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Sample records for vaccinia virus vaccine

  1. Antitumor efficacy of vaccinia virus-modified tumor cell vaccine

    SciTech Connect

    Ito, T.; Wang, D.Q.; Maru, M.; Nakajima, K.; Kato, S.; Kurimura, T.; Wakamiya, N. )

    1990-11-01

    The antitumor efficacies of vaccinia virus-modified tumor cell vaccines were examined in murine syngeneic MH134 and X5563 tumor cells. UV-inactivated vaccinia virus was inoculated i.p. into C3H/HeN mice that had received whole body X-irradiation at 150 rads. After 3 weeks, the vaccines were administered i.p. 3 times at weekly intervals. One week after the last injection, mice were challenged i.p. with various doses of syngeneic MH134 or X5563 viable tumor cells. Four methods were used for preparing tumor cell vaccines: X-ray irradiation; fixation with paraformaldehyde for 1 h or 3 months; and purification of the membrane fraction. All four vaccines were effective, but the former two vaccines were the most effective. A mixture of the membrane fraction of untreated tumor cells and UV-inactivated vaccinia virus also had an antitumor effect. These results indicate that vaccine with the complete cell structure is the most effective. The membrane fraction of UV-inactivated vaccinia virus-absorbed tumor cells was also effective. UV-inactivated vaccinia virus can react with not only intact tumor cells but also the purified membrane fraction of tumor cells and augment antitumor activity.

  2. Vaccinia Virus: A Tool for Research and Vaccine Development

    NASA Astrophysics Data System (ADS)

    Moss, Bernard

    1991-06-01

    Vaccinia virus is no longer needed for smallpox immunization, but now serves as a useful vector for expressing genes within the cytoplasm of eukaryotic cells. As a research tool, recombinant vaccinia viruses are used to synthesize biologically active proteins and analyze structure-function relations, determine the targets of humoral- and cell-mediated immunity, and investigate the immune responses needed for protection against specific infectious diseases. When more data on safety and efficacy are available, recombinant vaccinia and related poxviruses may be candidates for live vaccines and for cancer immunotherapy.

  3. Vaccinia virus as a vaccine delivery system for marsupial wildlife.

    PubMed

    Cross, Martin L; Fleming, Stephen B; Cowan, Phil E; Scobie, Susie; Whelan, Ellena; Prada, Diana; Mercer, Andrew A; Duckworth, Janine A

    2011-06-20

    Vaccines based on recombinant poxviruses have proved successful in controlling diseases such as rabies and plague in wild eutherian mammals. They have also been trialled experimentally as delivery agents for fertility-control vaccines in rodents and foxes. In some countries, marsupial mammals represent a wildlife disease reservoir or a threat to conservation values but, as yet there has been no bespoke study of efficacy or immunogenicity of a poxvirus-based vaccine delivery system in a marsupial. Here, we report a study of the potential for vaccination using vaccinia virus in the Australian brushtail possum Trichosurus vulpecula, an introduced pest species in New Zealand. Parent-strain vaccinia virus (Lister) infected 8/8 possums following delivery of virus to the oral cavity and outer nares surfaces (oronasal immunisation), and persisted in the mucosal epithelium around the palatine tonsils for up to 2 weeks post-exposure. A recombinant vaccinia virus construct (VV399, which expresses the Eg95 antigen of the hydatid disease parasite Echinococcus granulosus) was shown to infect 10/15 possums after a single-dose oronasal delivery and to also persist. Both parent vaccinia virus and the VV399 construct virus induced peripheral blood lymphocyte reactivity against viral antigens in possums, first apparent at 4 weeks post-exposure and still detectable at 4 months post-exposure. Serum antibody reactivity to Eg95 was recorded in 7/8 possums which received a single dose of the VV399 construct and 7/7 animals which received triple-dose delivery, with titre end-points in the latter case exceeding 1/4000 dilution. This study demonstrates that vaccinia virus will readily infect possums via a delivery means used to deploy wildlife vaccines, and in doing is capable of generating immune reactivity against viral and heterologous antigens. This highlights the future potential of recombinant vaccinia virus as a vaccine delivery system in marsupial wildlife. PMID:21570435

  4. Modified Vaccinia Ankara Virus Vaccination Provides Long-Term Protection against Nasal Rabbitpox Virus Challenge.

    PubMed

    Jones, Dorothy I; McGee, Charles E; Sample, Christopher J; Sempowski, Gregory D; Pickup, David J; Staats, Herman F

    2016-07-01

    Modified vaccinia Ankara virus (MVA) is a smallpox vaccine candidate. This study was performed to determine if MVA vaccination provides long-term protection against rabbitpox virus (RPXV) challenge, an animal model of smallpox. Two doses of MVA provided 100% protection against a lethal intranasal RPXV challenge administered 9 months after vaccination. PMID:27146001

  5. Vaccination of vampire bats using recombinant vaccinia-rabies virus.

    PubMed

    Aguilar-Setién, Alvaro; Leon, Yolanda Campos; Tesoro, Emiliano Cruz; Kretschmer, Roberto; Brochier, Bernard; Pastoret, Paul-Pierre

    2002-07-01

    Adult vampire bats (Desmodus rotundus) were vaccinated by intramuscular, scarification, oral, or aerosol routes (n = 8 in each group) using a vaccinia-rabies glycoprotein recombinant virus. Sera were obtained before and 30 days after vaccination. All animals were then challenged intramuscularly with a lethal dose of rabies virus. Neutralizing antirabies antibodies were measured by rapid fluorescent focus inhibition test (RFFIT). Seroconversion was observed with each of the routes employed, but some aerosol and orally vaccinated animals failed to seroconvert. The highest antibody titers were observed in animals vaccinated by intramuscular and scarification routes. All animals vaccinated by intramuscular, scarification, and oral routes survived the viral challenge, but one of eight vampire bats receiving aerosol vaccination succumbed to the challenge. Of 31 surviving vaccinated and challenged animals, nine lacked detectable antirabies antibodies by RFFIT (five orally and four aerosol immunized animals). In contrast, nine of 10 non-vaccinated control bats succumbed to viral challenge. The surviving control bat had antiviral antibodies 90 days after viral challenge. These results suggest that the recombinant vaccine is an adequate and safe immunogen for bats by all routes tested. PMID:12243138

  6. Recombinant modified vaccinia virus Ankara-based malaria vaccines.

    PubMed

    Sebastian, Sarah; Gilbert, Sarah C

    2016-01-01

    A safe and effective malaria vaccine is a crucial part of the roadmap to malaria elimination/eradication by the year 2050. Viral-vectored vaccines based on adenoviruses and modified vaccinia virus Ankara (MVA) expressing malaria immunogens are currently being used in heterologous prime-boost regimes in clinical trials for induction of strong antigen-specific T-cell responses and high-titer antibodies. Recombinant MVA is a safe and well-tolerated attenuated vector that has consistently shown significant boosting potential. Advances have been made in large-scale MVA manufacture as high-yield producer cell lines and high-throughput purification processes have recently been developed. This review describes the use of MVA as malaria vaccine vector in both preclinical and clinical studies in the past 5 years. PMID:26511884

  7. Recombinant Vaccinia Virus: Immunization against Multiple Pathogens

    NASA Astrophysics Data System (ADS)

    Perkus, Marion E.; Piccini, Antonia; Lipinskas, Bernard R.; Paoletti, Enzo

    1985-09-01

    The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.

  8. Oral Vaccination With Vaccinia Virus Expressing the Tick Antigen Subolesin Inhibits Tick Feeding and Transmission of Borrelia burgdorferi Vaccination

    PubMed Central

    Bensaci, Mekki; Bhattacharya, Debaditya; Clark, Roger; Hu, Linden T.

    2014-01-01

    Immunization with the Ixodes scapularis protein, subolesin, has previously been shown to protect hosts against tick infestation and to decrease acquisition of Anaplsma marginale and Babesia bigemina. Here we report the efficacy of subolesin expressed from Vaccinia virus for use as an orally delivered reservoir–targeted vaccine for prevention of tick infestation and acquisition/transmission of Borrelia burgdorferi to its tick and mouse hosts. We cloned subolesin into Vaccinia virus and showed that it is expressed from mammalian cells infected with the recombinant virus in vitro. We then vaccinated mice by oral gavage. A single dose of the vaccine was sufficient for mice to generate antibody response to subolesin. Vaccination with the subolesin expressing Vaccinia virus inhibited tick infestation by 52% compared to control vaccination with Vaccinia virus and reduced uptake of B. burgdorferi among the surviving ticks that fed to repletion by 34%. There was a reduction in transmission of B. burgdorferi to uninfected vaccinated mice of 40% compared to controls. These results suggest that subolesin has potential as a component of a reservoir targeted vaccine to decrease B. burgdorferi, Babesia and Anaplasma species infections in their natural hosts. PMID:22864146

  9. Oral vaccination of the fox against rabies using a live recombinant vaccinia virus.

    PubMed

    Blancou, J; Kieny, M P; Lathe, R; Lecocq, J P; Pastoret, P P; Soulebot, J P; Desmettre, P

    Rabies, a viral disease affecting all warm-blooded animals, is prevalent in most parts of the world, where it propagates amongst wild animals, particularly the fox and dog. The public health and economic consequences of infection in man and livestock are well known. Attempts to control the disease by vaccinating wild carnivores with inactivated or attenuated rabies virus remain controversial, and we have instead evaluated here the potential of a recombinant vaccinia virus to protect foxes against the disease. We have found that the administration of vaccinia virus (VV) or a recombinant harbouring the rabies surface antigen gene (VVTGgRAB) is innocuous to foxes. The recombinant virus can elicit the production of titers of rabies-neutralizing antibodies equal or superior to those obtained with conventional vaccine, and 10(8) plaque-forming units (PFU) of VVTGgRAB administered subcutaneously, intradermally or orally confers complete protection to severe challenge infection with street rabies virus. PMID:3736663

  10. Host range, growth property, and virulence of the smallpox vaccine: Vaccinia virus Tian Tan strain

    SciTech Connect

    Fang Qing; Yang Lin; Zhu Weijun; Liu Li; Wang Haibo; Yu Wenbo; Xiao Genfu; Tien Po; Zhang Linqi; Chen Zhiwei . E-mail: zchen@adarc.org

    2005-05-10

    Vaccinia Tian Tan (VTT) was used as a vaccine against smallpox in China for millions of people before 1980, yet the biological characteristics of the virus remain unclear. We have characterized VTT with respect to its host cell range, growth properties in vitro, and virulence in vivo. We found that 11 of the 12 mammalian cell lines studied are permissive to VTT infection whereas one, CHO-K1, is non-permissive. Using electron microscopy and sequence analysis, we found that the restriction of VTT replication in CHO-K1 is at a step before viral maturation probably due to the loss of the V025 gene. Moreover, VTT is significantly less virulent than vaccinia WR but remains neurovirulent in mice and causes significant body weight loss after intranasal inoculation. Our data demonstrate the need for further attenuation of VTT to serve either as a safer smallpox vaccine or as a live vaccine vector for other pathogens.

  11. Live Virus Smallpox Vaccine

    MedlinePlus

    ... Index SMALLPOX FACT SHEET The Live Virus Smallpox Vaccine The vaccinia virus is the "live virus" used ... cannot cause smallpox. What is a "live virus" vaccine? A "live virus" vaccine is a vaccine that ...

  12. Active vaccination with vaccinia virus A33 protects mice against lethal vaccinia and ectromelia viruses but not against cowpoxvirus; elucidation of the specific adaptive immune response

    PubMed Central

    2013-01-01

    Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104–120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope’s critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox. PMID:23842430

  13. A vaccinia virus renaissance

    PubMed Central

    Verardi, Paulo H.; Titong, Allison; Hagen, Caitlin J.

    2012-01-01

    In 1796, Edward Jenner introduced the concept of vaccination with cowpox virus, an Orthopoxvirus within the family Poxviridae that elicits cross protective immunity against related orthopoxviruses, including smallpox virus (variola virus). Over time, vaccinia virus (VACV) replaced cowpox virus as the smallpox vaccine, and vaccination efforts eventually led to the successful global eradication of smallpox in 1979. VACV has many characteristics that make it an excellent vaccine and that were crucial for the successful eradication of smallpox, including (1) its exceptional thermal stability (a very important but uncommon characteristic in live vaccines), (2) its ability to elicit strong humoral and cell-mediated immune responses, (3) the fact that it is easy to propagate, and (4) that it is not oncogenic, given that VACV replication occurs exclusively within the host cell cytoplasm and there is no evidence that the viral genome integrates into the host genome. Since the eradication of smallpox, VACV has experienced a renaissance of interest as a viral vector for the development of recombinant vaccines, immunotherapies, and oncolytic therapies, as well as the development of next-generation smallpox vaccines. This revival is mainly due to the successful use and extensive characterization of VACV as a vaccine during the smallpox eradication campaign, along with the ability to genetically manipulate its large dsDNA genome while retaining infectivity and immunogenicity, its wide mammalian host range, and its natural tropism for tumor cells that allows its use as an oncolytic vector. This review provides an overview of new uses of VACV that are currently being explored for the development of vaccines, immunotherapeutics, and oncolytic virotherapies. PMID:22777090

  14. A vaccinia virus renaissance: new vaccine and immunotherapeutic uses after smallpox eradication.

    PubMed

    Verardi, Paulo H; Titong, Allison; Hagen, Caitlin J

    2012-07-01

    In 1796, Edward Jenner introduced the concept of vaccination with cowpox virus, an Orthopoxvirus within the family Poxviridae that elicits cross protective immunity against related orthopoxviruses, including smallpox virus (variola virus). Over time, vaccinia virus (VACV) replaced cowpox virus as the smallpox vaccine, and vaccination efforts eventually led to the successful global eradication of smallpox in 1979. VACV has many characteristics that make it an excellent vaccine and that were crucial for the successful eradication of smallpox, including (1) its exceptional thermal stability (a very important but uncommon characteristic in live vaccines), (2) its ability to elicit strong humoral and cell-mediated immune responses, (3) the fact that it is easy to propagate, and (4) that it is not oncogenic, given that VACV replication occurs exclusively within the host cell cytoplasm and there is no evidence that the viral genome integrates into the host genome. Since the eradication of smallpox, VACV has experienced a renaissance of interest as a viral vector for the development of recombinant vaccines, immunotherapies, and oncolytic therapies, as well as the development of next-generation smallpox vaccines. This revival is mainly due to the successful use and extensive characterization of VACV as a vaccine during the smallpox eradication campaign, along with the ability to genetically manipulate its large dsDNA genome while retaining infectivity and immunogenicity, its wide mammalian host range, and its natural tropism for tumor cells that allows its use as an oncolytic vector. This review provides an overview of new uses of VACV that are currently being explored for the development of vaccines, immunotherapeutics, and oncolytic virotherapies. PMID:22777090

  15. Modified vaccinia virus Ankara as a vaccine against feline coronavirus: immunogenicity and efficacy.

    PubMed

    Hebben, Matthias; Duquesne, Véronique; Cronier, Joëlle; Rossi, Bernard; Aubert, André

    2004-04-01

    Feline infectious peritonitis virus (FIPV) is a coronavirus that induces a fatal systemic disease mediated by an inappropriate immune response. Most previous vaccination attempts against FIPV were unsuccessful because IgG antibodies against the surface protein enhance the infection. However, two studies have shown that poxvirus vectors (vaccinia WR and canarypox) expressing only the FIPV membrane (M) protein can elicit a partially protective immunity which is supposed to be cell-mediated (Virology 181 (1991) 327; International patent WO 97/20054 (1997)). In our study, we report the construction of another poxvirus, the modified vaccinia virus Ankara (MVA), as an expression vector for the FIPV M protein. In this vector, the M gene has been inserted downstream a strong early/late promoter, whereas the two previously described poxviruses expressed the M protein during their early stage only. The immunogenicity of the recombinant MVA-M was evaluated in the murine model which revealed an effect of the vector on the Th1/Th2 balance. The vaccine was then tested in cats to evaluate its efficacy in an FIPV 79-1146 challenge. Vaccinated kittens developed FIPV-specific antibodies after immunization, however, none of them was protected against FIPV. Our results suggest a crucial role for the type of poxviral promoter that must be used to induce an effective immune response against FIPV. PMID:15123156

  16. Attenuation of Vaccinia Virus.

    PubMed

    Yakubitskiy, S N; Kolosova, I V; Maksyutov, R A; Shchelkunov, S N

    2015-01-01

    Since 1980, in the post-smallpox vaccination era the human population has become increasingly susceptible compared to a generation ago to not only the variola (smallpox) virus, but also other zoonotic orthopoxviruses. The need for safer vaccines against orthopoxviruses is even greater now. The Lister vaccine strain (LIVP) of vaccinia virus was used as a parental virus for generating a recombinant 1421ABJCN clone defective in five virulence genes encoding hemagglutinin (A56R), the IFN-γ-binding protein (B8R), thymidine kinase (J2R), the complement-binding protein (C3L), and the Bcl-2-like inhibitor of apoptosis (N1L). We found that disruption of these loci does not affect replication in mammalian cell cultures. The isogenic recombinant strain 1421ABJCN exhibits a reduced inflammatory response and attenuated neurovirulence relative to LIVP. Virus titers of 1421ABJCN were 3 lg lower versus the parent VACV LIVP when administered by the intracerebral route in new-born mice. In a subcutaneous mouse model, 1421ABJCN displayed levels of VACV-neutralizing antibodies comparable to those of LIVP and conferred protective immunity against lethal challenge by the ectromelia virus. The VACV mutant holds promise as a safe live vaccine strain for preventing smallpox and other orthopoxvirus infections. PMID:26798498

  17. Genome Sequence of Vaccinia virus Strain Lister-Butantan, a Lister Vaccine Variant Used during a Smallpox Eradication Campaign in Brazil

    PubMed Central

    Assis, Felipe; Trindade, Giliane; Drumond, Betânia; Frace, Mike; Sammons, Scott; Emerson, Ginny; Li, Yu; Carroll, Darin; Batra, Dhwani; Kroon, Erna

    2016-01-01

    Here, we report the 187.8-kb genome sequence of Vaccinia virus Lister-Butantan, which was used in Brazil during the WHO smallpox eradication campaign. Its genome showed an average similarity of 98.18% with the original Lister isolate, highlighting the low divergence among related Vaccinia virus vaccine strains, even after several passages in animals and cell culture. PMID:27340056

  18. Genome Sequence of Vaccinia virus Strain Lister-Butantan, a Lister Vaccine Variant Used during a Smallpox Eradication Campaign in Brazil.

    PubMed

    Assis, Felipe; Trindade, Giliane; Drumond, Betânia; Frace, Mike; Sammons, Scott; Emerson, Ginny; Li, Yu; Carroll, Darin; Batra, Dhwani; Abrahão, Jonatas; Kroon, Erna

    2016-01-01

    Here, we report the 187.8-kb genome sequence of Vaccinia virus Lister-Butantan, which was used in Brazil during the WHO smallpox eradication campaign. Its genome showed an average similarity of 98.18% with the original Lister isolate, highlighting the low divergence among related Vaccinia virus vaccine strains, even after several passages in animals and cell culture. PMID:27340056

  19. Vaccinia virus transcription.

    PubMed

    Broyles, Steven S

    2003-09-01

    Vaccinia virus replication takes place in the cytoplasm of the host cell. The nearly 200 kbp genome owes part of its complexity to encoding most of the proteins involved in genome and mRNA synthesis. The multisubunit vaccinia virus RNA polymerase requires a separate set of virus-encoded proteins for the transcription of the early, intermediate and late classes of genes. Cell fractionation studies have provided evidence for a role for host cell proteins in the initiation and termination of vaccinia virus intermediate and late gene transcription. Vaccinia virus resembles nuclear DNA viruses in the integration of viral and host proteins for viral mRNA synthesis, yet is markedly less reliant on host proteins than its nuclear counterparts. PMID:12917449

  20. Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen

    NASA Astrophysics Data System (ADS)

    Smith, Geoffrey L.; Mackett, Michael; Moss, Bernard

    1983-04-01

    Potential live vaccines against hepatitis B virus have been produced. The coding sequence for hepatitis B virus surface antigen (HBsAg) has been inserted into the vaccinia virus genome under control of vaccinia virus early promoters. Cells infected with these vaccinia virus recombinants synthesize and excrete HBsAg and vaccinated rabbits rapidly produce antibodies to HBsAg.

  1. Protective Immunity to Vaccinia Virus Induced by Vaccination with Multiple Recombinant Outer Membrane Proteins of Intracellular and Extracellular Virions

    PubMed Central

    Fogg, Christiana; Lustig, Shlomo; Whitbeck, J. Charles; Eisenberg, Roselyn J.; Cohen, Gary H.; Moss, Bernard

    2004-01-01

    Infectious intracellular and extracellular forms of vaccinia virus have different outer membrane proteins, presenting multiple targets to the immune system. We investigated the immunogenicity of soluble forms of L1, an outer membrane protein of the intracellular mature virus, and of A33 and B5, outer membrane proteins of the extracellular enveloped virus. The recombinant proteins, in 10-μg amounts mixed with a Ribi- or saponin-type adjuvant, were administered subcutaneously to mice. Antibody titers to each protein rose sharply after the first and second boosts, reaching levels that surpassed those induced by percutaneous immunization with live vaccinia virus. Immunoglobulin G1 (IgG1) antibody predominated after the protein immunizations, indicative of a T-helper cell type 2 response, whereas live vaccinia virus induced mainly IgG2a, indicative of a T-helper cell type 1 response. Mice immunized with any one of the recombinant proteins survived an intranasal challenge with 5 times the 50% lethal dose of the pathogenic WR strain of vaccinia virus. Measurements of weight loss indicated that the A33 immunization most effectively prevented disease. The superiority of protein combinations was demonstrated when the challenge virus dose was increased 20-fold. The best protection was obtained with a vaccine made by combining recombinant proteins of the outer membranes of intracellular and extracellular virus. Indeed, mice immunized with A33 plus B5 plus L1 or with A33 plus L1 were better protected than mice immunized with live vaccinia virus. Three immunizations with the three-protein combination were necessary and sufficient for complete protection. These studies suggest the feasibility of a multiprotein smallpox vaccine. PMID:15367588

  2. Vaccinia virus infections in martial arts gym, Maryland, USA, 2008.

    PubMed

    Hughes, Christine M; Blythe, David; Li, Yu; Reddy, Ramani; Jordan, Carol; Edwards, Cindy; Adams, Celia; Conners, Holly; Rasa, Catherine; Wilby, Sue; Russell, Jamaal; Russo, Kelly S; Somsel, Patricia; Wiedbrauk, Danny L; Dougherty, Cindy; Allen, Christopher; Frace, Mike; Emerson, Ginny; Olson, Victoria A; Smith, Scott K; Braden, Zachary; Abel, Jason; Davidson, Whitni; Reynolds, Mary; Damon, Inger K

    2011-04-01

    Vaccinia virus is an orthopoxvirus used in the live vaccine against smallpox. Vaccinia virus infections can be transmissible and can cause severe complications in those with weakened immune systems. We report on a cluster of 4 cases of vaccinia virus infection in Maryland, USA, likely acquired at a martial arts gym. PMID:21470473

  3. [Modified vaccinia virus ankara (MVA)--development as recombinant vaccine and prospects for use in veterinary medicine].

    PubMed

    Volz, Asisa; Fux, Robert; Langenmayer, Martin C; Sutter, Gerd

    2015-01-01

    Poxviruses as expression vectors are widely used in medical research for the development of recombinant vaccines and molecular therapies. Here we review recent accomplishments in vaccine research using recombinant modified vaccinia virus ankara (MVA). MVA is a highly attenuated vaccinia virus strain that originated from serial tissue culture passage in chicken embryo fibroblasts more than 40 years ago. Growth adaptation to avian host cells caused deletions and mutations in the viral genome affecting about 15% of the original genetic information. In consequence, MVA is replication-deficient in cells of mammalian origin and fails to produce many of the virulence factors encoded by conventional vaccinia virus. Because of its safety for the general environment MVA can be handled under conditions of biosafety level one. Non-replicating MVA can enter any target cell and activate its molecular life cycle to express all classes of viral and recombinant genes. Therefore, recombinant MVA have been established as an extremely safe and efficient vector system for vaccine development in medical research. By now, various recombinant MVA vaccines have been found safe and immunogenic when used for phase I/II clinical testing in humans, and suitable for industrial scale production following good practice of manufacturing. Thus, there is an obvious usefulness of recombinant MVA vaccines for novel prophylactic and therapeutic approaches also in veterinary medicine. Results from first studies in companion and farm animals are highly promising. PMID:26697713

  4. Modified Vaccinia Virus Ankara (MVA) as Production Platform for Vaccines against Influenza and Other Viral Respiratory Diseases

    PubMed Central

    Altenburg, Arwen F.; Kreijtz, Joost H. C. M.; de Vries, Rory D.; Song, Fei; Fux, Robert; Rimmelzwaan, Guus F.; Sutter, Gerd; Volz, Asisa

    2014-01-01

    Respiratory viruses infections caused by influenza viruses, human parainfluenza virus (hPIV), respiratory syncytial virus (RSV) and coronaviruses are an eminent threat for public health. Currently, there are no licensed vaccines available for hPIV, RSV and coronaviruses, and the available seasonal influenza vaccines have considerable limitations. With regard to pandemic preparedness, it is important that procedures are in place to respond rapidly and produce tailor made vaccines against these respiratory viruses on short notice. Moreover, especially for influenza there is great need for the development of a universal vaccine that induces broad protective immunity against influenza viruses of various subtypes. Modified Vaccinia Virus Ankara (MVA) is a replication-deficient viral vector that holds great promise as a vaccine platform. MVA can encode one or more foreign antigens and thus functions as a multivalent vaccine. The vector can be used at biosafety level 1, has intrinsic adjuvant capacities and induces humoral and cellular immune responses. However, there are some practical and regulatory issues that need to be addressed in order to develop MVA-based vaccines on short notice at the verge of a pandemic. In this review, we discuss promising novel influenza virus vaccine targets and the use of MVA for vaccine development against various respiratory viruses. PMID:25036462

  5. Modified vaccinia virus ankara (MVA) as production platform for vaccines against influenza and other viral respiratory diseases.

    PubMed

    Altenburg, Arwen F; Kreijtz, Joost H C M; de Vries, Rory D; Song, Fei; Fux, Robert; Rimmelzwaan, Guus F; Sutter, Gerd; Volz, Asisa

    2014-07-01

    Respiratory viruses infections caused by influenza viruses, human parainfluenza virus (hPIV), respiratory syncytial virus (RSV) and coronaviruses are an eminent threat for public health. Currently, there are no licensed vaccines available for hPIV, RSV and coronaviruses, and the available seasonal influenza vaccines have considerable limitations. With regard to pandemic preparedness, it is important that procedures are in place to respond rapidly and produce tailor made vaccines against these respiratory viruses on short notice. Moreover, especially for influenza there is great need for the development of a universal vaccine that induces broad protective immunity against influenza viruses of various subtypes. Modified Vaccinia Virus Ankara (MVA) is a replication-deficient viral vector that holds great promise as a vaccine platform. MVA can encode one or more foreign antigens and thus functions as a multivalent vaccine. The vector can be used at biosafety level 1, has intrinsic adjuvant capacities and induces humoral and cellular immune responses. However, there are some practical and regulatory issues that need to be addressed in order to develop MVA-based vaccines on short notice at the verge of a pandemic. In this review, we discuss promising novel influenza virus vaccine targets and the use of MVA for vaccine development against various respiratory viruses. PMID:25036462

  6. Genomic analysis of the vaccinia virus strain variants found in Dryvax vaccine.

    PubMed

    Qin, Li; Upton, Chris; Hazes, Bart; Evans, David H

    2011-12-01

    Smallpox was eradicated using variant forms of vaccinia virus-based vaccines. One of these was Dryvax, a calf lymph vaccine derived from the New York City Board of Health strain. We used genome-sequencing technology to examine the genetic diversity of the population of viruses present in a sample of Dryvax. These studies show that the conserved cores of these viruses exhibit a lower level of sequence variation than do the telomeres. However, even though the ends of orthopoxviruses are more genetically plastic than the cores, there are still many telomeric genes that are conserved as intact open reading frames in the 11 genomes that we, and 4 genomes that others, have sequenced. Most of these genes likely modulate inflammation. Our sequencing also detected an evolving pattern of mutation, with some genes being highly fragmented by randomly assorting mutations (e.g., M1L), while other genes are intact in most viruses but have been disrupted in individual strains (e.g., I4L in strain DPP17). Over 85% of insertion and deletion mutations are associated with repeats, and a rare new isolate bearing a large deletion in the right telomere was identified. All of these strains cluster in dendrograms consistent with their origin but which also surprisingly incorporate horsepox virus. However, these viruses also exhibit a "patchy" pattern of polymorphic sites characteristic of recombinants. There is more genetic diversity detected within a vial of Dryvax than between variola virus major and minor strains, and our study highlights how propagation methods affect the genetics of orthopoxvirus populations. PMID:21976639

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

    PubMed

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

    2016-04-01

    West Nile virus (WNV) cycles between insects and wild birds, and is transmitted via mosquito vectors to horses and humans, potentially causing severe neuroinvasive disease. Modified Vaccinia virus Ankara (MVA) is an advanced viral vector for developing new recombinant vaccines against infectious diseases and cancer. Here, we generated and evaluated recombinant MVA candidate vaccines that deliver WNV envelope (E) antigens and fulfil all the requirements to proceed to clinical testing in humans. Infections of human and equine cell cultures with recombinant MVA demonstrated efficient synthesis and secretion of WNV envelope proteins in mammalian cells non-permissive for MVA replication. Prime-boost immunizations in BALB/c mice readily induced circulating serum antibodies binding to recombinant WNV E protein and neutralizing WNV in tissue culture infections. Vaccinations in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice elicited WNV E-specific CD8+ T cell responses. Moreover, the MVA-WNV candidate vaccines protected C57BL/6 mice against lineage 1 and lineage 2 WNV infection and induced heterologous neutralizing antibodies. Thus, further studies are warranted to evaluate these recombinant MVA-WNV vaccines in other preclinical models and use them as candidate vaccine in humans. PMID:26939903

  8. Genomic sequence and virulence of clonal isolates of vaccinia virus Tiantan, the Chinese smallpox vaccine strain.

    PubMed

    Zhang, Qicheng; Tian, Meijuan; Feng, Yi; Zhao, Kai; Xu, Jing; Liu, Ying; Shao, Yiming

    2013-01-01

    Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV) as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT) was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV) vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1) viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1) and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ∼190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs). ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH) strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector. PMID:23593246

  9. Reemergence of vaccinia virus during Zoonotic outbreak, Pará State, Brazil.

    PubMed

    de Assis, Felipe L; Vinhote, Wagner M; Barbosa, José D; de Oliveira, Cairo H S; de Oliveira, Carlos M G; Campos, Karinny F; Silva, Natália S; Trindade, Giliane de Souza

    2013-12-01

    In 2010, vaccinia virus caused an outbreak of bovine vaccinia that affected dairy cattle and rural workers in Pará State, Brazil. Genetic analyses identified the virus as distinct from BeAn58058 vaccinia virus (identified in 1960s) and from smallpox vaccine virus strains. These findings suggest spread of autochthonous group 1 vaccinia virus in this region. PMID:24274374

  10. Rabies vaccination: comparison of neutralizing antibody responses after priming and boosting with different combinations of DNA, inactivated virus, or recombinant vaccinia virus vaccines.

    PubMed

    Lodmell, D L; Ewalt, L C

    2000-05-01

    Long-term levels of neutralizing antibody were evaluated in mice after a single immunization with experimental DNA or recombinant vaccinia virus (RVV) vaccines encoding the rabies virus glycoprotein (G), or the commercially available inactivated virus human diploid cell vaccine (HDCV). Anamnestic antibody titers were also evaluated after two booster immunizations with vaccines that were identical to or different from the priming vaccine. Five hundred and forty days (1.5 year) after a single immunization with any of the three vaccines, neutralizing antibody titers remained greater than the minimal acceptable human level of antibody titer (0.5 International Units (IU)/ml). In addition, either an HDCV or DNA booster elicited early and elevated anamnestic antibody responses in mice that had been primed with any of the three vaccines. In contrast, RVV boosters failed to elevate titers in mice that had been previously primed with RVV, and elicited slowly rising titers in mice that had been primed with either DNA or HDCV. Thus, a single vaccination with any of the three different vaccines elicited long-term levels of neutralizing antibody that exceeded 0.5 IU/ml. In contrast, different prime-booster vaccine combinations elicited anamnestic neutralizing antibody responses that increased quickly, increased slowly or failed to increase. PMID:10738096

  11. Protection of rhesus monkeys from fatal Lassa fever by vaccination with a recombinant vaccinia virus containing the Lassa virus glycoprotein gene.

    PubMed Central

    Fisher-Hoch, S P; McCormick, J B; Auperin, D; Brown, B G; Castor, M; Perez, G; Ruo, S; Conaty, A; Brammer, L; Bauer, S

    1989-01-01

    Lassa fever is an acute febrile disease of West Africa, where there are as many as 300,000 infections a year and an estimated 3000 deaths. As control of the rodent host is impracticable at present, the best immediate prospect is vaccination. We tested as potential vaccines in rhesus monkeys a closely related virus, Mopeia virus (two monkeys), and a recombinant vaccinia virus containing the Lassa virus glycoprotein gene, V-LSGPC (four monkeys). Two monkeys vaccinated with the New York Board of Health strain of vaccinia virus as controls died after challenge with Lassa virus. The two monkeys vaccinated with Mopeia virus developed antibodies measurable by radioimmunoprecipitation prior to challenge, and they survived challenge by Lassa virus with minimal physical or physiologic disturbances. However, both showed a transient, low-titer Lassa viremia. Two of the four animals vaccinated with V-LSGPC had antibodies to both Lassa glycoproteins, as determined by radioimmunoprecipitation. All four animals survived a challenge of Lassa virus but experienced a transient febrile illness and moderate physiologic changes following challenge. Virus was recoverable from each of these animals, but at low titer and only during a brief period, as observed for the Mopeia-protected animals. We conclude that V-LSGPC can protect rhesus monkeys against death from Lassa fever. PMID:2911575

  12. Protective Properties of Vaccinia Virus-Based Vaccines: Skin Scarification Promotes a Nonspecific Immune Response That Protects against Orthopoxvirus Disease

    PubMed Central

    Rice, Amanda D.; Adams, Mathew M.; Lindsey, Scott F.; Swetnam, Daniele M.; Manning, Brandi R.; Smith, Andrew J.; Burrage, Andrew M.; Wallace, Greg; MacNeill, Amy L.

    2014-01-01

    ABSTRACT The process of vaccination introduced by Jenner generated immunity against smallpox and ultimately led to the eradication of the disease. Procedurally, in modern times, the virus is introduced into patients via a process called scarification, performed with a bifurcated needle containing a small amount of virus. What was unappreciated was the role that scarification itself plays in generating protective immunity. In rabbits, protection from lethal disease is induced by intradermal injection of vaccinia virus, whereas a protective response occurs within the first 2 min after scarification with or without virus, suggesting that the scarification process itself is a major contributor to immunoprotection. IMPORTANCE These results show the importance of local nonspecific immunity in controlling poxvirus infections and indicate that the process of scarification should be critically considered during the development of vaccination protocols for other infectious agents. PMID:24760885

  13. Induction of protective immunity in animals vaccinated with recombinant vaccinia viruses that express PreM and E glycoproteins of Japanese encephalitis virus.

    PubMed Central

    Yasuda, A; Kimura-Kuroda, J; Ogimoto, M; Miyamoto, M; Sata, T; Sato, T; Takamura, C; Kurata, T; Kojima, A; Yasui, K

    1990-01-01

    A cDNA clone representing the genome of structural proteins of Japanese encephalitis virus (JEV) was inserted into the thymidine kinase gene of vaccinia virus strains LC16mO and WR under the control of a strong early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. Indirect immunofluorescence and fluorescence-activated flow cytometric analysis revealed that the recombinant vaccinia viruses expressed JEV E protein on the membrane surface, as well as in the cytoplasm, of recombinant-infected cells. In addition, the E protein expressed from the JEV recombinants reacted to nine different characteristic monoclonal antibodies, some of which have hemagglutination-inhibiting and JEV-neutralizing activities. Radioimmunoprecipitation analysis demonstrated that two major proteins expressed in recombinant-infected cells were processed and glycosylated as the authentic PreM and E glycoproteins of JEV. Inoculation of rabbits with the infectious recombinant vaccinia virus resulted in rapid production of antiserum specific for the PreM and E glycoproteins of JEV. This antiserum had both hemagglutination-inhibiting and virus-neutralizing activities against JEV. Furthermore, mice vaccinated with the recombinant also produced JEV-neutralizing antibodies and were resistant to challenge with JEV. Images PMID:2159544

  14. Live-vaccinia virus encapsulation in pH-sensitive polymer increases safety of a reservoir-targeted Lyme disease vaccine by targeting gastrointestinal release.

    PubMed

    Kern, Aurelie; Zhou, Chensheng W; Jia, Feng; Xu, Qiaobing; Hu, Linden T

    2016-08-31

    The incidence of Lyme disease has continued to rise despite attempts to control its spread. Vaccination of zoonotic reservoirs of human pathogens has been successfully used to decrease the incidence of rabies in raccoons and foxes. We have previously reported on the efficacy of a vaccinia virus vectored vaccine to reduce carriage of Borrelia burgdorferi in reservoir mice and ticks. One potential drawback to vaccinia virus vectored vaccines is the risk of accidental infection of humans. To reduce this risk, we developed a process to encapsulate vaccinia virus with a pH-sensitive polymer that inactivates the virus until it is ingested and dissolved by stomach acids. We demonstrate that the vaccine is inactive both in vitro and in vivo until it is released from the polymer. Once released from the polymer by contact with an acidic pH solution, the virus regains infectivity. Vaccination with coated vaccinia virus confers protection against B. burgdorferi infection and reduction in acquisition of the pathogen by naïve feeding ticks. PMID:27502570

  15. Protection of Mice from Fatal Measles Encephalitis by Vaccination with Vaccinia Virus Recombinants Encoding Either the Hemagglutinin or the Fusion Protein

    NASA Astrophysics Data System (ADS)

    Drillien, Robert; Spehner, Daniele; Kirn, Andre; Giraudon, Pascale; Buckland, Robin; Wild, Fabian; Lecocq, Jean-Pierre

    1988-02-01

    Vaccinia virus recombinants encoding the hemagglutinin or fusion protein of measles virus have been constructed. Infection of cell cultures with the recombinants led to the synthesis of authentic measles proteins as judged by their electrophoretic mobility, recognition by antibodies, glycosylation, proteolytic cleavage, and presentation on the cell surface. Mice vaccinated with a single dose of the recombinant encoding the hemagglutinin protein developed antibodies capable of both inhibiting hemagglutination activity and neutralizing measles virus, whereas animals vaccinated with the recombinant encoding the fusion protein developed measles neutralizing antibodies. Mice vaccinated with either of the recombinants resisted a normally lethal intracerebral inoculation of a cell-associated measles virus subacute sclerosing panencephalitis strain.

  16. Complete Genome Sequence of Vaccinia Virus Strain L-IVP.

    PubMed

    Shvalov, Alexander N; Sivolobova, Galina F; Kuligina, Elena V; Kochneva, Galina V

    2016-01-01

    Most of the live vaccine doses of vaccinia virus donated to the Intensified Smallpox Eradication Programme after 1971 were prepared using the L-IVP strain. A mixture of three clones of the L-IVP strain was sequenced using MySEQ. Consensus sequence similarity with the vaccinia virus Lister strain is 99.5%. PMID:27174282

  17. Complete Genome Sequence of Vaccinia Virus Strain L-IVP

    PubMed Central

    Shvalov, Alexander N.; Sivolobova, Galina F.; Kuligina, Elena V.

    2016-01-01

    Most of the live vaccine doses of vaccinia virus donated to the Intensified Smallpox Eradication Programme after 1971 were prepared using the L-IVP strain. A mixture of three clones of the L-IVP strain was sequenced using MySEQ. Consensus sequence similarity with the vaccinia virus Lister strain is 99.5%. PMID:27174282

  18. Plasmodium knowlesi Sporozoite Antigen: Expression by Infectious Recombinant Vaccinia Virus

    NASA Astrophysics Data System (ADS)

    Smith, Geoffrey L.; Godson, G. Nigel; Nussenzweig, Victor; Nussenzweig, Ruth S.; Barnwell, John; Moss, Bernard

    1984-04-01

    The gene coding for the circumsporozoite antigen of the malaria parasite Plasmodium knowlesi was inserted into the vaccinia virus genome under the control of a defined vaccinia virus promoter. Cells infected with the recombinant virus synthesized polypeptides of 53,000 to 56,000 daltons that reacted with monoclonal antibody against the repeating epitope of the malaria protein. Furthermore, rabbits vaccinated with the recombinant virus produced antibodies that bound specifically to sporozoites. These data provide evidence for expression of a cloned malaria gene in mammalian cells and illustrate the potential of vaccinia virus recombinants as live malaria vaccines.

  19. Safety and Immunogenicity of Modified Vaccinia Ankara-Bavarian Nordic Smallpox Vaccine in Vaccinia-Naive and Experienced Human Immunodeficiency Virus-Infected Individuals: An Open-Label, Controlled Clinical Phase II Trial

    PubMed Central

    Overton, Edgar Turner; Stapleton, Jack; Frank, Ian; Hassler, Shawn; Goepfert, Paul A.; Barker, David; Wagner, Eva; von Krempelhuber, Alfred; Virgin, Garth; Meyer, Thomas Peter; Müller, Jutta; Bädeker, Nicole; Grünert, Robert; Young, Philip; Rösch, Siegfried; Maclennan, Jane; Arndtz-Wiedemann, Nathaly; Chaplin, Paul

    2015-01-01

    Background. First- and second-generation smallpox vaccines are contraindicated in individuals infected with human immunodeficiency virus (HIV). A new smallpox vaccine is needed to protect this population in the context of biodefense preparedness. The focus of this study was to compare the safety and immunogenicity of a replication-deficient, highly attenuated smallpox vaccine modified vaccinia Ankara (MVA) in HIV-infected and healthy subjects. Methods. An open-label, controlled Phase II trial was conducted at 36 centers in the United States and Puerto Rico for HIV-infected and healthy subjects. Subjects received 2 doses of MVA administered 4 weeks apart. Safety was evaluated by assessment of adverse events, focused physical exams, electrocardiogram recordings, and safety laboratories. Immune responses were assessed using enzyme-linked immunosorbent assay (ELISA) and a plaque reduction neutralization test (PRNT). Results. Five hundred seventy-nine subjects were vaccinated at least once and had data available for analysis. Rates of ELISA seropositivity were comparably high in vaccinia-naive healthy and HIV-infected subjects, whereas PRNT seropositivity rates were higher in healthy compared with HIV-infected subjects. Modified vaccinia Ankara was safe and well tolerated with no adverse impact on viral load or CD4 counts. There were no cases of myo-/pericarditis reported. Conclusions. Modified vaccinia Ankara was safe and immunogenic in subjects infected with HIV and represents a promising smallpox vaccine candidate for use in immunocompromised populations. PMID:26380340

  20. Protective Efficacy of the Conserved NP, PB1, and M1 Proteins as Immunogens in DNA- and Vaccinia Virus-Based Universal Influenza A Virus Vaccines in Mice.

    PubMed

    Wang, Wenling; Li, Renqing; Deng, Yao; Lu, Ning; Chen, Hong; Meng, Xin; Wang, Wen; Wang, Xiuping; Yan, Kexia; Qi, Xiangrong; Zhang, Xiangmin; Xin, Wei; Lu, Zhenhua; Li, Xueren; Bian, Tao; Gao, Yingying; Tan, Wenjie; Ruan, Li

    2015-06-01

    The conventional hemagglutinin (HA)- and neuraminidase (NA)-based influenza vaccines need to be updated most years and are ineffective if the glycoprotein HA of the vaccine strains is a mismatch with that of the epidemic strain. Universal vaccines targeting conserved viral components might provide cross-protection and thus complement and improve conventional vaccines. In this study, we generated DNA plasmids and recombinant vaccinia viruses expressing the conserved proteins nucleoprotein (NP), polymerase basic 1 (PB1), and matrix 1 (M1) from influenza virus strain A/Beijing/30/95 (H3N2). BALB/c mice were immunized intramuscularly with a single vaccine based on NP, PB1, or M1 alone or a combination vaccine based on all three antigens and were then challenged with lethal doses of the heterologous influenza virus strain A/PR/8/34 (H1N1). Vaccines based on NP, PB1, and M1 provided complete or partial protection against challenge with 1.7 50% lethal dose (LD50) of PR8 in mice. Of the three antigens, NP-based vaccines induced protection against 5 LD50 and 10 LD50 and thus exhibited the greatest protective effect. Universal influenza vaccines based on the combination of NP, PB1, and M1 induced a strong immune response and thus might be an alternative approach to addressing future influenza virus pandemics. PMID:25834017

  1. Extent of Systemic Spread Determines CD8+ T Cell Immunodominance for Laboratory Strains, Smallpox Vaccines, and Zoonotic Isolates of Vaccinia Virus.

    PubMed

    Flesch, Inge E A; Hollett, Natasha A; Wong, Yik Chun; Quinan, Bárbara Resende; Howard, Debbie; da Fonseca, Flávio G; Tscharke, David C

    2015-09-01

    CD8(+) T cells that recognize virus-derived peptides presented on MHC class I are vital antiviral effectors. Such peptides presented by any given virus vary greatly in immunogenicity, allowing them to be ranked in an immunodominance hierarchy. However, the full range of parameters that determine immunodominance and the underlying mechanisms remain unknown. In this study, we show across a range of vaccinia virus strains, including the current clonal smallpox vaccine, that the ability of a strain to spread systemically correlated with reduced immunodominance. Reduction in immunodominance was observed both in the lymphoid system and at the primary site of infection. Mechanistically, reduced immunodominance was associated with more robust priming and especially priming in the spleen. Finally, we show this is not just a property of vaccine and laboratory strains of virus, because an association between virulence and immunodominance was also observed in isolates from an outbreak of zoonotic vaccinia virus that occurred in Brazil. PMID:26195812

  2. Use of Vaccinia Virus Smallpox Vaccine in Laboratory and Health Care Personnel at Risk for Occupational Exposure to Orthopoxviruses - Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2015.

    PubMed

    Petersen, Brett W; Harms, Tiara J; Reynolds, Mary G; Harrison, Lee H

    2016-03-18

    On June 25, 2015, the Advisory Committee on Immunization Practices (ACIP) recommended routine vaccination with live smallpox (vaccinia) vaccine (ACAM2000) for laboratory personnel who directly handle 1) cultures or 2) animals contaminated or infected with replication-competent vaccinia virus, recombinant vaccinia viruses derived from replication-competent vaccinia strains (i.e., those that are capable of causing clinical infection and producing infectious virus in humans), or other orthopoxviruses that infect humans (e.g., monkeypox, cowpox, and variola) (recommendation category: A, evidence type 2 [Box]). Health care personnel (e.g., physicians and nurses) who currently treat or anticipate treating patients with vaccinia virus infections and whose contact with replication-competent vaccinia viruses is limited to contaminated materials (e.g., dressings) and persons administering ACAM2000 smallpox vaccine who adhere to appropriate infection prevention measures can be offered vaccination with ACAM2000 (recommendation category: B, evidence type 2 [Box]). These revised recommendations update the previous ACIP recommendations for nonemergency use of vaccinia virus smallpox vaccine for laboratory and health care personnel at risk for occupational exposure to orthopoxviruses (1). Since 2001, when the previous ACIP recommendations were developed, ACAM2000 has replaced Dryvax as the only smallpox vaccine licensed by the U.S. Food and Drug Administration (FDA) and available for use in the United States (2). These recommendations contain information on ACAM2000 and its use in laboratory and health care personnel at risk for occupational exposure to orthopoxviruses. PMID:26985679

  3. Direct comparison of antigen production and induction of apoptosis by canarypox virus- and modified vaccinia virus ankara-human immunodeficiency virus vaccine vectors.

    PubMed

    Zhang, Xiugen; Cassis-Ghavami, Farah; Eller, Mike; Currier, Jeff; Slike, Bonnie M; Chen, Xuemin; Tartaglia, James; Marovich, Mary; Spearman, Paul

    2007-07-01

    Recombinant poxvirus vectors are undergoing intensive evaluation as vaccine candidates for a variety of infectious pathogens. Avipoxviruses, such as canarypox virus, are replication deficient in mammalian cells by virtue of a poorly understood species-specific restriction. Highly attenuated vaccinia virus strains such as modified vaccinia virus Ankara (MVA) are similarly unable to complete replication in most mammalian cells but have an abortive-late phenotype, in that the block to replication occurs post-virus-specific DNA replication. In this study, an identical expression cassette for human immunodeficiency virus gag, pro, and env coding sequences was placed in canarypox virus and MVA vector backbones in order to directly compare vector-borne expression and to analyze differences in vector-host cell interactions. Antigen production by recombinant MVA was shown to be greater than that from recombinant canarypox virus in the mammalian cell lines and in the primary human cells tested. This observation was primarily due to a longer duration of antigen production in recombinant MVA-infected cells. Apoptosis induction was found to be more profound with the empty canarypox virus vector than with MVA. Remarkably, however, the inclusion of a gag/pro/env expression cassette altered the kinetics of apoptosis induction in recombinant MVA-infected cells to levels equal to those found in canarypox virus-infected cells. Antigen production by MVA was noted to be greater in human dendritic cells and resulted in enhanced T-cell stimulation in an in vitro antigen presentation assay. These results reveal differences in poxvirus vector-host cell interactions that should be relevant to their use as immunization vehicles. PMID:17409140

  4. Direct Comparison of Antigen Production and Induction of Apoptosis by Canarypox Virus- and Modified Vaccinia Virus Ankara-Human Immunodeficiency Virus Vaccine Vectors▿

    PubMed Central

    Zhang, Xiugen; Cassis-Ghavami, Farah; Eller, Mike; Currier, Jeff; Slike, Bonnie M.; Chen, Xuemin; Tartaglia, James; Marovich, Mary; Spearman, Paul

    2007-01-01

    Recombinant poxvirus vectors are undergoing intensive evaluation as vaccine candidates for a variety of infectious pathogens. Avipoxviruses, such as canarypox virus, are replication deficient in mammalian cells by virtue of a poorly understood species-specific restriction. Highly attenuated vaccinia virus strains such as modified vaccinia virus Ankara (MVA) are similarly unable to complete replication in most mammalian cells but have an abortive-late phenotype, in that the block to replication occurs post-virus-specific DNA replication. In this study, an identical expression cassette for human immunodeficiency virus gag, pro, and env coding sequences was placed in canarypox virus and MVA vector backbones in order to directly compare vector-borne expression and to analyze differences in vector-host cell interactions. Antigen production by recombinant MVA was shown to be greater than that from recombinant canarypox virus in the mammalian cell lines and in the primary human cells tested. This observation was primarily due to a longer duration of antigen production in recombinant MVA-infected cells. Apoptosis induction was found to be more profound with the empty canarypox virus vector than with MVA. Remarkably, however, the inclusion of a gag/pro/env expression cassette altered the kinetics of apoptosis induction in recombinant MVA-infected cells to levels equal to those found in canarypox virus-infected cells. Antigen production by MVA was noted to be greater in human dendritic cells and resulted in enhanced T-cell stimulation in an in vitro antigen presentation assay. These results reveal differences in poxvirus vector-host cell interactions that should be relevant to their use as immunization vehicles. PMID:17409140

  5. Strong, but Age-Dependent, Protection Elicited by a Deoxyribonucleic Acid/Modified Vaccinia Ankara Simian Immunodeficiency Virus Vaccine

    PubMed Central

    Chamcha, Venkateswarlu; Kannanganat, Sunil; Gangadhara, Sailaja; Nabi, Rafiq; Kozlowski, Pamela A.; Montefiori, David C.; LaBranche, Celia C.; Wrammert, Jens; Keele, Brandon F.; Balachandran, Harikrishnan; Sahu, Sujata; Lifton, Michelle; Santra, Sampa; Basu, Rahul; Moss, Bernard; Robinson, Harriet L.; Amara, Rama Rao

    2016-01-01

    Background. In this study, we analyzed the protective efficacy of a simian immunodeficiency virus (SIV) macaque 239 (SIVmac239) analogue of the clinically tested GOVX-B11 deoxyribonucleic acid (DNA)/modified vaccinia Ankara (MVA) human immunodeficiency virus vaccine. Methods. The tested vaccine used a DNA immunogen mutated to mimic the human vaccine and a regimen with DNA deliveries at weeks 0 and 8 and MVA deliveries at weeks 16 and 32. Twelve weekly rectal challenges with 0.3 animal infectious doses of SIV sootey mangabey E660 (SIVsmE660) were administered starting at 6 months after the last immunization. Results. Over the first 6 rectal exposures to SIVsmE660, <10-year-old tripartite motif-containing protein 5 (TRIM5)α-permissive rhesus macaques showed an 80% reduction in per-exposure risk of infection as opposed to a 46% reduction in animals over 10 years old; and, over the 12 challenges, they showed a 72% as opposed to a 10% reduction. Analyses of elicited immune responses suggested that higher antibody responses in the younger animals had played a role in protection. Conclusions. The simian analogue of the GOVX-B11 HIV provided strong protection against repeated rectal challenges in young adult macaques. PMID:27006959

  6. Strong, but Age-Dependent, Protection Elicited by a Deoxyribonucleic Acid/Modified Vaccinia Ankara Simian Immunodeficiency Virus Vaccine.

    PubMed

    Chamcha, Venkateswarlu; Kannanganat, Sunil; Gangadhara, Sailaja; Nabi, Rafiq; Kozlowski, Pamela A; Montefiori, David C; LaBranche, Celia C; Wrammert, Jens; Keele, Brandon F; Balachandran, Harikrishnan; Sahu, Sujata; Lifton, Michelle; Santra, Sampa; Basu, Rahul; Moss, Bernard; Robinson, Harriet L; Amara, Rama Rao

    2016-01-01

    Background.  In this study, we analyzed the protective efficacy of a simian immunodeficiency virus (SIV) macaque 239 (SIVmac239) analogue of the clinically tested GOVX-B11 deoxyribonucleic acid (DNA)/modified vaccinia Ankara (MVA) human immunodeficiency virus vaccine. Methods.  The tested vaccine used a DNA immunogen mutated to mimic the human vaccine and a regimen with DNA deliveries at weeks 0 and 8 and MVA deliveries at weeks 16 and 32. Twelve weekly rectal challenges with 0.3 animal infectious doses of SIV sootey mangabey E660 (SIVsmE660) were administered starting at 6 months after the last immunization. Results.  Over the first 6 rectal exposures to SIVsmE660, <10-year-old tripartite motif-containing protein 5 (TRIM5)α-permissive rhesus macaques showed an 80% reduction in per-exposure risk of infection as opposed to a 46% reduction in animals over 10 years old; and, over the 12 challenges, they showed a 72% as opposed to a 10% reduction. Analyses of elicited immune responses suggested that higher antibody responses in the younger animals had played a role in protection. Conclusions.  The simian analogue of the GOVX-B11 HIV provided strong protection against repeated rectal challenges in young adult macaques. PMID:27006959

  7. Lister vaccine strain of vaccinia virus armed with the endostatin-angiostatin fusion gene: an oncolytic virus superior to dl1520 (ONYX-015) for human head and neck cancer.

    PubMed

    Tysome, James R; Wang, Pengju; Alusi, Ghassan; Briat, Arnaud; Gangeswaran, Rathi; Wang, Jiwei; Bhakta, Vipul; Fodor, Istvan; Lemoine, Nick R; Wang, Yaohe

    2011-09-01

    Oncolytic viral therapy represents a promising strategy for the treatment of head and neck squamous cell carcinoma (HNSCC), with dl1520 (ONYX-015) the most widely used oncolytic adenovirus in clinical trials. This study aimed to determine the effectiveness of the Lister vaccine strain of vaccinia virus as well as a vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapy for HNSCC and to compare them with dl1520. The potency and replication of the Lister strain and VVhEA and the expression and function of the fusion protein were determined in human HNSCC cells in vitro and in vivo. Finally, the efficacy of VVhEA was compared with dl1520 in vivo in a human HNSCC model. The Lister vaccine strain of vaccinia virus was more effective than the adenovirus against all HNSCC cell lines tested in vitro. Although the potency of VVhEA was attenuated in vitro, the expression and function of the endostatin-angiostatin fusion protein was confirmed in HNSCC models both in vitro and in vivo. This novel vaccinia virus (VVhEA) demonstrated superior antitumor potency in vivo compared with both dl1520 and the control vaccinia virus. This study suggests that the Lister strain vaccinia virus armed with an endostatin-angiostatin fusion gene may be a potential therapeutic agent for HNSCC. PMID:21361787

  8. Vaccination of horses with a recombinant modified vaccinia Ankara virus (MVA) expressing African horse sickness (AHS) virus major capsid protein VP2 provides complete clinical protection against challenge

    PubMed Central

    Alberca, Berta; Bachanek-Bankowska, Katarzyna; Cabana, Marta; Calvo-Pinilla, Eva; Viaplana, Elisenda; Frost, Lorraine; Gubbins, Simon; Urniza, Alicia; Mertens, Peter; Castillo-Olivares, Javier

    2014-01-01

    African horse sickness virus (AHSV) is an arthropod-borne pathogen that infects all species of equidae and causes high mortality in horses. Previously, a recombinant modified vaccinia Ankara (MVA) virus expressing the protein VP2 of AHSV serotype 4 was shown to induce virus neutralising antibodies in horses and protected interferon alpha receptor gene knock-out mice (IFNAR −/−) against virulent AHSV challenge. This study builds on the previous work, examining the protective efficacy of MVA-VP2 vaccination in the natural host of AHSV infection. A study group of 4 horses was vaccinated twice with a recombinant MVA virus expressing the major capsid protein (VP2) of AHSV serotype 9. Vaccinated animals and a control group of unvaccinated horses were then challenged with a virulent strain of AHSV-9. The vaccinated animals were completely protected against clinical disease and also against viraemia as measured by standard end-point dilution assays. In contrast, all control horses presented viraemia after challenge and succumbed to the infection. These results demonstrate the potential of recombinant MVA viruses expressing the outer capsid VP2 of AHSV as a protective vaccine against AHSV infection in the field. PMID:24837765

  9. Prime-boost vaccination with plasmid DNA followed by recombinant vaccinia virus expressing BgGARP induced a partial protective immunity to inhibit Babesia gibsoni proliferation in dogs.

    PubMed

    Cao, Shinuo; Mousa, Ahmed Abdelmoniem; Aboge, Gabriel Oluga; Kamyingkird, Ketsarin; Zhou, Mo; Moumouni, Paul Franck Adjou; Terkawi, Mohamad Alaa; Masatani, Tatsunori; Nishikawa, Yoshifumi; Suzuki, Hiroshi; Fukumoto, Shinya; Xuan, Xuenan

    2013-12-01

    A heterologous prime-boost vaccination regime with DNA and recombinant vaccinia virus (rvv) vectors expressing relevant antigens has been shown to induce effective immune responses against several infectious pathogens. In this study, we describe the effectiveness of the prime-boost strategy by immunizing dogs with a recombinant plasmid followed by vaccinia virus, both of which expressed the glutamic acid-rich protein (BgGARP) of Babesia gibsoni. The dogs immunized with the prime-boost regime developed a significantly high level of specific antibodies against BgGARP when compared with the control groups. The antibody level was strongly increased after a booster immunization with a recombinant vaccinia virus. Two weeks after the booster immunization with a recombinant vaccinia virus expressing BgGARP, the dogs were challenged with B. gibsoni parasite. The dogs immunized with the prime-boost regime showed partial protection, manifested as a significantly low level of parasitemia. These results indicated that this type of DNA/rvv prime-boost immunization approach may have use against B. gibsoni infection in dogs. PMID:24338330

  10. Protective efficacy of a recombinant vaccinia virus in vaccinia-immune mice.

    PubMed

    Andrew, M E

    1989-10-01

    Recombinant viral vectors offer a potential means of vaccinating against diseases for which there are no current safe vaccines. One of the criteria on which a viral vaccine vector would be selected is that it either circulates in the human or livestock population without producing overt disease (e.g. adenovirus) or has a history as a safe vaccine (e.g. vaccinia virus). However, this selection criterion also means that the target population is likely to have circulating antibodies that are specific to the vaccine vector. Since a percentage of the world's population has been vaccinated during the World Health Organization's Smallpox Eradication Campaign, such antibody titres, which are likely to lower vaccine efficacy, have been raised as an objection to the use of recombinant vaccinia viruses as vaccines. We have tested the effect of vaccinia-specific immunity on the protective efficacy of a recombinant virus, VV-PR8-HA6 (1) which expresses the haemagglutinin of the influenza virus A/PR/8/34. PMID:2613281

  11. ACAM2000 clonal Vero cell culture vaccinia virus (New York City Board of Health strain)--a second-generation smallpox vaccine for biological defense.

