Expressing Long and Short Hairpins for Protection by RNA Interference.

    PubMed

    Chaudhari, Aparna; Pathakota, Gireesh-Babu; Annam, Pavan-Kumar

    2016-01-01

    DNA vaccines present the aquaculture industry with an effective and economically viable method of controlling viral pathogens that drastically affect productivity. Since specific immune response is rudimentary in invertebrates, the presence of RNA interference (RNAi) pathway in shrimps provides a promising new approach to vaccination. Plasmid DNA vaccines that express short or long double stranded RNA in vivo have shown protection against viral diseases. The design, construction and considerations for preparing such vaccines are discussed.

  4. 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

  5. Construction and Nonclinical Testing of a Puumala Virus Synthetic M Gene-Based DNA Vaccine

    DTIC Science & Technology

    2012-12-12

    nogenicity in hamsters, and the results indicated that there was no antibody response, even after five vaccinations delivered by gene gun (data not...infective doses [ID50]) (data not shown). In an earlier study , it was determined that the ANDV M gene-based DNA vaccine failed to elicit a de...data indicated that improve- ments in the quality of the immune response, i.e., the neutralizing antibody response, were needed. Synthetic, mammalian

  6. A comparative approach between heterologous prime-boost vaccination strategy and DNA vaccinations for rabies.

    PubMed

    Borhani, Kiandokht; Ajorloo, Mehdi; Bamdad, Taravat; Mozhgani, Sayed Hamid Reza; Ghaderi, Mostafa; Gholami, Ali Reza

    2015-04-01

    Rabies is a widespread neurological zoonotic disease causing significant mortality rates, especially in developing countries. Although a vaccine for rabies is available, its production and scheduling are costly in such countries. Advances in recombinant DNA technology have made it a good candidate for an affordable vaccine. Among the proteins of rabies virus, the Glycoprotein (RVG) has been the major target for new vaccine development which plays the principal role in providing complete protection against RV challenge. The aim of this study is to produce recombinant RVG which could be a DNA vaccine candidate and to evaluate the efficiency of this construct in a prime-boost vaccination regimen, compared to commercial vaccine. Cloning to pcDNA3.1(+) and expression of rabies virus glycoprotein gene in BSR cell  line were performed followed by SDS-PAGE and Western blot analysis of the expressed glycoprotein. The resulting genetic construct was used as a DNA vaccine by injecting 80 µg of the plasmid to MNRI mice twice. Prime-Boost vaccination strategy was performed using 80 µg plasmid construct as prime dose and the second dose of an inactivated rabies virus vaccine. Production of rabies virus neutralizing antibody (RVNA) titers of the serum samples were determined by RFFIT. In comparisons between heterologous prime-boost vaccination strategy and DNA vaccinations, the potency of group D that received Prime-Boost vaccine with the second dose of pcDNA3.1(+)-Gp was enhanced significantly compared to the group C which had received pcDNA3.1(+)-Gp as first injection. In this study, RVGP expressing construct was used in a comparative approach between Prime-Boost vaccination strategy and DNA vaccination and compared with the standard method of rabies vaccination. It was concluded that this strategy could lead to induction of acceptable humoral immunity.

  7. Selection and identification of malaria vaccine target molecule using bioinformatics and DNA vaccination.

    PubMed

    Shuaibu, M N; Kikuchi, M; Cherif, M S; Helegbe, G K; Yanagi, T; Hirayama, K

    2010-10-04

    Following a genome-wide search for a blood stage malaria DNA-based vaccine using web-based bioinformatic tools, 29 genes from the annotated Plasmodium yoelii genome sequence (www.PlasmoDB.org and www.tigr.org) were identified as encoding GPI-anchored proteins. Target genes were those with orthologues in P. falciparum, containing an N-terminal signal sequence containing hydrophobic amino acid stretch and signal P criteria, a transmembrane-like domain and GPI anchor motif. Focusing on the blood stage, we extracted mRNA from pRBCs, PCR-amplified 22 out of the 29 selected genes, and eventually cloned nine of these into a DNA vaccine plasmid, pVAX 200-DEST. Biojector-mediated delivery of the nine DNA vaccines was conducted using ShimaJET to C57BL/6 mice at a dose of 4 μg/mouse three times at an interval of 3 weeks. Two weeks after the second booster, immunized mice were challenged with P. y. yoelii 17XL-parasitized RBCs and the level of parasitaemia, protection and survival was assessed. Immunization with one gene (PY03470) resulted in 2-4 days of delayed onset and level of parasitaemia and was associated with increased survival compared to non-immunized mice. Antibody production was, however, low following DNA vaccination, as determined by immunofluorescence assay. Recombinant protein from this gene, GPI8p transamidase-related protein (rPyTAM) in PBS or emulsified with GERBU adjuvant was also used to immunize another set of C57BL/6 mice with 10-20 μg/mouse three times at 3-week interval. Higher antibody response was obtained as determined by ELISA with similar protective effects as observed after DNA vaccination.

  8. Dendritic Cell Targeting Effectively Boosts T Cell Responses Elicited by an HIV Multiepitope DNA Vaccine

    PubMed Central

    Apostólico, Juliana de Souza; Lunardelli, Victória Alves Santos; Yamamoto, Marcio Massao; Souza, Higo Fernando Santos; Cunha-Neto, Edecio; Boscardin, Silvia Beatriz; Rosa, Daniela Santoro

    2017-01-01

    Despite several efforts in the last decades, an efficacious HIV-1 vaccine is still not available. Different approaches have been evaluated, such as recombinant proteins, viral vectors, DNA vaccines, and, most recently, dendritic cell (DC) targeting. This strategy is based on DC features that place them as central for induction of immunity. Targeting is accomplished by the use of chimeric monoclonal antibodies directed to DC surface receptors fused to the antigen of interest. In this work, we targeted eight promiscuous HIV-derived CD4+ T cell epitopes (HIVBr8) to the DEC205+ DCs by fusing the multiepitope immunogen to the heavy chain of αDEC205 (αDECHIVBr8), in the presence of the TLR3 agonist poly (I:C). In addition, we tested a DNA vaccine encoding the same epitopes using homologous or heterologous prime-boost regimens. Our results showed that mice immunized with αDECHIVBr8 presented higher CD4+ and CD8+ T cell responses when compared to mice that received the DNA vaccine (pVAXHIVBr8). In addition, pVAXHIVBr8 priming followed by αDECHIVBr8 boosting induced higher polyfunctional proliferative and cytokine-producing T cell responses to HIV-1 peptides than homologous DNA immunization or heterologous αDEC prime/DNA boost. Based on these results, we conclude that homologous prime-boost and heterologous boosting immunization strategies targeting CD4+ epitopes to DCs are effective to improve HIV-specific cellular immune responses when compared to standalone DNA immunization. Moreover, our results indicate that antigen targeting to DC is an efficient strategy to boost immunity against a multiepitope immunogen, especially in the context of DNA vaccination. PMID:28223987

  9. Molecular adjuvants for malaria DNA vaccines based on the modulation of host-cell apoptosis.

    PubMed

    Bergmann-Leitner, Elke S; Leitner, Wolfgang W; Duncan, Elizabeth H; Savranskaya, Tatyana; Angov, Evelina

    2009-09-18

    Malaria represents a major global health problem but despite extensive efforts, no effective vaccine is available. Various vaccine candidates have been developed that provide protection in animal models, such as a gene gun-delivered DNA vaccine encoding the circumsporozoite protein (CSP) of Plasmodium berghei. A common shortcoming of most malaria vaccines is the requirement for multiple immunizations leaving room for improvement even for established vaccine candidates such as the CSP-DNA vaccine. In this study, we explored whether regulating apoptosis in DNA vaccine transfected host cells could accelerate the onset of protective immunity and provide significant protection after a single immunization. A pro-apoptotic gene (Bax) was used as a molecular adjuvant in an attempt to mimic the immunostimulatory apoptosis triggered by viral or virus-derived vaccines, while anti-apoptotic genes such as Bcl-XL may increase the life span of transfected cells thus prolonging antigen production. Surprisingly, co-delivery of either Bax or Bcl-XL greatly reduced CSP-DNA vaccine efficacy after a single immunization. Co-delivery of Bax for three immunizations still had a detrimental effect on protective immunity, while repeated co-delivery of Bcl-XL had no negative impact. The fine characterization of humoral and cellular immune response modulated by these two molecular adjuvants revealed a previously unknown effect, i.e., a shift in the Th-profile. These results demonstrate that pro- or anti-apoptotic molecules should not be used as molecular adjuvants without careful evaluation of the resulting immune response. This finding represents yet another example that strategies to enhance vaccine efficacy developed for other model systems such as viral diseases cannot easily be applied to any vaccine.

  10. Saccharomyces boulardii improves humoral immune response to DNA vaccines against leptospirosis.

    PubMed

    Silveira, Marcelle Moura; Conceição, Fabricio Rochedo; Mendonça, Marcelo; Moreira, Gustavo Marçal Schmidt Garcia; Da Cunha, Carlos Eduardo Pouey; Conrad, Neida Lucia; Oliveira, Patrícia Diaz de; Hartwig, Daiane Drawanz; De Leon, Priscila Marques Moura; Moreira, Ângela Nunes

    2017-02-01

    Saccharomyces boulardii may improve the immune response by enhancing the production of anti-inflammatory cytokines, T-cell proliferation and dendritic cell activation. The immunomodulator effect of this probiotic has never been tested with DNA vaccines, which frequently induce low antibody titers. This study evaluated the capacity of Saccharomyces boulardii to improve the humoral and cellular immune responses using DNA vaccines coding for the leptospiral protein fragments LigAni and LigBrep. BALB/c mice were fed with rodent-specific feed containing 108 c.f.u. of Saccharomycesboulardii per gram. Animals were immunized three times intramuscularly with 100 µg of pTARGET plasmids containing the coding sequences for the above mentioned proteins. Antibody titers were measured by indirect ELISA. Expression levels of IL-4, IL-10, IL-12, IL-17, IFN-γ and TGF-β were determined by quantitative real-time PCR from RNA extracted from whole blood, after an intraperitoneal boost with 50 µg of the recombinant proteins.Results/Key findings. Antibody titers increased significantly after the second and third application when pTARGET/ligAni and pTARGET/ligBrep were used to vaccinate the animals in comparison with the control group (P<0.05). In addition, there was a significant increase in the expression of the IL-10 in mice immunized with pTARGET/ligBrep and fed with Saccharomyces boulardii. The results suggested that Saccharomyces boulardii has an immunomodulator effect in DNA vaccines, mainly by stimulating the humoral response, which is often limited in this kind of vaccine. Therefore, the use of Saccharomyces boulardii as immunomodulator represents a new alternative strategy for more efficient DNA vaccination.

  11. Dendritic Cell Targeting Effectively Boosts T Cell Responses Elicited by an HIV Multiepitope DNA Vaccine.

    PubMed

    Apostólico, Juliana de Souza; Lunardelli, Victória Alves Santos; Yamamoto, Marcio Massao; Souza, Higo Fernando Santos; Cunha-Neto, Edecio; Boscardin, Silvia Beatriz; Rosa, Daniela Santoro

    2017-01-01

    Despite several efforts in the last decades, an efficacious HIV-1 vaccine is still not available. Different approaches have been evaluated, such as recombinant proteins, viral vectors, DNA vaccines, and, most recently, dendritic cell (DC) targeting. This strategy is based on DC features that place them as central for induction of immunity. Targeting is accomplished by the use of chimeric monoclonal antibodies directed to DC surface receptors fused to the antigen of interest. In this work, we targeted eight promiscuous HIV-derived CD4(+) T cell epitopes (HIVBr8) to the DEC205(+) DCs by fusing the multiepitope immunogen to the heavy chain of αDEC205 (αDECHIVBr8), in the presence of the TLR3 agonist poly (I:C). In addition, we tested a DNA vaccine encoding the same epitopes using homologous or heterologous prime-boost regimens. Our results showed that mice immunized with αDECHIVBr8 presented higher CD4(+) and CD8(+) T cell responses when compared to mice that received the DNA vaccine (pVAXHIVBr8). In addition, pVAXHIVBr8 priming followed by αDECHIVBr8 boosting induced higher polyfunctional proliferative and cytokine-producing T cell responses to HIV-1 peptides than homologous DNA immunization or heterologous αDEC prime/DNA boost. Based on these results, we conclude that homologous prime-boost and heterologous boosting immunization strategies targeting CD4(+) epitopes to DCs are effective to improve HIV-specific cellular immune responses when compared to standalone DNA immunization. Moreover, our results indicate that antigen targeting to DC is an efficient strategy to boost immunity against a multiepitope immunogen, especially in the context of DNA vaccination.

  12. Polyclonal antibody cocktails generated using DNA vaccine technology protect in murine models of orthopoxvirus disease

    PubMed Central

    2011-01-01

    Background Previously we demonstrated that DNA vaccination of nonhuman primates (NHP) with a small subset of vaccinia virus (VACV) immunogens (L1, A27, A33, B5) protects against lethal monkeypox virus challenge. The L1 and A27 components of this vaccine target the mature virion (MV) whereas A33 and B5 target the enveloped virion (EV). Results Here, we demonstrated that the antibodies produced in vaccinated NHPs were sufficient to confer protection in a murine model of lethal Orthopoxvirus infection. We further explored the concept of using DNA vaccine technology to produce immunogen-specific polyclonal antibodies that could then be combined into cocktails as potential immunoprophylactic/therapeutics. Specifically, we used DNA vaccines delivered by muscle electroporation to produce polyclonal antibodies against the L1, A27, A33, and B5 in New Zealand white rabbits. The polyclonal antibodies neutralized both MV and EV in cell culture. The ability of antibody cocktails consisting of anti-MV, anti-EV, or a combination of anti-MV/EV to protect BALB/c mice was evaluated as was the efficacy of the anti-MV/EV mixture in a mouse model of progressive vaccinia. In addition to evaluating weight loss and lethality, bioimaging technology was used to characterize the spread of the VACV infections in mice. We found that the anti-EV cocktail, but not the anti-MV cocktail, limited virus spread and lethality. Conclusions A combination of anti-MV/EV antibodies was significantly more protective than anti-EV antibodies alone. These data suggest that DNA vaccine technology could be used to produce a polyclonal antibody cocktail as a possible product to replace vaccinia immune globulin. PMID:21933385

  13. A novel non-integrative single-cycle chimeric HIV lentivector DNA vaccine.

    PubMed

    Moussa, Maha; Arrode-Brusés, Géraldine; Manoylov, Iliyan; Malogolovkin, Alexander; Mompelat, Dimitri; Ishimwe, Honorine; Smaoune, Amel; Ouzrout, Bilel; Gagnon, Jean; Chebloune, Yahia

    2015-05-05

    Novel HIV vaccine vectors and strategies are needed to control HIV/AIDS epidemic in humans and eradicate the infection. DNA vaccines alone failed to induce immune responses robust enough to control HIV-1. Development of lentivirus-based DNA vaccines deficient for integration and with a limited replication capacity is an innovative and promising approach. This type of vaccine mimics the early stages of virus infection/replication like the live-attenuated viruses but lacks the inconvenient integration and persistence associated with disease. We developed a novel lentivector DNA vaccine "CAL-SHIV-IN(-)" that undergoes a single round of replication in the absence of integration resulting in augmented expression of vaccine antigens in vivo. Vaccine gene expression is under control of the LTRs of a naturally attenuated lentivirus, Caprine arthritis encephalitis virus (CAEV) the natural goat lentivirus. The safety of this vaccine prototype was increased by the removal of the integrase coding sequences from the pol gene. We examined the functional properties of this lentivector DNA in cell culture and the immunogenicity in mouse models. Viral proteins were expressed in transfected cells, assembled into viral particles that were able to transduce once target permissive cells. Unlike the parental replication-competent SHIV-KU2 that was detected in DNA samples from any of the serial passage infected cells, CAL-SHIV-IN(-) DNA was detected only in target cells of the first round of infection, hence demonstrating the single cycle replication of the vaccine. A single dose DNA immunization of humanized NOD/SCID/β2 mice showed a substantial increase of IFN-γ-ELISPOT in splenocytes compared to the former replication and integration defective Δ4SHIV-KU2 DNA vaccine.

  14. Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA.

    PubMed

    Xu, Can; Li, Zhao-Shen; Du, Yi-Qi; Tu, Zhen-Xing; Gong, Yan-Fang; Jin, Jing; Wu, Hong-Yu; Xu, Guo-Ming

    2005-01-07

    To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity. Genomic DNA of the standard H pylori strain 17 874 was isolated as the template, hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified hpaA gene was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coli DH5alpha, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-hpaA was used to transform LB5000 and the recombinant plasmid isolated from LB5000 was finally used to transform SL7207. After that, the recombinant strain was grown in vitro repeatedly. In order to identify the immunogenicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using Lipofectamine2000, the immunogenicity of expressed HpaA protein was detected with SDS-PAGE and Western blot. The 750-base pair hpaA gene fragment was amplified from the genomic DNA and was consistent with the sequence of H pylori hpaA by sequence analysis. It was confirmed by PCR and restriction enzyme digestion that H pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying H pylori hpaA gene was successfully constructed and the specific strip of HpaA expressed by pIRES-hpaA was detected through Western blot. The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing HpaA protein with immunogenicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo.

  15. Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA

    PubMed Central

    Xu, Can; Li, Zhao-Shen; Du, Yi-Qi; Tu, Zhen-Xing; Gong, Yan-Fang; Jin, Jing; Wu, Hong-Yu; Xu, Guo-Ming

    2005-01-01

    AIM: To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity. METHODS: Genomic DNA of the standard H pylori strain 17 874 was isolated as the template, hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified hpaA gene was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coli DH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-hpaA was used to transform LB5000 and the recombinant plasmid isolated from LB5000 was finally used to transform SL7207. After that, the recombinant strain was grown in vitro repeatedly. In order to identify the immunogenicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using LipofectamineTM2000, the immunogenicity of expressed HpaA protein was detected with SDS-PAGE and Western blot. RESULTS: The 750-base pair hpaA gene fragment was amplified from the genomic DNA and was consistent with the sequence of H pylori hpaA by sequence analysis. It was confirmed by PCR and restriction enzyme digestion that H pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying H pylori hpaA gene was successfully constructed and the specific strip of HpaA expressed by pIRES-hpaA was detected through Western blot. CONCLUSION: The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing HpaA protein with immunogenicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo. PMID:15609408

  16. Immunization of olive flounder (Paralichthys olivaceus) with an auxotrophic Edwardsiella tarda mutant harboring the VHSV DNA vaccine.

    PubMed

    Choi, Seung Hyuk; Kim, Min Sun; Kim, Ki Hong

    2012-09-01

    The aims of the present study were to find more powerful promoter for DNA vaccines in olive flounder (Paralichthys olivaceus) and to evaluate the availability of the auxotrophic Edwardsiella tarda mutant (Δalr Δasd E. tarda) as a delivery vehicle for DNA vaccine against VHSV in olive flounder. The marine medaka (Oryzias dancena) β-actin promoter was clearly stronger than cytomegalovirus (CMV) promoter when the vectors were transfected to Epithelioma papulosum cyprini (EPC) cells or injected into the muscle of olive flounder, suggesting that marine medaka β-actin promoter would be more appropriate promoter for DNA vaccines in olive flounder than CMV promoter. Olive flounder immunized with the Δalr Δasd E. tarda harboring viral hemorrhagic septicemia virus (VHSV) DNA vaccine vector driven by the marine medaka β-actin promoter showed significantly higher serum neutralization titer and higher survival rates against challenge with VHSV than fish immunized with the bacteria carrying VHSV DNA vaccine vector driven by CMV promoter. These results indicate that auxotrophic E. tarda mutant harboring marine medaka β-actin promoter-driven DNA vaccine vectors would be a potential system for prophylactics of infectious diseases in olive flounder.

  17. Genetic Immunization With In Vivo Dendritic Cell-targeting Liposomal DNA Vaccine Carrier Induces Long-lasting Antitumor Immune Response

    PubMed Central

    Garu, Arup; Moku, Gopikrishna; Gulla, Suresh Kumar; Chaudhuri, Arabinda

    2016-01-01

    A major limiting factor retarding the clinical success of dendritic cell (DC)-based genetic immunizations (DNA vaccination) is the scarcity of biologically safe and effective carrier systems for targeting the antigen-encoded DNA vaccines to DCs under in vivo settings. Herein, we report on a potent, mannose receptor selective in vivo DC-targeting liposomes of a novel cationic amphiphile with mannose-mimicking shikimoyl head-group. Flow cytometric experiments with cells isolated from draining lymph nodes of mice s.c. immunized with lipoplexes of pGFP plasmid (model DNA vaccine) using anti-CD11c antibody-labeled magnetic beads revealed in vivo DC-targeting properties of the presently described liposomal DNA vaccine carrier. Importantly, s.c. immunizations of mice with electrostatic complex of the in vivo DC-targeting liposome and melanoma antigen-encoded DNA vaccine (p-CMV-MART1) induced long-lasting antimelanoma immune response (100 days post melanoma tumor challenge) with remarkable memory response (more than 6 months after the second tumor challenge). The presently described direct in vivo DC-targeting liposomal DNA vaccine carrier is expected to find future exploitations toward designing effective vaccines for various infectious diseases and cancers. PMID:26666450

  18. Hepatitis B DNA vaccine-polycation nano-complexes enhancing immune response by percutaneous administration with microneedle.

    PubMed

    Yin, Dongfeng; Liang, Wenqing; Xing, Shuxing; Gao, Zhixiang; Zhang, Wei; Guo, Zhili; Gao, Shen

    2013-01-01

    Percutaneous immune method is becoming an attractive alternative for DNA vaccine as a lot of antigen presenting cells are existed in the viable epidermis. However, due to the barrier function of stratum corneum, it would be hard for DNA vaccine to reach the viable epidermis of the skin. In order to deliver the DNA vaccine successfully cross the stratum corneum, pentagram silicon microneedle array was prepared in this study, and fluorescently labeled nanoparticle was taken as the model to observe the situation inside the skin processed by microneedle. Via microneedle nanoparticles could enter the skin through the micro-channel (diameter about 20-30 µm) and its amount is greatly larger than that enter though the hair follicle of intact skin. A new type of gene vector Pluronic P123-modified polyethyleneimine (P123-PEI) was synthesized by high molecular weight polyethylenimine and Pluronic P123 with the molar ratio of 1 : 1 to take the advantage of P123-PEI as low cytotoxicity and high transfection efficiency. Mice were immunized percutaneously with Hepatitis B DNA vaccine/P123-PEI nano-complexes by microneedle. The humoral and cellular immunity generated in percutaneously immunized mice through microneedle array by Hepatitis B DNA vaccine/P123-PEI nano-complex was significantly higher than that of DNA vaccine intramuscular administration.

  19. Do uncertainty analyses reveal uncertainties? Using the introduction of DNA vaccines to aquaculture as a case.

    PubMed

    Gillund, Frøydis; Kjølberg, Kamilla A; von Krauss, Martin Krayer; Myhr, Anne I

    2008-12-15

    The Walker and Harremoës (W&H) uncertainty framework is a tool to systematically identify scientific uncertainty. We applied the W&H uncertainty framework to elicit scientists' judgements of potential sources of uncertainty associated with the use of DNA vaccination in aquaculture. DNA vaccination is considered a promising solution to combat pathological fish diseases. There is, however, lack of knowledge regarding its ecological and social implications. Our findings indicate that scientists are open and aware of a number of uncertainties associated with DNA vaccination e.g. with regard to immune response, degradation and distribution of the DNA plasmid after injection and environmental release, and consider most of these uncertainties to be adequately reduced through more research. We proceed to discuss our experience of using the W&H uncertainty framework. Some challenges related to the application of the framework were recognised. This was especially related to the respondents' unfamiliarity with the concepts used and their lack of experience in discussing qualitative aspects of uncertainties. As we see it, the W&H framework should be considered as a useful tool to stimulate reflection on uncertainty and an important first step in a more extensive process of including and properly dealing with uncertainties in science and policymaking.

  20. Development of a suicidal DNA vaccine for infectious hematopoietic necrosis virus (IHNV).

    PubMed

    Alonso, Marta; Chiou, Peter P; Leong, Jo-Ann

    2011-03-01

    We developed a suicidal DNA vaccine (pIRF1A-G-pMT-M) for salmonid fish susceptible to Infectious Hematopoietic Necrosis Virus (IHNV). The suicidal vaccine consists of two operons: i) an inducible fish promoter, the interferon regulatory factor 1A promoter (pIRF1A), driving the expression of the IHNV viral glycoprotein (G) gene that induces protection, and ii) a ZnCl(2) inducible fish promoter, the metallothionein promoter (pMT), driving the expression of the IHNV matrix (M) protein that induces apoptosis. The vaccine induces an immune response to the G protein and then induces the cell to undergo apoptosis to eliminate the DNA vaccine-containing cell. Also developed is another suicidal construct (pCMV-luc-pMT-M) for monitoring the persistence of luciferase (luc) expression after induction of apoptosis. In this study, we evaluated the inducibility of the MT promoter with ZnCl(2) and the capacity of cells transfected with the suicidal vector pCMV-luc-pMT-M to undergo apoptosis after ZnCl(2) addition. We also demonstrated the protective immunity elicited by the suicidal DNA vaccine pIRF1A-G-pMT-M, the survival of fish after treatment with ZnCl(2), and the elimination of the suicidal vector in fish after ZnCl(2) treatment.

  1. DNA vaccines to attack cancer: Strategies for improving immunogenicity and efficacy.

    PubMed

    Tiptiri-Kourpeti, Angeliki; Spyridopoulou, Katerina; Pappa, Aglaia; Chlichlia, Katerina

    2016-09-01

    DNA vaccination represents a smart and promising approach to cancer immunotherapy. DNA vaccines for cancer immunotherapy are designed to deliver one or several genes encoding tumor antigens, thereby eliciting or augmenting antigen-specific immune responses against antigens that play a central role in tumor initiation, progression and metastasis. Vaccine efficacy can be significantly improved by implementing strategies for enhancing antigen presentation and immunogenicity, such as new delivery systems, addition of molecular adjuvants and immunostimulatory signals, optimized prime-boost strategies or blockade of immune checkpoints. Taken into consideration that innate immune responses are important in the induction and enhancement of antigen-specific adaptive responses, manipulations that integrate these approaches in the vaccine design can achieve activation of protective adaptive immune responses, thereby overcoming the self-tolerance towards many tumor antigens. Such approaches are employed in a number of clinical trials for DNA cancer immunotherapy and hold promise for prophylactic and therapeutic vaccine development. In this context, strategies that improve immunogenicity and enhance the efficacy of DNA vaccines for cancer immunotherapy are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Design of immunogenic and effective multi-epitope DNA vaccines for melanoma.

    PubMed

    Cho, Hyun-Il; Celis, Esteban

    2012-03-01

    Plasmid DNA vaccination is an attractive way to elicit T cell responses against infectious agents and tumor cells. DNA constructs can be designed to contain multiple T cell epitopes to generate a diverse immune response to incorporate numerous antigens and to reduce limitations due to MHC restriction into a single entity. We have prepared cDNA plasmid constructs containing several mouse T cell epitopes connected by either furin-sensitive or furin-resistant linkers and studied the effects of a cationic cell-penetrating sequence from HIV-tat. Significant CD8 T cell responses were obtained with multi-epitope DNA vaccines followed by in vivo electroporation regardless of the type of linker used and whether the construct had the HIV-tat sequence. The magnitude of immune responses was very similar to all CD8 T cell epitopes contained within each vaccine construct, indicating the absence of immunodominance. Incorporating a T helper epitope into the constructs increased the T cell responses. Prophylactic and therapeutic antitumor responses against B16 melanoma were obtained using a construct containing epitopes from melanosomal proteins, indicating that this vaccination was successful in generating responses to self-antigens that potentially may be subjected to immune tolerance. These findings are useful for designing DNA vaccines for a multitude of diseases where T lymphocytes play a protective or therapeutic role.

  3. Canine distemper virus DNA vaccination of mink can overcome interference by maternal antibodies.

    PubMed

    Jensen, Trine Hammer; Nielsen, Line; Aasted, Bent; Pertoldi, Cino; Blixenkrone-Møller, Merete

    2015-03-10

    Canine distemper virus (CDV) is highly contagious and can cause severe disease against which conventional live vaccines are ineffective in the presence of maternal antibodies. Vaccination in the presences of maternal antibodies was challenged by vaccination of 5 days old and 3 weeks old mink kits with CDV DNA vaccines. Virus neutralising (VN) antibody responses were induced in mink kits vaccinated with a plasmid encoding the haemaglutinin protein (H) of CDV (n=5, pCDV-H) or a combination of the H, fusion (F) and nucleoprotein (N) of CDV (n=5, pCDV-HFN). These DNA vaccinated kits were protected against virulent experimental infection with field strains of CDV. The pCDV-H was more efficient in inducing protective immunity in the presence of maternal antibodies compared to the pCDV-HFN. The results show that DNA vaccination with the pCDV-H or pCDV-HFN (n=4) only given once at 5 days of age induces virus specific immune response in neonatal mink and protection against virulent CDV exposure later in life.

  4. Designing and Development of a DNA Vaccine Based On Structural Proteins of Hepatitis C Virus

    PubMed Central

    Teimourpour, Roghayeh; Tajani, Amineh Sadat; Askari, Vahid Reza; Rostami, Sina; Meshkat, Zahra

    2016-01-01

    Background: Hepatitis C virus (HCV) infection is one of the most prevalent infectious diseases responsible for high morbidity and mortality worldwide. Therefore, designing new and effective therapeutics is of great importance. The aim of the current study was to construct a DNA vaccine containing structural proteins of HCV and evaluation of its expression in a eukaryotic system. Methods: Structural proteins of HCV (core, E1, and E2) were isolated and amplified from JFH strain of HCV genotype 2a using PCR method. The PCR product was cloned into pCDNA3.1 (+) vector and finally were confirmed by restriction enzyme analysis and sequencing methods. The eukaryotic expression of the vector was confirmed by RT-PCR. Results: A recombinant vector containing 2241bp fragment of HCV structural genes was constructed. The desired plasmid was sequenced and corresponded to 100% identity with the submitted sequences in GenBank. RT-PCR results indicated that the recombinant plasmid could be expressed efficiently in the eukaryotic expression system. Conclusion: Successful cloning of structural viral genes in pCDNA3.1 (+) vector and their expression in the eukaryotic expression system facilitates the development of new DNA vaccines against HCV. A DNA vaccine encoding core-E1-E2 antigens was designed. The desired expression vector can be used for further attempts in the development of vaccines. PMID:27799971

  5. Advances and challenges in the development of therapeutic DNA vaccines against hepatitis B virus infection.

    PubMed

    Cova, Lucyna

    2014-01-01

    Despite the existence of an effective prophylactic vaccine, chronic hepatitis B virus (HBV) infection remains a major public health problem. Because very weak and functionally impaired virus-specific immune responses play a key role in the persistence of HBV infection, the stimulation of these responses appears to be of particular importance for virus clearance. In this regard DNA-based vaccination has emerged as novel, promising therapeutic approach for chronic hepatitis B. This review provides an update of preclinical studies in animal models (mouse, chimpanzee, duck, woodchuck), which evaluated the ability of DNA vaccines targeting hepadnaviral proteins to induce potent and sustained immune responses in naïve animals and to enhance virus clearance and break immune tolerance in chronic virus-carriers. Different strategies have been developed and evaluated in these models to optimize DNA vaccine including genetic adjuvants, combination with antiviral drugs, prime-boost regimens and plasmid delivery. The delivery of DNA by in vivo electroporation appears to be of particular interest for increase of vaccine potency in both small and large animal models. Based on the promising results generated in preclinical studies, first clinical trials of DNA vaccines have been initiated, although effective therapy of chronic hepatitis B awaits further improvements in vaccine efficacy.

  6. CD4+ T cell–independent DNA vaccination against opportunistic infections

    PubMed Central

    Zheng, Mingquan; Ramsay, Alistair J.; Robichaux, Myles B.; Norris, Karen A.; Kliment, Corrine; Crowe, Christopher; Rapaka, Rekha R.; Steele, Chad; McAllister, Florencia; Shellito, Judd E.; Marrero, Luis; Schwarzenberger, Paul; Zhong, Qiu; Kolls, Jay K.

    2005-01-01

    Depletion or dysfunction of CD4+ T lymphocytes profoundly perturbs host defenses and impairs immunogenicity of vaccines. Here, we show that plasmid DNA vaccination with a cassette encoding antigen (OVA) and a second cassette encoding full-length CD40 ligand (CD40L), a molecule expressed on activated CD4+ T lymphocytes and critical for T cell helper function, can elicit significant titers of antigen-specific immunoglobulins in serum and Tc1 CD8+ T cell responses in CD4-deficient mice. To investigate whether this approach leads to CD4+ T cell–independent vaccine protection against a prototypic AIDS-defining infection, Pneumocystis (PC) pneumonia, we used serum from mice vaccinated with PC-pulsed, CD40L-modifed DCs to immunoprecipitate PC antigens. Kexin, a PC antigen identified by this approach, was used in a similar DNA vaccine strategy with or without CD40L. CD4-deficient mice receiving DNA vaccines encoding Kexin and CD40L showed significantly higher anti-PC IgG titers as well as opsonic killing of PC compared with those vaccinated with Kexin alone. Moreover, CD4-depleted, Kexin-vaccinated mice showed a 3-log greater protection in a PC challenge model. Adoptive transfer of CD19+ cells or IgG to SCID mice conferred protection against PC challenge, indicating a role of humoral immunity in the protection. The results of these studies show promise for CD4-independent vaccination against HIV-related or other opportunistic pathogens. PMID:16308571

  7. DNA Vaccine for West Nile Virus Infection in Fish Crows (Corvus ossifragus)

    PubMed Central

    Bunning, Michel; Ludwig, George V.; Ortman, Brian; Chang, Jeff; Speaker, Tully; Spielman, Andrew; McLean, Robert; Komar, Nicholas; Gates, Robert; McNamara, Tracey; Creekmore, Terry; Farley, Linda; Mitchell, Carl J.

    2003-01-01

    A DNA vaccine for West Nile virus (WNV) was evaluated to determine whether its use could protect fish crows (Corvus ossifragus) from fatal WNV infection. Captured adult crows were given 0.5 mg of the DNA vaccine either orally or by intramuscular (IM) inoculation; control crows were inoculated or orally exposed to a placebo. After 6 weeks, crows were challenged subcutaneously with 105 plaque-forming units of WNV (New York 1999 strain). None of the placebo inoculated–placebo challenged birds died. While none of the 9 IM vaccine–inoculated birds died, 5 of 10 placebo-inoculated and 4 of 8 orally vaccinated birds died within 15 days after challenge. Peak viremia titers in birds with fatal WNV infection were substantially higher than those in birds that survived infection. Although oral administration of a single DNA vaccine dose failed to elicit an immune response or protect crows from WNV infection, IM administration of a single dose prevented death and was associated with reduced viremia. PMID:14519243

  8. DNA vaccines encoding viral envelope proteins confer protective immunity against WSSV in black tiger shrimp.

    PubMed

    Rout, Namita; Kumar, Sudhir; Jaganmohan, Shanmugam; Murugan, Vadivel

    2007-04-12

    White Spot Syndrome Virus (WSSV) is a major cause of mortality in shrimp and poses a huge threat to aquaculture industry. Till now no comprehensive or individual strategy has been established to combat white spot disease. Previous efforts by other investigators have given insight of protein vaccination and its efficacy to protect shrimp against WSSV infection. In this study, we have explored the protective efficacy of DNA vaccination and tissue distribution of the immunised recombinant plasmid in black tiger shrimp (Penaeus monodon). Four recombinant constructs were generated by inserting four genes encoding the WSSV structural proteins VP15, VP28, VP35 and VP281 individually into DNA vaccine vector pVAX1. Expression of these proteins from the recombinant plasmids was confirmed in vitro in CHO cell lines. For vaccination experiments, shrimp were immunised with these DNA constructs and later challenged with WSSV. A significant level of protection was offered by the plasmids encoding VP28 or VP281 till 7 weeks whereas protein vaccination failed to protect vaccinated shrimp after 3 weeks of first immunisation. In addition, our tissue distribution study revealed the persistence of immunised DNA at least upto 2 months in the injected shrimp muscle. Thus, our results suggest that DNA vaccination strategy will have potential utility against WSSV infection in shrimp cultivation.

  9. Evaluation of a novel non-penetrating electrode for use in DNA vaccination.

    PubMed

    Donate, Amy; Coppola, Domenico; Cruz, Yolmari; Heller, Richard

    2011-04-29

    Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use with a wide variety of diseases. Alone, this technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our lab recently developed a non-invasive electrode known as the multi-electrode array (MEA), which lies flat on the surface of the skin without penetrating the tissue. In this study we evaluated the MEA for its use in DNA vaccination using Hepatitis B virus as the infectious model. We utilized the guinea pig model because their skin is similar in thickness and morphology to humans. The plasmid encoding Hepatitis B surface antigen (HBsAg) was delivered intradermally with the MEA to guinea pig skin. The results show increased protein expression resulting from plasmid delivery using the MEA as compared to injection alone. Within 48 hours of treatment, there was an influx of cellular infiltrate in experimental groups. Humoral responses were also increased significantly in both duration and intensity as compared to injection only groups. While this electrode requires further study, our results suggest that the MEA has potential for use in electrically mediated intradermal DNA vaccination.