    PubMed

    Monath, Thomas P; Caldwell, Joseph R; Mundt, Wolfgang; Fusco, Joan; Johnson, Casey S; Buller, Mark; Liu, Jian; Gardner, Bridget; Downing, Greg; Blum, Paul S; Kemp, Tracy; Nichols, Richard; Weltzin, Richard

    2004-10-01

    The threat of smallpox as a biological weapon has spurred efforts to create stockpiles of vaccine for emergency preparedness. In lieu of preparing vaccine in animal skin (the original method), we cloned vaccinia virus (New York City Board of Health strain, Dryvax by plaque purification and amplified the clone in cell culture. The overarching goal was to produce a modern vaccine that was equivalent to the currently licensed Dryvax in its preclinical and clinical properties, and could thus reliably protect humans against smallpox. A variety of clones were evaluated, and many were unacceptably virulent in animal models. One clonal virus (ACAM1000) was selected and produced at clinical grade in MRC-5 human diploid cells. ACAM1000 was comparable to Dryvax in immunogenicity and protective activity but was less neurovirulent for mice and nonhuman primates. To meet requirements for large quantities of vaccine after the events of September 11th 2001, the ACAM1000 master virus seed was used to prepare vaccine (designated ACAM2000) at large scale in Vero cells under serum-free conditions. The genomes of ACAM1000 and ACAM2000 had identical nucleotide sequences, and the vaccines had comparable biological phenotypes. ACAM1000 and ACAM2000 were evaluated in three Phase 1 clinical trials. The vaccines produced major cutaneous reactions and evoked neutralizing antibody and cell-mediated immune responses in the vast majority of subjects and had a reactogenicity profile similar to that of Dryvax. PMID:15491873

  12. Towards a universal vaccine for avian influenza: protective efficacy of modified Vaccinia virus Ankara and Adenovirus vaccines expressing conserved influenza antigens in chickens challenged with low pathogenic avian influenza virus.

    PubMed

    Boyd, Amy C; Ruiz-Hernandez, Raul; Peroval, Marylene Y; Carson, Connor; Balkissoon, Devanand; Staines, Karen; Turner, Alison V; Hill, Adrian V S; Gilbert, Sarah C; Butter, Colin

    2013-01-11

    Current vaccines targeting surface proteins can drive antigenic variation resulting either in the emergence of more highly pathogenic viruses or of antigenically distinct viruses that escape control by vaccination and thereby persist in the host population. Influenza vaccines typically target the highly mutable surface proteins and do not provide protection against heterologous challenge. Vaccines which induce immune responses against conserved influenza epitopes may confer protection against heterologous challenge. We report here the results of vaccination with recombinant modified Vaccinia virus Ankara (MVA) and Adenovirus (Ad) expressing a fusion construct of nucleoprotein and matrix protein (NP+M1). Prime and boost vaccination regimes were trialled in different ages of chicken and were found to be safe and immunogenic. Interferon-γ (IFN-γ) ELISpot was used to assess the cellular immune response post secondary vaccination. In ovo Ad prime followed by a 4 week post hatch MVA boost was identified as the most immunogenic regime in one outbred and two inbred lines of chicken. Following vaccination, one inbred line (C15I) was challenged with low pathogenic avian influenza (LPAI) H7N7 (A/Turkey/England/1977). Birds receiving a primary vaccination with Ad-NP+M1 and a secondary vaccination with MVA-NP+M1 exhibited reduced cloacal shedding as measured by plaque assay at 7 days post infection compared with birds vaccinated with recombinant viruses containing irrelevant antigen. This preliminary indication of efficacy demonstrates proof of concept in birds; induction of T cell responses in chickens by viral vectors containing internal influenza antigens may be a productive strategy for the development of vaccines to induce heterologous protection against influenza in poultry. PMID:23200938

  13. Brazilian Vaccinia Viruses and Their Origins

    PubMed Central

    Trindade, Giliane S.; Emerson, Ginny L.; Carroll, Darin S.; Kroon, Erna G.

    2007-01-01

    Although the World Health Organization (WHO) declared global smallpox eradicated in 1980, concerns over emergent poxvirus infections have increased. Most poxvirus infections are zoonotic; exploring their genetic diversity will illuminate the genetic and evolutionary aspects of poxvirus infections, ecology, and epidemiology. In recent decades, several strains of the orthopoxvirus vaccinia virus (VACV) have been isolated throughout Brazil, including many genetically distinct isolates within the same outbreak. To further investigate the diversity and origins of these viruses, we analyzed molecular data from 8 Brazilian VACV isolates and compared several genes involved in virus structure and pathogenicity. Genetic variation among isolates suggests that ancestral Brazilian VACVs existed before the beginning of the WHO smallpox eradication vaccination campaigns and that these viruses continue to circulate. PMID:18214166

  14. Vaccination of mice against canine distemper virus-induced encephalitis with vaccinia virus recombinants encoding measles or canine distemper virus antigens.

    PubMed

    Wild, T F; Bernard, A; Spehner, D; Villeval, D; Drillien, R

    1993-01-01

    Measles and canine distemper are caused by serologically related viruses. Although dogs immunized with measles virus (MV) do not elicit canine distemper virus (CDV) neutralizing antibodies, they are protected against the fatal disease. To investigate the potential role of the MV antigens in protection against CDV, we have immunized mice with vaccinia virus (VV) recombinants expressing the MV haemagglutinin (HA), fusion (F), nucleoprotein (NP) and matrix (M) antigens and challenged them with CDV. A partial protection was observed with the VV recombinants expressing the F, NP and M antigens, but not the HA. In contrast, immunization with a VV recombinant expressing the CDV F protein completely protected mice from CDV. PMID:8470428

  15. Mucosal Vaccination Overcomes the Barrier to Recombinant Vaccinia Immunization Caused by Preexisting Poxvirus Immunity

    NASA Astrophysics Data System (ADS)

    Belyakov, Igor M.; Moss, Bernard; Strober, Warren; Berzofsky, Jay A.

    1999-04-01

    Overcoming preexisting immunity to vaccinia virus in the adult population is a key requirement for development of otherwise potent recombinant vaccinia vaccines. Based on our observation that s.c. immunization with vaccinia induces cellular and antibody immunity to vaccinia only in systemic lymphoid tissue and not in mucosal sites, we hypothesized that the mucosal immune system remains naive to vaccinia and therefore amenable to immunization with recombinant vaccinia vectors despite earlier vaccinia exposure. We show that mucosal immunization of vaccinia-immune BALB/c mice with recombinant vaccinia expressing HIV gp160 induced specific serum antibody and strong HIV-specific cytotoxic T lymphocyte responses. These responses occurred not only in mucosal but also in systemic lymphoid tissue, whereas systemic immunization was ineffective under these circumstances. In this context, intrarectal immunization was more effective than intranasal immunization. Boosting with a second dose of recombinant vaccinia was also more effective via the mucosal route. The systemic HIV-specific cytotoxic T lymphocyte response was enhanced by coadministration of IL-12 at the mucosal site. These results also demonstrate the independent compartmentalization of the mucosal versus systemic immune systems and the asymmetric trafficking of lymphocytes between them. This approach to circumvent previous vaccinia immunity may be useful for induction of protective immunity against infectious diseases and cancer in the sizable populations with preexisting immunity to vaccinia from smallpox vaccination.

  16. Genomic Analysis, Phenotype, and Virulence of the Historical Brazilian Smallpox Vaccine Strain IOC: Implications for the Origins and Evolutionary Relationships of Vaccinia Virus

    PubMed Central

    Medaglia, Maria Luiza G.; Moussatché, Nissin; Nitsche, Andreas; Dabrowski, Pjotr Wojtek; Li, Yu; Damon, Inger K.; Lucas, Carolina G. O.; Arruda, Luciana B.

    2015-01-01

    ABSTRACT Smallpox was declared eradicated in 1980 after an intensive vaccination program using different strains of vaccinia virus (VACV; Poxviridae). VACV strain IOC (VACV-IOC) was the seed strain of the smallpox vaccine manufactured by the major vaccine producer in Brazil during the smallpox eradication program. However, little is known about the biological and immunological features as well as the phylogenetic relationships of this first-generation vaccine. In this work, we present a comprehensive characterization of two clones of VACV-IOC. Both clones had low virulence in infected mice and induced a protective immune response against a lethal infection comparable to the response of the licensed vaccine ACAM2000 and the parental strain VACV-IOC. Full-genome sequencing revealed the presence of several fragmented virulence genes that probably are nonfunctional, e.g., F1L, B13R, C10L, K3L, and C3L. Most notably, phylogenetic inference supported by the structural analysis of the genome ends provides evidence of a novel, independent cluster in VACV phylogeny formed by VACV-IOC, the Brazilian field strains Cantagalo (CTGV) and Serro 2 viruses, and horsepox virus, a VACV-like virus supposedly related to an ancestor of the VACV lineage. Our data strongly support the hypothesis that CTGV-like viruses represent feral VACV that evolved in parallel with VACV-IOC after splitting from a most recent common ancestor, probably an ancient smallpox vaccine strain related to horsepox virus. Our data, together with an interesting historical investigation, revisit the origins of VACV and propose new evolutionary relationships between ancient and extant VACV strains, mainly horsepox virus, VACV-IOC/CTGV-like viruses, and Dryvax strain. IMPORTANCE First-generation vaccines used to eradicate smallpox had rates of adverse effects that are not acceptable by current health care standards. Moreover, these vaccines are genetically heterogeneous and consist of a pool of quasispecies of VACV

  17. Adsorption of recombinant poxvirus L1-protein to aluminum hydroxide/CpG vaccine adjuvants enhances immune responses and protection of mice from vaccinia virus challenge

    PubMed Central

    Xiao, Yuhong; Zeng, Yuhong; Alexander, Edward; Mehta, Shyam; Joshi, Sangeeta B.; Buchman, George W.; Volkin, David B.; Middaugh, C. Russell; Isaacs, Stuart N.

    2012-01-01

    The stockpiling of live vaccinia virus vaccines has enhanced biopreparedness against the intentional or accidental release of smallpox. Ongoing research on future generation smallpox vaccines is providing key insights into protective immune responses as well as important information about subunit vaccine design strategies. For protein-based recombinant subunit vaccines, the formulation and stability of candidate antigens with different adjuvants are important factors to consider for vaccine design. In this work, a non-tagged secreted L1-protein, a target antigen on mature virus, was expressed using recombinant baculovirus technology and purified. To identify optimal formulation conditions for L1, a series of biophysical studies was performed over a range of pH and temperature conditions. The overall physical stability profile was summarized in an empirical phase diagram. Another critical question to address for development of an adjuvanted-vaccine was if immunogenicity and protection could be affected by the interactions and binding of L1 to aluminum salts (Alhydrogel) with and without a second adjuvant, CpG. We thus designed a series of vaccine formulations with different binding interactions between the L1 and the two adjuvants, and then performed a series of vaccination-challenge experiments in mice including measurement of antibody responses and post-challenge weight-loss and survival. We found that better humoral responses and protection were conferred with vaccine formulations when the L1-protein was adsorbed to Alhydrogel. These data demonstrate that designing vaccine formulation conditions to maximize antigen-adjuvant interactions is a key factor in smallpox subunit vaccine immunogenicity and protection. PMID:23153450

  18. Patterns of viral replication correlate with outcome in simian immunodeficiency virus (SIV)-infected macaques: effect of prior immunization with a trivalent SIV vaccine in modified vaccinia virus Ankara.

    PubMed Central

    Hirsch, V M; Fuerst, T R; Sutter, G; Carroll, M W; Yang, L C; Goldstein, S; Piatak, M; Elkins, W R; Alvord, W G; Montefiori, D C; Moss, B; Lifson, J D

    1996-01-01

    The dynamics of plasma viremia were explored in a group of 12 simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) that had received prior immunization with either nonrecombinant or trivalent (gag-pol, env) SIV-recombinant vaccinia viruses. Three distinct patterns of viral replication observed during and following primary viremia accounted for significant differences in survival times. High-level primary plasma viremia with subsequently increasing viremia was associated with rapid progression to AIDS (n = 2). A high-level primary plasma virus load with a transient decline and subsequent progressive increase in viremia in the post-acute phase of infection was associated with progression to AIDS within a year (n = 6). Low levels of primary plasma viremia followed by sustained restriction of virus replication were associated with maintenance of normal lymphocyte subsets and intact lymphoid architecture (n = 4), reminiscent of the profile observed in human immunodeficiency virus type 1-infected long-term nonprogressors. Three of four macaques that showed this pattern had been immunized with an SIV recombinant derived from the attenuated vaccinia virus, modified vaccinia virus Ankara. These data link the dynamics and extent of virus replication to disease course and suggest that sustained suppression of virus promotes long-term, asymptomatic survival of SIV-infected macaques. These findings also suggest that vaccine modulation of host immunity may have profound beneficial effects on the subsequent disease course, even if sterilizing immunity is not achieved. PMID:8648709

  19. Peptide vaccination of mice immune to LCMV or vaccinia virus causes serious CD8+ T cell-mediated, TNF-dependent immunopathology

    PubMed Central

    Liu, Fei; Feuer, Ralph; Hassett, Daniel E.; Whitton, J. Lindsay

    2006-01-01

    CD8+ T cells play a key role in clearing primary virus infections and in protecting against subsequent challenge. The potent antiviral effects of these cells make them important components of vaccine-induced immunity and, because of this, peptide vaccines often contain epitopes designed to induce strong CD8+ T cell responses. However, the same effector functions that protect the host also can be harmful if they are not tightly regulated, and virus-specific CD8+ T cells are a frequent cause of immunopathology. Here, we report that the administration of peptide to virus-immune recipient mice can lead to the synchronous activation of preexisting virus-specific CD8+ T cells with serious, and even lethal, consequences. Mice infected with LCMV or vaccinia virus developed rapid and profound hypothermia following injection of cognate synthetic peptides, and LCMV-infected mice frequently died within hours. Detailed analyses of the LCMV infected mice revealed enterocyte apoptosis and implicated TNF produced by peptide-specific CD8+ T cells as the major mediator of disease. The caspase inhibitor zVADfmk had no demonstrable effect on the development of hypothermia, but diminished enterocyte apoptosis and greatly reduced the number of deaths. These findings, if similarly observed in patients, counsel caution when administering powerful immunogens such as peptide vaccines to individuals who may have a large preexisting pool of epitope-specific CD8+ T cells. PMID:16424939

  20. Protective effects of a Modified Vaccinia Ankara-based vaccine candidate against Crimean-Congo Haemorrhagic Fever virus require both cellular and humoral responses

    PubMed Central

    Dowall, Stuart D.; Graham, Victoria A.; Rayner, Emma; Hunter, Laura; Watson, Robert; Taylor, Irene; Rule, Antony; Carroll, Miles W.; Hewson, Roger

    2016-01-01

    Crimean-Congo Haemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. There is no approved vaccine currently available against CCHF. The most promising candidate, which has previously been shown to confer protection in the small animal model, is a modified Vaccinia Ankara virus vector expressing the CCHF viral glycoprotein (MVA-GP). It has been shown that MVA-GP induces both humoral and cellular immunogenicity. In the present study, sera and T-lymphocytes were passively and adoptively transferred into recipient mice prior to challenge with CCHF virus. Results demonstrated that mediators from both arms of the immune system were required to demonstrate protective effects against lethal challenge. PMID:27272940

  1. Highly immunogenic variant of attenuated vaccinia virus.

    PubMed

    Yakubitskyi, S N; Kolosova, I V; Maksyutov, R A; Shchelkunov, S N

    2016-01-01

    The LIVPΔ6 strain of vaccinia virus (VACV) was created by genetic engineering on the basis of previously obtained attenuated 1421ABJCN strain by target deletion of the A35R gene encoding an inhibitor of antigen presentation by the major histocompatibility complex class II. 1421ABJCN is the LIVP strain of VACV with five inactivated virulence genes encoding hemagglutinin (A56R), γ-interferon-binding protein (B8R), thymidine kinase (J2R), complement-binding protein (C3L), and Bcl2-like inhibitor of apoptosis (N1L). The highly immunogenic LIVPΔ6 strain could be an efficient fourth-generation attenuated vaccine against smallpox and other orthopoxvirus infections. PMID:27025484

  2. Enhanced immunogenicity of CD4(+) t-cell responses and protective efficacy of a DNA-modified vaccinia virus Ankara prime-boost vaccination regimen for murine tuberculosis.

    PubMed

    McShane, H; Brookes, R; Gilbert, S C; Hill, A V

    2001-02-01

    DNA vaccines whose DNA encodes a variety of antigens from Mycobacterium tuberculosis have been evaluated for immunogenicity and protective efficacy. CD8(+) T-cell responses and protection achieved in other infectious disease models have been optimized by using a DNA immunization to prime the immune system and a recombinant virus encoding the same antigen(s) to boost the response. A DNA vaccine (D) and recombinant modified vaccinia virus Ankara (M) in which the DNA encodes early secreted antigenic target 6 and mycobacterial protein tuberculosis 63 synthesized, and each was found to generate specific gamma interferon (IFN-gamma)-secreting CD4(+) T cells. Enhanced CD4(+) IFN-gamma T-cell responses were produced by both D-M and M-D immunization regimens. Significantly higher levels of IFN-gamma were seen with a D-D-D-M immunization regimen. The most immunogenic regimens were assessed in a challenge study and found to produce protection equivalent to that produced by Mycobacterium bovis BCG. Thus, heterologous prime-boost regimens boost CD4(+) as well as CD8(+) T-cell responses, and the use of heterologous constructs encoding the same antigen(s) may improve the immunogenicity and protective efficacy of DNA vaccines against tuberculosis and other diseases. PMID:11159955

  3. Specificity and 6-Month Durability of Immune Responses Induced by DNA and Recombinant Modified Vaccinia Ankara Vaccines Expressing HIV-1 Virus-Like Particles

    PubMed Central

    Goepfert, Paul A.; Elizaga, Marnie L.; Seaton, Kelly; Tomaras, Georgia D.; Montefiori, David C.; Sato, Alicia; Hural, John; DeRosa, Stephen C.; Kalams, Spyros A.; McElrath, M. Juliana; Keefer, Michael C.; Baden, Lindsey R.; Lama, Javier R.; Sanchez, Jorge; Mulligan, Mark J.; Buchbinder, Susan P.; Hammer, Scott M.; Koblin, Beryl A.; Pensiero, Michael; Butler, Chris; Moss, Bernard; Robinson, Harriet L.; Donastorg, Yeycy; Qin, Li; Lawrence, Dale; Cardinali, Massimo; Bae, Jin; Holt, Renée; Redinger, Huguette; Johannessen, Jan; Broder, Gail; Moody-White, Jerri; McKay, Butch; Calazans, Gabriela; Bentley, Carter; Kakinami, Lisa; Skibinski, Katie; Estep, Scharla; Tseng, Jenny; Swenson, Molly; Madenwald, Tamra; Overton, Edgar Turner; Edupuganti, Srilatha; Rouphael, Nadine; Whitaker, Jennifer; Hay, C Mhorag; Bunce, Catherine A; Gonzales, Pedro; Hurtado, Juan Carlos; Dolin, Raphael; Mayer, Ken; Walsh, Steven; Johnson, Jennifer

    2014-01-01

    Background. Clade B DNA and recombinant modified vaccinia Ankara (MVA) vaccines producing virus-like particles displaying trimeric membrane-bound envelope glycoprotein (Env) were tested in a phase 2a trial in human immunodeficiency virus (HIV)–uninfected adults for safety, immunogenicity, and 6-month durability of immune responses. Methods. A total of 299 individuals received 2 doses of JS7 DNA vaccine and 2 doses of MVA/HIV62B at 0, 2, 4, and 6 months, respectively (the DDMM regimen); 3 doses of MVA/HIV62B at 0, 2, and 6 months (the MMM regimen); or placebo injections. Results. At peak response, 93.2% of the DDMM group and 98.4% of the MMM group had binding antibodies for Env. These binding antibodies were more frequent and of higher magnitude for the transmembrane subunit (gp41) than the receptor-binding subunit (gp120) of Env. For both regimens, response rates were higher for CD4+ T cells (66.4% in the DDMM group and 43.1% in the MMM group) than for CD8+ T cells (21.8% in the DDMM group and 14.9% in the MMM group). Responding CD4+ and CD8+ T cells were biased toward Gag, and >70% produced 2 or 3 of the 4 cytokines evaluated (ie, interferon γ, interleukin 2, tumor necrosis factor α, and granzyme B). Six months after vaccination, the magnitudes of antibodies and T-cell responses had decreased by <3-fold. Conclusions. DDMM and MMM vaccinations with virus-like particle–expressing immunogens elicited durable antibody and T-cell responses. PMID:24403557

  4. A Modified Vaccinia Ankara Virus (MVA) Vaccine Expressing African Horse Sickness Virus (AHSV) VP2 Protects Against AHSV Challenge in an IFNAR −/− Mouse Model

    PubMed Central

    Castillo-Olivares, Javier; Calvo-Pinilla, Eva; Casanova, Isabel; Bachanek-Bankowska, Katarzyna; Chiam, Rachael; Maan, Sushila; Nieto, Jose Maria; Ortego, Javier; Mertens, Peter Paul Clement

    2011-01-01

    African horse sickness (AHS) is a lethal viral disease of equids, which is transmitted by Culicoides midges that become infected after biting a viraemic host. The use of live attenuated vaccines has been vital for the control of this disease in endemic regions. However, there are safety concerns over their use in non-endemic countries. Research efforts over the last two decades have therefore focused on developing alternative vaccines based on recombinant baculovirus or live viral vectors expressing structural components of the AHS virion. However, ethical and financial considerations, relating to the use of infected horses in high biosecurity installations, have made progress very slow. We have therefore assessed the potential of an experimental mouse-model for AHSV infection for vaccine and immunology research. We initially characterised AHSV infection in this model, then tested the protective efficacy of a recombinant vaccine based on modified vaccinia Ankara expressing AHS-4 VP2 (MVA-VP2). PMID:21298069

  5. 42 CFR 102.21 - Smallpox (Vaccinia) Vaccine Injury Table.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... spinal cord (myelitis) such as paralysis or meningismus. Long term central nervous system impairments... 42 Public Health 1 2014-10-01 2014-10-01 false Smallpox (Vaccinia) Vaccine Injury Table. 102.21... SMALLPOX COMPENSATION PROGRAM Covered Injuries § 102.21 Smallpox (Vaccinia) Vaccine Injury Table....

  6. 42 CFR 102.21 - Smallpox (Vaccinia) Vaccine Injury Table.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... spinal cord (myelitis) such as paralysis or meningismus. Long term central nervous system impairments... 42 Public Health 1 2013-10-01 2013-10-01 false Smallpox (Vaccinia) Vaccine Injury Table. 102.21... SMALLPOX COMPENSATION PROGRAM Covered Injuries § 102.21 Smallpox (Vaccinia) Vaccine Injury Table....

  7. 42 CFR 102.21 - Smallpox (Vaccinia) Vaccine Injury Table.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... spinal cord (myelitis) such as paralysis or meningismus. Long term central nervous system impairments... 42 Public Health 1 2010-10-01 2010-10-01 false Smallpox (Vaccinia) Vaccine Injury Table. 102.21... SMALLPOX COMPENSATION PROGRAM Covered Injuries § 102.21 Smallpox (Vaccinia) Vaccine Injury Table....

  8. 42 CFR 102.21 - Smallpox (Vaccinia) Vaccine Injury Table.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... spinal cord (myelitis) such as paralysis or meningismus. Long term central nervous system impairments... 42 Public Health 1 2012-10-01 2012-10-01 false Smallpox (Vaccinia) Vaccine Injury Table. 102.21... SMALLPOX COMPENSATION PROGRAM Covered Injuries § 102.21 Smallpox (Vaccinia) Vaccine Injury Table....

  9. 42 CFR 102.21 - Smallpox (Vaccinia) Vaccine Injury Table.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... spinal cord (myelitis) such as paralysis or meningismus. Long term central nervous system impairments... 42 Public Health 1 2011-10-01 2011-10-01 false Smallpox (Vaccinia) Vaccine Injury Table. 102.21... SMALLPOX COMPENSATION PROGRAM Covered Injuries § 102.21 Smallpox (Vaccinia) Vaccine Injury Table....

  10. Easy and efficient protocols for working with recombinant vaccinia virus MVA.

    PubMed

    Kremer, Melanie; Volz, Asisa; Kreijtz, Joost H C M; Fux, Robert; Lehmann, Michael H; Sutter, Gerd

    2012-01-01

    Modified vaccinia virus Ankara (MVA) is a highly attenuated and replication-deficient strain of vaccinia virus that is increasingly used as vector for expression of recombinant genes in the research laboratory and in biomedicine for vaccine development. Major benefits of MVA include the clear safety advantage compared to conventional vaccinia viruses, the longstanding experience in the genetic engineering of the virus, and the availability of established procedures for virus production at an industrial scale. MVA vectors can be handled under biosafety level 1 conditions, and a multitude of recombinant MVA vaccines has proven to be immunogenic and protective when delivering various heterologous antigens in animals and humans. In this chapter we provide convenient state-of-the-art protocols for generation, amplification, and purification of recombinant MVA viruses. Importantly, we include methodology for rigid quality control to obtain best possible vector viruses for further investigations including clinical evaluation. PMID:22688761

  11. Vaccinia Virus Recombinants: Expression of VSV Genes and Protective Immunization of Mice and Cattle

    NASA Astrophysics Data System (ADS)

    Mackett, M.; Yilma, T.; Rose, J. K.; Moss, B.

    1985-01-01

    Vesicular stomatitis virus (VSV) causes a contagious disease of horses, cattle, and pigs. When DNA copies of messenger RNA's for the G or N proteins of VSV were linked to a vaccinia virus promoter and inserted into the vaccinia genome, the recombinants retained infectivity and synthesized VSV polypeptides. After intradermal vaccination with live recombinant virus expressing the G protein, mice produced VSV-neutralizing antibodies and were protected against lethal encephalitis upon intravenous challenge with VSV. In cattle, the degree of protection against intradermalingually injected VSV was correlated with the level of neutralizing antibody produced following vaccination.

  12. Vaccinia recombinant virus expressing the rabies virus glycoprotein: safety and efficacy trials in Canadian wildlife.

    PubMed Central

    Artois, M; Charlton, K M; Tolson, N D; Casey, G A; Knowles, M K; Campbell, J B

    1990-01-01

    Twenty-six meadow voles (Microtus pennsylvanicus), ten woodchucks (Marmota monax), thirteen grey squirrels (Sciurus carolinensis), thirteen ring-billed gulls (Larus delawarensis), six red-tailed hawks (Buteo jamaicensis) and eight great horned owls (Bubo virginianus) received vaccinia virus recombinant expressing the rabies virus glycoprotein (V-RG) by direct instillation into the oral cavity. Each of ten coyotes (Canis latrans) received the virus in two vaccine-laden baits. Several voles and most of the gulls died from diseases unrelated to vaccination during the observation period, but all other animals remained healthy and survived. These deaths from causes other than vaccination and the absence of any lesions suggestive of vaccinia infection indicate that it is unlikely that any animal suffered or died as a result of V-RG administration. In addition several animals showed an unexpected high level of rabies neutralizing antibodies. PMID:2249183

  13. Vaccinia recombinant virus expressing the rabies virus glycoprotein: safety and efficacy trials in Canadian wildlife.

    PubMed

    Artois, M; Charlton, K M; Tolson, N D; Casey, G A; Knowles, M K; Campbell, J B

    1990-10-01

    Twenty-six meadow voles (Microtus pennsylvanicus), ten woodchucks (Marmota monax), thirteen grey squirrels (Sciurus carolinensis), thirteen ring-billed gulls (Larus delawarensis), six red-tailed hawks (Buteo jamaicensis) and eight great horned owls (Bubo virginianus) received vaccinia virus recombinant expressing the rabies virus glycoprotein (V-RG) by direct instillation into the oral cavity. Each of ten coyotes (Canis latrans) received the virus in two vaccine-laden baits. Several voles and most of the gulls died from diseases unrelated to vaccination during the observation period, but all other animals remained healthy and survived. These deaths from causes other than vaccination and the absence of any lesions suggestive of vaccinia infection indicate that it is unlikely that any animal suffered or died as a result of V-RG administration. In addition several animals showed an unexpected high level of rabies neutralizing antibodies. PMID:2249183

  14. Vaccine Efficacy against Malaria by the Combination of Porcine Parvovirus-Like Particles and Vaccinia Virus Vectors Expressing CS of Plasmodium

    PubMed Central

    Rodríguez, Dolores; González-Aseguinolaza, Gloria; Rodríguez, Juan R.; Vijayan, Aneesh; Gherardi, Magdalena; Rueda, Paloma; Casal, J. Ignacio; Esteban, Mariano

    2012-01-01

    With the aim to develop an efficient and cost-effective approach to control malaria, we have generated porcine parvovirus-like particles (PPV-VLPs) carrying the CD8+ T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein from Plasmodium yoelii fused to the PPV VP2 capsid protein (PPV-PYCS), and tested in prime/boost protocols with poxvirus vectors for efficacy in a rodent malaria model. As a proof-of concept, we have characterized the anti-CS CD8+ T cell response elicited by these hybrid PPV-VLPs in BALB/c mice after immunizations with the protein PPV-PYCS administered alone or in combination with recombinant vaccinia virus (VACV) vectors from the Western Reserve (WR) and modified virus Ankara (MVA) strains expressing the entire P. yoelii CS protein. The results of different immunization protocols showed that the combination of PPV-PYCS prime/poxvirus boost was highly immunogenic, inducing specific CD8+ T cell responses to CS resulting in 95% reduction in liver stage parasites two days following sporozoite challenge. In contrast, neither the administration of PPV-PYCS alone nor the immunization with the vectors given in the order poxvirus/VLPs was as effective. The immune profile induced by VLPs/MVA boost was associated with polyfunctional and effector memory CD8+ T cell responses. These findings highlight the use of recombinant parvovirus PPV-PYCS particles as priming agents and poxvirus vectors, like MVA, as booster to enhance specific CD8+ T cell responses to Plasmodium antigens and to control infection. These observations are relevant in the design of T cell-inducing vaccines against malaria. PMID:22529915

  15. Nucleotide Phosphohydrolase in Purified Vaccinia Virus

    PubMed Central

    Munyon, William; Paoletti, Enzo; Ospina, Julio; Grace, James T.

    1968-01-01

    Purified infectious vaccinia virus has been shown to contain an enzyme or enzymes that remove the terminal phosphate group from adenosine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP). The Km for ATP of this enzyme is 5.5 × 10−4m, and the relative rates of the reaction with ATP, GTP, UTP, and CTP are 1.00, 0.34, 0.15, and 0.29, respectively. The virus enzyme does not react with any of the diphosphates. The rate of the reaction is proportional to the amount of virus added and is linear for 130 min. The virus nucleotide phosphohydrolase activity is greatly stimulated by Mg++ and very slightly stimulated by Ca++. The small residual activity observed in the absence of divalent cations is completely inhibited by ethylenediaminetetraacetic acid. Neither Na+ nor K+ ions, nor any mixture of these, was found to stimulate the reaction significantly, and ouabain, at 10−4m, inhibited the reaction by only 27%. The response of the vaccinia enzyme to mono- and divalent cations and to ouabain indicates that the vaccinia enzyme has different properties from those associated with microsomes and mitochondria. PMID:4986904

  16. Evolution of and Evolutionary Relationships between Extant Vaccinia Virus Strains

    PubMed Central

    Qin, Li; Favis, Nicole; Famulski, Jakub

    2014-01-01

    ABSTRACT Although vaccinia virus (VACV) was once used as a vaccine to eradicate smallpox on a worldwide scale, the biological origins of VACV are uncertain, as are the historical relationships between the different strains once used as smallpox vaccines. Here, we sequenced additional VACV strains that either represent relatively pristine examples of old vaccines (e.g., Dryvax, Lister, and Tashkent) or have been subjected to additional laboratory passage (e.g., IHD-W and WR). These genome sequences were compared with those previously reported for other VACVs as well as other orthopoxviruses. These extant VACVs do not always cluster in simple phylogenetic trees that are aligned with the known historical relationships between these strains. Rather, the pattern of deletions suggests that all existing strains likely come from a complex stock of viruses that has been passaged, distributed, and randomly sampled over time, thus obscuring simple historical or geographic links. We examined surviving nonclonal vaccine stocks, like Dryvax, which continue to harbor larger and now rare variants, including one that we have designated “clone DPP25.” DPP25 encodes genes not found in most VACV strains, including an ankyrin-F-box protein, a homolog of the variola virus (Bangladesh) B18R gene which we show can be deleted without affecting virulence in mice. We propose a simple common mechanism by which recombination of a larger and hypothetical DPP25-like ancestral strain, combined with selection for retention of critically important genes near the terminal inverted repeat boundaries (vaccinia virus growth factor gene and an interferon alpha/beta receptor homolog), could produce all known VACV variants. IMPORTANCE Smallpox was eradicated by using a combination of intensive disease surveillance and vaccination using vaccinia virus (VACV). Interestingly, little is known about the historical relationships between different strains of VACV and how these viruses may have evolved from a

  17. A vaccinia virus double recombinant expressing the F and H genes of rinderpest virus protects cattle against rinderpest and causes no pock lesions.

    PubMed Central

    Giavedoni, L; Jones, L; Mebus, C; Yilma, T

    1991-01-01

    Rinderpest is a highly contagious viral disease of ruminants with greater than 95% morbidity and mortality. We have constructed an infectious vaccinia virus recombinant that expresses both the fusion (F) gene and the hemagglutinin (H) gene of rinderpest virus. The Wyeth strain of vaccinia virus was used for the construction of the recombinant. Cattle vaccinated with the recombinant virus were 100% protected from challenge inoculation with greater than 1000 times the lethal dose of rinderpest virus. No transmission of recombinant vaccinia virus from vaccinated animals to contact animals was observed. The lyophilized form of vaccinia virus is thermostable and allows circumvention of the logistical problems associated with the distribution and administration of vaccines in the arid and hot regions of Asia and Africa. The insertional inactivation of both the thymidine kinase and the hemagglutinin genes of vaccinia virus led to increased attenuation of the virus; this was manifested by the lack of detectable pock lesions in vaccinated animals. This approach may have wide application in the development of safe and efficacious recombinant vaccines for humans and animals. This becomes quite relevant with the concern of the use of vaccinia virus in a population with high incidence of the human immunodeficiency virus. Images PMID:1896447

  18. Antiserum from mice vaccinated with modified vaccinia Ankara virus expressing African horse sickness virus (AHSV) VP2 provides protection when it is administered 48h before, or 48h after challenge.

    PubMed

    Calvo-Pinilla, Eva; de la Poza, Francisco; Gubbins, Simon; Mertens, Peter Paul Clement; Ortego, Javier; Castillo-Olivares, Javier

    2015-04-01

    Previous studies show that a recombinant modified vaccinia Ankara (MVA) virus expressing VP2 of AHSV serotype 4 (MVA-VP2) induced virus neutralising antibodies in horses and protected interferon alpha receptor gene knock-out mice (IFNAR -/-) against challenge. Follow up experiments indicated that passive transfer of antiserum, from MVA-VP2 immune donors to recipient mice 1h before challenge, conferred complete clinical protection and significantly reduced viraemia. These studies have been extended to determine the protective effect of MVA-VP2 vaccine-induced antiserum, when administered 48h before, or 48h after challenge. In addition, passive transfer of splenocytes was undertaken to assess if they confer any degree of immunity to immunologically naïve recipient mice. Thus, antisera and splenocytes were collected from groups of mice that had been vaccinated with MVA-VP2, or wild type MVA (MVA-wt), for passive immunisation of recipient mice. The latter were subsequently challenged with AHSV-4 (together with appropriate vaccinated or unvaccinated control animals) and protection was assessed by comparing clinical signs, lethality and viraemia between treated and control groups. All antiserum recipients showed high protection against disease (100% survival rates even in mice that were immunised 48h after challenge) and statistically significant reduction or viraemia in comparison with the control groups. The mouse group receiving splenocytes from MVA-VP2 vaccinates, showed only a 40% survival rate, with a small reduction in viraemia, compared to those mice that had received splenocytes from MVA-wt vaccinates. These results confirm the primarily humoral nature of protective immunity conferred by MVA-VP2 vaccination and show the potential of administering MVA-VP2 specific antiserum as an emergency treatment for AHSV. PMID:25643968

  19. Safety and Tolerability of Conserved Region Vaccines Vectored by Plasmid DNA, Simian Adenovirus and Modified Vaccinia Virus Ankara Administered to Human Immunodeficiency Virus Type 1-Uninfected Adults in a Randomized, Single-Blind Phase I Trial

    PubMed Central

    Hayton, Emma-Jo; Rose, Annie; Ibrahimsa, Umar; Del Sorbo, Mariarosaria; Capone, Stefania; Crook, Alison; Black, Antony P.; Dorrell, Lucy; Hanke, Tomáš

    2014-01-01

    Trial Design HIV-1 vaccine development has advanced slowly due to viral antigenic diversity, poor immunogenicity and recently, safety concerns associated with human adenovirus serotype-5 vectors. To tackle HIV-1 variation, we designed a unique T-cell immunogen HIVconsv from functionally conserved regions of the HIV-1 proteome, which were presented to the immune system using a heterologous prime-boost combination of plasmid DNA, a non-replicating simian (chimpanzee) adenovirus ChAdV-63 and a non-replicating poxvirus, modified vaccinia virus Ankara. A block-randomized, single-blind, placebo-controlled phase I trial HIV-CORE 002 administered for the first time candidate HIV-1- vaccines or placebo to 32 healthy HIV-1/2-uninfected adults in Oxford, UK and elicited high frequencies of HIV-1-specific T cells capable of inhibiting HIV-1 replication in vitro. Here, detail safety and tolerability of these vaccines are reported. Methods Local and systemic reactogenicity data were collected using structured interviews and study-specific diary cards. Data on all other adverse events were collected using open questions. Serum neutralizing antibody titres to ChAdV-63 were determined before and after vaccination. Results Two volunteers withdrew for vaccine-unrelated reasons. No vaccine-related serious adverse events or reactions occurred during 190 person-months of follow-up. Local and systemic events after vaccination occurred in 27/32 individuals and most were mild (severity grade 1) and predominantly transient (<48 hours). Myalgia and flu-like symptoms were more strongly associated with MVA than ChAdV63 or DNA vectors and more common in vaccine recipients than in placebo. There were no intercurrent HIV-1 infections during follow-up. 2/24 volunteers had low ChAdV-63-neutralizing titres at baseline and 7 increased their titres to over 200 with a median (range) of 633 (231-1533) post-vaccination, which is of no safety concern. Conclusions These data demonstrate safety and good

  20. Stability of vaccinia-vectored recombinant oral rabies vaccine under field conditions: A 3-year study

    PubMed Central

    Hermann, Joseph R.; Fry, Alethea M.; Siev, David; Slate, Dennis; Lewis, Charles; Gatewood, Donna M.

    2011-01-01

    Rabies is an incurable zoonotic disease caused by rabies virus, a member of the rhabdovirus family. It is transmitted through the bite of an infected animal. Control methods, including oral rabies vaccination (ORV) programs, have led to a reduction in the spread and prevalence of the disease in wildlife. This study evaluated the stability of RABORAL, a recombinant vaccinia virus vaccine that is used in oral rabies vaccination programs. The vaccine was studied in various field microenvironments in order to describe its viability and facilitate effective baiting strategies. Field microenvironments influenced the stability of this vaccine in this study. This study emphasizes the importance of understanding how vaccines perform under varying field conditions in order to plan effective baiting strategies. PMID:22468025

  1. Complement inhibition prevents oncolytic vaccinia virus neutralization in immune humans and cynomolgus macaques.

    PubMed

    Evgin, Laura; Acuna, Sergio A; Tanese de Souza, Christiano; Marguerie, Monique; Lemay, Chantal G; Ilkow, Carolina S; Findlay, C Scott; Falls, Theresa; Parato, Kelley A; Hanwell, David; Goldstein, Alyssa; Lopez, Roberto; Lafrance, Sandra; Breitbach, Caroline J; Kirn, David; Atkins, Harold; Auer, Rebecca C; Thurman, Joshua M; Stahl, Gregory L; Lambris, John D; Bell, John C; McCart, J Andrea

    2015-06-01

    Oncolytic viruses (OVs) have shown promising clinical activity when administered by direct intratumoral injection. However, natural barriers in the blood, including antibodies and complement, are likely to limit the ability to repeatedly administer OVs by the intravenous route. We demonstrate here that for a prototype of the clinical vaccinia virus based product Pexa-Vec, the neutralizing activity of antibodies elicited by smallpox vaccination, as well as the anamnestic response in hyperimmune virus treated cancer patients, is strictly dependent on the activation of complement. In immunized rats, complement depletion stabilized vaccinia virus in the blood and led to improved delivery to tumors. Complement depletion also enhanced tumor infection when virus was directly injected into tumors in immunized animals. The feasibility and safety of using a complement inhibitor, CP40, in combination with vaccinia virus was tested in cynomolgus macaques. CP40 pretreatment elicited an average 10-fold increase in infectious titer in the blood early after the infusion and prolonged the time during which infectious virus was detectable in the blood of animals with preexisting immunity. Capitalizing on the complement dependence of antivaccinia antibody with adjunct complement inhibitors may increase the infectious dose of oncolytic vaccinia virus delivered to tumors in virus in immune hosts. PMID:25807289

  2. Low-Resolution Structure of Vaccinia Virus DNA Replication Machinery

    PubMed Central

    Sèle, Céleste; Gabel, Frank; Gutsche, Irina; Ivanov, Ivan; Burmeister, Wim P.

    2013-01-01

    Smallpox caused by the poxvirus variola virus is a highly lethal disease that marked human history and was eradicated in 1979 thanks to a worldwide mass vaccination campaign. This virus remains a significant threat for public health due to its potential use as a bioterrorism agent and requires further development of antiviral drugs. The viral genome replication machinery appears to be an ideal target, although very little is known about its structure. Vaccinia virus is the prototypic virus of the Orthopoxvirus genus and shares more than 97% amino acid sequence identity with variola virus. Here we studied four essential viral proteins of the replication machinery: the DNA polymerase E9, the processivity factor A20, the uracil-DNA glycosylase D4, and the helicase-primase D5. We present the recombinant expression and biochemical and biophysical characterizations of these proteins and the complexes they form. We show that the A20D4 polymerase cofactor binds to E9 with high affinity, leading to the formation of the A20D4E9 holoenzyme. Small-angle X-ray scattering yielded envelopes for E9, A20D4, and A20D4E9. They showed the elongated shape of the A20D4 cofactor, leading to a 150-Å separation between the polymerase active site of E9 and the DNA-binding site of D4. Electron microscopy showed a 6-fold rotational symmetry of the helicase-primase D5, as observed for other SF3 helicases. These results favor a rolling-circle mechanism of vaccinia virus genome replication similar to the one suggested for tailed bacteriophages. PMID:23175373

  3. Cardiac Safety of Modified Vaccinia Ankara for Vaccination against Smallpox in a Young, Healthy Study Population

    PubMed Central

    Zitzmann-Roth, Eva-Maria; von Sonnenburg, Frank; de la Motte, Stephan; Arndtz-Wiedemann, Nathaly; von Krempelhuber, Alfred; Uebler, Nadine; Vollmar, Jens; Virgin, Garth; Chaplin, Paul

    2015-01-01

    Background Conventional smallpox vaccines based on replicating vaccinia virus (VV) strains (e.g. Lister Elstree, NYCBOH) are associated with a high incidence of myo-/pericarditis, a severe inflammatory cardiac complication. A new smallpox vaccine candidate based on a non-replicating Modified Vaccinia Ankara (MVA) poxvirus has been assessed for cardiac safety in a large placebo-controlled clinical trial. Methods Cardiac safety of one and two doses of MVA compared to placebo was assessed in 745 healthy subjects. Vaccinia-naïve subjects received either one dose of MVA and one dose of placebo, two doses of MVA, or two doses of placebo by subcutaneous injection four weeks apart; vaccinia-experienced subjects received a single dose of MVA. Solicited and unsolicited adverse events (AE) and cardiac safety parameters (recorded as Adverse Events of Special Interest, AESI) were monitored after each injection. Results A total of 5 possibly related AESI (3 cases of palpitations, 2 of tachycardia) were reported during the study. No case of myo- or pericarditis occurred. One possibly related serious AE (SAE) was reported during the 6-month follow-up period (sarcoidosis). The most frequently observed AEs were injection site reactions. Conclusions Vaccination with MVA was safe and well tolerated and did not increase the risk for development of myo-/pericarditis. Trial Registration ClinicalTrials.gov NCT00316524 PMID:25879867

  4. Vaccinia Virus Recombinant Expressing Herpes Simplex Virus Type 1 Glycoprotein D Prevents Latent Herpes in Mice

    NASA Astrophysics Data System (ADS)

    Cremer, Kenneth J.; Mackett, Michael; Wohlenberg, Charles; Notkins, Abner Louis; Moss, Bernard

    1985-05-01

    In humans, herpes simplex virus causes a primary infection and then often a latent ganglionic infection that persists for life. Because these latent infections can recur periodically, vaccines are needed that can protect against both primary and latent herpes simplex infections. Infectious vaccinia virus recombinants that contain the herpes simplex virus type 1 (HSV-1) glycoprotein D gene under control of defined early or late vaccinia virus promoters were constructed. Tissue culture cells infected with these recombinant viruses synthesized a glycosylated protein that had the same mass (60,000 daltons) as the glycoprotein D produced by HSV-1. Immunization of mice with one of these recombinant viruses by intradermal, subcutaneous, or intraperitoneal routes resulted in the production of antibodies that neutralized HSV-1 and protected the mice against subsequent lethal challenge with HSV-1 or HSV-2. Immunization with the recombinant virus also protected the majority of the mice against the development of a latent HSV-1 infection of the trigeminal ganglia. This is the first demonstration that a genetically engineered vaccine can prevent the development of latency.

  5. Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge.

    PubMed

    Taylor, J; Pincus, S; Tartaglia, J; Richardson, C; Alkhatib, G; Briedis, D; Appel, M; Norton, E; Paoletti, E

    1991-08-01

    cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge. PMID:1830113

  6. Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge.

    PubMed Central

    Taylor, J; Pincus, S; Tartaglia, J; Richardson, C; Alkhatib, G; Briedis, D; Appel, M; Norton, E; Paoletti, E

    1991-01-01

    cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge. Images PMID:1830113

  7. Protective Efficacy of Recombinant Modified Vaccinia Virus Ankara Delivering Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein

    PubMed Central

    Volz, Asisa; Kupke, Alexandra; Song, Fei; Jany, Sylvia; Fux, Robert; Shams-Eldin, Hosam; Schmidt, Jörg; Becker, Christin; Eickmann, Markus; Becker, Stephan

    2015-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans. We tested a recombinant modified vaccinia virus Ankara (MVA) vaccine expressing full-length MERS-CoV spike (S) glycoprotein by immunizing BALB/c mice with either intramuscular or subcutaneous regimens. In all cases, MVA-MERS-S induced MERS-CoV-specific CD8+ T cells and virus-neutralizing antibodies. Vaccinated mice were protected against MERS-CoV challenge infection after transduction with the human dipeptidyl peptidase 4 receptor. This MERS-CoV infection model demonstrates the safety and efficacy of the candidate vaccine. PMID:26018172

  8. Engineering the vaccinia virus L1 protein for increased neutralizing antibody response after DNA immunization

    PubMed Central

    Shinoda, Kaori; Wyatt, Linda S; Irvine, Kari R; Moss, Bernard

    2009-01-01

    Background The licensed smallpox vaccine, comprised of infectious vaccinia virus, has associated adverse effects, particularly for immunocompromised individuals. Therefore, safer DNA and protein vaccines are being investigated. The L1 protein, a component of the mature virion membrane that is conserved in all sequenced poxviruses, is required for vaccinia virus entry into host cells and is a target for neutralizing antibody. When expressed by vaccinia virus, the unglycosylated, myristoylated L1 protein attaches to the viral membrane via a C-terminal transmembrane anchor without traversing the secretory pathway. The purpose of the present study was to investigate modifications of the gene expressing the L1 protein that would increase immunogenicity in mice when delivered by a gene gun. Results The L1 gene was codon modified for optimal expression in mammalian cells and potential N-glycosylation sites removed. Addition of a signal sequence to the N-terminus of L1 increased cell surface expression as shown by confocal microscopy and flow cytometry of transfected cells. Removal of the transmembrane domain led to secretion of L1 into the medium. Induction of binding and neutralizing antibodies in mice was enhanced by gene gun delivery of L1 containing the signal sequence with or without the transmembrane domain. Each L1 construct partially protected mice against weight loss caused by intranasal administration of vaccinia virus. Conclusion Modifications of the vaccinia virus L1 gene including codon optimization and addition of a signal sequence with or without deletion of the transmembrane domain can enhance the neutralizing antibody response of a DNA vaccine. PMID:19257896

  9. A DNA Prime-Modified Vaccinia Virus Ankara Boost Vaccine Encoding Thrombospondin-Related Adhesion Protein but Not Circumsporozoite Protein Partially Protects Healthy Malaria-Naive Adults against Plasmodium falciparum Sporozoite Challenge

    PubMed Central

    Dunachie, S. J.; Walther, M.; Epstein, J. E.; Keating, S.; Berthoud, T.; Andrews, L.; Andersen, R. F.; Bejon, P.; Goonetilleke, N.; Poulton, I.; Webster, D. P.; Butcher, G.; Watkins, K.; Sinden, R. E.; Levine, G. L.; Richie, T. L.; Schneider, J.; Kaslow, D.; Gilbert, S. C.; Carucci, D. J.; Hill, A. V. S.

    2006-01-01

    The safety, immunogenicity, and efficacy of DNA and modified vaccinia virus Ankara (MVA) prime-boost regimes were assessed by using either thrombospondin-related adhesion protein (TRAP) with a multiple-epitope string ME (ME-TRAP) or the circumsporozoite protein (CS) of Plasmodium falciparum. Sixteen healthy subjects who never had malaria (malaria-naive subjects) received two priming vaccinations with DNA, followed by one boosting immunization with MVA, with either ME-TRAP or CS as the antigen. Immunogenicity was assessed by ex vivo gamma interferon (IFN-γ) enzyme-linked immunospot assay (ELISPOT) and antibody assay. Two weeks after the final vaccination, the subjects underwent P. falciparum sporozoite challenge, with six unvaccinated controls. The vaccines were well tolerated and immunogenic, with the DDM-ME TRAP regimen producing stronger ex vivo IFN-γ ELISPOT responses than DDM-CS. One of eight subjects receiving the DDM-ME TRAP regimen was completely protected against malaria challenge, with this group as a whole showing significant delay to parasitemia compared to controls (P = 0.045). The peak ex vivo IFN-γ ELISPOT response in this group correlated strongly with the number of days to parasitemia (P = 0.033). No protection was observed in the DDM-CS group. Prime-boost vaccination with DNA and MVA encoding ME-TRAP but not CS resulted in partial protection against P. falciparum sporozoite challenge in the present study. PMID:16988273

  10. Nonreplicating Vaccinia Virus Vectors Expressing the H5 Influenza Virus Hemagglutinin Produced in Modified Vero Cells Induce Robust Protection▿

    PubMed Central

    Mayrhofer, Josef; Coulibaly, Sogue; Hessel, Annett; Holzer, Georg W.; Schwendinger, Michael; Brühl, Peter; Gerencer, Marijan; Crowe, Brian A.; Shuo, Shen; Hong, Wanjing; Tan, Yee Joo; Dietrich, Barbara; Sabarth, Nicolas; Savidis-Dacho, Helga; Kistner, Otfried; Barrett, P. Noel; Falkner, Falko G.

    2009-01-01

    The timely development of safe and effective vaccines against avian influenza virus of the H5N1 subtype will be of the utmost importance in the event of a pandemic. Our aim was first to develop a safe live vaccine which induces both humoral and cell-mediated immune responses against human H5N1 influenza viruses and second, since the supply of embryonated eggs for traditional influenza vaccine production may be endangered in a pandemic, an egg-independent production procedure based on a permanent cell line. In the present article, the generation of a complementing Vero cell line suitable for the production of safe poxviral vaccines is described. This cell line was used to produce a replication-deficient vaccinia virus vector H5N1 live vaccine, dVV-HA5, expressing the hemagglutinin of a virulent clade 1 H5N1 strain. This experimental vaccine was compared with a formalin-inactivated whole-virus vaccine based on the same clade and with different replicating poxvirus-vectored vaccines. Mice were immunized to assess protective immunity after high-dose challenge with the highly virulent A/Vietnam/1203/2004(H5N1) strain. A single dose of the defective live vaccine induced complete protection from lethal homologous virus challenge and also full cross-protection against clade 0 and 2 challenge viruses. Neutralizing antibody levels were comparable to those induced by the inactivated vaccine. Unlike the whole-virus vaccine, the dVV-HA5 vaccine induced substantial amounts of gamma interferon-secreting CD8 T cells. Thus, the nonreplicating recombinant vaccinia virus vectors are promising vaccine candidates that induce a broad immune response and can be produced in an egg-independent and adjuvant-independent manner in a proven vector system. PMID:19279103

  11. Vaccinia Virus Infection Requires Maturation of Macropinosomes.

    PubMed

    Rizopoulos, Zaira; Balistreri, Giuseppe; Kilcher, Samuel; Martin, Caroline K; Syedbasha, Mohammedyaseen; Helenius, Ari; Mercer, Jason

    2015-08-01

    The prototypic poxvirus, vaccinia virus (VACV), occurs in two infectious forms, mature virions (MVs) and extracellular virions (EVs). Both enter HeLa cells by inducing macropinocytic uptake. Using confocal microscopy, live-cell imaging, targeted RNAi screening and perturbants of endosome maturation, we analyzed the properties and maturation pathway of the macropinocytic vacuoles containing VACV MVs in HeLa cells. The vacuoles first acquired markers of early endosomes [Rab5, early endosome antigen 1 and phosphatidylinositol(3)P]. Prior to release of virus cores into the cytoplasm, they contained markers of late endosomes and lysosomes (Rab7a, lysosome-associated membrane protein 1 and sorting nexin 3). RNAi screening of endocytic cell factors emphasized the importance of late compartments for VACV infection. Follow-up perturbation analysis showed that infection required Rab7a and PIKfyve, confirming that VACV is a late-penetrating virus dependent on macropinosome maturation. VACV EV infection was inhibited by depletion of many of the same factors, indicating that both infectious particle forms share the need for late vacuolar conditions for penetration. PMID:25869659

  12. Cryo-electron tomography of vaccinia virus

    PubMed Central

    Cyrklaff, Marek; Risco, Cristina; Fernández, Jose Jesús; Jiménez, Maria Victoria; Estéban, Mariano; Baumeister, Wolfgang; Carrascosa, José L.

    2005-01-01

    The combination of cryo-microscopy and electron tomographic reconstruction has allowed us to determine the structure of one of the more complex viruses, intracellular mature vaccinia virus, at a resolution of 4–6 nm. The tomographic reconstruction allows us to dissect the different structural components of the viral particle, avoiding projection artifacts derived from previous microscopic observations. A surface-rendering representation revealed brick-shaped viral particles with slightly rounded edges and dimensions of ≈360 × 270 × 250 nm. The outer layer was consistent with a lipid membrane (5–6 nm thick), below which usually two lateral bodies were found, built up by a heterogeneous material without apparent ordering or repetitive features. The internal core presented an inner cavity with electron dense coils of presumptive DNA–protein complexes, together with areas of very low density. The core was surrounded by two layers comprising an overall thickness of ≈18–19 nm; the inner layer was consistent with a lipid membrane. The outer layer was discontinuous, formed by a periodic palisade built by the side interaction of T-shaped protein spikes that were anchored in the lower membrane and were arranged into small hexagonal crystallites. It was possible to detect a few pore-like structures that communicated the inner side of the core with the region outside the layer built by the T-shaped spike palisade. PMID:15699328

  13. Modified Vaccinia Virus Ankara Encoding Influenza Virus Hemagglutinin Induces Heterosubtypic Immunity in Macaques

    PubMed Central

    Florek, Nicholas W.; Weinfurter, Jason T.; Jegaskanda, Sinthujan; Brewoo, Joseph N.; Powell, Tim D.; Young, Ginger R.; Das, Subash C.; Hatta, Masato; Broman, Karl W.; Hungnes, Olav; Dudman, Susanne G.; Kawaoka, Yoshihiro; Kent, Stephen J.; Stinchcomb, Dan T.

    2014-01-01

    ABSTRACT Current influenza virus vaccines primarily aim to induce neutralizing antibodies (NAbs). Modified vaccinia virus Ankara (MVA) is a safe and well-characterized vector for inducing both antibody and cellular immunity. We evaluated the immunogenicity and protective efficacy of MVA encoding influenza virus hemagglutinin (HA) and/or nucleoprotein (NP) in cynomolgus macaques. Animals were given 2 doses of MVA-based vaccines 4 weeks apart and were challenged with a 2009 pandemic H1N1 isolate (H1N1pdm) 8 weeks after the last vaccination. MVA-based vaccines encoding HA induced potent serum antibody responses against homologous H1 or H5 HAs but did not stimulate strong T cell responses prior to challenge. However, animals that received MVA encoding influenza virus HA and/or NP had high frequencies of virus-specific CD4+ and CD8+ T cell responses within the first 7 days of H1N1pdm infection, while animals vaccinated with MVA encoding irrelevant antigens did not. We detected little or no H1N1pdm replication in animals that received vaccines encoding H1 (homologous) HA, while a vaccine encoding NP from an H5N1 isolate afforded no protection. Surprisingly, H1N1pdm viral shedding was reduced in animals vaccinated with MVA encoding HA and NP from an H5N1 isolate. This reduced shedding was associated with cross-reactive antibodies capable of mediating antibody-dependent cellular cytotoxicity (ADCC) effector functions. Our results suggest that ADCC plays a role in cross-protective immunity against influenza. Vaccines optimized to stimulate cross-reactive antibodies with ADCC function may provide an important measure of protection against emerging influenza viruses when NAbs are ineffective. IMPORTANCE Current influenza vaccines are designed to elicit neutralizing antibodies (NAbs). Vaccine-induced NAbs typically are effective but highly specific for particular virus strains. Consequently, current vaccines are poorly suited for preventing the spread of newly emerging

  14. Successive site translocating inoculation potentiates DNA/recombinant vaccinia vaccination

    PubMed Central

    Ren, Yanqin; Wang, Na; Hu, Weiguo; Zhang, Xiaoyan; Xu, Jianqing; Wan, Yanmin

    2015-01-01

    DNA vaccines have advantages over traditional vaccine modalities; however the relatively low immunogenicity restrains its translation into clinical use. Further optimizations are needed to get the immunogenicity of DNA vaccine closer to the level required for human use. Here we show that intramuscularly inoculating into a different limb each time significantly improves the immunogenicities of both DNA and recombinant vaccinia vaccines during multiple vaccinations, compared to repeated vaccination on the same limb. We term this strategy successive site translocating inoculation (SSTI). SSTI could work in synergy with genetic adjuvant and DNA prime-recombinant vaccinia boost regimen. By comparing in vivo antigen expression, we found that SSTI avoided the specific inhibition of in vivo antigen expression, which was observed in the limbs being repeatedly inoculated. Employing in vivo T cell depletion and passive IgG transfer, we delineated that the inhibition was not mediated by CD8+ T cells but by specific antibodies. Finally, by using C3−/− mouse model and in vivo NK cells depletion, we identified that specific antibodies negatively regulated the in vivo antigen expression primarily in a complement depended way. PMID:26667202

  15. Development of a highly efficacious vaccinia-based dual vaccine against smallpox and anthrax, two important bioterror entities.