  10. Optimal designs of an HA-based DNA vaccine against H7 subtype influenza viruses

    PubMed Central

    Zhang, Lu; Jia, Na; Li, Jun; Han, Yaping; Cao, Wuchun; Wang, Shixia; Huang, Zuhu; Lu, Shan

    2014-01-01

    The outbreak of a novel H7N9 influenza virus in 2013 has raised serious concerns for the potential of another avian-source pandemic influenza. Effective vaccines against H7N9 virus are important in the prevention and control of any major outbreak. Novel vaccination technologies are useful additions to existing approaches. In the current report, DNA vaccine studies were conducted to identify the optimal design of an H7 HA antigen using the HA gene from a previously reported H7N7 virus that is lethal in humans as the model antigen. New Zealand White rabbits were immunized with DNA vaccines expressing 1 of 3 forms of H7 HA antigen inserts encoding the HA gene from the same H7N7 virus. High-level H7 HA-specific IgG was detected by ELISA, and functional antibodies were confirmed by hemagglutination inhibition assay and pseudotyped virus-based neutralization assay against viruses expressing HA antigens from either the previous H7N7 virus or the novel H7N9 virus. HA antigen design under the tissue plasminogen activator leader (tPA) was the most immunogenic. The data presented in the current report confirm the immunogenicity of the H7 HA antigen and provide useful guidance to prepare for an optimized H7 HA DNA vaccine to help to control the emerging H7N9 virus if and when it is needed. PMID:25424804

  11. Adjuvant effect of polysaccharide from fruits of Physalis alkekengi L. in DNA vaccine against systemic candidiasis.

    PubMed

    Yang, Huimin; Han, Shuying; Zhao, Danyang; Wang, Guiyun

    2014-08-30

    Adjuvant effect mediated by polysaccharide (PPSB) isolated from the fruits of Physalis alkekengi L. in DNA vaccine was evaluated in mice. Recombinant plasmid containing epitope C (LKVIRK) from heat shock protein 90 (HSP90) of Candida albicans (C. albican) was used as DNA vaccine (pD-HSP90C). The results indicated that PPSB significantly enhanced specific antibody titers IgG, IgG1, IgG2b, and concentration of IL-2 and IL-4 in sera of mice immunized with pD-HSP90C (p<0.05). More importantly, it was found that the mice immunized with pD-HSP90C/PPSB not only had fewer CFU (colony forming unites) in the kidneys than mice immunized with pD-HSP90C, but also a statistically significant higher survival rate over PBS-injected group (p<0.05) when the immunized mice were challenged with living C. albican cells. However, no statistically significant difference in survival rate was observed between pD-HSP90C-immunized group and PBS-injected group. Therefore, PPSB can be considered as a promising adjuvant eliciting both Th1 and Th2 responses to enhance the efficacy of DNA vaccines. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Rifapentine, Moxifloxacin, or DNA Vaccine Improves Treatment of Latent Tuberculosis in a Mouse Model

    PubMed Central

    Nuermberger, Eric; Tyagi, Sandeep; Williams, Kathy N.; Rosenthal, Ian; Bishai, William R.; Grosset, Jacques H.

    2005-01-01

    Rationale: Priorities for developing improved regimens for treatment of latent tuberculosis (TB) infection include (1) developing shorter and/or more intermittently administered regimens that are easier to supervise and (2) developing and evaluating regimens that are active against multidrug-resistant organisms. Objectives and Methods: By using a previously validated murine model that involves immunizing mice with Mycobacterium bovis bacillus Calmette-Guérin to augment host immunity before infection with virulent Mycobacterium tuberculosis, we evaluated new treatment regimens including rifapentine and moxifloxacin, and assessed the potential of the Mycobacterium leprae heat shock protein-65 DNA vaccine to augment the activity of moxifloxacin. Measurements: Quantitative spleen colony-forming unit counts, and the proportion of mice with culture-positive relapse after treatment, were determined. Main Results: Three-month, once-weekly regimens of rifapentine combined with either isoniazid or moxifloxacin were as active as daily isoniazid for 6–9 mo. Six-month daily combinations of moxifloxacin with pyrazinamide, ethionamide, or ethambutol were more active than pyrazinamide plus ethambutol, a regimen recommended for latent TB infection after exposure to multidrug-resistant TB. The combination of moxifloxacin with the experimental nitroimidazopyran PA-824 was especially active. Finally, the heat shock protein-65 DNA vaccine had no effect on colony-forming unit counts when given alone, but augmented the bactericidal activity of moxifloxacin. Conclusions: Together, these findings suggest that rifapentine, moxifloxacin, and, perhaps, therapeutic DNA vaccination have the potential to improve on the current treatment of latent TB infection. PMID:16151038

  13. Immunogenicity of RSV F DNA Vaccine in BALB/c Mice

    PubMed Central

    2016-01-01

    Respiratory syncytial virus (RSV) causes severe acute lower respiratory tract disease leading to numerous hospitalizations and deaths among the infant and elderly populations worldwide. There is no vaccine or a less effective drug available against RSV infections. Natural RSV infection stimulates the Th1 immune response and activates the production of neutralizing antibodies, while earlier vaccine trials that used UV-inactivated RSV exacerbated the disease due to the activation of the allergic Th2 response. With a focus on Th1 immunity, we developed a DNA vaccine containing the native RSV fusion (RSV F) protein and studied its immune response in BALB/c mice. High levels of RSV specific antibodies were induced during subsequent immunizations. The serum antibodies were able to neutralize RSV in vitro. The RSV inhibition by sera was also shown by immunofluorescence analyses. Antibody response of the RSV F DNA vaccine showed a strong Th1 response. Also, sera from RSV F immunized and RSV infected mice reduced the RSV infection by 50% and 80%, respectively. Our data evidently showed that the RSV F DNA vaccine activated the Th1 biased immune response and led to the production of neutralizing antibodies, which is the desired immune response required for protection from RSV infections. PMID:27688769

  14. Polylactide-co-glycolide (PLG) microparticles modify the immune response to DNA vaccination.

    PubMed

    Helson, Rebecca; Olszewska, Wieslawa; Singh, Manmohan; Megede, Jan Zur; Melero, Jose A; O'Hagan, Derek; Openshaw, Peter J M

    2008-02-06

    Priming with the major surface glycoprotein G of respiratory syncytial virus (RSV) expressed by recombinant vaccinia leads to strong Th2 responses and lung eosinophilia during viral challenge. We now show that DNA vaccination in BALB/c mice with plasmids encoding G attenuated RSV replication but also enhanced disease with lung eosinophilia and increased IL-4/5 production. However, formulating the DNA with PLG microparticles reduced the severity of disease during RSV challenge without significantly lessening protection against viral replication. PLG formulation greatly reduced lung eosinophilia and prevented the induction of IL-4 and IL-5 during challenge, accompanied by a less marked CD4+ T cell response and a restoration of the CD8+ T cell recruitment seen during infection of non-vaccinated animals. After RSV challenge, lung eosinophilia was enhanced and prolonged in mice vaccinated with DNA encoding a secreted form of G; this effect was virtually prevented by PLG formulation. Therefore, PLG microparticulate formulation modifies the pattern of immune responses induced by DNA vaccination boosts CD8+ T cell priming and attenuates Th2 responses. We speculate that PLG microparticles affect antigen uptake and processing, thereby influencing the outcome of DNA vaccination.

  15. Gene-gun DNA vaccination aggravates respiratory syncytial virus-induced pneumonitis.

    PubMed

    Bartholdy, Christina; Olszewska, Wieslawa; Stryhn, Anette; Thomsen, Allan Randrup; Openshaw, Peter J M

    2004-10-01

    A CD8+ T-cell memory response to respiratory syncytial virus (RSV) was generated by using a DNA vaccine construct encoding the dominant Kd-restricted epitope from the viral transcription anti-terminator protein M2 (M2(82-90)), linked covalently to human beta2-microglobulin (beta2m). Cutaneous gene-gun immunization of BALB/c mice with this construct induced an antigen-specific CD8+ T-cell memory. After intranasal RSV challenge, accelerated CD8+ T-cell responses were observed in pulmonary lymph nodes and virus clearance from the lungs was enhanced. The construct induced weaker CD8+ T-cell responses than those elicited with recombinant vaccinia virus expressing the complete RSV M2 protein, but stronger than those induced by a similar DNA construct without the beta2m gene. DNA vaccination led to enhanced pulmonary disease after RSV challenge, with increased weight loss and cell recruitment to the lung. Depletion of CD8+ T cells reduced, but did not abolish, enhancement of disease. Mice vaccinated with a construct encoding a class I-restricted lymphocytic choriomeningitis virus epitope and beta2m suffered more severe weight loss after RSV infection than unvaccinated RSV-infected mice, although RSV-specific CD8+ T-cell responses were not induced. Thus, in addition to specific CD8+ T cell-mediated immunopathology, gene-gun DNA vaccination causes non-specific enhancement of RSV disease without affecting virus clearance.

  16. Evaluation of Different DNA Vaccines against Porcine Reproductive and Respiratory Syndrome (PRRS) in Pigs

    PubMed Central

    Petrini, Stefano; Ramadori, Giorgio; Villa, Riccardo; Borghetti, Paolo; de Angelis, Elena; Cantoni, Anna Maria; Corradi, Attilio; Amici, Augusto; Ferrari, Maura

    2013-01-01

    In veterinary medicine, there have been different experiences with the plasmid DNA vaccination. In this area and with the hypothesis to demonstrate the effectiveness of different plasmids encoding porcine respiratory and reproductive syndrome (PRRS), five DNA vaccines against PRRS were evaluated for their innocuity and efficacy in pigs. Eighteen animals were divided into five groups which were injected with five (A, B, C, D, E) different DNA vaccines. Albeit, none of the proposed vaccines were able to protect the animals against PRRS virus. Only vaccines A and B were able to reduce the clinical signs of the infection. ELISA IgM were detected 30 days after the first vaccination in the pigs injected by Vaccine A or B. ELISA IgG were detected 90 days after the first vaccination in the pigs injected by Vaccine B or C. Neutralizing antibody were detected Post Challenge Days 61 (PCD) in all groups. In the pigs inoculated with Vaccine C, IFN-γ were detected 90 days after first vaccination, and after challenge exposure they increased. In the other groups, the IFN-γ were detected after challenge infection. Pigs injected with each of the vaccines A, B, C, D and E showed a significantly higher level of CD4−CD8+ lymphocytes (p < 0.001) after infection in comparison with their controls. PMID:26344342

  17. The attenuation of cockroach allergy by DNA vaccine encoding cockroach allergen Bla g 2.

    PubMed

    Zhou, Bin; Yuan, Jingdong; Zhou, Yixuan; Yang, Jun; James, Alan W; Nair, Usha; Shu, Xiji; Liu, Wei; Kanangat, Siva; Yoo, Tai June

    2012-01-01

    Bla g 2 is one of the most potent cockroach allergens. No effective treatment or vaccination strategies are yet available. We evaluated the prophylactic efficacy of Bla g 2 DNA vaccination in a mouse model of allergic airway inflammation. C57/BL6 mice were given Bla g 2 DNA vaccine prior to sensitization with recombinant Bla g 2 (rBla g 2) antigens, followed by nebulized rBla g 2 challenge. Bla g 2 vaccine could express at both transcriptional and translational levels in mammalian cells. Moreover, Bla g 2 vaccine significantly reduced the total inflammatory cell infiltrate and eosinophilia in bronchoalveolar lavage fluid, and markedly decreased allergen-induced inflammatory infiltrates in the lungs and Bla g 2-specific IgE in serum upon challenge with rBla g 2. Importantly, Bla g 2 vaccine could induce the production of antigen-specific IFN-γ and downregulated Th2 pro-inflammatory cytokines IL-4, IL-5, and IL-13. Thus, DNA vaccination showed protective efficacy against a clinically relevant allergen, Bla g 2.

  18. DNA vaccine protects ornamental koi (Cyprinus carpio koi) against North American spring viremia of carp virus

    USGS Publications Warehouse

    Emmenegger, E.J.; Kurath, G.

    2008-01-01

    The emergence of spring viremia of carp virus (SVCV) in the United States constitutes a potentially serious alien pathogen threat to susceptible fish stocks in North America. A DNA vaccine with an SVCV glycoprotein (G) gene from a North American isolate was constructed. In order to test the vaccine a challenge model utilizing a specific pathogen-free domestic koi stock and a cold water stress treatment was also developed. We have conducted four trial studies demonstrating that the pSGnc DNA vaccine provided protection in vaccinated fish against challenge at low, moderate, and high virus doses of the homologous virus. The protection was significant (p < 0.05) as compared to fish receiving a mock vaccine construct containing a luciferase reporter gene and to non-vaccinated controls in fish ranging in age from 3 to 14 months. In all trials, the SVCV-G DNA immunized fish were challenged 28-days post-vaccination (546 degree-days) and experienced low mortalities varying from 10 to 50% with relative percent survivals ranging from 50 to 88%. The non-vaccinated controls and mock construct vaccinated fish encountered high cumulative percent mortalities ranging from 70 to 100%. This is the first report of a SVCV DNA vaccine being tested successfully in koi. These experiments prove that the SVCV DNA (pSGnc) vaccine can elicit specific reproducible protection and validates its potential use as a prophylactic vaccine in koi and other vulnerable North American fish stocks.

  19. B lymphocytes as direct antigen-presenting cells for anti-tumor DNA vaccines

    PubMed Central

    Colluru, Viswa Teja; McNeel, Douglas G.

    2016-01-01

    In spite of remarkable preclinical efficacy, DNA vaccination has demonstrated low immunogenicity in humans. While efforts have focused on increasing cross-presentation of DNA-encoded antigens, efforts to increase DNA vaccine immunogenicity by targeting direct presentation have remained mostly unexplored. In these studies, we compared the ability of different APCs to present antigen to T cells after simple co-culture with plasmid DNA. We found that human primary peripheral B lymphocytes, and not monocytes or in vitro derived dendritic cells (DCs), were able to efficiently encode antigen mRNA and expand cognate tumor antigen-specific CD8 T cells ex vivo. Similarly, murine B lymphocytes co-cultured with plasmid DNA, and not DCs, were able to prime antigen-specific T cells in vivo. Moreover, B lymphocyte-mediated presentation of plasmid antigen led to greater Th1-biased immunity and was sufficient to elicit an anti-tumor effect in vivo. Surprisingly, increasing plasmid presentation by B cells, and not cross presentation of peptides by DCs, further augmented traditional plasmid vaccination. Together, these data suggest that targeting plasmid DNA to B lymphocytes, for example through transfer of ex vivo plasmidloaded B cells, may be novel means to achieve greater T cell immunity from DNA vaccines. PMID:27661128

  20. Recent advance in immunotherapies for Alzheimer disease: with special reference to DNA vaccination.

    PubMed

    Okura, Yoshio; Matsumoto, Yoh

    2009-06-01

    Alzheimer disease (AD) is the most common cause of dementia characterized by progressive neurodegeneration. Based on the amyloid cascade hypothesis, several immunotherapies for AD have been developed as curative treatment. In 1999, Schenk et al. reported for the first time that amyloid beta (Abeta) deposits in AD model mice could be reduced by active vaccination with Abeta peptide. Although clinical trials with the Abeta peptide were halted due to the development of meningoencephalitis in some treated patients, the vaccine therapy was judged to be effective on the basis of clinical and pathological analyses. Passive immunization using humanized anti-Abeta monoclonal antibodies is also under clinical trials; however they have some problems to be solved. As other strategies, DNA vaccines have been developed as immunotherapies for AD, which is simple, easily modified and can be administered without adjuvant. DNA vaccines were developed by several groups including our laboratory, which induced Abeta reduction in AD model mice without side effects. DNA vaccination may be open up new avenue of vaccine therapies for AD in the near future.

  1. Effects of DDA, CpG-ODN, and plasmid-encoded chicken IFN-γ on protective immunity by a DNA vaccine against IBDV in chickens

    PubMed Central

    Roh, Ha Jung; Sung, Haan Woo

    2006-01-01

    This study examined the adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA), CpG oligodeoxynucleotides (CpG-ODN), and chicken interferon-γ (ChIFN-γ) on a DNA vaccine (pcDNA-VP243) against the infectious bursal disease virus (IBDV). A plasmid encoding chicken IFN-ã was constructed. Twice at 2-week intervals, two-week-old chickens were injected intramuscularly and intraperitoneally with either a DNA vaccine alone or a DNA vaccine together with the respective adjuvants. On week 2 after the second immunization, the chickens were orally challenged with the highly virulent IBDV. The groups that received the DNA vaccines plus either DDA or CpG-ODN showed significantly lower survival rates than the group that received the DNA vaccine alone. However, the survival rates for the DNA vaccine alone and for the DNA vaccine plus ChIFN-γ were similar. The chickens had no detectable antibodies to the IBDV before the challenge but all the surviving chickens in all groups except for the normal control group showed the induction of antibodies to the IBDV at day 10 after the challenge. As judged by the lymphocyte proliferation assays using the a WST-8 solution performed on the peripheral blood and splenic lymphocytes, the stimulation indices (SI) of the peripheral blood lymphocytes in all groups except for the normal control group were similar immediately before the challenge. At 10 days post-challenge, the SI for DNA vaccine plus either CpG-ODN or ChIFN-γ was similar to that of the DNA vaccine control group. For splenic lymphocytes, the SI in the DNA vaccine plus CpG-ODN and DNA vaccine plus ChIFN-γ groups were higher than for the DNA vaccine control. These results suggest that DDA actually compromises the protection against the IBDV by DNA vaccine, and CpG-ODN and IFN-γ had no significant effect. PMID:17106228

  2. Early Activation of Teleost B Cells in Response to Rhabdovirus Infection

    PubMed Central

    Abós, Beatriz; Castro, Rosario; González Granja, Aitor; Havixbeck, Jeffrey J.; Barreda, Daniel R.

    2014-01-01

    ABSTRACT To date, the response of teleost B cells to specific pathogens has been only scarcely addressed. In this work, we have demonstrated that viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, has the capacity to infect rainbow trout spleen IgM-positive (IgM+) cells, although the infection is not productive. Consequently, we have studied the effects of VHSV on IgM+ cell functionality, comparing these effects to those elicited by a Toll-like receptor 3 (TLR3) ligand, poly(I·C). We found that poly(I·C) and VHSV significantly upregulated TLR3 and type I interferon (IFN) transcription in spleen and blood IgM+ cells. Further effects included the upregulated transcription of the CK5B chemokine. The significant inhibition of some of these effects in the presence of bafilomycin A1 (BAF), an inhibitor of endosomal acidification, suggests the involvement of an intracellular TLR in these responses. In the case of VHSV, these transcriptional effects were dependent on viral entry into B cells and the initiation of viral transcription. VHSV also provoked the activation of NF-κB and the upregulation of major histocompatibility complex class II (MHC-II) cell surface expression on IgM+ cells, which, along with the increased transcription of the costimulatory molecules CD80/86 and CD83, pointed to VHSV-induced IgM+ cell activation toward an antigen-presenting profile. Finally, despite the moderate effects of VHSV on IgM+ cell proliferation, a consistent effect on IgM+ cell survival was detected. IMPORTANCE Innate immune responses to pathogens established through their recognition by pattern recognition receptors (PRRs) have been traditionally ascribed to innate cells. However, recent evidence in mammals has revealed that innate pathogen recognition by B lymphocytes is a crucial factor in shaping the type of immune response that is mounted. In teleosts, these immediate effects of viral encounter on B lymphocytes have not been addressed to date. In our study, we

  3. Sigma viruses from three species of Drosophila form a major new clade in the rhabdovirus phylogeny

    PubMed Central

    Longdon, Ben; Obbard, Darren J.; Jiggins, Francis M.

    2010-01-01

    The sigma virus (DMelSV), which is a natural pathogen of Drosophila melanogaster, is the only Drosophila-specific rhabdovirus that has been described. We have discovered two new rhabdoviruses, D. obscura and D. affinis, which we have named DObsSV and DAffSV, respectively. We sequenced the complete genomes of DObsSV and DMelSV, and the L gene from DAffSV. Combining these data with sequences from a wide range of other rhabdoviruses, we found that the three sigma viruses form a distinct clade which is a sister group to the Dimarhabdovirus supergroup, and the high levels of divergence between these viruses suggest that they deserve to be recognized as a new genus. Furthermore, our analysis produced the most robustly supported phylogeny of the Rhabdoviridae to date, allowing us to reconstruct the major transitions that have occurred during the evolution of the family. Our data suggest that the bias towards research into plants and vertebrates means that much of the diversity of rhabdoviruses has been missed, and rhabdoviruses may be common pathogens of insects. PMID:19812076

  4. IgA response and protection following nasal vaccination of chickens with Newcastle disease virus DNA vaccine nanoencapsulated with Ag@SiO2 hollow nanoparticles

    PubMed Central

    Zhao, Kai; Rong, Guangyu; Hao, Yan; Yu, Lu; Kang, Hong; Wang, Xin; Wang, Xiaohua; Jin, Zheng; Ren, Zhiyu; Li, Zejun

    2016-01-01

    Newcastle disease caused by ND virus (NDV) is a highly contagious disease of birds. Vaccine for effective protection of poultry animals from NDV infection is urgently needed. Mucosal immunity plays a very important role in the antiviral immune response. In this study, a NDV F gene-containing DNA vaccine encapsulated in Ag@SiO2 hollow nanoparticles (pFDNA-Ag@SiO2-NPs) with an average diameter of 500 nm were prepared to assess the mucosal immune response. These nanoparticles exhibited low cytotoxicity and did not destroy the bioactivity of plasmid DNA, which could be expressed in vitro. The plasmid DNA was sustainably released after an initial burst release. In vivo immunization showed that the intranasal immunization of chickens with pFDNA-Ag@SiO2-NPs induced high titers of serum antibody, significantly promoted lymphocyte proliferation and induced higher expression levels of IL-2 and IFN-γ in a dose-dependent manner. These results indicated that the Ag@SiO2 hollow nanoparticles could serve as an efficient and safe delivery carrier for NDV DNA vaccine to induce mucosal immunity. This study has provided promising results for the further development of mucosal vaccines encapsulated in inorganic nanoparticles. PMID:27170532

  5. IgA response and protection following nasal vaccination of chickens with Newcastle disease virus DNA vaccine nanoencapsulated with Ag@SiO2 hollow nanoparticles

    NASA Astrophysics Data System (ADS)

    Zhao, Kai; Rong, Guangyu; Hao, Yan; Yu, Lu; Kang, Hong; Wang, Xin; Wang, Xiaohua; Jin, Zheng; Ren, Zhiyu; Li, Zejun

    2016-05-01

    Newcastle disease caused by ND virus (NDV) is a highly contagious disease of birds. Vaccine for effective protection of poultry animals from NDV infection is urgently needed. Mucosal immunity plays a very important role in the antiviral immune response. In this study, a NDV F gene-containing DNA vaccine encapsulated in Ag@SiO2 hollow nanoparticles (pFDNA-Ag@SiO2-NPs) with an average diameter of 500 nm were prepared to assess the mucosal immune response. These nanoparticles exhibited low cytotoxicity and did not destroy the bioactivity of plasmid DNA, which could be expressed in vitro. The plasmid DNA was sustainably released after an initial burst release. In vivo immunization showed that the intranasal immunization of chickens with pFDNA-Ag@SiO2-NPs induced high titers of serum antibody, significantly promoted lymphocyte proliferation and induced higher expression levels of IL-2 and IFN-γ in a dose-dependent manner. These results indicated that the Ag@SiO2 hollow nanoparticles could serve as an efficient and safe delivery carrier for NDV DNA vaccine to induce mucosal immunity. This study has provided promising results for the further development of mucosal vaccines encapsulated in inorganic nanoparticles.

  6. DNA Vaccines: MHC II-Targeted Vaccine Protein Produced by Transfected Muscle Fibres Induces a Local Inflammatory Cell Infiltrate in Mice

    PubMed Central

    Løvås, Tom-Ole; Gundersen, Kristian; Bogen, Bjarne

    2014-01-01

    Vaccination with naked DNA holds great promise but immunogenicity needs to be improved. DNA constructs encoding bivalent proteins that bind antigen-presenting cells (APC) for delivery of antigen have been shown to enhance T and B cell responses and protection in tumour challenge experiments. However, the mechanism for the increased potency remains to be determined. Here we have constructed DNA vaccines that express the fluorescent protein mCherry, a strategy which allowed tracking of vaccine proteins. Transfected muscle fibres in mice were visualized, and their relationship to infiltrating mononuclear cells could be determined. Interestingly, muscle fibers that produced MHC class II-specific dimeric vaccine proteins with mCherry were for weeks surrounded by a localized intense cellular infiltrate composed of CD45+, MHC class II+ and CD11b+ cells. Increasing numbers of eosinophils were observed among the infiltrating cells from day 7 after immunization. The local infiltrate surrounding mCherry+ muscle fibers was dependent on the MHC II-specificity of the vaccine proteins since the control, a non-targeted vaccine protein, failed to induce similar infiltrates. Chemokines measured on day 3 in immunized muscle indicate both a DNA effect and an electroporation effect. No influence of targeting was observed. These results contribute to our understanding for why targeted DNA vaccines have an improved immunogenicity. PMID:25299691

  7. DNA vaccines: MHC II-targeted vaccine protein produced by transfected muscle fibres induces a local inflammatory cell infiltrate in mice.

    PubMed

    Løvås, Tom-Ole; Bruusgaard, Jo C; Øynebråten, Inger; Gundersen, Kristian; Bogen, Bjarne

    2014-01-01

    Vaccination with naked DNA holds great promise but immunogenicity needs to be improved. DNA constructs encoding bivalent proteins that bind antigen-presenting cells (APC) for delivery of antigen have been shown to enhance T and B cell responses and protection in tumour challenge experiments. However, the mechanism for the increased potency remains to be determined. Here we have constructed DNA vaccines that express the fluorescent protein mCherry, a strategy which allowed tracking of vaccine proteins. Transfected muscle fibres in mice were visualized, and their relationship to infiltrating mononuclear cells could be determined. Interestingly, muscle fibers that produced MHC class II-specific dimeric vaccine proteins with mCherry were for weeks surrounded by a localized intense cellular infiltrate composed of CD45+, MHC class II+ and CD11b+ cells. Increasing numbers of eosinophils were observed among the infiltrating cells from day 7 after immunization. The local infiltrate surrounding mCherry+ muscle fibers was dependent on the MHC II-specificity of the vaccine proteins since the control, a non-targeted vaccine protein, failed to induce similar infiltrates. Chemokines measured on day 3 in immunized muscle indicate both a DNA effect and an electroporation effect. No influence of targeting was observed. These results contribute to our understanding for why targeted DNA vaccines have an improved immunogenicity.

  8. Development of avian influenza virus H5 DNA vaccine and MDP-1 gene of Mycobacterium bovis as genetic adjuvant

    PubMed Central

    2010-01-01

    Background Studies have shown that DNA vaccines can induce protective immunity, which demonstrated the high potential of DNA vaccines as an alternative to inactivated vaccines. Vaccines are frequently formulated with adjuvants to improve their release, delivery and presentation to the host immune system. Methods The H5 gene of H5N1 virus (A/Ck/Malaysia/5858/04) was cloned separately into pcDNA3.1 + vector. The immunogenicity of the cloned H5 DNA vaccine was tested on SPF chickens using two different approaches. First approach was using H5 DNA vaccine (pcDNA3.1/H5) and the second was using H5 DNA vaccine in addition to the pcDNA3.1/MDP1 vaccine. Ten days old chickens inoculated three times with two weeks intervals. The spleen and muscle samples from chickens immunized with H5 (pcDNA3.1/H5) and H5 + MDP1 (pcDNA3.1/H5 + pcDNA3.1/MDP1) vaccines were collected after sacrificing the chickens and successfully expressed H5 and MDP1 RNA transcripts. The sera of immunized chickens were collected prior to first immunization and every week after immunization; and analyzed using enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test. Results Results of competitive ELISA showed successful antibody responses two weeks post immunization. The HI test showed an increased in antibody titers during the course of experiment in group immunized with H5 and H5 + MDP1 vaccines. The result showed that the constructed DNA vaccines were able to produce detectable antibody titer in which the group immunized with H5 + MDP1 vaccine produced higher antibody comparing to H5 vaccine alone. Conclusions This study shows for the first time the usefulness of MDP1 as a genetic adjuvant for H5 DNA vaccine. PMID:20497569

  9. Distribution and expression in vitro and in vivo of DNA vaccine against lymphocystis disease virus in Japanese flounder ( Paralichthys olivaceus)

    NASA Astrophysics Data System (ADS)

    Zheng, Fengrong; Sun, Xiuqin; Liu, Hongzhan; Wu, Xingan; Zhong, Nan; Wang, Bo; Zhou, Guodong

    2010-01-01

    Lymphocystis disease, caused by the lymphocystis disease virus (LCDV), is a significant worldwide problem in fish industry causing substantial economic losses. In this study, we aimed to develop the DNA vaccine against LCDV, using DNA vaccination technology. We evaluated plasmid pEGFP-N2-LCDV1.3 kb as a DNA vaccine candidate. The plasmid DNA was transiently expressed after liposome transfection into the eukaryotic COS 7 cell line. The distribution and expression of the DNA vaccine (pEGFP-N2-LCDV1.3kb) were also analyzed in tissues of the vaccinated Japanese flounder by PCR, RT-PCR and fluorescent microscopy. Results from PCR analysis indicated that the vaccine-containing plasmids were distributed in injected muscle, the muscle opposite the injection site, the hind intestine, gill, spleen, head, kidney and liver, 6 and 25 days after vaccination. The vaccine plasmids disappeared 100 d post-vaccination. Fluorescent microscopy revealed green fluorescence in the injected muscle, the muscle opposite the injection site, the hind intestine, gill, spleen, head, kidney and liver of fish 48 h post-vaccination, green fluorescence did not appear in the control treated tissue. Green fluorescence became weak at 60 days post-vaccination. RT-PCR analysis indicated that the mcp gene was expressed in all tested tissues of vaccinated fish 6-50 days post-vaccination. These results demonstrate that the antigen encoded by the DNA vaccine is distributed and expressed in all of the tissues analyzed in the vaccinated fish. The antigen would therefore potentially initiate a specific immune response. the plasmid DNA was injected into Japanese flounder ( Paralichthys olivaceus) intramuscularly and antibodies against LCDV were evaluated. The results indicate that the plasmid encoded DNA vaccine could induce an immune response to LCDV and would therefore offer immune protection against LCD. Further studies are required for the development and application of this promising DNA vaccine.

  10. A ribonuclease protection assay can distinguish spring viremia of carp virus from pike fry rhabdovirus

    USGS Publications Warehouse

    Ahne, W.; Kurath, G.; Winton, J.R.

    1998-01-01

    Thirteen rhabdovirus isolates from 10 teleost fish species as well as reference strains of spring viraemia of carp virus (SVCV) and pike fry rhabdovirus (PFRV) cross-reacted in an indirect immunofluorescence assay and were thus indistinguishable by this method. A ribonuclease protection assay (RPA) using a super(32)P-labeled RNA probe made from a cloned copy of the full length SVCV glycoprotein (G) gene was able to discriminate clearly between the type strains of SVCV and PFRV and among the 13 rhabdovirus isolates. Results for the RPA were generally in agreement with standard serum neutralisation assays; however, the RPA was also able to detect genomic differences between isolates of SVCV. These results have implications for fish disease control programs for SVCV.

  11. Molecular Detection of Adenoviruses, Rhabdoviruses, and Paramyxoviruses in Bats from Kenya

    PubMed Central

    Conrardy, Christina; Tao, Ying; Kuzmin, Ivan V.; Niezgoda, Michael; Agwanda, Bernard; Breiman, Robert F.; Anderson, Larry J.; Rupprecht, Charles E.; Tong, Suxiang

    2014-01-01

    We screened 217 bats of at least 20 species from 17 locations in Kenya during July and August of 2006 for the presence of adenovirus, rhabdovirus, and paramyxovirus nucleic acids using generic reverse transcription polymerase chain reaction (RT-PCR) and PCR assays. Of 217 bat fecal swabs examined, 4 bats were adenovirus DNA-positive, 11 bats were paramyxovirus RNA-positive, and 2 bats were rhabdovirus RNA-positive. Three bats were coinfected by two different viruses. By sequence comparison and phylogenetic analysis, the Kenya bat paramyxoviruses and rhabdoviruses from this study may represent novel viral lineages within their respective families; the Kenya bat adenoviruses could not be confirmed as novel, because the same region sequences from other known bat adenovirus genomes for comparison were lacking. Our study adds to previous evidence that bats carry diverse, potentially zoonotic viruses and may be coinfected with more than one virus. PMID:24865685

  12. Suppression of antitumour protective cytotoxic T lymphocyte responses to a human papillomavirus 16 E7 DNA vaccine by coinjection of interleukin-12 complementary DNA: involvement of nitric oxide in immune suppression

    PubMed Central

    Sin, Jeong-Im

    2009-01-01

    Interleukin-12 (IL-12) has been shown to enhance cellular immunity in vitro and in vivo. The beneficial roles of IL-12 as a DNA vaccine adjuvant have been commonly observed. Here the impact of IL-12 complementary DNA (cDNA) as an adjuvant for a human papillomavirus (HPV) type 16 E7 DNA vaccine is investigated in a mouse tumour model. Coinjection of E7 DNA vaccine with IL-12 cDNA completely suppressed antigen-specific cytotoxic T-lymphocyte (CTL) responses, leading to a complete loss of antitumour protection from a tumour cell challenge. In addition, antigen-specific antibody and T helper cell proliferative responses were also suppressed by IL-12 cDNA coinjection. This inhibition was observed over different IL-12 cDNA doses. Furthermore, separate leg injections of IL-12 and E7 cDNAs suppressed antigen-specific CTL and tumour protective responses, but not antibody and T helper cell proliferative responses, suggesting different pathways for suppression of these two separate responses. Further knockout animal studies demonstrated that interferon-γ and nitric oxide are not directly associated with suppression of antigen-specific antibody responses by IL-12 cDNA coinjection. However, nitric oxide was found to be involved in suppression of antigen-specific CTL and tumour protective responses by IL-12 cDNA coinjection. These data suggest that coinjection of IL-12 cDNA results in suppression of E7-specific CTL responses through nitric oxide, leading to a loss of antitumour resistance in this DNA vaccine model. This study further shows that the adjuvant effect of IL-12 is dependent on the antigen types tested. PMID:19740332

  13. Suppression of antitumour protective cytotoxic T lymphocyte responses to a human papillomavirus 16 E7 DNA vaccine by coinjection of interleukin-12 complementary DNA: involvement of nitric oxide in immune suppression.

    PubMed

    Sin, Jeong-Im

    2009-09-01

    Interleukin-12 (IL-12) has been shown to enhance cellular immunity in vitro and in vivo. The beneficial roles of IL-12 as a DNA vaccine adjuvant have been commonly observed. Here the impact of IL-12 complementary DNA (cDNA) as an adjuvant for a human papillomavirus (HPV) type 16 E7 DNA vaccine is investigated in a mouse tumour model. Coinjection of E7 DNA vaccine with IL-12 cDNA completely suppressed antigen-specific cytotoxic T-lymphocyte (CTL) responses, leading to a complete loss of antitumour protection from a tumour cell challenge. In addition, antigen-specific antibody and T helper cell proliferative responses were also suppressed by IL-12 cDNA coinjection. This inhibition was observed over different IL-12 cDNA doses. Furthermore, separate leg injections of IL-12 and E7 cDNAs suppressed antigen-specific CTL and tumour protective responses, but not antibody and T helper cell proliferative responses, suggesting different pathways for suppression of these two separate responses. Further knockout animal studies demonstrated that interferon-gamma and nitric oxide are not directly associated with suppression of antigen-specific antibody responses by IL-12 cDNA coinjection. However, nitric oxide was found to be involved in suppression of antigen-specific CTL and tumour protective responses by IL-12 cDNA coinjection. These data suggest that coinjection of IL-12 cDNA results in suppression of E7-specific CTL responses through nitric oxide, leading to a loss of antitumour resistance in this DNA vaccine model. This study further shows that the adjuvant effect of IL-12 is dependent on the antigen types tested.

  14. DNA vaccine prime and recombinant FPV vaccine boost: an important candidate immunization strategy to control bluetongue virus type 1.

    PubMed

    Li, Junping; Yang, Tao; Xu, Qingyuan; Sun, Encheng; Feng, Yufei; Lv, Shuang; Zhang, Qin; Wang, Haixiu; Wu, Donglai

    2015-10-01

    Bluetongue virus (BTV) is the causative agent of bluetongue (BT), an important sheep disease that caused great economic loss to the sheep industry. There are 26 BTV serotypes based on the outer protein VP2. However, the serotypes BTV-1 and BTV-16 are the two most prevalent serotypes in China. Vaccination is the most effective method of preventing viral infections. Therefore, the need for an effective vaccine against BTV is urgent. In this study, DNA vaccines and recombinant fowlpox virus (rFPV) vaccines expressing VP2 alone or VP2 in combination with VP5 or co-expressing the VP2 and VP5 proteins of BTV-1 were evaluated in both mice and sheep. Several strategies were tested in mice, including DNA vaccine prime and boost, rFPV vaccine prime and boost, and DNA vaccine prime and rFPV vaccine boost. We then determined the best vaccine strategy in sheep. Our results indicated that a strategy combining a DNA vaccine prime (co-expressing VP2 and VP5) followed by an rFPV vaccine boost (co-expressing VP2 and VP5) induced a high titer of neutralizing antibodies in sheep. Therefore, our data suggest that a DNA vaccine consisting of a pCAG-(VP2+VP5) prime and an rFPV-(VP2+VP5) boost is an important candidate for the design of a novel vaccine against BTV-1.

  15. Antitumor Response to a Codon-Optimized HPV-16 E7/HSP70 Fusion Antigen DNA Vaccine.

    PubMed

    Soleimanjahi, Hoorieh; Razavinikoo, Hadi; Fotouhi, Fatemeh; Ardebili, Abdollah

    2017-09-01

    Vaccines based on virus-like particles are effective against Human Papilloma Virus (HPV) infection; however, they have not shown a therapeutic effect against HPV-associated diseases. New immunotherapy strategies based on immune responses against tumor antigens can positively affect the clearance of HPV-associated lesions. To generate two therapeutic fusion DNA vaccines (optimizedE7/mouseHSP70 and wildE7/mouseHSP70) to induce antitumor specific responses in mice models. Mice were immunized with recombinant DNA vaccines. The splenocytes of immunized mice were collected and lactate dehydrogenase and IFN-γ productions were measured after three injections in order to evaluate cytotoxic T lymphocytes (CTLs) activity. MTT assay was carried out for lymphocyte stimulation. The fusion DNA vaccines, specifically uE7-HSP70, elicited varying levels of IFN-γ and CTLs responses compared to the control group (P<0.05). Furthermore, antitumor response and tumor size reduction in fusion DNA vaccines groups were significantly higher than in the negative control group (P<0.05). It is concluded that our fusion DNA vaccines considerably enhanced specific cellular responses against HPV tumor model. In addition, optimized E7 showed a notable immunogenicity and inhibitory effect on the reduction of tumor size.

  16. Arg-Gingipain A DNA Vaccine Induces Protective Immunity against Infection by Porphyromonas gingivalis in a Murine Model

    PubMed Central

    Yonezawa, Hideo; Ishihara, Kazuyuki; Okuda, Katsuji

    2001-01-01

    Arginine-specific cysteine proteinases (RgpA and RgpB) produced by the periodontal pathogen Porphyromonas gingivalis are suspected virulence factors and are involved in interrupting host defense mechanisms as well as in penetrating and destroying periodontal connective tissues. To induce a protective immune response against P. gingivalis, we constructed an rgpA DNA vaccine. BALB/c mice were immunized intradermally by Gene Gun with plasmid DNA carrying rgpA. Antibody responses against P. gingivalis were determined by an enzyme-linked immunosorbent assay. The rgpA DNA vaccine induced high levels of serum antibodies against P. gingivalis. Sera from the rgpA DNA vaccine-immunized mice diminished the proteolytic activity of RgpA and RgpB and inhibited the binding of P. gingivalis to a type I collagen sponge. Moreover, the sera effectively reduced the hemagglutination of P. gingivalis, indicating that the hemagglutinin activity of the organism is associated with RgpA. We found with a murine abscess model that mice immunized with the rgpA DNA vaccine were resistant to an invasive P. gingivalis W50 challenge. These results suggest that the rgpA DNA vaccine induced specific antibodies against the enzyme and that this vaccine could confer protective immunity against P. gingivalis infection. PMID:11292699

  17. Efficacy of an autophagy-targeted DNA vaccine against avian leukosis virus subgroup J.

    PubMed

    Dai, Zhenkai; Huang, Jianfei; Lei, Xiaoya; Yan, Yiming; Lu, Piaopiao; Zhang, Huanmin; Lin, Wencheng; Chen, Weiguo; Ma, Jingyun; Xie, Qingmei

    2017-02-01

    Infection with the avian leukosis virus subgroup J (ALV-J) can lead to neoplastic disease in chickens, inflicting significant economic losses to the poultry industry. Recent reports have identified inhibitory effects of ALV-J on autophagy, a process involving in innate and adaptive immunity. Inspired by this connection between autophagy and immunity, we developed a novel DNA vaccine against ALV-J which includes co-administration of rapamycin to stimulate autophagy. To measure the efficacy of the developed prototype vaccine, five experimental groups of seven-day-old chickens was immunized three times at three-week intervals respectively with vector, pVAX1-gp85, pVAX1-gp85-LC3, pVAX1-gp85+rapamycin and pVAX1-gp85-LC3+rapamycin through electroporation. We then tested their antibody titers, cytokine levels and cellular immune responses. The immunoprotective efficacy of the prototype vaccines against the challenge of the ALV-J GD1109 strain was also examined. The results showed that the combination of pVAX1-gp85-LC3 and rapamycin was able to induce the highest antibody titers, and enhance interleukin(IL)-2, IL-10 and interferon (IFN)-γ expression, and the chickens immunized with the combination of pVAX1-gp85-LC3 and rapamycin showed the highest percentage of CD3+CD8+T lymphocytes. Based on our results, we suggest that stimulating autophagy can improve the efficacy of DNA vaccines and that our DNA vaccine shows the potential of being a candidate vaccine against ALV-J. This study provides a novel strategy for developing vaccines against ALV-J.

  18. DNA Vaccination Affords Significant Protection against Feline Immunodeficiency Virus Infection without Inducing Detectable Antiviral Antibodies

    PubMed Central

    Hosie, Margaret J.; Flynn, J. Norman; Rigby, Mark A.; Cannon, Celia; Dunsford, Thomas; Mackay, Nancy A.; Argyle, David; Willett, Brian J.; Miyazawa, Takayuki; Onions, David E.; Jarrett, Oswald; Neil, James C.

    1998-01-01

    To test the potential of a multigene DNA vaccine against lentivirus infection, we generated a defective mutant provirus of feline immunodeficiency virus (FIV) with an in-frame deletion in pol (FIVΔRT). In a first experiment, FIVΔRT DNA was administered intramuscularly to 10 animals, half of which also received feline gamma interferon (IFN-γ) DNA. The DNA was administered in four 100-μg doses at 0, 10, and 23 weeks. Immunization with FIVΔRT elicited cytotoxic T-cell (CTL) responses to FIV Gag and Env in the absence of a serological response. After challenge with homologous virus at week 26, all 10 of the control animals became seropositive and viremic but 4 of the 10 vaccinates remained seronegative and virus free. Furthermore, quantitative virus isolation and quantitative PCR analysis of viral DNA in peripheral blood mononuclear cells revealed significantly lower virus loads in the FIVΔRT vaccinates than in the controls. Immunization with FIVΔRT in conjunction with IFN-γ gave the highest proportion of protected cats, with only two of five vaccinates showing evidence of infection following challenge. In a second experiment involving two groups (FIVΔRT plus IFN-γ and IFN-γ alone), the immunization schedule was reduced to 0, 4, and 8 weeks. Once again, CTL responses were seen prior to challenge in the absence of detectable antibodies. Two of five cats receiving the proviral DNA vaccine were protected against infection, with an overall reduction in virus load compared to the five infected controls. These findings demonstrate that DNA vaccination can elicit protection against lentivirus infection in the absence of a serological response and suggest the need to reconsider efficacy criteria for lentivirus vaccines. PMID:9696827

  19. The swine CD81 enhances E2-based DNA vaccination against classical swine fever.

    PubMed

    Li, Wenliang; Mao, Li; Zhou, Bin; Liu, Xia; Yang, Leilei; Zhang, Wenwen; Jiang, Jieyuan

    2015-07-09

    Classical swine fever (CSF) is a highly contagious and economically important viral disease that affects the pig industry worldwide. The glycoprotein E2 of CSFV can induce neutralizing antibodies and protective immunity, and is widely used for novel vaccine development. The objective of this study was to explore whether a tetraspanin molecule CD81 could improve the immune responses of an E2-based DNA vaccine. Plasmids pVAX-CD81, pVAX-E2 and pVAX-CD81-E2 were constructed and the expression of target proteins was confirmed in BHK-21 cells by indirect immunofluorescence assay. BALB/c mice were divided into 5 groups and immunized with different plasmids (pVAX-E2, pVAX-CD81-E2, pVAX-E2+pVAX-CD81, pVAX-CD81 and PBS) three times with two weeks interval. The results showed that the introduction of CD81 promoted higher humoral and cellular immune responses than E2 expression alone (P<0.05). In addition, immunization with pVAX-CD81-E2 induced stronger immune responses than pVAX-E2+pVAX-CD81. Furthermore, four groups of pigs were immunized with pVAX-E2, pVAX-CD81-E2, pVAX-CD81 and PBS, respectively. Humoral and cellular immune responses detection showed similar results with those in mice. Compared to pVAX-E2, pVAX-CD81-E2 induced higher titers of neutralizing antibodies after viral challenge and conferred stronger protection. These results confirmed the capacity of swine CD81 enhancing the humoral and cellular responses with an adjuvant effect on CSFV DNA vaccine. This is the first report demonstrating the adjuvant effect of CD81 to enhance the DNA vaccination for swine pathogen.

  20. Induction of Potent Immune Responses by Cationic Microparticles with Adsorbed Human Immunodeficiency Virus DNA Vaccines

    PubMed Central

    O'Hagan, Derek; Singh, Manmohan; Ugozzoli, Mildred; Wild, Carl; Barnett, Susan; Chen, Minchao; Schaefer, Mary; Doe, Barbara; Otten, Gillis R.; Ulmer, Jeffrey B.

    2001-01-01

    The effectiveness of cationic microparticles with adsorbed DNA at inducing immune responses was investigated in mice, guinea pigs, and rhesus macaques. Plasmid DNA vaccines encoding human immunodeficiency virus (HIV) Gag and Env adsorbed onto the surface of cationic poly(lactide-coglycolide) (PLG) microparticles were shown to be substantially more potent than corresponding naked DNA vaccines. In mice immunized with HIV gag DNA, adsorption onto PLG increased CD8+ T-cell and antibody responses by ∼100- and ∼1,000-fold, respectively. In guinea pigs immunized with HIV env DNA adsorbed onto PLG, antibody responses showed a more rapid onset and achieved markedly higher enzyme-linked immunosorbent assay and neutralizing titers than in animals immunized with naked DNA. Further enhancement of antibody responses was observed in animals vaccinated with PLG/DNA microparticles formulated with aluminum phosphate. The magnitude of anti-Env antibody responses induced by PLG/DNA particles was equivalent to that induced by recombinant gp120 protein formulated with a strong adjuvant, MF-59. In guinea pigs immunized with a combination vaccine containing HIV env and HIV gag DNA plasmids on PLG microparticles, substantially superior antibody responses were induced against both components, as measured by onset, duration, and titer. Furthermore, PLG formulation overcame an apparent hyporesponsiveness of the env DNA component in the combination vaccine. Finally, preliminary data in rhesus macaques demonstrated a substantial enhancement of immune responses afforded by PLG/DNA. Therefore, formulation of DNA vaccines by adsorption onto PLG microparticles is a powerful means of increasing vaccine potency. PMID:11533167

  1. Protective immunity conferred by porcine circovirus 2 ORF2-based DNA vaccine in mice.

    PubMed

    Sylla, Seydou; Cong, Yan-Long; Sun, Yi-Xue; Yang, Gui-Lian; Ding, Xue-Mei; Yang, Zhan-Qing; Zhou, Yu-Long; Yang, Minnan; Wang, Chun-Feng; Ding, Zhuang

    2014-07-01

    Post-weaning multisystemic wasting syndrome (PMWS) associated with porcine circovirus type 2 (PCV2) has caused the swine industry significant health challenges and economic damage. Although inactivated and subunit vaccines against PMWS have been used widely, so far no DNA vaccine is available. In this study, with the aim of exploring a new route for developing a vaccine against PCV2, the immunogenicity of a DNA vaccine was evaluated in mice. The pEGFP-N1 vector was used to construct a PCV2 Cap gene recombinant vaccine. To assess the immunogenicity of pEGFP-Cap, 80 BALB/c mice were immunized three times at 2 weekly intervals with pEGFP-Cap, LG-strain vaccine, pEGFP-N1 vector or PBS and then challenged with PCV2. IgG and cytokines were assessed by indirect ELISA and ELISA, respectively. Specimens stained with hematoxylin and eosin (HE) and immunohistochemistry (IHC) techniques were examined histopathologically. It was found that vaccination of the mice with the pEGFP-Cap induced solid protection against PCV2 infection through induction of highly specific serum IgG antibodies and cytokines (IFN-γ and IL-10), and a small PCV2 viral load. The mice treated with the pEGFP-Cap and LG-strain developed no histopathologically detectable lesions (HE stain) and IHC techniques revealed only a few positive cells. Thus, this study demonstrated that recombinant pEGFP-Cap substantially alleviates PCV2 infection in mice and provides evidence that a DNA vaccine could be an alternative to PCV2 vaccines against PMWS. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

  2. Immunogenicity of Virus Like Particle Forming Baculoviral DNA Vaccine against Pandemic Influenza H1N1

    PubMed Central

    Gwon, Yong-Dae; Kim, Sehyun; Cho, Yeondong; Heo, Yoonki; Cho, Hansam; Park, Kihoon; Lee, Hee-Jung; Choi, Jiwon; Poo, Haryoung; Kim, Young Bong

    2016-01-01

    An outbreak of influenza H1N1 in 2009, representing the first influenza pandemic of the 21st century, was transmitted to over a million individuals and claimed 18,449 lives. The current status in many countries is to prepare influenza vaccine using cell-based or egg-based killed vaccine. However, traditional influenza vaccine platforms have several limitations. To overcome these limitations, many researchers have tried various approaches to develop alternative production platforms. One of the alternative approach, we reported the efficacy of influenza HA vaccination using a baculoviral DNA vaccine (AcHERV-HA). However, the immune response elicited by the AcHERV-HA vaccine, which only targets the HA antigen, was lower than that of the commercial killed vaccine. To overcome the limitations of this previous vaccine, we constructed a human endogenous retrovirus (HERV) envelope-coated, baculovirus-based, virus-like-particle (VLP)–forming DNA vaccine (termed AcHERV-VLP) against pandemic influenza A/California/04/2009 (pH1N1). BALB/c mice immunized with AcHERV-VLP (1×107 FFU AcHERV-VLP, i.m.) and compared with mice immunized with the killed vaccine or mice immunized with AcHERV-HA. As a result, AcHERV-VLP immunization produced a greater humoral immune response and exhibited neutralizing activity with an intrasubgroup H1 strain (PR8), elicited neutralizing antibody production, a high level of interferon-γ secretion in splenocytes, and diminished virus shedding in the lung after challenge with a lethal dose of influenza virus. In conclusion, VLP-forming baculovirus DNA vaccine could be a potential vaccine candidate capable of efficiently delivering DNA to the vaccinee and VLP forming DNA eliciting stronger immunogenicity than egg-based killed vaccines. PMID:27149064

  3. Efficacy of a DNA Vaccine Carrying Eimeria maxima Gam56 Antigen Gene against Coccidiosis in Chickens

    PubMed Central

    Xu, Jinjun; Zhang, Yan

    2013-01-01

    To control coccidiosis without using prophylactic medications, a DNA vaccine targeting the gametophyte antigen Gam56 from Eimeria maxima in chickens was constructed, and the immunogenicity and protective effects were evaluated. The ORF of Gam56 gene was cloned into an eukaryotic expression vector pcDNA3.1(zeo)+. Expression of Gam56 protein in COS-7 cells transfected with recombinant plasmid pcDNA-Gam56 was confirmed by indirect immunofluorescence assay. The DNA vaccine was injected intramuscularly to yellow feathered broilers of 1-week old at 3 dosages (25, 50, and 100 µg/chick). Injection was repeated once 1 week later. One week after the second injection, birds were challenged orally with 5×104 sporulated oocysts of E. maxima, then weighed and killed at day 8 post challenge. Blood samples were collected and examined for specific peripheral blood lymphocyte proliferation activity and serum antibody levels. Compared with control groups, the administration of pcDNA-Gam56 vaccine markedly increased the lymphocyte proliferation activity (P<0.05) at day 7 and 14 after the first immunization. The level of lymphocyte proliferation started to decrease on day 21 after the first immunization. A similar trend was seen in specific antibody levels. Among the 3 pcDNA-Gam56 immunized groups, the median dosage group displayed the highest lymphocyte proliferation and antibody levels (P<0.05). The median dosage group had the greatest relative body weight gain (89.7%), and the greatest oocyst shedding reduction (53.7%). These results indicate that median dosage of DNA vaccine had good immunogenicity and immune protection effects, and may be used in field applications for coccidiosis control. PMID:23710081

  4. DNA vaccination with VP2 gene fragment confers protection against Infectious Bursal Disease Virus in chickens.

    PubMed

    Pradhan, Satya Narayan; Prince, Prabhu Rajaiah; Madhumathi, Jayaprakasam; Arunkumar, Chakkaravarthy; Roy, Parimal; Narayanan, Rangarajan Badri; Antony, Usha

    2014-06-25

    Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens by destruction of antibody producing B cells in the Bursa of Fabricius and poses a potential threat to the poultry industry. We have examined the protective efficacy of a subunit DNA vaccine against IBDV infection in chickens in this study. An immunodominant VP2 gene fragment (VP252-417) was cloned into CMV promoter based DNA vaccine vector pVAX1 and in vitro expression of the DNA encoded antigens was confirmed by transfection of CHO cells with vaccine constructs followed by RT-PCR and western blot analysis using IBDV-antiserum. Two weeks old chickens were immunized intramuscularly with pVAXVP252-417 and the in vivo transcription of the plasmid DNA was confirmed by RT-PCR analysis of DNA injected muscle tissue at different intervals of post immunization. Tissue distribution analysis revealed that the plasmid DNA was extensively distributed in muscle, spleen, kidney, liver, and bursa tissues. Chickens immunized with pVAXVP252-417 developed high titer (1:12,000) of anti-VP252-417 antibodies. Further, chicken splenocytes from pVAXVP252-417 immunized group showed a significantly high proliferation to the whole viral and recombinant antigen (P<0.01) compared to control groups, which implies that pVAXVP252-417 codes for immunogenic fragment which has epitopes capable of eliciting both B and T cell responses. This is evident by the fact that, pVAXVP252-417 immunized chicken conferred 75% protection against virulent IBDV (vIBDV) challenge compared to the control group. Thus, the present study confirms that the immunodominant VP2 fragment can be used as a potential DNA vaccine against IBDV infection in chickens. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. DNA vaccination elicits protective immune responses against pandemic and classic swine influenza viruses in pigs.

    PubMed

    Gorres, J Patrick; Lager, Kelly M; Kong, Wing-Pui; Royals, Michael; Todd, John-Paul; Vincent, Amy L; Wei, Chih-Jen; Loving, Crystal L; Zanella, Eraldo L; Janke, Bruce; Kehrli, Marcus E; Nabel, Gary J; Rao, Srinivas S

    2011-11-01

    Swine influenza is a highly contagious viral infection in pigs that significantly impacts the pork industry due to weight loss and secondary infections. There is also the potential of a significant threat to public health, as was seen in 2009 when the pandemic H1N1 influenza virus strain emerged from reassortment events among avian, swine, and human influenza viruses within pigs. As classic and pandemic H1N1 strains now circulate in swine, an effective vaccine may be the best strategy to protect the pork industry and public health. Current inactivated-virus vaccines available for swine influenza protect only against viral strains closely related to the vaccine strain, and egg-based production of these vaccines is insufficient to respond to large outbreaks. DNA vaccines are a promising alternative since they can potentially induce broad-based protection with more efficient production methods. In this study we evaluated the potentials of monovalent and trivalent DNA vaccine constructs to (i) elicit both humoral and gamma interferon (IFN-γ) responses and (ii) protect pigs against viral shedding and lung disease after challenge with pandemic H1N1 or classic swine H1N1 influenza virus. We also compared the efficiency of a needle-free vaccine delivery method to that of a conventional needle/syringe injection. We report that DNA vaccination elicits robust serum antibody and cellular responses after three immunizations and confers significant protection against influenza virus challenge. Needle-free delivery elicited improved antibody responses with the same efficiency as conventional injection and should be considered for development as a practical alternative for vaccine administration.

  6. Induction of protective therapy for autoimmune diseases by targeted DNA vaccines encoding pro-inflammatory cytokines and chemokines.

    PubMed

    Karin, Nathan

    2004-02-01

    T-cell-mediated autoimmune diseases such as multiple sclerosis, rheumatoid arthritis or type 1 diabetes result from an aggressive attack of self-components by autoimmune T-cells. Pro-inflammatory mediators, particularly cytokines and chemokines, direct the homing and effectorfunction of these cells. It has recently been demonstrated that the immune system, which can attack self-components, also generates 'beneficial' autoimmunity against pro-inflammatory mediators. During the course of an autoimmune condition, and to a much lesser extent in response to microbial inflammation, the immune system produces auto-antibodies to pro-inflammatory mediators. This reduces the harm from these diseases. We also discovered that targeted DNA vaccines could effectively amplify these responses to provide protective immunity. The underlying mechanism is partially understood. At the site of immunization, the relevant gene product is produced and then presented by dendritic cells/macrophages, which undergo activation due to an interaction of plasmid CpG with toll-like receptor 9 on the dendritic cell. This then activates CD4+ T-cells, which help the production of T-cell-dependent antibodies against the gene product of the vaccines. These antibodies neutralize their target product and suppress inflammation. This review explores this interesting concept and its therapeutic implications.

  7. Bursal transcriptome of chickens protected by DNA vaccination versus those challenged with infectious bursal disease virus.

    PubMed

    Lee, Chih-Chun; Kim, Bong-Suk; Wu, Ching Ching; Lin, Tsang Long

    2015-01-01

    Infectious bursal disease virus (IBDV) infection destroys the bursa of Fabricius, causing immunosuppression and rendering chickens susceptible to secondary bacterial or viral infections. IBDV large-segment-protein-expressing DNA has been shown to confer complete protection of chickens from infectious bursal disease (IBD). The purpose of the present study was to compare DNA-vaccinated chickens and unvaccinated chickens upon IBDV challenge by transcriptomic analysis of bursa regarding innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. One-day-old specific-pathogen-free chickens were vaccinated intramuscularly three times at weekly intervals with IBDV large-segment-protein-expressing DNA. Chickens were challenged orally with 8.2 × 10(2) times the egg infective dose (EID)50 of IBDV strain variant E (VE) one week after the last vaccination. Bursae collected at 0.5, 1, 3, 5, 7, and 10 days post-challenge (dpc) were subjected to real-time RT-PCR quantification of bursal transcripts related to innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. The expression levels of granzyme K and CD8 in DNA-vaccinated chickens were significantly (p < 0.05) higher than those in unvaccinated chickens upon IBDV challenge at 0.5 or 1 dpc. The expression levels of other genes involved in innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport were not upregulated or downregulated in DNA-vaccinated chickens during IBDV challenge. Bursal transcripts related to innate immunity and inflammation, including TLR3, MDA5, IFN-α, IFN-β, IRF-1, IRF-10, IL-1β, IL-6, IL-8, iNOS, granzyme A, granzyme K and IL-10, were upregulated or significantly (p < 0.05) upregulated at 3 dpc and later in unvaccinated chickens challenged with IBDV. The expression levels of genes related to immune cell regulation, apoptosis and glucose transport, including CD4, CD8, IL-2, IFN-γ, IL-12(p40), IL-18, GM-CSF, GATA-3

  8. Induction of protection against porcine cysticercosis in growing pigs by DNA vaccination.

    PubMed

    Guo, Aijiang; Jin, Zhizhong; Zheng, Yadong; Hai, Gang; Yuan, Gailing; Li, Hailong; Cai, Xuepeng

    2007-01-02

    A DNA vaccine, pcDNA3-B, was developed by using the nucleotide sequence of Taenia solium B antigen and cloning into pcDNA3.1 plasmid. The growing pigs were vaccinated by one intramuscular infection of 200 or 1000 microg pcDNA3-B. The immunization with 1000 microg of pcDNA3-B showed 92.6% protection when the pigs were challenged by T. solium eggs and four of the five pigs vaccinated had no viable cysts. The results provide encouraging information on the use of pcDNA3-B vaccination for the prevention of cysticercosis.

  9. Enhanced neutralising antibody response to bovine viral diarrhoea virus (BVDV) induced by DNA vaccination in calves.

    PubMed

    R El-Attar, Laila M; Thomas, Carole; Luke, Jeremy; A Williams, James; Brownlie, Joe

    2015-07-31

    DNA vaccination is effective in inducing potent immunity in mice; however it appears to be less so in large animals. Increasing the dose of DNA plasmid to activate innate immunity has been shown to improve DNA vaccine adaptive immunity. Retinoic acid-inducible gene I (RIG-I) is a critical cytoplasmic double-stranded RNA pattern receptor required for innate immune activation in response to viral infection. RIG-I recognise viral RNA and trigger antiviral response, resulting in type I interferon (IFN) and inflammatory cytokine production. In an attempt to enhance the antibody response induced by BVDV DNA in cattle, we expressed BVDV truncated E2 (E2t) and NS3 codon optimised antigens from antibiotic free-plasmid vectors expressing a RIG-I agonist and designated either NTC E2t(co) and NTC NS3(co). To evaluate vaccine efficacy, groups of five BVDV-free calves were intramuscularly injected three times with NTC E2t(co) and NTC NS3(co) vaccine plasmids individually or in combination. Animals vaccinated with our (previously published) conventional DNA vaccines pSecTag/E2 and pTriExNS3 and plasmids expressing RIG-I agonist only presented both the positive and mock-vaccine groups. Our results showed that vaccines coexpressing E2t with a RIG-I agonist induced significantly higher E2 antigen specific antibody response (p<0.05). Additionally, E2t augmented the immune response to NS3 when the two vaccines were delivered in combination. Despite the lack of complete protection, on challenge day 4/5 calves vaccinated with NTC E2t(co) alone or NTC E2t(co) plus NTC NS3(co) had neutralising antibody titres exceeding 1/240 compared to 1/5 in the mock vaccine control group. Based on our results we conclude that co-expression of a RIG-I agonist with viral antigen could enhance DNA vaccine potency in cattle.

  10. Genetic diversity and associated pathology of rhabdovirus infections in farmed and wild perch Perca fluviatilis in Ireland.

    PubMed

    Ruane, N M; Rodger, H D; McCarthy, L J; Swords, D; Dodge, M; Kerr, R C; Henshilwood, K; Stone, D M

    2014-12-02

    Rhabdovirus infections are an emerging problem for both wild and farmed freshwater fish in Northern Europe. In October 2005, a clinical outbreak with an approximate mortality rate of 40% occurred in a single batch of juvenile perch on a farm in the Republic of Ireland. Clinical signs developed slowly and were consistent with a perch rhabdovirus infection: signs included haemorrhages at the base of the fins and apparent impairment of the central nervous system (manifested as loss of equilibrium and erratic swimming behaviour). Studies suggest that the infected fish originated from a hatchery within the country which relied on wild fish broodstock to supplement the production of perch juveniles. A related rhabdovirus was subsequently isolated from this hatchery. Virus isolation studies have shown that rhabdoviruses were often isolated from wild fish in the vicinity of the hatchery between 1993 and 2005. All isolates were analysed using a generic primer set specific for the L gene of fish vesiculotype viruses. Phylogenetic analysis revealed that all isolates recovered from perch clustered together with the European lake trout rhabdovirus (903/87) of the genus Perhabdovirus. In addition to this, anguillid rhabdovirus was isolated from eel, and the partial L-gene sequence of a previously reported isolate from tench clustered with the pike fry rhabdoviruses, in the genus Sprivivirus.

  11. Promoting effect of Antrodia camphorata as an immunomodulating adjuvant on the antitumor efficacy of HER-2/neu DNA vaccine.

    PubMed

    Huang, Chia-Hsin; Chang, Chia-Che; Lin, Chiu-Mei; Wang, Sin-Ting; Wu, Min-Tze; Li, Eric I C; Chang, Hsien-Chang; Lin, Chi-Chen

    2010-08-01

    It is well known that DNA vaccines induce protective humoral and cell-mediated immune responses in several animal models. Antrodia camphorata (AC) is a unique basidiomycete fungus of the Polyporaceae family that only grows on the aromatic tree Cinnamomum kanehirai Hayata (Lauraceae) endemic to Taiwan. Importantly, AC has been shown to be highly beneficial in the treatment and prevention of cancer. The goal of this study was to investigate whether AC is able to augment the antitumor immune properties of a HER-2/neu DNA vaccine in a mouse model in which p185neu is overexpressed in MBT-2 tumor cells. Compared with the mice that received the HER-2/neu DNA vaccine alone, co-treatment with AC suppressed tumor growth and extended the survival rate. This increase in the antitumor efficacy was attributed to the enhancement of the Th1-like cellular immune response by the HER-2/neu DNA vaccine-AC combination. Evidence for this came from the marked increase in the IFN-gamma mRNA expression in CD4+ T cells in the draining inguinal lymph nodes, an increase in the number of functional HER-2/neu-specific CTLs, and the increased tumor infiltration of both CD4+ and CD8+ T cells, depletion of which abolishes the antitumor effect of the HER-2/neu DNA vaccine-AC therapy. Our results further indicate that the treatment of mice with AC enhanced DC activation and production of Th1-activating cytokines (e.g. IL-12, and IFN-alpha) in the draining lymph nodes, which were sufficient to directly stimulate T cell proliferation and higher IFN-gamma production in response to ErbB2. Overall, these results clearly demonstrate that AC represents a promising immunomodulatory adjuvant that could enhance the therapeutic potency of HER-2/neu DNA vaccines in cancer therapy.

  12. Immunogenicity of bivalent human papillomavirus DNA vaccine using human endogenous retrovirus envelope-coated baculoviral vectors in mice and pigs.

    PubMed

    Lee, Hee-Jung; Hur, Yoon-Ki; Cho, Youn-Dong; Kim, Mi-Gyeong; Lee, Hoon-Taek; Oh, Yu-Kyoung; Kim, Young Bong

    2012-01-01

    Human papillomavirus is known to be the major pathogen of cervical cancer. Here, we report the efficacy of a bivalent human papillomavirus type 16 and 18 DNA vaccine system following repeated dosing in mice and pigs using a recombinant baculovirus bearing human endogenous retrovirus envelope protein (AcHERV) as a vector. The intramuscular administration of AcHERV-based HPV16L1 and HPV18L1 DNA vaccines induced antigen-specific serum IgG, vaginal IgA, and neutralizing antibodies to levels comparable to those achieved using the commercially marketed vaccine Cervarix. Similar to Cervarix, AcHERV-based bivalent vaccinations completely blocked subsequent vaginal challenge with HPV type-specific pseudovirions. However, AcHERV-based bivalent vaccinations induced significantly higher cell-mediated immune responses than Cervarix, promoting 4.5- (HPV16L1) and 3.9-(HPV18L1) fold higher interferon-γ production in splenocytes upon stimulation with antigen type-specific pseudovirions. Repeated dosing did not affect the immunogenicity of AcHERV DNA vaccines. Three sequential immunizations with AcHERV-HP18L1 DNA vaccine followed by three repeated dosing with AcHERV-HP16L1 over 11 weeks induced an initial production of anti-HPV18L1 antibody followed by subsequent induction of anti-HPV16L1 antibody. Finally, AcHERV-based bivalent DNA vaccination induced antigen-specific serum IgG immune responses in pigs. These results support the further development of AcHERV as a bivalent human papillomavirus DNA vaccine system for use in preventing the viral infection as well as treating the infected women by inducing both humoral and cell-mediated immune responses. Moreover, the possibility of repeated dosing indicates the utility of AcHERV system for reusable vectors of other viral pathogen vaccines.

  13. Enhanced non-inflammasome mediated immune responses by mannosylated zwitterionic-based cationic liposomes for HIV DNA vaccines.

    PubMed

    Qiao, Chenmeng; Liu, Jiandong; Yang, Jun; Li, Yan; Weng, Jie; Shao, Yiming; Zhang, Xin

    2016-04-01

    Human immunodeficiency virus (HIV) DNA vaccine can induce cellular and humoral immunity. A safe and effective HIV DNA vaccine is urgent need to prevent the spread of acquired immune deficiency syndrome (AIDS). The major drawback of DNA vaccines is the low immunogenicity, which is caused by the poor delivery to antigen presenting cells and insufficient antigen expression. Sparked by the capability of endosomal/lysosomal escape of the zwitterionic lipid distearoyl phosphoethanol-amine-polycarboxybetaine (DSPE-PCB), we attempted to develop a zwitterionic-based cationic liposome with enhanced immunogenicity of DNA vaccines. The mannosylated zwitterionic-based cationic liposome (man-ZCL) was constructed as a DNA vaccine adjuvant for HIV vaccination. Man-ZCL could complex with DNA antigens to form a tight structure and protect them from nuclei enzyme degradation. Benefited from the capability of the specific mannose receptor mediated antigen processing cells targeting and enhanced endosomal/lysosomal escape, the man-ZCL lipoplexes were supposed to promote antigen presentation and the immunogenicity of DNA vaccines. In vitro and in vivo results revealed that man-ZCL lipoplexes showed enhanced anti-HIV immune responses and lower toxicity compared with CpG/DNA and Lipo2k/DNA, and triggered a Th1/Th2 mixed immunity. An antigen-depot effect was observed in the administration site, and this resulted in enhanced retention of DNA antigens in draining lymph nodes. Most importantly, the man-ZCL could assist to activate T cells through a non-inflammasome pathway. These findings suggested that the man-ZCL could be potentially applied as a safe and efficient DNA adjuvant for HIV vaccines.

  14. Construction and immune effect of Haemophilus parasuis DNA vaccine encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in mice.

    PubMed

    Fu, Shulin; Zhang, Minmin; Ou, Jiwen; Liu, Huazhen; Tan, Chen; Liu, Jinlin; Chen, Huanchun; Bei, Weicheng

    2012-11-06

    Haemophilus parasuis, the causative agent of swine polyserositis, polyarthritis, and meningitis, is one of the most important bacterial diseases of pigs worldwide. The development of a vaccine against H. parasuis has been impeded due to the lack of induction of reliable cross-serotype protection. In this study the gapA gene that encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was shown to be present and highly conserved in various serotypes of H. parasuis and we constructed a novel DNA vaccine encoding GAPDH (pCgap) to evaluate the immune response and protective efficacy against infection with H. parasuis MD0322 serovar 4 or SH0165 serovar 5 in mice. A significant antibody response against GAPDH was generated following pCgap intramuscular immunization; moreover, antibodies to the pCgap DNA vaccine were bactericidal, suggesting that it was expressed in vivo. The gapA transcript was detected in muscle, liver, spleen, and kidney of the mice seven days post-vaccination. The IgG subclass (IgG1 and IgG2a) analysis indicated that the DNA vaccine induced both Th1 and Th2 immune responses, but the IgG1 response was greater than the IgG2a response. Moreover, the groups vaccinated with the pCgap vaccine exhibited 83.3% and 50% protective efficacy against the H. parasuis MD0322 serovar 4 or SH0165 serovar 5 challenges, respectively. The pCgap DNA vaccine provided significantly greater protective efficacy compared to the negative control groups or blank control groups (P<0.05 for both). Taken together, these findings indicate that the pCgap DNA vaccine provides a novel strategy against infection of H. parasuis and offer insight concerning the underlying immune mechanisms of a bacterial DNA vaccine.

  15. Isolation and characterization of a novel Rhabdovirus from a wild boar (Sus scrofa) in Japan.

    PubMed

    Sakai, Kouji; Hagiwara, Katsuro; Omatsu, Tsutomu; Hamasaki, Chinami; Kuwata, Ryusei; Shimoda, Hiroshi; Suzuki, Kazuo; Endoh, Daiji; Nagata, Noriyo; Nagai, Makoto; Katayama, Yukie; Oba, Mami; Kurane, Ichiro; Saijo, Masayuki; Morikawa, Shigeru; Mizutani, Tetsuya; Maeda, Ken

    2015-09-30

    A novel rhabdovirus was isolated from the serum of a healthy Japanese wild boar (Sus scrofa leucomystax) and identified using the rapid determination system for viral nucleic acid sequences (RDV), next-generation sequencing, and electron microscopy. The virus was tentatively named wild boar rhabdovirus 1 (WBRV1). Phylogenetic analysis of the entire genome sequence indicated that WBRV1 is closely related to Tupaia rhabdovirus (TRV), which was isolated from cultured cells of hepatocellular carcinoma tissue of tree shrew. TRV has not been assigned to any genus of Rhabdoviridae till date. Analysis of the L gene indicated that WBRV1 belongs to the genus Vesiculovirus. These observations suggest that both TRV and WBRV1 belong to a new genus of Rhabdoviridae. Next-generation genome sequencing of WBRV1 revealed 5 open reading frames of 1329, 765, 627, 1629, and 6336 bases in length. The WBRV1 gene sequences are similar to those of other rhabdoviruses. Epizootiological analysis of a population of wild boars in Wakayama prefecture in Japan indicated that 6.5% were positive for the WBRV1 gene and 52% were positive for WBRV1-neutralizing antibodies. Furthermore, such viral neutralizing antibodies were found in domestic pigs in another prefecture. WBRV1 was inoculated intranasally and intraperitoneally into SCID and BALB/c mice and viral RNA was detected in SCID mice, suggesting that WBRV1 can replicate in immunocompromised mice. These results indicate this novel virus is endemic in wild animals and livestock in Japan.

  16. In vitro inhibition of fish rhabdoviruses by Japanese flounder, Paralichthys olivaceus Mx.

    PubMed

    Caipang, Christopher Marlowe A; Hirono, Ikuo; Aoki, Takashi

    2003-12-20

    A homologous fish cell line stably expressing the recombinant Japanese flounder Mx (JFMx) was infected with hirame rhabdovirus (HIRRV) and viral hemorrhagic septicemia virus (VHSV), both of which are negative single-stranded RNA viruses belonging to the Rhabdoviridae family. Analysis of primary transcription of the two rhabdoviruses showed that there was lower expression level and copy number of the viral nucleoprotein transcript in the JFMx-transfected cell line than the infected, control cells, although no significant difference was observed. This suggests that JFMx may not be a potent inhibitor of rhabdoviral primary transcription. Kinetics of rhabdovirus expression by RT-PCR and quantitative real-time RT-PCR showed reduced levels of the rhabdoviral glycoprotein and nucleoprotein transcripts over time, indicating the possible role of JFMx in blocking rhabdoviral replication by interfering with the transcription of the viral subgenomic mRNAs. Significant inhibition in rhabdovirus replication consequently resulted in the synthesis of fewer viral particles. This may explain why JFMx-expressing cells are less susceptible to virus-induced cell lysis, and thus, why they would have a significantly higher survival than the infected, control cells. These results provide direct evidence that JFMx has an antiviral effect in vitro.