    PubMed

    Merkel, Tod J; Perera, Pin-Yu; Kelly, Vanessa K; Verma, Anita; Llewellyn, Zara N; Waldmann, Thomas A; Mosca, Joseph D; Perera, Liyanage P

    2010-10-19

    Bioterrorism poses a daunting challenge to global security and public health in the 21st century. Variola major virus, the etiological agent of smallpox, and Bacillus anthracis, the bacterial pathogen responsible for anthrax, remain at the apex of potential pathogens that could be used in a bioterror attack to inflict mass casualties. Although licensed vaccines are available for both smallpox and anthrax, because of inadequacies associated with each of these vaccines, serious concerns remain as to the deployability of these vaccines, especially in the aftermath of a bioterror attack involving these pathogens. We have developed a single vaccine (Wyeth/IL-15/PA) using the licensed Wyeth smallpox vaccine strain that is efficacious against both smallpox and anthrax due to the integration of immune-enhancing cytokine IL-15 and the protective antigen (PA) of B. anthracis into the Wyeth vaccinia virus. Integration of IL-15 renders Wyeth vaccinia avirulent in immunodeficient mice and enhances anti-vaccinia immune responses. Wyeth/IL-15/PA conferred sterile protection against a lethal challenge of B. anthracis Ames strain spores in rabbits. A single dose of Wyeth/IL-15/PA protected 33% of the vaccinated A/J mice against a lethal spore challenge 72 h later whereas a single dose of licensed anthrax vaccine protected only 10%. Our dual vaccine Wyeth/IL-15/PA remedies the inadequacies associated with the licensed vaccines, and the inherent ability of Wyeth vaccinia virus to be lyophilized without loss of potency makes it cold-chain independent, thus simplifying the logistics of storage, stockpiling, and field delivery in the event of a bioterror attack involving smallpox or anthrax. PMID:20921397

  16. A Novel High-Throughput Vaccinia Virus Neutralization Assay and Preexisting Immunity in Populations from Different Geographic Regions in China

    PubMed Central

    Liu, Qiang; Huang, Weijin; Nie, Jianhui; Zhu, Rong; Gao, Dongying; Song, Aijing; Meng, Shufang; Xu, Xuemei; Wang, Youchun

    2012-01-01

    Background Pre-existing immunity to Vaccinia Tian Tan virus (VTT) resulting from a large vaccination campaign against smallpox prior to the early 1980s in China, has been a major issue for application of VTT-vector based vaccines. It is essential to establish a sensitive and high-throughput neutralization assay to understand the epidemiology of Vaccinia-specific immunity in current populations in China. Methodology/Principal Findings A new anti-Vaccinia virus (VACV) neutralization assay that used the attenuated replication-competent VTT carrying the firefly luciferase gene of Photinus pyralis (rTV-Fluc) was established and standardized for critical parameters that included the choice of cell line, viral infection dose, and the infection time. The current study evaluated the maintenance of virus-specific immunity after smallpox vaccination by conducting a non-randomized, cross-sectional analysis of antiviral antibody-mediated immune responses in volunteers examined 30–55 years after vaccination. The rTV-Fluc neutralization assay was able to detect neutralizing antibodies (NAbs) against Vaccinia virus without the ability to differentiate strains of Vaccinia virus. We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT). Using this assay, we found a low prevalence of NAb to VTT (7.6%) in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low. No NAbs were detected in all 222 samples from individuals born after 1980. There was no significant difference observed for titer or prevalence by gender, age range and geographic origin. Conclusion A simplified, sensitive, standardized, reproducible, and high-throughput assay was developed for the quantitation of NAbs against different Vaccinia strains. The current study provides useful insights for the future development of VTT-based vaccination in Beijing and Anhui

  17. Hepatitis B virus large surface protein is not secreted but is immunogenic when selectively expressed by recombinant vaccinia virus.

    PubMed Central

    Cheng, K C; Smith, G L; Moss, B

    1986-01-01

    The envelope region of the hepatitis B virus (HBV) genome contains an open reading frame that begins upstream of the major surface protein gene. The two minor proteins that are initiated within this pre-s segment are immunogenic and may be involved in virus attachment to hepatocytes. We have constructed a recombinant vaccinia virus that contains the predicted coding segment for the large surface protein (LS) under control of a vaccinia virus that contains the predicted coding segment for the large surface protein (LS) under control of a vaccinia virus promoter. Cells infected with the recombinant virus synthesized HBV polypeptides of 39 and 42 kilodaltons, corresponding to the unglycosylated and glycosylated forms of LS, respectively. The presence of pre-s epitopes in the 39- and 42-kilodalton polypeptides was demonstrated by binding of antibody prepared against a synthetic peptide. Synthesis of the 42-kilodalton species was specifically inhibited by tunicamycin, suggesting that it is N-glycosylated. Despite apparent glycosylation, LS was not secreted into the medium of infected cells. Nevertheless, rabbits vaccinated with the purified recombinant virus made antibodies that recognized s and pre-s epitopes. Antibody to the NH2 terminus of LS appeared before or simultaneously with antibody that bound to the major surface protein. The additional immunogenicity provided by expression of LS may be advantageous for the development of an HBV vaccine. Images PMID:2430108

  18. Oncolytic and immunologic cancer therapy with GM-CSF-armed vaccinia virus of Tian Tan strain Guang9.

    PubMed

    Deng, Lili; Fan, Jun; Guo, Mingming; Huang, Biao

    2016-03-28

    Targeted oncolytic vaccinia viruses are being developed as a novel strategy in cancer therapy. Arming vaccinia viruses with immunostimulatory cytokines can enhance antitumor efficacy. Such engineered oncolytic viruses, like JX-594, a Wyeth strain vaccinia virus modified with human granulocyte-macrophage colony-stimulating factor (GM-CSF), have shown promising results and have proceeded rapidly in clinical trials. However, the oncolytic potential of the Chinese vaccine strain Tian Tan (VTT) has not been explored. In this study, we constructed a targeted oncolytic vaccinia virus of Tian Tan strain Guang9 (VG9) expressing murine GM-CSF (VG9-GMCSF) and evaluated the antitumor effect of this recombinant vaccinia virus in a murine melanoma model. In vitro, viral replication and cytotoxicity of VG9-GMCSF was as potent as VG9; in vivo, VG9-GMCSF significantly inhibited the growth of subcutaneously implanted melanoma tumors, prolonged the survival of tumor-bearing mice, and produced an antitumor cytotoxic response. Such antitumor effect may be due to the lytic nature of virus as well as the stimulation of immune activity by GM-CSF production. Our results indicate that VG9-GMCSF induces strong tumoricidal activity, providing a potential therapeutic strategy for combating cancer. PMID:26803055

  19. Use of Reporter Genes in the Generation of Vaccinia Virus-Derived Vectors

    PubMed Central

    Al Ali, Sally; Baldanta, Sara; Fernández-Escobar, Mercedes; Guerra, Susana

    2016-01-01

    Vaccinia virus (VACV) is one of the most extensively-studied viruses of the Poxviridae family. It is easy to genetically modify, so it has become a key tool for many applications. In this context, reporter genes facilitate the study of the role of foreign genes introduced into the genome of VACV. In this review, we describe the type of reporter genes that have been used to generate reporter-expressing VACV and the applications of the recombinant viruses obtained. Reporter-expressing VACV are currently employed in basic and immunology research, in the development of vaccines and cancer treatment. PMID:27213433

  20. Use of Reporter Genes in the Generation of Vaccinia Virus-Derived Vectors.

    PubMed

    Al Ali, Sally; Baldanta, Sara; Fernández-Escobar, Mercedes; Guerra, Susana

    2016-01-01

    Vaccinia virus (VACV) is one of the most extensively-studied viruses of the Poxviridae family. It is easy to genetically modify, so it has become a key tool for many applications. In this context, reporter genes facilitate the study of the role of foreign genes introduced into the genome of VACV. In this review, we describe the type of reporter genes that have been used to generate reporter-expressing VACV and the applications of the recombinant viruses obtained. Reporter-expressing VACV are currently employed in basic and immunology research, in the development of vaccines and cancer treatment. PMID:27213433

  1. Glycosylated and Nonglycosylated Complement Control Protein of the Lister Strain of Vaccinia Virus

    PubMed Central

    Kuhn, Jordan; Atukorale, Vajini; Campbell, Joseph; Weir, Jerry P.

    2014-01-01

    The vaccinia virus complement control protein (VCP) is a secreted viral protein that binds the C3b and C4b complement components and inhibits the classic and alternative complement pathways. Previously, we reported that an attenuated smallpox vaccine, LC16m8, which was derived from the Lister strain of vaccinia virus (VV-Lister), expressed a glycosylated form of VCP, whereas published sequence data at that time indicated that the VV-Lister VCP has no motif for N-linked glycosylation. We were interested in determining whether the glycosylation of VCP impairs its biological activity, possibly contributing to the attenuation of LC16m8, and the likely origin of the glycosylated VCP. Expression analysis indicated that VV-Lister contains substrains expressing glycosylated VCP and substrains expressing nonglycosylated VCP. Other strains of smallpox vaccine, as well as laboratory strains of vaccinia virus, all expressed nonglycosylated VCP. Individual Lister virus clones expressing either the glycosylated VCP or the nonglycosylated species were isolated, and partially purified VCP from the isolates were found to be functional equivalents in binding human C3b and C4b complement proteins and inhibiting hemolysis and in immunogenicity. Recombinant vaccinia viruses expressing FLAG-tagged glycosylated VCP (FLAG-VCPg) and nonglycosylated VCP (FLAG-VCP) were constructed based on the Western Reserve strain. Purified FLAG-VCP and FLAG-VCPg bind human C3b and C4b and blocked complement-mediated hemolysis. Our data suggest that glycosylation did not affect the biological activity of VCP and thus may not have contributed to the attenuation of LC16m8. In addition, the LC16m8 virus likely originated from a substrain of VV-Lister that expresses glycosylated VCP. PMID:25030055

  2. Vaccinia Virus Induces Programmed Necrosis in Ovarian Cancer Cells

    PubMed Central

    Whilding, Lynsey M; Archibald, Kyra M; Kulbe, Hagen; Balkwill, Frances R; Öberg, Daniel; McNeish, Iain A

    2013-01-01

    The mechanisms by which oncolytic vaccinia virus induces tumor cell death are poorly understood. We have evaluated cell death pathways following infection of ovarian cancer cells with both wild-type and thymidine kinase-deleted (dTK) Lister strain vaccinia. We show that death does not rely upon classical apoptosis despite the appearances of some limited apoptotic features, including phosphatidylserine externalization and appearance of sub-G1 DNA populations. Vaccinia infection induces marked lipidation of LC3 proteins, but there is no general activation of the autophagic process and cell death does not rely upon autophagy induction. We show that vaccinia induces necrotic morphology on transmission electron microscopy, accompanied by marked by reductions in intracellular adenosine triphosphate, altered mitochondrial metabolism, and release of high mobility group box 1 (HMGB1) protein. This necrotic cell death appears regulated, as infection induces formation of a receptor interacting protein (RIP1)/caspase-8 complex. In addition, pharmacological inhibition of both RIP1 and substrates downstream of RIP1, including MLKL, significantly attenuate cell death. Blockade of TNF-α, however, does not alter virus efficacy, suggesting that necrosis does not result from autocrine cytokine release. Overall, these results show that, in ovarian cancer cells, vaccinia virus causes necrotic cell death that is mediated through a programmed series of events. PMID:23985697

  3. Vaccinia Virus Induces Rapid Necrosis in Keratinocytes by a STAT3-Dependent Mechanism

    PubMed Central

    He, Yong; Fisher, Robert; Chowdhury, Soma; Sultana, Ishrat; Pereira, Claudia P.; Bray, Mike; Reed, Jennifer L.

    2014-01-01

    Rationale Humans with a dominant negative mutation in STAT3 are susceptible to severe skin infections, suggesting an essential role for STAT3 signaling in defense against cutaneous pathogens. Methods To focus on innate antiviral defenses in keratinocytes, we used a standard model of cutaneous infection of severe combined immunodeficient mice with the current smallpox vaccine, ACAM-2000. In parallel, early events post-infection with the smallpox vaccine ACAM-2000 were investigated in cultured keratinocytes of human and mouse origin. Results Mice treated topically with a STAT3 inhibitor (Stattic) developed larger vaccinia lesions with higher virus titers and died more rapidly than untreated controls. Cultured human and murine keratinocytes infected with ACAM-2000 underwent rapid necrosis, but when treated with Stattic or with inhibitors of RIP1 kinase or caspase-1, they survived longer, produced higher titers of virus, and showed reduced activation of type I interferon responses and inflammatory cytokines release. Treatment with inhibitors of RIP1 kinase and STAT3, but not caspase-1, also reduced the inflammatory response of keratinocytes to TLR ligands. Vaccinia growth properties in Vero cells, which are known to be defective in some antiviral responses, were unaffected by inhibition of RIP1K, caspase-1, or STAT3. Conclusions Our findings indicate that keratinocytes suppress the replication and spread of vaccinia virus by undergoing rapid programmed cell death, in a process requiring STAT3. These data offer a new framework for understanding susceptibility to skin infection in patients with STAT3 mutations. Interventions which promote prompt necroptosis/pyroptosis of infected keratinocytes may reduce risks associated with vaccination with live vaccinia virus. PMID:25419841

  4. Resistance to Human Respiratory Syncytial Virus (RSV) Infection Induced by Immunization of Cotton Rats with a Recombinant Vaccinia Virus Expressing the RSV G Glycoprotein

    NASA Astrophysics Data System (ADS)

    Elango, Narayanasamy; Prince, Gregory A.; Murphy, Brian R.; Venkatesan, Sundararajan; Chanock, Robert M.; Moss, Bernard

    1986-03-01

    A cDNA copy of the G glycoprotein gene of human respiratory syncytial virus (RSV) was placed under control of a vaccinia virus promoter and inserted into the thymidine kinase locus of the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and expressed a 93-kDa protein that migrated with the authentic RSV G glycoprotein upon polyacrylamide gel electrophoresis. Glycosylation of the expressed protein and transport to the cell surface were demonstrated in the absence of other RSV proteins. Cotton rats that were inoculated intradermally with the infectious recombinant virus produced serum antibody to the G glycoprotein that neutralized RSV in vitro. Furthermore, the vaccinated animals were resistant to lower respiratory tract infection upon intranasal inoculation with RSV and had reduced titers of RSV in the nose.

  5. Effect of suspending media on freeze-drying and preservation of vaccinia virus

    PubMed Central

    Suzuki, Masatoshi

    1970-01-01

    Unpurified and purified smallpox vaccines were prepared from calf dermal pulp, or chorioallantoic membrane (CAM) of hen eggs infected with vaccinia virus, and freeze-dried. The protective effect of various suspending media was investigated both in the course of the freeze-drying and in the period of subsequent storage of the dried product at different temperatures, including 100° C. Single media consisting of either sodium glutamate or peptone were effective in the preservation of both unpurified and purified vaccines prepared from calf dermal pulp or CAM. It was shown that there was an optimal concentration of sodium glutamate for the preservation of the vaccine preparations, especially of the purified vaccine. Combined media, consisting of soluble starch, polyvinylpyrrolidone or sodium carboxymethyl cellulose with sodium glutamate, were effective with the purified vaccine when the concentration of sodium glutamate exceeded the optimum necessary for preservation. PMID:5266585

  6. GMCSF-armed vaccinia virus induces an antitumor immune response.

    PubMed

    Parviainen, Suvi; Ahonen, Marko; Diaconu, Iulia; Kipar, Anja; Siurala, Mikko; Vähä-Koskela, Markus; Kanerva, Anna; Cerullo, Vincenzo; Hemminki, Akseli

    2015-03-01

    Oncolytic Western Reserve strain vaccinia virus selective for epidermal growth factor receptor pathway mutations and tumor-associated hypermetabolism was armed with human granulocyte-macrophage colony-stimulating factor (GMCSF) and a tdTomato fluorophore. As the assessment of immunological responses to human transgenes is challenging in the most commonly used animal models, we used immunocompetent Syrian golden hamsters, known to be sensitive to human GMCSF and semipermissive to vaccinia virus. Efficacy was initially tested in vitro on various human and hamster cell lines and oncolytic potency of transgene-carrying viruses was similar to unarmed virus. The hGMCSF-encoding virus was able to completely eradicate subcutaneous pancreatic tumors in hamsters, and to fully protect the animals from subsequent rechallenge with the same tumor. Induction of specific antitumor immunity was also shown by ex vivo co-culture experiments with hamster splenocytes. In addition, histological examination revealed increased infiltration of neutrophils and macrophages in GMCSF-virus-treated tumors. These findings help clarify the mechanism of action of GMCSF-armed vaccinia viruses undergoing clinical trials. PMID:25042001

  7. Construction and Characterization of an Infectious Vaccinia Virus Recombinant That Expresses the Influenza Hemagglutinin Gene and Induces Resistance to Influenza Virus Infection in Hamsters

    NASA Astrophysics Data System (ADS)

    Smith, Geoffrey L.; Murphy, Brian R.; Moss, Bernard

    1983-12-01

    A DNA copy of the influenza virus hemagglutinin gene, derived from influenza virus A/Jap/305/57 (H2N2) was inserted into the genome of vaccinia virus under the control of an early vaccinia virus promoter. Tissue culture cells infected with the purified recombinant virus synthesized influenza hemagglutinin, which was glycosylated and transported to the cell surface where it could be cleaved with trypsin into HA1 and HA2 subunits. Rabbits and hamsters inoculated intradermally with recombinant virus produced circulating antibodies that inhibited hemagglutination by influenza virus. Furthermore, vaccinated hamsters achieved levels of antibody similar to those obtained upon primary infection with influenza virus and were protected against respiratory infection with the A/Jap/305/57 influenza virus.

  8. Capturing the Natural Diversity of the Human Antibody Response against Vaccinia Virus

    PubMed Central

    Lantto, Johan; Haahr Hansen, Margit; Rasmussen, Søren Kofoed; Steinaa, Lucilla; Poulsen, Tine R.; Duggan, Jackie; Dennis, Mike; Naylor, Irene; Easterbrook, Linda; Bregenholt, Søren; Haurum, John; Jensen, Allan

    2011-01-01

    The eradication of smallpox (variola) and the subsequent cessation of routine vaccination have left modern society vulnerable to bioterrorism employing this devastating contagious disease. The existing, licensed vaccines based on live vaccinia virus (VACV) are contraindicated for a substantial number of people, and prophylactic vaccination of large populations is not reasonable when there is little risk of exposure. Consequently, there is an emerging need to develop efficient and safe therapeutics to be used shortly before or after exposure, either alone or in combination with vaccination. We have characterized the human antibody response to smallpox vaccine (VACV Lister) in immunized volunteers and isolated a large number of VACV-specific antibodies that recognize a variety of different VACV antigens. Using this broad antibody panel, we have generated a fully human, recombinant analogue to plasma-derived vaccinia immunoglobulin (VIG), which mirrors the diversity and specificity of the human antibody immune response and offers the advantage of unlimited supply and reproducible specificity and activity. The recombinant VIG was found to display a high specific binding activity toward VACV antigens, potent in vitro VACV neutralizing activity, and a highly protective efficacy against VACV challenge in the mouse tail lesion model when given either prophylactically or therapeutically. Altogether, the results suggest that this compound has the potential to be used as an effective postexposure prophylaxis or treatment of disease caused by orthopoxviruses. PMID:21147924

  9. Protein Composition of the Vaccinia Virus Mature Virion

    SciTech Connect

    Resch, Wolfgang; Hixson, Kim K.; Moore, Ronald J.; Lipton, Mary S.; Moss, Bernard

    2007-02-05

    The protein content of vaccinia virus mature virions, purified by rate zonal and isopycnic centrifugation and solubilized by SDS or a solution of urea and thiourea, was determined by the accurate mass and time tag technology which uses both tandem mass spectrometry and Fourier transform-ion cyclotron resonance mass spectrometry to detect tryptic peptides separated by high-resolution liquid chromatography. Eighty vaccinia virus-encoded proteins representing 37% of the 218 genes annotated in the complete genome sequence were detected in at least three analyses. Ten proteins accounted for approximately 80% of the mass, while the least abundant proteins made up 1% or less of the mass. Thirteen identified proteins were not previously reported as components of virions. On the other hand, 8 previously described virion proteins were not detected here, presumably due to technical reasons including small size and hydrophobicity. In addition to vaccinia virus-encoded proteins, 24 host proteins omitting isoforms were detected. The most abundant of these were cytoskeletal proteins, heat shock proteins, and proteins involved in translation.

  10. Phosphorylation of vaccinia virus core proteins during transcription in vitro.

    PubMed Central

    Moussatche, N; Keller, S J

    1991-01-01

    The phosphorylation of vaccinia virus core proteins has been studied in vitro during viral transcription. The incorporation of [gamma-32P]ATP into protein is linear for the first 2 min of the reaction, whereas incorporation of [3H]UTP into RNA lags for 1 to 2 min before linear synthesis. At least 12 different proteins are phosphorylated on autoradiograms of acrylamide gels, and the majority of label is associated with low-molecular-weight proteins. If the transcription reaction is reduced by dropping the pH to 7 from its optimal of 8.5, two proteins (70 and 80 kDa) are no longer phosphorylated. RNA isolated from the pH 7 transcription reaction hybridized primarily to the vaccinia virus HindIII DNA fragments D to F, whereas the transcripts synthesized at pH 8.5 hybridized to almost all of the HindIII-digested vaccinia virus DNA fragments. The differences between the pH 7.0 and 8.5 transcription reactions in phosphorylation and transcription could be eliminated by preincubating the viral cores with 2 mM ATP. In sum, the results suggest that the phosphorylation of the 70- and 80-kDa peptides may contribute to the regulation of early transcription. Images PMID:2016772

  11. A pilot study comparing the development of EIAV Env-specific antibodies induced by DNA/recombinant vaccinia-vectored vaccines and an attenuated Chinese EIAV vaccine.

    PubMed

    Meng, Qinglai; Lin, Yuezhi; Ma, Jian; Ma, Yan; Zhao, Liping; Li, Shenwei; Yang, Kai; Zhou, Jianhua; Shen, Rongxian; Zhang, Xiaoyan; Shao, Yiming

    2012-12-01

    Data from successful attenuated lentiviral vaccine studies indicate that fully mature Env-specific antibodies characterized by high titer, high avidity, and the predominant recognition of conformational epitopes are associated with protective efficacy. Although vaccination with a DNA prime/recombinant vaccinia-vectored vaccine boost strategy has been found to be effective in some trials with non-human primate/simian/human immunodeficiency virus (SHIV) models, it remains unclear whether this vaccination strategy could elicit mature equine infectious anemia virus (EIAV) Env-specific antibodies, thus protecting vaccinated horses against EIAV infection. Therefore, in this pilot study we vaccinated horses using a strategy based on DNA prime/recombinant Tiantan vaccinia (rTTV)-vectored vaccines encoding EIAV env and gag genes, and observed the development of Env-specific antibodies, neutralizing antibodies, and p26-specific antibodies. Vaccination with DNA induced low titer, low avidity, and the predominant recognition of linear epitopes by Env-specific antibodies, which was enhanced by boosting vaccinations with rTTV vaccines. However, the maturation levels of Env-specific antibodies induced by the DNA/rTTV vaccines were significantly lower than those induced by the attenuated vaccine EIAV(FDDV). Additionally, DNA/rTTV vaccines did not elicit broadly neutralizing antibodies. After challenge with a virulent EIAV strain, all of the vaccinees and control horses died from EIAV disease. These data indicate that the regimen of DNA prime/rTTV vaccine boost did not induce mature Env-specific antibodies, which might have contributed to immune protection failure. PMID:23171359

  12. Generation of Recombinant Modified Vaccinia Virus Ankara Encoding VP2, NS1, and VP7 Proteins of Bluetongue Virus.

    PubMed

    Marín-López, Alejandro; Ortego, Javier

    2016-01-01

    Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental vaccine vector for its lack of replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a high number of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination, and the capacity to deliver substantial amounts of heterologous antigens. Recombinant MVAs encoding proteins of bluetongue virus (BTV), an Orbivirus that infects domestic and wild ruminants transmitted by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of bluetongue virus as a model example for orbiviruses. The protocols included cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of recombinant MVAs, the titration of virus working stocks and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification. PMID:26458834

  13. Evaluation of modified vaccinia virus Ankara expressing VP2 protein of infectious bursal disease virus as an immunogen in chickens.

    PubMed

    Zanetti, Flavia Adriana; Del Médico Zajac, María Paula; Taboga, Oscar Alberto; Calamante, Gabriela

    2012-06-01

    A recombinant modified vaccinia Ankara (MVA) virus expressing mature viral protein 2 (VP2) of the infectious bursal disease virus (IBDV) was constructed to develop MVA-based vaccines for poultry. We demonstrated that this recombinant virus was able to induce a specific immune response by observing the production of anti-IBDV-seroneutralizing antibodies in specific pathogen-free chickens. Besides, as the epitopes of VP2 responsible to induce IBDV-neutralizing antibodies are discontinuous, our results suggest that VP2 protein expressed from MVA-VP2 maintained the correct conformational structure. To our knowledge, this is the first report on the usefulness of MVA-based vectors for developing recombinant vaccines for poultry. PMID:22705743

  14. Evaluation of modified vaccinia virus Ankara expressing VP2 protein of infectious bursal disease virus as an immunogen in chickens

    PubMed Central

    Zajac, María Paula Del Médico; Taboga, Oscar Alberto; Calamante, Gabriela

    2012-01-01

    A recombinant modified vaccinia Ankara (MVA) virus expressing mature viral protein 2 (VP2) of the infectious bursal disease virus (IBDV) was constructed to develop MVA-based vaccines for poultry. We demonstrated that this recombinant virus was able to induce a specific immune response by observing the production of anti-IBDV-seroneutralizing antibodies in specific pathogen-free chickens. Besides, as the epitopes of VP2 responsible to induce IBDV-neutralizing antibodies are discontinuous, our results suggest that VP2 protein expressed from MVA-VP2 maintained the correct conformational structure. To our knowledge, this is the first report on the usefulness of MVA-based vectors for developing recombinant vaccines for poultry. PMID:22705743

  15. [Hypoparathyroidismus following L-asparaginase and vaccinia virus infection. Effect of hypocalcemia on phagocytosis and the function of lymphocytes].

    PubMed

    Ricken, K H

    1975-11-21

    Rabbits, treated with injections of 4000 IU of L-Asparaginase, develop the clinical and chemical signs of hypoparathyroidism. A simultaneous vaccination with vaccinia virus (strain "Elstree") markedly increase the tetanic symptoms ("conditioned deficiency"). L-Asparaginase may influence the cellular immunity by hypocalcemia. Two mechanisms are discussed: 1. the suppression of the phagocytosis, recognizable by the absence of signs for vaccinal allergy by deficiency of macrophages in the intradermal test with inactivated small-pox vaccine. 2. the inhibition of the PHA-induced lymphocyte transformation caused by deficiency of calcium ions. PMID:1214695

  16. Initial characterization of Vaccinia Virus B4 suggests a role in virus spread

    SciTech Connect

    Burles, Kristin; Irwin, Chad R.; Burton, Robyn-Lee; Schriewer, Jill; Evans, David H.; Buller, R. Mark; Barry, Michele

    2014-05-15

    Currently, little is known about the ankyrin/F-box protein B4. Here, we report that B4R-null viruses exhibited reduced plaque size in tissue culture, and decreased ability to spread, as assessed by multiple-step growth analysis. Electron microscopy indicated that B4R-null viruses still formed mature and extracellular virions; however, there was a slight decrease of virions released into the media following deletion of B4R. Deletion of B4R did not affect the ability of the virus to rearrange actin; however, VACV811, a large vaccinia virus deletion mutant missing 55 open reading frames, had decreased ability to produce actin tails. Using ectromelia virus, a natural mouse pathogen, we demonstrated that virus devoid of EVM154, the B4R homolog, showed decreased spread to organs and was attenuated during infection. This initial characterization suggests that B4 may play a role in virus spread, and that other unidentified mediators of actin tail formation may exist in vaccinia virus. - Highlights: • B4R-null viruses show reduced plaque size, and decreased ability to spread. • B4R-null viruses formed mature and extracellular virions; and rearranged actin. • Virus devoid of EVM154, the B4R homolog, was attenuated during infection. • Initial characterization suggests that B4 may play a role in virus spread. • Unidentified mediators of actin tail formation may exist in vaccinia virus.

  17. Dissociation between Epitope Hierarchy and Immunoprevalence in CD8 Responses to Vaccinia Virus Western Reserve1

    PubMed Central

    Oseroff, Carla; Peters, Bjoern; Pasquetto, Valerie; Moutaftsi, Magdalini; Sidney, John; Panchanathan, Vijay; Tscharke, David C.; Maillere, Bernard; Grey, Howard; Sette, Alessandro

    2008-01-01

    Understanding immunity to vaccinia virus (VACV) is important for the development of safer vaccines for smallpox- and poxvirus-vectored recombinant vaccines. VACV is also emerging as an outstanding model for studying CD8+ T cell immunodominance because of the large number of CD8+ T cell epitopes known for this virus in both mice and humans. In this study, we characterize the CD8+ T cell response in vaccinated BALB/c mice by a genome-wide mapping approach. Responses to each of 54 newly identified H-2d-restricted T cell epitopes could be detected after i.p. and dermal vaccination routes. Analysis of these new epitopes in the context of those already known for VACV in mice and humans revealed two important findings. First, CD8+ T cell epitopes are not randomly distributed across the VACV proteome, with some proteins being poorly or nonimmunogenic, while others are immunoprevalent, being frequently recognized across diverse MHC haplotypes. Second, some proteins constituted the major targets of the immune response by a specific haplotype as they recruited the majority of the specific CD8+ T cells but these proteins did not correspond to the immunoprevalent Ags. Thus, we found a dissociation between immunoprevalence and immunodominance, implying that different sets of rules govern these two phenomena. Together, these findings have clear implications for the design of CD8+ T cell subunit vaccines and in particular raise the exciting prospect of being able to choose subunits without reference to MHC restriction. PMID:18490718

  18. Applications of pox virus vectors to vaccination: an update.

    PubMed

    Paoletti, E

    1996-10-15

    Recombinant pox viruses have been generated for vaccination against heterologous pathogens. Amongst these, the following are notable examples. (i) The engineering of the Copenhagen strain of vaccinia virus to express the rabies virus glycoprotein. When applied in baits, this recombinant has been shown to vaccinate the red fox in Europe and raccoons in the United States, stemming the spread of rabies virus infection in the wild. (ii) A fowlpox-based recombinant expressing the Newcastle disease virus fusion and hemagglutinin glycoproteins has been shown to protect commercial broiler chickens for their lifetime when the vaccine was administered at 1 day of age, even in the presence of maternal immunity against either the Newcastle disease virus or the pox vector. (iii) Recombinants of canarypox virus, which is restricted for replication to avian species, have provided protection against rabies virus challenge in cats and dogs, against canine distemper virus, feline leukemia virus, and equine influenza virus disease. In humans, canarypox virus-based recombinants expressing antigens from rabies virus, Japanese encephalitis virus, and HIV have been shown to be safe and immunogenic. (iv) A highly attenuated vaccinia derivative, NYVAC, has been engineered to express antigens from both animal and human pathogens. Safety and immunogenicity of NYVAC-based recombinants expressing the rabies virus glycoprotein, a polyprotein from Japanese encephalitis virus, and seven antigens from Plasmodium falciparum have been demonstrated to be safe and immunogenic in early human vaccine studies. PMID:8876138

  19. Expression of DAI by an oncolytic vaccinia virus boosts the immunogenicity of the virus and enhances antitumor immunity

    PubMed Central

    Hirvinen, Mari; Capasso, Cristian; Guse, Kilian; Garofalo, Mariangela; Vitale, Andrea; Ahonen, Marko; Kuryk, Lukasz; Vähä-Koskela, Markus; Hemminki, Akseli; Fortino, Vittorio; Greco, Dario; Cerullo, Vincenzo

    2016-01-01

    In oncolytic virotherapy, the ability of the virus to activate the immune system is a key attribute with regard to long-term antitumor effects. Vaccinia viruses bear one of the strongest oncolytic activities among all oncolytic viruses. However, its capacity for stimulation of antitumor immunity is not optimal, mainly due to its immunosuppressive nature. To overcome this problem, we developed an oncolytic VV that expresses intracellular pattern recognition receptor DNA-dependent activator of IFN-regulatory factors (DAI) to boost the innate immune system and to activate adaptive immune cells in the tumor. We showed that infection with DAI-expressing VV increases expression of several genes related to important immunological pathways. Treatment with DAI-armed VV resulted in significant reduction in the size of syngeneic melanoma tumors in mice. When the mice were rechallenged with the same tumor, DAI-VV-treated mice completely rejected growth of the new tumor, which indicates immunity established against the tumor. We also showed enhanced control of growth of human melanoma tumors and elevated levels of human T-cells in DAI-VV-treated mice humanized with human peripheral blood mononuclear cells. We conclude that expression of DAI by an oncolytic VV is a promising way to amplify the vaccine potency of an oncolytic vaccinia virus to trigger the innate—and eventually the long-lasting adaptive immunity against cancer. PMID:27626058

  20. Expression of DAI by an oncolytic vaccinia virus boosts the immunogenicity of the virus and enhances antitumor immunity.

    PubMed

    Hirvinen, Mari; Capasso, Cristian; Guse, Kilian; Garofalo, Mariangela; Vitale, Andrea; Ahonen, Marko; Kuryk, Lukasz; Vähä-Koskela, Markus; Hemminki, Akseli; Fortino, Vittorio; Greco, Dario; Cerullo, Vincenzo

    2016-01-01

    In oncolytic virotherapy, the ability of the virus to activate the immune system is a key attribute with regard to long-term antitumor effects. Vaccinia viruses bear one of the strongest oncolytic activities among all oncolytic viruses. However, its capacity for stimulation of antitumor immunity is not optimal, mainly due to its immunosuppressive nature. To overcome this problem, we developed an oncolytic VV that expresses intracellular pattern recognition receptor DNA-dependent activator of IFN-regulatory factors (DAI) to boost the innate immune system and to activate adaptive immune cells in the tumor. We showed that infection with DAI-expressing VV increases expression of several genes related to important immunological pathways. Treatment with DAI-armed VV resulted in significant reduction in the size of syngeneic melanoma tumors in mice. When the mice were rechallenged with the same tumor, DAI-VV-treated mice completely rejected growth of the new tumor, which indicates immunity established against the tumor. We also showed enhanced control of growth of human melanoma tumors and elevated levels of human T-cells in DAI-VV-treated mice humanized with human peripheral blood mononuclear cells. We conclude that expression of DAI by an oncolytic VV is a promising way to amplify the vaccine potency of an oncolytic vaccinia virus to trigger the innate-and eventually the long-lasting adaptive immunity against cancer. PMID:27626058

  1. Vaccinia virus strain differences in cell attachment and entry

    SciTech Connect

    Bengali, Zain; Townsley, Alan C.; Moss, Bernard

    2009-06-20

    Vaccinia virus (VACV) strain WR can enter cells by a low pH endosomal pathway or direct fusion with the plasma membrane at neutral pH. Here, we compared attachment and entry of five VACV strains in six cell lines and discovered two major patterns. Only WR exhibited pH 5-enhanced rate of entry following neutral pH adsorption to cells, which correlated with sensitivity to bafilomycin A1, an inhibitor of endosomal acidification. Entry of IHD-J, Copenhagen and Elstree strains were neither accelerated by pH 5 treatment nor prevented by bafilomycin A1. Entry of the Wyeth strain, although not augmented by pH 5, was inhibited by bafilomycin A1. WR and Wyeth were both relatively resistant to the negative effects of heparin on entry, whereas the other strains were extremely sensitive due to inhibition of cell binding. The relative sensitivities of individual vaccinia virus strains to heparin correlated inversely with their abilities to bind to and enter glycosaminoglycan-deficient sog9 cells but not other cell lines tested. These results suggested that that IHD-J, Copenhagen and Elstree have a more limited ability than WR and Wyeth to use the low pH endosomal pathway and are more dependent on binding to glycosaminoglycans for cell attachment.

  2. Vaccinia virus recombinants expressing chimeric proteins of human immunodeficiency virus and gamma interferon are attenuated for nude mice.

    PubMed Central

    Giavedoni, L D; Jones, L; Gardner, M B; Gibson, H L; Ng, C T; Barr, P J; Yilma, T

    1992-01-01

    We have developed a method for attenuating vaccinia virus recombinants by expressing a fusion protein of a lymphokine and an immunogen. Chimeric genes were constructed that coded for gamma interferon (IFN-gamma) and structural proteins of the human immunodeficiency virus type 1 (HIV-1). In this study, we describe the biological and immunological properties of vaccinia virus recombinants expressing chimeric genes of murine or human IFN-gamma with glycoprotein gp120, gag, and a fragment of gp41. All fusion proteins retained the antigenic characteristics of both IFN-gamma and HIV as shown by immunoblot analysis. However, the antiviral activity of IFN-gamma could be demonstrated only for the IFN-gamma-gag fusion protein. In contrast, the attenuating activity of IFN-gamma for nude mice was retained by all of the recombinants, albeit at various rates. Unlike the antiviral activity, the attenuating activity of IFN-gamma was not species specific. Implications for the development of attenuated live recombinant vaccines for AIDS are discussed. Images PMID:1565633

  3. Expression of Herpes Simplex Virus 1 Glycoprotein B by a Recombinant Vaccinia Virus and Protection of Mice against Lethal Herpes Simplex Virus 1 Infection

    NASA Astrophysics Data System (ADS)

    Cantin, Edouard M.; Eberle, Richard; Baldick, Joseph L.; Moss, Bernard; Willey, Dru E.; Notkins, Abner L.; Openshaw, Harry

    1987-08-01

    The herpes simplex virus 1 (HSV-1) strain F gene encoding glycoprotein gB was isolated and modified at the 5' end by in vitro oligonucleotide-directed mutagenesis. The modified gB gene was inserted into the vaccinia virus genome and expressed under the control of a vaccinia virus promoter. The mature gB glycoprotein produced by the vaccinia virus recombinant was glycosylated, was expressed at the cell surface, and was indistinguishable from authentic HSV-1 gB in terms of electrophoretic mobility. Mice immunized intradermally with the recombinant vaccinia virus produced gB-specific neutralizing antibodies and were resistant to a lethal HSV-1 challenge.

  4. Targeting the vaccinia virus L1 protein to the cell surface enhances production of neutralizing antibodies.

    PubMed

    Golden, Joseph W; Josleyn, Matthew D; Hooper, Jay W

    2008-06-25

    The current live-orthopoxvirus vaccine is associated with minor to serious adverse affects, and is contraindicated for use in a significant portion of the population. As an alternative vaccine, we have previously shown that a DNA subunit vaccine (4pox) based on four orthopoxvirus immunogens (L1R, B5R, A27L and A33R) can produce protective immunity against lethal orthopoxvirus challenges in mice and nonhuman primates. Because antibodies are critical for protection against secondary orthopoxvirus infections, we are now interested in strategies that will enhance the humoral immune response against vaccine targets. Here, we tested the immunogenicity of an L1R construct to which a tissue plasminogen activator signal sequence was placed in frame with the full-length L1R gene. The tPA-L1R construct produced a more robust neutralizing antibody response in vaccinated mice when the DNA vaccine was administered by gene-gun as a prime/single boost. When the tPA-L1R construct was substituted for the unmodified L1R gene in the 4pox vaccine, given as a prime and single boost, animals were better protected from lethal challenge with vaccinia virus (VACV). These findings indicate that adding a tPA-leader sequence can enhance the immunogenicity of the L1R gene when given as a DNA vaccine. Furthermore, our results demonstrate that a DNA-based vaccine is capable of establishing protection from lethal orthopoxvirus challenges when administered as a prime and single boost without requiring adjuvant. PMID:18485547

  5. Lister strain of vaccinia virus armed with endostatin-angiostatin fusion gene as a novel therapeutic agent for human pancreatic cancer.

    PubMed

    Tysome, J R; Briat, A; Alusi, G; Cao, F; Gao, D; Yu, J; Wang, P; Yang, S; Dong, Z; Wang, S; Deng, L; Francis, J; Timiryasova, T; Fodor, I; Lemoine, N R; Wang, Y

    2009-10-01

    Survival after pancreatic cancer remains poor despite incremental advances in surgical and adjuvant therapy, and new strategies for treatment are needed. Oncolytic virotherapy is an attractive approach for cancer treatment. In this study, we have evaluated the effectiveness of the Lister vaccine strain of vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapeutic approach for pancreatic cancer. The Lister vaccine strain of vaccinia virus was effective against all human pancreatic carcinoma cells tested in vitro, especially those insensitive to oncolytic adenovirus. The virus displayed inherently high selectivity for cancer cells, sparing normal cells both in vitro and in vivo, with effective infection of tumors after both intravenous (i.v.) and intratumoral (i.t.) administrations. The expression of the endostatin-angiostatin fusion protein was confirmed in a pancreatic cancer model both in vitro and in vivo, with evidence of inhibition of angiogenesis. This novel vaccinia virus showed significant antitumor potency in vivo against the Suit-2 model by i.t. administration. This study suggests that the novel Lister strain of vaccinia virus armed with the endostatin-angiostatin fusion gene is a potential therapeutic agent for pancreatic cancer. PMID:19587709

  6. The Lipid Raft-Associated Protein CD98 Is Required for Vaccinia Virus Endocytosis

    PubMed Central

    Schroeder, Nina; Chung, Che-Sheng; Chen, Chein-Hung; Liao, Chung-Lin

    2012-01-01

    Mature vaccinia virus (vaccinia MV) infects a broad range of animals in vivo and cell cultures in vitro; however, the cellular receptors that determine vaccinia MV tropism and entry pathways are poorly characterized. Here, we performed quantitative proteomic analyses of lipid raft-associated proteins upon vaccinia MV entry into HeLa cells. We found that a type II membrane glycoprotein, CD98, is enriched in lipid rafts upon vaccinia MV infection compared to mock-infected HeLa cells. The knockdown of CD98 expression in HeLa cells significantly reduced vaccinia MV entry. Furthermore, CD98 knockout (KO) mouse embryonic fibroblasts (MEFs) also exhibited reduced vaccinia MV infectivity without affecting MV attachment to cells, suggesting a role for CD98 in the postbinding step of virus entry. Further characterization with inhibitors and dominant negative proteins that block different endocytic pathways revealed that vaccinia MV entry into MEFs occurs through a clathrin-independent, caveolin-independent, dynamin-dependent, fluid-phase endocytic pathway, implying that CD98 plays a specific role in the vaccinia MV endocytic pathway. Infections of wild-type and CD98 KO MEF cells with different strains of vaccinia MV provided further evidence that CD98 plays a specific role in MV endocytosis but not in plasma membrane fusion. Finally, different CD98-C69 chimeric proteins were expressed in CD98 KO MEFs, but none were able to reconstitute MV infectivity, suggesting that the overall structure of the CD98 protein is required for vaccinia MV endocytosis. PMID:22345471

  7. The generation of CD8+ T-cell population specific for vaccinia virus epitope involved in the antiviral protection against ectromelia virus challenge.

    PubMed

    Gierynska, Malgorzata; Szulc-Dabrowska, Lidia; Dzieciatkowski, Tomasz; Golke, Anna; Schollenberger, Ada

    2015-12-01

    Eradication of smallpox has led to cessation of vaccination programs. This has rendered the human population increasingly susceptible not only to variola virus infection but also to infections with other representatives of Poxviridae family that cause zoonotic variola-like diseases. Thus, new approaches for designing improved vaccine against smallpox are required. Discovering that orthopoxviruses, e.g. variola virus, vaccinia virus, ectromelia virus, share common immunodominant antigen, may result in the development of such a vaccine. In our study, the generation of antigen-specific CD8(+) T cells in mice during the acute and memory phase of the immune response was induced using the vaccinia virus immunodominant TSYKFESV epitope and CpG oligodeoxynucleotides as adjuvants. The role of the generated TSYKFESV-specific CD8(+) T cells was evaluated in mice during ectromelia virus infection using systemic and mucosal model. Moreover, the involvement of dendritic cells subsets in the adaptive immune response stimulation was assessed. Our results indicate that the TSYKFESV epitope/TLR9 agonist approach, delivered systemically or mucosally, generated strong CD8(+) T-cell response when measured 10 days after immunization. Furthermore, the TSYKFESV-specific cell population remained functionally active 2 months post-immunization, and gave cross-protection in virally challenged mice, even though the numbers of detectable antigen-specific T cells decreased. PMID:26474845

  8. Transcriptional complexity of vaccinia virus in vivo and in vitro.

    PubMed Central

    Paoletti, E; Grady, L J

    1977-01-01

    The transcriptional complexity of vaccinia virus both in vivo and in vitro has been measured by using DNA:RNA hybridization with RNA in excess. In vivo, "early" or prereplicative RNA was found to saturate at 25% or one-half of the viral genome. "Late" or postreplicative RNA from infected HeLa cells saturated at 52% or essentially the entire genome. This well-regulated transcriptional pattern of the virus in vivo was not maintained in vitro. In a number of experiments a range of saturation values from 40 to 50% was obtained for in vitro synthesized RNA. The complexity of polyadenylated and non-polyadenylated RNA, as well as total purified 8 to 12S RNA released from the virus, was indistinguishable from purified high-molecular-weight virion-associated RNA with a sedimentation value of greater than 20S and equivalent to total in vitro synthesized RNA. No additional hybrid formation was observed in experiments in which total in vitro RNA and late in vivo RNA from infected HeLa cells were combined, suggesting that the virus does not transcribe in vitro DNA sequences that are not also transcribed during productive infection. Approximately 15% complementary RNA was detected when radiolabeled total in vitro RNA was allowed to reanneal with late in vivo RNA, while as much as 8% of the in vitro synthesized RNA was found to be complementary. PMID:894791

  9. Features of the Antitumor Effect of Vaccinia Virus Lister Strain

    PubMed Central

    Zonov, Evgeniy; Kochneva, Galina; Yunusova, Anastasiya; Grazhdantseva, Antonina; Richter, Vladimir; Ryabchikova, Elena

    2016-01-01

    Oncolytic abilities of vaccinia virus (VACV) served as a basis for the development of various recombinants for treating cancer; however, “natural” oncolytic properties of the virus are not examined in detail. Our study was conducted to know how the genetically unmodified L-IVP strain of VACV produces its antitumor effect. Human A431 carcinoma xenografts in nude mice and murine Ehrlich carcinoma in C57Bl mice were used as targets for VACV, which was injected intratumorally. A set of virological methods, immunohistochemistry, light and electron microscopy was used in the study. We found that in mice bearing A431 carcinoma, the L-IVP strain was observed in visceral organs within two weeks, but rapidly disappeared from the blood. The L-IVP strain caused decrease of sizes in both tumors, however, in different ways. Direct cell destruction by replicating virus plays a main role in regression of A431 carcinoma xenografts, while in Ehrlich carcinoma, which poorly supported VACV replication, the virus induced decrease of mitoses by pushing tumor cells into S-phase of cell cycle. Our study showed that genetically unmodified VACV possesses at least two mechanisms of antitumor effect: direct destruction of tumor cells and suppression of mitoses in tumor cells. PMID:26771631

  10. Features of the Antitumor Effect of Vaccinia Virus Lister Strain.

    PubMed

    Zonov, Evgeniy; Kochneva, Galina; Yunusova, Anastasiya; Grazhdantseva, Antonina; Richter, Vladimir; Ryabchikova, Elena

    2016-01-01

    Oncolytic abilities of vaccinia virus (VACV) served as a basis for the development of various recombinants for treating cancer; however, "natural" oncolytic properties of the virus are not examined in detail. Our study was conducted to know how the genetically unmodified L-IVP strain of VACV produces its antitumor effect. Human A431 carcinoma xenografts in nude mice and murine Ehrlich carcinoma in C57Bl mice were used as targets for VACV, which was injected intratumorally. A set of virological methods, immunohistochemistry, light and electron microscopy was used in the study. We found that in mice bearing A431 carcinoma, the L-IVP strain was observed in visceral organs within two weeks, but rapidly disappeared from the blood. The L-IVP strain caused decrease of sizes in both tumors, however, in different ways. Direct cell destruction by replicating virus plays a main role in regression of A431 carcinoma xenografts, while in Ehrlich carcinoma, which poorly supported VACV replication, the virus induced decrease of mitoses by pushing tumor cells into S-phase of cell cycle. Our study showed that genetically unmodified VACV possesses at least two mechanisms of antitumor effect: direct destruction of tumor cells and suppression of mitoses in tumor cells. PMID:26771631

  11. Analysis of canine herpesvirus gB, gC and gD expressed by a recombinant vaccinia virus.

    PubMed

    Xuan, X; Kojima, A; Murata, T; Mikami, T; Otsuka, H

    1997-01-01

    The genes encoding the canine herpesvirus (CHV) glycoprotein B (gB), gC and gD homologues have been reported already. However, products of these genes have not been identified yet. Previously, we have identified three CHV glycoproteins, gp 145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gB, gC or gD, the putative genes of gB, gC, and gD of CHV were inserted into the thymidine kinase gene of vaccinia virus LC16mO strain under the control of the early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. We demonstrated here that gp145/112, gp80 and gp47 were the translation products of the CHV gB, gC and gD genes, respectively. The antigenic authenticity of recombinant gB, gC and gD were confirmed by a panel of MAbs specific for each glycoprotein produced in CHV-infected cells. Immunization of mice with these recombinants produced high titers of neutralizing antibodies against CHV. These results suggest that recombinant vaccinia viruses expressing CHV gB, gC and gD may be useful to develop a vaccine to control CHV infection. PMID:9191864

  12. High level protein expression in mammalian cells using a safe viral vector: modified vaccinia virus Ankara.

    PubMed

    Hebben, Matthias; Brants, Jan; Birck, Catherine; Samama, Jean-Pierre; Wasylyk, Bohdan; Spehner, Danièle; Pradeau, Karine; Domi, Arban; Moss, Bernard; Schultz, Patrick; Drillien, Robert

    2007-12-01

    Vaccinia virus vectors are attractive tools to direct high level protein synthesis in mammalian cells. In one of the most efficient strategies developed so far, the gene to be expressed is positioned downstream of a bacteriophage T7 promoter within the vaccinia genome and transcribed by the T7 RNA polymerase, also encoded by the vaccinia virus genome. Tight regulation of transcription and efficient translation are ensured by control elements of the Escherichia coli lactose operon and the encephalomyocarditis virus leader sequence, respectively. We have integrated such a stringently controlled expression system, previously used successfully in a standard vaccinia virus backbone, into the modified vaccinia virus Ankara strain (MVA). In this manner, proteins of interest can be produced in mammalian cells under standard laboratory conditions because of the inherent safety of the MVA strain. Using this system for expression of beta-galactosidase, about 15 mg protein could be produced from 10(8) BHK21 cells over a 24-h period, a value 4-fold higher than the amount produced from an identical expression system based on a standard vaccinia virus strain. In another application, we employed the MVA vector to produce human tubulin tyrosine ligase and demonstrate that this protein becomes a major cellular protein upon induction conditions and displays its characteristic enzymatic activity. The MVA vector should prove useful for many other applications in which mammalian cells are required for protein production. PMID:17892951

  13. Construction of Poxviruses as Cloning Vectors: Insertion of the Thymidine Kinase Gene from Herpes Simplex Virus into the DNA of Infectious Vaccinia Virus

    NASA Astrophysics Data System (ADS)

    Panicali, Dennis; Paoletti, Enzo

    1982-08-01

    We have constructed recombinant vaccinia viruses containing the thymidine kinase gene from herpes simplex virus. The gene was inserted into the genome of a variant of vaccinia virus that had undergone spontaneous deletion as well as into the 120-megadalton genome of the large prototypic vaccinia variant. This was accomplished via in vivo recombination by contransfection of eukaryotic tissue culture cells with cloned BamHI-digested thymidine kinase gene from herpes simplex virus containing flanking vaccinia virus DNA sequences and infectious rescuing vaccinia virus. Pure populations of the recombinant viruses were obtained by replica filter techniques or by growth of the recombinant virus in biochemically selective medium. The herpes simplex virus thymidine kinase gene, as an insert in vaccinia virus, is transcribed in vivo and in vitro, and the fidelity of in vivo transcription into a functional gene product was detected by the phosphorylation of 5-[125I]iodo-2'-deoxycytidine.

  14. Oral vaccination of dogs with recombinant rabies virus vaccines.

    PubMed

    Rupprecht, Charles E; Hanlon, Cathleen A; Blanton, Jesse; Manangan, Jamie; Morrill, Patricia; Murphy, Staci; Niezgoda, Michael; Orciari, Lillian A; Schumacher, Carolin L; Dietzschold, Bernhard

    2005-07-01

    Oral rabies virus (RV) vaccines are used to immunize a diversity of mammalian carnivores, but no single biological is effective for all major species. Recently, advances in reverse genetics have allowed the design of recombinant RV for consideration as new vaccines. The objective of this experiment was to examine the safety, immunogenicity and efficacy of recombinant RV vaccines administered to captive dogs by the oral route, compared to a commercial vaccinia-rabies glycoprotein (V-RG) recombinant virus vaccine. Animals consisted of naive purpose-bred beagles of both sexes, and were 6 months of age or older. Dogs were randomly assigned to one of six groups, and received either diluent or vaccine (PBS; V-RG; RV SN10-333; RV SPBN-Cyto c; RV SPBNGA; RV SPBNGAGA), with at least six animals per group. On day 0, 1 ml of each vaccine (or PBS) was administered to the oral cavity of each dog, at an approximate concentration of 10(8) to 10(9) TCID50. After vaccination, dogs were observed daily and bled weekly, for 5 weeks, prior to RV challenge. No signs of illness related to vaccination were detected during the observation period. Excluding the controls, RV neutralizing antibodies were detected in the majority of animals within 1-2 weeks of primary vaccination. Thereafter, all dogs were inoculated in the masseter muscle with a street virus of canine origin. All control animals developed rabies, but no vaccinates succumbed, with the exception of a single dog in the V-RG group. Review of these preliminary data demonstrates the non-inferiority of recombinant RV products, as concerns both safety and efficacy, and supports the suggestion that these vaccines may hold promise for future development as oral immunogens for important carnivore species, such as dogs. PMID:15896409

  15. Vaccinia Virus Tropism for Primary Hematolymphoid Cells Is Determined by Restricted Expression of a Unique Virus Receptor

    PubMed Central

    Chahroudi, Ann; Chavan, Rahul; Koyzr, Natalia; Waller, Edmund K.; Silvestri, Guido; Feinberg, Mark B.

    2005-01-01

    The presumed broad tropism of poxviruses has stymied attempts to identify both the cellular receptor(s) and the viral determinant(s) for binding. Detailed studies of poxvirus binding to and infection of primary human cells have not been conducted. In particular, the determinants of target cell infection and the consequences of infection for cells involved in the generation of antiviral immune responses are incompletely understood. In this report, we show that vaccinia virus (VV) exhibits a more restricted tropism for primary hematolymphoid human cells than has been previously recognized. We demonstrate that vaccinia virus preferentially infects antigen-presenting cells (dendritic cells, monocytes/macrophages, and B cells) and activated T cells, but not resting T cells. The infection of activated T cells is permissive, with active viral replication and production of infectious progeny. Susceptibility to infection is determined by restricted expression of a cellular receptor that is induced de novo upon T-cell activation and can be removed from the cell surface by either trypsin or pronase treatment. The VV receptor expressed on activated T cells displays unique characteristics that distinguish it from the receptor used to infect cell lines in culture. The observed restricted tropism of VV may have significant consequences for the understanding of natural poxvirus infection and immunity and for poxvirus-based vaccine development. PMID:16051832

  16. Genomic Analysis of the Vaccinia Virus Strain Variants Found in Dryvax Vaccine▿†

    PubMed Central

    Qin, Li; Upton, Chris; Hazes, Bart; Evans, David H.

    2011-01-01

    Smallpox was eradicated using variant forms of vaccinia virus-based vaccines. One of these was Dryvax, a calf lymph vaccine derived from the New York City Board of Health strain. We used genome-sequencing technology to examine the genetic diversity of the population of viruses present in a sample of Dryvax. These studies show that the conserved cores of these viruses exhibit a lower level of sequence variation than do the telomeres. However, even though the ends of orthopoxviruses are more genetically plastic than the cores, there are still many telomeric genes that are conserved as intact open reading frames in the 11 genomes that we, and 4 genomes that others, have sequenced. Most of these genes likely modulate inflammation. Our sequencing also detected an evolving pattern of mutation, with some genes being highly fragmented by randomly assorting mutations (e.g., M1L), while other genes are intact in most viruses but have been disrupted in individual strains (e.g., I4L in strain DPP17). Over 85% of insertion and deletion mutations are associated with repeats, and a rare new isolate bearing a large deletion in the right telomere was identified. All of these strains cluster in dendrograms consistent with their origin but which also surprisingly incorporate horsepox virus. However, these viruses also exhibit a “patchy” pattern of polymorphic sites characteristic of recombinants. There is more genetic diversity detected within a vial of Dryvax than between variola virus major and minor strains, and our study highlights how propagation methods affect the genetics of orthopoxvirus populations. PMID:21976639

  17. Identification of Novel Antipoxviral Agents: Mitoxantrone Inhibits Vaccinia Virus Replication by Blocking Virion Assembly▿

    PubMed Central

    Deng, Liang; Dai, Peihong; Ciro, Anthony; Smee, Donald F.; Djaballah, Hakim; Shuman, Stewart

    2007-01-01

    The bioterror threat of a smallpox outbreak in an unvaccinated population has mobilized efforts to develop new antipoxviral agents. By screening a library of known drugs, we identified 13 compounds that inhibited vaccinia virus replication at noncytotoxic doses. The anticancer drug mitoxantrone is unique among the inhibitors identified in that it has no apparent impact on viral gene expression. Rather, it blocks processing of viral structural proteins and assembly of mature progeny virions. The isolation of mitoxantrone-resistant vaccinia strains underscores that a viral protein is the likely target of the drug. Whole-genome sequencing of mitoxantrone-resistant viruses pinpointed missense mutations in the N-terminal domain of vaccinia DNA ligase. Despite its favorable activity in cell culture, mitoxantrone administered intraperitoneally at the maximum tolerated dose failed to protect mice against a lethal intranasal infection with vaccinia virus. PMID:17928345

  18. Bioluminescent imaging of vaccinia virus infection in immunocompetent and immunodeficient rats as a model for human smallpox

    PubMed Central

    Liu, Qiang; Fan, Changfa; Zhou, Shuya; Guo, Yanan; Zuo, Qin; Ma, Jian; Liu, Susu; Wu, Xi; Peng, Zexu; Fan, Tao; Guo, Chaoshe; Shen, Yuelei; Huang, Weijin; Li, Baowen; He, Zhengming; Wang, Youchun

    2015-01-01

    Due to the increasing concern of using smallpox virus as biological weapons for terrorist attack, there is renewed interest in studying the pathogenesis of human smallpox and development of new therapies. Animal models are highly demanded for efficacy and safety examination of new vaccines and therapeutic drugs. Here, we demonstrated that both wild type and immunodeficient rats infected with an engineered vaccinia virus carrying Firefly luciferase reporter gene (rTV-Fluc) could recapitulate infectious and clinical features of human smallpox. Vaccinia viral infection in wild type Sprague-Dawley (SD) rats displayed a diffusible pattern in various organs, including liver, head and limbs. The intensity of bioluminescence generated from rTV-Fluc correlated well with viral loads in tissues. Moreover, neutralizing antibodies had a protective effect against virus reinfection. The recombination activating gene 2 (Rag2) knockout rats generated by transcription activator-like effector nucleases (TALENs) technology were further used to examine the infectivity of the rTV-Fluc in immunodeficient populations. Here we demonstrated that Rag2-/- rats were more susceptible to rTV-Fluc than SD rats with a slower virus clearance rate. Therefore, the rTV-Fluc/SD rats and rTV-Fluc/Rag2-/- rats are suitable visualization models, which recapitulate wild type or immunodeficient populations respectively, for testing human smallpox vaccine and antiviral drugs. PMID:26235050

  19. Bioluminescent imaging of vaccinia virus infection in immunocompetent and immunodeficient rats as a model for human smallpox.

    PubMed

    Liu, Qiang; Fan, Changfa; Zhou, Shuya; Guo, Yanan; Zuo, Qin; Ma, Jian; Liu, Susu; Wu, Xi; Peng, Zexu; Fan, Tao; Guo, Chaoshe; Shen, Yuelei; Huang, Weijin; Li, Baowen; He, Zhengming; Wang, Youchun

    2015-01-01

    Due to the increasing concern of using smallpox virus as biological weapons for terrorist attack, there is renewed interest in studying the pathogenesis of human smallpox and development of new therapies. Animal models are highly demanded for efficacy and safety examination of new vaccines and therapeutic drugs. Here, we demonstrated that both wild type and immunodeficient rats infected with an engineered vaccinia virus carrying Firefly luciferase reporter gene (rTV-Fluc) could recapitulate infectious and clinical features of human smallpox. Vaccinia viral infection in wild type Sprague-Dawley (SD) rats displayed a diffusible pattern in various organs, including liver, head and limbs. The intensity of bioluminescence generated from rTV-Fluc correlated well with viral loads in tissues. Moreover, neutralizing antibodies had a protective effect against virus reinfection. The recombination activating gene 2 (Rag2) knockout rats generated by transcription activator-like effector nucleases (TALENs) technology were further used to examine the infectivity of the rTV-Fluc in immunodeficient populations. Here we demonstrated that Rag2-/- rats were more susceptible to rTV-Fluc than SD rats with a slower virus clearance rate. Therefore, the rTV-Fluc/SD rats and rTV-Fluc/Rag2-/- rats are suitable visualization models, which recapitulate wild type or immunodeficient populations respectively, for testing human smallpox vaccine and antiviral drugs. PMID:26235050

  20. What to Do After You've Gotten the Smallpox Vaccine

    MedlinePlus

    ... to Do After You’ve Gotten the Smallpox Vaccine The smallpox vaccine contains a live virus called vaccinia. After vaccination, this live virus is present at the vaccine site and can be spread to other parts ...