  17. Evolutionary dynamics and genetic diversity from three genes of Anguillid rhabdovirus.

    PubMed

    Bellec, Laure; Cabon, Joelle; Bergmann, Sven; de Boisséson, Claire; Engelsma, Marc; Haenen, Olga; Morin, Thierry; Olesen, Niels Jørgen; Schuetze, Heike; Toffan, Anna; Way, Keith; Bigarré, Laurent

    2014-11-01

    Wild freshwater eel populations have dramatically declined in recent past decades in Europe and America, partially through the impact of several factors including the wide spread of infectious diseases. The anguillid rhabdoviruses eel virus European X (EVEX) and eel virus American (EVA) potentially play a role in this decline, even if their real contribution is still unclear. In this study, we investigate the evolutionary dynamics and genetic diversity of anguiillid rhabdoviruses by analysing sequences from the glycoprotein, nucleoprotein and phosphoprotein (P) genes of 57 viral strains collected from seven countries over 40 years using maximum-likelihood and Bayesian approaches. Phylogenetic trees from the three genes are congruent and allow two monophyletic groups, European and American, to be clearly distinguished. Results of nucleotide substitution rates per site per year indicate that the P gene is expected to evolve most rapidly. The nucleotide diversity observed is low (2-3 %) for the three genes, with a significantly higher variability within the P gene, which encodes multiple proteins from a single genomic RNA sequence, particularly a small C protein. This putative C protein is a potential molecular marker suitable for characterization of distinct genotypes within anguillid rhabdoviruses. This study provides, to our knowledge, the first molecular characterization of EVA, brings new insights to the evolutionary dynamics of two genotypes of Anguillid rhabdovirus, and is a baseline for further investigations on the tracking of its spread.

  18. RNA Splicing in a New Rhabdovirus from Culex Mosquitoes▿†

    PubMed Central

    Kuwata, Ryusei; Isawa, Haruhiko; Hoshino, Keita; Tsuda, Yoshio; Yanase, Tohru; Sasaki, Toshinori; Kobayashi, Mutsuo; Sawabe, Kyoko

    2011-01-01

    Among members of the order Mononegavirales, RNA splicing events have been found only in the family Bornaviridae. Here, we report that a new rhabdovirus isolated from the mosquito Culex tritaeniorhynchus replicates in the nuclei of infected cells and requires RNA splicing for viral mRNA maturation. The virus, designated Culex tritaeniorhynchus rhabdovirus (CTRV), shares a similar genome organization with other rhabdoviruses, except for the presence of a putative intron in the coding region for the L protein. Molecular phylogenetic studies indicated that CTRV belongs to the family Rhabdoviridae, but it is yet to be assigned a genus. Electron microscopic analysis revealed that the CTRV virion is extremely elongated, unlike virions of rhabdoviruses, which are generally bullet shaped. Northern hybridization confirmed that a large transcript (approximately 6,500 nucleotides [nt]) from the CTRV L gene was present in the infected cells. Strand-specific reverse transcription-PCR (RT-PCR) analyses identified the intron-exon boundaries and the 76-nt intron sequence, which contains the typical motif for eukaryotic spliceosomal intron-splice donor/acceptor sites (GU-AG), a predicted branch point, and a polypyrimidine tract. In situ hybridization exhibited that viral RNAs are primarily localized in the nucleus of infected cells, indicating that CTRV replicates in the nucleus and is allowed to utilize the host's nuclear splicing machinery. This is the first report of RNA splicing among the members of the family Rhabdoviridae. PMID:21507977

  19. Evaluation of the anti-tuberculosis activity generated by different multigene DNA vaccine constructs.

    PubMed

    Sali, Michela; Clarizio, Sandra; Pusceddu, Cinzia; Zumbo, Antonella; Pecorini, Giovanni; Rocca, Stefano; Zanetti, Stefania; Delogu, Giovanni; Fadda, Giovanni

    2008-05-01

    Development of multigenic constructs expressing Mycobacterium tuberculosis (Mtb) antigens may be a strategy to obtain improved DNA vaccines against tuberculosis (TB). Several multigenic constructs expressing two or three Mtb antigens as fusion proteins were developed, both as tPA- and ubiquitin-fusion proteins. To demonstrate proper protein expression and intracellular turnover all multiantigens were tagged with the HA epitope and constructs were used to transfect rhabdomyosarcoma (RD) cells. Antigen expression was demonstrated by immunofluorescence using anti-HA antibodies. C57Bl/6 mice were immunized with selected constructs and protective activity was assessed following aerogenic challenge with Mtb. Several of these constructs induced a significant level of protection in the lung and in the spleen. Immunization with the construct expressing tPA85B-ES6 induced a level of protection that approached that provided by BCG. Immunization with a combination of these constructs induced levels of protection that were not superior to those elicited by a single combination, and immunization with a construct expressing five Mtb antigens could not provide an improved level of protection compared to tPA85B-ES6. We conclude that the activity of a DNA vaccine based on tPA85B-ES6 cannot be enhanced by broadening the antigen repertoire with other highly immunogenic secreted Mtb proteins.

  20. Antiangiogenic immunotherapy targeting Flk-1, DNA vaccine and adoptive T cell transfer, inhibits ocular neovascularization

    SciTech Connect

    Zhang, Han; Sonoda, Koh-Hei; Hijioka, Kuniaki; Qiao, Hong; Oshima, Yuji; Ishibashi, Tatsuro

    2009-04-17

    Ocular neovascularization (NV) is the primary cause of blindness in a wide range of ocular diseases. The exact mechanism underlying the pathogenesis of ocular NV is not yet well understood, and so there is no satisfactory therapy for ocular NV. Here, we describe a strategy targeting Flk-1, a self-antigen overexpressed on proliferating endothelial cells in ocular NV, by antiangiogenic immunotherapy-DNA vaccine and adoptive T cell therapy. An oral DNA vaccine encoding Flk-1 carried by attenuated Salmonella typhimurium markedly suppressed development of laser-induced choroidal NV. We further demonstrated that adoptive transfer of vaccine-induced CD8{sup +} T cells reduced pathological preretinal NV, with a concomitant facilitation of physiological revascularization after oxygen-induced retinal vessel obliteration. However, physiological retinal vascular development was unaffected in neonatal mice transferred with vaccine-induced CD8{sup +} T cells. These findings suggested that antiangiogenic immunotherapy targeting Flk-1 such as vaccination and adoptive immunotherapy may contribute to future therapies for ocular NV.

  1. Immunogenicity and clinical protection against equine influenza by gene-based DNA vaccination of ponies

    PubMed Central

    Ault, Alida; Zajac, Alyse M.; Kong, Wing-Pui; Gorres, J. Patrick; Royals, Michael; Wei, Chih-Jen; Bao, Saran; Yang, Zhi-yong; Reedy, Stephanie E.; Sturgill, Tracy L.; Page, Allen E.; Donofrio-Newman, Jennifer; Adams, Amanda A.; Balasuriya, Udeni B.R.; Horohov, David W.; Chambers, Thomas M.; Nabel, Gary J.; Rao, Srinivas S.

    2012-01-01

    Equine influenza A (H3N8) virus is a leading cause of infectious respiratory disease in horses causing widespread morbidity and economic losses. As with influenza in other species, equine influenza strains continuously mutate, requiring constant re-evaluation of current vaccines and development of new vaccines. Current inactivated (killed) vaccines, while efficacious, only offer limited protection against multiple strains and require frequent boosts. Ongoing research into new vaccine technologies, including gene-based vaccines, aims to increase the neutralization potency, breadth, and duration of protective immunity of new or existing vaccines. In these hypothesis-generating experiments, we demonstrate that a DNA vaccine expressing the hemagglutinin protein of equine H3N8 influenza virus generates homologous and heterologous immune responses, and protects against clinical disease and viral replication following homologous H3N8 infection in horses. Furthermore, we demonstrate that a needle-free delivery device is as efficient and effective as conventional parenteral injection using a needle and syringe. The observed trends in this study drive the hypothesis that DNA vaccines offer a safe, effective, and promising alternative approach for veterinary vaccines against influenza, and applicable to combat equine influenza. PMID:22449425

  2. Construction and Nonclinical Testing of a Puumala Virus Synthetic M Gene-Based DNA Vaccine

    PubMed Central

    Brocato, R. L.; Josleyn, M. J.; Wahl-Jensen, V.; Schmaljohn, C. S.

    2013-01-01

    Puumala virus (PUUV) is a causative agent of hemorrhagic fever with renal syndrome (HFRS). Although PUUV-associated HFRS does not result in high case-fatality rates, the social and economic impact is considerable. There is no licensed vaccine or specific therapeutic to prevent or treat HFRS. Here we report the synthesis of a codon-optimized, full-length M segment open reading frame and its cloning into a DNA vaccine vector to produce the plasmid pWRG/PUU-M(s2). pWRG/PUU-M(s2) delivered by gene gun produced high-titer neutralizing antibodies in hamsters and nonhuman primates. Vaccination with pWRG/PUU-M(s2) protected hamsters against infection with PUUV but not against infection by related HFRS-associated hantaviruses. Unexpectedly, vaccination protected hamsters in a lethal disease model of Andes virus (ANDV) in the absence of ANDV cross-neutralizing antibodies. This is the first evidence that an experimental DNA vaccine for HFRS can provide protection in a hantavirus lethal disease model. PMID:23239797

  3. A DNA vaccine against dolphin morbillivirus is immunogenic in bottlenose dolphins.

    PubMed

    Vaughan, Kerrie; Del Crew, Jason; Hermanson, Gary; Wloch, Mary K; Riffenburgh, Robert H; Smith, Cynthia R; Van Bonn, William G

    2007-12-15

    The immunization of exotic species presents considerable challenges. Nevertheless, for facilities like zoos, animal parks, government facilities and non-profit conservation groups, the protection of valuable and endangered species from infectious disease is a growing concern. The rationale for immunization in these species parallels that for human and companion animals; to decrease the incidence of disease. The U.S. Navy Marine Mammal Program, in collaboration with industry and academic partners, has developed and evaluated a DNA vaccine targeting a marine viral pathogen - dolphin morbillivirus (DMV). The DMV vaccine consists of the fusion (F) and hemagglutinin (H) genes of DMV. Vaccine constructs (pVR-DMV-F and pVR-DMV-H) were evaluated for expression in vitro and then for immunogenicity in mice. Injection protocols were designed for application in Atlantic bottlenose dolphins (Tursiops truncatus) to balance vaccine effectiveness with clinical utility. Six dolphins were inoculated, four animals received both pDMV-F and pDMV-H and two animals received a mock vaccine (vector alone). All animals received an inoculation week 0, followed by two booster injections weeks 8 and 14. Vaccine-specific immune responses were documented in all four vaccinated animals. To our knowledge, this is the first report of pathogen-specific immunogenicity to a DNA vaccine in an aquatic mammal species.

  4. Optimized DNA Vaccines to Specifically Induce Therapeutic CD8 T Cell Responses Against Autochthonous Breast Tumors

    PubMed Central

    Cho, Hyun-Il; Niu, Guilian; Bradley, Norma; Celis, Esteban

    2008-01-01

    Vaccines capable of inducing CD8 T cell responses to antigens expressed by tumor cells are considered as attractive choices for the treatment and prevention of malignant diseases. Our group has previously reported that immunization with synthetic peptide corresponding to a CD8 T cell epitope derived from the rat neu oncogene administered together with a Toll-like receptor agonist as adjuvant, induced immune responses that translated into prophylactic and therapeutic benefit against autochthonous tumors in an animal model of breast cancer (BALB-neuT mice). DNA-based vaccines offer some advantages over peptide vaccines, such as the possibility of including multiple CD8 T cell epitopes in a single construct. Thus, we have evaluated the use of DNA vaccination for its ability to generate effective CD8 T cell responses against breast tumors expressing the rat neu oncogene. The results show that as with peptide vaccination, DNA-based vaccines were very effective in stimulating tumor-reactive CD8 T cell responses. Moreover, vaccination with modified DNA plasmids resulted in significant anti-tumor effects that were mediated by CD8 T cells without the requirement of generating antibodies to the product of rat neu. These results bear importance for the design of DNA vaccines for the treatment and prevention of cancer. PMID:18253731

  5. On the efficacy of malaria DNA vaccination with magnetic gene vectors.

    PubMed

    Nawwab Al-Deen, Fatin; Ma, Charles; Xiang, Sue D; Selomulya, Cordelia; Plebanski, Magdalena; Coppel, Ross L

    2013-05-28

    We investigated the efficacy and types of immune responses from plasmid malaria DNA vaccine encoding VR1020-PyMSP119 condensed on the surface of polyethyleneimine (PEI)-coated SPIONs. In vivo mouse studies were done firstly to determine the optimum magnetic vector composition, and then to observe immune responses elicited when magnetic vectors were introduced via different administration routes. Higher serum antibody titers against PyMSP119 were observed with intraperitoneal and intramuscular injections than subcutaneous and intradermal injections. Robust IgG2a and IgG1 responses were observed for intraperitoneal administration, which could be due to the physiology of peritoneum as a major reservoir of macrophages and dendritic cells. Heterologous DNA prime followed by single protein boost vaccination regime also enhanced IgG2a, IgG1, and IgG2b responses, indicating the induction of appropriate memory immunity that can be elicited by protein on recall. These outcomes support the possibility to design superparamagnetic nanoparticle-based DNA vaccines to optimally evoke desired antibody responses, useful for a variety of diseases including malaria.

  6. Advances in host and vector development for the production of plasmid DNA vaccines.

    PubMed

    Mairhofer, Juergen; Lara, Alvaro R

    2014-01-01

    Recent developments in DNA vaccine research provide a new momentum for this rather young and potentially disruptive technology. Gene-based vaccines are capable of eliciting protective immunity in humans to persistent intracellular pathogens, such as HIV, malaria, and tuberculosis, for which the conventional vaccine technologies have failed so far. The recent identification and characterization of genes coding for tumor antigens has stimulated the development of DNA-based antigen-specific cancer vaccines. Although most academic researchers consider the production of reasonable amounts of plasmid DNA (pDNA) for immunological studies relatively easy to solve, problems often arise during this first phase of production. In this chapter we review the current state of the art of pDNA production at small (shake flasks) and mid-scales (lab-scale bioreactor fermentations) and address new trends in vector design and strain engineering. We will guide the reader through the different stages of process design starting from choosing the most appropriate plasmid backbone, choosing the right Escherichia coli (E. coli) strain for production, and cultivation media and scale-up issues. In addition, we will address some points concerning the safety and potency of the produced plasmids, with special focus on producing antibiotic resistance-free plasmids. The main goal of this chapter is to make immunologists aware of the fact that production of the pDNA vaccine has to be performed with as much as attention and care as the rest of their research.

  7. Immunogenic and protective effects of an oral DNA vaccine against infectious pancreatic necrosis virus in fish.

    PubMed

    de las Heras, Ana I; Rodríguez Saint-Jean, S; Pérez-Prieto, Sara I

    2010-04-01

    DNA vaccines and oral DNA-based immunotherapy against infectious pancreatic necrosis virus (IPNV) have scarcely been studied in salmonid fish. Here, a vector with the capsid VP2 gene inserted was encapsulated in alginate microspheres to avoid the aggressive gastrointestinal conditions experienced following oral administration. Alginate microspheres were effective to protect the pDNA encoding VP2, which was expressed early in different organs of the vaccinated trout and that persisted for at least 60 days. The vaccine induces innate immune responses, raising the expression of IFN more than 10-fold relative to the fish vaccinated with the empty plasmid, at 7 and 15 days post-vaccination. Likewise, maximal expression of the IFN-induced antiviral Mx protein was recorded 15 days post-vaccination and neutralizing antibodies were also detected after 15 days, although their titre rose further at 21 days post-vaccination. Protection was high in the immunized fish, which showed around an 80% relative survival when challenged 15 and 30 days after vaccine delivery. Very low viral load with respect to the control group was detected in the vaccinated fish that survived 45 days after challenge. Thus, this study demonstrates the potential of the encapsulation technique for IPNV-DNA vaccine delivery and the relevance of the IPNV-VP2 gene for future plasmid constructs.

  8. Vaccination of rhesus macaques with a vif-deleted simian immunodeficiency virus proviral DNA vaccine

    SciTech Connect

    Sparger, Ellen E. Dubie, Robert A.; Shacklett, Barbara L.; Cole, Kelly S.; Chang, W.L.; Luciw, Paul A.

    2008-05-10

    Studies in non-human primates, with simian immunodeficiency virus (SIV) and simian/human immunodeficiency virus (SHIV) have demonstrated that live-attenuated viral vaccines are highly effective; however these vaccine viruses maintain a low level of pathogenicity. Lentivirus attenuation associated with deletion of the viral vif gene carries a significantly reduced risk for pathogenicity, while retaining the potential for virus replication of low magnitude in the host. This report describes a vif-deleted simian immunodeficiency virus (SIV)mac239 provirus that was tested as an attenuated proviral DNA vaccine by inoculation of female rhesus macaques. SIV-specific interferon-{gamma} enzyme-linked immunospot responses of low magnitude were observed after immunization with plasmid containing the vif-deleted SIV provirus. However, vaccinated animals displayed strong sustained virus-specific T cell proliferative responses and increasing antiviral antibody titers. These immune responses suggested either persistent vaccine plasmid expression or low level replication of vif-deleted SIV in the host. Immunized and unvaccinated macaques received a single high dose vaginal challenge with pathogenic SIVmac251. A transient suppression of challenge virus load and a greater median survival time was observed for vaccinated animals. However, virus loads for vaccinated and unvaccinated macaques were comparable by twenty weeks after challenge and overall survival curves for the two groups were not significantly different. Thus, a vif-deleted SIVmac239 proviral DNA vaccine is immunogenic and capable of inducing a transient suppression of pathogenic challenge virus, despite severe attenuation of the vaccine virus.

  9. Protection of tree shrews by pVAX-PS DNA vaccine against HBV infection.

    PubMed

    Zhou, Feng-Juan; Hu, Zhen-Lin; Dai, Jian-Xin; Chen, Rui-Wen; Shi, Ke; Lin, Yi; Sun, Shu-Han

    2003-07-01

    The immunological protection of pVAX-PS, a DNA vaccine, was assessed in the tree shrews model. pVAX-PS was constructed by inserting the gene encoding the middle (pre-S2 plus S) envelope protein of HBV into a plasmid vector pVAX1. Tree shrews (Tupaia belangeri chinenesis) were experimentally infected with human HBV by inoculation with human serum positive for HBV markers. DNA vaccination-induced seroconversion and antibody to HBV surface antigen (anti-HBs) were analyzed by ELISA, and protective effects elicited by pVAX-PS vaccination against subsequent HBV challenge were evaluated by detection of HBV seromarkers and observation of hepatic lesions in HBV-infected tree shrews. The results shown that anti-HBs were detectable in serum at week 2 after pVAX-PS vaccination and peaked at week 4, and immunization with pVAX-PS decreased the positive conversion rate of HBV seromarkers and relieved hepatic lesions in tree shrews challenged with HBV. These results indicated that pVAX-PS immunization could induce remarkable humoral immune response and prevent the experimental tree shrews from infection of HBV.

  10. Local gene expression and immune responses of vaginal DNA vaccination using a needle-free injector.

    PubMed

    Kanazawa, Takanori; Takashima, Yuuki; Tamura, Toshiaki; Tsuchiya, Miki; Shibata, Yasunori; Udagawa, Haruhide; Okada, Hiroaki

    2010-08-30

    The vaginal mucosa is the most common site of initiation of virus infections that are transmitted through heterosexual intercourse, including HIV and papillomavirus. Thus, in order to prevent or treat these infections, strong vaginal immunity is required as the first line of defense. In this study, to establish a less invasive, safe, convenient and effective immunization method, we examined the local (skin and vagina) gene transfection efficiency of a non-needle jet injector for daily insulin injection. In the skin experiment, the needle-free injector resulted in a marked increase in marker gene expression, compared to the conventional needle-syringe injection. In addition, intradermal DNA vaccination using the needle-free injector dramatically induced IFN-gamma and antibody systemic responses in mice. Furthermore, we investigated the applicability of the needle-free injector as a vaginal vaccination tool in rabbits. Vaginal gene expression using the needle-free injector was significantly greater than that using needle-syringe injection. Moreover, intravaginal vaccination by the needle-free injector promoted vaginal IgA secretion and IFN-gamma mRNA expression in the blood lymphocytes, to a degree significantly higher than that by needle-syringe injection. In conclusion, local vaginal DNA vaccination using a needle-free jet injector is a promising approach for the prevention and treatment of mucosal infectious diseases.

  11. Production and purification of plasmid DNA vaccines: is there scope for further innovation?

    PubMed

    Xenopoulos, Alex; Pattnaik, Priyabrata

    2014-12-01

    The demand for plasmid DNA (pDNA) has vastly increased over the past decade in response to significant advances that have been made in its application for gene therapy and vaccine development. Plasmid DNA-based vaccines are experiencing a resurgence due to success with prime-boost immunization strategies. The challenge has always been poor productivity and delivery of pDNA. Plasmid DNA-based vaccines have traditionally required milligram scale of GMP-grade product for vaccination due to the relatively low efficacy and duration of gene expression. However, efforts to increase pDNA vaccine effectiveness are evolving in genetic manipulations of bacterial host, improvements in product recovery and innovative delivery methods. This review summarizes recent advances in large-scale pDNA vaccine manufacturing, ranging from upstream processing, downstream processing and formulation, as such information is usually not available to the scientific community. The article will highlight technology gaps and offer insight on further scope of innovation.

  12. Protective immunity induced by a DNA vaccine encoding Eimeria tenella rhomboid against homologous challenge.

    PubMed

    Liu, Yingli; Zheng, Jun; Li, Jianhua; Gong, Pengtao; Zhang, Xichen

    2013-01-01

    Rhomboid protein in Apicomplexa was associated with the process of host cell invasion. To evaluate the potential of the protein in eliciting protective immunity against challenge, a DNA vaccine pVAX1-Rho encoding Eimeria tenella rhomboid was constructed. Recombinant protein was expressed in Hela cells and verified by indirect immunofluorescence and western blotting analysis. In vivo experiments, 1-week-old chickens were randomly divided into three groups. Experimental group of chickens were immunized with DNA vaccines while control group of chickens were injected with pVAX1 plasmid alone or sterile water. Two weeks following the booster dose, all chickens were inoculated orally with 5 × 10(4) sporulated oocysts of E. tenella. The host immunity and protective efficacy of this vaccine against E. tenella challenge in broilers were evaluated. Results showed that specific antibody, the levels of interleukin-2 (IL-2), interferon-γ (IFN-γ), and the percentages of CD4(+) and CD8(+) T lymphocyte cells were significantly increased in the pVAX1-Rho group. Challenge experiments demonstrated that pVAX1-Rho vaccination could reduce oocyst excretion, decrease cecal lesion, increase bodyweight gains and provide chickens with oocysts decrease ratio around 75.8 %. These results suggest that the pVAX1-Rho was able to induce humoral and cellular responses and generate protective immunity against E. tenella infection.

  13. Concentration of infectious aquatic rhabdoviruses from freshwater and seawater using ultrafiltration.

    PubMed

    Grant, Amelia A M; Jakob, Eva; Richard, Jon; Garver, Kyle A

    2011-12-01

    Infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus, and spring viremia of carp virus were concentrated and detected from freshwater and seawater samples by using hollow-fiber ultrafiltration. Within 60 min, virus in a 50-L freshwater or saltwater sample was concentrated more than 70-fold, and virus retention efficiencies were consistently greater than 88%. Retention efficiency was highly dependent upon concentrations of column blocking and sample stabilization solutions. A large column with a surface area of 1.15 m2 and a filtration capacity of 5-200 L exhibited optimal viral retention when blocked with 2% fetal bovine serum (FBS) and when the samples were supplemented with 0.1% FBS. Conversely, a small column with 100-fold less surface area and a filtering capacity of 0.5-2.0 L was optimized when blocked with 1% FBS and when the samples were supplemented with 0.1% FBS. The optimized ultrafiltration procedure was further validated with water from a tank that contained IHNV-exposed juvenile sockeye salmon Oncorhynchus nerka, resulting in an average virus retention efficiency of 91.6 +/- 4.1% (mean +/- SE). Virus quantification of concentrated samples demonstrated that IHNV shedding in sockeye salmon preceded mortality; shedding of the virus was observed to increase significantly as early as 7 d postchallenge and peaked at day 14, when virus levels reached 4.87 x 10(3) plaque-forming units/mL. We conclude that ultrafiltration is a reliable and effective method for concentrating viable aquatic rhabdoviruses from large volumes of water and has application for the analysis of environmental water samples.

  14. Immunomodulation of bivalent Newcastle disease DNA vaccine induced immune response by co-delivery of chicken IFN-γ and IL-4 genes.

    PubMed

    Sawant, P M; Verma, P C; Subudhi, P K; Chaturvedi, U; Singh, M; Kumar, Rajeev; Tiwari, A K

    2011-11-15

    The basic objective of this study was to enumerate whether co-administration of interferon-γ (IFN-γ) and/or interleukin-4 (IL-4) gene along with a bivalent Newcastle disease (ND) DNA vaccine construct could modulate the immune response to the DNA vaccine in chickens. pVIVO2 vector carrying Haemaglutinin-Neuraminidase (HN) and Fusion (F) genes of Newcastle disease virus (NDV) at its two cloning sites was used as a DNA vaccine. The same vector was used to clone the chicken IFN-γ and IL-4 genes at the multiple cloning site-1 separately. In vitro expression of IFN-γ and IL-4 gene constructs was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and that of HN and F genes by indirect fluorescent antibody technique (IFAT) in addition to RT-PCR. The chickens were immunized thrice intramuscularly at 21, 36 and 46 days of age with the bivalent DNA vaccine alone, or in combination with IFN-γ/IL-4 or both cytokine gene constructs. The bivalent DNA vaccine led to increase in both NDV specific antibodies as assessed by enzyme linked immunosorbent assay (ELISA) and haemagglutination inhibition test (HI) and cell mediated immune (CMI) response as assessed by lymphocyte transformation test (LTT) employing MTT assay. Co-administration of the DNA vaccine with IL-4 gene resulted in highest IgY levels while IFN-γ produced highest CMI response. The DNA vaccine alone could afford only 10% protection against challenge infection by velogenic NDV. This protection was increased to 40% when IL-4 gene construct was co-administered with the DNA vaccine. Co-injection of IFN-γ as well as the combination of IFN-γ and IL-4 gene constructs with the DNA vaccine yielded 20% protection. Our study suggests that IL-4 may prove to be more appropriate as a genetic adjuvant than IFN-γ for ND DNA vaccine.

  15. Suppression of breast tumor growth by DNA vaccination against phosphatase of regenerating liver 3.

    PubMed

    Lv, J; Liu, C; Huang, H; Meng, L; Jiang, B; Cao, Y; Zhou, Z; She, T; Qu, L; Wei Song, S; Shou, C

    2013-08-01

    Phosphatase of regenerating liver (PRL)-3 is highly expressed in multiple cancers and has important roles in cancer development. Some small-molecule inhibitors and antibodies targeting PRL-3 have been recently reported to inhibit tumor growth effectively. To determine whether PRL-3-targeted DNA vaccination can induce immune response to prevent or inhibit the tumor growth, we established mouse D2F2 breast cancer cells expressing PRL-3 (D2F2/PRL-3) and control cells (D2F2/NC) with lentivirus, and constructed pVAX1-Igκ-PRL-3 plasmid (named as K-P3) as DNA vaccine to immunize BALB/c mice. We found that the K-P3 vaccine delivered by gene gun significantly prevented the growth of D2F2/PRL-3 compared with pVAX1-vector (P<0.01), but not of D2F2/NC, and improved the survival of D2F2/PRL-3-innoculated mice. Both PRL-3-targeted cytotoxic T lymphocytes (CTLs) and T-helper type 1 cell immune response (production of high levels of interferon-γ and tumor necrosis factor-α) were found to be involved in the preventive effect. Furthermore, PRL-3-targeted DNA immunization inhibited tumor growth of D2F2/PRL-3 cells in mice. We also evaluated the potential of immunization with PRL-3 protein, but no significant therapeutic or preventive effect was obtained on tumor growth. To enhance the immunity of PRL-3, we incorporated different molecular adjuvants, such as Mycobacterium tuberculosis heat-shock protein, CTL antigen 4 and M. tuberculosis T-cell stimulatory epitope (MT), into K-P3 vaccine for expressing the fusion proteins. We found that these adjuvant molecules did not significantly improve the antitumor activity of PRL-3 vaccine, but enhanced the production of PRL-3 antibodies in immunized mice. Summarily, our findings demonstrate that PRL-3-targeted DNA vaccine can generate significantly preventive and therapeutic effects on the growth of breast cancer expressing PRL-3 through the induction of cellular immune responses to PRL-3.

  16. Immunotherapy with an HIV-DNA Vaccine in Children and Adults

    PubMed Central

    Palma, Paolo; Gudmundsdotter, Lindvi; Finocchi, Andrea; Eriksson, Lars E.; Mora, Nadia; Santilli, Veronica; Aquilani, Angela; Manno, Emma C.; Zangari, Paola; Romiti, Maria Luisa; Montesano, Carla; Grifoni, Alba; Brave, Andreas; Ljungberg, Karl; Blomberg, Pontus; Bernardi, Stefania; Sandström, Eric; Hejdeman, Bo; Rossi, Paolo; Wahren, Britta

    2014-01-01

    Therapeutic HIV immunization is intended to induce new HIV-specific cellular immune responses and to reduce viral load, possibly permitting extended periods without antiretroviral drugs. A multigene, multi-subtype A, B, C HIV-DNA vaccine (HIVIS) has been used in clinical trials in both children and adults with the aim of improving and broadening the infected individuals’ immune responses. Despite the different country locations, different regimens and the necessary variations in assays performed, this is, to our knowledge, the first attempt to compare children’s and adults’ responses to a particular HIV vaccine. Ten vertically HIV-infected children aged 4–16 years were immunized during antiretroviral therapy (ART). Another ten children were blindly recruited as controls. Both groups continued their antiretroviral treatment during and after vaccinations. Twelve chronically HIV-infected adults were vaccinated, followed by repeated structured therapy interruptions (STI) of their antiretroviral treatment. The adult group included four controls, receiving placebo vaccinations. The HIV-DNA vaccine was generally well tolerated, and no serious adverse events were registered in any group. In the HIV-infected children, an increased specific immune response to Gag and RT proteins was detected by antigen-specific lymphoproliferation. Moreover, the frequency of HIV-specific CD8+ T-cell lymphocytes releasing perforin was significantly higher in the vaccinees than the controls. In the HIV-infected adults, increased CD8+ T-cell responses to Gag, RT and viral protease peptides were detected. No augmentation of HIV-specific lymphoproliferative responses were detected in adults after vaccination. In conclusion, the HIV-DNA vaccine can elicit new HIV-specific cellular immune responses, particularly to Gag antigens, in both HIV-infected children and adults. Vaccinated children mounted transient new HIV-specific immune responses, including both CD4+ T-cell lymphoproliferation

  17. Limited Protection Conferred by a DNA Vaccine against a Lethal Pseudorabies Virus Infection at Day 5 Postvaccination▿

    PubMed Central

    Dory, Daniel; Torché, Anne-Marie; Béven, Véronique; Cariolet, Roland; Jestin, André

    2007-01-01

    No pseudorabies virus (PRV)-specific neutralizing or immunoglobulin G1-type antibodies were detected in sera 5 days after injection of a DNA vaccine against PRV infection in pigs. PRV-stimulated peripheral blood mononuclear cells produced gamma interferon mRNA in vitro. Two out of five pigs recovered from lethal PRV infection without attenuation of nasal viral excretion. PMID:17301217

  18. Ebola Vaccination Using a DNA Vaccine Coated on PLGA-PLL/γPGA Nanoparticles Administered Using a Microneedle Patch.

    PubMed

    Yang, Hung-Wei; Ye, Ling; Guo, Xin Dong; Yang, Chinglai; Compans, Richard W; Prausnitz, Mark R

    2017-01-01

    Ebola DNA vaccine is incorporated into PLGA-PLL/γPGA nanoparticles and administered to skin using a microneedle (MN) patch. The nanoparticle delivery system increases vaccine thermostability and immunogenicity compared to free vaccine. Vaccination by MN patch produces stronger immune responses than intramuscular administration. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Long-Term Reduction of High Blood Pressure by Angiotensin II DNA Vaccine in Spontaneously Hypertensive Rats.

    PubMed

    Koriyama, Hiroshi; Nakagami, Hironori; Nakagami, Futoshi; Osako, Mariana Kiomy; Kyutoku, Mariko; Shimamura, Munehisa; Kurinami, Hitomi; Katsuya, Tomohiro; Rakugi, Hiromi; Morishita, Ryuichi

    2015-07-01

    Recent research on vaccination has extended its scope from infectious diseases to chronic diseases, including Alzheimer disease, dyslipidemia, and hypertension. The aim of this study was to design DNA vaccines for high blood pressure and eventually develop human vaccine therapy to treat hypertension. Plasmid vector encoding hepatitis B core-angiotensin II (Ang II) fusion protein was injected into spontaneously hypertensive rats using needleless injection system. Anti-Ang II antibody was successfully produced in hepatitis B core-Ang II group, and antibody response against Ang II was sustained for at least 6 months. Systolic blood pressure was consistently lower in hepatitis B core-Ang II group after immunization, whereas blood pressure reduction was continued for at least 6 months. Perivascular fibrosis in heart tissue was also significantly decreased in hepatitis B core-Ang II group. Survival rate was significantly improved in hepatitis B core-Ang II group. This study demonstrated that Ang II DNA vaccine to spontaneously hypertensive rats significantly lowered high blood pressure for at least 6 months. In addition, Ang II DNA vaccines induced an adequate humoral immune response while avoiding the activation of self-reactive T cells, assessed by ELISPOT assay. Future development of DNA vaccine to treat hypertension may provide a new therapeutic option to treat hypertension.

  20. Use of Staby(®) technology for development and production of DNA vaccines free of antibiotic resistance gene.

    PubMed

    Reschner, Anca; Scohy, Sophie; Vandermeulen, Gaëlle; Daukandt, Marc; Jacques, Céline; Michel, Benjamin; Nauwynck, Hans; Xhonneux, Florence; Préat, Véronique; Vanderplasschen, Alain; Szpirer, Cédric

    2013-10-01

    The appearance of new viruses and the cost of developing certain vaccines require that new vaccination strategies now have to be developed. DNA vaccination seems to be a particularly promising method. For this application, plasmid DNA is injected into the subject (man or animal). This plasmid DNA encodes an antigen that will be expressed by the cells of the subject. In addition to the antigen, the plasmid also encodes a resistance to an antibiotic, which is used during the construction and production steps of the plasmid. However, regulatory agencies (FDA, USDA and EMA) recommend to avoid the use of antibiotics resistance genes. Delphi Genetics developed the Staby(®) technology to replace the antibiotic-resistance gene by a selection system that relies on two bacterial genes. These genes are small in size (approximately 200 to 300 bases each) and consequently encode two small proteins. They are naturally present in the genomes of bacteria and on plasmids. The technology is already used successfully for production of recombinant proteins to achieve higher yields and without the need of antibiotics. In the field of DNA vaccines, we have now the first data validating the innocuousness of this Staby(®) technology for eukaryotic cells and the feasibility of an industrial production of an antibiotic-free DNA vaccine. Moreover, as a proof of concept, mice have been successfully vaccinated with our antibiotic-free DNA vaccine against a deadly disease, pseudorabies (induced by Suid herpesvirus-1).

  1. Protective effect of DNA vaccine encoding pseudomonas exotoxin A and PcrV against acute pulmonary P. aeruginosa Infection.

    PubMed

    Jiang, Mingzi; Yao, Jing; Feng, Ganzhu

    2014-01-01

    Infections with Pseudomonas aeruginosa have been a long-standing challenge for clinical therapy because of complex pathogenesis and resistance to antibiotics, thus attaching importance to explore effective vaccines for prevention and treatment. In the present study, we constructed a novel DNA vaccine by inserting mutated gene toxAm encoding Pseudomonas Exotoxin A and gene pcrV encoding tip protein of the type III secretion system into respective sites of a eukaryotic plasmid pIRES, named pIRES-toxAm-pcrV, and next evaluated the efficacy of the vaccine in murine acute Pseudomonas pneumonia models. Compared to DNA vaccines encoding single antigen, mice vaccinated with pIRES-toxAm-pcrV elicited higher levels of antigen-specific serum immunoglobulin G (IgG), enhanced splenic cell proliferation and cytokine secretion in response to Pseudomonas aeruginosa antigens, additionally PAO1 challenge in mice airway resulted in reduced bacteria burden and milder pathologic changes in lungs. Besides, it was observed that immunogenicity and protection could be promoted by the CpG ODN 1826 adjuvant. Taken together, it's revealed that recombinant DNA vaccine pIRES-toxAm-pcrV was a potential candidate for immunotherapy of Pseudomonas aeruginosa infection and the CpG ODN 1826 a potent stimulatory adjuvant for DNA vaccination.

  2. DNA vaccine encoding Middle East respiratory syndrome coronavirus S1 protein induces protective immune responses in mice.

    PubMed

    Chi, Hang; Zheng, Xuexing; Wang, Xiwen; Wang, Chong; Wang, Hualei; Gai, Weiwei; Perlman, Stanley; Yang, Songtao; Zhao, Jincun; Xia, Xianzhu

    2017-04-11

    The Middle East respiratory syndrome coronavirus (MERS-CoV), is an emerging pathogen that continues to cause outbreaks in the Arabian peninsula and in travelers from this region, raising the concern that a global pandemic could occur. Here, we show that a DNA vaccine encoding the first 725 amino acids (S1) of MERS-CoV spike (S) protein induces antigen-specific humoral and cellular immune responses in mice. With three immunizations, high titers of neutralizing antibodies (up to 1: 10(4)) were generated without adjuvant. DNA vaccination with the MERS-CoV S1 gene markedly increased the frequencies of antigen-specific CD4(+) and CD8(+) T cells secreting IFN-γ and other cytokines. Both pcDNA3.1-S1 DNA vaccine immunization and passive transfer of immune serum from pcDNA3.1-S1 vaccinated mice protected Ad5-hDPP4-transduced mice from MERS-CoV challenge. These results demonstrate that a DNA vaccine encoding MERS-CoV S1 protein induces strong protective immune responses against MERS-CoV infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Preclinical safety assessment of a DNA vaccine using particle-mediated epidermal delivery in domestic pig, minipig and mouse.