  1. Immunogenicity and virulence of attenuated vaccinia virus Tian Tan encoding HIV-1 muti-epitope genes, p24 and cholera toxin B subunit in mice.

    PubMed

    Du, Shouwen; Wang, Yuhang; Liu, Cunxia; Wang, Maopeng; Zhu, Yilong; Tan, Peng; Ren, Dayong; Li, Xiao; Tian, Mingyao; Yin, Ronglan; Li, Chang; Jin, Ningyi

    2015-07-01

    No effective prophylactic or therapeutic vaccine against HIV-1 in humans is currently available. This study analyzes the immunogenicity and safety of a recombinant attenuated vaccinia virus. A chimeric gene of HIV-1 multi-epitope genes containing CpG ODN and cholera toxin B subunit (CTB) was inserted into Chinese vaccinia virus Tian Tan strain (VTT) mutant strain. The recombinant virus rddVTT(-CCMp24) was assessed for immunogenicity and safety in mice. Results showed that the protein CCMp24 was expressed stably in BHK-21 infected with rddVTT(-CCMp24). And the recombinant virus induced the production of HIV-1 p24 specific immunoglobulin G (IgG), IL-2 and IL-4. The recombinant vaccine induced γ-interferon secretion against HIV peptides, and elicited a certain levels of immunological memory. Results indicated that the recombinant virus had certain immunogenicity to HIV-1. Additionally, the virulence of the recombinant virus was been attenuated in vivo of mice compared with wild type VTT (wtVTT), and the introduction of CTB and HIV Mp24 did not alter the infectivity and virulence of defective vaccinia virus. PMID:25796990

  2. Locally Produced IL-10 Limits Cutaneous Vaccinia Virus Spread

    PubMed Central

    Cush, Stephanie S.; Reynoso, Glennys V.; Kamenyeva, Olena; Bennink, Jack R.; Yewdell, Jonathan W.; Hickman, Heather D.

    2016-01-01

    Skin infection with the poxvirus vaccinia (VV) elicits a powerful, inflammatory cellular response that clears virus infection in a coordinated, spatially organized manner. Given the high concentration of pro-inflammatory effectors at areas of viral infection, it is unclear how tissue pathology is limited while virus-infected cells are being eliminated. To better understand the spatial dynamics of the anti-inflammatory response to a cutaneous viral infection, we first screened cytokine mRNA expression levels after epicutaneous (ec.) VV infection and found a large increase the anti-inflammatory cytokine IL-10. Ex vivo analyses revealed that T cells in the skin were the primary IL-10-producing cells. To understand the distribution of IL-10-producing T cells in vivo, we performed multiphoton intravital microscopy (MPM) of VV-infected mice, assessing the location and dynamic behavior of IL-10 producing cells. Although virus-specific T cells were distributed throughout areas of the inflamed skin lacking overt virus-infection, IL-10+ cells closely associated with large keratinocytic foci of virus replication where they exhibited similar motility patterns to bulk antigen-specific CD8+ T cells. Paradoxically, neutralizing secreted IL-10 in vivo with an anti-IL-10 antibody increased viral lesion size and viral replication. Additional analyses demonstrated that IL-10 antibody administration decreased recruitment of CCR2+ inflammatory monocytes, which were important for reducing viral burden in the infected skin. Based upon these findings, we conclude that spatially concentrated IL-10 production limits cutaneous viral replication and dissemination, likely through modulation of the innate immune repertoire at the site of viral growth. PMID:26991092

  3. Live-Cell Imaging of Vaccinia Virus Recombination

    PubMed Central

    Paszkowski, Patrick; Noyce, Ryan S.; Evans, David H.

    2016-01-01

    Recombination between co-infecting poxviruses provides an important mechanism for generating the genetic diversity that underpins evolution. However, poxviruses replicate in membrane-bound cytoplasmic structures known as factories or virosomes. These are enclosed structures that could impede DNA mixing between co-infecting viruses, and mixing would seem to be essential for this process. We hypothesize that virosome fusion events would be a prerequisite for recombination between co-infecting poxviruses, and this requirement could delay or limit viral recombination. We have engineered vaccinia virus (VACV) to express overlapping portions of mCherry fluorescent protein fused to a cro DNA-binding element. In cells also expressing an EGFP-cro fusion protein, this permits live tracking of virus DNA and genetic recombination using confocal microscopy. Our studies show that different types of recombination events exhibit different timing patterns, depending upon the relative locations of the recombining elements. Recombination between partly duplicated sequences is detected soon after post-replicative genes are expressed, as long as the reporter gene sequences are located in cis within an infecting genome. The same kinetics are also observed when the recombining elements are divided between VACV and transfected DNA. In contrast, recombination is delayed when the recombining sequences are located on different co-infecting viruses, and mature recombinants aren’t detected until well after late gene expression is well established. The delay supports the hypothesis that factories impede inter-viral recombination, but even after factories merge there remain further constraints limiting virus DNA mixing and recombinant gene assembly. This delay could be related to the continued presence of ER-derived membranes within the fused virosomes, membranes that may once have wrapped individual factories. PMID:27525721

  4. Non-coding RNAs and heme oxygenase-1 in vaccinia virus infection

    SciTech Connect

    Meseda, Clement A.; Srinivasan, Kumar; Wise, Jasen; Catalano, Jennifer; Yamada, Kenneth M.; Dhawan, Subhash

    2014-11-07

    Highlights: • Heme oxygenase-1 (HO-1) induction inhibited vaccinia virus infection of macrophages. • Reduced infectivity inversely correlated with increased expression of non-coding RNAs. • The regulation of HO-1 and ncRNAs suggests a novel host defense response against vaccinia virus infection. - Abstract: Small nuclear RNAs (snRNAs) are <200 nucleotide non-coding uridylate-rich RNAs. Although the functions of many snRNAs remain undetermined, a population of snRNAs is produced during the early phase of infection of cells by vaccinia virus. In the present study, we demonstrate a direct correlation between expression of the cytoprotective enzyme heme oxygenase-1 (HO-1), suppression of selective snRNA expression, and inhibition of vaccinia virus infection of macrophages. Hemin induced HO-1 expression, completely reversed virus-induced host snRNA expression, and suppressed vaccinia virus infection. This involvement of specific virus-induced snRNAs and associated gene clusters suggests a novel HO-1-dependent host-defense pathway in poxvirus infection.

  5. Redundancy complicates the definition of essential genes for vaccinia virus.

    PubMed

    Dobson, Bianca M; Tscharke, David C

    2015-11-01

    Vaccinia virus (VACV) genes are characterized as either essential or non-essential for growth in culture. It seems intuitively obvious that if a gene can be deleted without imparting a growth defect in vitro it does not have a function related to basic replication or spread. However, this interpretation relies on the untested assumption that there is no redundancy across the genes that have roles in growth in cell culture. First, we provide a comprehensive summary of the literature that describes the essential genes of VACV. Next, we looked for interactions between large blocks of non-essential genes located at the ends of the genome by investigating sets of VACVs with large deletions at the genomic termini. Viruses with deletions at either end of the genome behaved as expected, exhibiting only mild or host-range defects. In contrast, combining deletions at both ends of the genome for the VACV Western Reserve (WR) strain caused a devastating growth defect on all cell lines tested. Unexpectedly, we found that the well-studied VACV growth factor homologue encoded by C11R has a role in growth in vitro that is exposed when 42 genes are absent from the left end of the VACV WR genome. These results demonstrate that some non-essential genes contribute to basic viral growth, but redundancy means these functions are not revealed by single-gene-deletion mutants. PMID:26290187

  6. Atomic Force Microscopy Investigation of Vaccinia Virus Structure▿

    PubMed Central

    Kuznetsov, Y.; Gershon, P. D.; McPherson, A.

    2008-01-01

    Vaccinia virus was treated in a controlled manner with various combinations of nonionic detergents, reducing agents, and proteolytic enzymes, and successive products of the reactions were visualized using atomic force microscopy (AFM). Following removal of the outer lipid/protein membrane, a layer 20 to 40 nm in thickness was encountered that was composed of fibrous elements which, under reducing conditions, rapidly decomposed into individual monomers on the substrate. Beneath this layer was the virus core and its prominent lateral bodies, which could be dissociated or degraded with proteases. The core, in addition to the lateral bodies, was composed of a thick, multilayered shell of proteins of diverse sizes and shapes. The shell, which was readily etched with proteases, was thoroughly permeated with pores, or channels. Prolonged exposure to proteases and reductants produced disgorgement of the viral DNA from the remainders of the cores and also left residual, flattened, protease-resistant sacs on the imaging substrate. The DNA was readily visualized by AFM, which revealed some regions to be “soldered” by proteins, others to be heavily complexed with protein, and yet other parts to apparently exist as bundled, naked DNA. Prolonged exposure to proteases deproteinized the DNA, leaving masses of extended, free DNA. Estimates of the interior core volume suggest moderate but not extreme compaction of the genome. PMID:18508898

  7. Multiple Viral Ligands Naturally Presented by Different Class I Molecules in Transporter Antigen Processing-Deficient Vaccinia Virus-Infected Cells

    PubMed Central

    Lorente, Elena; Infantes, Susana; Barnea, Eilon; Beer, Ilan; García, Ruth; Lasala, Fátima; Jiménez, Mercedes; Vilches, Carlos; Lemonnier, François A.; Admon, Arie

    2012-01-01

    The transporter associated with antigen processing (TAP) delivers the viral proteolytic products generated by the proteasome in the cytosol to the endoplasmic reticulum lumen that are subsequently recognized by cytotoxic T lymphocytes (CTLs). However, several viral epitopes have been identified in TAP-deficient models. Using mass spectrometry to analyze complex human leukocyte antigen (HLA)-bound peptide pools isolated from large numbers of TAP-deficient vaccinia virus-infected cells, we identified 11 ligands naturally presented by four different HLA-A, HLA-B, and HLA-C class I molecules. Two of these ligands were presented by two different HLA class I alleles, and, as a result, 13 different HLA-peptide complexes were formed simultaneously in the same vaccinia virus-infected cells. In addition to the high-affinity ligands, one low-affinity peptide restricted by each of the HLA-A, HLA-B, and HLA-C class I molecules was identified. Both high- and low-affinity ligands generated long-term memory CTL responses to vaccinia virus in an HLA-A2-transgenic mouse model. The processing and presentation of two vaccinia virus-encoded HLA-A2-restricted antigens took place via proteasomal and nonproteasomal pathways, which were blocked in infected cells with chemical inhibitors specific for different subsets of metalloproteinases. These data have implications for the study of the effectiveness of early empirical vaccination with cowpox virus against smallpox disease. PMID:22031944

  8. Nigericin is a potent inhibitor of the early stage of vaccinia virus replication.

    PubMed

    Myskiw, Chad; Piper, Jessica; Huzarewich, Rhiannon; Booth, Tim F; Cao, Jingxin; He, Runtao

    2010-12-01

    Poxviruses remain a significant public health concern due to their potential use as bioterrorist agents and the spread of animal borne poxviruses, such as monkeypox virus, to humans. Thus, the identification of small molecule inhibitors of poxvirus replication is warranted. Vaccinia virus is the prototypic member of the Orthopoxvirus genus, which also includes variola and monkeypox virus. In this study, we demonstrate that the carboxylic ionophore nigericin is a potent inhibitor of vaccinia virus replication in several human cell lines. In HeLa cells, we found that the 50% inhibitory concentration of nigericin against vaccinia virus was 7.9 nM, with a selectivity index of 1038. We present data demonstrating that nigericin targets vaccinia virus replication at a post-entry stage. While nigericin moderately inhibits both early vaccinia gene transcription and translation, viral DNA replication and intermediate and late gene expression are severely compromised in the presence of nigericin. Our results demonstrate that nigericin has the potential to be further developed into an effective antiviral to treat poxvirus infections. PMID:20951746

  9. Expanding the Repertoire of Modified Vaccinia Ankara-Based Vaccine Vectors via Genetic Complementation Strategies

    PubMed Central

    Garber, David A.; O'Mara, Leigh A.; Zhao, Jun; Gangadhara, Sailaja; An, InChul; Feinberg, Mark B.

    2009-01-01

    Background Modified Vaccinia virus Ankara (MVA) is a safe, highly attenuated orthopoxvirus that is being developed as a recombinant vaccine vector for immunization against a number of infectious diseases and cancers. However, the expression by MVA vectors of large numbers of poxvirus antigens, which display immunodominance over vectored antigens-of-interest for the priming of T cell responses, and the induction of vector-neutralizing antibodies, which curtail the efficacy of subsequent booster immunizations, remain as significant impediments to the overall utility of such vaccines. Thus, genetic approaches that enable the derivation of MVA vectors that are antigenically less complex may allow for rational improvement of MVA-based vaccines. Principal Findings We have developed a genetic complementation system that enables the deletion of essential viral genes from the MVA genome, thereby allowing us to generate MVA vaccine vectors that are antigenically less complex. Using this system, we deleted the essential uracil-DNA-glycosylase (udg) gene from MVA and propagated this otherwise replication-defective variant on a complementing cell line that constitutively expresses the poxvirus udg gene and that was derived from a newly identified continuous cell line that is permissive for growth of wild type MVA. The resulting virus, MVAΔudg, does not replicate its DNA genome or express late viral gene products during infection of non-complementing cells in culture. As proof-of-concept for immunological ‘focusing’, we demonstrate that immunization of mice with MVAΔudg elicits CD8+ T cell responses that are directed against a restricted repertoire of vector antigens, as compared to immunization with parental MVA. Immunization of rhesus macaques with MVAΔudg-gag, a udg− recombinant virus that expresses an HIV subtype-B consensus gag transgene, elicited significantly higher frequencies of Gag-specific CD8 and CD4 T cells following both primary (2–4-fold) and booster (2

  10. Initial characterization of vaccinia virus B4 suggests a role in virus spread.

    PubMed

    Burles, Kristin; Irwin, Chad R; Burton, Robyn-Lee; Schriewer, Jill; Evans, David H; Buller, R Mark; Barry, Michele

    2014-05-01

    Currently, little is known about the ankyrin/F-box protein B4. Here, we report that B4R-null viruses exhibited reduced plaque size in tissue culture, and decreased ability to spread, as assessed by multiple-step growth analysis. Electron microscopy indicated that B4R-null viruses still formed mature and extracellular virions; however, there was a slight decrease of virions released into the media following deletion of B4R. Deletion of B4R did not affect the ability of the virus to rearrange actin; however, VACV811, a large vaccinia virus deletion mutant missing 55 open reading frames, had decreased ability to produce actin tails. Using ectromelia virus, a natural mouse pathogen, we demonstrated that virus devoid of EVM154, the B4R homolog, showed decreased spread to organs and was attenuated during infection. This initial characterization suggests that B4 may play a role in virus spread, and that other unidentified mediators of actin tail formation may exist in vaccinia virus. PMID:24889230

  11. Animal movement and establishment of vaccinia virus Cantagalo strain in Amazon biome, Brazil.

    PubMed

    Quixabeira-Santos, Jociane Cristina; Medaglia, Maria Luiza G; Pescador, Caroline A; Damaso, Clarissa R

    2011-04-01

    To understand the emergence of vaccinia virus Cantagalo strain in the Amazon biome of Brazil, during 2008-2010 we conducted a molecular and epidemiologic survey of poxvirus outbreaks. Data indicate that animal movement was the major cause of virus dissemination within Rondonia State, leading to the establishment and spread of this pathogen. PMID:21470472

  12. Oncolytic Immunotherapy Using Recombinant Vaccinia Virus GLV-1h68 Efficiently Kills Sorafenib-Resistant Hepatocellular Carcinoma

    PubMed Central

    Ady, Justin W.; Heffner, Jacqueline; Mojica, Kelly; Johnsen, Clark; Belin, Laurence J.; Love, Damon; Pugalenthi, Amudhan; Klein, Elizabeth; Chen, Nanhai G.; Yu, Yong A.; Szalay, Aladar A.; Fong, Yuman

    2014-01-01

    Background Sorafenib is the standard systemic therapy for un-resectable or recurrent hepatocellular carcinoma (HCC) with minimal increase in survival. Therefore, there is a great need to develop novel therapies for advanced or recurrent HCC. One emerging field of cancer treatment involves oncolytic viruses that specifically infect, replicate within, and kill cancer cells. In this study we look at the ability of GLV-1h68, a recombinant vaccinia virus derived from the vaccine strain that was used to eradicate smallpox, to kill sorafenib-resistant HCC. Methods Four sorafenib-resistant HCC cell lines were generated by repeated passage in the presence of sorafenib. Median inhibitory concentration was determined for all cell lines. The infectivity, viral replication and cytotoxicity of GLV-1h68 were assayed for both parental and sorafenib-resistant HCC cells. Results Infectivity increased in a time and concentration dependent manner in all cell lines. All cell lines supported efficient replication of virus. No significant difference between the rates of cell death between the parental and sorafenib-resistant cell lines was observed. Conclusions Our results demonstrate that oncolytic vaccinia virus GLV-1h68 efficiently kills both parental and sorafenib-resistant HCC cell lines. This study indicates that patients who have failed treatment with sorafenib remain viable candidates for oncolytic therapy. PMID:24957667

  13. Myxoma and Vaccinia Viruses Bind Differentially to Human Leukocytes

    PubMed Central

    Chan, Winnie M.; Bartee, Eric C.; Moreb, Jan S.; Dower, Ken; Connor, John H.

    2013-01-01

    Myxoma virus (MYXV) and vaccinia virus (VACV), two distinct members of the family Poxviridae, are both currently being developed as oncolytic virotherapeutic agents. Recent studies have demonstrated that ex vivo treatment with MYXV can selectively recognize and kill contaminating cancerous cells from autologous bone marrow transplants without perturbing the engraftment of normal CD34+ hematopoietic stem and progenitor cells. However, the mechanism(s) by which MYXV specifically recognizes and eliminates the cancer cells in the autografts is not understood. While little is known about the cellular attachment factor(s) exploited by MYXV for entry into any target cells, VACV has been shown to utilize cell surface glycosaminoglycans such as heparan sulfate (HS), the extracellular matrix protein laminin, and/or integrin β1. We have constructed MYXV and VACV virions tagged with the Venus fluorescent protein and compared their characteristics of binding to various human cancer cell lines as well as to primary human leukocytes. We report that the binding of MYXV or VACV to some adherent cell lines could be partially inhibited by heparin, but laminin blocked only VACV binding. In contrast to cultured fibroblasts, the binding of MYXV and VACV to a wide spectrum of primary human leukocytes could not be competed by either HS or laminin. Additionally, MYXV and VACV exhibited very different binding characteristics against certain select human leukocytes, suggesting that the two poxviruses utilize different cell surface determinants for the attachment to these cells. These results indicate that VACV and MYXV can exhibit very different oncolytic tropisms against some cancerous human leukocytes. PMID:23388707

  14. Systemically administered DNA and fowlpox recombinants expressing four vaccinia virus genes although immunogenic do not protect mice against the highly pathogenic IHD-J vaccinia strain.

    PubMed

    Bissa, Massimiliano; Pacchioni, Sole Maria; Zanotto, Carlo; De Giuli Morghen, Carlo; Illiano, Elena; Granucci, Francesca; Zanoni, Ivan; Broggi, Achille; Radaelli, Antonia

    2013-12-26

    The first-generation smallpox vaccine was based on live vaccinia virus (VV) and it successfully eradicated the disease worldwide. Therefore, it was not administered any more after 1980, as smallpox no longer existed as a natural infection. However, emerging threats by terrorist organisations has prompted new programmes for second-generation vaccine development based on attenuated VV strains, which have been shown to cause rare but serious adverse events in immunocompromised patients. Considering the closely related animal poxviruses that might also be used as bioweapons, and the increasing number of unvaccinated young people and AIDS-affected immunocompromised subjects, a safer and more effective smallpox vaccine is still required. New avipoxvirus-based vectors should improve the safety of conventional vaccines, and protect from newly emerging zoonotic orthopoxvirus diseases and from the threat of deliberate release of variola or monkeypox virus in a bioterrorist attack. In this study, DNA and fowlpox recombinants expressing the L1R, A27L, A33R and B5R genes were constructed and evaluated in a pre-clinical trial in mouse, following six prime/boost immunisation regimens, to compare their immunogenicity and protective efficacy against a challenge with the lethal VV IHD-J strain. Although higher numbers of VV-specific IFNγ-producing T lymphocytes were observed in the protected mice, the cytotoxic T-lymphocyte response and the presence of neutralising antibodies did not always correlate with protection. In spite of previous successful results in mice, rabbits and monkeys, where SIV/HIV transgenes were expressed by the fowlpox vector, the immune response elicited by these recombinants was low, and most of the mice were not protected. PMID:24050999

  15. The vaccinia virus E6 protein influences virion protein localization during virus assembly.

    PubMed

    Condit, Richard C; Moussatche, Nissin

    2015-08-01

    Vaccinia virus mutants in which expression of the virion core protein gene E6R is repressed are defective in virion morphogenesis. E6 deficient infections fail to properly package viroplasm into viral membranes, resulting in an accumulation of empty immature virions and large aggregates of viroplasm. We have used immunogold electron microscopy and immunofluorescence confocal microscopy to assess the intracellular localization of several virion structural proteins and enzymes during E6R mutant infections. We find that during E6R mutant infections virion membrane proteins and virion transcription enzymes maintain a normal localization within viral factories while several major core and lateral body proteins accumulate in aggregated virosomes. The results support a model in which vaccinia virions are assembled from at least three substructures, the membrane, the viroplasm and a "pre-nucleocapsid", and that the E6 protein is essential for maintaining proper localization of the seven-protein complex and the viroplasm during assembly. PMID:25863879

  16. Dengue virus vaccine development.

    PubMed

    Yauch, Lauren E; Shresta, Sujan

    2014-01-01

    Dengue virus (DENV) is a significant cause of morbidity and mortality in tropical and subtropical regions, causing hundreds of millions of infections each year. Infections range from asymptomatic to a self-limited febrile illness, dengue fever (DF), to the life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The expanding of the habitat of DENV-transmitting mosquitoes has resulted in dramatic increases in the number of cases over the past 50 years, and recent outbreaks have occurred in the United States. Developing a dengue vaccine is a global health priority. DENV vaccine development is challenging due to the existence of four serotypes of the virus (DENV1-4), which a vaccine must protect against. Additionally, the adaptive immune response to DENV may be both protective and pathogenic upon subsequent infection, and the precise features of protective versus pathogenic immune responses to DENV are unknown, complicating vaccine development. Numerous vaccine candidates, including live attenuated, inactivated, recombinant subunit, DNA, and viral vectored vaccines, are in various stages of clinical development, from preclinical to phase 3. This review will discuss the adaptive immune response to DENV, dengue vaccine challenges, animal models used to test dengue vaccine candidates, and historical and current dengue vaccine approaches. PMID:24373316

  17. Vaccinia virus telomeres: interaction with the viral I1, I6, and K4 proteins.

    PubMed

    DeMasi, J; Du, S; Lennon, D; Traktman, P

    2001-11-01

    The 192-kb linear DNA genome of vaccinia virus has covalently closed hairpin termini that are extremely AT rich and contain 12 extrahelical bases. Vaccinia virus telomeres have previously been implicated in the initiation of viral genome replication; therefore, we sought to determine whether the telomeres form specific protein-DNA complexes. Using an electrophoretic mobility shift assay, we found that extracts prepared from virions and from the cytoplasm of infected cells contain telomere binding activity. Four shifted complexes were detected using hairpin probes representing the viral termini, two of which represent an interaction with the "flip" isoform and two with the "flop" isoform. All of the specificity for protein binding lies within the terminal 65-bp hairpin sequence. Viral hairpins lacking extrahelical bases cannot form the shifted complexes, suggesting that DNA structure is crucial for complex formation. Using an affinity purification protocol, we purified the proteins responsible for hairpin-protein complex formation. The vaccinia virus I1 protein was identified as being necessary and sufficient for the formation of the upper doublet of shifted complexes, and the vaccinia virus I6 protein was shown to form the lower doublet of shifted complexes. Competition and challenge experiments confirmed that the previously uncharacterized I6 protein binds tightly and with great specificity to the hairpin form of the viral telomeric sequence. Incubation of viral hairpins with extracts from infected cells also generates a smaller DNA fragment that is likely to reflect specific nicking at the apex of the hairpin; we show that the vaccinia virus K4 protein is necessary and sufficient for this reaction. We hypothesize that these telomere binding proteins may play a role in the initiation of vaccinia virus genome replication and/or genome encapsidation. PMID:11581377

  18. Smallpox Vaccine Overview

    MedlinePlus

    ... complications from the vaccinia virus can be severe. Benefit of Vaccine Following Exposure Vaccination within 3 days ... Policies About CDC.gov Link to Us All Languages Contact CDC Centers for Disease Control and Prevention ...

  19. Virus-Vectored Influenza Virus Vaccines

    PubMed Central

    Tripp, Ralph A.; Tompkins, S. Mark

    2014-01-01

    Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. PMID:25105278

  20. Oncolytic vaccinia virus synergizes with irinotecan in colorectal cancer.

    PubMed

    Ottolino-Perry, Kathryn; Acuna, Sergio A; Angarita, Fernando A; Sellers, Clara; Zerhouni, Siham; Tang, Nan; McCart, J Andrea

    2015-10-01

    Metastatic colorectal cancer (CRC) is complex clinical challenge for which there are limited treatment options. Chemotherapy with or without surgery provides moderate improvements in overall survival and quality of life; nevertheless the 5-year survival remains below 30%. Oncolytic vaccinia virus (VV) shows strong anti-tumour activity in models of CRC, however transient delays in disease progression are insufficient to lead to long-term survival. Here we examined the efficacy of VV with oxaliplatin or SN-38 (active metabolite of irinotecan) in CRC cell lines in vitro and VV with irinotecan in an orthotopic model of metastatic CRC. Synergistic improvements in in vitro cell killing were observed in multiple cell lines. Combination therapy was well tolerated in tumour-bearing mice and the median survival was significantly increased relative to monotherapy despite a drug-dependent decrease in the mean tumour titer. Increased apoptosis following in vitro and in vivo combination therapy was observed. In vitro cell cycle analysis showed increases in S-phase cells following infection occurred in both infected and uninfected cell populations. This corresponded to a 4-fold greater increase in apoptosis in the uninfected compared to infected cells following combination therapy. Combination treatment strategies are among the best options for patients with advanced cancers. VV is currently under clinical investigation in patients with CRC and the data presented here suggest that its combination with irinotecan may provide benefit to a subset of CRC patients. Further, investigation of this combination is necessary to determine the tumour characteristics responsible for mediating synergy. PMID:26004084

  1. The vaccinia virus E6 protein influences virion protein localization during virus assembly

    SciTech Connect

    Condit, Richard C. Moussatche, Nissin

    2015-08-15

    Vaccinia virus mutants in which expression of the virion core protein gene E6R is repressed are defective in virion morphogenesis. E6 deficient infections fail to properly package viroplasm into viral membranes, resulting in an accumulation of empty immature virions and large aggregates of viroplasm. We have used immunogold electron microscopy and immunofluorescence confocal microscopy to assess the intracellular localization of several virion structural proteins and enzymes during E6R mutant infections. We find that during E6R mutant infections virion membrane proteins and virion transcription enzymes maintain a normal localization within viral factories while several major core and lateral body proteins accumulate in aggregated virosomes. The results support a model in which vaccinia virions are assembled from at least three substructures, the membrane, the viroplasm and a “pre-nucleocapsid”, and that the E6 protein is essential for maintaining proper localization of the seven-protein complex and the viroplasm during assembly. - Highlights: • Mutation of E6 disrupts association of viral membranes with viral core proteins • Mutation of E6 does not perturb viral membrane biosynthesis • Mutation of E6 does not perturb localization of viral transcription enzymes • Mutation of E6 causes mis-localization and aggregation of viral core proteins • Vaccinia assembly uses three subassemblies: membranes, viroplasm, prenucleocapsid.

  2. Selecting Viruses for the Seasonal Influenza Vaccine

    MedlinePlus

    ... which viruses are selected for use in vaccine production? The influenza viruses in the seasonal flu vaccine ... to get a good vaccine virus for vaccine production? There are a number of factors that can ...

  3. High, broad, polyfunctional, and durable T cell immune responses induced in mice by a novel hepatitis C virus (HCV) vaccine candidate (MVA-HCV) based on modified vaccinia virus Ankara expressing the nearly full-length HCV genome.

    PubMed

    Gómez, Carmen E; Perdiguero, Beatriz; Cepeda, María Victoria; Mingorance, Lidia; García-Arriaza, Juan; Vandermeeren, Andrea; Sorzano, Carlos Óscar S; Esteban, Mariano

    2013-07-01

    A major goal in the control of hepatitis C infection is the development of a vaccine. Here, we have developed a novel HCV vaccine candidate based on the highly attenuated poxvirus vector MVA (referred to as MVA-HCV) expressing the nearly full-length (7.9-kbp) HCV sequence, with the aim to target almost all of the T and B cell determinants described for HCV. In infected cells, MVA-HCV produces a polyprotein that is subsequently processed into the structural and nonstructural HCV proteins, triggering the cytoplasmic accumulation of dense membrane aggregates. In both C57BL/6 and transgenic HLA-A2-vaccinated mice, MVA-HCV induced high, broad, polyfunctional, and long-lasting HCV-specific T cell immune responses. The vaccine-induced T cell response was mainly mediated by CD8 T cells; however, although lower in magnitude, the CD4(+) T cells were highly polyfunctional. In homologous protocol (MVA-HCV/MVA-HCV) the main CD8(+) T cell target was p7+NS2, whereas in heterologous combination (DNA-HCV/MVA-HCV) the main target was NS3. Antigenic responses were also detected against other HCV proteins (Core, E1-E2, and NS4), but the magnitude of the responses was dependent on the protocol used. The majority of the HCV-induced CD8(+) T cells were triple or quadruple cytokine producers. The MVA-HCV vaccine induced memory CD8(+) T cell responses with an effector memory phenotype. Overall, our data showed that MVA-HCV induced broad, highly polyfunctional, and durable T cell responses of a magnitude and quality that might be associated with protective immunity and open the path for future considerations of MVA-HCV as a prophylactic and/or therapeutic vaccine candidate against HCV. PMID:23596307

  4. Anti-tumour activity of oncolytic Western Reserve vaccinia viruses in canine tumour cell lines, xenografts, and fresh tumour biopsies.

    PubMed

    Autio, K; Knuuttila, A; Kipar, A; Ahonen, M; Parviainen, S; Diaconu, I; Kanerva, A; Hakonen, T; Vähä-Koskela, M; Hemminki, A

    2014-10-10

    Cancer is one of the most common reasons for death in dogs. One promising approach is oncolytic virotherapy. We assessed the oncolytic effect of genetically modified vaccinia viruses in canine cancer cells, in freshly excised tumour biopsies, and in mice harbouring canine tumour xenografts. Tumour transduction efficacy was assessed using virus expressing luciferase or fluorescent marker genes and oncolysis was quantified by a colorimetric cell viability assay. Oncolytic efficacy in vivo was evaluated in a nude mouse xenograft model. Vaccinia virus was shown to infect most tested canine cancer cell lines and primary surgical tumour tissues. Virus infection significantly reduced tumour growth in the xenograft model. Oncolytic vaccinia virus has antitumour effects against canine cancer cells and experimental tumours and is able to replicate in freshly excised patient tumour tissue. Our results suggest that oncolytic vaccinia virus may offer an effective treatment option for otherwise incurable canine tumours. PMID:25302859

  5. Ultraviolet-irradiated vaccinia virus recombinants, exposing HIV-envelope on their outer membrane, induce antibodies against this antigen in rabbits.

    PubMed

    Loewinger, M; Katz, E

    2002-01-01

    The construction and isolation of recombinants of vaccinia virus (IHD-J strain), bearing on their outer membrane a chimeric protein consisting of the cytoplasmic and transmembrane domains of vaccinia B5R protein and the external domain of HIV envelope, has been previously described by us. The present study aimed to investigate the potential use of such recombinants as a vaccine, following inactivation of their infectivity by ultraviolet (UV) irradiation. The minimal dose of UV irradiation, required for the complete inactivation of the infectivity of these recombinants, was determined. Injections of rabbits with the irradiated noninfectious recombinant viruses successfully induced specific antibodies against the HIV envelope antigen, in addition to those against the poxvirus. PMID:12479396

  6. One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

    PubMed Central

    Nitsche, Andreas; Kurth, Andreas; Dunkhorst, Anna; Pänke, Oliver; Sielaff, Hendrik; Junge, Wolfgang; Muth, Doreen; Scheller, Frieder; Stöcklein, Walter; Dahmen, Claudia; Pauli, Georg; Kage, Andreas

    2007-01-01

    Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method. PMID:17697378

  7. Vaccinia Virus Entry, Exit, and Interaction with Differentiated Human Airway Epithelia▿

    PubMed Central

    Vermeer, Paola D.; McHugh, Julia; Rokhlina, Tatiana; Vermeer, Daniel W.; Zabner, Joseph; Welsh, Michael J.

    2007-01-01

    Variola virus, the causative agent of smallpox, enters and exits the host via the respiratory route. To better understand the pathogenesis of poxvirus infection and its interaction with respiratory epithelia, we used vaccinia virus and examined its interaction with primary cultures of well-differentiated human airway epithelia. We found that vaccinia virus preferentially infected the epithelia through the basolateral membrane and released viral progeny across the apical membrane. Despite infection and virus production, epithelia retained tight junctions, transepithelial electrical conductance, and a steep transepithelial concentration gradient of virus, indicating integrity of the epithelial barrier. In fact, during the first four days of infection, epithelial height and cell number increased. These morphological changes and maintenance of epithelial integrity required vaccinia virus growth factor, which was released basolaterally, where it activated epidermal growth factor 1 receptors. These data suggest a complex interaction between the virus and differentiated airway epithelia; the virus preferentially enters the cells basolaterally, exits apically, and maintains epithelial integrity by stimulating growth factor receptors. PMID:17581984

  8. Vaccinia virus infection induces dendritic cell maturation but inhibits antigen presentation by MHC class II

    PubMed Central

    Yao, Yongxue; Li, Ping; Singh, Pratibha; Thiele, Allison T.; Wilkes, David S.; Renukaradhya, Gourapura J.; Brutkiewicz, Randy R.; Travers, Jeffrey B.; Luker, Gary D.; Hong, Soon-Cheol; Blum, Janice S.; Chang, Cheong-Hee

    2007-01-01

    Vaccinia virus (VV) infection is known to inhibit dendritic cells (DC) functions in vitro. Paradoxically, VV is also highly immunogenic and thus has been used as a vaccine. In the present study, we investigated the effects of an in vivo VV infection on DC function by focusing on early innate immunity. Our data indicated that DC are activated upon in vivo VV infection of mice. Splenic DC from VV-infected mice expressed elevated levels of MHC class I and co-stimulatory molecules on their cell surface and exhibited the enhanced potential to produce cytokines upon LPS stimulation. DC from VV-infected mice also expressed a high level of interferon-β. However, a VV infection resulted in the down-regulation of MHC class II expression and the impairment of antigen presentation to CD4 T cells by DC. Thus, during the early stage of a VV infection, although DC are impaired in some of the critical antigen presentation functions, they can promote innate immune defenses against viral infection. PMID:17678637

  9. Enhancement of CD8+ T-cell memory by removal of a vaccinia virus nuclear factor-κB inhibitor

    PubMed Central

    Ren, Hongwei; Ferguson, Brian J; de Motes, Carlos Maluquer; Sumner, Rebecca P; Harman, Laura E R; Smith, Geoffrey L

    2015-01-01

    Factors influencing T-cell responses are important for vaccine development but are incompletely understood. Here, vaccinia virus (VACV) protein N1 is shown to impair the development of both effector and memory CD8+ T cells and this correlates with its inhibition of nuclear factor-κB (NF-κB) activation. Infection with VACVs that either have the N1L gene deleted (vΔN1) or contain a I6E mutation (vN1.I6E) that abrogates its inhibition of NF-κB resulted in increased central and memory CD8+ T-cell populations, increased CD8+ T-cell cytotoxicity and lower virus titres after challenge. Furthermore, CD8+ memory T-cell function was increased following infection with vN1.I6E, with more interferon-γ production and greater protection against VACV infection following passive transfer to naive mice, compared with CD8+ T cells from mice infected with wild-type virus (vN1.WT). This demonstrates the importance of NF-κB activation within infected cells for long-term CD8+ T-cell memory and vaccine efficacy. Further, it provides a rationale for deleting N1 from VACV vectors to enhance CD8+ T-cell immunogenicity, while simultaneously reducing virulence to improve vaccine safety. PMID:25382035

  10. An update on approaches to the development of respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3) vaccines.

    PubMed

    Murphy, B R; Hall, S L; Kulkarni, A B; Crowe, J E; Collins, P L; Connors, M; Karron, R A; Chanock, R M

    1994-04-01

    RSV and PIV3 are responsible for about 30% of severe viral respiratory tract disease leading to hospitalization of infants and children. For this reason, there is a need to develop vaccines effective against these viruses. Since these viruses cause severe disease in early infancy, vaccines must be effective in the presence of maternal antibody. Currently, several strategies for immunization against these viruses are being explored including peptide vaccines, subunit vaccines, vectored vaccines (e.g., vaccinia-RSV or adenovirus-RSV recombinants), and live attenuated virus vaccines. The current status of these approaches is reviewed. In addition, the immunologic basis for the disease potentiation seen in vaccinees immunized with formalin-inactivated RSV during subsequent RSV infection is reviewed. The efficacy of immunization in the presence of maternal antibody is discussed. Much progress for a RSV and PIV3 vaccine has been made and successful immunization against each of these pathogens should be achieved within this decade. PMID:8030364

  11. Disparity between Levels of In Vitro Neutralization of Vaccinia Virus by Antibody to the A27 Protein and Protection of Mice against Intranasal Challenge▿

    PubMed Central

    Fogg, Christiana N.; Americo, Jeffrey L.; Earl, Patricia L.; Resch, Wolfgang; Aldaz-Carroll, Lydia; Eisenberg, Roselyn J.; Cohen, Gary H.; Moss, Bernard

    2008-01-01

    Immunization with recombinant proteins may provide a safer alternative to live vaccinia virus for prophylaxis of poxvirus infections. Although antibody protects against vaccinia virus infection, the mechanism is not understood and the selection of immunogens is daunting as there are dozens of surface proteins and two infectious forms known as the mature virion (MV) and the enveloped virion (EV). Our previous studies showed that mice immunized with soluble forms of EV membrane proteins A33 and B5 and MV membrane protein L1 or passively immunized with antibodies to these proteins survived an intranasal challenge with vaccinia virus. The present study compared MV protein A27, which has a role in virus attachment to glycosaminoglycans on the cell surface, to L1 with respect to immunogenicity and protection. Although mice developed similar levels of neutralizing antibody after immunizations with A27 or L1, A27-immunized mice exhibited more severe disease upon an intranasal challenge with vaccinia virus. In addition, mice immunized with A27 and A33 were not as well protected as mice receiving L1 and A33. Polyclonal rabbit anti-A27 and anti-L1 IgG had equivalent MV-neutralizing activities when measured by the prevention of infection of human or mouse cells or cells deficient in glycosaminoglycans or by adding antibody prior to or after virus adsorption. Nevertheless, the passive administration of antibody to A27 was poorly protective compared to the antibody to L1. These studies raise questions regarding the basis for antibody protection against poxvirus disease and highlight the importance of animal models for the early evaluation of vaccine candidates. PMID:18524827

  12. Analysis of vaccinia virus-host protein-protein interactions: validations of yeast two-hybrid screenings.

    PubMed

    Zhang, Leiliang; Villa, Nancy Y; Rahman, Masmudur M; Smallwood, Sherin; Shattuck, Donna; Neff, Chris; Dufford, Max; Lanchbury, Jerry S; Labaer, Joshua; McFadden, Grant

    2009-09-01

    Vaccinia virus, a large double-stranded DNA virus, is the prototype of the Orthopoxvirus genus, which includes several pathogenic poxviruses of humans, such as monkeypox virus and variola virus. Here, we report a comprehensive yeast two-hybrid (Y2H) screening for the protein-protein interactions between vaccinia and human proteins. A total of 109 novel vaccinia-human protein interactions were detected among 33 viral proteins. To validate subsets of those interactions, we constructed an ORFeome library of vaccinia virus strain WR using the Gateway plasmid cloning system. By co-expressing selected vaccinia and host proteins in a variety of expression systems, we found that at least 17 of the Y2H hits identified between vaccinia and human proteins can be verified by independent methods using GST pull-down assays, representing a 63% validation rate for the Y2H hits examined (17/27). Because the cloned ORFs are conveniently transferable from the entry vectors to various destination expression vectors, the vaccinia ORFeome library will be a useful resource for future high-throughput functional proteomic experiments. PMID:19637933

  13. Novel vaccines against influenza viruses

    PubMed Central

    Kang, Sang-Moo; Song, Jae-Min; Compans, Richard W.

    2011-01-01

    Killed and live attenuated influenza virus vaccines are effective in preventing and curbing the spread of influenza epidemics when the strains present in the vaccines are closely matched with the predicted epidemic strains. These vaccines are primarily targeted to induce immunity to the variable major target antigen, hemagglutinin (HA) of influenza virus. However, current vaccines are not effective in preventing the emergence of new pandemic or highly virulent viruses. New approaches are being investigated to develop universal influenza virus vaccines as well as to apply more effective vaccine delivery methods. Conserved vaccine targets including the influenza M2 ion channel protein and HA stalk domains are being developed using recombinant technologies to improve the level of cross protection. In addition, recent studies provide evidence that vaccine supplements can provide avenues to further improve current vaccination. PMID:21968298

  14. Relationship between RNA polymerase II and efficiency of vaccinia virus replication.

    PubMed Central

    Wilton, S; Dales, S

    1989-01-01

    It is clear from previous studies that host transcriptase or RNA polymerase II (pol II) has a role in poxvirus replication. To elucidate the participation of this enzyme further, in this study we examined several parameters related to pol II during the cycle of vaccinia virus infection in L-strain fibroblasts, HeLa cells, and L6H9 rat myoblasts. Nucleocytoplasmic transposition of pol II into virus factories and virions was assessed by immunofluorescence and immunoblotting by using anti-pol II immunoglobulin G. RNA polymerase activities were compared in nuclear extracts containing crude enzyme preparations. Rates of translation into cellular or viral polypeptides were ascertained by labeling with [35S]methionine. In L and HeLa cells, which produced vaccinia virus more abundantly, the rates of RNA polymerase and translation in controls and following infection were higher than in myoblasts. The data on synthesis and virus formation could be correlated with observations on transmigration of pol II, which was more efficient and complete in L and HeLa cells. The stimulus for pol II to leave the nucleus required the expression of both early and late viral functions. On the basis of current and past information, we suggest that mobilization of pol II depends on the efficiency of vaccina virus replication and furthermore that control over vaccinia virus production by the host is related to the content or availability (or both) of pol II in different cell types. Images PMID:2648021

  15. Myristoylation increases the CD8+T-cell response to a GFP prototype antigen delivered by modified vaccinia virus Ankara.

    PubMed

    Marr, Lisa; Lülf, Anna-Theresa; Freudenstein, Astrid; Sutter, Gerd; Volz, Asisa

    2016-04-01

    Activation of CD8(+)T-cells is an essential part of immune responses elicited by recombinant modified vaccinia virus Ankara (MVA). Strategies to enhance T-cell responses to antigens may be particularly necessary for broadly protective immunization against influenza A virus infections or for candidate vaccines targeting chronic infections and cancer. Here, we tested recombinant MVAs that targeted a model antigen, GFP, to different localizations in infected cells. In vitro characterization demonstrated that GFP accumulated in the nucleus (MVA-nls-GFP), associated with cellular membranes (MVA-myr-GFP) or was equally distributed throughout the cell (MVA-GFP). On vaccination, we found significantly higher levels of GFP-specific CD8(+)T-cells in MVA-myr-GFP-vaccinated BALB/c mice than in those immunized with MVA-GFP or MVA-nls-GFP. Thus, myristoyl modification may be a useful strategy to enhance CD8(+)T-cell responses to MVA-delivered target antigens. PMID:26864442

  16. Targeting vaccinia virus-expressed secretory beta subunit of human chorionic gonadotropin to the cell surface induces antibodies.

    PubMed Central

    Srinivasan, J; Singh, O; Chakrabarti, S; Talwar, G P

    1995-01-01

    We carried out experiments designed to study the effect of a protein's localization on its immunogenicity. A novel cell-surface protein was generated from a small, glycosylated secretory protein. The DNA sequence encoding the entire precursor of the human chorionic gonadotropin beta (beta hCG) subunit was fused in the correct reading frame to the DNA sequence encoding the transmembrane and cytoplasmic domains of vesicular stomatitis virus glycoprotein. This chimeric gene was introduced into the vaccinia virus genome to generate a recombinant virus. The recombinant virus, when used to infect animal cells, expressed a 135-amino-acid beta hCG subunit anchored in cellular membranes by the 48 carboxy-terminal amino acids of vesicular stomatitis virus glycoprotein. The immunogenicity of this recombinant virus with respect to its ability to generate anti-hCG antibodies was compared with that of a second recombinant vaccinia virus expressing the native secretory form of beta hCG. All animals immunized with the vaccinia virus expressing beta hCG on the cell surface elicited high titers of anti-hCG antibodies. Even after a single immunization with the recombinant vaccinia virus, the anti-hCG antibody titers persisted for a long period of time (more than 6 months). None of the animals immunized with vaccinia virus expressing the native secretory form of beta hCG showed any hCG-specific antibody response. PMID:7591154

  17. Induction of Potent Humoral and Cell-Mediated Immune Responses by Attenuated Vaccinia Virus Vectors with Deleted Serpin Genes

    PubMed Central

    Legrand, Fatema A.; Verardi, Paulo H.; Jones, Leslie A.; Chan, Kenneth S.; Peng, Yue; Yilma, Tilahun D.

    2004-01-01

    Vaccinia virus (VV) has been effectively utilized as a live vaccine against smallpox as well as a vector for vaccine development and immunotherapy. Increasingly there is a need for a new generation of highly attenuated and efficacious VV vaccines, especially in light of the AIDS pandemic and the threat of global bioterrorism. We therefore developed recombinant VV (rVV) vaccines that are significantly attenuated and yet elicit potent humoral and cell-mediated immune responses. B13R (SPI-2) and B22R (SPI-1) are two VV immunomodulating genes with sequence homology to serine protease inhibitors (serpins) that possess antiapoptotic and anti-inflammatory properties. We constructed and characterized rVVs that have the B13R or B22R gene insertionally inactivated (vΔB13R and vΔB22R) and coexpress the vesicular stomatitis virus glycoprotein (v50ΔB13R and v50ΔB22R). Virulence studies with immunocompromised BALB/cBy nude mice indicated that B13R or B22R gene deletion decreases viral replication and significantly extends time of survival. Viral pathogenesis studies in immunocompetent CB6F1 mice further demonstrated that B13R or B22R gene inactivation diminishes VV virulence, as measured by decreased levels of weight loss and limited viral spread. Finally, rVVs with B13R and B22R deleted elicited potent humoral, T-helper, and cytotoxic T-cell immune responses, revealing that the observed attenuation did not reduce immunogenicity. Therefore, inactivation of immunomodulating genes such as B13R or B22R represents a general method for enhancing the safety of rVV vaccines while maintaining a high level of immunogenicity. Such rVVs could serve as effective vectors for vaccine development and immunotherapy. PMID:14990697

  18. Dominant negative selection of vaccinia virus using a thymidine kinase/thymidylate kinase fusion gene and the prodrug azidothymidine

    SciTech Connect

    Holzer, Georg W. . E-mail: falknef@baxter.com

    2005-07-05

    The Escherichia coli thymidine kinase/thymidylate kinase (tk/tmk) fusion gene encodes an enzyme that efficiently converts the prodrug 3'-azido-2',3'-dideoxythymidine (AZT) into its toxic triphosphate derivative, a substance which stops DNA chain elongation. Integration of this marker gene into vaccinia virus that normally is not inhibited by AZT allowed the establishment of a powerful selection procedure for recombinant viruses. In contrast to the conventional vaccinia thymidine kinase (tk) selection that is performed in tk-negative cell lines, AZT selection can be performed in normal (tk-positive) cell lines. The technique is especially useful for the generation of replication-deficient vaccinia viruses and may also be used for gene knock-out studies of essential vaccinia genes.

  19. Identification of a Bohle iridovirus thymidine kinase gene and demonstration of activity using vaccinia virus.

    PubMed

    Coupar, B E H; Goldie, S G; Hyatt, A D; Pallister, J A

    2005-09-01

    In recent years interest in the family Iridoviridae has been renewed by the identification of a number of viruses, particularly from the genus Ranavirus, associated with disease in a range of poikilotherms. Ranaviruses have been isolated from amphibian, piscine and reptilian species. Here we describe an open reading frame (ORF) identified in the genome of Bohle iridovirus (BIV) which contains a nucleotide binding motif conserved within the thymidine kinase (TK) genes of iridoviruses from other genera (lymphocystis disease virus, LCDV, type species of the genus Lymphocystivirus; Chilo iridescent virus, CIV, type species of the genus Iridovirus). The ability of this putative gene to express a functional TK was confirmed by rescue of a TK negative mutant vaccinia virus in the presence of selective media, when expression was controlled by a vaccinia virus promoter. The sequence of the BIV TK was compared with the homologous sequences from epizootic haematopoietic necrosis virus (EHNV), a virus associated with disease in fish, from Wamena iridovirus (WIV) associated with systemic disease in green pythons, and from frog virus 3 (FV3) the ranavirus type species. Comparisons between these sequences and those available from other ranaviruses, other iridoviruses, other DNA viruses and cellular TKs are presented. PMID:15883656

  20. Human cytomegalovirus UL97 kinase confers ganciclovir susceptibility to recombinant vaccinia virus.

    PubMed Central

    Metzger, C; Michel, D; Schneider, K; Lüske, A; Schlicht, H J; Mertens, T

    1994-01-01

    We analyzed whether the phosphotransferase encoded by the UL97 open reading frame of human cytomegalovirus (HCMV) alone is sufficient to confer ganciclovir (GCV) susceptibility to a foreign virus. Two vaccinia virus recombinants (T1 and A5) containing the UL97 open reading frames from a GCV-sensitive HCMV and from a GCV-resistant strain were constructed. T1 exhibited a GCV-sensitive phenotype in plaque reduction assays, whereas A5 did not. Moreover, T1-infected cell cultures showed a strongly increased incorporation of [14C]GCV triphosphate into macromolecular DNA, compared with recombinant A5 or vaccinia virus controls, which could be inhibited by the addition of guanosine. This shows that UL97 kinase is the only additional gene product required to make vaccinia virus susceptible to GCV, and guanosine seems to be one natural substrate for the enzyme. The system described here should be very helpful for fast and detailed functional analyses of UL97 mutations found in GCV-resistant HCMV isolates. Images PMID:7966639

  1. Prospective Surveillance for Cardiac Adverse Events in Healthy Adults Receiving Modified Vaccinia Ankara Vaccines: A Systematic Review

    PubMed Central

    Elizaga, Marnie L.; Vasan, Sandhya; Marovich, Mary A.; Sato, Alicia H.; Lawrence, Dale N.; Chaitman, Bernard R.; Frey, Sharon E.; Keefer, Michael C.

    2013-01-01

    Background Vaccinia-associated myo/pericarditis was observed during the US smallpox vaccination (DryVax) campaign initiated in 2002. A highly-attenuated vaccinia strain, modified vaccinia Ankara (MVA) has been evaluated in clinical trials as a safer alternative to DryVax and as a vector for recombinant vaccines. Due to the lack of prospectively collected cardiac safety data, the US Food and Drug Administration required cardiac screening and surveillance in all clinical trials of MVA since 2004. Here, we report cardiac safety surveillance from 6 phase I trials of MVA vaccines. Methods Four clinical research organizations contributed cardiac safety data using common surveillance methods in trials administering MVA or recombinant MVA vaccines to healthy participants. ‘Routine cardiac investigations’ (ECGs and cardiac enzymes obtained 2 weeks after injections of MVA or MVA-HIV recombinants, or placebo-controls), and ‘Symptom-driven cardiac investigations’ are reported. The outcome measure is the number of participants who met the CDC-case definition for vaccinia-related myo/pericarditis or who experienced cardiac adverse events from an MVA vaccine. Results Four hundred twenty-five study participants had post-vaccination safety data analyzed, 382 received at least one MVA-containing vaccine and 43 received placebo; 717 routine ECGs and 930 cardiac troponin assays were performed. Forty-five MVA recipients (12%) had additional cardiac testing performed; 22 for cardiac symptoms, 19 for ECG/laboratory changes, and 4 for cardiac symptoms with an ECG/laboratory change. No participant had evidence of symptomatic or asymptomatic myo/pericarditis meeting the CDC-case definition and judged to be related to an MVA vaccine. Conclusions Prospective surveillance of MVA recipients for myo/pericarditis did not detect cardiac adverse reactions in 382 study participants. Trial Registration ClinicalTrials.gov NCT00082446 NCT003766090 NCT00252148 NCT00083603 NCT00301184

  2. A Pandemic Influenza H1N1 Live Vaccine Based on Modified Vaccinia Ankara Is Highly Immunogenic and Protects Mice in Active and Passive Immunizations

    PubMed Central

    Hessel, Annett; Schwendinger, Michael; Fritz, Daniela; Coulibaly, Sogue; Holzer, Georg W.; Sabarth, Nicolas; Kistner, Otfried; Wodal, Walter; Kerschbaum, Astrid; Savidis-Dacho, Helga; Crowe, Brian A.; Kreil, Thomas R.; Barrett, P. Noel; Falkner, Falko G.

    2010-01-01

    Background The development of novel influenza vaccines inducing a broad immune response is an important objective. The aim of this study was to evaluate live vaccines which induce both strong humoral and cell-mediated immune responses against the novel human pandemic H1N1 influenza virus, and to show protection in a lethal animal challenge model. Methodology/Principal Findings For this purpose, the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A/California/07/2009 (H1N1) strain (CA/07) were inserted into the replication-deficient modified vaccinia Ankara (MVA) virus - a safe poxviral live vector – resulting in MVA-H1-Ca and MVA-N1-Ca vectors. These live vaccines, together with an inactivated whole virus vaccine, were assessed in a lung infection model using immune competent Balb/c mice, and in a lethal challenge model using severe combined immunodeficient (SCID) mice after passive serum transfer from immunized mice. Balb/c mice vaccinated with the MVA-H1-Ca virus or the inactivated vaccine were fully protected from lung infection after challenge with the influenza H1N1 wild-type strain, while the neuraminidase virus MVA-N1-Ca induced only partial protection. The live vaccines were already protective after a single dose and induced substantial amounts of neutralizing antibodies and of interferon-γ-secreting (IFN-γ) CD4- and CD8 T-cells in lungs and spleens. In the lungs, a rapid increase of HA-specific CD4- and CD8 T cells was observed in vaccinated mice shortly after challenge with influenza swine flu virus, which probably contributes to the strong inhibition of pulmonary viral replication observed. In addition, passive transfer of antisera raised in MVA-H1-Ca vaccinated immune-competent mice protected SCID mice from lethal challenge with the CA/07 wild-type virus. Conclusions/Significance The non-replicating MVA-based H1N1 live vaccines induce a broad protective immune response and are promising vaccine candidates for pandemic influenza

  3. Attenuated recombinant vaccinia virus expressing oncofetal antigen (tumor-associated antigen) 5T4 induces active therapy of established tumors.

    PubMed

    Mulryan, Kate; Ryan, Matthew G; Myers, Kevin A; Shaw, David; Wang, Who; Kingsman, Susan M; Stern, Peter L; Carroll, Miles W

    2002-10-01

    The human oncofetal antigen 5T4 (h5T4) is a transmembrane glycoprotein overexpressed by a wide spectrum of cancers, including colorectal, ovarian, and gastric, but with a limited normal tissue expression. Such properties make 5T4 an excellent putative target for cancer immunotherapy. The murine homologue of 5T4 (m5T4) has been cloned and characterized, which allows for the evaluation of immune intervention strategies in "self-antigen" in vivo tumor models. We have constructed recombinant vaccinia viruses based on the highly attenuated and modified vaccinia virus ankara (MVA strain), expressing h5T4 (MVA-h5T4), m5T4 (MVA-m5T4), and Escherichia coli LacZ (MVA-LacZ). Immunization of BALB/c and C57BL/6 mice with MVA-h5T4 and MVA-m5T4 constructs induced antibody responses to human and mouse 5T4, respectively. C57BL/6 and BALB/c mice vaccinated with MVA-h5T4 were challenged with syngeneic tumor line transfectants, B16 melanoma, and CT26 colorectal cells that express h5T4. MVA-h5T4-vaccinated mice showed significant tumor retardation compared with mice vaccinated with MVA-LacZ or PBS. In active treatment studies, inoculation with MVA-h5T4 was able to treat established CT26-h5T4 lung tumor and to a lesser extent B16.h5T4 s.c. tumors. Additionally, when C57BL/6 mice vaccinated with MVA-m5T4 were challenged with B16 cells expressing m5T4, resulting growth of the tumors was significantly retarded compared with control animals. Furthermore, mice vaccinated with MVA-m5T4 showed no signs of autoimmune toxicity. These data support the use of MVA-5T4 for tumor immunotherapy. PMID:12481437

  4. Identification of the vaccinia virus gene encoding nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase.

    PubMed Central

    Broyles, S S; Moss, B

    1987-01-01

    Vaccinia virus encapsidates a DNA-dependent ATPase known as nucleoside triphosphate phosphohydrolase I (NPH I). A bacteriophage lambda gt11 expression library of poxvirus DNA was screened with antibodies specific for NPH I. Positive clones were used to probe restriction fragments of vaccinia virus genomic DNA to locate the NPH I gene. The identity of the open reading frame (ORF) was confirmed by placing it downstream of a bacteriophage T7 promoter, transcribing the ORF in vitro, and translating the RNA in a reticulocyte lysate. A polypeptide of the correct molecular weight, which was recognized by anti-NPH I antibody, was synthesized. Inspection of the deduced amino acid sequence of the NPH I ORF revealed consensus ATP-binding sites. Images PMID:2437324

  5. Isolation of cis-acting vaccinia virus DNA fragments promoting the expression of herpes simplex virus thymidine kinase by recombinant viruses.

    PubMed Central

    Vassef, A; Mars, M; Dru, A; Plucienniczak, A; Streeck, R E; Beaud, G

    1985-01-01

    Recombinant TK- vaccinia viruses containing the pBR322 sequence inserted in either orientation within the coding sequence of the viral thymidine kinase gene were constructed. They were characterized by genomic analysis, hybridization studies, reversion to wild-type virus by in vivo recombination, and rescue from their genomes of plasmids which contained all or parts of the pBR322 sequence. TK- cells were infected with one of these recombinant viruses and then transfected with pools of chimeric plasmids composed of a cloned herpes simplex virus thymidine kinase gene which contained upstream inserts of different vaccinia DNA fragments prepared by restriction or sonication. Recombination between homologous pBR322 sequences within infected cells generated selectable recombinant viruses in which expression of the herpes simplex virus thymidine kinase gene was promoted by the upstream vaccinia insert. These viruses were characterized by genomic analysis, hybridization, and in vivo or in vitro phosphorylation of (5-[125I]deoxycytidine as a specific assay for the expressed herpes simplex virus thymidine kinase. Vaccinia DNA inserts were isolated conveniently for transfer to bacteria by rescuing appropriate plasmids from the genome of recombinant viruses. The sequence of 100 nucleotides adjacent to the upstream region of the herpes simplex virus gene was determined in nine different inserts measuring 0.17 to 1.07 kilobase pairs. Images PMID:2989553

  6. Oncolytic vaccinia virus as a vector for therapeutic sodium iodide symporter gene therapy in prostate cancer

    PubMed Central

    Mansfield, D C; Kyula, J N; Rosenfelder, N; Chao-Chu, J; Kramer-Marek, G; Khan, A A; Roulstone, V; McLaughlin, M; Melcher, A A; Vile, R G; Pandha, H S; Khoo, V; Harrington, K J

    2016-01-01

    Oncolytic strains of vaccinia virus are currently in clinical development with clear evidence of safety and promising signs of efficacy. Addition of therapeutic genes to the viral genome may increase the therapeutic efficacy of vaccinia. We evaluated the therapeutic potential of vaccinia virus expressing the sodium iodide symporter (NIS) in prostate cancer models, combining oncolysis, external beam radiotherapy and NIS-mediated radioiodide therapy. The NIS-expressing vaccinia virus (VV-NIS), GLV-1h153, was tested in in vitro analyzes of viral cell killing, combination with radiotherapy, NIS expression, cellular radioiodide uptake and apoptotic cell death in PC3, DU145, LNCaP and WPMY-1 human prostate cell lines. In vivo experiments were carried out in PC3 xenografts in CD1 nude mice to assess NIS expression and tumor radioiodide uptake. In addition, the therapeutic benefit of radioiodide treatment in combination with viral oncolysis and external beam radiotherapy was measured. In vitro viral cell killing of prostate cancers was dose- and time-dependent and was through apoptotic mechanisms. Importantly, combined virus therapy and iodizing radiation did not adversely affect oncolysis. NIS gene expression in infected cells was functional and mediated uptake of radioiodide both in vitro and in vivo. Therapy experiments with both xenograft and immunocompetent Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse models showed that the addition of radioiodide to VV-NIS-infected tumors was more effective than each single-agent therapy, restricting tumor growth and increasing survival. In conclusion, VV-NIS is effective in prostate cancer models. This treatment modality would be an attractive complement to existing clinical radiotherapy practice. PMID:26814609

  7. Oncolytic vaccinia virus as a vector for therapeutic sodium iodide symporter gene therapy in prostate cancer.

    PubMed

    Mansfield, D C; Kyula, J N; Rosenfelder, N; Chao-Chu, J; Kramer-Marek, G; Khan, A A; Roulstone, V; McLaughlin, M; Melcher, A A; Vile, R G; Pandha, H S; Khoo, V; Harrington, K J

    2016-04-01

    Oncolytic strains of vaccinia virus are currently in clinical development with clear evidence of safety and promising signs of efficacy. Addition of therapeutic genes to the viral genome may increase the therapeutic efficacy of vaccinia. We evaluated the therapeutic potential of vaccinia virus expressing the sodium iodide symporter (NIS) in prostate cancer models, combining oncolysis, external beam radiotherapy and NIS-mediated radioiodide therapy. The NIS-expressing vaccinia virus (VV-NIS), GLV-1h153, was tested in in vitro analyzes of viral cell killing, combination with radiotherapy, NIS expression, cellular radioiodide uptake and apoptotic cell death in PC3, DU145, LNCaP and WPMY-1 human prostate cell lines. In vivo experiments were carried out in PC3 xenografts in CD1 nude mice to assess NIS expression and tumor radioiodide uptake. In addition, the therapeutic benefit of radioiodide treatment in combination with viral oncolysis and external beam radiotherapy was measured. In vitro viral cell killing of prostate cancers was dose- and time-dependent and was through apoptotic mechanisms. Importantly, combined virus therapy and iodizing radiation did not adversely affect oncolysis. NIS gene expression in infected cells was functional and mediated uptake of radioiodide both in vitro and in vivo. Therapy experiments with both xenograft and immunocompetent Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse models showed that the addition of radioiodide to VV-NIS-infected tumors was more effective than each single-agent therapy, restricting tumor growth and increasing survival. In conclusion, VV-NIS is effective in prostate cancer models. This treatment modality would be an attractive complement to existing clinical radiotherapy practice. PMID:26814609

  8. Oral vaccination of raccoons (Procyon lotor) with genetically modified rabies virus vaccines

    PubMed Central

    Blanton, Jesse D.; Self, Joshua; Niezgoda, Michael; Faber, Marie-Luise; Dietzschold, Bernhard; Rupprecht, Charles

    2007-01-01

    Oral vaccination is an important tool currently in use to control the spread of rabies in wildlife populations in various programs around the world. Oral rabies vaccination (ORV) of raccoons represents the largest targeted program to control wildlife rabies in the United States. Currently, the vaccinia-rabies glycoprotein recombinant virus vaccine (V-RG) is the only licensed oral rabies vaccine in the US. In the current study, captive raccoons were used to evaluate two previously described constructs of a rabies virus vaccine developed by reverse genetics (SPBNGAS and SPBNGAS-GAS) for immunogenicity and efficacy compared to the V-RG vaccine. Four of five control animals succumbed to rabies virus after severe challenge, while three of five animals vaccinated orally with SPBNGAS succumbed. No mortality was observed for animals administered SPBNGAS-GAS or the V-RG vaccine. The results of this preliminary study suggest that SPBNGAS-GAS provides comparable efficacy to V-RG. Additional studies will be needed to determine the duration of immunity and optimal dosage of SPBNGAS-GAS and to examine its efficacy in other reservoir species. PMID:17826874

  9. Oral vaccination of raccoons (Procyon lotor) with genetically modified rabies virus vaccines.

    PubMed

    Blanton, Jesse D; Self, Joshua; Niezgoda, Michael; Faber, Marie-Luise; Dietzschold, Bernhard; Rupprecht, Charles

    2007-10-16

    Oral vaccination is an important tool currently in use to control the spread of rabies in wildlife populations in various programs around the world. Oral rabies vaccination (ORV) of raccoons represents the largest targeted program to control wildlife rabies in the United States. Currently, the vaccinia-rabies glycoprotein recombinant virus vaccine (V-RG) is the only licensed oral rabies vaccine in the US. In the current study, captive raccoons were used to evaluate two previously described constructs of a rabies virus vaccine developed by reverse genetics (SPBNGAS and SPBNGAS-GAS) for immunogenicity and efficacy compared to the V-RG vaccine. Four of five control animals succumbed to rabies virus after severe challenge, while three of five animals vaccinated orally with SPBNGAS succumbed. No mortality was observed for animals administered SPBNGAS-GAS or the V-RG vaccine. The results of this preliminary study suggest that SPBNGAS-GAS provides comparable efficacy to V-RG. Additional studies will be needed to determine the duration of immunity and optimal dosage of SPBNGAS-GAS and to examine its efficacy in other reservoir species. PMID:17826874

  10. Seven major genomic deletions of vaccinia virus Tiantan strain are sufficient to decrease pathogenicity.

    PubMed

    Li, Yiquan; Sheng, Yuan; Chu, Yunjie; Ji, Huifan; Jiang, Shuang; Lan, Tian; Li, Min; Chen, Shuang; Fan, Yuanyuan; Li, Wenjie; Li, Xiao; Sun, Lili; Jin, Ningyi

    2016-05-01

    Attenuated strain TTVAC7, as a multi-gene-deleted vaccinia virus Tiantan strain (VTT), was constructed by knocking out parts of non-essential genes related to virulence, host range and immunomodulation of VTT, and by combining double marker screening with exogenous selectable marker knockout techniques. In this study, shuttle vector plasmids pTC-EGFP, pTA35-EGFP, pTA66-EGFP, pTE-EGFP, pTB-EGFP, pTI-EGFP and pTJ-EGFP were constructed, which contained seven pairs of recombinant arms linked to the early and late strong promoter pE/L, as well as to enhanced green fluorescent protein (EGFP) as an exogenous selectable marker. BHK cells were co-transfected/infected successively with the above plasmids and VTT or gene-deleted VTT, and homologous recombination and fluorescence plaque screening methods were used to knock out the gene fragments (TC: TC7L ∼ TK2L; TA35: TA35L; TA66: TA66R; TE: TE3L ∼ TE4L; TB: TB13R; TI: TI4L; TJ: TJ2R). The Cre/LoxP system was then applied to knock out the exogenous selectable marker, and ultimately the gene-deleted attenuated strain TTVAC7 was obtained. A series of in vivo and in vitro experiments demonstrated that not only the host range of TTVAC7 could be narrowed and its toxicity weakened significantly, but its high immunogenicity was maintained at the same time. These results support the potential of TTVAC7 to be developed as a safe viral vector or vaccine. PMID:26821204

  11. Enteric Immunization of Mice Against Influenza with Recombinant Vaccinia

    NASA Astrophysics Data System (ADS)

    Meitin, Catherine A.; Bender, Bradley S.; Small, Parker A., Jr.

    1994-11-01

    Intrajejunal administration to mice of a recombinant vaccinia virus containing the influenza virus hemagglutinin gene induced IgA antibody in nasal, gut, and vaginal secretions. It also induced IgG antibody in serum and cell-mediated immunity. The immunization provided significant protection against an influenza virus challenge. This work suggests that enteric-coated recombinant vaccinia could be an orally administered, inexpensive, multivalent, temperature-stable, safe, and effective vaccine for children that could be particularly useful in developing nations, where multiple injections are not easily administered. Oral administration of vaccines should also reduce children's fear of shots at the doctor's office.

  12. Potent and Broadly Reactive HIV-2 Neutralizing Antibodies Elicited by a Vaccinia Virus Vector Prime-C2V3C3 Polypeptide Boost Immunization Strategy▿ †

    PubMed Central

    Marcelino, José Maria; Borrego, Pedro; Rocha, Cheila; Barroso, Helena; Quintas, Alexandre; Novo, Carlos; Taveira, Nuno

    2010-01-01

    Human immunodeficiency virus type 2 (HIV-2) infection affects about 1 to 2 million individuals, the majority living in West Africa, Europe, and India. As for HIV-1, new strategies for the prevention of HIV-2 infection are needed. Our aim was to produce new vaccine immunogens that elicit the production of broadly reactive HIV-2 neutralizing antibodies (NAbs). Native and truncated envelope proteins from the reference HIV-2ALI isolate were expressed in vaccinia virus or in bacteria. This source isolate was used due to its unique phenotype combining CD4 independence and CCR5 usage. NAbs were not elicited in BALB/c mice by single immunization with a truncated and fully glycosylated envelope gp125 (gp125t) or a recombinant polypeptide comprising the C2, V3, and C3 envelope regions (rpC2-C3). A strong and broad NAb response was, however, elicited in mice primed with gp125t expressed in vaccinia virus and boosted with rpC2-C3. Serum from these animals potently neutralized (median 50% neutralizing titer, 3,200) six of six highly divergent primary HIV-2 isolates. Coreceptor usage and the V3 sequence of NAb-sensitive isolates were similar to that of the vaccinating immunogen (HIV-2ALI). In contrast, NAbs were not reactive on three X4 isolates that displayed major changes in V3 loop sequence and structure. Collectively, our findings demonstrate that broadly reactive HIV-2 NAbs can be elicited by using a vaccinia virus vector-prime/rpC2-C3-boost immunization strategy and suggest a potential relationship between escape to neutralization and cell tropism. PMID:20844029

  13. Potent and broadly reactive HIV-2 neutralizing antibodies elicited by a vaccinia virus vector prime-C2V3C3 polypeptide boost immunization strategy.

    PubMed

    Marcelino, José Maria; Borrego, Pedro; Rocha, Cheila; Barroso, Helena; Quintas, Alexandre; Novo, Carlos; Taveira, Nuno

    2010-12-01

    Human immunodeficiency virus type 2 (HIV-2) infection affects about 1 to 2 million individuals, the majority living in West Africa, Europe, and India. As for HIV-1, new strategies for the prevention of HIV-2 infection are needed. Our aim was to produce new vaccine immunogens that elicit the production of broadly reactive HIV-2 neutralizing antibodies (NAbs). Native and truncated envelope proteins from the reference HIV-2ALI isolate were expressed in vaccinia virus or in bacteria. This source isolate was used due to its unique phenotype combining CD4 independence and CCR5 usage. NAbs were not elicited in BALB/c mice by single immunization with a truncated and fully glycosylated envelope gp125 (gp125t) or a recombinant polypeptide comprising the C2, V3, and C3 envelope regions (rpC2-C3). A strong and broad NAb response was, however, elicited in mice primed with gp125t expressed in vaccinia virus and boosted with rpC2-C3. Serum from these animals potently neutralized (median 50% neutralizing titer, 3,200) six of six highly divergent primary HIV-2 isolates. Coreceptor usage and the V3 sequence of NAb-sensitive isolates were similar to that of the vaccinating immunogen (HIV-2ALI). In contrast, NAbs were not reactive on three X4 isolates that displayed major changes in V3 loop sequence and structure. Collectively, our findings demonstrate that broadly reactive HIV-2 NAbs can be elicited by using a vaccinia virus vector-prime/rpC2-C3-boost immunization strategy and suggest a potential relationship between escape to neutralization and cell tropism. PMID:20844029

  14. Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes.

    PubMed Central

    Ink, B S; Gilbert, C S; Evan, G I

    1995-01-01

    The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family. PMID:7815529

  15. Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid.

    PubMed Central

    Bolden, A; Aucker, J; Weissbach, A

    1975-01-01

    Purified nuclei, isolated from appropriately infected HeLa cells, are shown to synthesize large amounts of either herpes simplex virus (HSV) or vaccinia virus DNA in vitro. The rate of synthesis of DNA by nuclei from infected cells is up to 30 times higher than the synthesis of host DNA in vitro by nuclei isolated from uninfected HeLa cells. Thus HSV nuclei obtained from HSV-infected cells make DNA in vitro at a rate comparable to that seen in the intact, infected cell. Molecular hybridization studies showed that 80% of the DNA sequences synthesized in vitro by nuclei from herpesvirus-infected cells are herpesvirus specific. Vaccinia virus nuclei from vaccinia virus-infected cells, also produce comparable percentages of vaccinia virus-specific DNA sequences. Adenovirus nuclei from adenovirus 2-infected HeLa cells, which also synthesize viral DNA in vitro, have been included in this study. Synthesis of DNA by HSV or vaccinia virus nuclei is markedly inhibited by the corresponding viral-specific antisera. These antisera inhibit in a similar fashion the purified herpesvirus-induced or vaccinia virus-induced DNA polymerase isolated from infected cells. Phosphonoacetic acid, reported to be a specific inhibitor of herpesvirus formation and the herpesvirus-induced DNA polymerase, is equally effective as an inhibitor of HSV DNA synthesis in isolated nuclei in vitro. However, we also find phosphonoacetic acid to be an effective inhibitor of vaccinia virus nuclear DNA synthesis and the purified vaccinia virus-induced DNA polymerase. In addition, this compound shows significant inhibition of DNA synthesis in isolated nuclei obtained from adenovirus-infected or uninfected cells and is a potent inhibitor of HeLa cell DNA polymerase alpha. PMID:172658

  16. Inhibition of Translation Initiation by Protein 169: A Vaccinia Virus Strategy to Suppress Innate and Adaptive Immunity and Alter Virus Virulence

    PubMed Central

    Strnadova, Pavla; Ren, Hongwei; Valentine, Robert; Mazzon, Michela; Sweeney, Trevor R.; Brierley, Ian; Smith, Geoffrey L.

    2015-01-01

    Vaccinia virus (VACV) is the prototypic orthopoxvirus and the vaccine used to eradicate smallpox. Here we show that VACV strain Western Reserve protein 169 is a cytoplasmic polypeptide expressed early during infection that is excluded from virus factories and inhibits the initiation of cap-dependent and cap-independent translation. Ectopic expression of protein 169 causes the accumulation of 80S ribosomes, a reduction of polysomes, and inhibition of protein expression deriving from activation of multiple innate immune signaling pathways. A virus lacking 169 (vΔ169) replicates and spreads normally in cell culture but is more virulent than parental and revertant control viruses in intranasal and intradermal murine models of infection. Intranasal infection by vΔ169 caused increased pro-inflammatory cytokines and chemokines, infiltration of pulmonary leukocytes, and lung weight. These alterations in innate immunity resulted in a stronger CD8+ T-cell memory response and better protection against virus challenge. This work illustrates how inhibition of host protein synthesis can be a strategy for virus suppression of innate and adaptive immunity. PMID:26334635

  17. Inhibition of Translation Initiation by Protein 169: A Vaccinia Virus Strategy to Suppress Innate and Adaptive Immunity and Alter Virus Virulence.

    PubMed

    Strnadova, Pavla; Ren, Hongwei; Valentine, Robert; Mazzon, Michela; Sweeney, Trevor R; Brierley, Ian; Smith, Geoffrey L

    2015-09-01

    Vaccinia virus (VACV) is the prototypic orthopoxvirus and the vaccine used to eradicate smallpox. Here we show that VACV strain Western Reserve protein 169 is a cytoplasmic polypeptide expressed early during infection that is excluded from virus factories and inhibits the initiation of cap-dependent and cap-independent translation. Ectopic expression of protein 169 causes the accumulation of 80S ribosomes, a reduction of polysomes, and inhibition of protein expression deriving from activation of multiple innate immune signaling pathways. A virus lacking 169 (vΔ169) replicates and spreads normally in cell culture but is more virulent than parental and revertant control viruses in intranasal and intradermal murine models of infection. Intranasal infection by vΔ169 caused increased pro-inflammatory cytokines and chemokines, infiltration of pulmonary leukocytes, and lung weight. These alterations in innate immunity resulted in a stronger CD8+ T-cell memory response and better protection against virus challenge. This work illustrates how inhibition of host protein synthesis can be a strategy for virus suppression of innate and adaptive immunity. PMID:26334635

  18. Advances in virus research

    SciTech Connect

    Maramorosch, K. ); Murphy, F.A. ); Shatkin, A.J. )

    1988-01-01

    This book contains eight chapters. Some of the titles are: Initiation of viral DNA replication; Vaccinia: virus, vector, vaccine; The pre-S region of hepadnavirus envelope proteins; and Archaebacterial viruses.

  19. Assembly of vaccinia virus: effects of rifampin on the intracellular distribution of viral protein p65.

    PubMed Central

    Sodeik, B; Griffiths, G; Ericsson, M; Moss, B; Doms, R W

    1994-01-01

    The cytoplasmic assembly of vaccinia virus is reversibly blocked by the antibiotic rifampin, leading to the accumulation of partially membrane-delineated rifampin bodies in infected cells. Rifampin-resistant vaccinia virus mutants have point mutations in the D13L gene, which is controlled by a late promoter and expresses a 65-kDa protein, designated p65. To further characterize the mechanism of rifampin inhibition and the function of p65 in virus assembly, we raised antibodies to this protein. Immunoreactive p65 was expressed at late times of infection, and neither its expression nor its turnover was affected by rifampin. Virus-associated p65 could be extracted only with denaturing detergents from purified virions, suggesting that it is an integral viral component. Immunofluorescence studies showed that p65 is localized to the sites of virus assembly. Also, immunoelectron microscopy showed p65 to be associated with viral crescents as well as spherical, immature virions, in both cases predominantly on the inner or concave surface. In the presence of rifampin, p65 was found in large, cytoplasmic inclusion bodies that were distinct from rifampin bodies. The rifampin bodies themselves were labeled with p65 antibodies only after reversal of the rifampin block, predominantly on the viral crescents which rapidly formed following removal of the drug. We propose that p65 functions as an internal scaffold in the formation of viral crescents and immature virions, analogously to the matrix proteins of other viruses. Images PMID:8289340

  20. Isolation and characterization of a Chinese hamster ovary mutant cell line with altered sensitivity to vaccinia virus killing.

    PubMed Central

    Bair, C H; Chung, C S; Vasilevskaya, I A; Chang, W

    1996-01-01

    The Chinese hamster ovary (CHO) cell line is nonpermissive for vaccinia virus, and translation of viral intermediate genes was reported to be blocked (A. Ramsey-Ewing and B. Moss, Virology 206:984-993, 1995). However, cells are readily killed by vaccinia virus. A vaccinia virus-resistant CHO mutant, VV5-4, was isolated by retroviral insertional mutagenesis. Parental CHO cells, upon infection with vaccinia virus, die within 2 to 3 days, whereas VV5-4 cells preferentially survive this cytotoxic effect. The survival phenotype of VV5-4 is partial and in inverse correlation with the multiplicity of infection used. In addition, viral infection fails to shut off host protein synthesis in VV5-4. VV5-4 was used to study the relationship of progression of the virus life cycle and cell fate. We found that in parental CHO cells, vaccinia virus proceeds through expression of viral early genes, uncoating, viral DNA replication, and expression of intermediate and late promoters. In contrast, we detect only expression of early genes and uncoating in VV5-4 cells, whereas viral DNA replication appears to be blocked. Consistent with the cascade regulation model of viral gene expression, we detect little intermediate- and late-gene expression in VV5-4 cells. Since vaccinia virus is known to be cytolytic, isolation of this mutant therefore demonstrates a new mode of the cellular microenvironment that affects progression of the virus life cycle, resulting in a different cell fate. This process appears to be mediated by a general mechanism, since VV5-4 is also resistant to Shope fibroma virus and myxoma virus killing. On the other hand, VV5-4 remains sensitive to cowpox virus killing. To examine the mechanism of VV5-4 survival, we investigated whether apoptosis is involved. DNA laddering and staining of apoptotic nuclei with Hoechst 33258 were observed in both CHO and VV5-4 cells infected with vaccinia virus. We concluded that the cellular pathway, which blocks viral DNA replication and

  1. A vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles.

    PubMed

    Sánchez-Puig, Juana M; Lorenzo, María M; Blasco, Rafael

    2013-01-01

    Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV) are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV) are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses. PMID:24130722

  2. The immune response to vaccinia virus is significantly reduced after scarification with TK- recombinants as compared to wild-type virus.

    PubMed

    Phillpotts, R J; Lescott, T; Gates, A J; Jones, L

    2000-01-01

    Although it is unlikely that large-scale vaccination against smallpox will ever be required again, it is conceivable that the need may arise to vaccinate against a human orthopoxvirus infection. A possible example could be the emergence of monkey poxvirus (MPV) as a significant human disease in Africa. Vaccinia virus (VV) recombinants, genetically modified to carry the immunogenic proteins of other pathogenic organisms, have potential use as vaccines against other diseases present in this region. The immune response to parental wild-type (wt) or recombinant VV was examined by binding and functional assays, relevant to protection: total IgG, IgG subclass profile, B5R gene product (gp42)-specific IgG, neutralizing antibodies and class 1-mediated cytotoxic lymphocyte activity. There was a substantial reduction in the immune response to VV after scarification with about 10(8) PFU of recombinant as compared to wt virus. These data suggest that to achieve the levels of immunity associated with protection against human orthopoxvirus infection, and to control a possible future outbreak of orthopoxvirus disease, the use of wt VV would be necessary. PMID:11155357

  3. Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus

    SciTech Connect

    Schlehofer, J.R.; Ehrbar, M.; zur Hausen, H.

    1986-07-15

    The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens (initiators) as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. As reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells.

  4. Suitability of vaccinia virus and bovine viral diarrhea virus (BVDV) for determining activities of three commonly-used alcohol-based hand rubs against enveloped viruses

    PubMed Central

    Kampf, Günter; Steinmann, Jochen; Rabenau, Holger

    2007-01-01

    Background A procedure for including activity against enveloped viruses in the post-contamination treatment of hands has been recommended, but so far no European standard is available to implement it. In 2004, the German Robert Koch-Institute (RKI) and the German Association for the Control of Virus Disease (DVV) suggested that vaccinia virus and bovine viral diarrhea virus (BVDV) should be used as test viruses in a quantitative suspension test to determine the activity of a disinfectant against all enveloped viruses. Methods We have studied the activities of three commonly-used alcohol-based hand rubs (hand rub A, based on 45% propan-2-ol, 30% propan-1-ol and 0.2% mecetronium etilsulfate; hand rub B, based on 80% ethanol; hand rub C, based on 95% ethanol) against vaccinia virus and BVDV, and in addition against four other clinically relevant enveloped viruses: herpes simplex virus (HSV) types 1 and 2, and human and avian influenza A virus. The hand rubs were challenged with different organic loads at exposure time of 15, 30 and 60 s. According to the guidelines of both BGA/RKI and DVV, and EN 14476:2005, the reduction of infectivity of each test virus was measured on appropriate cell lines using a quantitative suspension test. Results All three alcohol-based hand rubs reduced the infectivity of vaccinia virus and BVDV by ≥ 4 log10-steps within 15 s, irrespective of the type of organic load. Similar reductions of infectivity were seen against the other four enveloped viruses within 15 s in the presence of different types of organic load. Conclusion Commonly used alcohol-based hand rubs with a total alcohol concentration ≥ 75% can be assumed to be active against clinically relevant enveloped viruses if they effectively reduce the infectivities of vaccinia virus and BVDV in a quantitative suspension test. PMID:17291338

  5. Structural Insight into BH3 Domain Binding of Vaccinia Virus Antiapoptotic F1L

    PubMed Central

    Campbell, Stephanie; Thibault, John; Mehta, Ninad; Colman, Peter M.

    2014-01-01

    ABSTRACT Apoptosis is a tightly regulated process that plays a crucial role in the removal of virus-infected cells, a process controlled by both pro- and antiapoptotic members of the Bcl-2 family. The proapoptotic proteins Bak and Bax are regulated by antiapoptotic Bcl-2 proteins and are also activated by a subset of proteins known as BH3-only proteins that perform dual functions by directly activating Bak and Bax or by sequestering and neutralizing antiapoptotic family members. Numerous viruses express proteins that prevent premature host cell apoptosis. Vaccinia virus encodes F1L, an antiapoptotic protein essential for survival of infected cells that bears no discernible sequence homology to mammalian cell death inhibitors. Despite the limited sequence similarities, F1L has been shown to adopt a novel dimeric Bcl-2-like fold that enables hetero-oligomeric binding to both Bak and the proapoptotic BH3-only protein Bim that ultimately prevents Bak and Bax homo-oligomerization. However, no structural data on the mode of engagement of F1L and its Bcl-2 counterparts are available. Here we solved the crystal structures of F1L in complex with two ligands, Bim and Bak. Our structures indicate that F1L can engage two BH3 ligands simultaneously via the canonical Bcl-2 ligand binding grooves. Furthermore, by structure-guided mutagenesis, we generated point mutations within the binding pocket of F1L in order to elucidate the residues responsible for both Bim and Bak binding and prevention of apoptosis. We propose that the sequestration of Bim by F1L is primarily responsible for preventing apoptosis during vaccinia virus infection. IMPORTANCE Numerous viruses have adapted strategies to counteract apoptosis by encoding proteins responsible for sequestering proapoptotic components. Vaccinia virus, the prototypical member of the family Orthopoxviridae, encodes a protein known as F1L that functions to prevent apoptosis by interacting with Bak and the BH3-only protein Bim. Despite

  6. New vaccines against influenza virus

    PubMed Central

    Lee, Young-Tae; Kim, Ki-Hye; Ko, Eun-Ju; Lee, Yu-Na; Kim, Min-Chul; Kwon, Young-Man; Tang, Yinghua; Cho, Min-Kyoung; Lee, Youn-Jeong

    2014-01-01

    Vaccination is one of the most effective and cost-benefit interventions that prevent the mortality and reduce morbidity from infectious pathogens. However, the licensed influenza vaccine induces strain-specific immunity and must be updated annually based on predicted strains that will circulate in the upcoming season. Influenza virus still causes significant health problems worldwide due to the low vaccine efficacy from unexpected outbreaks of next epidemic strains or the emergence of pandemic viruses. Current influenza vaccines are based on immunity to the hemagglutinin antigen that is highly variable among different influenza viruses circulating in humans and animals. Several scientific advances have been endeavored to develop universal vaccines that will induce broad protection. Universal vaccines have been focused on regions of viral proteins that are highly conserved across different virus subtypes. The strategies of universal vaccines include the matrix 2 protein, the hemagglutinin HA2 stalk domain, and T cell-based multivalent antigens. Supplemented and/or adjuvanted vaccination in combination with universal target antigenic vaccines would have much promise. This review summarizes encouraging scientific advances in the field with a focus on novel vaccine designs. PMID:24427759

  7. Computer Bytes, Viruses and Vaccines.

    ERIC Educational Resources Information Center

    Palmore, Teddy B.

    1989-01-01

    Presents a history of computer viruses, explains various types of viruses and how they affect software or computer operating systems, and describes examples of specific viruses. Available vaccines are explained, and precautions for protecting programs and disks are given. (nine references) (LRW)

  8. A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen

    PubMed Central

    Vijayan, Aneesh; García-Arriaza, Juan; C. Raman, Suresh; Conesa, José Javier; Chichón, Francisco Javier; Santiago, César; Sorzano, Carlos Óscar S.; Carrascosa, José L.; Esteban, Mariano

    2015-01-01

    In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs), gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1β, IFN-γ, IL-6, IL-8, IL-12, RANTES). Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1-specific adaptive

  9. Genome Sequence of WAU86/88-1, a New Variant of Vaccinia Virus Lister Strain from Poland.

    PubMed

    Mavian, Carla; López-Bueno, Alberto; Alcamí, Antonio

    2014-01-01

    The poxviruses Warsaw Agricultural University 86 (WAU86) and 88-1 (WAU88-1) were isolated in 1986 to 1988 from separate outbreaks in laboratory mice in Poland and described as ectromelia virus isolates. The genome sequences of these poxviruses reveal that they are almost identical and represent a novel variant of the vaccinia virus Lister strain. PMID:24407630

  10. Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

    PubMed Central

    Hsiao, Jye-Chian; Chu, Li-Wei; Lo, Yung-Tsun; Lee, Sue-Ping; Chen, Tzu-Jung; Huang, Cheng-Yen

    2015-01-01

    ABSTRACT Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex

  11. An improved high pressure freezing and freeze substitution method to preserve the labile vaccinia virus nucleocapsid.

    PubMed

    Jesus, Desyree Murta; Moussatche, Nissin; Condit, Richard C

    2016-07-01

    In recent years, high pressure freezing and freeze substitution have been widely used for electron microscopy to reveal viral and cellular structures that are difficult to preserve. Vaccinia virus, a member of the Poxviridae family, presents one of the most complex viral structures. The classical view of vaccinia virus structure consists of an envelope surrounding a biconcave core, with a lateral body in each concavity of the core. This classical view was challenged by Peters and Muller (1963), who demonstrated the presence of a folded tubular structure inside the virus core and stated the difficulty in visualizing this structure, possibly because it is labile and cannot be preserved by conventional sample preparation. Therefore, this tubular structure, now called the nucleocapsid, has been mostly neglected over the years. Earlier studies were able to preserve the nucleocapsid, but with low efficiency. In this study, we report the protocol (and troubleshooting) that resulted in preservation of the highest numbers of nucleocapsids in several independent preparations. Using this protocol, we were able to demonstrate an interdependence between the formation of the virus core wall and the nucleocapsid, leading to the hypothesis that an interaction exists between the major protein constituents of these compartments, A3 (core wall) and L4 (nucleocapsid). Our results show that high pressure freezing and freeze substitution can be used in more in-depth studies concerning the nucleocapsid structure and function. PMID:27155322

  12. Granzyme B Inhibits Vaccinia Virus Production through Proteolytic Cleavage of Eukaryotic Initiation Factor 4 Gamma 3

    PubMed Central

    Marcet-Palacios, Marcelo; Duggan, Brenda Lee; Shostak, Irene; Barry, Michele; Geskes, Tracy; Wilkins, John A.; Yanagiya, Akiko; Sonenberg, Nahum; Bleackley, R. Chris

    2011-01-01

    Cytotoxic T lymphocytes (CTLs) are the major killer of virus-infected cells. Granzyme B (GrB) from CTLs induces apoptosis in target cells by cleavage and activation of substrates like caspase-3 and Bid. However, while undergoing apoptosis, cells are still capable of producing infectious viruses unless a mechanism exists to specifically inhibit viral production. Using proteomic approaches, we identified a novel GrB target that plays a major role in protein synthesis: eukaryotic initiation factor 4 gamma 3 (eIF4G3). We hypothesized a novel role for GrB in translation of viral proteins by targeting eIF4G3, and showed that GrB cleaves eIF4G3 specifically at the IESD1408S sequence. Both GrB and human CTL treatment resulted in degradation of eIF4G3 and reduced rates of translation. When Jurkat cells infected with vaccinia virus were treated with GrB, there was a halt in viral protein synthesis and a decrease in production of infectious new virions. The GrB-induced inhibition of viral translation was independent of the activation of caspases, as inhibition of protein synthesis still occurred with addition of the pan-caspase inhibitor zVAD-fmk. This demonstrated for the first time that GrB prevents the production of infectious vaccinia virus by targeting the host translational machinery. PMID:22194691

  13. A novel naturally occurring tandem promoter in modified vaccinia virus ankara drives very early gene expression and potent immune responses.

    PubMed

    Wennier, Sonia T; Brinkmann, Kay; Steinhäußer, Charlotte; Mayländer, Nicole; Mnich, Claudia; Wielert, Ursula; Dirmeier, Ulrike; Hausmann, Jürgen; Chaplin, Paul; Steigerwald, Robin

    2013-01-01

    Modified vaccinia virus Ankara (MVA) has been shown to be suitable for the generation of experimental vaccines against cancer and infectious diseases, eliciting strong humoral and cellular immune responses. In viral vectored vaccines, strong recombinant antigen expression and timing of expression influence the quantity and quality of the immune response. Screening of synthetic and native poxvirus promoters for strong protein expression in vitro and potent immune responses in vivo led to the identification of the MVA13.5L promoter, a unique and novel naturally occurring tandem promoter in MVA composed of two 44 nucleotide long repeated motifs, each containing an early promoter element. The MVA13.5L gene is highly conserved across orthopoxviruses, yet its function is unknown. The unique structure of its promoter is not found for any other gene in the MVA genome and is also conserved in other orthopoxviruses. Comparison of the MVA13.5L promoter activity with synthetic poxviral promoters revealed that the MVA13.5L promoter produced higher levels of protein early during infection in HeLa cells and particularly in MDBK cells, a cell line in which MVA replication stops at an early stage before the expression of late genes. Finally, a recombinant antigen expressed under the control of this novel promoter induced high antibody titers and increased CD8 T cell responses in homologous prime-boost immunization compared to commonly used promoters. In particular, the recombinant antigen specific CD8 T cell responses dominated over the immunodominant B8R vector-specific responses after three vaccinations and even more during the memory phase. These results have identified the native MVA13.5L promoter as a new potent promoter for use in MVA vectored preventive and therapeutic vaccines. PMID:23951355

  14. A Novel Naturally Occurring Tandem Promoter in Modified Vaccinia Virus Ankara Drives Very Early Gene Expression and Potent Immune Responses

    PubMed Central

    Wennier, Sonia T.; Brinkmann, Kay; Steinhäußer, Charlotte; Mayländer, Nicole; Mnich, Claudia; Wielert, Ursula; Dirmeier, Ulrike; Hausmann, Jürgen; Chaplin, Paul; Steigerwald, Robin

    2013-01-01

    Modified vaccinia virus Ankara (MVA) has been shown to be suitable for the generation of experimental vaccines against cancer and infectious diseases, eliciting strong humoral and cellular immune responses. In viral vectored vaccines, strong recombinant antigen expression and timing of expression influence the quantity and quality of the immune response. Screening of synthetic and native poxvirus promoters for strong protein expression in vitro and potent immune responses in vivo led to the identification of the MVA13.5L promoter, a unique and novel naturally occurring tandem promoter in MVA composed of two 44 nucleotide long repeated motifs, each containing an early promoter element. The MVA13.5L gene is highly conserved across orthopoxviruses, yet its function is unknown. The unique structure of its promoter is not found for any other gene in the MVA genome and is also conserved in other orthopoxviruses. Comparison of the MVA13.5L promoter activity with synthetic poxviral promoters revealed that the MVA13.5L promoter produced higher levels of protein early during infection in HeLa cells and particularly in MDBK cells, a cell line in which MVA replication stops at an early stage before the expression of late genes. Finally, a recombinant antigen expressed under the control of this novel promoter induced high antibody titers and increased CD8 T cell responses in homologous prime-boost immunization compared to commonly used promoters. In particular, the recombinant antigen specific CD8 T cell responses dominated over the immunodominant B8R vector-specific responses after three vaccinations and even more during the memory phase. These results have identified the native MVA13.5L promoter as a new potent promoter for use in MVA vectored preventive and therapeutic vaccines. PMID:23951355

  15. The heterogeneity of human antibody responses to vaccinia virus revealed through use of focused protein arrays.

    PubMed

    Duke-Cohan, Jonathan S; Wollenick, Kristin; Witten, Elizabeth A; Seaman, Michael S; Baden, Lindsey R; Dolin, Raphael; Reinherz, Ellis L

    2009-02-18

    The renewed interest in strategies to combat infectious agents with epidemic potential has led to a re-examination of vaccination protocols against smallpox. To help define which antigens elicit a human antibody response, we have targeted proteins known or predicted to be presented on the surface of the intracellular mature virion (IMV) or the extracellular enveloped virion (EEV). The predicted ectodomains were expressed in a mammalian in vitro coupled transcription/translation reaction using tRNA(lys) precharged with lysine-epsilon-biotin followed by solid phase immobilization on 384-well neutravidin-coated plates. The generated array is highly specific and sensitive in a micro-ELISA format. By comparison of binding of vaccinia-immune sera to the reticulocyte lysate-produced proteins and to secreted post-translationally modified proteins, we demonstrate that for several proteins including the EEV proteins B5 and A33, proper recognition is dependent upon appropriate folding, with little dependence upon glycosylation per se. We further demonstrate that the humoral immune response to vaccinia among different individuals is not uniform in specificity or strength, as different IMV and EEV targets predominate within the group of immunogenic proteins. This heterogeneity likely results from the diversity of HLA Class II alleles and CD4 T helper cell epitopes stimulating B cell antibody production. Our findings have important implications both for design of new recombinant subunit vaccines as well as for methods of assaying the human antibody response utilizing recombinant proteins produced in vitro. PMID:19146908

  16. Fine structure marker rescue of temperature-sensitive mutations of vaccinia virus within a central conserved region of the genome.

    PubMed Central

    Ensinger, M J; Weir, J P; Moss, B

    1985-01-01

    Fine structure marker rescue involving the use of subfragments of vaccinia virus HindIII DNA fragments L, J, and H has been used to map the mutations in eight temperature-sensitive mutants of vaccinia virus representing four complementation groups. Comparison of their map locations with the positions of the open reading frames and RNA transcripts that have been mapped within this region has allowed the identification of one or two polypeptides as the temperature-sensitive gene product of each mutant. PMID:4068140

  17. Nucleotide sequence of a cluster of early and late genes in a conserved segment of the vaccinia virus genome.

    PubMed Central

    Plucienniczak, A; Schroeder, E; Zettlmeissl, G; Streeck, R E

    1985-01-01

    The nucleotide sequence of a 7.6 kb vaccinia DNA segment from a genomic region conserved among different orthopox virus has been determined. This segment contains a tight cluster of 12 partly overlapping open reading frames most of which can be correlated with previously identified early and late proteins and mRNAs. Regulatory signals used by vaccinia virus have been studied. Presumptive promoter regions are rich in A, T and carry the consensus sequences TATA and AATAA spaced at 20-24 base pairs. Tandem repeats of a CTATTC consensus sequence are proposed to be involved in the termination of early transcription. PMID:2987815

  18. Vaccination of chimpanzees against infection by the hepatitis C virus.

    PubMed Central

    Choo, Q L; Kuo, G; Ralston, R; Weiner, A; Chien, D; Van Nest, G; Han, J; Berger, K; Thudium, K; Kuo, C

    1994-01-01

    A high incidence of community-acquired hepatitis C virus infection that can lead to the progressive development of chronic active hepatitis, liver cirrhosis, and primary hepatocellular carcinoma occurs throughout the world. A vaccine to control the spread of this agent that represents a major cause of chronic liver disease is therefore needed. Seven chimpanzees (Pan troglodytes) have been immunized with both putative envelope glycoproteins [E1 (gp33) and E2 (gp72)] that were copurified from HeLa cells infected with a recombinant vaccinia virus expression vector. Despite the induction of a weak humoral immune response to these viral glycoproteins in experimentally infected chimpanzees, a strong humoral immune response was obtained in all vaccines. The five highest responders showed complete protection against an i.v. challenge with homologous hepatitis C virus 1. The remaining two vaccines became infected, but both infection and disease may have been ameliorated in comparison with four similarly challenged control chimpanzees, all of which developed acute hepatitis and chronic infections. These results provide considerable encouragement for the eventual control of hepatitis C virus infection by vaccination. PMID:7509068

  19. Vaccination of chimpanzees against infection by the hepatitis C virus.

    PubMed

    Choo, Q L; Kuo, G; Ralston, R; Weiner, A; Chien, D; Van Nest, G; Han, J; Berger, K; Thudium, K; Kuo, C

    1994-02-15

    A high incidence of community-acquired hepatitis C virus infection that can lead to the progressive development of chronic active hepatitis, liver cirrhosis, and primary hepatocellular carcinoma occurs throughout the world. A vaccine to control the spread of this agent that represents a major cause of chronic liver disease is therefore needed. Seven chimpanzees (Pan troglodytes) have been immunized with both putative envelope glycoproteins [E1 (gp33) and E2 (gp72)] that were copurified from HeLa cells infected with a recombinant vaccinia virus expression vector. Despite the induction of a weak humoral immune response to these viral glycoproteins in experimentally infected chimpanzees, a strong humoral immune response was obtained in all vaccines. The five highest responders showed complete protection against an i.v. challenge with homologous hepatitis C virus 1. The remaining two vaccines became infected, but both infection and disease may have been ameliorated in comparison with four similarly challenged control chimpanzees, all of which developed acute hepatitis and chronic infections. These results provide considerable encouragement for the eventual control of hepatitis C virus infection by vaccination. PMID:7509068

  20. Efficiently Editing the Vaccinia Virus Genome by Using the CRISPR-Cas9 System

    PubMed Central

    Yuan, Ming; Zhang, Wensheng; Wang, Jun; Al Yaghchi, Chadwan; Ahmed, Jahangir; Chard, Louisa

    2015-01-01

    Vaccinia virus (VACV) continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer since its use for the eradication of smallpox. However, the current method of editing the VACV genome is not efficient. Here, we demonstrate that the CRISPR-Cas9 system can be used to edit the VACV genome rapidly and efficiently. Additionally, a set of 8,964 computationally designed unique guide RNAs (gRNAs) targeting all VACV genes will be valuable for the study of VACV gene functions. PMID:25741005

  1. Efficiently editing the vaccinia virus genome by using the CRISPR-Cas9 system.

    PubMed

    Yuan, Ming; Zhang, Wensheng; Wang, Jun; Al Yaghchi, Chadwan; Ahmed, Jahangir; Chard, Louisa; Lemoine, Nick R; Wang, Yaohe

    2015-05-01

    Vaccinia virus (VACV) continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer since its use for the eradication of smallpox. However, the current method of editing the VACV genome is not efficient. Here, we demonstrate that the CRISPR-Cas9 system can be used to edit the VACV genome rapidly and efficiently. Additionally, a set of 8,964 computationally designed unique guide RNAs (gRNAs) targeting all VACV genes will be valuable for the study of VACV gene functions. PMID:25741005

  2. Mapping of a gene coding for a major late structural polypeptide on the vaccinia virus genome.

    PubMed Central

    Wittek, R; Hänggi, M; Hiller, G

    1984-01-01

    Cell-free translation of total RNA isolated from vaccinia virus-infected cells late in infection results in a complex mixture of polypeptides. A monospecific antibody directed against one of the major structural proteins of the virus particle immunoprecipitated a single polypeptide with a molecular weight of 11,000 (11K) from this mixture. Immunoprecipitation was therefore used to identify the structural polypeptide among the in vitro translation products of RNA purified by hybridization selection to restriction fragments of the vaccinia virus genome. This allowed us to map the mRNA coding for the 11K polypeptide to the extreme left-hand end of the HindIII E fragment. Detailed transcriptional mapping of this region of the genome by nuclease S1 analysis revealed the presence of a late RNA transcribed from the rightward-reading strand. Its 5' end mapped at ca. 130 base pairs to the left of the HindIII site at the junction between the HindIII F and E fragments. The map position of this RNA coincided precisely with the map position of the late message coding for the 11K polypeptide. Images PMID:6319738

  3. Whole Cell Cryo-Electron Tomography Reveals Distinct Disassembly Intermediates of Vaccinia Virus

    PubMed Central

    Cyrklaff, Marek; Linaroudis, Alexandros; Boicu, Marius; Chlanda, Petr; Baumeister, Wolfgang; Griffiths, Gareth; Krijnse-Locker, Jacomine

    2007-01-01

    At each round of infection, viruses fall apart to release their genome for replication, and then reassemble into stable particles within the same host cell. For most viruses, the structural details that underlie these disassembly and assembly reactions are poorly understood. Cryo-electron tomography (cryo-ET), a unique method to investigate large and asymmetric structures at the near molecular resolution, was previously used to study the complex structure of vaccinia virus (VV). Here we study the disassembly of VV by cryo-ET on intact, rapidly frozen, mammalian cells, infected for up to 60 minutes. Binding to the cell surface induced distinct structural rearrangements of the core, such as a shape change, the rearrangement of its surface spikes and de-condensation of the viral DNA. We propose that the cell surface induced changes, in particular the decondensation of the viral genome, are a prerequisite for the subsequent release of the vaccinia DNA into the cytoplasm, which is followed by its cytoplasmic replication. Generally, this is the first study that employs whole cell cryo-ET to address structural details of pathogen-host cell interaction. PMID:17487274

  4. Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses.

    PubMed

    Ralston, R; Thudium, K; Berger, K; Kuo, C; Gervase, B; Hall, J; Selby, M; Kuo, G; Houghton, M; Choo, Q L

    1993-11-01

    We constructed recombinant vaccinia virus vectors for expression of the structural region of hepatitis C virus (HCV). Infection of mammalian cells with a vector (vv/HCV1-906) encoding C-E1-E2-NS2 generated major protein species of 22 kDa (C), 33 to 35 kDa (E1), and 70 to 72 kDa (E2), as observed previously with other mammalian expression systems. The bulk of the E1 and E2 expressed by vv/HCV1-906 was found integrated into endoplasmic reticulum membranes as core-glycosylated species, suggesting that these E1 and E2 species represent intracellular forms of the HCV envelope proteins. HCV E1 and E2 formed E1-E2 complexes which were precipitated by either anti-E1 or anti-E2 serum and which sedimented at approximately 15 S on glycerol density gradients. No evidence of intermolecular disulfide bonding between E1 and E2 was detected. E1 and E2 were copurified to approximately 90% purity by mild detergent extraction followed by chromatography on Galanthus nivalus lectin-agarose and DEAE-Fractogel. Immunization of chimpanzees with purified E1-E2 generated high titers of anti-E1 and anti-E2 antibodies. Further studies, to be reported separately, demonstrated that purified E1-E2 complexes were recognized at high frequency by HCV+ human sera (D. Y. Chien, Q.-L. Choo, R. Ralston, R. Spaete, M. Tong, M. Houghton, and G. Kuo, Lancet, in press) and generated protective immunity in chimpanzees (Q.-L. Choo, G. Kuo, R. Ralston, A. Weiner, D. Chien, G. Van Nest, J. Han, K. Berger, K. Thudium, J. Kansopon, J. McFarland, A. Tabrizi, K. Ching, B. Mass, L. B. Cummins, E. Muchmore, and M. Houghton, submitted for publication), suggesting that these purified HCV envelope proteins display native HCV epitopes. PMID:8411378

  5. New approaches to the development of virus vaccines for veterinary use.

    PubMed

    Yamanouchi, K; Barrett, T; Kai, C

    1998-12-01

    The marked progress in recombinant deoxyribonucleic acid (DNA) technology during the past decade has led to the development of a variety of safe new vaccine vectors which are capable of efficiently expressing foreign immunogens. These have been based on a variety of virus types--poxviruses, herpesviruses and adenoviruses--and have led to the production of many new potential recombinant vaccines. Of these recombinant vaccines, the rabies vaccine, in which the rabies G protein is expressed in a vaccinia vector, has been widely used in the field to prevent the spread of rabies both in Europe and in the United States of America. A recombinant Newcastle disease virus vaccine, using fowlpox virus as the vector to express immunogenic proteins from the Newcastle disease virus, has been licensed as the first commercial recombinant vectored vaccine. Many other recombinant virus vaccines are still at the stage of laboratory or field testing. The most recent breakthrough in vaccinology has been the success with the use of naked DNA as a means of vaccination. This approach has shown great promise in mouse model systems and has now become the most active field in new vaccine development. Molecular redesigning of conventional ribonucleic acid (RNA) viruses to obtain more stable attenuated vaccines was previously possible only for positive-strand RNA viruses, such as poliovirus. However, recent advances in molecular biological techniques have enabled the rescuing of negative-strand viruses from DNA copies of their genomes. This has made it possible to engineer specific changes in the genomes of Rhabdoviridae and Paramyxoviridae, both of which include several viruses of veterinary importance. The authors describe the current progress in the development of vector vaccines, DNA vaccines and vaccines based on engineered positive- and negative-strand RNA virus genomes, with special emphasis on their application to diseases of veterinary importance. PMID:9850535

  6. Inhibition of Vaccinia virus entry by a broad spectrum antiviral peptide

    SciTech Connect

    Altmann, S.E.; Jones, J.C.; Schultz-Cherry, S.; Brandt, C.R.

    2009-06-05

    Concerns about the possible use of Variola virus, the causative agent of smallpox, as a weapon for bioterrorism have led to renewed efforts to identify new antivirals against orthopoxviruses. We identified a peptide, EB, which inhibited infection by Vaccinia virus with an EC{sub 50} of 15 muM. A control peptide, EBX, identical in composition to EB but differing in sequence, was inactive (EC{sub 50} > 200 muM), indicating sequence specificity. The inhibition was reversed upon removal of the peptide, and EB treatment had no effect on the physical integrity of virus particles as determined by electron microscopy. Viral adsorption was unaffected by the presence of EB, and the addition of EB post-entry had no effect on viral titers or on early gene expression. The addition of EB post-adsorption resulted in the inhibition of beta-galactosidase expression from an early viral promoter with an EC{sub 50} of 45 muM. A significant reduction in virus entry was detected in the presence of the peptide when the number of viral cores released into the cytoplasm was quantified. Electron microscopy indicated that 88% of the virions remained on the surface of cells in the presence of EB, compared to 37% in the control (p < 0.001). EB also blocked fusion-from-within, suggesting that virus infection is inhibited at the fusion step. Analysis of EB derivatives suggested that peptide length may be important for the activity of EB. The EB peptide is, to our knowledge, the first known small molecule inhibitor of Vaccinia virus entry.

  7. Chemokine (C-C Motif) Receptor 1 Is Required for Efficient Recruitment of Neutrophils during Respiratory Infection with Modified Vaccinia Virus Ankara

    PubMed Central

    Price, Philip J. R.; Luckow, Bruno; Torres-Domínguez, Lino E.; Brandmüller, Christine; Zorn, Julia; Kirschning, Carsten J.; Sutter, Gerd

    2014-01-01

    ABSTRACT Modified vaccinia virus Ankara (MVA) serves as a versatile platform in vaccine development. This highly attenuated orthopoxvirus, which cannot replicate in mammalian cells, triggers strong innate immune responses, including cell migration. Previously, we have shown that induction of chemokine (C-C motif) ligand 2 (CCL2) by MVA is necessary for the recruitment of monocytes and T cells, but not neutrophils, to the lung. Here, we identified neutrophil-attracting chemokines produced by MVA-infected primary murine lung fibroblasts and murine bone marrow-derived macrophages. We demonstrate that MVA, but not vaccinia virus (VACV) strain WR, induces chemokine expression, which is independent of Toll-like receptor 2 (TLR2) signaling. Additionally, we show that both chemokine (C-C motif) receptor 1 (CCR1) and chemokine (C-X-C motif) receptor 2 (CXCR2) are involved in MVA-induced neutrophil chemotaxis in vitro. Finally, intranasal infection of Ccr1−/− mice with MVA, as well as application of the CCR1 antagonist J-113863, revealed a role for CCR1 in leukocyte recruitment, including neutrophils, into the lung. IMPORTANCE Rapid attraction of leukocytes to the site of inoculation is unique to MVA in comparison to other VACV strains. The findings here extend current knowledge about the regulation of MVA-induced leukocyte migration, particularly regarding neutrophils, which could potentially be exploited to improve other VACV strains currently in development as oncolytic viruses and viral vectors. Additionally, the data presented here indicate that the inflammatory response may vary depending on the cell type infected by MVA, highlighting the importance of the site of vaccine application. Moreover, the rapid recruitment of neutrophils and other leukocytes can directly contribute to the induction of adaptive immune responses elicited by MVA inoculation. Thus, a better understanding of leukocyte migration upon MVA infection is particularly relevant for further

  8. Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins

    SciTech Connect

    Zheng Min; Jin Ningyi; Liu Qi; Huo Xiaowei; Li Yang; Hu Bo; Ma Haili; Zhu Zhanbo; Cong Yanzhao; Li Xiao; Jin Minglan; Zhu Guangze

    2009-08-15

    Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AAL and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.

  9. Structural and Biochemical Characterization of the Vaccinia Virus Envelope Protein D8 and Its Recognition by the Antibody LA5

    PubMed Central

    Matho, Michael H.; Maybeno, Matt; Benhnia, Mohammed Rafii-El-Idrissi; Becker, Danielle; Meng, Xiangzhi; Xiang, Yan; Crotty, Shane; Peters, Bjoern

    2012-01-01

    Smallpox vaccine is considered a gold standard of vaccines, as it is the only one that has led to the complete eradication of an infectious disease from the human population. B cell responses are critical for the protective immunity induced by the vaccine, yet their targeted epitopes recognized in humans remain poorly described. Here we describe the biochemical and structural characterization of one of the immunodominant vaccinia virus (VACV) antigens, D8, and its binding to the monoclonal antibody LA5, which is capable of neutralizing VACV in the presence of complement. The full-length D8 ectodomain was found to form a tetramer. We determined the crystal structure of the LA5 Fab-monomeric D8 complex at a resolution of 2.1 Å, as well as the unliganded structures of D8 and LA5-Fab at resolutions of 1.42 Å and 1.6 Å, respectively. D8 features a carbonic anhydrase (CAH) fold that has evolved to bind to the glycosaminoglycan (GAG) chondroitin sulfate (CS) on host cells. The central positively charged crevice of D8 was predicted to be the CS binding site by automated docking experiments. Furthermore, sequence alignment of various poxvirus D8 orthologs revealed that this crevice is structurally conserved. The D8 epitope is formed by 23 discontinuous residues that are spread across 80% of the D8 protein sequence. Interestingly, LA5 binds with a high-affinity lock-and-key mechanism above this crevice with an unusually large antibody-antigen interface, burying 2,434 Å2 of protein surface. PMID:22623786

  10. Detection of Vaccinia virus in blood and faeces of experimentally infected cows.

    PubMed

    Guedes, M I M C; Rehfeld, I S; de Oliveira, T M L; Assis, F L; Matos, A C D; Abrahão, J S; Kroon, E G; Lobato, Z I P

    2013-12-01

    Bovine vaccinia (BV), a zoonosis caused by Vaccinia virus (VACV), affects dairy cattle and milkers, causing economic, veterinary and human health impacts. Despite such impacts, there are no experimental studies about the pathogenesis of BV in cows to assess whether there is a systemic spread of the virus and whether there are different ways of VACV shedding. Trying to answer some of these questions, a study was proposed using experimental inoculation of VACV in cows. All experimentally infected cows developed lesions compatible with VACV infection in cattle. Two of the six animals presented VACV DNA in blood and faecal samples, starting at the 2nd and the 3rd day post-infection (d.p.i.), respectively, and lasting until the 36th d.p.i., in an intermittent way. This study provides new evidence that VACV can be detected in blood and faeces of infected cows, suggesting that BV could be a systemic disease, and also bringing new information about the epidemiology and pathogenesis of BV. PMID:22909142

  11. Safety, Immunogenicity and Efficacy of Modified Vaccinia Ankara (MVA) Against Dryvax® Challenge in Vaccinia-Naïve and Vaccinia-Immune Individuals

    PubMed Central

    Parrino, Janie; McCurdy, Lewis H.; Larkin, Brenda D.; Gordon, Ingelise J.; Rucker, Steven E.; Enama, Mary E.; Koup, Richard A.; Roederer, Mario; Bailer, Robert T.; Moodie, Zoe; Gu, Lin; Yan, Lihan; Graham, Barney S.

    2007-01-01

    Modified vaccinia Ankara (MVA) was evaluated as an alternative to Dryvax® in vaccinia-naïve and immune adult volunteers. Subjects received intramuscular MVA or placebo followed by Dryvax® challenge at 3 months. Two or more doses of MVA prior to Dryvax® reduced severity of lesion formation, decreased magnitude and duration of viral shedding, and augmented post-Dryvax® vaccinia-specific CD8+ T cell responses and extracellular enveloped virus protein-specific antibody responses. MVA vaccination is safe and immunogenic and improves the safety and immunogenicity of subsequent Dryvax® vaccination supporting the potential for using MVA as a vaccine in the general population to improve immunity to orthopoxviruses. PMID:17126963

  12. Dogs and Opossums Positive for Vaccinia Virus during Outbreak Affecting Cattle and Humans, São Paulo State, Brazil

    PubMed Central

    Peres, Marina G.; Barros, Claudenice B.; Appolinário, Camila M.; Antunes, João M.A.P.; Mioni, Mateus S.R.; Bacchiega, Thais S.; Allendorf, Susan D.; Vicente, Acácia F.; Fonseca, Clóvis R.

    2016-01-01

    During a vaccinia virus (VACV) outbreak in São Paulo State, Brazil, blood samples were collected from cows, humans, other domestic animals, and wild mammals. Samples from 3 dogs and 3 opossums were positive for VACV by PCR. Results of gene sequencing yielded major questions regarding other mammalian species acting as reservoirs of VACV. PMID:26812352

  13. Dogs and Opossums Positive for Vaccinia Virus during Outbreak Affecting Cattle and Humans, São Paulo State, Brazil.

    PubMed

    Peres, Marina G; Barros, Claudenice B; Appolinário, Camila M; Antunes, João M A P; Mioni, Mateus S R; Bacchiega, Thais S; Allendorf, Susan D; Vicente, Acácia F; Fonseca, Clóvis R; Megid, Jane

    2016-02-01

    During a vaccinia virus (VACV) outbreak in São Paulo State, Brazil, blood samples were collected from cows, humans, other domestic animals, and wild mammals. Samples from 3 dogs and 3 opossums were positive for VACV by PCR. Results of gene sequencing yielded major questions regarding other mammalian species acting as reservoirs of VACV. PMID:26812352

  14. Human immunodeficiency virus vaccines.

    PubMed

    Goepfert, Paul; Bansal, Anju

    2014-12-01

    Although some success was achieved in recent years in HIV prevention, an effective vaccine remains the means with the most potential of curtailing HIV-1 infections worldwide. Despite multiple failed attempts, a recent HIV vaccine regimen demonstrated modest protection from infection. Although the protective efficacy in this trial was not sufficient to warrant licensure, it spurred renewed optimism in the field and has provided valuable insights for improving future vaccine designs. This review summarizes the pertinent details of vaccine development and discusses ways the field is moving forward to develop a vaccine to prevent HIV infection and disease progression. PMID:25287587

  15. Expression of the highly conserved vaccinia virus E6 protein is required for virion morphogenesis

    SciTech Connect

    Resch, Wolfgang; Weisberg, Andrea S.; Moss, Bernard

    2009-04-10

    The vaccinia virus E6R gene (VACVWR062) is conserved in all members of the poxvirus family and encodes a protein associated with the mature virion. We confirmed this association and provided evidence for an internal location. An inducible mutant that conditionally expresses E6 was constructed. In the absence of inducer, plaque formation and virus production were severely inhibited in several cell lines, whereas some replication occurred in others. This difference could be due to variation in the stringency of repression, since we could not isolate a stable deletion mutant even in the more 'permissive' cells. Under non-permissive conditions, viral late proteins were synthesized but processing of core proteins was inefficient, indicative of an assembly block. Transmission electron microscopy of sections of cells infected with the mutant in the absence of inducer revealed morphogenetic defects with crescents and empty immature virions adjacent to dense inclusions of viroplasm. Mature virions were infrequent and cores appeared to have lucent centers.

  16. The E6 protein from vaccinia virus is required for the formation of immature virions

    SciTech Connect

    Boyd, Olga; Turner, Peter C.; Moyer, Richard W.; Condit, Richard C.; Moussatche, Nissin

    2010-04-10

    An IPTG-inducible mutant in the E6R gene of vaccinia virus was used to study the role of the E6 virion core protein in viral replication. In the absence of the inducer, the mutant exhibited a normal pattern DNA replication, concatemer resolution and late gene expression, but it showed an inhibition of virion structural protein processing it failed to produce infectious particles. Electron microscopic analysis showed that in the absence of IPTG viral morphogenesis was arrested before IV formation: crescents, aberrant or empty IV-like structures, and large aggregated virosomes were observed throughout the cytoplasm. The addition of IPTG to release a 12-h block showed that virus infectious particles could be formed in the absence of de novo DNA synthesis. Our observations show that in the absence of E6 the association of viroplasm with viral membrane crescents is impaired.