    PubMed

    Dincer, Zuhal; Jones, Stewart; Haworth, Richard

    2006-07-01

    DNA vaccination involves the direct injection of genes coding for specific antigenic proteins. One technique known as particle-mediated epidermal delivery (PMED) is a practical approach for epidermal delivery and provides a strong immune response. An important aspect of the preclinical safety assessment of DNA vaccines is the selection of a pharmacologically relevant animal model for the assessment of antigen expression, optimization of delivery and formulation of the plasmid. This paper describes a comparative study of domestic pig, minipig and mouse in regard to local tolerance and antigen expression of HIV immunotherapeutic using PMED. Pig/minipig is considered a good model for the safety assessment of DNA vaccines due to the similarity to human skin. Local reactions were evaluated at 10 min, 4, 24 and 48 h. Histology of administration sites revealed epidermal necrosis with associated dermal inflammation at 10 min and 4h, and subsequent regeneration with repair at 24 and 48 h. The degree and extent of these changes varied according to species. Domestic pig and minipig showed superficial epidermal necrosis and complete repair, while the mouse showed full-thickness epidermal necrosis and partial repair. Expression of HIV antigen was confirmed using immunohistochemistry in all three species at 4, 24 and 48 h. The results showed that PMED is an effective system for DNA vaccine delivery as demonstrated by the antigen expression seen as early as 4 h.

  4. Studies on the protective immunity of Schistosoma japonicum bivalent DNA vaccine encoding Sj23 and Sj14.

    PubMed

    Yuan, Hu; You-En, Shi; Long-Jiang, Yu; Xiao-Hua, Zhu; Liu-Zhe, Li; Cash, Melanie; Lu, Zhu; Zhi, Liu; Deng-Xin, Song

    2007-04-01

    In order to explore the high performance bivalent DNA vaccine of Schistosoma japonicum, the fatty-acid-binding protein (Sj14) and the 23 kDa transmembrane protein (Sj23) two proteins were selected to construct the DNA-based vaccine. It was successful to construct a bivalent DNA vaccine using three strategies: the co-expression of two genes, a fusion gene expression and two kinds of plasmids in combination (cocktail vaccine). The bivalent DNA was proven to express well in vitro and in vivo by indirect immunofluorescence test (IIF) and reverse transcriptase-polymerase chain reaction (RT-PCR). The protective immunity of bivalent DNA vaccine was higher than that of univalent DNA vaccine (p<0.05). There were four groups of bivalent vaccine whose protective immunity was higher than 50%. Granuloma diameter reduction rates were in the range of 18-39%. There was no significant impact on immunity protection exerted by the four factors including dosage, inoculated times, inoculated routes and challenge time after the last immunization in three levels (p>0.05).

  5. The site of administration influences both the type and the magnitude of the immune response induced by DNA vaccine electroporation.

    PubMed

    Vandermeulen, Gaëlle; Vanvarenberg, Kevin; De Beuckelaer, Ans; De Koker, Stefaan; Lambricht, Laure; Uyttenhove, Catherine; Reschner, Anca; Vanderplasschen, Alain; Grooten, Johan; Préat, Véronique

    2015-06-22

    We investigated the influence of the site of administration of DNA vaccine on the induced immune response. DNA vaccines were administered by electroporation at three different sites: tibial cranial muscle, abdominal skin and ear pinna. Aiming to draw general conclusions about DNA vaccine delivery, we successively used several plasmids encoding either luciferase and ovalbumin as models or gp160 and P1A as vaccines against HIV and P815 mastocytoma, respectively. Low levels and duration of luciferase transgene expression were observed after electroporation of the abdominal skin, partly explaining its lower immunogenic performance as compared to the other sites of administration. Analyses of OT-I CD8+ and OT-II CD4+ T cell responses highlighted the differential impact of the delivery site on the elicited immune response. Muscle electroporation induced the strongest humoral immune response and both muscle and ear pinna sites induced cellular immunity against gp160. Ear pinna delivery generated the highest level of CTL responses against P1A but electroporation of muscle and ear pinna were equally efficient in delaying P815 growth and improving mice survival. The present study demonstrated that the site of administration is a key factor to be tested in the development of DNA vaccine.

  6. Dual recombinant Lactococcus lactis for enhanced delivery of DNA vaccine reporter plasmid pPERDBY.

    PubMed

    Yagnik, Bhrugu; Sharma, Drashya; Padh, Harish; Desai, Priti

    2017-03-04

    Food grade Lactococcus lactis (L. lactis) has been widely used as an antigen and DNA delivery vehicle. We had previously reported the use of non-invasive L. lactis for the delivery of newly constructed immunostimulatory DNA vaccine reporter plasmid, pPERDBY. In the present report, we outline the construction of dual recombinant L. lactis expressing Internalin A of Listeria monocytogenes and harbouring pPERDBY (LL InlA+ pPERDBY) to enhance the DNA delivery efficiency of L. lactis. After confirmation and validation of LL InlA+ pPERDBY, its DNA delivery potential was compared with previously developed non-invasive r- L. lactis::pPERDBY. The use of invasive L. lactis resulted in around three fold increase in number of Enhanced Green Fluorescent Protein expressing Caco- cells. Thus, these findings reinforce the prospective application of invasive strain of L. lactis in delivery of DNA/RNA and antigens.

  7. Expression of tak1 and tram induces synergistic pro-inflammatory signalling and adjuvants DNA vaccines.

    PubMed

    Larsen, Karen Colbjørn; Spencer, Alexandra J; Goodman, Anna L; Gilchrist, Ashley; Furze, Julie; Rollier, Christine S; Kiss-Toth, Endre; Gilbert, Sarah C; Bregu, Migena; Soilleux, Elizabeth J; Hill, Adrian V S; Wyllie, David H

    2009-09-18

    Improving vaccine immunogenicity remains a major challenge in the fight against developing country diseases like malaria and AIDS. We describe a novel strategy to identify new DNA vaccine adjuvants. We have screened components of the Toll-like receptor signalling pathways for their ability to activate pro-inflammatory target genes in transient transfection assays and assessed in vivo adjuvant activity by expressing the activators from the DNA backbone of vaccines. We find that a robust increase in the immune response necessitates co-expression of two activators. Accordingly, the combination of tak1 and tram elicits synergistic reporter activation in transient transfection assays. In a mouse model this combination, but not the individual molecules, induced approximately twofold increases in CD8+ T-cell immune responses. These results indicate that optimal immunogenicity may require activation of distinct innate immune signalling pathways. Thus this strategy offers a novel route to the discovery of a new generation of adjuvants.

  8. Myostatin DNA vaccine increases skeletal muscle mass and endurance in mice.

    PubMed

    Tang, Liang; Yan, Zhen; Wan, Yi; Han, Wei; Zhang, Yingqi

    2007-09-01

    Myostatin is a transforming growth factor-beta family member that acts as a negative regulator of skeletal muscle growth. In mice, genetic disruption of the myostatin gene leads to a marked increase in body weight and muscle mass. Similarly, pharmacological interference with myostatin in vivo in mdx knockout mice results in a functional improvement of the dystrophic phenotype. Consequently, myostatin is an important therapeutic target for treatment of diseases associated with muscle wasting. To construct a therapeutic DNA vaccine against myostatin, we coupled the foreign, immunodominant T-helper epitope of tetanus toxin to the N terminus of myostatin, and BALB/c mice were immunized with the recombinant vector. Sera from vaccinated mice showed the presence of specific antibodies against the recombinant protein. In addition, body weight, muscle mass, and grip endurance of vaccinated mice were significantly increased. Our study provides a novel, pharmacological strategy for treatment of diseases associated with muscle wasting.

  9. Immunogenicity of a lentiviral-based DNA vaccine driven by the 5'LTR of the naturally attenuated caprine arthritis encephalitis virus (CAEV) in mice and macaques.

    PubMed

    Arrode-Brusés, Géraldine; Hegde, Ramakrishna; Jin, Yuhuai; Liu, Zhengian; Narayan, Opendra; Chebloune, Yahia

    2012-04-19

    Increasing the safety and the efficacy of existing HIV vaccines is one of the strategies that could help to promote the development of a vaccine for human use. We developed a HIV DNA vaccine (Δ4-SHIVKU2) that has been shown to induce potent polyfunctional HIV-specific T cell responses following a single dose immunization of mice and macaques. Δ4-SHIVKU2 also induced protection when immunized macaques were challenged with homologous pathogenic viruses. In the present study, our aim was to examine whether a chimeric HIV DNA vaccine (CAL-Δ4-SHIVKU2) whose genome is driven by the LTR of the goat lentivirus, caprine arthritis encephalitis (CAEV) expresses efficiently the vaccine antigens and induces potent immune responses in animal models for HIV vaccine. Data of radioimmunoprecipitation assays clearly show that this chimeric genome drives efficient expression of all HIV antigens in the construct. In addition, evaluation of the p24 Gag protein in the supernatant of HEK-293-T cells transfected in parallel with Δ4-SHIVKU2 and CAL-Δ4-SHIVKU2 showed no difference suggesting that these two LTRs are inducing equally the expression of the viral genes. Immunization of mice and macaques using our single dose immunization regimen resulted in induction of similar IFN-γ ELISPOT responses in Δ4-SHIVKU2- and CAL-Δ4-SHIVKU2-treated mice. Similar profiles of T cell responses were also detected both in mice and macaques when multiparametric flow cytometry analyses were performed. Since CAEV LTR is not dependent of Tat to drive viral gene expression and is not functional for integration with HIV integrase, this new vector increases the safety and efficacy of our vaccine vectors and vaccination strategy.

  10. Characterization of a rhabdovirus isolated from carpione Salmo trutta carpio in Italy

    USGS Publications Warehouse

    Bovo, G.; Olesen, N.J.; Jorgensen, P.E.V.; Ahne, W.; Winton, J.R.

    1995-01-01

    A virus, strain 583, was isolated from carpione Salmo trutta carpio fry exhibiting high mortality. The virus was not neutralized by rabbit antisera against the fish rhabdoviruses viral haemorrhagic septicaemia virus (VHSV), infectious hematopoietic necrosis virus, eel rhabdovirus European X, spring viraemia of carp virus or pike fry rhabdovirus, or against the birnavirus infectious pancreatic necrosis virus. The virus replicated in several fish cell lines incubated at 20 to 25*C and grew optimally in the bluegill fry (BF-2) and fathead minnow (FHM) cell lines. Electron microscopy of infected BF-2 cell cultures revealed the presence of typical rhabdovirus particles, and immunofluorescent staining was observed using various polyclonal and monoclonal antibodies (MAbs) against Egtved virus, the causative agent of viral haemorrhagic septicaemia. The staining by a MAb against the nucleoprotein (N) of VHSV was particularly strong, a MAb against the glycoprotein (G) gave a moderate reaction, whereas a second MAb against the G protein and MAbs against the matrix proteins, M_(1) and M_(2), of VHSV did not react. Fluorescence titres using 3 rabbit antisera against whole Egtved virus varied between negative and moderately positive. Western blotting using polyclonal and monoclonal sera confirmed that both the N and G proteins of the carpione virus shared some epitopes with those of VHSV, but the M_(1) and M_(2) proteins did not. SDS-PAGE showed the structural proteins of the carpione virus produced a pattern typical of members of the Lyssavirus genus of the Rhabdoviridae and the molecular weights were very similar to those of VHSV, except for the M_(2) protein which was somewhat smaller. Infection trials showed the carpione virus induced high mortalities in carpione fry but not in rainbow trout Oncorhynchus mykiss fry. The carpione virus was clearly distinguishable from Egtved virus despite limited serological cross reaction. Since it was also easily distinguishable by

  11. A DNA Vaccine Formulated with Chemical Adjuvant Provides Partial Protection against Bovine Herpes Virus Infection in Cattle.

    PubMed

    Quattrocchi, Valeria; Soria, Ivana; Langellotti, Cecilia Ana; Gnazzo, Victoria; Gammella, Mariela; Moore, Dadin P; Zamorano, Patricia I

    2017-01-01

    Bovine herpesvirus-1 (BoHV-1) is the causative agent of bovine infectious rhinotracheitis, an important disease worldwide. Although conventional BoHV-1 vaccines, including those based on the use of modified live virus and also inactivated vaccines, are currently used in many countries, they have several disadvantages. DNA vaccines have emerged as an attractive approach since they have the potential to induce both humoral and cellular immune response; nevertheless, it is largely known that potency of naked DNA vaccines is limited. We demonstrated previously, in the murine model, that the use of adjuvants in combination with a DNA vaccine against BoHV-1 is immunologically beneficial. In this study, we evaluate the immune response and protection against challenge elicited in bovines, by a DNA vaccine carrying the sequence of secreted version of glycoprotein D (gD) of BoHV-1 formulated with chemical adjuvants. Bovines were vaccinated with formulations containing the sequence of gD alone or in combination with adjuvants ESSAI 903110 or Montanide™ 1113101PR. After prime vaccination and two boosters, animals were challenged with infectious BoHV-1. Formulations containing adjuvants Montanide™ 1113101PR and ESSAI 903110 were both, capable of increasing humoral immune response against the virus and diminishing clinical symptoms. Nevertheless, only formulations containing adjuvant Montanide™ 1113101PR was capable of improving cellular immune response and diminishing viral excretion. To our knowledge, it is the first time that a BoHV-1 DNA vaccine is combined with adjuvants and tested in cattle. These results could be useful to design a vaccine for the control of bovine rhinotracheitis.

  12. A DNA Vaccine Formulated with Chemical Adjuvant Provides Partial Protection against Bovine Herpes Virus Infection in Cattle

    PubMed Central

    Quattrocchi, Valeria; Soria, Ivana; Langellotti, Cecilia Ana; Gnazzo, Victoria; Gammella, Mariela; Moore, Dadin P.; Zamorano, Patricia I.

    2017-01-01

    Bovine herpesvirus-1 (BoHV-1) is the causative agent of bovine infectious rhinotracheitis, an important disease worldwide. Although conventional BoHV-1 vaccines, including those based on the use of modified live virus and also inactivated vaccines, are currently used in many countries, they have several disadvantages. DNA vaccines have emerged as an attractive approach since they have the potential to induce both humoral and cellular immune response; nevertheless, it is largely known that potency of naked DNA vaccines is limited. We demonstrated previously, in the murine model, that the use of adjuvants in combination with a DNA vaccine against BoHV-1 is immunologically beneficial. In this study, we evaluate the immune response and protection against challenge elicited in bovines, by a DNA vaccine carrying the sequence of secreted version of glycoprotein D (gD) of BoHV-1 formulated with chemical adjuvants. Bovines were vaccinated with formulations containing the sequence of gD alone or in combination with adjuvants ESSAI 903110 or Montanide™ 1113101PR. After prime vaccination and two boosters, animals were challenged with infectious BoHV-1. Formulations containing adjuvants Montanide™ 1113101PR and ESSAI 903110 were both, capable of increasing humoral immune response against the virus and diminishing clinical symptoms. Nevertheless, only formulations containing adjuvant Montanide™ 1113101PR was capable of improving cellular immune response and diminishing viral excretion. To our knowledge, it is the first time that a BoHV-1 DNA vaccine is combined with adjuvants and tested in cattle. These results could be useful to design a vaccine for the control of bovine rhinotracheitis. PMID:28179907

  13. The larval specific lymphatic filarial ALT-2: induction of protection using protein or DNA vaccination.

    PubMed

    Ramachandran, Sabarinathan; Kumar, Mishra Pankaj; Rami, Reddy Maryada Venkata; Chinnaiah, Harinath Basker; Nutman, Thomas; Kaliraj, Perumal; McCarthy, James

    2004-01-01

    Genes from the infective stage of lymphatic filarial parasites expressed at the time of host invasion have been identified as potential vaccine candidates. By screening an L3 cDNA library with sera from uninfected longstanding residents of an area endemic for onchocerciasis, so-called "endemic normals" (EN), we have cloned and characterized one such gene termed the abundant larval transcript two (ALT-2). The stage specificity of ALT-2 gene transcription and protein synthesis was confirmed by PCR using genespecific primers, and by western blot analysis of protein extracts from various stages of the parasite life cycle using specific antisera. Significant differences in antibody response to the recombinant ALT-2 were observed in endemic populations with differing clinical manifestations of lymphatic filariasis with an antibody response present in sera from 18 of 25 (72%) EN subjects compared to 9 of 25 (36%) with subclinical microfilaracmia (MF) and 14 of 25 (52%) of those with chronic lymphatic obstruction (CP) (P=0.01 for comparison of EN to CP or to MF). This differential responsiveness suggests that the protective immunity postulated to account for their uninfected status might be associated with a response to this protein. When the utility of ALT-2 as a vaccine candidate was tested in a murine model using either recombinant protein or a DNA vaccine construct, statistically significant protection was observed when compared to a control filarial gene product expressed across all stages of the parasite lifecycle (SXP-1; P=0.02 for protein and P=0.01 for the DNA vaccine) or compared to adjuvant alone. This level of protection indicates that this vaccine is a promising candidate for further development.

  14. pH-sensitive carbonate apatite nanoparticles as DNA vaccine carriers enhance humoral and cellular immunity.

    PubMed

    He, Pan; Takeshima, Shin-nosuke; Tada, Seiichi; Akaike, Toshihiro; Ito, Yoshihiro; Aida, Yoko

    2014-10-29

    To demonstrate the potential of pH-sensitive carbonate apatite (CO₃Ap) nanoparticles as DNA vaccine carriers to enhance vaccination efficacy, we examined the humoral and cellular immune responses of C57BL/6 mice immunized with the plasmid expression vector pCI-neo encoding the full-length soluble ovalbumin (OVA) (pCI-neo-sOVA), pCI-neo-sOVA/CO₃Ap complexes, or pCI-neo/CO₃Ap complexes as a control. Mice immunized with a low dose of pCI-neo-sOVA-loaded CO₃Ap (10 μg) produced ex vivo splenocyte proliferation after stimulation with CD8 T-cell but not CD4 T-cell epitopes and a delayed-type-hypersensitivity reaction more efficiently than mice in the other groups. Furthermore, mice receiving this immunization generated the same levels of OVA-specific antibodies and interferon (IFN)-γ secretion after CD8 T-cell and CD4 T-cell epitope challenges as those in mice treated with 100 μg of free pCI-neo-sOVA, whereas mice injected with a high dose of pCI-neo-sOVA-loaded CO₃Ap (100 μg) or with control plasmids produced negligible levels of OVA-specific antibodies or IFN-γ. Therefore, our results showed that 10 μg of pCI-neo-sOVA delivered by CO₃Ap strongly elicited humoral and cellular immune responses. This study is the first to demonstrate the promising potential of CO₃Ap nanoparticles for DNA vaccine delivery.

  15. Evaluation of attenuated Salmonella choleraesuis-mediated inhibin recombinant DNA vaccine in rats.

    PubMed

    Hui, F M; Meng, C L; Guo, N N; Yang, L G; Shi, F X; Mao, D G

    2014-08-07

    DNA vaccination has been studied intensively as a potential vaccine technology. We evaluated the effect of an attenuated Salmonella choleraesuis-mediated inhibin DNA vaccine in rats. First, 15 rats were treated with different doses of an inhibin vaccine to evaluate vaccine safety. Next, 30 rats were divided into 3 groups and injected intramuscularly with the inhibin vaccine two (T1) or three times (T2) or with control bacteria (Con) at 4-week intervals. The inhibin antibody levels increased [positive/negative well (P/N) value: T1 vs Con = 2.39 ± 0.01 vs 1.08 ± 0.1; T2 vs Con = 2.36 ± 0.1 vs 1.08 ± 0.1, P < 0.05] at week 2 and were maintained at a high level in T1 and T2 until week 8, although a small decrease in T2 was observed at week 10. Rats in the T1 group showed more corpora lutea compared with the Con group (10.50 ± 0.87 vs 7.4 ± 0.51, P < 0.05). Estradiol (0.439 ± 0.052 vs 0.719 ± 0.063 ng/mL, P < 0.05) and progesterone (1.315 ± 0.2 vs 0.737 ± 0.11 ng/mL, P < 0.05) levels differed significantly at metestrus after week 10 between rats in the T1 and Con groups. However, there were no significant differences in body, ovary, uterus weights, or pathological signs in the ovaries after immunization, indicating that this vaccine is safe. In conclusion, the attenuated S. choleraesuis-mediated inhibin vaccine may be an alternative to naked inhibin plasmids for stimulating ovarian follicular development to increase the ovulation rate in rats.

  16. Designing and modeling of complex DNA vaccine based on tropomyosin protein of Boophilus genus tick.

    PubMed

    Ranjbar, Mohamamd Mahdi; Gupta, Shishir K; Ghorban, Khodayar; Nabian, Sedigheh; Sazmand, Alireza; Taheri, Mohammad; Esfandyari, Sahar; Taheri, Maryam

    2015-01-01

    Boophilus tick is a bloodsucking ectoparasite that transfers some pathogens, reducing production and thus leading to economical losses in the cattle industry. Tropomyosin (TPM) protein is a salivary protein, has actin regulator activity, and plays an important role in immune reactions against parasites. In the current study, besides developing a safe, effective, and broad spectrum protective measure against Boophilus genus tick based on TPM protein, we attempted to minimize possible problems occurring in the design of polytopic vaccines. Briefly, the steps that were followed in the present study were as follows: retrieving sequences and finding the mutational/conservative regions, selecting consensus and high immunogenic epitopes of B and CD4(+) T cells by different approaches, three-dimensional structure (3D structure) prediction and representation of epitopes and highly variable/conserve regions, designing vaccinal construct by fusion of B and T cell epitopes by special patterns and improving immunogenicity, evaluation of the constructs' primary structure and posttranslational modification, calculation of hydrophobic regions, reverse translation, codon optimization, open reading frame checking, insertion of start/end codon, Kozak sequence, and finally constructing the DNA vaccine. Variation plot showed some shared epitopes among the ticks' and mites' species that some might be effective only in some species. Finally, by following the steps mentioned above, two constructs for B and T cells were achieved. Checking constructs revealed their reliability and efficacy for in vitro production and utilization. Successful in silico modeling is an essential step of designing vigorous vaccines. We developed a novel protective and therapeutic vaccine against Boophilus genus (based on TPM protein). At the next step, constructed DNA vaccine would be produced in vitro and administrated to cattle, and its potency to induction of immune response and protection against Boophilus

  17. Rapid DNA vaccination against Burkholderia pseudomallei flagellin by tattoo or intranasal application.

    PubMed

    Lankelma, Jacqueline M; Wagemakers, Alex; Birnie, Emma; Haak, Bastiaan W; Trentelman, Jos J A; Weehuizen, Tassili A F; Ersöz, Jasmin; Roelofs, Joris J T H; Hovius, Joppe W; Wiersinga, W Joost; Bins, Adriaan D

    2017-03-21

    Melioidosis is a severe infectious disease with a high mortality that is endemic in South-East Asia and Northern Australia. The causative pathogen, Burkholderia pseudomallei, is listed as potential bioterror weapon due to its high virulence and potential for easy dissemination. Currently, there is no licensed vaccine for prevention of melioidosis. Here, we explore the use of rapid plasmid DNA vaccination against B. pseudomallei flagellin for protection against respiratory challenge. We tested three flagellin DNA vaccines with different subcellular targeting designs. C57BL/6 mice were vaccinated via skin tattoo on day 0, 3 and 6 before intranasal challenge with B. pseudomallei on day 21. Next, the most effective construct was used as single vaccination on day 0 by tattoo or intranasal formulation. Mice were sacrificed 72 hours post-challenge to assess bacterial loads, cytokine responses, inflammation and microscopic lesions. A construct encoding a cellular secretion signal resulted in the most effective protection against melioidosis via tattooing, with a 10-fold reduction in bacterial loads in lungs and distant organs compared to the empty vector. Strikingly, a single intranasal administration of the same vaccine resulted in >1000-fold lower bacterial loads and increased survival. Pro-inflammatory cytokine responses were significantly diminished and strong reductions in markers for distant organ damage were observed. A rapid vaccination scheme using flagellin DNA tattoo provides significant protection against intranasal challenge with B. pseudomallei, markedly improved by a single administration via airway mucosa. Hence intranasal vaccination with flagellin-encoding DNA may be applicable when acute mass vaccination is indicated and warrants further testing.

  18. Prevention and synergistic control of Ph(+) ALL by a DNA vaccine and 6-mercaptopurine.

    PubMed

    Köchling, Joachim; Rott, Yvonne; Arndt, Stefanie; Marschke, Christina; Schmidt, Manuel; Wittig, Burghardt; Kalies, Katrin; Westermann, Jürgen; Henze, Günter

    2012-09-07

    Although the outcome of patients with acute lymphoblastic leukemia (ALL) has been improved continuously by chemotherapy and tyrosine kinase inhibitors, prognosis of patients with Philadelphia chromosome positive (Ph(+)) ALL still remains poor. Since further intensification of chemotherapy is limited by toxic side effects and patients with high risk of transplant-related mortality are not eligible for allogeneic stem cell transplantation new treatment strategies are urgently needed for the prevention of Ph(+) ALL relapse. There is increasing evidence that the immune system plays an essential role for the eradication or immunologic control of remaining leukemia cells. We developed several DNA-based vaccines encoding a BCR-ABL(p185) specific peptide and GM-CSF, and CD40-L, IL-27 or IL-12 and evaluated the preventive and therapeutic efficacy against a lethal challenge of syngeneic Ph(+) ALL in Balb/c mice. In vivo cell depletion assays and cytokine expression studies were performed and the efficacy of the DNA vaccine was compared with 6-mercaptopurine (6-MP) alone and the combination of the DNA vaccine and 6-MP. Preventive immunization with the vaccine BCR-ABL/GM-CSF/IL-12 and the TLR-9 agonist dSLIM induced an innate and adaptive immune response mediated by NK-cells, CD4(+) T-cells and CD8(+) T-cells leading to a survival rate of 80%. Therapeutic vaccination resulted in a significantly longer leukemia-free survival (40.7 days vs. 20.4 days) and a higher survival rate (56% vs. 10%) compared to chemotherapy with 6-MP. Remarkably, in combination with the vaccine 6-MP acted synergistically and led to 100% survival. These results demonstrate that minimal residual disease of Ph(+) ALL can be significantly better controlled by a combined treatment approach of immunotherapy and chemotherapy. This provides a rationale for improving maintenance therapy in order to reduce the relapse rate in patients with Ph(+) ALL. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries.

    PubMed

    Yang, Ya-ping; Li, Yu-hong; Zhang, Ai-hua; Bi, Lan; Fan, Ming-wen

    2009-11-01

    To prepare a clinical-grade anti-caries DNA vaccine pGJA-P/VAX and explore its immune effect and protective efficacy against a cariogenic bacterial challenge. A large-scale industrial production process was developed under Good Manufacturing Practices (GMP) by combining and optimizing common unit operations such as alkaline lysis, precipitation, endotoxin removal and column chromatography. Quality controls of the purified bulk and final lyophilized vaccine were conducted according to authoritative guidelines. Mice and gnotobiotic rats were intranasally immunized with clinical-grade pGJA-P/VAX with chitosan. Antibody levels of serum IgG and salivary SIgA were assessed by an enzyme-linked immunosorbent assay (ELISA), and caries activity was evaluated by the Keyes method. pGJA-P/VAX and pVAX1 prepared by a laboratory-scale commercial kit were used as controls. The production process proved to be scalable and reproducible. Impurities including host protein, residual RNA, genomic DNA and endotoxin in the purified plasmid were all under the limits of set specifications. Intranasal vaccination with clinical-grade pGJA-P/VAX induced higher serum IgG and salivary SIgA in both mice and gnotobiotic rats. While in the experimental caries model, the enamel (E), dentinal slight (Ds), and dentinal moderate (Dm) caries lesions were reduced by 21.1%, 33.0%, and 40.9%, respectively. The production process under GMP was efficient in preparing clinical-grade pGJA-P/VAX with high purity and intended effectiveness, thus facilitating future clinical trials for the anti-caries DNA vaccine.

  20. Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries

    PubMed Central

    Yang, Ya-ping; Li, Yu-hong; Zhang, Ai-hua; Bi, Lan; Fan, Ming-wen

    2009-01-01

    Aim: To prepare a clinical-grade anti-caries DNA vaccine pGJA-P/VAX and explore its immune effect and protective efficacy against a cariogenic bacterial challenge. Methods: A large-scale industrial production process was developed under Good Manufacturing Practices (GMP) by combining and optimizing common unit operations such as alkaline lysis, precipitation, endotoxin removal and column chromatography. Quality controls of the purified bulk and final lyophilized vaccine were conducted according to authoritative guidelines. Mice and gnotobiotic rats were intranasally immunized with clinical-grade pGJA-P/VAX with chitosan. Antibody levels of serum IgG and salivary SIgA were assessed by an enzyme-linked immunosorbent assay (ELISA), and caries activity was evaluated by the Keyes method. pGJA-P/VAX and pVAX1 prepared by a laboratory-scale commercial kit were used as controls. Results: The production process proved to be scalable and reproducible. Impurities including host protein, residual RNA, genomic DNA and endotoxin in the purified plasmid were all under the limits of set specifications. Intranasal vaccination with clinical-grade pGJA-P/VAX induced higher serum IgG and salivary SIgA in both mice and gnotobiotic rats. While in the experimental caries model, the enamel (E), dentinal slight (Ds), and dentinal moderate (Dm) caries lesions were reduced by 21.1%, 33.0%, and 40.9%, respectively. Conclusion: The production process under GMP was efficient in preparing clinical-grade pGJA-P/VAX with high purity and intended effectiveness, thus facilitating future clinical trials for the anti-caries DNA vaccine. PMID:19890359

  1. DNA Vaccines against Dengue Virus Type 2 Based on Truncate Envelope Protein or Its Domain III

    PubMed Central

    Azevedo, Adriana S.; Yamamura, Anna M. Y.; Freire, Marcos S.; Trindade, Gisela F.; Bonaldo, Myrna; Galler, Ricardo; Alves, Ada M. B.

    2011-01-01

    Two DNA vaccines were constructed encoding the ectodomain (domains I, II and III) of the DENV2 envelope protein (pE1D2) or only its domain III (pE2D2), fused to the human tissue plasminogen activator signal peptide (t-PA). The expression and secretion of recombinant proteins was confirmed in vitro in BHK cells transfected with the two plasmids, detected by immunofluorescence or immunoprecipitation of metabolically labeled gene products, using polyclonal and monoclonal antibodies against DENV2. Besides, results reveal that the ectodomain of the E protein can be efficiently expressed in vivo, in a mammalian system, without the prM protein that is hypothesized to act as a chaperonin during dengue infection. Balb/c mice were immunized with the DNA vaccines and challenged with a lethal dose of DENV2. All pE1D2-vaccinated mice survived challenge, while 45% of animals immunized with the pE2D2 died after infection. Furthermore, only 10% of pE1D2-immunized mice presented some clinical signs of infection after challenge, whereas most of animals inoculated with the pE2D2 showed effects of the disease with high morbidity degrees. Levels of neutralizing antibodies were significantly higher in pE1D2-vaccinated mice than in pE2D2-immunized animals, also suggesting that the pE1D2 vaccine was more protective than the pE2D2. PMID:21779317

  2. Skewing the T-cell repertoire by combined DNA vaccination, host conditioning, and adoptive transfer.

    PubMed

    Jorritsma, Annelies; Bins, Adriaan D; Schumacher, Ton N M; Haanen, John B A G

    2008-04-01

    Approaches for T-cell-based immunotherapy that have shown substantial effects in clinical trials are generally based on the adoptive transfer of high numbers of antigen-specific cells, and the success of these approaches is thought to rely on the high magnitude of the tumor-specific T-cell responses that are induced. In this study, we aimed to develop strategies that also yield a T-cell repertoire that is highly skewed toward tumor recognition but do not rely on ex vivo generation of tumor-specific T cells. To this end, the tumor-specific T-cell repertoire was first expanded by DNA vaccination and then infused into irradiated recipients. Subsequent vaccination of the recipient mice with the same antigen resulted in peak CD8(+) T-cell responses of approximately 50%. These high T-cell responses required the presence of antigen-experienced tumor-specific T cells within the graft because only mice that received cells of previously vaccinated donor mice developed effective responses. Tumor-bearing mice treated with this combined therapy showed a significant delay in tumor outgrowth, compared with mice treated by irradiation or vaccination alone. Furthermore, this antitumor effect was accompanied by an increased accumulation of activated and antigen-specific T cells within the tumor. In summary, the combination of DNA vaccination with host conditioning and adoptive transfer generates a marked, but transient, skewing of the T-cell repertoire toward tumor recognition. This strategy does not require ex vivo expansion of cells to generate effective antitumor immunity and may therefore easily be translated to clinical application.

  3. Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization

    PubMed Central

    2014-01-01

    Background Pulmonary-delivered gene therapy promises to mitigate vaccine safety issues and reduce the need for needles and skilled personnel to use them. While plasmid DNA (pDNA) offers a rapid route to vaccine production without side effects or reliance on cold chain storage, its delivery to the lung has proved challenging. Conventional methods, including jet and ultrasonic nebulizers, fail to deliver large biomolecules like pDNA intact due to the shear and cavitational stresses present during nebulization. Methods In vitro structural analysis followed by in vivo protein expression studies served in assessing the integrity of the pDNA subjected to surface acoustic wave (SAW) nebulisation. In vivo immunization trials were then carried out in rats using SAW nebulized pDNA (influenza A, human hemagglutinin H1N1) condensate delivered via intratracheal instillation. Finally, in vivo pulmonary vaccinations using pDNA for influenza was nebulized and delivered via a respirator to sheep. Results The SAW nebulizer was effective at generating pDNA aerosols with sizes optimal for deep lung delivery. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to male Swiss mice via intratracheal instillation. Effective systemic and mucosal antibody responses were found in rats via post-nebulized, condensed fluid instillation. Significantly, we demonstrated the suitability of the SAW nebulizer to administer unprotected pDNA encoding an influenza A virus surface glycoprotein to respirated sheep via aerosolized inhalation. Conclusion Given the difficulty of inducing functional antibody responses for DNA vaccination in large animals, we report here the first instance of successful aerosolized inhalation delivery of a pDNA vaccine in a large animal model relevant to human lung development, structure, physiology, and disease, using a novel, low-power (<1 W) surface acoustic wave (SAW) hand-held nebulizer to produce droplets of p

  4. Recombinant Saccharomyces cerevisiae serves as novel carrier for oral DNA vaccines in Carassius auratus.

    PubMed

    Yan, Nana; Xu, Kun; Li, Xinyi; Liu, Yuwan; Bai, Yichun; Zhang, Xiaohan; Han, Baoquan; Chen, Zhilong; Zhang, Zhiying

    2015-12-01

    Oral delivery of DNA vaccines represents a promising vaccinating method for fish. Recombinant yeast has been proved to be a safe carrier for delivering antigen proteins and DNAs to some species in vivo. However, whether recombinant yeast can be used to deliver functional DNAs for vaccination to fish is still unknown. In this study, red crucian carp (Carassius auratus) was orally administrated with recombinant Saccharomyces cerevisiae harboring CMV-EGFP expression cassette. On day 5 post the first vaccination, EGFP expression in the hindgut was detected under fluorescence microscope. To further study whether the delivered gene could induce specific immune responses, the model antigen ovalbumin (OVA) was used as immunogen, and oral administrations were conducted with recombinant S. cerevisiae harboring pCMV-OVA mammalian gene expression cassette as gene delivery or pADH1-OVA yeast gene expression cassette as protein delivery. Each administration was performed with three different doses, and the OVA-specific serum antibody was detected in all the experimental groups by western blotting and enzyme-linked immunosorbent assay (ELISA). ELISA assay also revealed that pCMV-OVA group with lower dose (pCMV-OVA-L) and pADH1-OVA group with moderate dose (pADH1-OVA-M) triggered relatively stronger antibody response than the other two doses. Moreover, the antibody level induced by pCMV-OVA-L group was significantly higher than pADH1-OVA-M group at the same serum dilutions. All the results suggested that recombinant yeast can be used as a potential carrier for oral DNA vaccines and would help to develop more practical strategies to control infectious diseases in aquaculture.

  5. Protective immunity to H7N9 influenza viruses elicited by synthetic DNA vaccine.

    PubMed

    Yan, Jian; Villarreal, Daniel O; Racine, Trina; Chu, Jaemi S; Walters, Jewell N; Morrow, Matthew P; Khan, Amir S; Sardesai, Niranjan Y; Kim, J Joseph; Kobinger, Gary P; Weiner, David B

    2014-05-19

    Despite an intensive vaccine program influenza infections remain a major health problem, due to the viruses' ability to change its envelope glycoprotein hemagglutinin (HA), through shift and drift, permitting influenza to escape protection induced by current vaccines or natural immunity. Recently a new variant, H7N9, has emerged in China causing global concern. First, there have been more than 130 laboratory-confirmed human infections resulting in an alarmingly high death rate (32.3%). Second, genetic changes found in H7N9 appear to be associated with enabling avian influenza viruses to spread more effectively in mammals, thus transmitting infections on a larger scale. Currently, no vaccines or drugs are effectively able to target H7N9. Here, we report the rapid development of a synthetic consensus DNA vaccine (pH7HA) to elicit potent protective immunity against the H7N9 viruses. We show that pH7HA induces broad antibody responses that bind to divergent HAs from multiple new members of the H7N9 family. These antibody responses result in high-titer HAI against H7N9. Simultaneously, this vaccine induces potent polyfunctional effector CD4 and CD8T cell memory responses. Animals vaccinated with pH7HA are completely protected from H7N9 virus infection and any morbidity associated with lethal challenge. This study establishes that this synthetic consensus DNA vaccine represents a new tool for targeting emerging infection, and more importantly, its design, testing and development into seed stock for vaccine production in a few days in the pandemic setting has significant implications for the rapid deployment of vaccines protecting against emerging infectious diseases.