  17. Molecular genetic analysis of a vaccinia virus gene with an essential role in DNA replication.

    PubMed Central

    Evans, E; Traktman, P

    1987-01-01

    We have identified a gene encoded by vaccinia virus which is essential for DNA replication. The gene, located in the HindIII D fragment of the viral genome, is transcribed early after infection into two transcripts of 3.0 and 3.7 kilobases which share a 3' terminus. The lesions of three temperature-sensitive DNA replication mutants with defects in this gene have been localized by marker rescue with progressively smaller DNA fragments. We have determined by hybrid selection that the gene encodes an 82-kilodalton protein. An antibody has been prepared against this polypeptide and used to quantitate expression of the protein after infection with wild-type virus or with a viral mutant whose lesion maps within this gene. The temporal pattern of expression in the mutant is unaffected, but the product encoded by the mutant is significantly more thermolabile than the wild-type protein. Images PMID:3041037

  18. Vaccinia viruses with a serpin gene deletion and expressing IFN-γ induce potent immune responses without detectable replication in vivo

    PubMed Central

    Legrand, Fatema A.; Verardi, Paulo H.; Chan, Kenneth S.; Peng, Yue; Jones, Leslie A.; Yilma, Tilahun D.

    2005-01-01

    In a continuing effort to develop safe and efficacious vaccine and immunotherapeutic vectors, we constructed recombinant vaccinia virus (rVV) vaccines lacking either the B13R (SPI-2) or the B22R (SPI-1) immune-modulating gene and coexpressing IFN-γ. B13R and B22R are nonessential VV immune-modulating genes that have antiapoptotic and antiinflammatory properties with sequence homology to serine protease inhibitors (serpins). IFN-γ is a cytokine with potent immunoregulatory, antineoplastic, and antiviral properties. We observed that these rVVs with a deletion in a serpin gene and expressing IFN-γ replicated to high titers in tissue culture yet were avirulent in both immunocompromised and immunocompetent mice with no detectable viral replication in these animals. A single immunization elicited potent humoral, T helper, and cytotoxic T cell immune responses in mice despite the absence of any detectable virus replication in vivo. IFN-γ coexpression and the inactivation of one or more VV immune-modulating genes provide an optimized method for increasing the safety while maintaining the efficacy of rVV vaccines. This strategy provides a method for developing highly safe and efficacious vaccines for smallpox and other diseases and immunotherapeutic vectors. PMID:15705716

  19. Vaccination of prostate cancer patients with modified vaccinia ankara delivering the tumor antigen 5T4 (TroVax): a phase 2 trial.

    PubMed

    Amato, Robert J; Drury, Noel; Naylor, Stuart; Jac, Jaroslaw; Saxena, Somya; Cao, Amy; Hernandez-McClain, Joan; Harrop, Richard

    2008-01-01

    The attenuated vaccinia virus, modified vaccinia Ankara, has been engineered to deliver the tumor antigen 5T4 (TroVax). TroVax has been evaluated in an open-label phase 2 trial in hormone refractory prostate cancer patients in which the vaccine was administered either alone or in combination with granulocyte macrophage-colony stimulating factor (GM-CSF). The comparative safety and immunologic and clinical efficacy of TroVax alone or in combination with GM-CSF was determined. Twenty-seven patients with metastatic hormone refractory prostate cancer were treated with TroVax alone (n=14) or TroVax+GM-CSF (n=13). 5T4-specific cellular and humoral responses were monitored throughout the study. Clinical responses were assessed by quantifying prostate-specific antigen concentrations and measuring changes in tumor burden by computer-assisted tomography scan. TroVax was well tolerated in all patients with no serious adverse events attributed to vaccination. Of 24 immunologically evaluable patients, all mounted 5T4-specific antibody responses. Periods of disease stabilization from 2 to >10 months were observed. Time to progression was significantly greater in patients who mounted 5T4-specific cellular responses compared with those who did not (5.6 vs. 2.3 mo, respectively). There were no objective clinical responses seen in this study. In this study, the combination of GM-CSF with TroVax showed similar clinical and immunologic responses to TroVax alone. The high frequency of 5T4-specific immune responses and relationship with enhanced time to progression is encouraging and warrants further investigation. PMID:18528296

  20. Vaccinia Virus Virulence Factor N1L is a Novel Promising Target for Antiviral Therapeutic Intervention

    PubMed Central

    Cheltsov, Anton V.; Aoyagi, Mika; Aleshin, Alexander; Chi-Wang, Yu Eric; Gilliland, Taylor; Zhai, Dayong; Bobkov, Andrey A.; Reed, John C.; Liddington, Robert C.; Abagyan, Ruben

    2010-01-01

    The 14 kDa homodimeric N1L protein is a potent vaccinia and variola (smallpox) virulence factor. It is not essential for viral replication, but it causes a strong attenuation of viral production in culture when deleted. The N1L protein is predicted to contain the BH3-like binding domain characteristic of Bcl-2 family proteins, and it is able to bind the BH3 peptides. Its overexpression has been reported to prevent infected cells from committing apoptosis. Therefore, interfering with the N1L apoptotic blockade may be a legitimate therapeutic strategy affecting the viral growth. By using in silico ligand docking and an array of in vitro assays, we have identified sub-micromolar (600 nM) N1L antagonists, belonging to the family of polyphenols. Their affinity is comparable to that of the BH3 peptides (70 nM ÷ 1000 nM). We have also identified the natural polyphenol resveratrol as a moderate N1L inhibitor. Finally, we show that our ligands efficiently inhibit growth of vaccinia virus. PMID:20441222

  1. Incomplete protection, but suppression of virus burden, elicited by subunit simian immunodeficiency virus vaccines.

    PubMed Central

    Israel, Z R; Edmonson, P F; Maul, D H; O'Neil, S P; Mossman, S P; Thiriart, C; Fabry, L; Van Opstal, O; Bruck, C; Bex, F

    1994-01-01

    We compared the efficacy of immunization with either simian immunodeficiency virus (SIV) Env glycoprotein (Env), Env plus Gag proteins (Gag-Env), or whole inactivated virus (WIV), with or without recombinant live vaccinia vector (VV) priming, in protecting 23 rhesus macaques (six vaccine and two control groups) from challenge with SIVmac251 clone BK28. Vaccination elicited high titers of syncytium-inhibiting and anti-Env (gp120/gp160) antibodies in all vaccinated macaques and anti-Gag (p27) antibodies in groups immunized with WIV or Gag-Env. Only WIV-immunized macaques developed anticell (HuT78) antibodies. After homologous low-dose intravenous virus challenge, we used frequency of virus isolation, provirus burden, and change in antibody titers to define four levels of resistance to SIV infection as follows. (i) No infection ("sterilizing" immunity) was induced only in WIV-immunized animals. (ii) Abortive infection (strong immunity) was defined when virus or provirus were detected early in the postchallenge period but not thereafter and no evidence of virus or provirus was detected in terminal tissues. This response was observed in two animals (one VV-Env and one Gag-Env). (iii) Suppression of infection (incomplete or partial immunity) described a gradient of virus suppression manifested by termination of viremia, declining postchallenge antibody titers, and low levels (composite mean = 9.1 copies per 10(6) cells) of provirus detectable in peripheral blood mononuclear cells or lymphoid tissues at termination (40 weeks postchallenge). This response occurred in the majority (8 of 12) of subunit-vaccinated animals. (iv) Active infection (no immunity) was characterized by persistent virus isolation from blood mononuclear cells, increasing viral antibody titers postchallenge, and high levels (composite mean = 198 copies per 10(6) cells) of provirus in terminal tissues and blood. Active infection developed in all controls and two of three VV-Gag-Env-immunized animals

  2. Vaccines, viruses, and voodoo.

    PubMed

    Borchers, Andrea T; Keen, Carl L; Shoenfeld, Yehuda; Silva, Joseph; Gershwin, M Eric

    2002-01-01

    Vaccinations are invaluable in protection from a wide variety of diseases that can cause substantial morbidity and mortality. Although a rare complication of vaccination, autoimmune disorders represent one of these morbidities. Recently, widespread public concern has arisen from case reports suggesting that--similar to what has been observed after natural viral infections--there might be an association between specific immunizations and autoimmune diseases. Herein we address the biological plausibility of such a connection, focusing particularly on the examples of hepatitis B, rubella, and measles-mumps-rubella (MMR) vaccinations, and the autoimmune diseases they are potentially associated with. Our review of the available data suggests that, for the general population, the risk: benefit ratio is overwhelmingly in favor of vaccinations. However, the possibility cannot be ruled out that, in genetically susceptible individuals, vaccination can result in the unmasking of an autoimmune disease triggered by the immunization. We also critically examine the existing data suggesting a link between immunization against MMR and autism, and briefly discuss the controversial evidence pointing to a possible relationship between mercury exposure from vaccines and autistic disorders. There is a continued urgent need for rigorously designed and executed studies addressing these potential associations, although the use of vaccinations remains a critical public health tool for protection against infectious disease. PMID:12530114

  3. Regulation of Vaccinia Virus E3 Protein by Small Ubiquitin-Like Modifier Proteins ▿ †

    PubMed Central

    González-Santamaría, José; Campagna, Michela; García, María Angel; Marcos-Villar, Laura; González, Dolores; Gallego, Pedro; Lopitz-Otsoa, Fernando; Guerra, Susana; Rodríguez, Manuel S.; Esteban, Mariano; Rivas, Carmen

    2011-01-01

    The vaccinia virus (VACV) E3 protein is essential for virulence and has antiapoptotic activity and the ability to impair the host innate immune response. Here we demonstrate that E3 interacts with SUMO1 through a small ubiquitin-like modifier (SUMO)-interacting motif (SIM). SIM integrity is required for maintaining the stability of the viral protein and for the covalent conjugation of E3 to SUMO1 or SUMO2, a modification that has a negative effect on the E3 transcriptional transactivation of the p53-upregulated modulator of apoptosis (PUMA) and APAF-1 genes. We also demonstrate that E3 is ubiquitinated, a modification that does not destabilize the wild-type protein but triggers the degradation of an E3-ΔSIM mutant. This report constitutes the first demonstration of the important roles that both SUMO and ubiquitin play in the regulation of the VACV protein E3. PMID:21957283

  4. Vaccinia Virus A35R Inhibits MHC Class II Antigen Presentation

    PubMed Central

    Rehm, Kristina E.; Connor, Ramsey F.; Jones, Gwendolyn J.B.; Yimbu, Kenneth; Roper, Rachel L.

    2009-01-01

    The Vaccinia virus gene A35R (Copenhagen designation) is highly conserved in mammalian-tropic poxviruses and is an important virulence factor, but its function was unknown. We show herein that A35 does not affect viral infectivity, apoptosis induction, or replication; however, we found that A35 significantly inhibited MHC class II-restricted antigen presentation, immune priming of T lymphocytes, and subsequent chemokine and cytokine synthesis. A35 localized to endosomes and reduced the amount of a model antigenic peptide displayed in the cleft of class II MHC. In addition, A35 decreased VV specific T cell responses in vivo. Thus, this is the first report identifying a function for the A35 protein in virulence as well as the first report identifying a VV gene that inhibits peptide antigen presentation. PMID:19954808

  5. Myxoma and vaccinia viruses exploit different mechanisms to enter and infect human cancer cells

    SciTech Connect

    Villa, Nancy Y.; Bartee, Eric; Mohamed, Mohamed R.; Rahman, Masmudur M.; Barrett, John W.; McFadden, Grant

    2010-06-05

    Myxoma (MYXV) and vaccinia (VACV) viruses have recently emerged as potential oncolytic agents that can infect and kill different human cancer cells. Although both are structurally similar, it is unknown whether the pathway(s) used by these poxviruses to enter and cause oncolysis in cancer cells are mechanistically similar. Here, we compared the entry of MYXV and VACV-WR into various human cancer cells and observed significant differences: 1 - low-pH treatment accelerates fusion-mediated entry of VACV but not MYXV, 2 - the tyrosine kinase inhibitor genistein inhibits entry of VACV, but not MYXV, 3 - knockdown of PAK1 revealed that it is required for a late stage event downstream of MYXV entry into cancer cells, whereas PAK1 is required for VACV entry into the same target cells. These results suggest that VACV and MYXV exploit different mechanisms to enter into human cancer cells, thus providing some rationale for their divergent cancer cell tropisms.

  6. Respiratory syncytial virus vaccine development

    PubMed Central

    Hurwitz, Julia L

    2011-01-01

    Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract viral disease in infants and young children. Presently, there are no explicit recommendations for RSV treatment apart from supportive care. The virus is therefore responsible for an estimated 160,000 deaths per year worldwide. Despite half a century of dedicated research, there remains no licensed vaccine product. Herein are described past and current efforts to harness innate and adaptive immune potentials to combat RSV. A plethora of candidate vaccine products and strategies are reviewed. The development of a successful RSV vaccine may ultimately stem from attention to historical lessons, in concert with an integral partnering of immunology and virology research fields. PMID:21988307

  7. Immunological Characterization of a Modified Vaccinia Virus Ankara Vector Expressing the Human Papillomavirus 16 E1 Protein

    PubMed Central

    Remy-Ziller, Christelle; Germain, Claire; Spindler, Anita; Hoffmann, Chantal; Silvestre, Nathalie; Rooke, Ronald; Bonnefoy, Jean-Yves

    2014-01-01

    Women showing normal cytology but diagnosed with a persistent high-risk human papillomavirus (HR-HPV) infection have a higher risk of developing high-grade cervical intraepithelial neoplasia and cervical cancer than noninfected women. As no therapeutic management other than surveillance is offered to these women, there is a major challenge to develop novel targeted therapies dedicated to the treatment of these patients. As such, E1 and E2 antigens, expressed early in the HPV life cycle, represent very interesting candidates. Both proteins are necessary for maintaining coordinated viral replication and gene synthesis during the differentiation process of the epithelium and are essential for the virus to complete its normal and propagative replication cycle. In the present study, we evaluated a new active targeted immunotherapeutic, a modified vaccinia virus Ankara (MVA) vector containing the E1 sequence of HPV16, aimed at inducing cellular immune responses with the potential to help and clear persistent HPV16-related infection. We carried out an extensive comparative time course analysis of the cellular immune responses induced by different schedules of immunization in C57BL/6 mice. We showed that multiple injections of MVA-E1 allowed sustained HPV16 E1-specific cellular immune responses in vaccinated mice and had no impact on the exhaustion phenotype of the generated HPV16 E1-specific CD8+ T cells, but they led to the differentiation of multifunctional effector T cells with high cytotoxic capacity. This study provides proof of concept that an MVA expressing HPV16 E1 can induce robust and long-lasting E1-specific responses and warrants further development of this candidate. PMID:24307238

  8. Immunological characterization of a modified vaccinia virus Ankara vector expressing the human papillomavirus 16 E1 protein.

    PubMed

    Remy-Ziller, Christelle; Germain, Claire; Spindler, Anita; Hoffmann, Chantal; Silvestre, Nathalie; Rooke, Ronald; Bonnefoy, Jean-Yves; Préville, Xavier

    2014-02-01

    Women showing normal cytology but diagnosed with a persistent high-risk human papillomavirus (HR-HPV) infection have a higher risk of developing high-grade cervical intraepithelial neoplasia and cervical cancer than noninfected women. As no therapeutic management other than surveillance is offered to these women, there is a major challenge to develop novel targeted therapies dedicated to the treatment of these patients. As such, E1 and E2 antigens, expressed early in the HPV life cycle, represent very interesting candidates. Both proteins are necessary for maintaining coordinated viral replication and gene synthesis during the differentiation process of the epithelium and are essential for the virus to complete its normal and propagative replication cycle. In the present study, we evaluated a new active targeted immunotherapeutic, a modified vaccinia virus Ankara (MVA) vector containing the E1 sequence of HPV16, aimed at inducing cellular immune responses with the potential to help and clear persistent HPV16-related infection. We carried out an extensive comparative time course analysis of the cellular immune responses induced by different schedules of immunization in C57BL/6 mice. We showed that multiple injections of MVA-E1 allowed sustained HPV16 E1-specific cellular immune responses in vaccinated mice and had no impact on the exhaustion phenotype of the generated HPV16 E1-specific CD8⁺ T cells, but they led to the differentiation of multifunctional effector T cells with high cytotoxic capacity. This study provides proof of concept that an MVA expressing HPV16 E1 can induce robust and long-lasting E1-specific responses and warrants further development of this candidate. PMID:24307238

  9. T-cell Engager-armed Oncolytic Vaccinia Virus Significantly Enhances Antitumor Therapy

    PubMed Central

    Yu, Feng; Wang, Xingbing; Guo, Z Sheng; Bartlett, David L; Gottschalk, Stephen M; Song, Xiao-Tong

    2014-01-01

    Oncolytic vaccinia virus (VV) therapy has shown promise in preclinical models and in clinical studies. However, complete responses have rarely been observed. This lack of efficacy is most likely due to suboptimal virus spread through the tumor resulting in limited tumor cell destruction. We reasoned that redirecting T cells to the tumor has the potential to improve the antitumor activity of oncolytic VVs. We, therefore, constructed a VV encoding a secretory bispecific T-cell engager consisting of two single- chain variable fragments specific for CD3 and the tumor cell surface antigen EphA2 (EphA2-T-cell engager-armed VV (EphA2-TEA-VV)). In vitro, EphA2-TEA-VV's ability to replicate and induce oncolysis was similar to that of unmodified virus. However, only tumor cells infected with EphA2-TEA-VV induced T-cell activation as judged by the secretion of interferon-γ and interleukin-2. In coculture assays, EphA2-TEA-VV not only killed infected tumor cells, but in the presence of T cells, it also induced bystander killing of noninfected tumor cells. In vivo, EphA2-TEA-VV plus T cells had potent antitumor activity in comparison with control VV plus T cells in a lung cancer xenograft model. Thus, arming oncolytic VVs with T-cell engagers may represent a promising approach to improve oncolytic virus therapy. PMID:24135899

  10. Vaccinia virus Transmission through Experimentally Contaminated Milk Using a Murine Model

    PubMed Central

    Rehfeld, Izabelle Silva; Guedes, Maria Isabel Maldonado Coelho; Fraiha, Ana Luiza Soares; Costa, Aristóteles Gomes; Matos, Ana Carolina Diniz; Fiúza, Aparecida Tatiane Lino; Lobato, Zélia Inês Portela

    2015-01-01

    Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects dairy cattle and humans. Previous studies have detected the presence of viable virus particles in bovine milk samples naturally and experimentally contaminated with VACV. However, it is not known whether milk contaminated with VACV could be a route of viral transmission. However, anti-Orthopoxvirus antibodies were detected in humans from BV endemic areas, whom had no contact with affected cows, which suggest that other VACV transmission routes are possible, such as consumption of contaminated milk and dairy products. Therefore, it is important to study the possibility of VACV transmission by contaminated milk. This study aimed to examine VACV transmission, pathogenesis and shedding in mice orally inoculated with experimentally contaminated milk. Thirty mice were orally inoculated with milk containing 107 PFU/ml of VACV, and ten mice were orally inoculated with uncontaminated milk. Clinical examinations were performed for 30 consecutive days, and fecal samples and oral swabs (OSs) were collected every other day. Mice were euthanized on predetermined days, and tissue and blood samples were collected. Nested-PCR, plaque reduction neutralization test (PRNT), viral isolation, histopathology, and immunohistochemistry (IHC) methods were performed on the collected samples. No clinical changes were observed in the animals. Viral DNA was detected in feces, blood, OSs and tissues, at least in one of the times tested. The lungs displayed moderate to severe interstitial lymphohistiocytic infiltrates, and only the heart, tonsils, tongue, and stomach did not show immunostaining at the IHC analysis. Neutralizing antibodies were detected at the 20th and 30th days post infection in 50% of infected mice. The results revealed that VACV contaminated milk could be a route of viral transmission in mice experimentally infected, showing systemic distribution and shedding through feces and oral mucosa, albeit

  11. Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA.

    PubMed

    Burmeister, Wim P; Tarbouriech, Nicolas; Fender, Pascal; Contesto-Richefeu, Céline; Peyrefitte, Christophe N; Iseni, Frédéric

    2015-07-17

    Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A201-50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A201-50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a Kd of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action. PMID:26045555

  12. From lesions to viral clones: biological and molecular diversity amongst autochthonous Brazilian vaccinia virus.

    PubMed

    Oliveira, Graziele; Assis, Felipe; Almeida, Gabriel; Albarnaz, Jonas; Lima, Maurício; Andrade, Ana Cláudia; Calixto, Rafael; Oliveira, Cairo; Diomedes Neto, José; Trindade, Giliane; Ferreira, Paulo César; Kroon, Erna Geessien; Abrahão, Jônatas

    2015-03-01

    Vaccinia virus (VACV) has had an important role for humanity because of its use during the smallpox eradication campaign. VACV is the etiologic agent of the bovine vaccinia (BV), an emerging zoonosis that has been associated with economic, social, veterinary and public health problems, mainly in Brazil and India. Despite the current and historical VACV importance, there is little information about its circulation, prevalence, origins and maintenance in the environment, natural reservoirs and diversity. Brazilian VACV (VACV-BR) are grouped into at least two groups based on genetic and biological diversity: group 1 (G1) and group 2 (G2). In this study, we went to the field and investigated VACV clonal diversity directly from exanthemous lesions, during BV outbreaks. Our results demonstrate that the G1 VACV-BR were more frequently isolated. Furthermore, we were able to co-detect the two variants (G1 and G2) in the same sample. Molecular and biological analysis corroborated previous reports and confirmed the co-circulation of two VACV-BR lineages. The detected G2 clones presented exclusive genetic and biological markers, distinct to reference isolates, including VACV-Western Reserve. Two clones presented a mosaic profile, with both G1 and G2 features based on the molecular analysis of A56R, A26L and C23L genes. Indeed, some SNPs and INDELs in A56R nucleotide sequences were observed among clones of the same virus population, maybe as a result of an increased mutation rate in a mixed population. These results provide information about the diversity profile in VACV populations, highlighting its importance to VACV evolution and maintenance in the environment. PMID:25785515

  13. From Lesions to Viral Clones: Biological and Molecular Diversity amongst Autochthonous Brazilian Vaccinia Virus

    PubMed Central

    Oliveira, Graziele; Assis, Felipe; Almeida, Gabriel; Albarnaz, Jonas; Lima, Maurício; Andrade, Ana Cláudia; Calixto, Rafael; Oliveira, Cairo; Neto, José Diomedes; Trindade, Giliane; Ferreira, Paulo César; Kroon, Erna Geessien; Abrahão, Jônatas

    2015-01-01

    Vaccinia virus (VACV) has had an important role for humanity because of its use during the smallpox eradication campaign. VACV is the etiologic agent of the bovine vaccinia (BV), an emerging zoonosis that has been associated with economic, social, veterinary and public health problems, mainly in Brazil and India. Despite the current and historical VACV importance, there is little information about its circulation, prevalence, origins and maintenance in the environment, natural reservoirs and diversity. Brazilian VACV (VACV-BR) are grouped into at least two groups based on genetic and biological diversity: group 1 (G1) and group 2 (G2). In this study, we went to the field and investigated VACV clonal diversity directly from exanthemous lesions, during BV outbreaks. Our results demonstrate that the G1 VACV-BR were more frequently isolated. Furthermore, we were able to co-detect the two variants (G1 and G2) in the same sample. Molecular and biological analysis corroborated previous reports and confirmed the co-circulation of two VACV-BR lineages. The detected G2 clones presented exclusive genetic and biological markers, distinct to reference isolates, including VACV-Western Reserve. Two clones presented a mosaic profile, with both G1 and G2 features based on the molecular analysis of A56R, A26L and C23L genes. Indeed, some SNPs and INDELs in A56R nucleotide sequences were observed among clones of the same virus population, maybe as a result of an increased mutation rate in a mixed population. These results provide information about the diversity profile in VACV populations, highlighting its importance to VACV evolution and maintenance in the environment. PMID:25785515

  14. Vaccination with a Fusion Protein That Introduces HIV-1 Gag Antigen into a Multitrimer CD40L Construct Results in Enhanced CD8+ T Cell Responses and Protection from Viral Challenge by Vaccinia-Gag

    PubMed Central

    Gupta, Sachin; Termini, James M.; Raffa, Francesca N.; Williams, Cindi-Ann; Kornbluth, Richard S.

    2014-01-01

    CD40 ligand (CD40L, CD154) is a membrane protein that is important for the activation of dendritic cells (DCs) and DC-induced CD8+ T cell responses. To be active, CD40L must cluster CD40 receptors on responding cells. To produce a soluble form of CD40L that clusters CD40 receptors necessitates the use of a multitrimer construct. With this in mind, a tripartite fusion protein was made from surfactant protein D (SPD), HIV-1 Gag as a test antigen, and CD40L, where SPD serves as a scaffold for the multitrimer protein complex. This SPD-Gag-CD40L protein activated CD40-bearing cells and bone marrow-derived DCs in vitro. Compared to a plasmid for Gag antigen alone (pGag), DNA vaccination of mice with pSPD-Gag-CD40L induced an increased number of Gag-specific CD8+ T cells with increased avidity for major histocompatibility complex class I-restricted Gag peptide and improved vaccine-induced protection from challenge by vaccinia-Gag virus. The importance of the multitrimeric nature of the complex was shown using a plasmid lacking the N terminus of SPD that produced a single trimer fusion protein. This plasmid, pTrimer-Gag-CD40L, was only weakly active on CD40-bearing cells and did not elicit strong CD8+ T cell responses or improve protection from vaccinia-Gag challenge. An adenovirus 5 (Ad5) vaccine incorporating SPD-Gag-CD40L was much stronger than Ad5 expressing Gag alone (Ad5-Gag) and induced complete protection (i.e., sterilizing immunity) from vaccinia-Gag challenge. Overall, these results show the potential of a new vaccine design in which antigen is introduced into a construct that expresses a multitrimer soluble form of CD40L, leading to strongly protective CD8+ T cell responses. PMID:24227853

  15. Identification of the DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus

    SciTech Connect

    Morgan, J.R.; Cohen, L.K.; Roberts, B.E.

    1984-10-01

    The DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus were located on the viral genome. The formation of an enzyme-guanylate covalent intermediate labeled with (alpha-/sup 32/P)GTP allowed the identification of the large subunit of the capping enzyme and was used to monitor the appearance of the enzyme during the infectious cycle. This assay confirmed that after vaccinia infection, a novel 84,000-molecular-weight polypeptide corresponding to the large subunit was rapidly synthesized before viral DNA replication. Hybrid-selected cell-free translation of early viral mRNA established that vaccinia virus encoded a polypeptide identical in molecular weight with the /sup 32/P-labeled 84,000-molecular-weight polypeptide found in vaccinia virions. Like the authentic capping enzyme, this virus-encoded cell-free translation product bound specifically to DNA-cellulose. A comparison of the partial proteolytic digestion fragments generated by V8 protease, chymotrypsin, and trypsin demonstrated that the /sup 32/P-labeled large subunit and the (/sup 35/S)methionine-labeled cell-free translation product were identical. The mRNA encoding the large subunit of the capping enzyme was located 3.1 kilobase pairs to the left of the HindIII D restriction fragment of the vaccinia genome. Furthermore, the mRNA was determined to be 3.0 kilobases in size, and its 5 and 3 termini were precisely located by S1 nuclease analysis.

  16. Safety and biodistribution of a double-deleted oncolytic vaccinia virus encoding CD40 ligand in laboratory Beagles.

    PubMed

    Autio, Karoliina; Knuuttila, Anna; Kipar, Anja; Pesonen, Sari; Guse, Kilian; Parviainen, Suvi; Rajamäki, Minna; Laitinen-Vapaavuori, Outi; Vähä-Koskela, Markus; Kanerva, Anna; Hemminki, Akseli

    2014-01-01

    We evaluated adverse events, biodistribution and shedding of oncolytic vaccinia virus encoding CD40 ligand in two Beagles, in preparation for a phase 1 trial in canine cancer patients. Dog 1 received one dose of vaccinia virus and was euthanized 24 hours afterwards, while dog 2 received virus four times once weekly and was euthanized 7 days after that. Dogs were monitored for adverse events and underwent a detailed postmortem examination. Blood, saliva, urine, feces, and organs were collected for virus detection. Dog 1 had mild fever and lethargy while dog 2 experienced a possible seizure 5.5 hours after first virus administration. Viral DNA declined quickly in the blood after virus administration in both dogs but was still detectable 1 week later by quantitative polymerase chain reaction. Only samples taken directly after virus infusion contained infectious virus. Small amounts of viral DNA, but no infectious virus, were detected in a few saliva and urine samples. Necropsies did not reveal any relevant pathological changes and virus DNA was detected mainly in the spleen. The dogs in the study did not have cancer, and thus adverse events could be more common and viral load higher in dogs with tumors which allow viral amplification. PMID:27119092

  17. Safety and biodistribution of a double-deleted oncolytic vaccinia virus encoding CD40 ligand in laboratory Beagles

    PubMed Central

    Autio, Karoliina; Knuuttila, Anna; Kipar, Anja; Pesonen, Sari; Guse, Kilian; Parviainen, Suvi; Rajamäki, Minna; Laitinen-Vapaavuori, Outi; Vähä-Koskela, Markus; Kanerva, Anna; Hemminki, Akseli

    2014-01-01

    We evaluated adverse events, biodistribution and shedding of oncolytic vaccinia virus encoding CD40 ligand in two Beagles, in preparation for a phase 1 trial in canine cancer patients. Dog 1 received one dose of vaccinia virus and was euthanized 24 hours afterwards, while dog 2 received virus four times once weekly and was euthanized 7 days after that. Dogs were monitored for adverse events and underwent a detailed postmortem examination. Blood, saliva, urine, feces, and organs were collected for virus detection. Dog 1 had mild fever and lethargy while dog 2 experienced a possible seizure 5.5 hours after first virus administration. Viral DNA declined quickly in the blood after virus administration in both dogs but was still detectable 1 week later by quantitative polymerase chain reaction. Only samples taken directly after virus infusion contained infectious virus. Small amounts of viral DNA, but no infectious virus, were detected in a few saliva and urine samples. Necropsies did not reveal any relevant pathological changes and virus DNA was detected mainly in the spleen. The dogs in the study did not have cancer, and thus adverse events could be more common and viral load higher in dogs with tumors which allow viral amplification. PMID:27119092

  18. Recent advances in the development of vaccines for Ebola virus disease.

    PubMed

    Ohimain, Elijah Ige

    2016-01-01

    Ebola virus is one of the most dangerous microorganisms in the world causing hemorrhagic fevers in humans and non-human primates. Ebola virus (EBOV) is a zoonotic infection, which emerges and re-emerges in human populations. The 2014 outbreak was caused by the Zaire strain, which has a kill rate of up to 90%, though 40% was recorded in the current outbreak. The 2014 outbreak is larger than all 20 outbreaks that have occurred since 1976, when the virus was first discovered. It is the first time that the virus was sustained in urban centers and spread beyond Africa into Europe and USA. Thus far, over 22,000 cases have been reported with about 50% mortality in one year. There are currently no approved therapeutics and preventive vaccines against Ebola virus disease (EVD). Responding to the devastating effe1cts of the 2014 outbreak and the potential risk of global spread, has spurred research for the development of therapeutics and vaccines. This review is therefore aimed at presenting the progress of vaccine development. Results showed that conventional inactivated vaccines produced from EBOV by heat, formalin or gamma irradiation appear to be ineffective. However, novel vaccines production techniques have emerged leading to the production of candidate vaccines that have been demonstrated to be effective in preclinical trials using small animal and non-human primates (NHP) models. Some of the promising vaccines have undergone phase 1 clinical trials, which demonstrated their safety and immunogenicity. Many of the candidate vaccines are vector based such as Vesicular Stomatitis Virus (VSV), Rabies Virus (RABV), Adenovirus (Ad), Modified Vaccinia Ankara (MVA), Cytomegalovirus (CMV), human parainfluenza virus type 3 (HPIV3) and Venezuelan Equine Encephalitis Virus (VEEV). Other platforms include virus like particle (VLP), DNA and subunit vaccines. PMID:26596227

  19. A comparison of DNA vaccines for the rabies-related virus, Mokola.

    PubMed

    Nel, L H; Niezgoda, M; Hanlon, C A; Morril, P A; Yager, P A; Rupprecht, C E

    2003-06-01

    Mokola virus, a rabies-related virus, has been reported to date from the African continent only. Like rabies virus, it is highly pathogenic, causes acute encephalitis, and zoonotic events have been documented. Although believed to be rare, there has been an unexplained increase in the number of isolations of the virus in South Africa in recent years. We have cloned and sequenced the glycoprotein (G) and nucleoprotein (N) genes from a South African Mokola virus, and used these in the construction of different DNA vaccines for immunization against Mokola virus. Four vaccines, utilizing different promoters and DNA backbone compositions, were generated and compared for efficacy in protection against Mokola virus. In one of these, both the Mokola virus G and N genes were co-expressed. Two of the single G-expressing DNA vaccines (based on pSG5 and pCI-neo, respectively) protected laboratory mice against lethal challenge, despite major differences in their promoters. However, neither vaccine was fully protective in a single immunization only. Serological assays confirmed titers of virus-neutralizing antibodies after immunization, which increased upon booster vaccine administration. A third construct (based on pBudCE4) was less effective in inducing a protective immune response, despite employing a strong CMV enhancer/promoter also used in the pCI-neo plasmid. Dual expression of Mokola virus G and N genes in pBudCE4 did not enhance its efficacy, under the conditions described. In addition, no significant utility could be demonstrated for a combined prime-boost approach, as no cross-protective immunity was observed against rabies or Mokola viruses from the use of pSG5-mokG or vaccinia-rabies glycoprotein recombinant virus vaccines, respectively, even though both vaccines provided 60-100% protection against homologous virus challenge. PMID:12744896

  20. Rapid Expansion of CD8+ T Cells in Wild-Type and Type I Interferon Receptor-Deficient Mice Correlates with Protection after Low-Dose Emergency Immunization with Modified Vaccinia Virus Ankara

    PubMed Central

    Volz, Asisa; Langenmayer, Martin; Jany, Sylvia; Kalinke, Ulrich

    2014-01-01

    ABSTRACT Immunization with modified vaccinia virus Ankara (MVA) can rapidly protect mice against lethal ectromelia virus (ECTV) infection, serving as an experimental model for severe systemic infections. Importantly, this early protective capacity of MVA vaccination completely depends on virus-specific cytotoxic CD8+ T cell responses. We used MVA vaccination in the mousepox challenge model using ECTV infection to investigate the previously unknown factors required to elicit rapid protective T cell immunity in normal C57BL/6 mice and in mice lacking the interferon alpha/beta receptor (IFNAR−/−). We found a minimal dose of 105 PFU of MVA vaccine fully sufficient to allow robust protection against lethal mousepox, as assessed by the absence of disease symptoms and failure to detect ECTV in organs from vaccinated animals. Moreover, MVA immunization at low dosage also protected IFNAR−/− mice, indicating efficient activation of cellular immunity even in the absence of type I interferon signaling. When monitoring for virus-specific CD8+ T cell responses in mice vaccinated with the minimal protective dose of MVA, we found significantly enhanced levels of antigen-specific T cells in animals that were MVA vaccinated and ECTV challenged compared to mice that were only vaccinated. The initial priming of naive CD8+ T cells by MVA immunization appears to be highly efficient and, even at low doses, mediates a rapid in vivo burst of pathogen-specific T cells upon challenge. Our findings define striking requirements for protective emergency immunization against severe systemic infections with orthopoxviruses. IMPORTANCE We demonstrate that single-shot low-dose immunizations with vaccinia virus MVA can rapidly induce T cell-mediated protective immunity against lethal orthopoxvirus infections. Our data provide new evidence for an efficient protective capacity of vaccination with replication-deficient MVA. These data are of important practical relevance for public health, as

  1. Comparison of the Cowpox Virus and Vaccinia Virus Mature Virion Proteome: Analysis of the Species- and Strain-Specific Proteome.

    PubMed

    Doellinger, Joerg; Schaade, Lars; Nitsche, Andreas

    2015-01-01

    Cowpox virus (CPXV) causes most zoonotic orthopoxvirus (OPV) infections in Europe and Northern as well as Central Asia. The virus has the broadest host range of OPV and is transmitted to humans from rodents and other wild or domestic animals. Increasing numbers of human CPXV infections in a population with declining immunity have raised concerns about the virus' zoonotic potential. While there have been reports on the proteome of other human-pathogenic OPV, namely vaccinia virus (VACV) and monkeypox virus (MPXV), the protein composition of the CPXV mature virion (MV) is unknown. This study focused on the comparative analysis of the VACV and CPXV MV proteome by label-free single-run proteomics using nano liquid chromatography and high-resolution tandem mass spectrometry (nLC-MS/MS). The presented data reveal that the common VACV and CPXV MV proteome contains most of the known conserved and essential OPV proteins and is associated with cellular proteins known to be essential for viral replication. While the species-specific proteome could be linked mainly to less genetically-conserved gene products, the strain-specific protein abundance was found to be of high variance in proteins associated with entry, host-virus interaction and protein processing. PMID:26556597

  2. Hepatitis virus vaccines: present status.

    PubMed Central

    Krugman, S.

    1982-01-01

    During the past decade there has been extraordinary progress toward the development of vaccines for the prevention of type A and type B hepatitis. The successful propagation of hepatitis A virus in cell culture in 1979 was followed by the preparation of experimental live attenuated hepatitis A vaccines that have been shown to induce antibody in marmosets and chimpanzees and protect immunized marmosets against challenge with hepatitis A virus. The first human immunization trials will begin in mid-1982. An inactivated hepatitis B vaccine that was licensed in the United States in November 1981 has been shown to be safe, immunogenic, and effective. When this vaccine becomes available for use in July 1982, it will be recommended for persons who are considered to be at increased risk of contracting hepatitis B infection. Future generations of hepatitis B vaccines may be prepared from hepatitis B surface antigen derived from DNA recombinant technology or by in vitro synthesis of HBs Ag determinants by chemical means. PMID:6295013

  3. Silk-elastin-like protein polymer matrix for intraoperative delivery of an oncolytic vaccinia virus

    PubMed Central

    Price, Daniel L.; Li, Pingdong; Chen, Chun-Hao; Wong, Danni; Yu, Zhenkun; Chen, Nanhai G.; Yu, Yong A.; Szalay, Aladar A.; Cappello, Joseph; Fong, Yuman; Wong, Richard J.

    2016-01-01

    Background Oncolytic viral efficacy may be limited by the penetration of the virus into tumors. This may be enhanced by intraoperative application of virus immediately after surgical resection. Methods Oncolytic vaccinia virus GLV-1h68 was delivered in silk-elastin-like protein polymer (SELP) in vitro and in vivo in anaplastic thyroid carcinoma cell line 8505c in nude mice. Results GLV-1h68 in SELP infected and lysed anaplastic thyroid cancer cells in vitro equally as effectively as in phosphate-buffered saline (PBS), and at 1 week retains a thousand fold greater infectious plaque-forming units. In surgical resection models of residual tumor, GLV-1h68 in SELP improves tumor control and shows increased viral β-galactosidase expression as compared to PBS. Conclusion The use of SELP matrix for intraoperative oncolytic viral delivery protects infectious viral particles from degradation, facilitates sustained viral delivery and transgene expression, and improves tumor control. Such optimization of methods of oncolytic viral delivery may enhance therapeutic outcomes. PMID:25244076

  4. RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

    SciTech Connect

    Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.; Brown, Helen K.; James, John; Prescott, Alan; Haga, Ismar R.; Beard, Philippa M.

    2015-01-15

    Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. - Highlights: • Characterisation of the role of the small GTPase RAB1A in VACV replication. • RAB1A is not required for production of the primary virion form (IMV). • RAB1A is required for production of processed virion forms (IEVs, CEVs and EEVs). • Consistent with known role of RAB1A in ER to Golgi transport.

  5. Efficacy of a Plasmodium vivax Malaria Vaccine Using ChAd63 and Modified Vaccinia Ankara Expressing Thrombospondin-Related Anonymous Protein as Assessed with Transgenic Plasmodium berghei Parasites

    PubMed Central

    Bauza, Karolis; Malinauskas, Tomas; Pfander, Claudia; Anar, Burcu; Jones, E. Yvonne; Billker, Oliver; Hill, Adrian V. S.

    2014-01-01

    Plasmodium vivax is the world's most widely distributed malaria parasite and a potential cause of morbidity and mortality for approximately 2.85 billion people living mainly in Southeast Asia and Latin America. Despite this dramatic burden, very few vaccines have been assessed in humans. The clinically relevant vectors modified vaccinia virus Ankara (MVA) and the chimpanzee adenovirus ChAd63 are promising delivery systems for malaria vaccines due to their safety profiles and proven ability to induce protective immune responses against Plasmodium falciparum thrombospondin-related anonymous protein (TRAP) in clinical trials. Here, we describe the development of new recombinant ChAd63 and MVA vectors expressing P. vivax TRAP (PvTRAP) and show their ability to induce high antibody titers and T cell responses in mice. In addition, we report a novel way of assessing the efficacy of new candidate vaccines against P. vivax using a fully infectious transgenic Plasmodium berghei parasite expressing P. vivax TRAP to allow studies of vaccine efficacy and protective mechanisms in rodents. Using this model, we found that both CD8+ T cells and antibodies mediated protection against malaria using virus-vectored vaccines. Our data indicate that ChAd63 and MVA expressing PvTRAP are good preerythrocytic-stage vaccine candidates with potential for future clinical application. PMID:24379295

  6. A Randomized, Double-Blind, Placebo-Controlled Phase II Trial Investigating the Safety and Immunogenicity of Modified Vaccinia Ankara Smallpox Vaccine (MVA-BN®) in 56-80-Year-Old Subjects

    PubMed Central

    Greenberg, Richard N.; Hay, Christine M.; Stapleton, Jack T.; Marbury, Thomas C.; Wagner, Eva; Kreitmeir, Eva; von Krempelhuber, Alfred; Young, Philip; Nichols, Richard; Meyer, Thomas P.; Weigl, Josef; Virgin, Garth; Arndtz-Wiedemann, Nathaly; Chaplin, Paul

    2016-01-01

    Background Modified Vaccinia Ankara MVA-BN® is a live, highly attenuated, viral vaccine under advanced development as a non-replicating smallpox vaccine. In this Phase II trial, the safety and immunogenicity of Modified Vaccinia Ankara MVA-BN® (MVA) was assessed in a 56–80 years old population. Methods MVA with a virus titer of 1 x 108 TCID50/dose was administered via subcutaneous injection to 56–80 year old vaccinia-experienced subjects (N = 120). Subjects received either two injections of MVA (MM group) or one injection of Placebo and one injection of MVA (PM group) four weeks apart. Safety was evaluated by assessment of adverse events (AE), focused physical exams, electrocardiogram recordings and safety laboratories. Solicited AEs consisted of a set of pre-defined expected local reactions (erythema, swelling, pain, pruritus, and induration) and systemic symptoms (body temperature, headache, myalgia, nausea and fatigue) and were recorded on a memory aid for an 8-day period following each injection. The immunogenicity of the vaccine was evaluated in terms of humoral immune responses measured with a vaccinia-specific enzyme-linked immunosorbent assay (ELISA) and a plaque reduction neutralization test (PRNT) before and at different time points after vaccination. Results Vaccinations were well tolerated by all subjects. No serious adverse event related to MVA and no case of myopericarditis was reported. The overall incidence of unsolicited AEs was similar in both groups. For both groups immunogenicity responses two weeks after the final vaccination (i.e. Visit 4) were as follows: Seroconversion (SC) rates (doubling of titers from baseline) in vaccine specific antibody titers measured by ELISA were 83.3% in Group MM and 82.8% in Group PM (difference 0.6% with 95% exact CI [-13.8%, 15.0%]), and 90.0% for Group MM and 77.6% for Group PM measured by PRNT (difference 12.4% with 95% CI of [-1.1%, 27.0%]). Geometric mean titers (GMT) measured by ELISA two weeks after

  7. Comparison of the Cowpox Virus and Vaccinia Virus Mature Virion Proteome: Analysis of the Species- and Strain-Specific Proteome

    PubMed Central

    Doellinger, Joerg; Schaade, Lars; Nitsche, Andreas

    2015-01-01

    Cowpox virus (CPXV) causes most zoonotic orthopoxvirus (OPV) infections in Europe and Northern as well as Central Asia. The virus has the broadest host range of OPV and is transmitted to humans from rodents and other wild or domestic animals. Increasing numbers of human CPXV infections in a population with declining immunity have raised concerns about the virus’ zoonotic potential. While there have been reports on the proteome of other human-pathogenic OPV, namely vaccinia virus (VACV) and monkeypox virus (MPXV), the protein composition of the CPXV mature virion (MV) is unknown. This study focused on the comparative analysis of the VACV and CPXV MV proteome by label-free single-run proteomics using nano liquid chromatography and high-resolution tandem mass spectrometry (nLC-MS/MS). The presented data reveal that the common VACV and CPXV MV proteome contains most of the known conserved and essential OPV proteins and is associated with cellular proteins known to be essential for viral replication. While the species-specific proteome could be linked mainly to less genetically-conserved gene products, the strain-specific protein abundance was found to be of high variance in proteins associated with entry, host-virus interaction and protein processing. PMID:26556597

  8. Local Production of Tumor Necrosis Factor Encoded by Recombinant Vaccinia Virus is Effective in Controlling Viral Replication in vivo

    NASA Astrophysics Data System (ADS)

    Sambhi, Sharan K.; Kohonen-Corish, Maija R. J.; Ramshaw, Ian A.

    1991-05-01

    Tumor necrosis factor (TNF) has pleiotropic effects on a wide variety of cell types. In vitro studies have demonstrated that TNF has antiviral properties and is induced in response to viral infections. However, a role for TNF in the antiviral immune response of the host has yet to be demonstrated. Here we describe the construction of and studies using a recombinant vaccinia virus that encodes the gene for murine TNF-α. By comparing the replication of and immune responses elicited by the TNF-encoding virus to a similarly constructed control virus, we hoped to observe immunobiological effects of TNF in the host. The in vivo experiments with this recombinant virus demonstrate that the localized production of TNF-α during a viral infection leads to the rapid and efficient clearance of the virus in normal mice and attenuates the otherwise lethal pathogenicity of the virus in immunodeficient animals. This attenuation occurs early in the infection (by postinfection hour 24) and is not due to the enhancement of cellular or antibody responses by the vaccinia virus-encoded TNF. This evidence suggests that attenuation of the recombinant virus is due to a direct antiviral effect of TNF on cells at the site of infection. Therefore, these results support the suggestion that TNF produced by immune cells may be an important effector mechanism of viral clearance in vivo.

  9. The Orthopoxvirus 68-Kilodalton Ankyrin-Like Protein Is Essential for DNA Replication and Complete Gene Expression of Modified Vaccinia Virus Ankara in Nonpermissive Human and Murine Cells▿

    PubMed Central

    Sperling, Karin M.; Schwantes, Astrid; Staib, Caroline; Schnierle, Barbara S.; Sutter, Gerd

    2009-01-01

    Modified vaccinia virus Ankara (MVA) is a highly attenuated and replication-deficient vaccinia virus (VACV) that is being evaluated as replacement smallpox vaccine and candidate viral vector. MVA lacks many genes associated with virulence and/or regulation of virus tropism. The 68-kDa ankyrin-like protein (68k-ank) is the only ankyrin repeat-containing protein that is encoded by the MVA genome and is highly conserved throughout the Orthopoxvirus genus. We showed previously that 68k-ank is composed of ankyrin repeats and an F-box-like domain and forms an SCF ubiquitin ligase complex together with the cellular proteins Skp1a and Cullin-1. We now report that 68k-ank (MVA open reading frame 186R) is an essential factor for completion of the MVA intracellular life cycle in nonpermissive human and murine cells. Infection of mouse NIH 3T3 and human HaCaT cells with MVA with a deletion of the 68k-ank gene (MVA-Δ68k-ank) was characterized by an extensive reduction of viral intermediate RNA and protein, as well as late transcripts and drastically impaired late protein synthesis. Furthermore, infections with MVA-Δ68k-ank failed to induce the host protein shutoff that is characteristic of VACV infections. Although we demonstrated that proteasome function in general is essential for the completion of the MVA molecular life cycle, we found that a mutant 68k-ank protein with a deletion of the F-box-like domain was able to fully complement the deficiency of MVA-Δ68k-ank to express all classes of viral genes. Thus, our data demonstrate that the 68k-ank protein contains another critical domain that may function independently of SCF ubiquitin ligase complex formation, suggesting multiple activities of this interesting regulatory protein. PMID:19357172

  10. Co-administration of the broad-spectrum antiviral, brincidofovir (CMX001), with smallpox vaccine does not compromise vaccine protection in mice challenged with ectromelia virus.

    PubMed

    Parker, Scott; Crump, Ryan; Foster, Scott; Hartzler, Hollyce; Hembrador, Ed; Lanier, E Randall; Painter, George; Schriewer, Jill; Trost, Lawrence C; Buller, R Mark

    2014-11-01

    Natural orthopoxvirus outbreaks such as vaccinia, cowpox, cattlepox and buffalopox continue to cause morbidity in the human population. Monkeypox virus remains a significant agent of morbidity and mortality in Africa. Furthermore, monkeypox virus's broad host-range and expanding environs make it of particular concern as an emerging human pathogen. Monkeypox virus and variola virus (the etiological agent of smallpox) are both potential agents of bioterrorism. The first line response to orthopoxvirus disease is through vaccination with first-generation and second-generation vaccines, such as Dryvax and ACAM2000. Although these vaccines provide excellent protection, their widespread use is impeded by the high level of adverse events associated with vaccination using live, attenuated virus. It is possible that vaccines could be used in combination with antiviral drugs to reduce the incidence and severity of vaccine-associated adverse events, or as a preventive in individuals with uncertain exposure status or contraindication to vaccination. We have used the intranasal mousepox (ectromelia) model to evaluate the efficacy of vaccination with Dryvax or ACAM2000 in conjunction with treatment using the broad spectrum antiviral, brincidofovir (BCV, CMX001). We found that co-treatment with BCV reduced the severity of vaccination-associated lesion development. Although the immune response to vaccination was quantifiably attenuated, vaccination combined with BCV treatment did not alter the development of full protective immunity, even when administered two days following ectromelia challenge. Studies with a non-replicating vaccine, ACAM3000 (MVA), confirmed that BCV's mechanism of attenuating the immune response following vaccination with live virus was, as expected, by limiting viral replication and not through inhibition of the immune system. These studies suggest that, in the setting of post-exposure prophylaxis, co-administration of BCV with vaccination should be considered

  11. Temperature-sensitive vaccinia virus mutants identify a gene with an essential role in viral replication.

    PubMed Central

    Rempel, R E; Anderson, M K; Evans, E; Traktman, P

    1990-01-01

    Vaccinia virus mutants ts2 and ts25, members of the same complementation group, exhibit a temperature-dependent arrest at the stage of viral DNA replication. The lesions responsible for the mutant phenotypes have been localized to the far left region of the HindIII B genomic fragment by marker rescue studies. Hybrid selection analyses established that the DNA fragments positive for rescue represented the first open reading frame of the HindIII B fragment and encoded a 30-kilodalton protein. The gene is expressed early after infection as a rightwardly transcribed 1-kilobase-pair mRNA whose coordinates were determined by S1 nuclease mapping. To further the phenotypic analysis of the mutants, the accumulation of viral DNA sequences during permissive and nonpermissive infections was quantitated. The extent of the DNA- phenotype was shown to vary in different cell types. In mouse L cells at either high or low multiplicity of infection, nonpermissive DNA synthesis was less than 5% of that seen in permissive infections. This severe defect was mirrored by correspondingly low viral yields. In infections of BSC40 monkey cells, however, the deficiencies in both DNA synthesis and virus production were far less severe. For one mutant (ts2), the temperature sensitivity in BSC40 cells varied inversely with the multiplicity of infection. Images PMID:2296077

  12. High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus.

    PubMed Central

    Stunnenberg, H G; Lange, H; Philipson, L; van Miltenburg, R T; van der Vliet, P C

    1988-01-01

    Initiation of Adenovirus (Ad) DNA replication occurs by a protein-priming mechanism in which the viral precursor terminal protein (pTP) and DNA polymerase (pol) as well as two nuclear DNA-binding proteins from uninfected HeLa cells are required. Biochemical studies on the pTP and DNA polymerase proteins separately have been hampered due to their low abundance and their presence as a pTP-pol complex in Ad infected cells. We have constructed a genomic sequence containing the large open reading frame from the Ad5 pol gene to which 9 basepairs from a putative exon were ligated. When inserted behind a modified late promoter of vaccinia virus the resulting recombinant virus produced enzymatically active 140 kDa Ad DNA polymerase. The same strategy was applied to express the 80 kDa pTP gene in a functional form. Both proteins were overexpressed at least 30-fold compared to extracts from Adenovirus infected cells and, when combined, were fully active for initiation in an in vitro Adenovirus DNA replication system. Images PMID:3362670

  13. Vaccinia viruses isolated from cutaneous disease in horses are highly virulent for rabbits.

    PubMed

    Felipetto Cargnelutti, Juliana; Schmidt, Candice; Masuda, Eduardo Kenji; Braum, Lisiane Danusa; Weiblen, Rudi; Furtado Flores, Eduardo

    2012-03-01

    Two genotypically distinct Vaccinia viruses (VACV), named P1V and P2V, were isolated from an outbreak of cutaneous disease in horses in Southern Brazil. We herein investigated the susceptibility of rabbits, a proposed animal model, to P1V and P2V infection. Groups of weanling rabbits were inoculated intranasally (IN) with P1V or P2V at low (10(2.5) TCID50), medium (10(4.5)TCID50), or high titer (10(6.5)TCID50). Rabbits inoculated with medium and high titers shed virus in nasal secretions and developed serous to hemorrhagic nasal discharge and severe respiratory distress, followed by progressive apathy and high lethality. Clinical signs appeared around days 3-6 post-inoculation (pi) and lasted up to the day of death or euthanasia (around days 5-10). Virus shedding and clinical signs were less frequent in rabbits inoculated with low virus titers. Viremia was detected in all groups, with different frequencies. Viral DNA was detected in the feces of a few animals inoculated with P1V and P2V, low titer, and with P2V at high titer. Gross necropsy findings and histological examination showed diffuse interstitial fibrousing pneumonia with necrosuppurative bronchopneumonia and intestinal liquid content. Neutralizing antibodies were detected in all inoculated animals surviving beyond day 9 pi. These results show that rabbits are highly susceptible to VACV isolated from horses, and develop severe respiratory and systemic disease upon IN inoculation. Thus, rabbits may be used to study selected aspects of VACV infection and disease. PMID:22226666

  14. Involvement of the Cellular Phosphatase DUSP1 in Vaccinia Virus Infection

    PubMed Central

    Cáceres, Ana; Perdiguero, Beatriz; Gómez, Carmen E.; Cepeda, Maria Victoria; Caelles, Carme; Sorzano, Carlos Oscar; Esteban, Mariano

    2013-01-01

    Poxviruses encode a large variety of proteins that mimic, block or enhance host cell signaling pathways on their own benefit. It has been reported that mitogen-activated protein kinases (MAPKs) are specifically upregulated during vaccinia virus (VACV) infection. Here, we have evaluated the role of the MAPK negative regulator dual specificity phosphatase 1 (DUSP1) in the infection of VACV. We demonstrated that DUSP1 expression is enhanced upon infection with the replicative WR virus and with the attenuated VACV viruses MVA and NYVAC. This upregulation is dependent on early viral gene expression. In the absence of DUSP1 in cultured cells, there is an increased activation of its molecular targets JNK and ERK and an enhanced WR replication. Moreover, DUSP1 knock-out (KO) mice are more susceptible to WR infection as a result of enhanced virus replication in the lungs. Significantly, MVA, which is known to produce non-permissive infections in most mammalian cell lines, is able to grow in DUSP1 KO immortalized murine embryo fibroblasts (MEFs). By confocal and electron microscopy assays, we showed that in the absence of DUSP1 MVA morphogenesis is similar as in permissive cell lines and demonstrated that DUSP1 is involved at the stage of transition between IVN and MV in VACV morphogenesis. In addition, we have observed that the secretion of pro-inflammatory cytokines at early times post-infection in KO mice infected with MVA and NYVAC is increased and that the adaptive immune response is enhanced in comparison with WT-infected mice. Altogether, these findings reveal that DUSP1 is involved in the replication and host range of VACV and in the regulation of host immune responses through the modulation of MAPKs. Thus, in this study we demonstrate that DUSP1 is actively involved in the antiviral host defense mechanism against a poxvirus infection. PMID:24244156

  15. Vaccinia Virus E3 Protein Prevents the Antiviral Action of ISG15

    PubMed Central

    Guerra, Susana; Cáceres, Ana; Knobeloch, Klaus-Peter; Horak, Ivan; Esteban, Mariano

    2008-01-01

    The ubiquitin-like modifier ISG15 is one of the most predominant proteins induced by type I interferons (IFN). In this study, murine embryo fibroblast (MEFs) and mice lacking the gene were used to demonstrate a novel role of ISG15 as a host defense molecule against vaccinia virus (VACV) infection. In MEFs, the growth of replication competent Western Reserve (WR) VACV strain was affected by the absence of ISG15, but in addition, virus lacking E3 protein (VVΔE3L) that is unable to grow in ISG15+/+ cells replicated in ISG15-deficient cells. Inhibiting ISG15 with siRNA or promoting its expression in ISG15−/− cells with a lentivirus vector showed that VACV replication was controlled by ISG15. Immunoprecipitation analysis revealed that E3 binds ISG15 through its C-terminal domain. The VACV antiviral action of ISG15 and its interaction with E3 are events independent of PKR (double-stranded RNA-dependent protein kinase). In mice lacking ISG15, infection with VVΔE3L caused significant disease and mortality, an effect not observed in VVΔE3L-infected ISG15+/+ mice. Pathogenesis in ISG15-deficient mice infected with VVΔE3L or with an E3L deletion mutant virus lacking the C-terminal domain triggered an enhanced inflammatory response in the lungs compared with ISG15+/+-infected mice. These findings showed an anti-VACV function of ISG15, with the virus E3 protein suppressing the action of the ISG15 antiviral factor. PMID:18604270

  16. Efficacy of a virus-vectored vaccine against human and bovine respiratory syncytial virus infections.

    PubMed

    Taylor, Geraldine; Thom, Michelle; Capone, Stefania; Pierantoni, Angiolo; Guzman, Efrain; Herbert, Rebecca; Scarselli, Elisa; Napolitano, Federico; Giuliani, Alessandro; Folgori, Antonella; Colloca, Stefano; Cortese, Riccardo; Nicosia, Alfredo; Vitelli, Alessandra

    2015-08-12

    Human respiratory syncytial virus (HRSV) is a major cause of lower respiratory tract disease in children and the elderly for which there is still no effective vaccine. We have previously shown that PanAd3-RSV, which is a chimpanzee adenovirus-vectored vaccine candidate that expresses a secreted form of the HRSV F protein together with the N and M2-1 proteins of HRSV, is immunogenic in rodents and nonhuman primates, and protects mice and cotton rats from HRSV challenge. Because the extent to which protection demonstrated in rodent models will translate to humans is unclear, we have exploited the calf model of bovine RSV (BRSV) infection, which mimics HRSV disease in children more closely than do experimental models of unnatural laboratory hosts, to evaluate the safety and efficacy of the PanAd3-RSV vaccine. We show that PanAd3-RSV alone and in combination with a modified vaccinia Ankara expressing the same HRSV antigens (MVA-RSV) induced neutralizing antibodies and cellular immunity in young seronegative calves and protected against upper and lower respiratory tract infection and pulmonary disease induced by heterologous BRSV challenge. There was no evidence either of enhanced pulmonary pathology or of enhanced respiratory disease in vaccinated calves after BRSV challenge. These findings support the continued evaluation of the vectored RSV vaccines in man. PMID:26268314

  17. 9 CFR 113.206 - Wart Vaccine, Killed Virus.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Wart Vaccine, Killed Virus. 113.206... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.206 Wart Vaccine, Killed Virus. Wart Vaccine, Killed Virus, shall be...

  18. 9 CFR 113.206 - Wart Vaccine, Killed Virus.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Wart Vaccine, Killed Virus. 113.206... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.206 Wart Vaccine, Killed Virus. Wart Vaccine, Killed Virus, shall be...

  19. 9 CFR 113.206 - Wart Vaccine, Killed Virus.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Wart Vaccine, Killed Virus. 113.206... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.206 Wart Vaccine, Killed Virus. Wart Vaccine, Killed Virus, shall be...

  20. 9 CFR 113.206 - Wart Vaccine, Killed Virus.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Wart Vaccine, Killed Virus. 113.206... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.206 Wart Vaccine, Killed Virus. Wart Vaccine, Killed Virus, shall be...

  1. 9 CFR 113.206 - Wart Vaccine, Killed Virus.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Wart Vaccine, Killed Virus. 113.206... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.206 Wart Vaccine, Killed Virus. Wart Vaccine, Killed Virus, shall be...

  2. Inducible Gene Expression in Tumors Colonized by Modified Oncolytic Vaccinia Virus Strains

    PubMed Central

    Huppertz, Sascha; Zhang, Qian; Geissinger, Ulrike; Härtl, Barbara; Gentschev, Ivaylo

    2014-01-01

    ABSTRACT Exogenous gene induction of therapeutic, diagnostic, and safety mechanisms could be a considerable improvement in oncolytic virotherapy. Here, we introduced a doxycycline-inducible promoter system (comprised of a tetracycline repressor, several promoter constructs, and a tet operator sequence) into oncolytic recombinant vaccinia viruses (rVACV), which were further characterized in detail. Experiments in cell cultures as well as in tumor-bearing mice were analyzed to determine the role of the inducible-system components. To accomplish this, we took advantage of the optical reporter construct, which resulted in the production of click-beetle luciferase as well as a red fluorescent protein. The results indicated that each of the system components could be used to optimize the induction rates and had an influence on the background expression levels. Depending on the given gene to be induced in rVACV-colonized tumors of patients, we discuss the doxycycline-inducible promoter system adjustment and further optimization. IMPORTANCE Oncolytic virotherapy of cancer can greatly benefit from the expression of heterologous genes. It is reasonable that some of those heterologous gene products could have detrimental effects either on the cancer patient or on the oncolytic virus itself if they are expressed at the wrong time or if the expression levels are too high. Therefore, exogenous control of gene expression levels by administration of a nontoxic inducer will have positive effects on the safety as well as the therapeutic outcome of oncolytic virotherapy. In addition, it paves the way for the introduction of new therapeutic genes into the genome of oncolytic viruses that could not have been tested otherwise. PMID:25056902

  3. Ectopic Expression of Vaccinia Virus E3 and K3 Cannot Rescue Ectromelia Virus Replication in Rabbit RK13 Cells

    PubMed Central

    Peng, Chen; Rothenburg, Stefan; Hersperger, Adam R.

    2015-01-01

    As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV. PMID:25734776

  4. Ectopic expression of vaccinia virus E3 and K3 cannot rescue ectromelia virus replication in rabbit RK13 cells.

    PubMed

    Hand, Erin S; Haller, Sherry L; Peng, Chen; Rothenburg, Stefan; Hersperger, Adam R

    2015-01-01

    As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV. PMID:25734776

  5. Anchoring a secreted plasmodium antigen on the surface of recombinant vaccinia virus-infected cells increases its immunogenicity.

    PubMed Central

    Langford, C J; Edwards, S J; Smith, G L; Mitchell, G F; Moss, B; Kemp, D J; Anders, R F

    1986-01-01

    We show that the subcellular location of foreign antigens expressed in recombinant vaccinia viruses influences their effectiveness as immunogens. Live recombinant viruses induced very poor antibody responses to a secreted repetitive plasmodial antigen (the S-antigen) in rabbits and mice. The poor response accords with epidemiological data suggesting that S-antigens are poorly immunogenic. Appending the transmembrane domain of a membrane immunoglobulin (immunoglobulin G1) to its carboxy terminus produced a hybrid S-antigen that was no longer secreted but was located on the surface of virus-infected cells. This recombinant virus elicited high antibody titers to the S-antigen. This approach will facilitate the use of live virus delivery systems to immunize against a wide range of foreign nonsurface antigens. Images PMID:3537732

  6. Combinations of Polyclonal or Monoclonal Antibodies to Proteins of the Outer Membranes of the Two Infectious Forms of Vaccinia Virus Protect Mice against a Lethal Respiratory Challenge

    PubMed Central

    Lustig, Shlomo; Fogg, Christiana; Whitbeck, J. Charles; Eisenberg, Roselyn J.; Cohen, Gary H.; Moss, Bernard

    2005-01-01

    Previous studies demonstrated that antibodies to live vaccinia virus infection are needed for optimal protection against orthopoxvirus infection. The present report is the first to compare the protective abilities of individual and combinations of specific polyclonal and monoclonal antibodies that target proteins of the intracellular (IMV) and extracellular (EV) forms of vaccinia virus. The antibodies were directed to one IMV membrane protein, L1, and to two outer EV membrane proteins, A33 and B5. In vitro studies showed that the antibodies to L1 neutralized IMV and that the antibodies to A33 and B5 prevented the spread of EV in liquid medium. Prophylactic administration of individual antibodies to BALB/c mice partially protected them against disease following intranasal challenge with lethal doses of vaccinia virus. Combinations of antibodies, particularly anti-L1 and -A33 or -L1 and -B5, provided enhanced protection when administered 1 day before or 2 days after challenge. Furthermore, the protection was superior to that achieved with pooled immune gamma globulin from human volunteers inoculated with live vaccinia virus. In addition, single injections of anti-L1 plus anti-A33 antibodies greatly delayed the deaths of severe combined immunodeficiency mice challenged with vaccinia virus. These studies suggest that antibodies to two or three viral membrane proteins optimally derived from the outer membranes of IMV and EV, may be beneficial for prophylaxis or therapy of orthopoxvirus infections. PMID:16227266

  7. Introduction of the six major genomic deletions of modified vaccinia virus Ankara (MVA) into the parental vaccinia virus is not sufficient to reproduce an MVA-like phenotype in cell culture and in mice.

    PubMed

    Meisinger-Henschel, Christine; Späth, Michaela; Lukassen, Susanne; Wolferstätter, Michael; Kachelriess, Heike; Baur, Karen; Dirmeier, Ulrike; Wagner, Markus; Chaplin, Paul; Suter, Mark; Hausmann, Jürgen

    2010-10-01

    Modified vaccinia virus Ankara (MVA) has a highly restricted host range in cell culture and is apathogenic in vivo. MVA was derived from the parental chorioallantois vaccinia virus Ankara (CVA) by more than 570 passages in chicken embryo fibroblast (CEF) cells. During CEF cell passaging, six major deletions comprising 24,668 nucleotides occurred in the CVA genome. We have cloned both the MVA and the parental CVA genome as bacterial artificial chromosomes (BACs) and have sequentially introduced the six major MVA deletions into the cloned CVA genome. Reconstituted mutant CVA viruses containing up to six major MVA deletions showed no detectable replication restriction in 12 of 14 mammalian cell lines tested; the exceptions were rabbit cell lines RK13 and SIRC. In mice, CVA mutants with up to three deletions showed slightly enhanced virulence, suggesting that gene deletion in replicating vaccinia virus (VACV) can result in gain of fitness in vivo. CVA mutants containing five or all six deletions were still pathogenic, with a moderate degree of attenuation. Deletion V was mainly responsible for the attenuated phenotype of these mutants. In conclusion, loss or truncation of all 31 open reading frames in the six major deletions is not sufficient to reproduce the specific MVA phenotype of strong attenuation and highly restricted host range. Mutations in viral genes outside or in association with the six major deletions appear to contribute significantly to this phenotype. Host range restriction and avirulence of MVA are most likely a cooperative effect of gene deletions and mutations involving the major deletions. PMID:20668072

  8. Introduction of the Six Major Genomic Deletions of Modified Vaccinia Virus Ankara (MVA) into the Parental Vaccinia Virus Is Not Sufficient To Reproduce an MVA-Like Phenotype in Cell Culture and in Mice▿

    PubMed Central

    Meisinger-Henschel, Christine; Späth, Michaela; Lukassen, Susanne; Wolferstätter, Michael; Kachelriess, Heike; Baur, Karen; Dirmeier, Ulrike; Wagner, Markus; Chaplin, Paul; Suter, Mark; Hausmann, Jürgen

    2010-01-01

    Modified vaccinia virus Ankara (MVA) has a highly restricted host range in cell culture and is apathogenic in vivo. MVA was derived from the parental chorioallantois vaccinia virus Ankara (CVA) by more than 570 passages in chicken embryo fibroblast (CEF) cells. During CEF cell passaging, six major deletions comprising 24,668 nucleotides occurred in the CVA genome. We have cloned both the MVA and the parental CVA genome as bacterial artificial chromosomes (BACs) and have sequentially introduced the six major MVA deletions into the cloned CVA genome. Reconstituted mutant CVA viruses containing up to six major MVA deletions showed no detectable replication restriction in 12 of 14 mammalian cell lines tested; the exceptions were rabbit cell lines RK13 and SIRC. In mice, CVA mutants with up to three deletions showed slightly enhanced virulence, suggesting that gene deletion in replicating vaccinia virus (VACV) can result in gain of fitness in vivo. CVA mutants containing five or all six deletions were still pathogenic, with a moderate degree of attenuation. Deletion V was mainly responsible for the attenuated phenotype of these mutants. In conclusion, loss or truncation of all 31 open reading frames in the six major deletions is not sufficient to reproduce the specific MVA phenotype of strong attenuation and highly restricted host range. Mutations in viral genes outside or in association with the six major deletions appear to contribute significantly to this phenotype. Host range restriction and avirulence of MVA are most likely a cooperative effect of gene deletions and mutations involving the major deletions. PMID:20668072

  9. Structural analysis of point mutations at the Vaccinia virus A20/D4 interface.

    PubMed

    Contesto-Richefeu, Céline; Tarbouriech, Nicolas; Brazzolotto, Xavier; Burmeister, Wim P; Peyrefitte, Christophe N; Iseni, Frédéric

    2016-09-01

    The Vaccinia virus polymerase holoenzyme is composed of three subunits: E9, the catalytic DNA polymerase subunit; D4, a uracil-DNA glycosylase; and A20, a protein with no known enzymatic activity. The D4/A20 heterodimer is the DNA polymerase cofactor, the function of which is essential for processive DNA synthesis. The recent crystal structure of D4 bound to the first 50 amino acids of A20 (D4/A201-50) revealed the importance of three residues, forming a cation-π interaction at the dimerization interface, for complex formation. These are Arg167 and Pro173 of D4 and Trp43 of A20. Here, the crystal structures of the three mutants D4-R167A/A201-50, D4-P173G/A201-50 and D4/A201-50-W43A are presented. The D4/A20 interface of the three structures has been analysed for atomic solvation parameters and cation-π interactions. This study confirms previous biochemical data and also points out the importance for stability of the restrained conformational space of Pro173. Moreover, these new structures will be useful for the design and rational improvement of known molecules targeting the D4/A20 interface. PMID:27599859

  10. Products and substrate/template usage of vaccinia virus DNA primase

    SciTech Connect

    De Silva, Frank S.; Paran, Nir; Moss, Bernard

    2009-01-05

    Vaccinia virus encodes a 90-kDa protein conserved in all poxviruses, with DNA primase and nucleoside triphosphatase activities. DNA primase products, synthesized with a single stranded {phi}X174 DNA template, were resolved as dinucleotides and long RNAs on denaturing polyacrylamide and agarose gels. Following phosphatase treatment, the dinucleotides GpC and ApC in a 4:1 ratio were identified by nearest neighbor analysis in which {sup 32}P was transferred from [{alpha}-{sup 32}P]CTP to initiating purine nucleotides. Differences in the nucleotide binding sites for initiation and elongation were suggested by the absence of CpC and UpC dinucleotides as well as the inability of deoxynucleotides to mediate primer synthesis despite their incorporation into mixed RNA/DNA primers. Strong primase activity was detected with an oligo(dC) template. However, there was only weak activity with an oligo(dT) template and none with oligo(dA) or oligo(dG). The absence of stringent template specificity is consistent with a role for the enzyme in priming DNA synthesis at the replication fork.

  11. Poly(A) Polymerase from Vaccinia Virus-Infected Cells I. Partial Purification and Characterization

    PubMed Central

    Brakel, Christine; Kates, Joseph R.

    1974-01-01

    Poly(A) polymerase activity is induced during vaccinia virus infection of HeLa cells. The enzyme is maximally induced at 3.5 h postinfection. Partial purification frees the preparation of RNase activity and RNA polymerase activity. ATP is the substrate for poly(A) synthesis. A small amount of poly(A) is produced from added adenosine diphosphate due to the production of ATP by an adenylate kinase present in the preparation. The incorporation of ATP into poly(A) is dependent on divalent cations (Mg2+ or Mn2+) and is not inhibited by UTP, CTP, or GTP. Poly(U) stimulates ATP incorporation; poly(A) and poly(C) have little effect on ATP incorporation, and poly(dT) is extremely inhibitory. RNA prepared from HeLa cells and from the partially purified poly(A) polymerase (the enzyme preparation contains endogenous RNA [Brakel and Kates]) stimulates ATP incorporation by poly(A) polymerase which was subjected to DEAE-cellulose chromatography. RNase's, pancreatic and T1, inhibit the production of poly(A). DNase has little effect. Poly(U) is able to stimulate poly(A) production in the presence of T1 RNase. PMID:4417406

  12. Role of the vaccinia virus O3 protein in cell entry can be fulfilled by its Sequence flexible transmembrane domain

    SciTech Connect

    Satheshkumar, P.S.; Chavre, James; Moss, Bernard

    2013-09-15

    The vaccinia virus O3 protein, a component of the entry–fusion complex, is encoded by all chordopoxviruses. We constructed truncation mutants and demonstrated that the transmembrane domain, which comprises two-thirds of this 35 amino acid protein, is necessary and sufficient for interaction with the entry–fusion complex and function in cell entry. Nevertheless, neither single amino acid substitutions nor alanine scanning mutagenesis revealed essential amino acids within the transmembrane domain. Moreover, replication-competent mutant viruses were generated by randomization of 10 amino acids of the transmembrane domain. Of eight unique viruses, two contained only two amino acids in common with wild type and the remainder contained one or none within the randomized sequence. Although these mutant viruses formed normal size plaques, the entry–fusion complex did not co-purify with the mutant O3 proteins suggesting a less stable interaction. Thus, despite low specific sequence requirements, the transmembrane domain is sufficient for function in entry. - Highlights: • The 35 amino acid O3 protein is required for efficient vaccinia virus entry. • The transmembrane domain of O3 is necessary and sufficient for entry. • Mutagenesis demonstrated extreme sequence flexibility compatible with function.

  13. The highly attenuated vaccinia virus strain modified virus Ankara induces apoptosis in melanoma cells and allows bystander dendritic cells to generate a potent anti-tumoral immunity

    PubMed Central

    Greiner, S; Humrich, J Y; Thuman, P; Sauter, B; Schuler, G; Jenne, L

    2006-01-01

    Vaccinia virus (VV) has been tested as oncolytic virus against malignant melanoma in clinical trials for more than 40 years. Until now, mainly strains comparable to viral strains used for smallpox vaccination have been probed for anti-tumoral therapy. We have shown recently that the wild-type strain Western Reserve (WR) can interfere with crucial functions of monocyte-derived dendritic cells (DCs). Our aim was to examine whether viral immune evasion mechanisms might be responsible for the ineffectiveness of WR-based vaccination strategies and whether the highly attenuated strain modified virus Ankara (MVA) differs from WR with respect to its possible immunostimulatory capacity after intratumoral injection. Using in vitro experiments, we compared the effect of both strains on melanoma cells and on local bystander DCs. We found that both VV-strains infected melanoma cells efficiently and caused disintegration of the actin cytoskeleton, as shown by fluorescence microscopy. In addition, both VV-strains caused apoptotic cell death in melanoma cells after infection. In contrast to MVA, WR underwent a complete viral replication cycle in melanoma cells. Bystander DCs were consecutively infected by newly generated WR virions and lost their capacity to induce allogeneic T cell proliferation. DCs in contact with MVA-infected melanoma cells retained their capacity to induce T cell proliferation. Immature DCs were capable of phagocytosing MVA-infected melanoma cells. Priming of autologous CD8+ T cells by DCs that had phagocytosed MVA-infected, MelanA positive melanoma cells resulted in the induction of T cell clones specifically reactive against the model antigen MelanA as shown by enzyme-linked immunospot (ELISPOT) analysis. We conclude that the clinical trials with oncolytic wild-type VV failed probably because of suppression of bystander DCs and consecutive suppression of T cell-mediated anti-melanoma immunity. The attenuated VV-strain MVA facilitates the generation of

  14. Newcastle disease virus vaccine potency determination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potency of inactivated Newcastle disease virus (NDV) vaccines is determined using vaccination and challenge. If the minimum killed viral antigen necessary for clinical protection can be determined, vaccines meeting or exceeding this dose might be considered of adequate potency. In these studies, c...

  15. Recombinant Swinepox Virus for Veterinary Vaccine Development.

    PubMed

    Fan, Hong-Jie; Lin, Hui-Xing

    2016-01-01

    Poxvirus-vectors have been widely used in vaccine development for several important human and animal diseases; some of these vaccines have been licensed and used extensively. Swinepox virus (SPV) is well suited to develop recombinant vaccines because of its large packaging capacity for recombinant DNA, its host range specificity, and its ability to induce appropriate immune responses. PMID:26458836

  16. Vaccine Therapy, Oncolytic Viruses, and Gliomas.

    PubMed

    Desjardins, Annick; Vlahovic, Gordana; Friedman, Henry S

    2016-03-01

    After years of active research and refinement, vaccine therapy and oncolytic viruses are becoming part of the arsenal in the treatment of gliomas. In contrast to standard treatment with radiation therapy and chemotherapy, vaccines are more specific to the patient and the tumor. The majority of ongoing vaccine trials are investigating peptide, heat shock protein, and dendritic cell vaccines. The immunosuppression triggered by the tumor itself and by its treatment is a major obstacle to vaccine and oncolytic virus therapy. Thus, combination therapy with different agents that affect the immune system will probably be necessary. PMID:26984213

  17. The effects of targeting the vaccinia virus B5R protein to the endoplasmic reticulum on virus morphogenesis and dissemination.

    PubMed

    Mathew, E C; Sanderson, C M; Hollinshead, R; Hollinshead, M; Grimley, R; Smith, G L

    1999-12-01

    The consequence of redirecting the vaccinia virus (VV) B5R protein to the endoplasmic reticulum (ER) has been investigated by the addition of an ER retrieval signal KKSL (K(2)X(2)) to the B5R C-terminus. This mutant B5R gene and a version of the gene with the inactive ER retrieval sequence KKSLAL (K(2)X(4)) were inserted into the thymidine kinase locus of a VV mutant lacking the B5R gene, vDeltaB5R. Similar levels of B5R protein were made by each virus, but the B5R-K(2)X(2) protein remained sensitive to endoglycosidase H and colocalised with protein disulphide isomerase in the ER. In contrast, the B5R-K(2)X(4) protein colocalised with 1, 4-galactosyltransferase in the trans-Golgi network. Electron microscopy revealed that even when the B5R protein was redirected to the ER, intracellular mature virus particles were wrapped by cellular membranes to form intracellular enveloped virus particles, although more incompletely wrapped particles were evident compared with wild type. These intracellular enveloped virus particles were, however, unable to efficiently induce the polymerisation of actin and the plaque size formed by vB5R-K(2)X(2) was small. Nevertheless, the amount and specific infectivity of EEV produced by vB5R-K(2)X(2) were similar to those of wild type, despite the dramatic reduction in the amount of B5R protein present in vB5R-K(2)X(2) EEV. PMID:10603324

  18. Evaluation of swinepox virus as a vaccine vector in pigs using an Aujeszky's disease (pseudorabies) virus gene insert coding for glycoproteins gp50 and gp63.

    PubMed

    van der Leek, M L; Feller, J A; Sorensen, G; Isaacson, W; Adams, C L; Borde, D J; Pfeiffer, N; Tran, T; Moyer, R W; Gibbs, E P

    1994-01-01

    Pigs were vaccinated by scarification or intramuscular injection with a swinepox virus-Aujeszky's disease (pseudorabies) recombinant (rSPV-AD) constructed by inserting the linked Aujeszky's disease virus genes coding for glycoproteins gp50 and gp63, attached to a vaccinia virus p7.5 promoter, into the thymidine kinase gene of swinepox virus. By 21 days after vaccination, 90 and 100 per cent of the animals vaccinated by scarification or intramuscular injection, respectively, had developed serum neutralising antibodies to Aujeszky's disease virus. Upon challenge with virulent virus, significantly fewer vaccinated pigs developed clinical Aujeszky's disease, nasal shedding of challenge virus was markedly reduced, and the vaccinated groups of pigs maintained or gained weight during the week after challenge whereas the unvaccinated control group lost weight. No transmission of rSPV-AD to in-contact controls was detected during the three weeks before challenge. In a second experiment, serum neutralising antibodies to Aujeszky's disease virus persisted for 150 days after the pigs were vaccinated with rSPV-AD by scarification or intramuscular injection and all the pigs showed an anamnestic response when they were revaccinated. PMID:8128561

  19. Primary Human Leukocyte Subsets Differentially Express Vaccinia Virus Receptors Enriched in Lipid Rafts

    PubMed Central

    Byrd, Daniel; Amet, Tohti; Hu, Ningjie; Lan, Jie; Hu, Sishun

    2013-01-01

    Poxviruses, including vaccinia virus (VV) and canarypox virus (ALVAC), do not indiscriminately infect all cell types of the primary human leukocytes (PHLs) that they encounter but instead demonstrate an extremely strong bias toward infection of monocytes and monocyte lineage cells. We studied the specific molecular events that determine the VV tropism for major PHL subsets including monocytes, B cells, neutrophils, NK cells, and T cells. We found that VV exhibited an extremely strong bias of cell surface protein-dependent binding to monocytes, B cells, and activated T cells to a similar degree and to neutrophils to a much lesser extent. Resting T cells and resting NK cells exhibited only trace amounts of VV binding. Activated T cells, however, became permissive to VV binding, infection, and replication, while activated NK cells still resisted VV binding. VV binding strongly colocalized with lipid rafts on the surfaces of all VV binding-susceptible PHL subsets, even when lipid rafts were relocated to cell uropods upon cell polarization. Immunosera raised against detergent-resistant membranes (DRMs) from monocytes or activated T cells, but not resting T cells, effectively cross-blocked VV binding to and infection of PHL subsets. CD29 and CD98, two lipid raft-associated membrane proteins that had been found to be important for VV entry into HeLa cells, had no effect on VV binding to and infection of primary activated T cells. Our data indicate that PHL subsets express VV protein receptors enriched in lipid rafts and that receptors are cross-presented on all susceptible PHLs. PMID:23785200

  20. Photodynamic therapy augments the efficacy of oncolytic vaccinia virus against primary and metastatic tumours in mice

    PubMed Central

    Gil, M; Bieniasz, M; Seshadri, M; Fisher, D; Ciesielski, M J; Chen, Y; Pandey, R K; Kozbor, D

    2011-01-01

    Background: Therapies targeted towards the tumour vasculature can be exploited for the purpose of improving the systemic delivery of oncolytic viruses to tumours. Photodynamic therapy (PDT) is a clinically approved treatment for cancer that is known to induce potent effects on tumour vasculature. In this study, we examined the activity of PDT in combination with oncolytic vaccinia virus (OVV) against primary and metastatic tumours in mice. Methods: The effect of 2-[1-hexyloxyethyl-]-2-devinyl pyropheophorbide-a (HPPH)-sensitised-PDT on the efficacy of oncolytic virotherapy was investigated against subcutaneously implanted syngeneic murine NXS2 neuroblastoma and human FaDu head and neck squamous cell carcinoma xenografts in nude mice. Treatment efficacy was evaluated by monitoring tumour growth and survival. The effects of combination treatment on vascular function were examined using magnetic resonance imaging (MRI) and immunohistochemistry, whereas viral replication in tumour cells was analysed by a standard plaque assay. Normal tissue phototoxicity following PDT-OV treatment was studied using the mouse foot response assay. Results: Combination of PDT with OVV resulted in inhibition of primary and metastatic tumour growth compared with either monotherapy. PDT-induced vascular disruption resulted in higher intratumoural viral titres compared with the untreated tumours. Five days after delivery of OVV, there was a loss of blood flow to the interior of tumour that was associated with infiltration of neutrophils. Administration of OVV did not result in any additional photodynamic damage to normal mouse foot tissue. Conclusion: These results provide evidence into the usefulness of PDT as a means of enhancing intratumoural replication and therapeutic efficacy of OV. PMID:21989183

  1. Towards a universal influenza vaccine: volunteer virus challenge studies in quarantine to speed the development and subsequent licensing

    PubMed Central

    Oxford, John S

    2013-01-01

    There are now more than 5 experimental vaccine formulations which induce T and B cell immunity towards the internally situated virus proteins matrix (M1 and M2e) and nucleoprotein (NP), and towards stem and stalk regions of the HA which have a shared antigenic structure amongst many of the 17 influenza A virus sub types. Such ‘universal vaccines’ could be used, at least in theory, as a prophylactic stockpile vaccine for newly emerged epidemic and novel pandemic influenza A viruses or as a supplement to conventional HA/NA vaccines. My own laboratory has approached the problem from the clinical viewpoint by identifying CD4+ cells which are present in influenza infected volunteers who resist influenza infection. We have established precisely which peptides in M and NP proteins react with these immune CD4 cells. These experimental vaccines induce immunity in animal models but with a single exception no data have been published on protection against influenza virus infection in humans. The efficacy of the latter vaccine is based on vaccinia virus (MVA) as a carrier and was analyzed in a quarantine unit. Given the absence of induced HI antibody in the new universal vaccines a possible licensing strategy is a virus challenge model in quarantine whereby healthy volunteers can be immunized with the new vaccine and thereafter deliberately infected and clinical signs recorded alongside quantities of virus excreted and compared with unvaccinated controls. PMID:23617282

  2. RNA Virus Reverse Genetics and Vaccine Design

    PubMed Central

    Stobart, Christopher C.; Moore, Martin L.

    2014-01-01

    RNA viruses are capable of rapid spread and severe or potentially lethal disease in both animals and humans. The development of reverse genetics systems for manipulation and study of RNA virus genomes has provided platforms for designing and optimizing viral mutants for vaccine development. Here, we review the impact of RNA virus reverse genetics systems on past and current efforts to design effective and safe viral therapeutics and vaccines. PMID:24967693

  3. Genetic characterisation of attenuated SAD rabies virus strains used for oral vaccination of wildlife.

    PubMed

    Geue, Lutz; Schares, Susann; Schnick, Christina; Kliemt, Jeannette; Beckert, Aline; Freuling, Conrad; Conraths, Franz J; Hoffmann, Bernd; Zanoni, Reto; Marston, Denise; McElhinney, Lorraine; Johnson, Nicholas; Fooks, Anthony R; Tordo, Noel; Müller, Thomas

    2008-06-19

    The elimination of rabies from the red fox (Vulpes vulpes) in Western Europe has been achieved by the oral rabies vaccination (ORV) of wildlife with a range of attenuated rabies virus strains. With the exception of the vaccinia rabies glycoprotein recombinant vaccine (VRG), all strains were originally derived from a common ancestor; the Street Alabama Dufferin (SAD) field strain. However, after more than 30 years of ORV it is still not possible to distinguish these vaccine strains and there is little information on the genetic basis for their attenuation. We therefore sequenced and compared the full-length genome of five commercially available SAD vaccine viruses (SAD B19, SAD P5/88, SAG2, SAD VA1 and SAD Bern) and four other SAD strains (the original SAD Bern, SAD VA1, ERA and SAD 1-3670 Wistar). Nucleotide sequencing allowed identifying each vaccine strain unambiguously. Phylogenetic analysis revealed that the majority of the currently used commercial attenuated rabies virus vaccines appear to be derived from SAD B19 rather than from SAD Bern. One commercially available vaccine virus did not contain the SAD strain mentioned in the product information of the producer. Two SAD vaccine strains appeared to consist of mixed genomic sequences. Furthermore, in-del events targeting A-rich sequences (in positive strand) within the 3' non-coding regions of M and G genes were observed in SAD-derivates developed in Europe. Our data also supports the idea of a possible recombination that had occurred during the derivation of the European branch of SAD viruses. If confirmed, this recombination event would be the first one reported among RABV vaccine strains. PMID:18485548

  4. Structure and Function of A41, a Vaccinia Virus Chemokine Binding Protein

    PubMed Central

    Abrescia, Nicola G. A; Pease, James E; Wise, Emma L; Stuart, David I; Smith, Geoffrey L; Grimes, Jonathan M

    2008-01-01

    The vaccinia virus (VACV) A41L gene encodes a secreted 30 kDa glycoprotein that is nonessential for virus replication but affects the host response to infection. The A41 protein shares sequence similarity with another VACV protein that binds CC chemokines (called vCKBP, or viral CC chemokine inhibitor, vCCI), and strains of VACV lacking the A41L gene induced stronger CD8+ T-cell responses than control viruses expressing A41. Using surface plasmon resonance, we screened 39 human and murine chemokines and identified CCL21, CCL25, CCL26 and CCL28 as A41 ligands, with Kds of between 8 nM and 118 nM. Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors. However the interaction of A41 and chemokines was inhibited in a dose-dependent manner by heparin, suggesting that A41 and heparin bind to overlapping sites on these chemokines. To better understand the mechanism of action of A41 its crystal structure was solved to 1.9 Å resolution. The protein has a globular β sandwich structure similar to that of the poxvirus vCCI family of proteins, but there are notable structural differences, particularly in surface loops and electrostatic charge distribution. Structural modelling suggests that the binding paradigm as defined for the vCCI–chemokine interaction is likely to be conserved between A41 and its chemokine partners. Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding. The biological and structural data suggest that A41 functions by forming moderately strong (nM) interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (μM–nM) and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM) chemokine–chemokine receptor interactions. PMID:18208323

  5. Immunization with vaccinia virus recombinants that express the surface glycoproteins of human parainfluenza virus type 3 (PIV3) protects patas monkeys against PIV3 infection.

    PubMed Central

    Spriggs, M K; Collins, P L; Tierney, E; London, W T; Murphy, B R

    1988-01-01

    Patas monkeys (Eryphrocebus patas) were immunized intradermally with two vaccinia virus recombinants that individually express the hemagglutinin-neuraminidase glycoprotein or the fusion glycoprotein of human parainfluenza virus type 3 (PIV3). These immunizations induced a high titer of PIV3 serum-neutralizing antibodies. At 1 month after immunization, monkeys were challenged intratracheally with PIV3. Subsequent virus replication was reduced in these monkeys by 3.2 log10 and 1.9 log10 (mean peak virus titers) in the upper and lower respiratory tracts, respectively, compared with control animals. The average duration of virus shedding was also reduced from 9.0 to 3.4 days in the upper respiratory tract and from 5.3 to 1.2 days in the lower respiratory tract. These findings demonstrate that a single intradermal dose of live recombinant vaccinia viruses can significantly restrict the replication of a virus which primarily infects the epithelial cells of the respiratory tract. PMID:2831389

  6. A cancer-favoring oncolytic vaccinia virus shows enhanced suppression of stem-cell like colon cancer

    PubMed Central

    Yoo, So Young; Bang, Seo Young; Jeong, Su-Nam; Kang, Dae Hwan; Heo, Jeong

    2016-01-01

    Stem cell-like colon cancer cells (SCCs) pose a major challenge in colon cancer treatment because of their resistance to chemotherapy and radiotherapy. Oncolytic virus-based therapy has shown promising results in uncured cancer patients; however, its effects on SCCs are not well studied yet. Here, we engineered a cancer-favoring oncolytic vaccinia virus (CVV) as a potent biotherapeutic and investigated its therapeutic efficacy in terms of killing SCCs. CVV is an evolved Wyeth strain vaccinia virus (EVV) lacking the viral thymidine kinase. SCC models were established using human or mouse colon cancer spheres, which continuously expressed stemness markers. The cancer-favoring characteristics and different cytotoxic pathways for killing cancer cells successfully overrode general drug resistance, thereby killing colon cancer cells regardless of the presence of SCCs. Subcutaneously injected HT29 spheres showed lower growth in CVV-treated models than in 5-Fu-treated models. Intraperitoneally injected CT26 spheres induced tumor masses in the abdominal region. CVV-treated groups showed higher survival rates and smaller tumor mass formation, compared to 5-Fu-treated groups. Interestingly, the combined treatment of CVV with 5-Fu showed improved survival rates and complete suppression of tumor mass. The CVV developed in this study, thus, effectively suppresses SCCs, which can be synergistically enhanced by simultaneous treatment with the anticancer drug 5-Fu. Our novel CVV is highly advantageous as a next-generation therapeutic for treating colon cancer. PMID:26918725

  7. A cancer-favoring oncolytic vaccinia virus shows enhanced suppression of stem-cell like colon cancer.

    PubMed

    Yoo, So Young; Bang, Seo Young; Jeong, Su-Nam; Kang, Dae Hwan; Heo, Jeong

    2016-03-29

    Stem cell-like colon cancer cells (SCCs) pose a major challenge in colon cancer treatment because of their resistance to chemotherapy and radiotherapy. Oncolytic virus-based therapy has shown promising results in uncured cancer patients; however, its effects on SCCs are not well studied yet. Here, we engineered a cancer-favoring oncolytic vaccinia virus (CVV) as a potent biotherapeutic and investigated its therapeutic efficacy in terms of killing SCCs. CVV is an evolved Wyeth strain vaccinia virus (EVV) lacking the viral thymidine kinase. SCC models were established using human or mouse colon cancer spheres, which continuously expressed stemness markers. The cancer-favoring characteristics and different cytotoxic pathways for killing cancer cells successfully overrode general drug resistance, thereby killing colon cancer cells regardless of the presence of SCCs. Subcutaneously injected HT29 spheres showed lower growth in CVV-treated models than in 5-Fu-treated models. Intraperitoneally injected CT26 spheres induced tumor masses in the abdominal region. CVV-treated groups showed higher survival rates and smaller tumor mass formation, compared to 5-Fu-treated groups. Interestingly, the combined treatment of CVV with 5-Fu showed improved survival rates and complete suppression of tumor mass. The CVV developed in this study, thus, effectively suppresses SCCs, which can be synergistically enhanced by simultaneous treatment with the anticancer drug 5-Fu. Our novel CVV is highly advantageous as a next-generation therapeutic for treating colon cancer. PMID:26918725

  8. Marker rescue of temperature-sensitive mutations of vaccinia virus WR: correlation of genetic and physical maps.

    PubMed Central

    Ensinger, M J; Rovinsky, M

    1983-01-01

    The physical map locations of 62 temperature-sensitive mutations of vaccinia virus WR have been determined by marker rescue experiments, using cloned HindIII fragments of wild-type DNA. Since vaccinia virus DNA is not infectious, marker rescue was performed by infecting monolayers of cells at the nonpermissive temperature with a low multiplicity of the mutant to be rescued and transfecting with calcium phosphate-precipitated recombinant DNA. Wild-type recombinants were measured by using either a direct plaque assay technique or a two-step procedure in which the final yield of virus from the transfected cells was assayed at the permissive and nonpermissive temperatures. Mutants that had been previously assigned to the same complementation-recombination group were rescued by the same HindIII fragment, with the exception of three mutants in one group that were rescued by either one of two adjacent fragments. A comparison between the genetic linkage map of the temperature-sensitive mutations in 30 mutants with their physical locations demonstrated that not only was the order of the genetic map correct but also recombination frequencies generally reflected actual physical distances. PMID:6312100

  9. The vaccinia virus E8R gene product is required for formation of transcriptionally active virions.

    PubMed

    Kato, Sayuri E M; Condit, Richard C; Moussatché, Nissin

    2007-10-25

    Two vaccinia virus temperature-sensitive mutants were mapped to the E8R gene and subjected to phenotypic characterization. Dts23 contains a missense mutation in the coding region of E8R (L81F), and in Cts19 the initiating methionine codon is changed from ATG to ATA (M1I). The two ts mutants display normal patterns of gene expression and DNA replication during infection. The E8 protein is synthesized exclusively late during infection and packaged into virion cores Western blot analysis revealed that E8 synthesis is reduced in Dts23 infected cells at permissive (31 degrees C) and non-permissive temperature (39.7 degrees C) and absent in Cts19 infection under both conditions. Dts23 virions produced at 39.7 degrees C were indistinguishable in appearance from wt virions. Cts19 fails to produce identifiable viral structures when incubated at 39.7 degrees C. Purified Dts23 virions produced at 39.7 degrees C contain reduced amounts of E8 and have a high particle to infectivity ratio; purified Cts19 virions grown at 31 degrees C also show reduced infectivity and do not contain detectable E8. Dts23 grown at 39.7 degrees C could enter cells but failed to synthesize early mRNA or produce CPE. Soluble extracts from mutant virions were active in a promoter dependent in vitro transcription assay, however intact mutant cores were defective in transcription. We suggest that E8 plays a subtle role in virion core structure that impacts directly or indirectly on core transcription. PMID:17619043

  10. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

    SciTech Connect

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; Chattopadhyay, Debasish

    2015-06-02

    Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This also represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.

  11. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

    DOE PAGESBeta

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; Chattopadhyay, Debasish

    2015-06-02

    Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

  12. Vaccinia Virus Extracellular Enveloped Virion Neutralization In Vitro and Protection In Vivo Depend on Complement▿

    PubMed Central

    Benhnia, Mohammed Rafii-El-Idrissi; McCausland, Megan M.; Moyron, Juan; Laudenslager, John; Granger, Steven; Rickert, Sandra; Koriazova, Lilia; Kubo, Ralph; Kato, Shinichiro; Crotty, Shane

    2009-01-01

    Antibody neutralization is an important component of protective immunity against vaccinia virus (VACV). Two distinct virion forms, mature virion and enveloped virion (MV and EV, respectively), possess separate functions and nonoverlapping immunological properties. In this study we examined the mechanics of EV neutralization, focusing on EV protein B5 (also called B5R). We show that neutralization of EV is predominantly complement dependent. From a panel of high-affinity anti-B5 monoclonal antibodies (MAbs), the only potent neutralizer in vitro (90% at 535 ng/ml) was an immunoglobulin G2a (IgG2a), and neutralization was complement mediated. This MAb was the most protective in vivo against lethal intranasal VACV challenge. Further studies demonstrated that in vivo depletion of complement caused a >50% loss of anti-B5 IgG2a protection, directly establishing the importance of complement for protection against the EV form. However, the mechanism of protection is not sterilizing immunity via elimination of the inoculum as the viral inoculum consisted of a purified MV form. The prevention of illness in vivo indicated rapid control of infection. We further demonstrate that antibody-mediated killing of VACV-infected cells expressing surface B5 is a second protective mechanism provided by complement-fixing anti-B5 IgG. Cell killing was very efficient, and this effector function was highly isotype specific. These results indicate that anti-B5 antibody-directed cell lysis via complement is a powerful mechanism for clearance of infected cells, keeping poxvirus-infected cells from being invisible to humoral immune responses. These findings highlight the importance of multiple mechanisms of antibody-mediated protection against VACV and point to key immunobiological differences between MVs and EVs that impact the outcome of infection. PMID:19019965

  13. Genetically Engineered Poxviruses for Recombinant Gene Expression, Vaccination, and Safety

    NASA Astrophysics Data System (ADS)

    Moss, Bernard

    1996-10-01

    Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure--function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

  14. Substrate specificity of three viral thymidine kinases (TK): vaccinia virus TK, feline herpesvirus TK, and canine herpesvirus TK.

    PubMed

    Solaroli, N; Johansson, M; Balzarini, J; Karlsson, A

    2006-01-01

    In search of novel suicide gene candidates we have cloned and characterized thymidine kinases from three viruses; vaccinia virus TK (VVTK), feline herpesvirus TK (FHV-TK), and canine herpesvirus TK (CHV-TK). Our studies showed that VVTK primarily is a thymidine kinase, with a substrate specificity mainly restricted to dThd and only minor affinity for dCyd. VVTK also is related closely to mammalian thymidine kinase 1 (TK1), with 66% identity and 75% general homology. Although CHV-TK and FHV-TK are sequence related to herpes simplex virus types 1 thymidine kinase (HSV1-TK), with 31% and 35% identity and a general similarity of 54%, the substrate specificity of these enzymes was restricted to dThd and thymidine analogs. PMID:17065088

  15. Live-attenuated influenza A virus vaccines using a B virus backbone

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The currently FDA-licensed live attenuated influenza virus vaccine contains a trivalent mixture of types A (H1N1 and H3N2) and B vaccine viruses. The two A virus vaccines have the backbone of a cold-adapted influenza A virus and the B virus vaccine has the six backbone segments derived from a cold-...

  16. Production of HIV-1 gp120 in packed-bed bioreactor using the vaccinia virus/T7 expression system.

    PubMed

    Hu, Y C; Kaufman, J; Cho, M W; Golding, H; Shiloach, J

    2000-01-01

    The HeLa cell-vaccinia virus system is an attractive method for producing recombinant mammalian proteins with proper post-translation modifications. This approach is especially important for the production of HIV-1 envelope glycoprotein, gp120, since more than half of its total mass is due to carbohydrates. A recombinant vaccinia virus/T7 RNA polymerase expression system was developed to express and produce large amounts of gp120 tagged with six histidine residues. In this system, the expressed T7 RNA polymerase from one virus drives the transcription of the gp120 encoded in the second virus. During the process development phase, the following parameters were studied: infection time, infection duration, multiplicity of infection, ratio of the two viruses, medium composition, and medium replacement strategy during the infection phase. The chosen production method was based on using the packed-bed bioreactor. The HeLa cells were immobilized on fibrous disks (Fibra-Cel) packed in an internal basket positioned in a vertically mixed bioreactor (Celligen Plus), and 25 g of carriers were packed in a 1.6-L (working volume) reactor. The process included a growth stage followed by a production stage. In the growth stage, the bed was perfused with a serum-containing medium, allowing the cells to grow to saturation, and in the production stage, done using serum-free medium, the cells were infected with the two recombinant viruses. The expressed protein was secreted, collected from the culture fluid, and purified. The specific production was found to be between 2 and 3 microg of protein/10(6) cells, and the volumetric production was around 10 mg/50 g carriers. PMID:11027165

  17. An orthopoxvirus-based vaccine reduces virus excretion after MERS-CoV infection in dromedary camels.

    PubMed

    Haagmans, Bart L; van den Brand, Judith M A; Raj, V Stalin; Volz, Asisa; Wohlsein, Peter; Smits, Saskia L; Schipper, Debby; Bestebroer, Theo M; Okba, Nisreen; Fux, Robert; Bensaid, Albert; Solanes Foz, David; Kuiken, Thijs; Baumgärtner, Wolfgang; Segalés, Joaquim; Sutter, Gerd; Osterhaus, Albert D M E

    2016-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) infections have led to an ongoing outbreak in humans, which was fueled by multiple zoonotic MERS-CoV introductions from dromedary camels. In addition to the implementation of hygiene measures to limit further camel-to-human and human-to-human transmissions, vaccine-mediated reduction of MERS-CoV spread from the animal reservoir may be envisaged. Here we show that a modified vaccinia virus Ankara (MVA) vaccine expressing the MERS-CoV spike protein confers mucosal immunity in dromedary camels. Compared with results for control animals, we observed a significant reduction of excreted infectious virus and viral RNA transcripts in vaccinated animals upon MERS-CoV challenge. Protection correlated with the presence of serum neutralizing antibodies to MERS-CoV. Induction of MVA-specific antibodies that cross-neutralize camelpox virus would also provide protection against camelpox. PMID:26678878

  18. De novo Fatty Acid Biosynthesis Contributes Significantly to Establishment of a Bioenergetically Favorable Environment for Vaccinia Virus Infection

    PubMed Central

    Greseth, Matthew D.; Traktman, Paula

    2014-01-01

    The poxvirus life cycle, although physically autonomous from the host nucleus, is nevertheless dependent upon cellular functions. A requirement for de novo fatty acid biosynthesis was implied by our previous demonstration that cerulenin, a fatty acid synthase inhibitor, impaired vaccinia virus production. Here we show that additional inhibitors of this pathway, TOFA and C75, reduce viral yield significantly, with partial rescue provided by exogenous palmitate, the pathway's end-product. Palmitate's major role during infection is not for phospholipid synthesis or protein palmitoylation. Instead, the mitochondrial import and β-oxidation of palmitate are essential, as shown by the impact of etomoxir and trimetazidine, which target these two processes respectively. Moreover, the impact of these inhibitors is exacerbated in the absence of exogenous glucose, which is otherwise dispensable for infection. In contrast to glucose, glutamine is essential for productive viral infection, providing intermediates that sustain the TCA cycle (anaplerosis). Cumulatively, these data suggest that productive infection requires the mitochondrial β-oxidation of palmitate which drives the TCA cycle and energy production. Additionally, infection causes a significant rise in the cellular oxygen consumption rate (ATP synthesis) that is ablated by etomoxir. The biochemical progression of the vaccinia life cycle is not impaired in the presence of TOFA, C75, or etomoxir, although the levels of viral DNA and proteins synthesized are somewhat diminished. However, by reversibly arresting infections at the onset of morphogenesis, and then monitoring virus production after release of the block, we determined that virion assembly is highly sensitive to TOFA and C75. Electron microscopic analysis of cells released into C75 revealed fragmented aggregates of viroplasm which failed to be enclosed by developing virion membranes. Taken together, these data indicate that vaccinia infection, and in

  19. De novo fatty acid biosynthesis contributes significantly to establishment of a bioenergetically favorable environment for vaccinia virus infection.

    PubMed

    Greseth, Matthew D; Traktman, Paula

    2014-03-01

    The poxvirus life cycle, although physically autonomous from the host nucleus, is nevertheless dependent upon cellular functions. A requirement for de novo fatty acid biosynthesis was implied by our previous demonstration that cerulenin, a fatty acid synthase inhibitor, impaired vaccinia virus production. Here we show that additional inhibitors of this pathway, TOFA and C75, reduce viral yield significantly, with partial rescue provided by exogenous palmitate, the pathway's end-product. Palmitate's major role during infection is not for phospholipid synthesis or protein palmitoylation. Instead, the mitochondrial import and β-oxidation of palmitate are essential, as shown by the impact of etomoxir and trimetazidine, which target these two processes respectively. Moreover, the impact of these inhibitors is exacerbated in the absence of exogenous glucose, which is otherwise dispensable for infection. In contrast to glucose, glutamine is essential for productive viral infection, providing intermediates that sustain the TCA cycle (anaplerosis). Cumulatively, these data suggest that productive infection requires the mitochondrial β-oxidation of palmitate which drives the TCA cycle and energy production. Additionally, infection causes a significant rise in the cellular oxygen consumption rate (ATP synthesis) that is ablated by etomoxir. The biochemical progression of the vaccinia life cycle is not impaired in the presence of TOFA, C75, or etomoxir, although the levels of viral DNA and proteins synthesized are somewhat diminished. However, by reversibly arresting infections at the onset of morphogenesis, and then monitoring virus production after release of the block, we determined that virion assembly is highly sensitive to TOFA and C75. Electron microscopic analysis of cells released into C75 revealed fragmented aggregates of viroplasm which failed to be enclosed by developing virion membranes. Taken together, these data indicate that vaccinia infection, and in

  20. Vaccinia virus recombinants encoding the truncated structural gene region of Venezuelan equine encephalitis virus (VEEV) give solid protection against peripheral challenge but only partial protection against airborne challenge with virulent VEEV.

    PubMed

    Phillpotts, R J; Lescott, T L; Jacobs, S C

    2000-10-01

    Vaccinia virus (VV) recombinants that contain the genes encoding the Venezuelan equine encephalitis virus (VEEV) structural gene region (C-E3-E2-6 K-E1) solidly protect mice against peripheral challenge with virulent VEEV, but provide only partial protection against airborne challenge. To improve upon these results we focussed on the principal antigens involved in protection. VV recombinants encoding the structural genes E3-E2-6 K-E1, E3-E2-6 K or 6 K-E1 were prepared and evaluated for their ability to protect Balb/c mice after a single dorsal scarification with 10(8) PFU against peripheral or airborne challenge with virulent VEEV. The antibody response was also examined. Our experiments provide new evidence that truncates of the VEEV structural region (E3-E2-6 K-E1, E3-E2-6 K), cloned and expressed in VV, protect against challenge with virulent virus. They also confirm the important role of E2 in protection. However, we were unable to improve upon previously reported levels of protection against airborne challenge. A substantial level of circulating antibodies and the presence of local IgA (not always induced by mucosal immunization) (Greenway et al., 1992) appear essential for protection against the airborne virus. Current VV-VEEV recombinants seem unable to elicit this level of immune response and further improvements are therefore required to increase the immunogenicity of VV-VEEV vaccines. PMID:11252667

  1. Attacking Postoperative Metastases using Perioperative Oncolytic Viruses and Viral Vaccines

    PubMed Central

    Tai, Lee-Hwa; Auer, Rebecca

    2014-01-01

    Surgical resection of solid primary malignancies is a mainstay of therapy for cancer patients. Despite being the most effective treatment for these tumors, cancer surgery has been associated with impaired metastatic clearance due to immunosuppression. In preclinical surgery models and human cancer patients, we and others have demonstrated a profound suppression of both natural killer (NK) and T cell function in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Oncolytic viruses (OV) were originally designed to selectively infect and replicate in tumors, with the primary objective of directly lysing cancer cells. It is becoming increasingly clear, however, that OV infection results in a profound inflammatory reaction within the tumor, initiating innate and adaptive immune responses against it that is critical for its therapeutic benefit. This anti-tumor immunity appears to be mediated predominantly by NK and cytotoxic T cells. In preclinical models, we found that preoperative OV prevents postoperative NK cell dysfunction and attenuates tumor dissemination. Due to theoretical safety concerns of administering live virus prior to surgery in cancer patients, we characterized safe, attenuated versions of OV, and viral vaccines that could stimulate NK cells and reduce metastases when administered in the perioperative period. In cancer patients, we observed that in vivo infusion with oncolytic vaccinia virus and ex vivo stimulation with viral vaccines promote NK cell activation. These preclinical studies provide a novel and clinically relevant setting for OV therapy. Our challenge is to identify safe and promising OV therapies that will activate NK and T cells in the perioperative period preventing the establishment of micrometastatic disease in cancer patients. PMID:25161958

  2. Functional analysis of N-linked glycosylation mutants of the measles virus fusion protein synthesized by recombinant vaccinia virus vectors.

    PubMed Central

    Alkhatib, G; Shen, S H; Briedis, D; Richardson, C; Massie, B; Weinberg, R; Smith, D; Taylor, J; Paoletti, E; Roder, J

    1994-01-01

    The role of N-linked glycosylation in the biological activity of the measles virus (MV) fusion (F) protein was analyzed by expressing glycosylation mutants with recombinant vaccinia virus vectors. There are three potential N-linked glycosylation sites located on the F2 subunit polypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant proteins was reduced by 50% relative to the wild-type level when glycosylation at either Asn-29 or Asn-61 was abolished. Despite the similar levels of cell surface expression, the Asn-29 and Asn-61 mutant proteins had different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not exhibit any significant cell fusion activity. Inactivation of the Asn-67 glycosylation site also reduced cell surface transport of mutant protein but had little effect on its ability to cause cell fusion. However, when the Asn-67 mutation was combined with mutations at either of the other two sites, cleavage-dependent activation, cell surface expression, and cell fusion activity were completely abolished. Our data show that the loss of N-linked oligosaccharides markedly impaired the proteolytic cleavage, stability, and biological activity of the MV F protein. The oligosaccharide side chains in MV F are thus essential for optimum conformation of the extracellular F2 subunit that is presumed to bind cellular membranes. Images PMID:8107215

  3. A Heterologous Prime/Boost Vaccination Strategy Enhances the Immunogenicity of Therapeutic Vaccines for Hepatitis C Virus

    PubMed Central

    Fournillier, Anne; Frelin, Lars; Jacquier, Emilie; Ahlén, Gustaf; Brass, Anette; Gerossier, Estelle; Holmström, Fredrik; Broderick, Kate E.; Sardesai, Niranjan Y.; Bonnefoy, Jean-Yves; Inchauspé, Geneviève; Sällberg, Matti

    2013-01-01

    Background. We explored the concept of heterologous prime/boost vaccination using 2 therapeutic vaccines currently in clinical development aimed at treating chronically infected hepatitis C virus (HCV) patients: prime with a DNA-based vaccine expressing HCV genotype 1a NS3/4A proteins (ChronVac-C) and boost with a modified vaccinia virus Ankara vaccine expressing genotype 1b NS3/4/5B proteins (MVATG16643). Methods. Two ChronVac-C immunizations 4 weeks apart were delivered intramuscularly in combination with in vivo electroporation and subsequently 5 or 12 weeks later boosted by 3 weekly subcutaneous injections of MVATG16643. Two mouse strains were used, and we evaluated quality, magnitude, and functionality of the T cells induced. Results. DNA prime/MVA boost regimen induced significantly higher levels of interferon γ (IFN-γ) or interleukin 2 (IL-2) ELISpot responses compared with each vaccine alone, independent of the time of analysis and the time interval between vaccinations. Both CD8+ and CD4+ T-cell responses as well as the spectrum of epitopes recognized was improved. A significant increase in polyfunctional IFN-γ/tumor necrosis factor α (TNF-α)/CD107a+ CD8+ T cells was detected following ChronVac-C/MVATG16643 vaccination (from 3% to 25%), and prime/boost was the only regimen that activated quadrifunctional T cells (IFN-γ/TNF-α/CD107a/IL-2). In vivo functional protective capacity of DNA prime/MVA boost was demonstrated in a Listeria-NS3-1a challenge model. Conclusions. We provide a proof-of-concept that immunogenicity of 2 HCV therapeutic vaccines can be improved using their combination, which merits further clinical development. PMID:23776192

  4. Clinical experience with respiratory syncytial virus vaccines.

    PubMed

    Piedra, Pedro A

    2003-02-01

    Respiratory syncytial virus (RSV) infection is at times associated with life-threatening lower respiratory tract illness in infancy. Severe infection during the first year of life may be an important risk factor or indicator for the development of asthma in early childhood. Severe infections primarily occur in healthy infants, and young infants and children with specific risk factors. However, RSV causes respiratory infections in all age groups. Indeed it is now recognized that RSV disease is responsible for significant morbidity and mortality in the geriatric population. RSV infection remains difficult to treat, and prevention is a worldwide goal. For this reason there has been an intensive effort to develop an effective and safe RSV vaccine. Initial infection with RSV affords limited protection to reinfection, yet repeated episodes decrease the risk for lower respiratory tract illness. In the 20 years from 1960 to 1980, trials of several candidate RSV vaccines failed to attain the desired safety and protection against natural infection. Some vaccine types either failed to elicit immunogenicity, as with the live subcutaneous vaccine, or resulted in exaggerated disease on natural exposure to the virus, as with the formalin-inactivated (FI) type. Currently vaccine candidates are being developed based on the molecular virology of RSV. Recent formulations of candidate RSV vaccines have focused on subunit vaccines [such as purified fusion protein (PFP)], subunit vaccines combined with nonspecific immune activating adjuvants, live attenuated vaccines (including cold passaged, temperature-sensitive or cpts mutants), genetically engineered live attenuated vaccines and polypeptide vaccines. PMID:12671459

  5. Vaccinia virus entry/fusion complex subunit A28 is a target of neutralizing and protective antibodies

    SciTech Connect

    Nelson, Gretchen E.; Sisler, Jerry R.; Chandran, Dev; Moss, Bernard

    2008-10-25

    The vaccinia virus entry/fusion complex (EFC) is comprised of at least eight transmembrane proteins that are conserved in all poxviruses. However, neither the physical structure of the EFC nor the immunogenicity of the individual components has been determined. We prepared soluble forms of two EFC components, A28 and H2, by replacing the transmembrane domain with a signal peptide and adding a polyhistidine tail. The proteins were expressed by baculoviruses, secreted from insect cells, purified by affinity chromatography and used to raise antibodies in rabbits. The antibodies recognized the viral proteins but only the antibody to recombinant A28 bound intact virions and neutralized infectivity. Analyses with a set of overlapping peptides revealed a neutralizing epitope between residues 73 and 92 of A28. Passive immunization of mice with IgG purified from the anti-A28 serum provided partial protection against a vaccinia virus intranasal challenge, whereas IgG from the anti-H2 serum did not.

  6. Vaccines in development against West Nile virus.

    PubMed

    Brandler, Samantha; Tangy, Frederic

    2013-10-01

    West Nile encephalitis emerged in 1999 in the United States, then rapidly spread through the North American continent causing severe disease in human and horses. Since then, outbreaks appeared in Europe, and in 2012, the United States experienced a new severe outbreak reporting a total of 5,387 cases of West Nile virus (WNV) disease in humans, including 243 deaths. So far, no human vaccine is available to control new WNV outbreaks and to avoid worldwide spreading. In this review, we discuss the state-of-the-art of West Nile vaccine development and the potential of a novel safe and effective approach based on recombinant live attenuated measles virus (MV) vaccine. MV vaccine is a live attenuated negative-stranded RNA virus proven as one of the safest, most stable and effective human vaccines. We previously described a vector derived from the Schwarz MV vaccine strain that stably expresses antigens from emerging arboviruses, such as dengue, West Nile or chikungunya viruses, and is strongly immunogenic in animal models, even in the presence of MV pre-existing immunity. A single administration of a recombinant MV vaccine expressing the secreted form of WNV envelope glycoprotein elicited protective immunity in mice and non-human primates as early as two weeks after immunization, indicating its potential as a human vaccine. PMID:24084235

  7. Clinical development of Ebola vaccines.

    PubMed

    Sridhar, Saranya

    2015-09-01

    The ongoing outbreak of Ebola virus disease in West Africa highlighted the lack of a licensed drug or vaccine to combat the disease and has renewed the urgency to develop a pipeline of Ebola vaccines. A number of different vaccine platforms are being developed by assessing preclinical efficacy in animal models and expediting clinical development. Over 15 different vaccines are in preclinical development and 8 vaccines are now in different stages of clinical evaluation. These vaccines include DNA vaccines, virus-like particles and viral vectors such as live replicating vesicular stomatitis virus (rVSV), human and chimpanzee adenovirus, and vaccinia virus. Recently, in preliminary results reported from the first phase III trial of an Ebola vaccine, the rVSV-vectored vaccine showed promising efficacy. This review charts this rapidly advancing area of research focusing on vaccines in clinical development and discusses the future opportunities and challenges faced in the licensure and deployment of Ebola vaccines. PMID:26668751

  8. Clinical development of Ebola vaccines

    PubMed Central

    Sridhar, Saranya

    2015-01-01

    The ongoing outbreak of Ebola virus disease in West Africa highlighted the lack of a licensed drug or vaccine to combat the disease and has renewed the urgency to develop a pipeline of Ebola vaccines. A number of different vaccine platforms are being developed by assessing preclinical efficacy in animal models and expediting clinical development. Over 15 different vaccines are in preclinical development and 8 vaccines are now in different stages of clinical evaluation. These vaccines include DNA vaccines, virus-like particles and viral vectors such as live replicating vesicular stomatitis virus (rVSV), human and chimpanzee adenovirus, and vaccinia virus. Recently, in preliminary results reported from the first phase III trial of an Ebola vaccine, the rVSV-vectored vaccine showed promising efficacy. This review charts this rapidly advancing area of research focusing on vaccines in clinical development and discusses the future opportunities and challenges faced in the licensure and deployment of Ebola vaccines. PMID:26668751

  9. [Immunogenicity and heterologous protection in mice with a recombinant adenoviral-based vaccine carrying a hepatitis C virus truncated NS3 and core fusion protein].

    PubMed

    Guan, Jie; Deng, Yao; Chen, Hong; Yang, Yang; Wen, Bo; Tan, Wenjie

    2015-01-01

    To develop a safe and broad-spectrum effective hepatitis C virus (HCV) T cell vaccine,we constructed the recombinant adenovirus-based vaccine that carried the hepatitis C virus truncated NS3 and core fusion proteins. The expression of the fusion antigen was confirmed by in vitro immunofluorescence and western blotting assays. Our results indicated that this vaccine not only stimulated antigen-specific antibody responses,but also activated strong NS3-specific T cell immune responses. NS3-specific IFN-γ+ and TNF-α+ CD4+ T cell subsets were also detected by a intracellular cytokine secretion assay. In a surrogate challenge assay based on a recombinant heterologous HCV (JFH1,2a) vaccinia virus,the recombinant adenovirus-based vaccine was capable of eliciting effective levels of cross-protection. These findings have im- portant implications for the study of HCV immune protection and the future development of a novel vaccine. PMID:25997323

  10. Biophysical analysis of bacterial and viral systems. A shock tube study of bio-aerosols and a correlated AFM/nanosims investigation of vaccinia virus

    SciTech Connect

    Gates, Sean Damien

    2013-05-01

    The work presented herein is concerned with the development of biophysical methodology designed to address pertinent questions regarding the behavior and structure of select pathogenic agents. Two distinct studies are documented: a shock tube analysis of endospore-laden bio-aerosols and a correlated AFM/NanoSIMS study of the structure of vaccinia virus.

  11. Vaccinia viruses isolated from skin infection in horses produced cutaneous and systemic disease in experimentally infected rabbits.