  6. DNA Vaccination Partially Protects Muskellunge against Viral Hemorrhagic Septicemia Virus (VHSV-IVb).

    PubMed

    Millard, Elena V; Bourke, Ashley M; LaPatra, Scott E; Brenden, Travis O; Fitzgerald, Scott D; Faisal, Mohamed

    2017-03-01

    A DNA vaccine containing the glycoprotein (G) gene of the North American viral hemorrhagic septicemia virus (VHSV) genotype IVb was developed to evaluate the immune response of fish following vaccination and evaluate its efficacy in protecting a susceptible species, the Muskellunge Esox masquinongy, against VHSV-IVb challenge. Seven weeks (539 degree-days) following vaccination with 10 μg of either pVHSivb-G or a control plasmid, Muskellunge were challenged by immersion with 10(5) plaque-forming units (pfu)/mL of VHSV-IVb. Fish vaccinated with pVHSivb-G had a relative percent survival (RPS) of 45%. Vaccinated fish also had significantly lower mean viral titers in tissues (4.2 × 10(2) pfu/g) and viral prevalence (4%) than fish receiving the plasmid control vaccine (3.3 × 10(5) pfu/g; 82%). Neutralizing antibodies were detected 28 d (308 degree-days) postchallenge (11 weeks postvaccination) in 100% of Muskellunge vaccinated with pVHSivb-G compared with only 12% of plasmid-control-vaccinated Muskellunge, suggesting robust induction of a secondary, adaptive immune response. In addition, pVHSivb-G-vaccinated Rainbow Trout Oncorhynchus mykiss challenged 7 d (100 degree-days) postvaccination with the heterologous novirhabdovirus, infectious hematopoietic necrosis virus (IHNV), experienced an RPS of 61%, compared to control fish, suggesting induction of an early and transient nonspecific antiviral immune response. This study provides an important starting point for VHSV-IVb vaccine development and useful information about the antiviral immune response elicited by DNA vaccination in a nondomesticated fish species. Received May 1, 2016; accepted September 1, 2016.

  7. Efficacy and safety of an oral somatostatin DNA vaccine without antibiotic resistance gene in promoting growth of piglets.

    PubMed

    Han, Y -G; Liang, A -X; Han, L; Guo, A -Z; Jiang, X -P; Yang, L -G

    2014-04-01

    This study aimed to evaluate the efficacy and safety of an oral DNA vaccine against somatostatin (SS) (pGS/2SS-asd, encoding two copies of somatostatin genes) mediated by attenuated Salmonella choleraesuis C500 without antibiotic resistance gene on piglets growth. A total of 50 piglets were uniformly divided into five groups. The animals in the first three groups were orally given vaccine in dose of either 5 9 1010, 5 9 109 or 5 9 108 colony-forming units (CFU).The remaining two groups were orally administered with either bacteria C500(containing pVAX-asd plasmid without somatostatin gene) or phosphate buffered saline (PBS) as controls. The results indicated that the vaccine induced SS-specific antibodies in a dose-dependent pattern. Compared with the PBS control, animals in the high-dose group showed lower SS levels and higher growth hormone (GH) levels in sera. Average daily gain of animals in the high dose group was increased by 32.88% and 26.46% during 4 and 8 weeks,respectively. Anti-SS antibodies were positively correlated with either GH levels or average daily gain at week 8 after primary immunization (P < 0.05). Faecal,soil and water samples originating from immunized piglets and surrounding environment were collected. The target gene (the fusion gene GS/2SS) of C500(pGS/2SS-asd) was not detected by PCR amplification in these samples,indicating that the surrounding environment was not contaminated by residual recombinant bacteria. In conclusion, the vaccine without antibiotic resistance gene is attributable to improve growth performance of piglets through an influence on GH secretion. Moreover, the immunization did not contaminate the surrounding environment of animals.

  8. Influenza nucleoprotein DNA vaccination by a skin targeted, dry coated, densely packed microprojection array (Nanopatch) induces potent antibody and CD8(+) T cell responses.

    PubMed

    Fernando, Germain J P; Zhang, Jin; Ng, Hwee-Ing; Haigh, Oscar L; Yukiko, Sally R; Kendall, Mark A F

    2016-09-10

    DNA vaccines have many advantages such as thermostability and the ease and rapidity of manufacture; for example, in an influenza pandemic situation where rapid production of vaccine is essential. However, immunogenicity of DNA vaccines was shown to be poor in humans unless large doses of DNA are used. If a highly efficacious DNA vaccine delivery system could be identified, then DNA vaccines have the potential to displace protein vaccines. In this study, we show in a C57BL/6 mouse model, that the Nanopatch, a microprojection array of high density (>21,000 projections/cm(2)), could be used to deliver influenza nucleoprotein DNA vaccine to skin, to generate enhanced antigen specific antibody and CD8(+) T cell responses compared to the conventional intramuscular (IM) delivery by the needle and syringe. Antigen specific antibody was measured using ELISA assays of mice vaccinated with a DNA plasmid containing the nucleoprotein gene of influenza type A/WSN/33 (H1N1). Antigen specific CD8(+) T cell responses were measured ex-vivo in splenocytes of mice using IFN-γ ELISPOT assays. These results and our previous antibody and CD4(+) T cell results using the Nanopatch delivered HSV DNA vaccine indicate that the Nanopatch is an effective delivery system of general utility that could potentially be used in humans to increase the potency of the DNA vaccines.

  9. Delivery of noncarrier naked DNA vaccine into the skin by supersonic flow induces a polarized T helper type 1 immune response to cancer.

    PubMed

    Lin, Chi-Chen; Yen, Meng-Chi; Lin, Chiu-Mei; Huang, Shih-Shien; Yang, Huei-Jiun; Chow, Nan-Haw; Lai, Ming-Derg

    2008-06-01

    DNA vaccine is a new and powerful approach to generate immunological responses against infectious disease and cancer. The T helper type (Th)1 immune response is usually required for generating effective anti-tumor responses. A microparticulate bombardment system can induce an immune response using very low amounts of DNA. Using nozzle aerodynamics, a low pressure gene gun has been developed to decrease the noise associated with high pressure gene guns. Particles are propelled by supersonic flow through this novel nozzle. To test whether this gun could inoculate a DNA vaccine that stimulates an anti-tumor Th1 immune response, we examined the effect of direct delivery of naked DNA (i.e. without any carrier) on the anti-tumor immune response of mice. The luciferase reporter plasmid DNA was delivered using a low-pressure biolistic device and expressed in C3H/HeN, BALB/c, and C57BL/6 mice. Plasmid DNA expression was mainly in the epidermis. Noncarrier naked neu DNA vaccine and gold particle-coated neu DNA vaccine (at 1 microg per mouse) had similar anti-tumor effects in C3H mice. However, cytokine profile examination showed the Th1-bias of the response induced by naked DNA vaccine and the Th2-bias of the response induced by coated DNA vaccine. A shift in the immune response to favour enhanced tumor rejection can be achieved by skin delivery of naked DNA vaccine.

  10. [The adjuvant effect of granulocyte macrophage colony stimulating factor (GM-CSF) in dengue virus and hepatitis C virus DNA vaccines].

    PubMed

    Wu, Jiang-Man; Chen, Hui; Sheng, Zi-Yang; Wang, Juan; Fan, Dong-Ying; Gao, Na; An, Jing

    2012-05-01

    To investigate the adjuvant effect of granulocyte macrophage colony stimulating factor (GM-CSF) in Flaviviridae virus DNA vaccines. After DNA immunization, the antibody levels of serum from mice were detected by ELISA and indirect immunofluorescence assay. Co-immunization of GM-CSF suppressed the immune responses induced by DV1 and DV2 candidate vaccines whereas enhanced the immune response induced by HCV C and E1 DNA vaccines. As genetic adjuvant for DNA vaccines, GM-CSF might display complex diversity on the immune responses: an augmentation or suppression due to different immunogens. Therefore, GM-CSF should be used with some cautions in clinic.

  11. First isolation of a rhabdovirus from perch Perca fluviatilis in Switzerland.

    PubMed

    Wahli, Thomas; Bellec, Laure; von Siebenthal, Beat; Cabon, Joëlle; Schmidt-Posthaus, Heike; Morin, Thierry

    2015-10-16

    Perca fluviatilis is a fish species of increasing interest to the Swiss fish farming industry. In recent years, recirculation systems have been specifically set up to increase production. In one of these farms, abnormal spiral swimming associated with elevated mortalities occurred in repeated batches of imported perch shortly after stocking on several occasions. No bacterial or parasitic etiology was detected, but a virus grown in bluegill fry (BF-2) cells was identified as perch rhabdovirus. Subsequent investigations of other samples suggested a viral tropism for the central nervous system (CNS). Phylogenetic analysis of the partial N and entire G gene sequences positioned this isolate in genogroup C of the species Perch rhabdovirus, with high nucleotide and amino acid (aa) sequence identities with the DK5533 strain isolated in Denmark in 1989. Comparative studies using other closely related isolates allowed the distinction of 2 serological patterns among perch rhabdoviruses and the identification of a proline substitution by a serine in position 147 of the glycoprotein potentially involved in antigenic differentiation. Even if perch imported onto the farm tested negative by virus isolation prior to transport, they may have been the origin of this outbreak since CNS tissue was not included in the samples that were analyzed. Another possibility might be a sub-clinical infection with a viral load in resident fish too low to be detected. This study reports the first isolation of a perch rhabdovirus in Switzerland, and emphasizes the necessity of optimizing diagnostic tools that facilitate better control of the risks associated with fish translocation.

  12. Simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative RT-PCR assay.

    PubMed

    Liu, Zongxiao; Teng, Yong; Liu, Hong; Jiang, Yulin; Xie, Xiayang; Li, Huifang; Lv, Jiangqiang; Gao, Longying; He, Junqiang; Shi, Xiujie; Tian, Feiyan; Yang, Jingshun; Xie, Congxin

    2008-04-01

    Spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are three important fish rhabdoviruses, causing serious Office International des Epizooties (OIE) classified diseases in wild and farmed fish. Here, a new multiplex real-time quantitative RT-PCR (mqRT-PCR) assay was developed for simultaneous detection, identification and quantification of these three rhabdoviruses. The sets of primers and probes were targeted to conserved regions of glycoprotein (G) gene of SVCV, nucleoprotein (N) gene of IHNV and G gene of VHSV and used to amplify. The sensitivity, specificity and interference test of mqRT-PCR assay was analyzed. It was shown that the detection levels of 100 copies of SVCV, 220 copies of IHNV and 140 copies of VHSV were achieved, and there was no non-specific amplification and cross-reactivity using RNA of pike fry rhabdovirus (PFRV), infectious pancreatic necrosis virus (IPNV) and grass carp reovirus (GCRV). A total of 80 clinical fish samples were tested using the mqRT-PCR assay and the results were confirmed by antigen-capture ELISA and cell culture assay. This assay has the potential to be used for both research applications and diagnosis.

  13. Safety and Immunogenicity of an Anti-Zika Virus DNA Vaccine - Preliminary Report.

    PubMed

    Tebas, Pablo; Roberts, Christine C; Muthumani, Kar; Reuschel, Emma L; Kudchodkar, Sagar B; Zaidi, Faraz I; White, Scott; Khan, Amir S; Racine, Trina; Choi, Hyeree; Boyer, Jean; Park, Young K; Trottier, Sylvie; Remigio, Celine; Krieger, Diane; Spruill, Susan E; Bagarazzi, Mark; Kobinger, Gary P; Weiner, David B; Maslow, Joel N

    2017-10-04

    Background Although Zika virus (ZIKV) infection is typically self-limiting, other associated complications such as congenital birth defects and the Guillain-Barré syndrome are well described. There are no approved vaccines against ZIKV infection. Methods In this phase 1, open-label clinical trial, we evaluated the safety and immunogenicity of a synthetic, consensus DNA vaccine (GLS-5700) encoding the ZIKV premembrane and envelope proteins in two groups of 20 participants each. The participants received either 1 mg or 2 mg of vaccine intradermally, with each injection followed by electroporation (the use of a pulsed electric field to introduce the DNA sequence into cells) at baseline, 4 weeks, and 12 weeks. Results The median age of the participants was 38 years, and 60% were women; 78% were white, and 22% black; in addition, 30% were Hispanic. At the interim analysis at 14 weeks (i.e., after the third dose of vaccine), no serious adverse events were reported. Local reactions at the vaccination site (e.g., injection-site pain, redness, swelling, and itching) occurred in approximately 50% of the participants. After the third dose of vaccine, binding antibodies (as measured on enzyme-linked immunosorbent assay) were detected in all the participants, with geometric mean titers of 1642 and 2871 in recipients of 1 mg and 2 mg of vaccine, respectively. Neutralizing antibodies developed in 62% of the samples on Vero-cell assay. On neuronal-cell assay, there was 90% inhibition of ZIKV infection in 70% of the serum samples and 50% inhibition in 95% of the samples. The intraperitoneal injection of postvaccination serum protected 103 of 112 IFNAR knockout mice (bred with deletion of genes encoding interferon-α and interferon-β receptors) (92%) that were challenged with a lethal dose of ZIKV-PR209 strain; none of the mice receiving baseline serum survived the challenge. Survival was independent of the neutralization titer. Conclusions In this phase 1, open-label clinical

  14. A multiagent filovirus DNA vaccine delivered by intramuscular electroporation completely protects mice from ebola and Marburg virus challenge.

    PubMed

    Grant-Klein, Rebecca J; Van Deusen, Nicole M; Badger, Catherine V; Hannaman, Drew; Dupuy, Lesley C; Schmaljohn, Connie S

    2012-11-01

    We evaluated the immunogenicity and protective efficacy of DNA vaccines expressing the codon-optimized envelope glycoprotein genes of Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus (Musoke and Ravn). Intramuscular or intradermal delivery of the vaccines in BALB/c mice was performed using the TriGrid™ electroporation device. Mice that received DNA vaccines against the individual viruses developed robust glycoprotein-specific antibody titers as determined by ELISA and survived lethal viral challenge with no display of clinical signs of infection. Survival curve analysis revealed there was a statistically significant increase in survival compared to the control groups for both the Ebola and Ravn virus challenges. These data suggest that further analysis of the immune responses generated in the mice and additional protection studies in nonhuman primates are warranted.

  15. DNA vaccines expressing pneumococcal surface protein A (PspA) elicit protection levels comparable to recombinant protein.

    PubMed

    Ferreira, Daniela M; Miyaji, Eliane N; Oliveira, Maria Leonor S; Darrieux, Michelle; Arêas, Ana Paula M; Ho, Paulo L; Leite, Luciana C C

    2006-04-01

    Pneumococcal surface protein A (PspA) is a promising candidate for the development of cost-effective vaccines against Streptococcus pneumoniae. In the present study, BALB/c mice were immunized with DNA vaccine vectors expressing the N-terminal region of PspA. Animals immunized with a vector expressing secreted PspA developed higher levels of antibody than mice immunized with the vector expressing the antigen in the cytosol. However, both immunogens elicited similar levels of protection against intraperitoneal challenge. Furthermore, immunization with exactly the same fragment in the form of a recombinant protein, with aluminium hydroxide as an adjuvant, elicited even higher antibody levels, but this increased humoral response did not correlate with enhanced protection. These results show that DNA vaccines expressing PspA are able to elicit protection levels comparable to recombinant protein, even though total anti-PspA IgG response is considerably lower.

  16. Altered Response Hierarchy and Increased T-Cell Breadth upon HIV-1 Conserved Element DNA Vaccination in Macaques

    PubMed Central

    Kulkarni, Viraj; Valentin, Antonio; Rosati, Margherita; Alicea, Candido; Singh, Ashish K.; Jalah, Rashmi; Broderick, Kate E.; Sardesai, Niranjan Y.; Le Gall, Sylvie; Mothe, Beatriz; Brander, Christian; Rolland, Morgane; Mullins, James I.; Pavlakis, George N.; Felber, Barbara K.

    2014-01-01

    HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24gag elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55gag increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist. PMID:24465991

  17. Presence of CpG DNA and the local cytokine milieu determine the efficacy of suppressive DNA vaccination in experimental autoimmune encephalomyelitis.

    PubMed

    Lobell, A; Weissert, R; Eltayeb, S; Svanholm, C; Olsson, T; Wigzell, H

    1999-11-01

    We here study the adjuvant properties of immunostimulatory DNA sequences (ISS) and coinjected cytokine-coding cDNA in suppressive vaccination with DNA encoding an autoantigenic peptide, myelin basic protein peptide 68-85, against Lewis rat experimental autoimmune encephalomyelitis (EAE). EAE is an autoaggressive, T1-mediated disease of the CNS. ISS are unmethylated CpG motifs found in bacterial DNA, which can induce production of type 1 cytokines in vertebrates through the innate immune system. Because ISS in the plasmid backbone are necessary for efficient DNA vaccination, we studied the effect of one such ISS, the 5'-AACGTT-3' motif, in our system. Treatment with a DNA vaccine encoding myelin basic protein peptide 68-85 and containing three ISS of 5'-AACGTT-3' sequence suppressed clinical signs of EAE, while a corresponding DNA vaccine without such ISS had no effect. We further observed reduced proliferative T cell responses in rats treated with the ISS-containing DNA vaccine, compared with controls. We also studied the possible impact of coinjection of plasmid DNA encoding rat cytokines IL-4, IL-10, GM-CSF, and TNF-alpha with the ISS-containing DNA vaccine. Coinjection of IL-4-, IL-10-, or TNF-alpha-coding cDNA inhibited the suppressive effect of the DNA vaccine on EAE, whereas GM-CSF-coding cDNA had no effect. Coinjection of cytokine-coding cDNA with the ISS-deficient DNA vaccine failed to alter clinical signs of EAE. We conclude that the presence of ISS and induction of a local T1 cytokine milieu is decisive for specific protective DNA vaccination in EAE.

  18. Improvement of the Immunogenicity of Porcine Circovirus Type 2 DNA Vaccine by Recombinant ORF2 Gene and CpG Motifs.

    PubMed

    Li, Jun; Shi, Jian-Li; Wu, Xiao-Yan; Fu, Fang; Yu, Jiang; Yuan, Xiao-Yuan; Peng, Zhe; Cong, Xiao-Yan; Xu, Shao-Jian; Sun, Wen-Bo; Cheng, Kai-Hui; Du, Yi-Jun; Wu, Jia-Qiang; Wang, Jin-Bao; Huang, Bao-Hua

    2015-06-01

    Nowadays, adjuvant is still important for boosting immunity and improving resistance in animals. In order to boost the immunity of porcine circovirus type 2 (PCV2) DNA vaccine, CpG motifs were inserted. In this study, the dose-effect was studied, and the immunity of PCV2 DNA vaccines by recombinant open reading frame 2 (ORF2) gene and CpG motifs was evaluated. Three-week-old Changbai piglets were inoculated intramuscularly with 200 μg, 400 μg, and 800 μg DNA vaccines containing 14 and 18 CpG motifs, respectively. Average gain and rectum temperature were recorded everyday during the experiments. Blood was collected from the piglets after vaccination to detect the changes of specific antibodies, interleukin-2, and immune cells every week. Tissues were collected for histopathology and polymerase chain reaction. The results indicated that compared to those of the control piglets, all concentrations of two DNA vaccines could induce PCV2-specific antibodies. A cellular immunity test showed that PCV2-specific lymphocytes proliferated the number of TH, TC, and CD3+ positive T-cells raised in the blood of DNA vaccine immune groups. There was no distinct pathological damage and viremia occurring in pigs that were inoculated with DNA vaccines, but there was some minor pathological damage in the control group. The results demonstrated that CpG motifs as an adjuvant could boost the humoral and cellular immunity of pigs to PCV2, especially in terms of cellular immunity. Comparing two DNA vaccines that were constructed, the one containing 18 CpG motifs was more effective. This is the first report that CpG motifs as an adjuvant insert to the PCV2 DNA vaccine could boost immunity.

  19. Emergence of a new rhabdovirus associated with mass mortalities in eelpout (Zoarces viviparous) in the Baltic Sea.

    PubMed

    Axén, C; Hakhverdyan, M; Boutrup, T S; Blomkvist, E; Ljunghager, F; Alfjorden, A; Hagström, Å; Olesen, N J; Juremalm, M; Leijon, M; Valarcher, J-F

    2017-02-01

    We report the first description of a new Rhabdoviridae tentatively named eelpout rhabdovirus (EpRV genus Perhabdovirus). This virus was associated with mass mortalities in eelpout (Zoarces viviparous, Linnaeus) along the Swedish Baltic Sea coast line in 2014. Diseased fish showed signs of central nervous system infection, and brain lesions were confirmed by histology. A cytopathogenic effect was observed in cell culture, but ELISAs for the epizootic piscine viral haemorrhagic septicaemia virus (VHSV), infectious pancreas necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and spring viraemia of carp virus (SVCV) were negative. Further investigations by chloroform inactivation, indirect fluorescence antibody test and electron microscopy indicated the presence of a rhabdovirus. By deep sequencing of original tissue suspension and infected cell culture supernatant, the full viral genome was assembled and we confirmed the presence of a rhabdovirus with 59.5% nucleotide similarity to the closest relative Siniperca chuatsi rhabdovirus. The full-genome sequence of this new virus, eelpout rhabdovirus (EpRV), has been deposited in GenBank under accession number KR612230. An RT-PCR based on the L-gene sequence confirmed the presence of EpRV in sick/dead eelpout, but the virus was not found in control fish. Additional investigations to characterize the pathogenicity of EpRV are planned.

  20. Hantaan/Andes virus DNA Vaccine Elicits a Broadly Cross-Reactive Neutralizing Antibody Response in Nonhuman Primates

    DTIC Science & Technology

    2006-01-01

    pulmonary syndrome (HPS). The most prevalent and lethal hantaviruses associated with HFRS and HPS are Hantaan virus (HTNV) and Andes virus (ANDV...Published by Elsevier Inc.Keywords: Hantavirus; DNA vaccine; Hantaan virus; Andes virus; Neutralizing antibodiesIntroduction Hantaviruses are rodent...borne viruses that cause hemor- rhagic fever in humans. Different hantaviruses are associated with different disease syndromes with varying degrees of

  1. Quantitative expression profiling of immune response genes in rainbow trout following infectious haematopoietic necrosis virus (IHNV) infection or DNA vaccination.

    PubMed

    Purcell, Maureen K; Kurath, Gael; Garver, Kyle A; Herwig, Russell P; Winton, James R

    2004-11-01

    Infectious haematopoietic necrosis virus (IHNV) is a well-studied virus of salmonid fishes. A highly efficacious DNA vaccine has been developed against this virus and studies have demonstrated that this vaccine induces both an early and transient non-specific anti-viral phase as well as long-term specific protection. The mechanisms of the early anti-viral phase are not known, but previous studies noted changes in Mx gene expression, suggesting a role for type I interferon. This study used quantitative real-time reverse transcriptase PCR methodology to compare expression changes over time of a number of cytokine or cytokine-related genes in the spleen of rainbow trout following injection with poly I:C, live IHNV, the IHNV DNA vaccine or a control plasmid encoding the non-antigenic luciferase gene. The target genes included Mx-1, viral haemorrhagic septicaemia virus induced gene 8 (Vig-8), TNF-alpha1, TNF-alpha2, IL-1beta1, IL-8, TGF-beta1 and Hsp70. Poly I:C stimulation induced several genes but the strongest and significant response was observed in the Mx-1 and Vig-8 genes. The live IHN virus induced a significant response in all genes examined except TGF-beta1. The control plasmid construct and the IHNV DNA vaccine marginally induced a number of genes, but the main difference between these two groups was a statistically significant induction of the Mx-1 and Vig-8 genes by the IHNV vaccine only. The gene expression profiles elicited by the live virus and the IHNV DNA vaccine differed in a number of aspects but this study confirms the clear role for a type I interferon-like response in early anti-viral defence.

  2. Chimeric DNA vaccines encoding Eimeria acervulina macrophage migration inhibitory factor (E.MIF) induce partial protection against experimental Eimeria infection.

    PubMed

    Song, Xiaokai; Zhang, Ruirui; Xu, Lixin; Yan, Ruofeng; Li, Xiangrui

    2015-09-01

    Chimeric DNA vaccines co-expressing Eimeria acervulina macrophage migration inhibitory factor (E.MIF) and chicken IL-2 (IL-2) or interferon-γ (IFN-γ) were constructed and their efficacies against E. acervulina were evaluated. The open reading frame (ORF) of E.MIF was cloned from E. acervulina merozoites and subcloned into the eukaryotic expression vector pVAX1 with chicken cytokine gene IFN-γ or IL-2 to construct the DNA vaccines pVAX-E.MIF-IFN-γ, pVAX-E.MIF-IL-2 and pVAX-E.MIF. The in vivo transfection of the target genes was detected by use of reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Immunizations were carried out by vaccinating chickens twice with a dose rate of 100 μg intramuscularly. Seven days post second immunization, all chickens except the unchallenged control group were challenged orally with 1 × 105 sporulated oocysts of E. acervulina. Seven days later, the duodenum was collected. The results showed that the target genes were expressed effectively in vivo. DNA vaccines and the recombinant E.MIF protein could alleviate body weight loss and duodenal lesions significantly compared to the control groups. Furthermore, pVAX-E.MIF-IL-2 and pVAX-E.MIF-IFN-γ induced anticoccidial indexs (ACIs) of 179.12 and 170, respectively, which were significantly higher than that of pVAX-E.MIF (ACI = 162.31). Our results demonstrated that E.MIF is a potential vaccine candidate against E. acervulina and chicken IFN-γ or IL- 2 may be used as genetic adjuvants to improve the efficacies of DNA vaccines against avian coccidiosis.

  3. Recombinant invasive Lactococcus lactis can transfer DNA vaccines either directly to dendritic cells or across an epithelial cell monolayer.

    PubMed

    de Azevedo, Marcela; Meijerink, Marjolein; Taverne, Nico; Pereira, Vanessa Bastos; LeBlanc, Jean Guy; Azevedo, Vasco; Miyoshi, Anderson; Langella, Philippe; Wells, Jerry M; Chatel, Jean-Marc

    2015-09-11

    Lactococcus lactis (L. lactis), a generally regarded as safe (GRAS) bacterium has recently been investigated as a mucosal delivery vehicle for DNA vaccines. Because of its GRAS status, L. lactis represents an attractive alternative to attenuated pathogens. Previous studies showed that eukaryotic expression plasmids could be delivered into intestinal epithelial cells (IECs) by L. lactis, or recombinant invasive strains of L. lactis, leading to heterologous protein expression. Although expression of antigens in IECs might lead to vaccine responses, it would be of interest to know whether uptake of L. lactis DNA vaccines by dendritic cells (DCs) could lead to antigen expression as they are unique in their ability to induce antigen-specific T cell responses. To test this, we incubated mouse bone marrow-derived DCs (BMDCs) with invasive L. lactis strains expressing either Staphylococcus aureus Fibronectin Binding Protein A (LL-FnBPA+), or Listeria monocytogenes mutated Internalin A (LL-mInlA+), both strains carrying a plasmid DNA vaccine (pValac) encoding for the cow milk allergen β-lactoglobulin (BLG). We demonstrated that they can transfect BMDCs, inducing the secretion of the pro-inflammatory cytokine IL-12. We also measured the capacity of strains to invade a polarized monolayer of IECs, mimicking the situation encountered in the gastrointestinal tract. Gentamycin survival assay in these cells showed that LL-mInlA+ is 100 times more invasive than L. lactis. The cross-talk between differentiated IECs, BMDCs and bacteria was also evaluated using an in vitro transwell co-culture model. Co-incubation of strains in this model showed that DCs incubated with LL-mInlA+ containing pValac:BLG could express significant levels of BLG. These results suggest that DCs could sample bacteria containing the DNA vaccine across the epithelial barrier and express the antigen.

  4. Synergistic and additive effects of cimetidine and levamisole on cellular immune responses to hepatitis B virus DNA vaccine in mice.

    PubMed

    Niu, X; Yang, Y; Wang, J

    2013-02-01

    We and others have previously shown that both cimetidine (CIM) and levamisole (LMS) enhance humoral and cellular responses to DNA vaccines via different mechanisms. In this study, we investigated the synergistic and additive effects of CIM and LMS on the potency of antigen-specific immunities generated by a DNA vaccine encoding the hepatitis B surface antigen (HBsAg, pVax-S2). Compared with CIM or LMS alone, the combination of CIM and LMS elicited a robust HBsAg-specific cellular response that was characterized by higher IgG2a, but did not further increase HBsAg-specific antibody IgG and IgG1 production. Consistent with these results, the combination of CIM and LMS produced the highest level of IL-2 and IFN-γ in antigen-specific CD4(+) T cells, whereas the combination of CIM and LMS did not further increase IL-4 production. Significantly, a robust HBsAg-specific cytotoxic response was also observed in the animals immunized with pVax-S2 in the presence of the combination of CIM and LMS. Further mechanistic studies demonstrated that the combination of CIM and LMS promoted dendritic cell (DC) activation and blocked anti-inflammatory cytokine IL-10 and TGF-β production in CD4(+) CD25(+) T cells. These findings suggest that CIM and LMS have the synergistic and additive ability to enhance cellular response to hepatitis B virus DNA vaccine, which may be mediated by DC activation and inhibition of anti-inflammatory cytokine expression. Thus, the combination of cimetidine and levamisole may be useful as an effective adjuvant in DNA vaccinations for chronic hepatitis B virus infection.

  5. DNA vaccine-derived human IgG produced in transchromosomal bovines protect in lethal models of hantavirus pulmonary syndrome.

    PubMed

    Hooper, Jay W; Brocato, Rebecca L; Kwilas, Steven A; Hammerbeck, Christopher D; Josleyn, Matthew D; Royals, Michael; Ballantyne, John; Wu, Hua; Jiao, Jin-an; Matsushita, Hiroaki; Sullivan, Eddie J

    2014-11-26

    Polyclonal immunoglobulin-based medical products have been used successfully to treat diseases caused by viruses for more than a century. We demonstrate the use of DNA vaccine technology and transchromosomal bovines (TcBs) to produce fully human polyclonal immunoglobulins (IgG) with potent antiviral neutralizing activity. Specifically, two hantavirus DNA vaccines [Andes virus (ANDV) DNA vaccine and Sin Nombre virus (SNV) DNA vaccine] were used to produce a candidate immunoglobulin product for the prevention and treatment of hantavirus pulmonary syndrome (HPS). A needle-free jet injection device was used to vaccinate TcB, and high-titer neutralizing antibodies (titers >1000) against both viruses were produced within 1 month. Plasma collected at day 10 after the fourth vaccination was used to produce purified α-HPS TcB human IgG. Treatment with 20,000 neutralizing antibody units (NAU)/kg starting 5 days after challenge with ANDV protected seven of eight animals, whereas zero of eight animals treated with the same dose of normal TcB human IgG survived. Likewise, treatment with 20,000 NAU/kg starting 5 days after challenge with SNV protected immunocompromised hamsters from lethal HPS, protecting five of eight animals. Our findings that the α-HPS TcB human IgG is capable of protecting in animal models of lethal HPS when administered after exposure provides proof of concept that this approach can be used to develop candidate next-generation polyclonal immunoglobulin-based medical products without the need for human donors, despeciation protocols, or inactivated/attenuated vaccine antigen. Copyright © 2014, American Association for the Advancement of Science.

  6. Mixing of M Segment DNA Vaccines to Hantaan Virus and Puumala Virus Reduces Their Immunogenicity in Hamsters

    DTIC Science & Technology

    2008-01-01

    with renal syndrome (HFRS). In merica, several other hantaviruses cause hantavirus pulmonary yndrome (HPS) [1]. There are currently no U.S. licensed vac...online 25 April 2008 eywords: a b s t r a c t To determine if DNA vaccines for two hantaviruses causing hemorrhagic fever with renal syndrome, Hantaan...twoantaviruses emorrhagic fever with renal syndrome NA vaccines hantaviruses could be detected. In contrast, if the DNAs were given as separate vaccinations to

  7. Alum adjuvanted rabies DNA vaccine confers 80% protection against lethal 50 LD50 rabies challenge virus standard strain.

    PubMed

    Garg, Rajni; Kaur, Manpreet; Saxena, Ankur; Prasad, Rajendra; Bhatnagar, Rakesh

    2017-03-03

    Rabies is a serious concern world-wide. Despite availability of rabies vaccines for long; their efficacy, safety, availability and cost effectiveness has been a tremendous issue. This calls for improvement of rabies vaccination strategies. DNA vaccination has immense potential in this regard. The DNA vaccine pgp.LAMP-1 conferred 60% protection to BALB/c mice against 20 LD50 rabies challenge virus standard (CVS) strain challenge. Upon supplementation with Emulsigen-D, the vaccine formulation conferred complete protection against lethal challenge. To assess the feasibility of this vaccine formulation for human use, it was tested along with other FDA approved adjuvants, namely, Alum, Immuvac, Montanide ISA720 VG. Enhanced immune response correlated with high IgG antibody titer, Th2 biased response with a high level of rabies virus neutralizing antibodies (RVNAs) and IgG1/IgG2a ratio >1, observed upon alum supplementation of the rabies DNA vaccine. The total IgG antibody titer was 2IU/ml and total RVNA titer was observed to be 4IU/ml which is eight times higher than the minimum protective titer recommended by WHO. Furthermore, it conferred 80% protection against challenge with 50 LD50 of the rabies CVS strain, conducted in compliance with the potency test for rabies recommended by the National Institutes of Health (NIH), USA. Previously, we have established pre-clinical safety of this vaccine as per the guidelines of Schedule Y, FDA as well as The European Agency for evaluation of Medicinal Products. The vaccine showed no observable toxicity at the site of injection as well as at systemic level in Wistar rats when administered with 10X recommended dose. Therefore, supplementation of rabies DNA vaccine, pgp.LAMP-1 with alum would lead to development of a non-toxic, efficacious, stable and affordable vaccine that can be used to combat high numbers of fatal rabies infections tormenting developing countries.

  8. Enhancing the Immunogenicity of a Dengue-2 DNA Vaccine With Adjuvants and Anti-FCyRI Antibodies

    DTIC Science & Technology

    2005-10-01

    antibody responses to the vaccine given IM were enhanced slightly by combining it with TT. However, the ELISA and neutralizing antibody levels were...aluminum phosphate, tetanus toxoid and DNA vaccine linked with anti-FcγR antibody . 3. Analyze antibody response by indirect ELISA and plaque reduction...FcγR antibody to the DEN-1 DNA. 3. Analyze antibody response by indirect ELISA and plaque reduction neutralization tests. Perform ELISPOT

  9. PD-1 or PD-L1 Blockade Restores Antitumor Efficacy Following SSX2 Epitope-Modified DNA Vaccine Immunization.

    PubMed

    Rekoske, Brian T; Smith, Heath A; Olson, Brian M; Maricque, Brett B; McNeel, Douglas G

    2015-08-01

    DNA vaccines have demonstrated antitumor efficacy in multiple preclinical models, but low immunogenicity has been observed in several human clinical trials. This has led to many approaches seeking to improve the immunogenicity of DNA vaccines. We previously reported that a DNA vaccine encoding the cancer-testis antigen SSX2, modified to encode altered epitopes with increased MHC class I affinity, elicited a greater frequency of cytolytic, multifunctional CD8(+) T cells in non-tumor-bearing mice. We sought to test whether this optimized vaccine resulted in increased antitumor activity in mice bearing an HLA-A2-expressing tumor engineered to express SSX2. We found that immunization of tumor-bearing mice with the optimized vaccine elicited a surprisingly inferior antitumor effect relative to the native vaccine. Both native and optimized vaccines led to increased expression of PD-L1 on tumor cells, but antigen-specific CD8(+) T cells from mice immunized with the optimized construct expressed higher PD-1. Splenocytes from immunized animals induced PD-L1 expression on tumor cells in vitro. Antitumor activity of the optimized vaccine could be increased when combined with antibodies blocking PD-1 or PD-L1, or by targeting a tumor line not expressing PD-L1. These findings suggest that vaccines aimed at eliciting effector CD8(+) T cells, and DNA vaccines in particular, might best be combined with PD-1 pathway inhibitors in clinical trials. This strategy may be particularly advantageous for vaccines targeting prostate cancer, a disease for which antitumor vaccines have demonstrated clinical benefit and yet PD-1 pathway inhibitors alone have shown little efficacy to date.

  10. Induction of a protective response in mice by the dengue virus NS3 protein using DNA vaccines.

    PubMed

    Costa, Simone M; Yorio, Anna Paula; Gonçalves, Antônio J S; Vidale, Mariana M; Costa, Emmerson C B; Mohana-Borges, Ronaldo; Motta, Marcia A; Freire, Marcos S; Alves, Ada M B

    2011-01-01

    The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection.

  11. Immunogenicity of a plasmid DNA vaccine encoding 42kDa fragment of Plasmodium vivax merozoite surface protein-1.

    PubMed

    Sheikh, Inayat Hussain; Kaushal, Deep C; Chandra, Deepak; Kaushal, Nuzhat A

    2016-10-01

    Plasmodium vivax is the second major human malaria parasite that inflicts debilitating morbidity and consequent economic impact in South-East Asian countries. The relapsing nature of P. vivax along with the emergence of drug-resistant P. vivax strains has emphasized the urgent need for a vaccine. However, the development of an effective vivax vaccine is seriously hampered due to the diversity and variation in parasite antigens and non-availability of suitable animal models. DNA based vaccines represent an alternative approach in inducing immunity to multiple targets from different stages of malaria parasite. DNA prime-boosting strategies induce both antibody mediated and cell-mediated immune responses that are the major mechanisms of protection against malaria parasites. We have earlier studied the immunogenicity and protective efficacy of the soluble and refolded forms of recombinant 42kDa fragment of Plasmodium vivax merozoite surface protein-1 (PvMSP-142) using P. cynomolgi rhesus monkey model. In the present study, we have constructed a recombinant DNA vaccine encoding 42kDa fragment of P. vivax MSP-1 and studied the immunogenicity of PvMSP-142 DNA vaccine construct in mice. The 42kDa gene fragment of PvMSP-1 was PCR amplified using gene specific primers and subcloned into pcDNA 3.1 (+) eukaryotic expression vector. In vitro expression of PvMSP-142 plasmid construct was checked by transfection in COS-1 cell line. Indirect immunofluorescence of transfected COS-1 cells probed with monoclonal antibodies against PvMSP-142 exhibited positive fluorescence. Immunization of BALB/c mice with PvMSP-142-pcDNA vaccine construct revealed the immunogenicity of recombinant vaccine plasmid that can be enhanced by prime boosting with recombinant protein corresponding to the DNA vaccine as evidenced by significant elevation of antibody and the cytokines responses.

  12. Quantitative expression profiling of immune response genes in rainbow trout following infectious haematopoietic necrosis virus (IHNV) infection or DNA vaccination

    USGS Publications Warehouse

    Purcell, Maureen K.; Kurath, Gael; Garver, Kyle A.; Herwig, Russell P.; Winton, James R.

    2004-01-01

    Infectious haematopoietic necrosis virus (IHNV) is a well-studied virus of salmonid fishes. A highly efficacious DNA vaccine has been developed against this virus and studies have demonstrated that this vaccine induces both an early and transient non-specific anti-viral phase as well as long-term specific protection. The mechanisms of the early anti-viral phase are not known, but previous studies noted changes in Mx gene expression, suggesting a role for type I interferon. This study used quantitative real-time reverse transcriptase PCR methodology to compare expression changes over time of a number of cytokine or cytokine-related genes in the spleen of rainbow trout following injection with poly I:C, live IHNV, the IHNV DNA vaccine or a control plasmid encoding the non-antigenic luciferase gene. The target genes included Mx-1, viral haemorrhagic septicaemia virus induced gene 8 (Vig-8), TNF-α1, TNF-α2, IL-1β1, IL-8, TGF-β1 and Hsp70. Poly I:C stimulation induced several genes but the strongest and significant response was observed in the Mx-1 and Vig-8 genes. The live IHN virus induced a significant response in all genes examined except TGF-β1. The control plasmid construct and the IHNV DNA vaccine marginally induced a number of genes, but the main difference between these two groups was a statistically significant induction of the Mx-1 and Vig-8 genes by the IHNV vaccine only. The gene expression profiles elicited by the live virus and the IHNV DNA vaccine differed in a number of aspects but this study confirms the clear role for a type I interferon-like response in early anti-viral defence.

  13. Topical Administration Is a Promising Inoculating Route versus Intramuscular Inoculation for the Nanoparticle-Carried DNA Vaccine to Prevent Corneal Infections.

    PubMed

    Hu, Kai; Malla, Tejsu; Zhai, Yujia; Dong, Lili; Tang, Ru

    2015-01-01

    To evaluate the comparative effect of topical versus intramuscular administration of nanoparticle-carried DNA vaccine in preventing corneal herpes simplex virus type 1 (HSV-1) infection. Nanoparticle [polyethylenimine (PEI)-Fe3O4]-carried DNA vaccine (PEI-Fe3O4-pRSC-gD-IL-21) or DNA vaccine (pRSC-gD-IL-21) alone were topically versus intramuscularly inoculated into one eye each of mice on days 0, 14 and 28. Three weeks after the final immunization, the specific immune responses and clinical degrees of primary herpes simplex keratitis were evaluated. Topical inoculation of nanoparticle-carried DNA vaccine induced mice to generate similar levels of specific HSV-1-neutralizing antibody, IFN-γ and IL-4 in serum and specific killing (cytotoxicity) and proliferative activities of the splenic lymphocytes, but a significantly higher level of secretory IgA in tears compared to those of intramuscular inoculation. More importantly, the mice inoculated topically showed a significantly decreased herpes simplex keratitis severity than the mice inoculated intramuscularly after HSV-1 challenge on the corneas of the mice. Topical inoculation of nanoparticle-carried DNA vaccine elicits a stronger specific local immune response and more effectively inhibits herpes simplex keratitis as compared to intramuscular inoculation in an HSV-1 ocular challenge mouse model. Thus, topical administration may be a promising inoculating route for the nanoparticle-carried DNA vaccine to prevent corneal infections. © 2015 S. Karger AG, Basel.

  14. Construction of glutathione peroxidase (GPx) DNA vaccine and its protective efficiency on the orange-spotted grouper (Epinephelus coioides) challenged with Vibrio harveyi.

    PubMed

    Wang, Haiyang; Zhu, Fan; Huang, Yucong; Ding, Yu; Jian, Jichang; Wu, Zaohe

    2017-01-01

    The main aims of this study were to construct glutathione peroxidase (GPx) DNA vaccine of Vibrio harveyi ZJ0603 and to investigate its immune protective efficiency as a vaccine candidate on the orange-spotted grouper (Epinephelus coioides) treated with V. harveyi. Base on the cloning of ZJ0603 GPx gene, a DNA vaccine, named as pcDNA-GPx, was constructed by inserting GPx gene into pcDNA3.1 (+) plasmid. Orange-spotted groupers were immunized with the pcDNA-GPx plasmid by injection intramuscularly. The relative percent of survival (RPS) of fish vaccinated with the DNA vaccine against pathogenic V. harveyi infection was 77.5%. The expression of DNA vaccine was analyzed in the tissues of orange-spotted grouper by PCR and RT-PCR. The results indicated that pcDNA-GPx distributed and expressed in the head kidney, liver, spleen, gill and injected muscle at 7 and 28 days after vaccination. Significant specific antibody responses were also detected in the vaccinated orange-spotted groupers by indirect ELISA method. In a conclusion, DNA vaccine pcDNA-GPx showed an effective immune protection to the orange-spotted grouper treated with V. harveyi. The GPx can be used as a candidate DNA vaccine for the control of vibriosis.

  15. Mutual enhancement of IL-2 and IL-7 on DNA vaccine immunogenicity mainly involves regulations on their receptor expression and receptor-expressing lymphocyte generation.

    PubMed

    Zhang, Yonghong; Liang, Shuang; Li, Xiujin; Wang, Liyue; Zhang, Jianlou; Xu, Jian; Huo, Shanshan; Cao, Xuebin; Zhong, Zhenyu; Zhong, Fei

    2015-07-09

    Our previous study showed that IL-2 and IL-7 could mutually enhance the immunogenicity of canine parvovirus VP2 DNA vaccine, although the underlying mechanism remained unknown. Here, we used the OVA gene as a DNA vaccine in a mouse model to test their enhancement on DNA vaccine immunogenicity and to explore the molecular mechanism. Results showed that both IL-2 and IL-7 genes significantly increased the immunogenicity of OVA DNA vaccine in mice. Co-administration of IL-2 and IL-7 genes with OVA DNA significantly increased OVA-specific antibody titers, T cell proliferation and IFN-γ production compared with IL-2 or IL-7 alone, confirming that IL-2 and IL-7 mutually enhanced DNA vaccine immunogenicity. Mechanistically, we have shown that IL-2 significantly stimulated generation of IL-7 receptor-expressing lymphocytes, and that IL-7 significantly induced IL-2 receptor expression. These results contribute to an explanation of the mechanism of the mutual effects of IL-2 and IL-7 on enhancing DNA vaccine immunogenicity and provided a basis for further investigation on their mutual effects on adjuvant activity and immune regulation.

  16. MultiTEP platform-based DNA vaccines for alpha-synucleinopathies: preclinical evaluation of immunogenicity and therapeutic potency.

    PubMed

    Davtyan, Hayk; Zagorski, Karen; Petrushina, Irina; Kazarian, Konstantin; Goldberg, Natalie R S; Petrosyan, Janet; Blurton-Jones, Mathew; Masliah, Eliezer; Cribbs, David H; Agadjanyan, Michael G; Ghochikyan, Anahit

    2017-08-10

    We have previously demonstrated that anti-beta amyloid DNA vaccine (AV-1959D) based on our proprietary MultiTEP platform technology is extremely immunogenic in mice, rabbits, and monkeys. Importantly, MultiTEP platform enables development of vaccines targeting pathological molecules involved in various neurodegenerative disorders. Taking advantage of the universality of MultiTEP platform, we developed DNA vaccines targeting 3 B-cell epitopes (amino acids [aa]85-99, aa109-126, and aa126-140) of human alpha-synuclein (hα-Syn) separately or all 3 epitopes simultaneously. All 4 DNA vaccines (1) generate high titers of anti-hα-Syn antibodies and (2) induce robust MultiTEP-specific T-helper cell responses without activation of potentially detrimental autoreactive anti-hα-Syn T-helper cells. Generated antibodies recognize misfolded hα-Syn produced by neuroblastoma cells, hα-Syn in the brain tissues of transgenic mouse strains and in the brain tissues of dementia with Lewy body cases. Based on these results, the most promising vaccine targeting 3 B-cell epitopes of hα-Syn simultaneously (PV-1950D) has been chosen for ongoing preclinical assessment in mouse models of hα-Syn with the aim to translate it to the human clinical trials. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Optimization of HIV-1 Envelope DNA Vaccine Candidates within Three Different Animal Models, Guinea Pigs, Rabbits and Cynomolgus Macaques

    PubMed Central

    Borggren, Marie; Vinner, Lasse; Andresen, Betina Skovgaard; Grevstad, Berit; Repits, Johanna; Melchers, Mark; Elvang, Tara Laura; Sanders, Rogier W; Martinon, Frédéric; Dereuddre-Bosquet, Nathalie; Bowles, Emma Joanne; Stewart-Jones, Guillaume; Biswas, Priscilla; Scarlatti, Gabriella; Jansson, Marianne; Heyndrickx, Leo; Le Grand, Roger; Fomsgaard, Anders

    2013-01-01

    HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often starts in small animal models before macaque and human trials. Here, we selected and optimized DNA vaccine candidates through systematic testing in rabbits for the induction of broadly neutralizing antibodies (bNAb). We compared three different animal models: guinea pigs, rabbits and cynomolgus macaques. Envelope genes from the prototype isolate HIV-1 Bx08 and two elite neutralizers were included. Codon-optimized genes, encoded secreted gp140 or membrane bound gp150, were modified for expression of stabilized soluble trimer gene products, and delivered individually or mixed. Specific IgG after repeated i.d. inoculations with electroporation confirmed in vivo expression and immunogenicity. Evaluations of rabbits and guinea pigs displayed similar results. The superior DNA construct in rabbits was a trivalent mix of non-modified codon-optimized gp140 envelope genes. Despite NAb responses with some potency and breadth in guinea pigs and rabbits, the DNA vaccinated macaques displayed less bNAb activity. It was concluded that a trivalent mix of non-modified gp140 genes from rationally selected clinical isolates was, in this study, the best option to induce high and broad NAb in the rabbit model, but this optimization does not directly translate into similar responses in cynomolgus macaques. PMID:26344115

  18. Assessment of delivery parameters with the multi-electrode array for development of a DNA vaccine against Bacillus anthracis.

    PubMed

    Donate, Amy; Heller, Richard

    2013-12-01

    Gene electrotransfer (GET) enhances delivery of DNA vaccines by increasing both gene expression and immune responses. Our lab has developed the multi-electrode array (MEA) for DNA delivery to skin. The MEA was used at constant pulse duration (150 ms) and frequency (6.67 Hz). In this study, delivery parameters including applied voltage (5-45 V), amount of plasmid (100-300 μg), and number of treatments (2-3) were evaluated for delivery of a DNA vaccine. Mice were intradermally injected with plasmid expressing Bacillus anthracis protective antigen with or without GET and αPA serum titers measured. Within this experiment no significant differences were noted in antibody levels from varying dose or treatment number. However, significant differences were measured from applied voltages of 25 and 35 V. These voltages generated antibody levels between 20,000 and 25,000. Serum from animals vaccinated with these conditions also resulted in toxin neutralization in 40-60% of animals. Visual damage was noted at MEA conditions of 40 V. No damage was noted either visually or histologically from conditions of 35 V or below. These results reflect the importance of establishing appropriate electrical parameters and the potential for the MEA in non-invasive DNA vaccination against B. anthracis.

  19. DNA vaccine initiates replication of live attenuated chikungunya virus in vitro and elicits protective immune response in mice.

    PubMed

    Tretyakova, Irina; Hearn, Jason; Wang, Eryu; Weaver, Scott; Pushko, Peter

    2014-06-15

    Chikungunya virus (CHIKV) causes outbreaks of chikungunya fever worldwide and represents an emerging pandemic threat. Vaccine development against CHIKV has proved challenging. Currently there is no approved vaccine or specific therapy for the disease. To develop novel experimental CHIKV vaccine, we used novel immunization DNA (iDNA) infectious clone technology, which combines the advantages of DNA and live attenuated vaccines. Here we describe an iDNA vaccine composed of plasmid DNA that encode the full-length infectious genome of live attenuated CHIKV clone 181/25 downstream from a eukaryotic promoter. The iDNA approach was designed to initiate replication of live vaccine virus from the plasmid in vitro and in vivo. Experimental CHIKV iDNA vaccines were prepared and evaluated in cultured cells and in mice. Transfection with 10 ng of iDNA was sufficient to initiate replication of vaccine virus in vitro. Vaccination of BALB/c mice with a single 10 μg of CHIKV iDNA plasmid resulted in seroconversion, elicitation of neutralizing antibodies, and protection from experimental challenge with a neurovirulent CHIKV. Live attenuated CHIKV 181/25 vaccine can be delivered in vitro and in vivo by using DNA vaccination. The iDNA approach appears to represent a promising vaccination strategy for CHIK and other alphaviral diseases. © The Author 2014. 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.

  20. Protective antibody responses against Clostridium difficile elicited by a DNA vaccine expressing the enzymatic domain of toxin B.

    PubMed

    Jin, Ke; Wang, Shixia; Zhang, Chunhua; Xiao, Yanling; Lu, Shan; Huang, Zuhu

    2013-01-01

    A DNA vaccination approach was used in the current study to screen for the immunogenicity of different fragments of toxin A and toxin B from Clostridium difficile. With this approach, protein antigens do not need to be produced in vitro and the immunogenicity of candidate C. difficile antigens can be identified directly in animals. Codon optimized toxin gene fragments were individually cloned into the DNA vaccine vector and tested in mice and rabbits for their ability to elicit C. difficile toxin-specific antibody responses. Only a subset of the C. difficile toxin fragments, including the C-terminal receptor binding domain of toxin A and a novel N-terminal enzymatic domain of toxin B, were able to elicit protective antibody responses as determined by protection of target cells in a cytotoxicity assay or by preventing death of mice in a passive antibody protection study. Significantly, antibodies elicited by the novel N-terminus of the toxin B DNA vaccine were able to increase the level of protection when used in combination with anti-toxin A antibodies in a toxin challenge model in mice.

  1. 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.

  2. A DNA Vaccine Encoding for TcSSP4 Induces Protection against Acute and Chronic Infection in Experimental Chagas Disease

    PubMed Central

    Arce-Fonseca, Minerva; Ramos-Ligonio, Angel; López-Monteón, Aracely; Salgado-Jiménez, Berenice; Talamás-Rohana, Patricia; Rosales-Encina, José Luis

    2011-01-01

    Immunization of mice with plasmids containing genes of Trypanosoma cruzi induces protective immunity in the murine model of Chagas disease. A cDNA clone that codes for an amastigote-specific surface protein (TcSSP4) was used as a candidate to develop a DNA vaccine. Mice were immunized with the recombinant protein rTcSSP4 and with cDNA for TcSSP4, and challenged with bloodstream trypomastigotes. Immunization with rTcSSP4 protein makes mice more susceptible to trypomastigote infection, with high mortality rates, whereas mice immunized with a eukaryotic expression plasmid containing the TcSSP4 cDNA were able to control the acute phase of infection. Heart tissue of gene-vaccinated animals did not show myocarditis and tissue damage at 365 days following infection, as compared with control animals. INF-γ was detected in sera of DNA vaccinated mice shortly after immunization, suggesting the development of a Th1 response. The TcSSP4 gene is a promising candidate for the development of an anti-T. cruzi DNA vaccine. PMID:22110377

  3. Characterization of guinea pig T cell responses elicited after EP-assisted delivery of DNA vaccines to the skin.

    PubMed

    Schultheis, Katherine; Schaefer, Hubert; Yung, Bryan S; Oh, Janet; Muthumani, Karuppiah; Humeau, Laurent; Broderick, Kate E; Smith, Trevor R F

    2017-01-03

    The skin is an ideal target tissue for vaccine delivery for a number of reasons. It is highly accessible, and most importantly, enriched in professional antigen presenting cells. Possessing strong similarities to human skin physiology and displaying a defined epidermis, the guinea pig is an appropriate model to study epidermal delivery of vaccine. However, whilst we have characterized the humoral responses in the guinea pig associated with skin vaccine protocols we have yet to investigate the T cell responses. In response to this inadequacy, we developed an IFN-γ ELISpot assay to characterize the cellular immune response in the peripheral blood of guinea pigs. Using a nucleoprotein (NP) influenza pDNA vaccination regimen, we characterized host T cell responses. After delivery of the DNA vaccine to the guinea pig epidermis we detected robust and rapid T cell responses. The levels of IFN-γ spot-forming units averaged approximately 5000 per million cells after two immunizations. These responses were broad in that multiple regions across the NP antigen elicited a T cell response. Interestingly, we identified a number of NP immunodominant T cell epitopes to be conserved across an outbred guinea pig population, a phenomenon which was also observed after immunization with a RSV DNA vaccine. We believe this data enhances our understanding of the cellular immune response elicited to a vaccine in guinea pigs, and globally, will advance the use of this model for vaccine development, especially those targeting skin as a delivery site.

  4. Characterization of guinea pig T cell responses elicited after EP-assisted delivery of DNA vaccines to the skin

    PubMed Central

    Schultheis, Katherine; Schaefer, Hubert; Yung, Bryan S.; Oh, Janet; Muthumani, Karuppiah; Humeau, Laurent; Broderick, Kate E.

    2016-01-01

    The skin is an ideal target tissue for vaccine delivery for a number of reasons. It is highly accessible, and most importantly, enriched in professional antigen presenting cells. Possessing strong similarities to human skin physiology and displaying a defined epidermis, the guinea pig is an appropriate model to study epidermal delivery of vaccine. However, whilst we have characterized the humoral responses in the guinea pig associated with skin vaccine protocols we have yet to investigate the T cell responses. In response to this inadequacy, we developed an IFN-γ ELISpot assay to characterize the cellular immune response in the peripheral blood of guinea pigs. Using a nucleoprotein (NP) influenza pDNA vaccination regimen, we characterized host T cell responses. After delivery of the DNA vaccine to the guinea pig epidermis we detected robust and rapid T cell responses. The levels of IFN-γ spot-forming units averaged approximately 5000 per million cells after two immunizations. These responses were broad in that multiple regions across the NP antigen elicited a T cell response. Interestingly, we identified a number of NP immunodominant T cell epitopes to be conserved across an outbred guinea pig population, a phenomenon which was also observed after immunization with a RSV DNA vaccine. We believe this data enhances our understanding of the cellular immune response elicited to a vaccine in guinea pigs, and globally, will advance the use of this model for vaccine development, especially those targeting skin as a delivery site. PMID:27894716

  5. Recombinant DNA technology for melanoma immunotherapy: anti-Id DNA vaccines targeting high molecular weight melanoma-associated antigen.

    PubMed

    Barucca, A; Capitani, M; Cesca, M; Tomassoni, D; Kazmi, U; Concetti, F; Vincenzetti, L; Concetti, A; Venanzi, F M

    2014-11-01

    Anti-idiotypic MK2-23 monoclonal antibody (anti-Id MK2-23 mAb), which mimics the high molecular weight melanoma-associated antigen (HMW-MAA), has been used to implement active immunotherapy against melanoma. However, due to safety and standardization issues, this approach never entered extensive clinical trials. In the present study, we investigated the usage of DNA vaccines as an alternative to MK2-23 mAb immunization. MK2-23 DNA plasmids coding for single chain (scFv) MK2-23 antibody were constructed via the insertion of variable heavy (V H) and light (V L) chains of MK2-23 into the pVAC-1mcs plasmids. Two alternative MK2-23 plasmids format V H/V L, and V L/V H were assembled. We demonstrate that both polypeptides expressed by scFv plasmids in vitro retained the ability to mimic HMW-MAA antigen, and to elicit specific anti-HMW-MAA humoral and cellular immunoresponses in immunized mice. Notably, MK2-23 scFv DNA vaccines impaired the onset and growth of transplantable B16 melanoma cells not engineered to express HMW-MAA. This pilot study suggests that optimized MK2-23 scFv DNA vaccines could potentially provide a safer and cost-effective alternative to anti-Id antibody immunization, for melanoma immunotherapy.

  6. Protective immune response in mice induced by a suicidal DNA vaccine encoding NTPase-II gene of Toxoplasma gondii.

    PubMed

    Zheng, Lina; Hu, Yue; Hua, Qianqian; Luo, Fangjun; Xie, Guizhen; Li, Xiangzhi; Lin, Jiaxin; Wan, Yujing; Ren, Shoufeng; Pan, Changwang; Tan, Feng

    2017-02-01

    DNA-based alphaviral RNA replicon vectors, also called suicidal DNA vectors, have been employed to alleviate biosafety concerns attribution to its ability to induce apoptotic cell death of the transfected cells. Toxoplasma gondii nucleoside triphosphate hydrolase-II (TgNTPase-II), which facilitates the parasite to salvage purines from the host cell for survival and replication, have been demonstrated to be a potential vaccine candidate for toxoplasmosis. Herein, we evaluated the immunogenic potential of a suicidal DNA vaccine encoding TgNTPase-II gene, pDREP-TgNTPase-II, delivered intramuscularly in combination with electroporation. Immunization of mice with pDREP-TgNTPase-II elicited specific humoral responses, with high IgG antibody titers and a mixed IgG1/IgG2a response. The cellular immune response was associated with high level production of IFN-γ, IL-2, IL-10 cytokines and low level IL-4 production as well as the increase of the percentage of CD8+ T cells, indicating that a Th1 predominant response was elicited. Furthermore, mice vaccinated with this suicidal DNA vaccine displayed partial protection against acute infection with the virulent RH strain as well as chronic infection with PRU cyst, which shows 77.7% and 71.4% reduction in brain cyst burden in comparison to PBS and pDREP-eGFP control group, respectively. Based on the cellular and antibody responses, the suicidal DNA vaccine elicited a Th1-predominant immune response against T. gondii challenge.

  7. Induction of broad cytotoxic T cells by protective DNA vaccination against Marburg and Ebola.

    PubMed

    Shedlock, Devon J; Aviles, Jenna; Talbott, Kendra T; Wong, Gary; Wu, Stephan J; Villarreal, Daniel O; Myles, Devin Jf; Croyle, Maria A; Yan, Jian; Kobinger, Gary P; Weiner, David B

    2013-07-01

    Marburg and Ebola hemorrhagic fevers have been described as the most virulent viral diseases known to man due to associative lethality rates of up to 90%. Death can occur within days to weeks of exposure and there is currently no licensed vaccine or therapeutic. Recent evidence suggests an important role for antiviral T cells in conferring protection, but little detailed analysis of this response as driven by a protective vaccine has been reported. We developed a synthetic polyvalent-filovirus DNA vaccine against Marburg marburgvirus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus (SUDV). Preclinical efficacy studies were performed in guinea pigs and mice using rodent-adapted viruses, whereas murine T-cell responses were extensively analyzed using a novel modified assay described herein. Vaccination was highly potent, elicited robust neutralizing antibodies, and completely protected against MARV and ZEBOV challenge. Comprehensive T-cell analysis revealed cytotoxic T lymphocytes (CTLs) of great magnitude, epitopic breadth, and Th1-type marker expression. This model provides an important preclinical tool for studying protective immune correlates that could be applied to existing platforms. Data herein support further evaluation of this enhanced gene-based approach in nonhuman primate studies for in depth analyses of T-cell epitopes in understanding protective efficacy.

  8. Screening of novel malaria DNA vaccine candidates using full-length cDNA library.

    PubMed

    Shibui, Akiko; Nakae, Susumu; Watanabe, Junichi; Sato, Yoshitaka; Tolba, Mohammed E M; Doi, Junko; Shiibashi, Takashi; Nogami, Sadao; Sugano, Sumio; Hozumi, Nobumichi

    2013-11-01

    No licensed malaria vaccine exists, in spite of intensive development efforts. We have been investigating development of a DNA vaccine to prevent malaria infection. To date, we have established a full-length cDNA expression library from the erythrocytic-stage murine malaria parasite, Plasmodium berghei. We found that immunization of mice with combined 2000 clones significantly prolonged survival after challenge infection and that splenocytes from the immunized mice showed parasite-specific cytokine production. We determined the 5'-end one-pass sequence of these clones and mapped a draft genomic sequence for P. berghei for use in screening vaccine candidates for efficacy. In this study, we annotated these cDNA clones by comparing them with the genomic sequence of Plasmodium falciparum. We then divided them into several subsets based on their characteristics and examined their protective effects against malaria infection. Consequently, we selected 104 clones that strongly induced specific IgG production and decreased the mortality rate in the early phase. Most of these 104 clones coded for unknown proteins. The results suggest that these clones represent potential novel malaria vaccine candidates.

  9. Chemokine-adjuvanted electroporated DNA vaccine induces substantial protection from simian immunodeficiency virus vaginal challenge.

    PubMed

    Kutzler, M A; Wise, M C; Hutnick, N A; Moldoveanu, Z; Hunter, M; Reuter, M A; Yuan, S; Yan, J; Ginsberg, A A; Sylvester, A; Pahar, B; Carnathan, D G; Kathuria, N; Khan, A S; Montefiori, D; Sardesai, N Y; Betts, M R; Mestecky, J; Marx, P A; Weiner, D B

    2016-01-01

    There have been encouraging results for the development of an effective HIV vaccine. However, many questions remain regarding the quality of immune responses and the role of mucosal antibodies. We addressed some of these issues by using a simian immunodeficiency virus (SIV) DNA vaccine adjuvanted with plasmid-expressed mucosal chemokines combined with an intravaginal SIV challenge in rhesus macaque (RhM) model. We previously reported on the ability of CCR9 and CCR10 ligand (L) adjuvants to enhance mucosal and systemic IgA and IgG responses in small animals. In this study, RhMs were intramuscularly immunized five times with either DNA or DNA plus chemokine adjuvant delivered by electroporation followed by challenge with SIVsmE660. Sixty-eight percent of all vaccinated animals (P<0.01) remained either uninfected or had aborted infection compared with only 14% in the vaccine naïve group. The highest protection was observed in the CCR10L chemokines group, where six of nine animals had aborted infection and two remained uninfected, leading to 89% protection (P<0.001). The induction of mucosal SIV-specific antibodies and neutralization titers correlated with trends in protection. These results indicate the need to further investigate the contribution of chemokine adjuvants to modulate immune responses and the role of mucosal antibodies in SIV/HIV protection.

  10. Modular multiantigen T cell epitope-enriched DNA vaccine against human leishmaniasis.

    PubMed

    Das, Shantanabha; Freier, Anja; Boussoffara, Thouraya; Das, Sushmita; Oswald, Detlef; Losch, Florian O; Selka, Melanie; Sacerdoti-Sierra, Nina; Schönian, Gabriele; Wiesmüller, Karl-Heinz; Seifert, Karin; Schroff, Matthias; Juhls, Christiane; Jaffe, Charles L; Roy, Syamal; Das, Pradeep; Louzir, Hechmi; Croft, Simon L; Modabber, Farrokh; Walden, Peter

    2014-04-30

    The leishmaniases are protozoal diseases that severely affect large populations in tropical and subtropical regions. There are only limited treatment options and preventative measures. Vaccines will be important for prevention, control and elimination of leishmaniasis, and could reduce the transmission and burden of disease in endemic populations. We report the development of a DNA vaccine against leishmaniasis that induced T cell-based immunity and is a candidate for clinical trials. The vaccine antigens were selected as conserved in various Leishmania species, different endemic regions, and over time. They were tested with T cells from individuals cured of leishmaniasis, and shown to be immunogenic and to induce CD4(+) and CD8(+) T cell responses in genetically diverse human populations of different endemic regions. The vaccine proved protective in a rodent model of infection. Thus, the immunogenicity of candidate vaccine antigens in human populations of endemic regions, as well as proof of principle for induction of specific immune responses and protection against Leishmania infection in mice, provides a viable strategy for T cell vaccine development.

  11. Development of an ultrasound-responsive and mannose-modified gene carrier for DNA vaccine therapy.

    PubMed

    Un, Keita; Kawakami, Shigeru; Suzuki, Ryo; Maruyama, Kazuo; Yamashita, Fumiyoshi; Hashida, Mitsuru

    2010-10-01

    Development of a gene delivery system to transfer the gene of interest selectively and efficiently into targeted cells is essential for achievement of sufficient therapeutic effects by gene therapy. Here, we succeeded in developing the gene transfection method using ultrasound (US)-responsive and mannose-modified gene carriers, named Man-PEG(2000) bubble lipoplexes. Compared with the conventional lipofection method using mannose-modified carriers, this transfection method using Man-PEG(2000) bubble lipoplexes and US exposure enabled approximately 500-800-fold higher gene expressions in the antigen presenting cells (APCs) selectively in vivo. This enhanced gene expression was contributed by the improvement of delivering efficiency of nucleic acids to the targeted organs, and by the increase of introducing efficiency of nucleic acids into the cytoplasm followed by US exposure. Moreover, high anti-tumor effects were demonstrated by applying this method to DNA vaccine therapy using ovalbumin (OVA)-expressing plasmid DNA (pDNA). This US-responsive and cell-specific gene delivery system can be widely applied to medical treatments such as vaccine therapy and anti-inflammation therapy, which its targeted cells are APCs, and our findings may help in establishing innovative methods for in-vivo gene delivery to overcome the poor introducing efficiency of carriers into cytoplasm which the major obstacle associated with gene delivery by non-viral carriers.

  12. C3d enhanced DNA vaccination induced humoral immune response to glycoprotein C of pseudorabies virus

    SciTech Connect

    Tong Tiezhu; Fan Huiying; Tan Yadi; Xiao Shaobo; Ling Jieyu; Chen Huanchun; Guo Aizhen . E-mail: aizhen@mail.hzau.edu.cn

    2006-09-08

    Murine C3d were utilized to enhance immunogenicity of pseudorabies virus (PrV) gC DNA vaccination. Three copies of C3d and four copies of CR2-binding domain M28{sub 4} were fused, respectively, to truncated gC gene encoding soluble glycoprotein C (sgC) in pcDNA3.1. BALB/c mice were, respectively, immunized with recombinant plasmids, blank vector, and inactivated vaccine. The antibody ELISA titer for sgC-C3d{sub 3} DNA was 49-fold more than that for sgC DNA, and the neutralizing antibody obtained 8-fold rise. Protection of mice from death after lethal PrV (316 LD{sub 5}) challenge was augmented from 25% to 100%. Furthermore, C3d fusion increased Th2-biased immune response by inducing IL-4 production. The IL-4 level for sgC-C3d{sub 3} DNA immunization approached that for the inactivated vaccine. Compared to C3d, M28 enhanced sgC DNA immunogenicity to a lesser extent. In conclusion, we demonstrated that murine C3d fusion significantly enhanced gC DNA immunity by directing Th1-biased to a balanced and more effective Th1/Th2 response.

  13. Effects of menstrual cycle on gene transfection through mouse vagina for DNA vaccine.

    PubMed

    Kanazawa, T; Takashima, Y; Hirayama, S; Okada, H

    2008-08-06

    Human immunodeficiency virus (HIV) infections mainly occur through the vaginal and rectal mucosal membranes. In the present study, to develop a DNA vaginal vaccine against viral and bacterial infections, the effects of the menstrual cycle on DNA transfection through the vaginal mucosa in female mice and transfection enhancement by electroporation, a chelating agent, cell-penetrating peptides (CPP) and nuclear localizing signals (NLS) were investigated. The transfection efficiencies of a marker plasmid DNA (pDNA), pCMV-Luc, on the vaginal mucosal membrane in mice at the stages of metestrus and diestrus were significantly higher than those at the stages of proestrus and estrus. The gene expression was markedly enhanced by electroporation and by pretreatment with the chelating agent. The highest level of expression was obtained by 2h pretreatment with 5% citric acid solution combined with electroporation with 15 pulses at 250 V/cm for 5 milliseconds (ms). Furthermore, a synergistic promoting effect on pDNA transfection was obtained by co-administration of CPP, the Tat peptide analog, and NLS, the NF-kappaB analog. These results indicate that effective DNA vaccination administered through the vaginal tract is possible by selecting the menstrual stage and overcoming the mucosal barrier using a combination of methods that promotes uptake.

  14. Epitope analysis and protection by a ROP19 DNA vaccine against Toxoplasma gondii

    PubMed Central

    Zhou, Jian; Wang, Lin; Lu, Gang; Zhou, Aihua; Zhu, Meiyan; Li, Qihang; Wang, Zhilin; Arken, Miradel; Wang, Ao; He, Shenyi

    2016-01-01

    We used bioinformatics approaches to identify B-cell and T-cell epitopes on the ROP19 protein of Toxoplasma gondii. Then, we constructed plasmids with ROP19 (pEGFP-C1-ROP19) and injected them into BALB/c mice to test the immunoprotection induced by this vaccine candidate. The results showed that immunization with pEGFP-C1-ROP19 induced effective cellular and humoral immune responses in mice; specifically, high serum levels of T. gondii-specific IgG and increased interferon-gamma production by splenocytes. Furthermore, the mice vaccinated with pROP19 had significantly fewer brain cysts (583 ± 160) than the mice injected with phosphate-buffered saline (1350 ± 243) or with the control plasmid, pEGFP-C1 (1300 ± 167). Compared with PBS-treated mice, those immunized with pROP19 had only 43% of the number of brain cysts. These results suggest that the DNA vaccine encoding ROP19 induced a significant immune response and provided protection against a challenge with T. gondii strain PRU cysts. PMID:27055564

  15. Immunogenicity of Candidate MERS-CoV DNA Vaccines Based on the Spike Protein

    PubMed Central

    Al-amri, Sawsan S.; Abbas, Ayman T.; Siddiq, Loai A.; Alghamdi, Abrar; Sanki, Mohammad A.; Al-Muhanna, Muhanna K.; Alhabbab, Rowa Y.; Azhar, Esam I.; Li, Xuguang; Hashem, Anwar M.

    2017-01-01

    MERS-coronavirus is a novel zoonotic pathogen which spread rapidly to >25 countries since 2012. Its apparent endemicity and the wide spread of its reservoir host (dromedary camels) in the Arabian Peninsula highlight the ongoing public health threat of this virus. Therefore, development of effective prophylactic vaccine needs to be urgently explored given that there are no approved prophylactics or therapeutics for humans or animals to date. Different vaccine candidates have been investigated but serious safety concerns remain over protein or full-length spike (S) protein-based vaccines. Here, we investigated the immunogenicity of naked DNA vaccines expressing different fragments of MERS-CoV S protein in mice. We found that plasmids expressing full-length (pS) or S1-subunit (pS1) could induce significant levels of S1-specific antibodies (Abs) but with distinct IgG isotype patterns. Specifically, pS1 immunization elicited a balanced Th1/Th2 response and generally higher levels of all IgG isotypes compared to pS vaccination. Interestingly, only mice immunized with pS1 demonstrated significant S1-specific cellular immune response. Importantly, both constructs induced cross-neutralizing Abs against multiple strains of human and camel origins. These results indicate that vaccines expressing S1-subunit of the MERS-CoV S protein could represent a potential vaccine candidate without the possible safety concerns associated with full-length protein-based vaccines. PMID:28332568

  16. Evaluating the immunogenicity and protective efficacy of a DNA vaccine encoding Lassa virus nucleoprotein.

    PubMed

    Rodriguez-Carreno, Maria P; Nelson, Michael S; Botten, Jason; Smith-Nixon, Kim; Buchmeier, Michael J; Whitton, J Lindsay

    2005-04-25

    Several viruses in the Arenavirus genus of the family Arenaviridae cause severe, often fatal, hemorrhagic fever. One such virus, Lassa virus (LV), is a frequent cause of disease in Africa, and survivors often are left with substantial neurological impairment. The feasibility of protective immunization against LV infection, and the associated disease, has been demonstrated in animal models, using recombinant vaccinia viruses to deliver Lassa proteins. Circumstantial evidence implicates cellular immunity in this Lassa-induced protection, but this has not been confirmed. Here, we describe DNA vaccines that encode LV proteins. A single inoculation of a plasmid encoding full-length Lassa nucleoprotein (LNP) can induce CD8(+) T cell responses in mice and can protect against challenge with two arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV). A DNA minigene vaccine encoding a 9 amino acid sequence from LNP also induces CD8(+) T cells and protects against arenavirus challenge, thus confirming prior speculation that protective cellular immunity is induced by LV proteins.

  17. Immunogenicity and protective efficacy of DNA vaccine against visceral leishmaniasis in BALB/c mice

    PubMed Central

    Kaur, Sukhbir; Kaur, Tejinder; Joshi, Jyoti

    2016-01-01

    Abstract The current study was designed to examine the protective efficacy of DNA vaccines based on gp63 and Hsp70 against murine visceral leishmaniasis. Inbred BALB/c mice were immunized subcutaneously twice at an interval of three weeks with pcDNA3.1(+) encoding T cell epitopes of gp63 and Hsp70 individually and in combination. Animals were challenged intracardially with 107 promastigotes of Leishmania donovani 10 days post immunization and sacrificed 1, 2 and 3 months post challenge. The immunized animals revealed a significant reduction (P < 0.05) in splenic and hepatic parasite burden as compared to the infected controls. Maximum reduction in parasite load (P < 0.05) was observed in animals treated with a combination of pcDNA/gp63 and pcDNA/Hsp70. These animals also showed heightened DTH response, increased IgG2a, elevated Th1 cytokines (IFN-γ and IL-2) and reduced IgG1 and IL-10 levels. Thus, mice immunized with the cocktail vaccine exhibited significantly greater protection in comparison to those immunized with individual antigens. PMID:27533939

  18. Preparation and characterization of novel PBAE/PLGA polymer blend microparticles for DNA vaccine delivery.

    PubMed

    Balashanmugam, Meenashi Vanathi; Nagarethinam, Sivagurunathan; Jagani, Hitesh; Josyula, Venkata Rao; Alrohaimi, Abdulmohsen; Udupa, Nayanabhirama

    2014-01-01

    Poly(beta-amino ester) (PBAE) with its pH sensitiveness and Poly(lactic-co-glycolic acid) (PLGA) with huge DNA cargo capacity in combination prove to be highly efficient as DNA delivery system. To study the effectiveness of novel synthesized PBAE polymer with PLGA blend at different ratios in DNA vaccine delivery. In the present study, multifunctional polymer blend microparticles using a combination of PLGA and novel PBAE polymers A1 (bis(3-(propionyloxy)propyl)3,3'-(propane-1,3-diyl-bis(methylazanediyl))dipropanoate) and A2 (bis(4-(propionyloxy)butyl)3,3'-(ethane-1,2-diyl-bis(isopropylazanediyl))dipropanoate) at different ratios (85:15, 75:25, and 50:50) were prepared by double emulsion solvent removal method. The microparticles were characterized for cytotoxicity, transfection efficiency, and DNA encapsulation efficiency. It was evident from results that among the microparticles prepared with PLGA/PBAE blend the PLGA:PBAE at 85:15 ratio was found to be more effective combination than the microparticles prepared with PLGA alone in terms of transfection efficiency and better DNA integrity. Microparticles made of PLGA and PBAE A1 at 85:15 ratio, respectively, were found to be less toxic when compared with microparticles prepared with A2 polymer. The results encourage the use of the synthesized PBAE polymer in combination with PLGA as an effective gene delivery system.

  19. A novel DNA vaccine containing four mimicry epitopes for gastric cancer.

    PubMed

    Chen, Yu; Wu, Kaichun; Guo, Changcun; Liu, Changjiang; Han, Shuang; Lin, Tao; Ning, Xiaoxuan; Shi, Rui; Shi, Yongquan; Fan, Daiming

    2005-03-01

    Gastric cancer is one of the most common malignant tumors in China. This paper focuses on the development of a DNA vaccine containing four mimotopes of MG7Ag for gastric cancer (multi-epitope vaccine). By inoculating BALB/c mice, the vaccine was characterized and compared with a similar vaccine containing only one mimotope (mono-epitope vaccine) and other controls. Cellular ELISA indicated that serum titer of antibody against MG7Ag was significantly higher in mice immunized with the multi-epitope vaccine than that in the group immunized with the mono-epitope vaccine (0.8627 vs 0.6754, P < 0.05). And ELISPOT assay showed that the number of INF-gamma spots induced by multi-epitope vaccine was significantly larger than that of the group immunized with mono-epitope vaccine(93.3 vs 70.7, P < 0.05). Two weeks after tumor challenge, the weight of tumor in each mouse was evaluated, and the tumor masses formed in the mice immunized with multi-epitope vaccine were markedly smaller than those formed in the mice immunized with mono-epitope vaccine. These studies demonstrated that both humoral and cellular response were induced by the two vaccines and the efficiency of multi-epitope vaccine is stronger than that of the mono-epitope vaccine.

  20. Plasmid DNA Vaccine vector design: impact on efficacy, safety and upstream production

    PubMed Central

    Williams, James A; Carnes, Aaron E; Hodgson, Clague P

    2009-01-01

    Critical molecular and cellular biological factors impacting design of licensable DNA vaccine vectors that combine high yield and integrity during bacterial production with increased expression in mammalian cells are reviewed. Food and Drug Administration (FDA), World Health Organization (WHO) and European Medical Agencies (EMEA) regulatory guidance’s are discussed, as they relate to vector design and plasmid fermentation. While all new vectors will require extensive preclinical testing to validate safety and performance prior to clinical use, regulatory testing burden for follow-on products can be reduced by combining carefully designed synthetic genes with existing validated vector backbones. A flowchart for creation of new synthetic genes, combining rationale design with bioinformatics, is presented. The biology of plasmid replication is reviewed, and process engineering strategies that reduce metabolic burden discussed. Utilizing recently developed low metabolic burden seed stock and fermentation strategies, optimized vectors can now be manufactured in high yields exceeding 2 g/L, with specific plasmid yields of 5% total dry cell weight. PMID:19233255

  1. Chemokine Adjuvanted Electroporated-DNA Vaccine Induces Substantial Protection from Simian Immunodeficiency Virus Vaginal Challenge

    PubMed Central

    Hutnick, N A; Moldoveanu, Z; Hunter, M; Reuter, M; Yuan, S; Yan, J; Ginsberg, A; Sylvester, A; Pahar, B; Carnathan, D; Kathuria, N; Khan, A S; Montefiori, D; Sardesai, N Y; Betts, M R; Mestecky, J; Marx, P; Weiner, D B

    2015-01-01

    There have been encouraging results for the development of an effective HIV vaccine. However, many questions remain regarding the quality of immune responses and the role of mucosal antibodies. We addressed some of these issues by using a simian immunodeficiency virus (SIV) DNA vaccine adjuvanted with plasmid-expressed mucosal chemokines combined with an intravaginal SIV challenge in rhesus macaque (RhM) model. We previously reported on the ability of CCR9 and CCR10 ligand (L) adjuvants to enhance mucosal and systemic IgA and IgG in small animals. In this study, RhMs were intramuscularly immunized five times with either DNA or DNA plus chemokine adjuvant delivered by electroporation followed by challenge with SIVsmE660. Sixty-eight percent of all vaccinated animals (P=0.0016) remained either uninfected or had aborted infection compared to only 14% in the vaccine naïve group. The highest protection was observed in the CCR10L chemokines group, where 6 of 9 animals had aborted infection and two remained uninfected, leading to 89% protection (P=0.0003). The induction of mucosal SIV-specific antibodies and neutralization titers correlated with trends in protection. These results indicate the need to further investigate the contribution of chemokine adjuvants to modulate immune responses and the role of mucosal antibodies in SIV/HIV protection. PMID:25943275

  2. Expanded breadth of the T-cell response to mosaic HIV-1 envelope DNA vaccination

    SciTech Connect

    Korber, Bette; Fischer, William; Wallstrom, Timothy

    2009-01-01

    An effective AIDS vaccine must control highly diverse circulating strains of HIV-1. Among HIV -I gene products, the envelope (Env) protein contains variable as well as conserved regions. In this report, an informatic approach to the design of T-cell vaccines directed to HIV -I Env M group global sequences was tested. Synthetic Env antigens were designed to express mosaics that maximize the inclusion of common potential Tcell epitope (PTE) 9-mers and minimize the inclusion of rare epitopes likely to elicit strain-specific responses. DNA vaccines were evaluated using intracellular cytokine staining (ICS) in inbred mice with a standardized panel of highly conserved 15-mer PTE peptides. I, 2 and 3 mosaic sets were developed that increased theoretical epitope coverage. The breadth and magnitude ofT-cell immunity stimulated by these vaccines were compared to natural strain Env's; additional comparisons were performed on mutant Env's, including gpl60 or gpl45 with or without V regions and gp41 deletions. Among them, the 2 or 3 mosaic Env sets elicited the optimal CD4 and CD8 responses. These responses were most evident in CD8 T cells; the 3 mosaic set elicited responses to an average of 8 peptide pools compared to 2 pools for a set of3 natural Env's. Synthetic mosaic HIV -I antigens can therefore induce T-cell responses with expanded breadth and may facilitate the development of effective T -cell-based HIV -1 vaccines.

  3. Stable and long-lasting immune response in horses after DNA vaccination against equine arteritis virus.

    PubMed

    Giese, M; Bahr, U; Jakob, N J; Kehm, R; Handermann, M; Müller, H; Vahlenkamp, T H; Spiess, C; Schneider, T H; Schusse, G; Darai, G

    2002-10-01

    Equine arteritis virus (EAV) is the causative agent of the equine viral arteritis. It is a small RNA virus with a linear, non-segmented plus RNA genome. EAV is a member of the Arteriviridae family that includes porcine reproductive and respiratory syndrome virus (PRSSV), simian haemorrhagic fever virus (SHFV) and lactate dehydrogenase virus (LDV). The viral transmission is via respiratory and reproductive routes. Clinical signs in horses vary, and severe infection can lead to abortions in pregnant mares or neonatal foal death. The aim of this study was to investigate the development of the immune response in horses after immunization with a DNA vaccine harbouring and expressing EAV Open Reading Frames (ORF) 2, 5, and 7, in combination with equine interleukin 2 (eqIL2). Three boosters followed the basic immunization in two-week intervals. Each immunization was a combination of gene gun and intramuscular injection. All horses developed a high titer of neutralizing antibodies after basic immunization within 2 weeks. Remarkably, this immune response was found to be independent of the age of animals. The youngest horse was six-years old, and the oldest twenty-two years old. A remarkable difference in the immune response between the young and old were not observed. The duration of immunity was investigated during a period of one year. After 12 months, neutralizing antibodies were still detectable in all the vaccinated horses.

  4. Preparation and efficacy of Newcastle disease virus DNA vaccine encapsulated in chitosan nanoparticles.

    PubMed

    Zhao, Kai; Zhang, Yang; Zhang, Xiaoyan; Li, Wei; Shi, Ci; Guo, Chen; Dai, Chunxiao; Chen, Qian; Jin, Zheng; Zhao, Yan; Cui, Hongyu; Wang, Yunfeng

    2014-01-01

    Optimal preparation conditions of Newcastle disease virus (NDV) F gene deoxyribonucleic acid (DNA) vaccine encapsulated in chitosan nanoparticles (pFNDV-CS-NPs) were determined. The pFNDV-CS-NPs were prepared according to a complex coacervation method. The pFNDV-CS-NPs were produced with good morphology, high stability, a mean diameter of 199.5 nm, encapsulation efficiency of 98.37% ± 0.87%, loading capacity of 36.12% ± 0.19%, and a zeta potential of +12.11 mV. The in vitro release assay showed that the plasmid DNA was sustainably released from the pFNDV-CS-NPs, up to 82.9% ± 2.9% of the total amount. Cell transfection test indicated that the vaccine expressed the F gene in cells and maintained good bioactivity. Additionally, the safety of mucosal immunity delivery system of the pFNDV-CS-NPs was also tested in vitro by cell cytotoxicity and in vivo by safety test in chickens. In vivo immunization showed that better immune responses of specific pathogen-free chickens immunized with the pFNDV-CS-NPs were induced, and prolonged release of the plasmid DNA was achieved compared to the chickens immunized with the control plasmid. This study lays the foundation for the further development of mucosal vaccines and drugs encapsulated in chitosan nanoparticles.

  5. Tumor prevention in HPV8 transgenic mice by HPV8-E6 DNA vaccination.

    PubMed

    Marcuzzi, Gian Paolo; Awerkiew, Sabine; Hufbauer, Martin; Schädlich, Lysann; Gissmann, Lutz; Eming, Sabine; Pfister, Herbert

    2014-06-01

    The genus beta human papillomavirus 8 (HPV8) is involved in the development of cutaneous squamous cell carcinomas (SCCs) in individuals with epidermodysplasia verruciformis. Immunosuppressed transplant recipients are prone to harbor particularly high betapapillomavirus DNA loads, which may contribute to their highly increased risk of SCC. Tumor induction in HPV8 transgenic mice correlates with increased expression of viral oncogenes E6 and E2. In an attempt to prevent skin tumor development, we evaluated an HPV8-E6-DNA vaccine, which was able to stimulate a detectable HPV8-E6-specific cell-mediated immune response in 8/15 immunized mice. When skin of HPV8 transgenic mice was grafted onto non-transgenic littermates, the grafted HPV8 transgenic tissue was not rejected and papillomas started to grow within 14 days all over the transplant of 9/9 non-vaccinated and 7/15 not successfully vaccinated mice. In contrast, no papillomas developed in 6/8 successfully vaccinated mice. In the other two of these eight mice, a large ulcerative lesion developed within the initial papilloma growth or papilloma development was highly delayed. As the vaccine completely or partially prevented papilloma development without rejecting the transplanted HPV8 positive skin, the immune system appears to attack only keratinocytes with increased levels of E6 protein, which would give rise to papillomas.

  6. DNA Vaccine-Generated Duck Polyclonal Antibodies as a Postexposure Prophylactic to Prevent Hantavirus Pulmonary Syndrome (HPS)

    PubMed Central

    Brocato, Rebecca; Josleyn, Matthew; Ballantyne, John; Vial, Pablo; Hooper, Jay W.

    2012-01-01

    Andes virus (ANDV) is the predominant cause of hantavirus pulmonary syndrome (HPS) in South America and the only hantavirus known to be transmitted person-to-person. There are no vaccines, prophylactics, or therapeutics to prevent or treat this highly pathogenic disease (case-fatality 35–40%). Infection of Syrian hamsters with ANDV results in a disease that closely mimics human HPS in incubation time, symptoms of respiratory distress, and disease pathology. Here, we evaluated the feasibility of two postexposure prophylaxis strategies in the ANDV/hamster lethal disease model. First, we evaluated a natural product, human polyclonal antibody, obtained as fresh frozen plasma (FFP) from a HPS survivor. Second, we used DNA vaccine technology to manufacture a polyclonal immunoglobulin-based product that could be purified from the eggs of vaccinated ducks (Anas platyrhynchos). The natural “despeciation" of the duck IgY (i.e., Fc removed) results in an immunoglobulin predicted to be minimally reactogenic in humans. Administration of ≥5,000 neutralizing antibody units (NAU)/kg of FFP-protected hamsters from lethal disease when given up to 8 days after intranasal ANDV challenge. IgY/IgYΔFc antibodies purified from the eggs of DNA-vaccinated ducks effectively neutralized ANDV in vitro as measured by plaque reduction neutralization tests (PRNT). Administration of 12,000 NAU/kg of duck egg-derived IgY/IgYΔFc protected hamsters when administered up to 8 days after intranasal challenge and 5 days after intramuscular challenge. These experiments demonstrate that convalescent FFP shows promise as a postexposure HPS prophylactic. Moreover, these data demonstrate the feasibility of using DNA vaccine technology coupled with the duck/egg system to manufacture a product that could supplement or replace FFP. The DNA vaccine-duck/egg system can be scaled as needed and obviates the necessity of using limited blood products obtained from a small number of HPS survivors. This is the

  7. Influence of methisoprinol on the replication of rhabdoviruses isolated from carp (Cyprinus carpio) and catfish (Ictalurus melas): in vitro study.

    PubMed

    Siwicki, A K; Pozet, F; Morand, M; Kazuń, B; Trapkowska, S; Małaczewska, J

    2003-01-01

    Rhabdoviruses constitute one of the most pathogenic viruses isolated from rainbow trout and carp culture. Several viruses were also isolated from other species of fish. These viruses are mostly associated with epizootics and heavy losses. Spring viraemia of carp virus (SVCV) and pike fry rhabdovirus (PFRV) have been the most extensively studied, due to their significant economic impact. Significant progress has been made towards controlling the major bacterial fish diseases using vaccines, but this approach has not yet been successful in preventing viral diseases in fish culture. However, for an effective therapeutic approach, specific drugs should be developed to selectively inhibit virus replication and/or stimulate antiviral protection. In this investigation we examined the in vitro influence of methisoprinol on the SVCV and virus isolated from catfish (Ictalurus melas) replication by measuring their RNA synthesis. The viruses were propagated in EPC cells and cell cultures containing methisoprinol were followed by infection with SVCV or catfish rhabdovirus suspension containing 10(7) TCID50/ml. Methisoprinol (Polfa, Poland) at concentrations of 0, 100, 200, 300, 400 and 500 microg/ml of medium (Glasgow MEM) was used in this study. The results of this study show the strong inhibition of incorporation (cpm) of [3H]-uridine into SVCV and catfish rhabdovirus RNA in cell culture exposed to methisoprinol at various concentrations. The highest percent of inhibition of viral RNA at 72 h after infection with two rhabdoviruses were observed in doses of 400 and 500 microg/ml of methisoprinol in medium. The results of this in vitro study showed that methisoprinol inhibits the rhabdoviruses isolated from carp and catfish.

  8. A strain of Siniperca chuatsi rhabdovirus causes high mortality among cultured Largemouth Bass in South China.

    PubMed

    Ma, Dongmei; Deng, Guocheng; Bai, Junjie; Li, Shengjie; Yu, Lingyun; Quan, Yingchun; Yang, Xiaojing; Jiang, Xiaoyan; Zhu, Zemin; Ye, Xing

    2013-09-01

    In April 2011, 40% mortality of Largemouth Bass Micropterus salmoides juveniles occurred at a farm of Zhongshan City, Guangdong Province, China. Infected fish became lethargic, exhibited corkscrew and irregular swimming, and developed a distended abdomen and crooked body. Fish began to die within 2 d after the appearance of clinical signs. In order to analyze the pathogeny and diagnose the disease earlier, observation of clinical signs, cell infection, titer calculation, electron microscopy, immersion infection assay for fish, and nucleotide sequence analysis were carried out. Fathead minnow (FHM) cell cultures, inoculated with filtrate of liver and spleen homogenates from the diseased fish, developed the obvious cytopathic effect 46 h after inoculation in the primary culture and 24 h at the first passage. Typical rhabdovirus particles, 115-143 nm in length and 62-78 nm in diameter, were observed in infected FHM cells by direct transmission electron microscopy. The isolated virus produced a titer of 10(7.15) TCID50/mL. Immersion-Fish infected with the virus had similar clinical signs and 80% mortality with 10(2.5) LD50/mL. The data indicated that the rhabdovirus was the lethal pathogeny of the current disease. Based on nucleoprotein-gene nucleotide sequence multiple alignment analysis, the newly isolated virus is a strain of Siniperca chuatsi rhabdovirus (SCRV) under family Rhabdoviridae, which was initially isolated from Mandarin Fish Siniperca chuatsi. Up to the present, at least four virus strains have been isolated from diseased Largemouth Bass, which have had different clinical signs. Comparison of the clinical signs can help in an early diagnosis of the disease.

  9. Isolation and identification of a lethal rhabdovirus from farmed rice field eels Monopterus albus.

    PubMed

    Ou, Tong; Zhu, Ruo-Lin; Chen, Zhong-Yuan; Zhang, Qi-Ya

    2013-11-06

    We provide the first description of a virus responsible for a systemic hemorrhagic disease causing high mortality in farmed rice field eels Monopterus albus in China. Typical signs exhibited by the diseased fish were extensive hemorrhages in the skin and viscera and some neurological signs, such as loss of equilibrium and disorganized swimming. Histopathological examination revealed various degrees of necrosis within the spleen and liver. Virus isolation was attempted from visceral tissues of diseased fish by inoculation on 6 fish cell lines. Typical cytopathic effects (CPE) were produced in bluegill fry (BF2) cells, so this cell line was chosen for further isolation and propagation of the virus. Electron microscopy observation showed that the negative stained viral particles had the characteristic bullet shape of rhabdoviruses and an estimated size of 60 × 120 nm. We therefore tentatively refer to this virus as Monopterus albus rhabdovirus (MoARV). Molecular characterization of MoARV, including sequence analysis of the nucleoprotein (N), phosphoprotein (P), and glycoprotein (G) genes, revealed 94.5 to 97.3% amino acid similarity to that of Siniperca chuatsi rhabdovirus. Phylogenetic analysis based on the amino acid sequences of N and G proteins indicated that MoARV should be a member of the genus Vesiculovirus. Koch's postulates were fulfilled by infecting healthy rice field eels with MoARV, which produced an acute infection. RT-PCR analysis demonstrated that MoARV RNA could be detected in both naturally and experimentally infected fish. The data suggest that MoARV was the causative pathogen of the disease.

  10. Protective immunity elicited by a divalent DNA vaccine encoding both the L7/L12 and Omp16 genes of Brucella abortus in BALB/c mice.

    PubMed

    Luo, Deyan; Ni, Bing; Li, Peng; Shi, Wei; Zhang, Songle; Han, Yue; Mao, Liwei; He, Yangdong; Wu, Yuzhang; Wang, Xiliang

    2006-05-01

    This study was designed to evaluate the immunogenicity and the protective efficacy of a divalent fusion DNA vaccine encoding both the Brucella abortus L7/L12 protein (ribosomal protein) and Omp16 protein (outer membrane lipoprotein), designated pcDNA3.1-L7/L12-Omp16. Intramuscular injection of this divalent DNA vaccine into BALB/c mice elicited markedly both humoral and cellular immune responses. The specific antibodies exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the dual-gene DNA vaccine elicited a strong T-cell proliferative response and induced a large amount of gamma interferon-producing T cells upon restimulation in vitro with recombinant fusion protein L7/L12-Omp16, suggesting the induction of a typical T-helper-1-dominated immune response in vivo. This divalent DNA vaccine could also induce a significant level of protection against challenge with the virulent strain B. abortus 544 in BALB/c mice. Furthermore, the protection level induced by the divalent DNA vaccine was significantly higher than that induced by the univalent DNA vaccines pcDNA3.1-L7/L12 or pcDNA3.1-Omp16. Taken together, the results of this study verify for the first time that the Omp16 gene can be a candidate target for a DNA vaccine against brucellosis. Additionally, a divalent genetic vaccine based on the L7/L12 and Omp16 genes can elicit a stronger cellular immune response and better immunoprotection than the relevant univalent vaccines can.

  11. Evaluation of the persistence, integration, histopathology and environmental release of DNA vaccine encoding Eimeria tenella TA4 and chicken IL-2.

    PubMed

    Song, Xiaokai; Zhang, Zeyang; Liu, Chang; Xu, Lixin; Yan, Ruofeng; Li, Xiangrui

    2016-10-15

    In a previous study, the construction of the Eimeria tenella DNA vaccine pVAX1.0-TA4-IL-2 which provides effective protection against coccidiosis was described and the immunization procedure was optimized. However, the persistence, integration, histopathology and environmental release of the DNA vaccine remain unknown. In this study, the persistence, integration and histopathology of the DNA vaccine pVAX1.0-TA4-IL-2 was evaluated in chickens in the following immunization studies: (1) single-dose immunization in one-day-old chickens; (2) repeat-dose immunization in chickens; and (3) single-high-dose immunization of three batches of plasmid in chickens. The persistence, integration, histopathology of the DNA vaccine was also evaluated in mice. At 1, 1.5, 2-4 months post immunization, blood, duodenum, heart, liver, spleen, kidneys and the immunized muscle tissue were collected from ten animals of each group. Persistence and integration were evaluated using PCR with a confirmed sensitivity of 30 plasmid copies. Hematoxylin and eosin stained sections were examined for the presence of inflammation or abnormalities that may result from vaccination. Water and fecal samples were also collected from the chicken enclosures to evaluate the potential for environmental release of the DNA vaccine. Testing various tissues by PCR confirmed that plasmid DNA persisted 1.5 months in blood, heart, liver and spleen, 2 months in kidneys and muscle of injected site. Furthermore, the vaccine did not integrate with the host genome. The histopathological examinations did not show obvious inflammation or pathological damage in any tissue of the immunized chickens. Similar results were observed in mice. Moreover, the DNA vaccine was not released into the surrounding environment. These results indicate that the DNA vaccine pVAX1.0-TA4-IL-2 has potential as safe vaccine against coccidiosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. PAMAM-Lys, a Novel Vaccine Delivery Vector, Enhances the Protective Effects of the SjC23 DNA Vaccine against Schistosoma japonicum Infection

    PubMed Central

    Wang, Xiaoting; Dai, Yang; Zhao, Song; Tang, Jianxia; Li, Hongjun; Xing, Yuntian; Qu, Guoli; Li, Xinsong; Dai, Jianrong; Zhu, Yinchang; Zhang, Xueguang

    2014-01-01

    Background Schistosomiasis japonica remains a major public-health concern in China. Praziquantel-based chemotherapy effectively reduces both infections and intensity; however, it can not prevent re-infection. Furthermore, there is an increasing concern about praziquantel resistance following long-term repeated use of the drug in endemic areas. Therefore, development of a schistosomiasis vaccine, as a strategy to prevent and control schistosomiasis japonica, has been given high priority. The present study was conducted to develop PAMAM dendrimers as a novel vaccine delivery vector for a schistosomiasis japonica DNA vaccine and evaluate its ability to enhance protective effects against Schistosoma japonicum infection. Methodology/Principal Findings Lysine was used to modify 4.0G PAMAM, and the modified product PAMAM-Lys was synthesized. PAMAM-Lys showed both high transfection and low cytotocity for gene delivery in vitro. DNA vaccines combined with PAMAM-Lys produced higher level of protection compare with naked DNA vaccines against S. japonicum infection in a mouse model. Futhermore,antibodies from mice immunized with PAMAM-Lys combined DNA vaccines were significantly higher than those of mice immunized with the naked DNA vaccines. The PAMAM-Lys vector elicited a predominantly IgG2a antibody response and a tremendously increase in the production of IL-2 and IFN-γ. Conclusion/Significance Lysine-modified PAMAM-Lys is an excellent vector. PAMAM-Lys may enhance the immunoreactivity of DNA vaccine and increase the protective effect of the SjC23 DNA vaccine against S. japonicum infection. PMID:24497955

  13. Mucosal application of cationic poly(D,L-lactide-co-glycolide) microparticles as carriers of DNA vaccine and adjuvants to protect chickens against infectious bursal disease.

    PubMed

    Negash, Tamiru; Liman, Martin; Rautenschlein, Silke

    2013-08-12

    Infectious bursal disease virus (IBDV) is an immunosuppressive virus of chickens. The virus protein (VP) 2 induces neutralizing antibodies, which protect chickens against the disease. The aim of this study was to develop a cationic poly(d,l-lactide-co-glycolide) (PLGA) microparticle (MP) based IBDV-VP2 DNA vaccine (MP-IBDV-DNA) for chickens to be delivered orally and by eye drop route. The tested IBDV-VP2 DNA vaccines were immunogenic for specific-pathogen-free chickens and induced an antibody response after intramuscular application. Co-inoculation with a plasmid encoding chicken IL-2 (chIL-2) or CpG-ODN did not significantly improve protection against IBDV challenge. However, the application of a MP-IBDV-DNA vaccine alone or in combination with a delayed oral and eye drop application of cationic MP loaded with CpG-ODN or chIL-2 improved protection against challenge. The MP-IBDV-DNA-vaccinated chickens showed less pathological and histopathological bursal lesions, a reduced IBDV antigen load as well as T-cell influx into the bursa of Fabricius (BF) compared to the other groups (p<0.05). The addition of chIL-2 loaded MP improved challenge virus clearance from the BF as demonstrated by lower neutralizing antibody titers and reduced IL-4 and IFN-α mRNA expression in the bursa at 7 days postchallenge compared to the other challenged groups. Overall, the efficacy of the IBDV-DNA vaccine was improved by adsorption of the DNA vaccine onto cationic PLGA-MP, which also allowed mucosal application of the DNA vaccine.

  14. A polyvalent influenza A DNA vaccine induces heterologous immunity and protects pigs against pandemic A(H1N1)pdm09 virus infection.

    PubMed

    Bragstad, Karoline; Vinner, Lasse; Hansen, Mette Sif; Nielsen, Jens; Fomsgaard, Anders

    2013-04-26

    The composition of current influenza protein vaccines has to be reconsidered every season to match the circulating influenza viruses, continuously changing antigenicity. Thus, influenza vaccines inducing a broad cross-reactive immune response would be a great advantage for protection against both seasonal and emerging influenza viruses. We have developed an alternative influenza vaccine based on DNA expressing selected influenza proteins of pandemic and seasonal origin. In the current study, we investigated the protection of a polyvalent influenza DNA vaccine approach in pigs. We immunised pigs intradermally with a combination of influenza DNA vaccine components based on the pandemic 1918 H1N1 (M and NP genes), pandemic 2009 H1N1pdm09 (HA and NA genes) and seasonal 2005 H3N2 genes (HA and NA genes) and investigated the protection against infection with virus both homologous and heterologous to the DNA vaccine components. We found that pigs challenged with a virus homologous to the HA and NA DNA vaccine components were well protected from infection. In addition, heterologous challenge virus was cleared rapidly compared to the unvaccinated control pigs. Immunisation by electroporation induced HI antibodies >40 HAU/ml seven days after second vaccination. Heterologous virus challenge as long as ten weeks after last immunisation was able to trigger a vaccine antibody HI response 26 times higher than in the control pigs. The H3N2 DNA vaccine HA and NA genes also triggered an effective vaccine response with protective antibody titres towards heterologous H3N2 virus. The described influenza DNA vaccine is able to induce broadly protective immune responses even in a larger animal, like the pig, against both heterologous and homologous virus challenges despite relatively low HI titres after vaccination. The ability of this DNA vaccine to limit virus shedding may have an impact on virus spread among pigs which could possibly extend to humans as well, thereby diminishing the

  15. Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system.

    PubMed

    Meerak, Jomkhwan; Wanichwecharungruang, Supason P; Palaga, Tanapat

    2013-01-21

    DNA vaccines are a promising new generation of vaccines that can elicit an immune response using DNA encoding the antigen of interest. The efficacy of these vaccines, however, still needs to be improved. In this study, we investigated the effect of autophagy on increasing the efficacy of a candidate DNA vaccine against Mycobacterium tuberculosis (MTB), a causative agent of tuberculosis. Low molecular weight chitosan was used to encapsulate plasmid DNA containing a gene encoding MTB Antigen 85B (Ag85B), a secreted fibronectin-binding protein. To induce autophagy upon DNA vaccination, the kinase defective mTOR (mTOR-KD) was transfected into cells, and autophagy was detected based on the presence of LC3II. To investigate whether autophagy enhances an immune response upon DNA vaccination, we coencapsualted the Ag85B-containing plasmid with a plasmid encoding mTOR-KD. Plasmids encapsulated by chitosan particles were used for primary subcutaneous immunization and for intranasal boost in mice. After the boost vaccination, sera from the mice were measured for humoral immune response. The DNA vaccine with the autophagy-inducing construct elicited significantly higher Ag85B-specific antibody levels than the control group treated with the Ag85B plasmid alone or with the Ag85B plasmid plus the wild type mTOR construct. Upon in vitro stimulation of splenocytes from mice immunized with recombinant Ag85B, the highest levels of secreted IFN-γ and IL-2 were detected in mice immunized with the autophagy-inducing plasmid, while no differences in IL-4 levels were detected between the groups, suggesting that the DNA vaccine regimen with autophagy induction induced primarily a Th1 immune response. Furthermore, the enhanced proliferation of CD4+ T cells from mice receiving the autophagy-inducing vaccine was observed in vitro. Based on the evidence presented, we conclude that incorporating an autophagy-inducing element into a DNA vaccine may help to improve immune response.

  16. A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

    PubMed Central

    Franco, Luís H; Wowk, Pryscilla F; Silva, Célio L; Trombone, Ana PF; Coelho-Castelo, Arlete AM; Oliver, Constance; Jamur, Maria C; Moretto, Edson L; Bonato, Vânia LD

    2008-01-01

    Background A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses. Methods To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity. Results It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes. Conclusion Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy. PMID:18208592

  17. Rabies Group-Specific Ribonucleoprotein Antigen and a Test System for Grouping and Typing of Rhabdoviruses

    PubMed Central

    Schneider, L. G.; Dietzschold, B.; Dierks, R. E.; Matthaeus, W.; Enzmann, P.-J.; Strohmaier, K.

    1973-01-01

    Cell-associated ribonucleoprotein (RNP) was isolated from BHK-21 cells infected with several strains of rabies and rabies-related viruses. The RNP-antigen from rabies and related viruses induced the formation of complement-fixing, precipitating, and immunofluorescent antibodies, and proved to be the group-specific antigen common to all rabies viruses. Antigens of the envelope which induce virus-neutralizing antibodies are apparently determinative for the serotype of a virus as evidenced by two-way neutralization tests. A combination of these methods seems to be a useful approach to the serological grouping and typing of rhabdoviruses. Images PMID:4196634

  18. 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.

  19. Targeted DNA vaccines for enhanced induction of idiotype-specific B and T cells

    PubMed Central

    Fredriksen, Agnete B.; Sandlie, Inger; Bogen, Bjarne

    2012-01-01

    Background: Idiotypes (Id) are antigenic determinants localized in variable (V) regions of Ig. Id-specific T and B cells (antibodies) play a role in immunotherapy of Id+ tumors. However, vaccine strategies that enhance Id-specific responses are needed. Methods: Id+ single-chain fragment variable (scFv) from multiple myelomas and B cell lymphomas were prepared in a fusion format that bivalently target surface molecules on antigen-presenting cells (APC). APC-specific targeting units were either scFv from APC-specific mAb (anti-MHC II, anti-CD40) or chemokines (MIP-1α, RANTES). Homodimeric Id-vaccines were injected intramuscularly or intradermally as plasmids in mice, combined with electroporation. Results: (i) Transfected cells secreted plasmid-encoded Id+ fusion proteins to extracellular fluid followed by binding of vaccine molecules to APC. (ii) Targeted vaccine molecules increased Id-specific B and T cell responses. (iii) Bivalency and xenogeneic sequences both contributed to enhanced responses. (iv) Targeted Id DNA vaccines induced tumor resistance against challenges with Id+ tumors. (v) Human MIP-1α targeting units enhanced Id-specific responses in mice, due to a cross reaction with murine chemokine receptors. Thus, targeted vaccines designed for humans can be quality tested in mice. (vi) Human Id+ scFv from four multiple myeloma patients were inserted into the vaccine format and were successfully tested in mice. (vii) Human MIP-1α vaccine proteins enhanced human T cell responses in vitro. (viii) A hypothetical model for how the APC-targeted vaccine molecules enhance Id-specific T and B cells is presented. Conclusion: Targeted DNA Id-vaccines show promising results in preclinical studies, paving the way for testing in patients. PMID:23115759

  20. DNA vaccination regimes against Schmallenberg virus infection in IFNAR(-/-) mice suggest two targets for immunization.

    PubMed

    Boshra, Hani Y; Charro, Diego; Lorenzo, Gema; Sánchez, Isbene; Lazaro, Beatriz; Brun, Alejandro; Abrescia, Nicola G A

    2017-05-01

    Schmallenberg virus (SBV) is an RNA virus of the Bunyaviridae family, genus Orthobunyavirus that infects wild and livestock species of ruminants. While inactivated and attenuated vaccines have been shown to prevent SBV infection, little is known about their mode of immunity; specifically, which components of the virus are responsible for inducing immunological responses in the host. As previous DNA vaccination experiments on other bunyaviruses have found that glycoproteins, as well as modified (i.e. ubiquitinated) nucleoproteins (N) can confer immunity against virulent viral challenge, constructs encoding for fragments of SBV glycoproteins GN and GC, as well as ubiquitinated and non-ubiquitinated N were cloned in mammalian expression vectors, and vaccinated intramuscularly in IFNAR(-/-) mice. Upon viral challenge with virulent SBV, disease progression was monitored. Both the ubiquitinated and non-ubiquitinated nucleoprotein candidates elicited high titers of antibodies against SBV, but only the non-ubiquitinated candidate induced statistically significant protection of the vaccinated mice from viral challenge. Another construct encoding for a putative ectodomain of glycoprotein GC (segment aa. 678-947) also reduced the SBV-viremia in mice after SBV challenge. When compared to other experimental groups, both the nucleoprotein and GC-ectodomain vaccinated groups displayed significantly reduced viremia, as well as exhibiting no clinical signs of SBV infection. These results show that both the nucleoprotein and the putative GC-ectodomain can serve as protective immunological targets against SBV infection, highlighting that viral glycoproteins, as well as nucleoproteins are potent targets in vaccination strategies against bunyaviruses. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Preparation and Characterization of Novel PBAE/PLGA Polymer Blend Microparticles for DNA Vaccine Delivery

    PubMed Central

    Balashanmugam, Meenashi Vanathi; Nagarethinam, Sivagurunathan; Jagani, Hitesh; Josyula, Venkata Rao; Alrohaimi, Abdulmohsen; Udupa, Nayanabhirama

    2014-01-01

    Context. Poly(beta-amino ester) (PBAE) with its pH sensitiveness and Poly(lactic-co-glycolic acid) (PLGA) with huge DNA cargo capacity in combination prove to be highly efficient as DNA delivery system. Objective. To study the effectiveness of novel synthesized PBAE polymer with PLGA blend at different ratios in DNA vaccine delivery. Methods. In the present study, multifunctional polymer blend microparticles using a combination of PLGA and novel PBAE polymers A1 (bis(3-(propionyloxy)propyl)3,3′-(propane-1,3-diyl-bis(methylazanediyl))dipropanoate) and A2 (bis(4-(propionyloxy)butyl)3,3′-(ethane-1,2-diyl-bis(isopropylazanediyl))dipropanoate) at different ratios (85 : 15, 75 : 25, and 50 : 50) were prepared by double emulsion solvent removal method. The microparticles were characterized for cytotoxicity, transfection efficiency, and DNA encapsulation efficiency. Result. It was evident from results that among the microparticles prepared with PLGA/PBAE blend the PLGA : PBAE at 85 : 15 ratio was found to be more effective combination than the microparticles prepared with PLGA alone in terms of transfection efficiency and better DNA integrity. Microparticles made of PLGA and PBAE A1 at 85 : 15 ratio, respectively, were found to be less toxic when compared with microparticles prepared with A2 polymer. Conclusion. The results encourage the use of the synthesized PBAE polymer in combination with PLGA as an effective gene delivery system. PMID:25401137

  2. Neutralizing antibodies respond to a bivalent dengue DNA vaccine or/and a recombinant bivalent antigen.

    PubMed

    Zhang, Zhi-Shan; Weng, Yu-Wei; Huang, Hai-Long; Zhang, Jian-Ming; Yan, Yan-Sheng

    2015-02-01

    There is currently no effective vaccine to prevent dengue infection, despite the existence of multiple studies on potential methods of immunization. The aim of the present study was to explore the effect of DNA and/or recombinant protein on levels of neutralizing antibodies. For this purpose, envelope domain IIIs of dengue serotypes 1 and 2 (DEN-1/2)were spliced by a linker (Gly‑Gly‑Ser‑Gly‑Ser)3 and cloned into the prokaryotic expression plasmid pET30a (+) and eukaryotic vector pcDNA3.1 (+). The chimeric bivalent protein was expressed in Escherichia coli, and one‑step purification by high‑performance liquid chromatography was conducted. Protein expression levels of the DNA plasmid were tested in BHK‑21 cells by indirect immunofluorescent assay. In order to explore a more effective immunization strategy and to develop neutralizing antibodies against the two serotypes, mice were inoculated with recombinant bivalent protein, the DNA vaccine, or the two given simultaneously. Presence of the specific antibodies was tested by ELISA and the presence of the neutralizing antibodies was determined by plaque reduction neutralization test. Results of the analysis indicat