    PubMed

    Cargnelutti, Juliana Felipetto; Schmidt, Candice; Masuda, Eduardo Kenji; Nogueira, Paula Rochelle Kurrle; Weiblen, Rudi; Flores, Eduardo Furtado

    2012-10-01

    The susceptibility of rabbits to two isolates of Vaccinia virus (VACV) recovered from cutaneous disease in horses in Southern Brazil was investigated. Rabbits were inoculated in the ear skin with both VACV isolates, either in single or mixed infection. All inoculated animals presented local skin lesions characterized by hyperaemia, papules, vesicles, pustules and ulcers. Infectious virus was detected in the lungs and intestine of rabbits that died during acute disease. Histological examination of the skin revealed changes characteristic of those associated with members of the genus Orthopoxvirus. These results demonstrate that rabbits develop skin disease accompanied by systemic signs upon intradermal inoculation of these two equine VACV isolates, either alone or in combination, opening the way for using rabbits to study selected aspects of the biology and pathogenesis of VACV infection. PMID:22244689

  12. Evaluation of radiation effects against C6 glioma in combination with vaccinia virus-p53 gene therapy

    NASA Technical Reports Server (NTRS)

    Gridley, D. S.; Andres, M. L.; Li, J.; Timiryasova, T.; Chen, B.; Fodor, I.; Nelson, G. A. (Principal Investigator)

    1998-01-01

    The primary objective of this study was to evaluate the antitumor effects of recombinant vaccinia virus-p53 (rVV-p53) in combination with radiation therapy against the C6 rat glioma, a p53 deficient tumor that is relatively radioresistant. VV-LIVP, the parental virus (Lister strain), was used as a control. Localized treatment of subcutaneous C6 tumors in athymic mice with either rVV-p53 or VV-LIVP together with tumor irradiation resulted in low tumor incidence and significantly slower tumor progression compared to the agents given as single modalities. Assays of blood and spleen indicated that immune system activation may account, at least partly, for the enhance tumor inhibition seen with combined treatment. No overt signs of treatment-related toxicity were noted.

  13. 9 CFR 113.204 - Mink Enteritis Vaccine, Killed Virus.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Mink Enteritis Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.204 Mink Enteritis Vaccine, Killed Virus. Mink Enteritis...

  14. 9 CFR 113.212 - Bursal Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Bursal Disease Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.212 Bursal Disease Vaccine, Killed Virus. Bursal Disease...

  15. 9 CFR 113.212 - Bursal Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Bursal Disease Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.212 Bursal Disease Vaccine, Killed Virus. Bursal Disease...

  16. 9 CFR 113.204 - Mink Enteritis Vaccine, Killed Virus.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Mink Enteritis Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.204 Mink Enteritis Vaccine, Killed Virus. Mink Enteritis...

  17. 9 CFR 113.212 - Bursal Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Bursal Disease Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.212 Bursal Disease Vaccine, Killed Virus. Bursal Disease...

  18. 9 CFR 113.204 - Mink Enteritis Vaccine, Killed Virus.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Mink Enteritis Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.204 Mink Enteritis Vaccine, Killed Virus. Mink Enteritis...

  19. 9 CFR 113.204 - Mink Enteritis Vaccine, Killed Virus.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Mink Enteritis Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.204 Mink Enteritis Vaccine, Killed Virus. Mink Enteritis...

  20. 9 CFR 113.212 - Bursal Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Bursal Disease Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.212 Bursal Disease Vaccine, Killed Virus. Bursal Disease...

  1. 9 CFR 113.212 - Bursal Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Bursal Disease Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.212 Bursal Disease Vaccine, Killed Virus. Bursal Disease...

  2. 9 CFR 113.204 - Mink Enteritis Vaccine, Killed Virus.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Mink Enteritis Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.204 Mink Enteritis Vaccine, Killed Virus. Mink Enteritis...

  3. Characterization and Structure of the Vaccinia Virus NF-κB Antagonist A46 *

    PubMed Central

    Fedosyuk, Sofiya; Grishkovskaya, Irina; de Almeida Ribeiro, Euripedes; Skern, Tim

    2014-01-01

    Successful vaccinia virus (VACV) replication in the host requires expression of viral proteins that interfere with host immunity, such as antagonists of the activation of the proinflammatory transcription factor NF-κB. Two such VACV proteins are A46 and A52. A46 interacts with the Toll-like receptor/interleukin-1R (TIR) domain of Toll-like receptors and intracellular adaptors such as MAL (MyD88 adapter-like), TRAM (TIR domain-containing adapter-inducing interferon-β (TRIF)-related adaptor molecule), TRIF, and MyD88, whereas A52 binds to the downstream signaling components TRAF6 and IRAK2. Here, we characterize A46 biochemically, determine by microscale thermophoresis binding constants for the interaction of A46 with the TIR domains of MyD88 and MAL, and present the 2.0 Å resolution crystal structure of A46 residues 87–229. Full-length A46 behaves as a tetramer; variants lacking the N-terminal 80 residues are dimeric. Nevertheless, both bind to the Toll-like receptor domains of MAL and MyD88 with KD values in the low μm range. Like A52, A46 also shows a Bcl-2-like fold but with biologically relevant differences from that of A52. Thus, A46 uses helices α4 and α6 to dimerize, compared with the α1-α6 face used by A52 and other Bcl-2 like VACV proteins. Furthermore, the loop between A46 helices α4-α5 is flexible and shorter than in A52; there is also evidence for an intramolecular disulfide bridge between consecutive cysteine residues. We used molecular docking to propose how A46 interacts with the BB loop of the TRAM TIR domain. Comparisons of A46 and A52 exemplify how subtle changes in viral proteins with the same fold lead to crucial differences in biological activity. PMID:24356965

  4. A Novel Replication-Competent Vaccinia Vector MVTT Is Superior to MVA for Inducing High Levels of Neutralizing Antibody via Mucosal Vaccination

    PubMed Central

    Yu, Wenbo; Fang, Qing; Liu, Li; Zhuang, Ke; Shen, Tingting; Wang, Haibo; Tian, Po; Zhang, Linqi; Chen, Zhiwei

    2009-01-01

    Mucosal vaccination offers great advantage for inducing protective immune response to prevent viral transmission and dissemination. Here, we report our findings of a head-to-head comparison of two viral vectors modified vaccinia Ankara (MVA) and a novel replication-competent modified vaccinia Tian Tan (MVTT) for inducing neutralizing antibodies (Nabs) via intramuscular and mucosal vaccinations in mice. MVTT is an attenuated variant of the wild-type VTT, which was historically used as a smallpox vaccine for millions of Chinese people. The spike glycoprotein (S) of SARS-CoV was used as the test antigen after the S gene was constructed in the identical genomic location of two vectors to generate vaccine candidates MVTT-S and MVA-S. Using identical doses, MVTT-S induced lower levels (∼2-3-fold) of anti- SARS-CoV neutralizing antibodies (Nabs) than MVA-S through intramuscular inoculation. MVTT-S, however, was capable of inducing consistently 20-to-100-fold higher levels of Nabs than MVA-S when inoculated via either intranasal or intraoral routes. These levels of MVTT-S-induced Nab responses were substantially (∼10-fold) higher than that induced via the intramuscular route in the same experiments. Moreover, pre-exposure to the wild-type VTT via intranasal or intraoral route impaired the Nab response via the same routes of MVTT-S vaccination probably due to the pre-existing anti-VTT Nab response. The efficacy of intranasal or intraoral vaccination, however, was still 20-to-50-fold better than intramuscular inoculation despite the subcutaneous pre-exposure to wild-type VTT. Our data have implications for people who maintain low levels of anti-VTT Nabs after historical smallpox vaccination. MVTT is therefore an attractive live viral vector for mucosal vaccination. PMID:19159014

  5. The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surfaces.

    PubMed Central

    Lai, C F; Gong, S C; Esteban, M

    1991-01-01

    The nature of interaction between vaccinia virus and the surface of host cells as the first step in virus infection is undefined. A 32-kDa virus envelope protein has been identified as a cell surface binding protein (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:1569-1577, 1990). To carry out studies on the structure-function relationship of this protein, the 32-kDa protein was obtained from Escherichia coli cells harboring the expression plasmid pT7Ek32. The recombinant polypeptide was found to have structural properties similar to those of the native virus envelope protein. Binding studies of 125I-labeled 32-kDa protein to cultured cells of various origins revealed that the E. coli-produced 32-kDa protein exhibited selectivity, specificity, and saturability. Scatchard analysis indicated about 4.5 x 10(4) sites per cell with a high affinity (Kd = 1.8 x 10(-9) M), suggesting interaction of the 32-kDa protein with a specific receptor. The availability of large quantities of the 32-kDa virus protein in bacteria will permit further structural and functional studies of this virus envelope protein and facilitate identification of the specific cell surface receptor. Images PMID:1985213

  6. Duplication of the A17L Locus of Vaccinia Virus Provides an Alternate Route to Rifampin Resistance

    PubMed Central

    Erlandson, Karl J.; Cotter, Catherine A.; Charity, James C.; Martens, Craig; Fischer, Elizabeth R.; Ricklefs, Stacy M.; Porcella, Stephen F.

    2014-01-01

    ABSTRACT Specific gene duplications can enable double-stranded DNA viruses to adapt rapidly to environmental pressures despite the low mutation rate of their high-fidelity DNA polymerases. We report on the rapid positive selection of a novel vaccinia virus genomic duplication mutant in the presence of the assembly inhibitor rifampin. Until now, all known rifampin-resistant vaccinia virus isolates have contained missense mutations in the D13L gene, which encodes a capsid-like scaffold protein required for stabilizing membrane curvature during the early stage of virion assembly. Here we describe a second pathway to rifampin resistance involving A17, a membrane protein that binds and anchors D13 to the immature virion. After one round of selection, a rifampin-resistant virus that contained a genomic duplication in the A17L-A21L region was recovered. The mutant had both C-terminally truncated and full-length A17L open reading frames. Expression of the truncated A17 protein was retained when the virus was passaged in the presence of rifampin but was lost in the absence of the drug, suggesting that the duplication decreased general fitness. Both forms of A17 were bound to the virion membrane and associated with D13. Moreover, insertion of an additional truncated or inducible full-length A17L open reading frame into the genome of the wild-type virus was sufficient to confer rifampin resistance. In summary, this report contains the first evidence of an alternate mechanism for resistance of poxviruses to rifampin, indicates a direct relationship between A17 levels and the resistance phenotype, and provides further evidence of the ability of double-stranded DNA viruses to acquire drug resistance through gene duplication. IMPORTANCE The present study provides the first evidence of a new mechanism of resistance of a poxvirus to the antiviral drug rifampin. In addition, it affirms the importance of the interaction between the D13 scaffold protein and the A17 membrane protein

  7. Natural Killer Cells and Innate Interferon Gamma Participate in the Host Defense against Respiratory Vaccinia Virus Infection

    PubMed Central

    Abboud, Georges; Tahiliani, Vikas; Desai, Pritesh; Varkoly, Kyle; Driver, John; Hutchinson, Tarun E.

    2015-01-01

    ABSTRACT In establishing a respiratory infection, vaccinia virus (VACV) initially replicates in airway epithelial cells before spreading to secondary sites of infection, mainly the draining lymph nodes, spleen, gastrointestinal tract, and reproductive organs. We recently reported that interferon gamma (IFN-γ) produced by CD8 T cells ultimately controls this disseminated infection, but the relative contribution of IFN-γ early in infection is unknown. Investigating the role of innate immune cells, we found that the frequency of natural killer (NK) cells in the lung increased dramatically between days 1 and 4 postinfection with VACV. Lung NK cells displayed an activated cell surface phenotype and were the primary source of IFN-γ prior to the arrival of CD8 T cells. In the presence of an intact CD8 T cell compartment, depletion of NK cells resulted in increased lung viral load at the time of peak disease severity but had no effect on eventual viral clearance, disease symptoms, or survival. In sharp contrast, RAG−/− mice devoid of T cells failed to control VACV and succumbed to infection despite a marked increase in NK cells in the lung. Supporting an innate immune role for NK cell-derived IFN-γ, we found that NK cell-depleted or IFN-γ-depleted RAG−/− mice displayed increased lung VACV titers and dissemination to ovaries and a significantly shorter mean time to death compared to untreated NK cell-competent RAG−/− controls. Together, these findings demonstrate a role for IFN-γ in aspects of both the innate and adaptive immune response to VACV and highlight the importance of NK cells in T cell-independent control of VACV in the respiratory tract. IMPORTANCE Herein, we provide the first systematic evaluation of natural killer (NK) cell function in the lung after infection with vaccinia virus, a member of the Poxviridae family. The respiratory tract is an important mucosal site for entry of many human pathogens, including poxviruses, but precisely how our

  8. Zika Virus: Diagnosis, Therapeutics, and Vaccine.

    PubMed

    Shan, Chao; Xie, Xuping; Barrett, Alan D T; Garcia-Blanco, Mariano A; Tesh, Robert B; Vasconcelos, Pedro Fernando da Costa; Vasilakis, Nikos; Weaver, Scott C; Shi, Pei-Yong

    2016-03-11

    The current explosive epidemic of Zika virus in South and Central America, as well as the Caribbean, poses a global public health emergency. Here we comment on the challenges on development of better diagnosis and potential therapeutics and vaccine for Zika virus. PMID:27623030

  9. Yellow fever vector live-virus vaccines: West Nile virus vaccine development.

    PubMed

    Arroyo, J; Miller, C A; Catalan, J; Monath, T P

    2001-08-01

    By combining molecular-biological techniques with our increased understanding of the effect of gene sequence modification on viral function, yellow fever 17D, a positive-strand RNA virus vaccine, has been manipulated to induce a protective immune response against viruses of the same family (e.g. Japanese encephalitis and dengue viruses). Triggered by the emergence of West Nile virus infections in the New World afflicting humans, horses and birds, the success of this recombinant technology has prompted the rapid development of a live-virus attenuated candidate vaccine against West Nile virus. PMID:11516995

  10. Gold nanorod vaccine for respiratory syncytial virus

    NASA Astrophysics Data System (ADS)

    Stone, John W.; Thornburg, Natalie J.; Blum, David L.; Kuhn, Sam J.; Wright, David W.; Crowe, James E., Jr.

    2013-07-01

    Respiratory syncytial virus (RSV) is a major cause of pneumonia and wheezing in infants and the elderly, but to date there is no licensed vaccine. We developed a gold nanorod construct that displayed the major protective antigen of the virus, the fusion protein (F). Nanorods conjugated to RSV F were formulated as a candidate vaccine preparation by covalent attachment of viral protein using a layer-by-layer approach. In vitro studies using ELISA, electron microscopy and circular dichroism revealed that conformation-dependent epitopes were maintained during conjugation, and transmission electron microscopy studies showed that a dispersed population of particles could be achieved. Human dendritic cells treated with the vaccine induced immune responses in primary human T cells. These results suggest that this vaccine approach may be a potent method for immunizing against viruses such as RSV with surface glycoproteins that are targets for the human immune response.

  11. Emerging Respiratory Viruses: Challenges and Vaccine Strategies

    PubMed Central

    Gillim-Ross, Laura; Subbarao, Kanta

    2006-01-01

    The current threat of avian influenza to the human population, the potential for the reemergence of severe acute respiratory syndrome (SARS)-associated coronavirus, and the identification of multiple novel respiratory viruses underline the necessity for the development of therapeutic and preventive strategies to combat viral infection. Vaccine development is a key component in the prevention of widespread viral infection and in the reduction of morbidity and mortality associated with many viral infections. In this review we describe the different approaches currently being evaluated in the development of vaccines against SARS-associated coronavirus and avian influenza viruses and also highlight the many obstacles encountered in the development of these vaccines. Lessons learned from current vaccine studies, coupled with our increasing knowledge of the host and viral factors involved in viral pathogenesis, will help to increase the speed with which efficacious vaccines targeting newly emerging viral pathogens can be developed. PMID:17041137

  12. Phase 1 Study of Intratumoral Pexa-Vec (JX-594), an Oncolytic and Immunotherapeutic Vaccinia Virus, in Pediatric Cancer Patients

    PubMed Central

    Cripe, Timothy P; Ngo, Minhtran C; Geller, James I; Louis, Chrystal U; Currier, Mark A; Racadio, John M; Towbin, Alexander J; Rooney, Cliona M; Pelusio, Adina; Moon, Anne; Hwang, Tae-Ho; Burke, James M; Bell, John C; Kirn, David H; Breitbach, Caroline J

    2015-01-01

    Pexa-Vec (pexastimogene devacirepvec, JX-594) is an oncolytic and immunotherapeutic vaccinia virus designed to destroy cancer cells through viral lysis and induction of granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven tumor-specific immunity. Pexa-Vec has undergone phase 1 and 2 testing alone and in combination with other therapies in adult patients, via both intratumoral and intravenous administration routes. We sought to determine the safety of intratumoral administration in pediatric patients. In a dose-escalation study using either 106 or 107 plaque-forming units per kilogram, we performed one-time injections in up to three tumor sites in five pediatric patients and two injections in one patient. Ages at study entry ranged from 4 to 21 years, and their cancer diagnoses included neuroblastoma, hepatocellular carcinoma, and Ewing sarcoma. All toxicities were ≤ grade 3. The most common side effects were sinus fever and sinus tachycardia. All three patients at the higher dose developed asymptomatic grade 1 treatment-related skin pustules that resolved within 3–4 weeks. One patient showed imaging evidence suggestive of antitumor biological activity. The two patients tested for cellular immunoreactivity to vaccinia antigens showed strong responses. Overall, our study suggests Pexa-Vec is safe to administer to pediatric patients by intratumoral administration and could be studied further in this patient population. PMID:25531693

  13. The new ACAM2000 vaccine and other therapies to control orthopoxvirus outbreaks and bioterror attacks.

    PubMed

    Handley, Lauren; Buller, Robert Mark; Frey, Sharon E; Bellone, Clifford; Parker, Scott

    2009-07-01

    Quarantine, case tracing and population vaccination facilitated the global eradication, in 1980, of variola virus, the causative agent of smallpox. The vaccines used during the eradication period, including Dryvax, the smallpox vaccine used in the USA, were live vaccinia and cowpoxvirus-based vaccines, which induced long-lasting and cross-protective immunity against variola and other related poxviruses. These vaccine viruses were produced by serial propagation in domesticated animals. The drawbacks to such serially propagated live viral vaccines include the level of postvaccination local and systemic reactions and contraindications to their use in immunocompromised individuals, individuals with certain skin and cardiac diseases, and pregnant women. In the latter stages of the population-based smallpox vaccination campaign, research began with ways to improve safety and modernizing the manufacture of vaccinia vaccines; however, with the eradication of variola this work stopped. Outbreaks of monkeypoxvirus in humans and the bioterrorist threat of monkeypox and variola virus renewed the need for improved vaccinia vaccines. ACAM2000 is one of the new generation of smallpox vaccines. It is produced in cell culture from a clonally purified master seed stock of vaccinia derived from the New York City Board of Health strain of vaccinia. The clonally purified master seed assures a more homogeneous vaccine without the inherent mutations associated with serial propagation and the cell culture limits adventitious and bacterial contamination in vaccine production. In preclinical and clinical trials, ACAM2000 demonstrated an immunogenicity and safety profile similar to that of Dryvax. PMID:19538111

  14. Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis.

    PubMed Central

    Falgout, B; Bray, M; Schlesinger, J J; Lai, C J

    1990-01-01

    The protective immunity conferred by a set of recombinant vaccinia viruses containing the entire coding sequence of dengue virus type 4 nonstructural glycoprotein NS1 plus various flanking sequences was evaluated by using a mouse encephalitis model. Mice immunized with recombinant vNS1-NS2a, which expresses authentic NS1, were solidly protected against intracerebral dengue virus challenge. However, mice immunized with recombinants vNS1-15%NS2a and vRSVG/NS1-15%NS2a, which express aberrant forms of NS1, were only partially protected (63 to 67% survival rate). Serologic analysis showed that mice immunized with vNS1-NS2a developed high titers of antibodies to NS1 as measured by radioimmunoprecipitation, enzyme-linked immunosorbent assay, and complement-mediated cytolytic assays. In addition, a pool of sera from these animals was protective in a passive transfer experiment. Lower titers of NS1-specific antibodies were detected in sera of animals immunized with vNS1-15%NS2a or vRSVG/NS1-15%NS2a by all three assays. These data support the view that protection against dengue virus infection in mice may be mediated at least in part by NS1-specific antibodies through a mechanism of complement-mediated lysis of infected cells. Additionally, immunization with two recombinant viruses expressing authentic NS1 of dengue virus type 2 conferred partial protection (30-50%) against dengue virus type 2 challenge. Images PMID:2143542

  15. Canine distemper virus (CDV) infection of ferrets as a model for testing Morbillivirus vaccine strategies: NYVAC- and ALVAC-based CDV recombinants protect against symptomatic infection.

    PubMed

    Stephensen, C B; Welter, J; Thaker, S R; Taylor, J; Tartaglia, J; Paoletti, E

    1997-02-01

    Canine distemper virus (CDV) infection of ferrets causes an acute systemic disease involving multiple organ systems, including the respiratory tract, lymphoid system, and central nervous system (CNS). We have tested candidate CDV vaccines incorporating the fusion (F) and hemagglutinin (HA) proteins in the highly attenuated NYVAC strain of vaccinia virus and in the ALVAC strain of canarypox virus, which does not productively replicate in mammalian hosts. Juvenile ferrets were vaccinated twice with these constructs, or with an attenuated live-virus vaccine, while controls received saline or the NYVAC and ALVAC vectors expressing rabies virus glycoprotein. Control animals did not develop neutralizing antibody and succumbed to distemper after developing fever, weight loss, leukocytopenia, decreased activity, conjunctivitis, an erythematous rash typical of distemper, CNS signs, and viremia in peripheral blood mononuclear cells (as measured by reverse transcription-PCR). All three CDV vaccines elicited neutralizing titers of at least 1:96. All vaccinated ferrets survived, and none developed viremia. Both recombinant vaccines also protected against the development of symptomatic distemper. However, ferrets receiving the live-virus vaccine lost weight, became lymphocytopenic, and developed the erythematous rash typical of CDV. These data show that ferrets are an excellent model for evaluating the ability of CDV vaccines to protect against symptomatic infection. Because the pathogenesis and clinical course of CDV infection of ferrets is quite similar to that of other Morbillivirus infections, including measles, this model will be useful in testing new candidate Morbillivirus vaccines. PMID:8995676

  16. Canine distemper virus (CDV) infection of ferrets as a model for testing Morbillivirus vaccine strategies: NYVAC- and ALVAC-based CDV recombinants protect against symptomatic infection.

    PubMed Central

    Stephensen, C B; Welter, J; Thaker, S R; Taylor, J; Tartaglia, J; Paoletti, E

    1997-01-01

    Canine distemper virus (CDV) infection of ferrets causes an acute systemic disease involving multiple organ systems, including the respiratory tract, lymphoid system, and central nervous system (CNS). We have tested candidate CDV vaccines incorporating the fusion (F) and hemagglutinin (HA) proteins in the highly attenuated NYVAC strain of vaccinia virus and in the ALVAC strain of canarypox virus, which does not productively replicate in mammalian hosts. Juvenile ferrets were vaccinated twice with these constructs, or with an attenuated live-virus vaccine, while controls received saline or the NYVAC and ALVAC vectors expressing rabies virus glycoprotein. Control animals did not develop neutralizing antibody and succumbed to distemper after developing fever, weight loss, leukocytopenia, decreased activity, conjunctivitis, an erythematous rash typical of distemper, CNS signs, and viremia in peripheral blood mononuclear cells (as measured by reverse transcription-PCR). All three CDV vaccines elicited neutralizing titers of at least 1:96. All vaccinated ferrets survived, and none developed viremia. Both recombinant vaccines also protected against the development of symptomatic distemper. However, ferrets receiving the live-virus vaccine lost weight, became lymphocytopenic, and developed the erythematous rash typical of CDV. These data show that ferrets are an excellent model for evaluating the ability of CDV vaccines to protect against symptomatic infection. Because the pathogenesis and clinical course of CDV infection of ferrets is quite similar to that of other Morbillivirus infections, including measles, this model will be useful in testing new candidate Morbillivirus vaccines. PMID:8995676

  17. West Nile virus seroconversion in penguins after vaccination with a killed virus vaccine or a DNA vaccine.

    PubMed

    Davis, Michelle R; Langan, Jennifer N; Johnson, Yvette J; Ritchie, Branson W; Van Bonn, William

    2008-12-01

    To investigate the serologic response of penguins to West Nile virus (WNV) vaccines, four species of exclusively indoor-housed penguins, negative for WNV by serology, were evaluated: Humboldt (Spheniscus humboldti), Magellanic (Spheniscus magellanicus), Gentoo (Pygoscelis papua), and Rockhopper (Eudyptes chrysoscome) penguins. Birds were inoculated with either a killed virus vaccine or a plasmid-mediated DNA WNV vaccine, and postinoculation serology was evaluated. Both vaccines induced seroconversion in all four species, and no adverse reactions were noted. Postvaccination serology results varied across species and vaccine types. However, in all four species, the killed virus vaccine resulted in a greater seroconversion rate than the DNA vaccine and in a significantly shorter time period. Additionally, the duration of the seropositive titer was significantly longer in those birds vaccinated with the killed virus vaccine compared with those vaccinated with the DNA vaccine. A subset of unvaccinated penguins serving as negative controls remained negative throughout the duration of the study despite the presence of WNV in the geographic locations of the study, suggesting that indoor housing may minimize exposure to the virus and may be an additional means of preventing WNV infection in penguins. PMID:19110700

  18. Modified Vaccinia Virus Ankara-Infected Dendritic Cells Present CD4+ T-Cell Epitopes by Endogenous Major Histocompatibility Complex Class II Presentation Pathways

    PubMed Central

    Thiele, Frank; Tao, Sha; Zhang, Yi; Muschaweckh, Andreas; Zollmann, Tina; Protzer, Ulrike; Abele, Rubert

    2014-01-01

    ABSTRACT CD4+ T lymphocytes play a central role in the immune system and mediate their function after recognition of their respective antigens presented on major histocompatibility complex II (MHCII) molecules on antigen-presenting cells (APCs). Conventionally, phagocytosed antigens are loaded on MHCII for stimulation of CD4+ T cells. Certain epitopes, however, can be processed directly from intracellular antigens and are presented on MHCII (endogenous MHCII presentation). Here we characterized the MHCII antigen presentation pathways that are possibly involved in the immune response upon vaccination with modified vaccinia virus Ankara (MVA), a promising live viral vaccine vector. We established CD4+ T-cell lines specific for MVA-derived epitopes as tools for in vitro analysis of MHCII antigen processing and presentation in MVA-infected APCs. We provide evidence that infected APCs are able to directly transfer endogenous viral proteins into the MHCII pathway to efficiently activate CD4+ T cells. By using knockout mice and chemical inhibitory compounds, we further elucidated the molecular basis, showing that among the various subcellular pathways investigated, proteasomes and autophagy are key players in the endogenous MHCII presentation during MVA infection. Interestingly, although proteasomal processing plays an important role, neither TAP nor LAMP-2 was found to be involved in the peptide transport. Defining the molecular mechanism of MHCII presentation during MVA infection provides a basis for improving MVA-based vaccination strategies by aiming for enhanced CD4+ T-cell activation by directing antigens into the responsible pathways. IMPORTANCE This work contributes significantly to our understanding of the immunogenic properties of pathogens by deciphering antigen processing pathways contributing to efficient activation of antigen-specific CD4+ T cells. We identified autophagosome formation, proteasomal activity, and lysosomal integrity as being crucial for

  19. [The effectiveness of immunization with vaccinia virus type "MVA" against an infection with cowpox virus type "OPV 85" in rabbits].

    PubMed

    Munz, E; Linckh, S; Renner-Müller, I C; Reimann, M

    1993-03-01

    The immunological efficacy of vacciniavirus "MVA" was tested against a dermal and intradermal infection with cowpoxvirus "OPV 85" in rabbits: A single vaccination with "MVA" provided only insufficient immunity, a revaccination induced good immunity. Intramuscular immunizations protected better than subcutaneous applications. Immunized rabbits showed "revaccination reactions" after infection with "OPV 85" indicating a cellular immunity. After immunization with "MVA" and after infection with cowpoxvirus "OPV 85" all rabbits developed N- and ELISA-antibodies. HAI-antibodies were not found after immunization, but indicated a multiplication of cowpoxvirus after challenge. Vacciniavirus "MVA" is suggested for immunization of man and animal against possible infections with cowpoxvirus and cowpoxlike viruses. PMID:8322545

  20. Characterization of a temperature-sensitive mutant of vaccinia virus reveals a novel function that prevents virus-induced breakdown of RNA.

    PubMed Central

    Pacha, R F; Condit, R C

    1985-01-01

    We have attempted to characterize the molecular defect in a temperature-sensitive mutant of vaccinia virus, ts22, which has an abortive late phenotype. At the nonpermissive temperature, ts22 displays normal viral protein synthesis until 8 h postinfection. Between 8 and 10 h after infection all viral protein synthesis ceases abruptly. Characterization of ts22 revealed that (i) primary transcription of late viral genes was not grossly impaired, (ii) late viral mRNA was biologically inactive since it could not stimulate in vitro protein synthesis, and (iii) extensive cleavage of rRNA and late viral mRNA occurred at the time that viral protein synthesis aborted in vivo. These data suggest that ts22 is defective in a function which prevents host rRNA and viral mRNA from being degraded. Inhibitor studies with cytosine arabinoside and cycloheximide showed that induction of and protection from rRNA breakdown occurred at approximately the same time during infection and required late viral gene expression. The viral protein synthesis pattern observed in vaccinia virus-infected cells treated with the drug isatin-beta-thiosemicarbazone was strikingly similar to that observed in ts22-infected cells at the nonpermissive temperature (J. Cooper, B. Moss, and E. Katz, Virology 96:381-392, 1979). Analysis of rRNA integrity in isatin-beta-thiosemicarbazone-treated, vaccinia virus-infected cells revealed extensive cleavage of rRNA, suggesting that the ts22 and drug inhibitor may function in the same pathway. Images PMID:4057355

  1. Vaccination of renal cell cancer patients with modified vaccinia Ankara delivering the tumor antigen 5T4 (TroVax) alone or administered in combination with interferon-alpha (IFN-alpha): a phase 2 trial.

    PubMed

    Amato, Robert J; Shingler, William; Goonewardena, Madusha; de Belin, Jackie; Naylor, Stuart; Jac, Jaroslaw; Willis, James; Saxena, Somyata; Hernandez-McClain, Joan; Harrop, Richard

    2009-09-01

    Attenuated vaccinia virus, modified vaccinia Ankara (MVA) has been engineered to deliver the tumor antigen 5T4 (TroVax). MVA-5T4 has been evaluated in an open-label phase 2 trial in metastatic renal cell cancer patients in which the vaccine was administered alone or in combination with interferon-alpha-2b (IFN-alpha). The safety, immunologic, and clinical efficacy of MVA-5T4 with or without IFN-alpha was determined. Twenty-eight patients with metastatic renal cell cancer were treated with MVA-5T4 alone (13) or plus IFN-alpha (15). The 5T4-specific cellular and humoral responses were monitored throughout the study. Clinical responses were assessed by measuring changes in tumor burden by computed tomography or magnetic resonance imaging scan. MVA-5T4 was well tolerated with no serious adverse event attributed to vaccination. Of 23 intent-to-treat patients tested for immune responses postvaccination, 22 (96%) mounted 5T4-specific antibody and/or cellular responses. One patient treated with MVA-5T4 plus IFN-alpha showed a partial response for >7 months, whereas an additional 14 patients (7 receiving MVA-5T4 plus IFN and 7 receiving MVA-5T4 alone) showed periods of disease stabilization ranging from 1.73 to 9.60 months. Median progression free survival and overall survival for all intent-to-treat patients was 3.8 months (range: 1 to 11.47 mo) and 12.1 months (range: 1 to 27 mo), respectively. MVA-5T4 administered alone or in combination with IFN-alpha was well tolerated in all patients. Despite the high frequency of 5T4-specific immune responses, it is not possible to conclude that patients are receiving clinical benefit. The results are encouraging and warrant further investigation. PMID:19561532

  2. Kinetics and intracellular location of intramolecular disulfide bond formation mediated by the cytoplasmic redox system encoded by vaccinia virus

    SciTech Connect

    Bisht, Himani; Brown, Erica; Moss, Bernard

    2010-03-15

    Poxviruses encode a redox system for intramolecular disulfide bond formation in cytoplasmic domains of viral proteins. Our objectives were to determine the kinetics and intracellular location of disulfide bond formation. The vaccinia virus L1 myristoylated membrane protein, used as an example, has three intramolecular disulfide bonds. Reduced and disulfide-bonded forms of L1 were distinguished by electrophoretic mobility and reactivity with monoclonal and polyclonal antibodies. Because disulfide bonds formed during 5 min pulse labeling with radioactive amino acids, a protocol was devised in which dithiothreitol was present at this step. Disulfide bond formation was detected by 2 min after removal of reducing agent and was nearly complete in 10 min. When the penultimate glycine residue was mutated to prevent myristoylation, L1 was mistargeted to the endoplasmic reticulum and disulfide bond formation failed to occur. These data suggested that viral membrane association was required for oxidation of L1, providing specificity for the process.

  3. Primary pulmonary cytotoxic T lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza A virus nucleoprotein peptide do not protect mice against challenge.

    PubMed

    Lawson, C M; Bennink, J R; Restifo, N P; Yewdell, J W; Murphy, B R

    1994-06-01

    The nucleoprotein (NP) of influenza A virus is the dominant antigen recognized by influenza virus-specific cytotoxic T lymphocytes (CTLs), and adoptive transfer of NP-specific CTLs protects mice from influenza A virus infection. BALB/c mouse cells (H-2d) recognize a single Kd-restricted CTL epitope of NP consisting of amino acids 147 to 155. In the present study, mice were immunized with various vaccinia virus recombinant viruses to examine the effect of the induction of primary pulmonary CTLs on resistance to challenge with influenza A/Puerto Rico/8/34 virus. The minigene ESNP(147-155)-VAC construct, composed of a signal sequence from the adenovirus E3/19K glycoprotein (designated ES) and expressing the 9-amino-acid NP natural determinant (amino acids 147 to 155) preceded by an alanine residue, a similar minigene NP(Met 147-155)-VAC lacking ES, and a full-length NP-VAC recombinant of influenza virus were analyzed. The two minigene NP-VAC recombinants induced a greater primary pulmonary CTL response than the full-length NP-VAC recombinant. However, NP-specific CTLs induced by immunization with ESNP(147-155)-VAC did not decrease peak virus titer or accelerate clearance of virus in the lungs of mice challenged intranasally with A/PR/8/34. Furthermore, NP-specific CTLs induced by immunization did not protect mice challenged intranasally with a lethal dose of A/PR/8/34. Sequence analysis of the NP CTL epitope of A/PR/8/34 challenge virus obtained from lungs after 8 days of replication in ESNP(147-155)-VAC-immunized mice showed identity with that of the input virus, demonstrating that an escape mutant had not emerged during replication in vivo. Thus, in contrast to adoptively transferred CTLs, pulmonary NP-specific CTLs induced by recombinant vaccinia virus immunization do not have protective in vivo antiviral activity against influenza virus infection. PMID:7514677

  4. Vaccinia virus protein K7 is a virulence factor that alters the acute immune response to infection

    PubMed Central

    Benfield, Camilla T. O.; Ren, Hongwei; Lucas, Stuart J.; Bahsoun, Basma

    2013-01-01

    Vaccinia virus (VACV) encodes many proteins that antagonize the innate immune system including a family of intracellular proteins with a B-cell lymphoma (Bcl)-2-like structure. One of these Bcl-2 proteins called K7 binds Toll-like receptor-adaptor proteins and the DEAD-box RNA helicase DDX3 and thereby inhibits the activation of NF-κB and interferon regulatory factor 3. However, the contribution of K7 to virus virulence is not known. Here a VACV lacking the K7R gene (vΔK7) was constructed and compared with control viruses that included a plaque purified wt (vK7), a revertant with the K7R gene reinserted (vK7-rev) and a frame-shifted virus in which the translational initiation codon was mutated to prevent K7 protein expression (vK7-fs). Data presented show that loss of K7 does not affect virus replication in cell culture or in vivo; however, viruses lacking the K7 protein were less virulent than controls in murine intradermal (i.d.) and intranasal (i.n.) infection models and there was an altered acute immune response to infection. In the i.d. model, vΔK7 induced smaller lesions than controls, and after i.n. infection vΔK7 induced a reduced weight loss and signs of illness, and more rapid clearance of virus from infected tissue. Concomitantly, the intrapulmonary innate immune response to infection with vΔK7 showed increased infiltration of NK cells and CD8+ T-cells, enhanced MHC class II expression by macrophages, and enhanced cytolysis of target cells by NK cells and VACV-specific CD8+ T-cells. Thus protein K7 is a virulence factor that affects the acute immune response to infection. PMID:23580427

  5. Development of high-yield influenza A virus vaccine viruses.

    PubMed

    Ping, Jihui; Lopes, Tiago J S; Nidom, Chairul A; Ghedin, Elodie; Macken, Catherine A; Fitch, Adam; Imai, Masaki; Maher, Eileen A; Neumann, Gabriele; Kawaoka, Yoshihiro

    2015-01-01

    Vaccination is one of the most cost-effective ways to prevent infection. Influenza vaccines propagated in cultured cells are approved for use in humans, but their yields are often suboptimal. Here, we screened A/Puerto Rico/8/34 (PR8) virus mutant libraries to develop vaccine backbones (defined here as the six viral RNA segments not encoding haemagglutinin and neuraminidase) that support high yield in cell culture. We also tested mutations in the coding and regulatory regions of the virus, and chimeric haemagglutinin and neuraminidase genes. A combination of high-yield mutations from these screens led to a PR8 backbone that improved the titres of H1N1, H3N2, H5N1 and H7N9 vaccine viruses in African green monkey kidney and Madin-Darby canine kidney cells. This PR8 backbone also improves titres in embryonated chicken eggs, a common propagation system for influenza viruses. This PR8 vaccine backbone thus represents an advance in seasonal and pandemic influenza vaccine development. PMID:26334134

  6. Development of high-yield influenza A virus vaccine viruses

    PubMed Central

    Ping, Jihui; Lopes, Tiago J.S.; Nidom, Chairul A.; Ghedin, Elodie; Macken, Catherine A.; Fitch, Adam; Imai, Masaki; Maher, Eileen A.; Neumann, Gabriele; Kawaoka, Yoshihiro

    2015-01-01

    Vaccination is one of the most cost-effective ways to prevent infection. Influenza vaccines propagated in cultured cells are approved for use in humans, but their yields are often suboptimal. Here, we screened A/Puerto Rico/8/34 (PR8) virus mutant libraries to develop vaccine backbones (defined here as the six viral RNA segments not encoding haemagglutinin and neuraminidase) that support high yield in cell culture. We also tested mutations in the coding and regulatory regions of the virus, and chimeric haemagglutinin and neuraminidase genes. A combination of high-yield mutations from these screens led to a PR8 backbone that improved the titres of H1N1, H3N2, H5N1 and H7N9 vaccine viruses in African green monkey kidney and Madin–Darby canine kidney cells. This PR8 backbone also improves titres in embryonated chicken eggs, a common propagation system for influenza viruses. This PR8 vaccine backbone thus represents an advance in seasonal and pandemic influenza vaccine development. PMID:26334134

  7. Vaccine protection against Zika virus from Brazil.

    PubMed

    Larocca, Rafael A; Abbink, Peter; Peron, Jean Pierre S; Zanotto, Paolo M de A; Iampietro, M Justin; Badamchi-Zadeh, Alexander; Boyd, Michael; Ng'ang'a, David; Kirilova, Marinela; Nityanandam, Ramya; Mercado, Noe B; Li, Zhenfeng; Moseley, Edward T; Bricault, Christine A; Borducchi, Erica N; Giglio, Patricia B; Jetton, David; Neubauer, George; Nkolola, Joseph P; Maxfield, Lori F; De La Barrera, Rafael A; Jarman, Richard G; Eckels, Kenneth H; Michael, Nelson L; Thomas, Stephen J; Barouch, Dan H

    2016-08-25

    Zika virus (ZIKV) is a flavivirus that is responsible for the current epidemic in Brazil and the Americas. ZIKV has been causally associated with fetal microcephaly, intrauterine growth restriction, and other birth defects in both humans and mice. The rapid development of a safe and effective ZIKV vaccine is a global health priority, but very little is currently known about ZIKV immunology and mechanisms of immune protection. Here we show that a single immunization with a plasmid DNA vaccine or a purified inactivated virus vaccine provides complete protection in susceptible mice against challenge with a strain of ZIKV involved in the outbreak in northeast Brazil. This ZIKV strain has recently been shown to cross the placenta and to induce fetal microcephaly and other congenital malformations in mice. We produced DNA vaccines expressing ZIKV pre-membrane and envelope (prM-Env), as well as a series of deletion mutants. The prM-Env DNA vaccine, but not the deletion mutants, afforded complete protection against ZIKV, as measured by absence of detectable viraemia following challenge, and protective efficacy correlated with Env-specific antibody titers. Adoptive transfer of purified IgG from vaccinated mice conferred passive protection, and depletion of CD4 and CD8 T lymphocytes in vaccinated mice did not abrogate this protection. These data demonstrate that protection against ZIKV challenge can be achieved by single-shot subunit and inactivated virus vaccines in mice and that Env-specific antibody titers represent key immunologic correlates of protection. Our findings suggest that the development of a ZIKV vaccine for humans is likely to be achievable. PMID:27355570

  8. Vaccinia Virus Mutations in the L4R Gene Encoding a Virion Structural Protein Produce Abnormal Mature Particles Lacking a Nucleocapsid

    PubMed Central

    Moussatche, Nissin; Condit, Richard C.

    2014-01-01

    ABSTRACT Electron micrographs from the 1960s revealed the presence of an S-shaped tubular structure in the center of the vaccinia virion core. Recently, we showed that packaging of virus transcription enzymes is necessary for the formation of the tubular structure, suggesting that the structure is equivalent to a nucleocapsid. Based on this study and on what is known about nucleocapsids of other viruses, we hypothesized that in addition to transcription enzymes, the tubular structure also contains the viral DNA and a structural protein as a scaffold. The vaccinia virion structural protein L4 stands out as the best candidate for the role of a nucleocapsid structural protein because it is abundant, it is localized in the center of the virion core, and it binds DNA. In order to gain more insight into the structure and relevance of the nucleocapsid, we analyzed thermosensitive and inducible mutants in the L4R gene. Using a cryo-fixation method for electron microscopy (high-pressure freezing followed by freeze-substitution) to preserve labile structures like the nucleocapsid, we were able to demonstrate that in the absence of functional L4, mature particles with defective internal structures are produced under nonpermissive conditions. These particles do not contain a nucleocapsid. In addition, the core wall of these virions is abnormal. This suggests that the nucleocapsid interacts with the core wall and that the nucleocapsid structure might be more complex than originally assumed. IMPORTANCE The vaccinia virus nucleocapsid has been neglected since the 1960s due to a lack of electron microscopy techniques to preserve this labile structure. With the advent of cryo-fixation techniques, like high-pressure freezing/freeze-substitution, we are now able to consistently preserve and visualize the nucleocapsid. Because vaccinia virus early transcription is coupled to the viral core structure, detailing the structure of the nucleocapsid is indispensable for determining the

  9. Vaccinia Virus N1l Protein Resembles a B Cell Lymphoma-2 (Bcl-2) Family Protein

    SciTech Connect

    Aoyagi, M.; Zhai, D.; Jin, C.; Aleshin, A.E.; Stec, B.; Reed, J.C.; Liddington, R.C.; /Burnham Inst.

    2007-07-03

    Poxviruses encode immuno-modulatory proteins capable of subverting host defenses. The poxvirus vaccinia expresses a small 14-kDa protein, N1L, that is critical for virulence. We report the crystal structure of N1L, which reveals an unexpected but striking resemblance to host apoptotic regulators of the B cell lymphoma-2 (Bcl-2) family. Although N1L lacks detectable Bcl-2 homology (BH) motifs at the sequence level, we show that N1L binds with high affinity to the BH3 peptides of pro-apoptotic Bcl-2 family proteins in vitro, consistent with a role for N1L in modulating host antiviral defenses.

  10. Structure Function Studies of Vaccinia Virus Host Range Protein K1 Reveal a Novel Functional Surface for Ankyrin Repeat Proteins

    SciTech Connect

    Li, Yongchao; Meng, Xiangzhi; Xiang, Yan; Deng, Junpeng

    2010-06-15

    Poxvirus host tropism at the cellular level is regulated by virus-encoded host range proteins acting downstream of virus entry. The functioning mechanisms of most host range proteins are unclear, but many contain multiple ankyrin (ANK) repeats, a motif that is known for ligand interaction through a concave surface. We report here the crystal structure of one of the ANK repeat-containing host range proteins, the vaccinia virus K1 protein. The structure, at a resolution of 2.3 {angstrom}, showed that K1 consists entirely of ANK repeats, including seven complete ones and two incomplete ones, one each at the N and C terminus. Interestingly, Phe82 and Ser83, which were previously shown to be critical for K1's function, are solvent exposed and located on a convex surface, opposite the consensus ANK interaction surface. The importance of this convex surface was further supported by our additional mutagenesis studies. We found that K1's host range function was negatively affected by substitution of either Asn51 or Cys47 and completely abolished by substitution of both residues. Cys47 and Asn51 are also exposed on the convex surface, spatially adjacent to Phe82 and Ser83. Altogether, our data showed that K1 residues on a continuous convex ANK repeat surface are critical for the host range function, suggesting that K1 functions through ligand interaction and does so with a novel ANK interaction surface.

  11. Structure Function Studies of Vaccinia Virus Host Range Protein K1 Reveal a Novel Functional Surface for Ankyrin Repeat Proteins▿

    PubMed Central

    Li, Yongchao; Meng, Xiangzhi; Xiang, Yan; Deng, Junpeng

    2010-01-01

    Poxvirus host tropism at the cellular level is regulated by virus-encoded host range proteins acting downstream of virus entry. The functioning mechanisms of most host range proteins are unclear, but many contain multiple ankyrin (ANK) repeats, a motif that is known for ligand interaction through a concave surface. We report here the crystal structure of one of the ANK repeat-containing host range proteins, the vaccinia virus K1 protein. The structure, at a resolution of 2.3 Å, showed that K1 consists entirely of ANK repeats, including seven complete ones and two incomplete ones, one each at the N and C terminus. Interestingly, Phe82 and Ser83, which were previously shown to be critical for K1's function, are solvent exposed and located on a convex surface, opposite the consensus ANK interaction surface. The importance of this convex surface was further supported by our additional mutagenesis studies. We found that K1's host range function was negatively affected by substitution of either Asn51 or Cys47 and completely abolished by substitution of both residues. Cys47 and Asn51 are also exposed on the convex surface, spatially adjacent to Phe82 and Ser83. Altogether, our data showed that K1 residues on a continuous convex ANK repeat surface are critical for the host range function, suggesting that K1 functions through ligand interaction and does so with a novel ANK interaction surface. PMID:20089642

  12. A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9

    PubMed Central

    Yuan, Ming; Gao, Xuefei; Chard, Louisa S; Ali, Zarah; Ahmed, Jahangir; Li, Yunqing; Liu, Pentao; Lemoine, Nick R; Wang, Yaohe

    2015-01-01

    The current method for creation of vaccinia virus (VACV) vectors involves using a selection and purification marker, however inclusion of a gene without therapeutic value in the resulting vector is not desirable for clinical use. The Cre-LoxP system has been used to make marker-free Poxviruses, but the efficiency was very low. To obtain a marker-free VACV vector, we developed marker gene excision systems to modify the thymidine kinase (TK) region and N1L regions using Cre-Loxp and Flp-FRET systems respectively. CRISPR-Cas9 system significantly resulted in a high efficiency (~90%) in generation of marker gene-positive TK-mutant VACV vector. The marker gene (RFP) could be excised from the recombinant virus using Cre recombinase. To make a marker-free VV vector with double gene deletions targeting the TK and N1L gene, we constructed a donor repair vector targeting the N1L gene, which can carry a therapeutic gene and the marker (RFP) that could be excised from the recombinant virus using Flp recombinase. The marker-free system developed here can be used to efficiently construct VACV vectors armed with any therapeutic genes in the TK region or N1L region without marker genes. Our marker-free system platform has significant potential for development of new marker-free VACV vectors for clinical application. PMID:26417609

  13. 9 CFR 113.205 - Newcastle Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Virus. 113.205 Section 113.205 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.205 Newcastle Disease Vaccine, Killed Virus. Newcastle Disease...

  14. 9 CFR 113.210 - Feline Calicivirus Vaccine, Killed Virus.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Virus. 113.210 Section 113.210 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.210 Feline Calicivirus Vaccine, Killed Virus. Feline...

  15. 9 CFR 113.210 - Feline Calicivirus Vaccine, Killed Virus.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Virus. 113.210 Section 113.210 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.210 Feline Calicivirus Vaccine, Killed Virus. Feline...

  16. 9 CFR 113.205 - Newcastle Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Virus. 113.205 Section 113.205 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.205 Newcastle Disease Vaccine, Killed Virus. Newcastle Disease...

  17. 9 CFR 113.208 - Avian Encephalomyelitis Vaccine, Killed Virus.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ..., Killed Virus. 113.208 Section 113.208 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.208 Avian Encephalomyelitis Vaccine, Killed Virus....

  18. 9 CFR 113.210 - Feline Calicivirus Vaccine, Killed Virus.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Virus. 113.210 Section 113.210 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.210 Feline Calicivirus Vaccine, Killed Virus. Feline...

  19. 9 CFR 113.208 - Avian Encephalomyelitis Vaccine, Killed Virus.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., Killed Virus. 113.208 Section 113.208 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.208 Avian Encephalomyelitis Vaccine, Killed Virus....

  20. 9 CFR 113.211 - Feline Rhinotracheitis Vaccine, Killed Virus.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Virus. 113.211 Section 113.211 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.211 Feline Rhinotracheitis Vaccine, Killed Virus....

  1. 9 CFR 113.211 - Feline Rhinotracheitis Vaccine, Killed Virus.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Virus. 113.211 Section 113.211 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.211 Feline Rhinotracheitis Vaccine, Killed Virus....

  2. 9 CFR 113.208 - Avian Encephalomyelitis Vaccine, Killed Virus.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., Killed Virus. 113.208 Section 113.208 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.208 Avian Encephalomyelitis Vaccine, Killed Virus....

  3. 9 CFR 113.211 - Feline Rhinotracheitis Vaccine, Killed Virus.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Virus. 113.211 Section 113.211 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.211 Feline Rhinotracheitis Vaccine, Killed Virus....

  4. 9 CFR 113.210 - Feline Calicivirus Vaccine, Killed Virus.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Virus. 113.210 Section 113.210 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.210 Feline Calicivirus Vaccine, Killed Virus. Feline...

  5. 9 CFR 113.205 - Newcastle Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Virus. 113.205 Section 113.205 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.205 Newcastle Disease Vaccine, Killed Virus. Newcastle Disease...

  6. 9 CFR 113.205 - Newcastle Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Virus. 113.205 Section 113.205 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.205 Newcastle Disease Vaccine, Killed Virus. Newcastle Disease...

  7. 9 CFR 113.211 - Feline Rhinotracheitis Vaccine, Killed Virus.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Virus. 113.211 Section 113.211 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.211 Feline Rhinotracheitis Vaccine, Killed Virus....

  8. 9 CFR 113.208 - Avian Encephalomyelitis Vaccine, Killed Virus.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ..., Killed Virus. 113.208 Section 113.208 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.208 Avian Encephalomyelitis Vaccine, Killed Virus....

  9. 9 CFR 113.210 - Feline Calicivirus Vaccine, Killed Virus.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Virus. 113.210 Section 113.210 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.210 Feline Calicivirus Vaccine, Killed Virus. Feline...

  10. 9 CFR 113.205 - Newcastle Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Virus. 113.205 Section 113.205 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.205 Newcastle Disease Vaccine, Killed Virus. Newcastle Disease...

  11. 9 CFR 113.211 - Feline Rhinotracheitis Vaccine, Killed Virus.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Virus. 113.211 Section 113.211 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.211 Feline Rhinotracheitis Vaccine, Killed Virus....

  12. 9 CFR 113.208 - Avian Encephalomyelitis Vaccine, Killed Virus.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ..., Killed Virus. 113.208 Section 113.208 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.208 Avian Encephalomyelitis Vaccine, Killed Virus....

  13. Prime-boost vaccination with chimpanzee adenovirus and modified vaccinia Ankara encoding TRAP provides partial protection against Plasmodium falciparum infection in Kenyan adults.

    PubMed

    Ogwang, Caroline; Kimani, Domtila; Edwards, Nick J; Roberts, Rachel; Mwacharo, Jedidah; Bowyer, Georgina; Bliss, Carly; Hodgson, Susanne H; Njuguna, Patricia; Viebig, Nicola K; Nicosia, Alfredo; Gitau, Evelyn; Douglas, Sandy; Illingworth, Joe; Marsh, Kevin; Lawrie, Alison; Imoukhuede, Egeruan B; Ewer, Katie; Urban, Britta C; S Hill, Adrian V; Bejon, Philip

    2015-05-01

    Protective immunity to the liver stage of the malaria parasite can be conferred by vaccine-induced T cells, but no subunit vaccination approach based on cellular immunity has shown efficacy in field studies. We randomly allocated 121 healthy adult male volunteers in Kilifi, Kenya, to vaccination with the recombinant viral vectors chimpanzee adenovirus 63 (ChAd63) and modified vaccinia Ankara (MVA), both encoding the malaria peptide sequence ME-TRAP (the multiple epitope string and thrombospondin-related adhesion protein), or to vaccination with rabies vaccine as a control. We gave antimalarials to clear parasitemia and conducted PCR (polymerase chain reaction) analysis on blood samples three times a week to identify infection with the malaria parasite Plasmodium falciparum. On Cox regression, vaccination reduced the risk of infection by 67% [95% confidence interval (CI), 33 to 83%; P = 0.002] during 8 weeks of monitoring. T cell responses to TRAP peptides 21 to 30 were significantly associated with protection (hazard ratio, 0.24; 95% CI, 0.08 to 0.75; P = 0.016). PMID:25947165

  14. DIVA vaccination strategies for avian influenza virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vaccination for both low pathogenic and highly pathogenic avian influenza is commonly used for countries that have been endemic for avian influenza influenza virus, but stamping out policies are common for countries that are normally free of the disease. Stamping out policies of euthanizing infecte...

  15. Therapeutics and vaccines against chikungunya virus.

    PubMed

    Ahola, Tero; Couderc, Therese; Courderc, Therese; Ng, Lisa F P; Hallengärd, David; Powers, Ann; Lecuit, Marc; Esteban, Mariano; Merits, Andres; Roques, Pierre; Liljeström, Peter

    2015-04-01

    Currently, there are no licensed vaccines or therapies available against chikungunya virus (CHIKV), and these were subjects discussed during a CHIKV meeting recently organized in Langkawi, Malaysia. In this review, we chart the approaches taken in both areas. Because of a sharp increase in new data in these fields, the present paper is complementary to previous reviews by Weaver et al. in 2012 and Kaur and Chu in 2013 . The most promising antivirals so far discovered are reviewed, with a special focus on the virus-encoded replication proteins as potential targets. Within the vaccines in development, our review emphasizes the various strategies in parallel development that are unique in the vaccine field against a single disease. PMID:25897811

  16. Persistence of antibodies and anamnestic response in calves vaccinated with inactivated infectious bovine rhinotracheitis virus and parainfluenza-3 virus vaccines.

    PubMed

    Sweat, R L

    1983-04-15

    Persistence of antibodies in calves vaccinated with 2 types of inactivated infectious bovine rhinotracheitis (IBR) virus and parainfluenza-3 (PI-3) virus vaccines were determined. Calves seronegative for IBR and PI-3 viruses were inoculated with 2 doses of inactivated IBR virus-PI-3 virus vaccines administered 2 weeks apart. Blood samples were obtained from the calves for serum at 2 weeks, 6 months, and 1 year after vaccination. The serums were tested by serum-neutralization tests. Antibody response to the vaccines persisted on a declining scale for 1 year. The anamnestic responses to the vaccines were determined by inoculating the same calves with a booster dose of vaccine 1 year after the original 2 doses were given. Blood samples were obtained from the calves for serum 2 weeks later. The serums were tested by serum-neutralization tests. The single booster dose of vaccine elicited an anamnestic response to both IBR and PI-3 viruses. PMID:6303996

  17. Chimpanzee adenovirus- and MVA-vectored respiratory syncytial virus vaccine is safe and immunogenic in adults.

    PubMed

    Green, Christopher A; Scarselli, Elisa; Sande, Charles J; Thompson, Amber J; de Lara, Catherine M; Taylor, Kathryn S; Haworth, Kathryn; Del Sorbo, Mariarosaria; Angus, Brian; Siani, Loredana; Di Marco, Stefania; Traboni, Cinzia; Folgori, Antonella; Colloca, Stefano; Capone, Stefania; Vitelli, Alessandra; Cortese, Riccardo; Klenerman, Paul; Nicosia, Alfredo; Pollard, Andrew J

    2015-08-12

    Respiratory syncytial virus (RSV) causes respiratory infection in annual epidemics, with infants and the elderly at particular risk of developing severe disease and death. However, despite its importance, no vaccine exists. The chimpanzee adenovirus, PanAd3-RSV, and modified vaccinia virus Ankara, MVA-RSV, are replication-defective viral vectors encoding the RSV fusion (F), nucleocapsid (N), and matrix (M2-1) proteins for the induction of humoral and cellular responses. We performed an open-label, dose escalation, phase 1 clinical trial in 42 healthy adults in which four different combinations of prime/boost vaccinations were investigated for safety and immunogenicity, including both intramuscular (IM) and intranasal (IN) administration of the adenovirus-vectored vaccine. The vaccines were safe and well tolerated, with the most common reported adverse events being mild injection site reactions. No vaccine-related serious adverse events occurred. RSV neutralizing antibody titers rose in response to IM prime with PanAd3-RSV and after IM boost for individuals primed by the IN route. Circulating anti-F immunoglobulin G (IgG) and IgA antibody-secreting cells (ASCs) were observed after the IM prime and IM boost. RSV-specific T cell responses were increased after the IM PanAd3-RSV prime and were most efficiently boosted by IM MVA-RSV. Interferon-γ (IFN-γ) secretion after boost was from both CD4(+) and CD8(+) T cells, without detectable T helper cell 2 (TH2) cytokines that have been previously associated with immune pathogenesis following exposure to RSV after the formalin-inactivated RSV vaccine. In conclusion, PanAd3-RSV and MVA-RSV are safe and immunogenic in healthy adults. These vaccine candidates warrant further clinical evaluation of efficacy to assess their potential to reduce the burden of RSV disease. PMID:26268313

  18. Redirected cellular cytotoxicity by infection of effector cells with a recombinant vaccinia virus encoding a tumor-specific monoclonal antibody.

    PubMed

    Paul, S; Bizouarne, N; Dott, K; Ruet, L; Dufour, P; Acres, R B; Kieny, M P

    2000-04-01

    Cytotoxicity is an important function of the immune system that results in the destruction of cellular targets by humoral and/or cellular mechanisms. We wanted to assess the possibility of targeting the lytic function of immune cells toward cancer cells, which express the gene coding for a known tumor antigen (Ag) (GA733-2/epithelial cell adhesion molecule), using a viral vector encoding a monoclonal antibody (mAb) specific for said tumor Ag (CO17-1A). To this end, we have constructed recombinant vaccinia viruses expressing the sequences corresponding to mAb CO17-1A, which recognizes a specific Ag (GA733-2) that is present on the surface of most gastrointestinal carcinomas. The recombinant vectors encoding either a secreted or membrane-anchored form of CO17-1A mAb were used to infect effector cells, which were subsequently assessed for their cytotoxic activity. The recombinant viruses were able to infect both granulocyte-macrophage colony-stimulating factor-activated human macrophages and Ag-stimulated murine cytotoxic T lymphocytes. Infected granulocyte-macrophage colony-stimulating factor-activated macrophages were found to be able to kill GA733-2-expressing tumor cells. Likewise, infected cytotoxic T lymphocytes, although conserving their original alloreactivity, gained the capability of killing GA733-2-expressing cancer cells. PMID:10811480

  19. Antiviral immunity and virus vaccines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As obligate intracellular organisms, viruses have co-evolved with their respective host species, which in turn have evolved diverse and sophisticated capabilities to protect themselves against viral infections and their associated diseases. Viruses have also evolved a remarkable variety of strategie...

  20. The effect of vaccination on Marek's disease virus shedding

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Current Marek’s Disease vaccines are efficient at preventing disease. However, vaccination can reduce but not completely eliminate virus shedding. Our hypothesis is that an efficient vaccine will result in fewer viruses being shed. To test this hypothesis, we developed a real-time PCR to measure Mar...

  1. Vaccines for herpes simplex virus infections.

    PubMed

    Koelle, David M

    2006-02-01

    Infections with herpes simplex virus (HSV) type 1 (HSV-1) and type 2 (HSV-2) can have serious medical consequences. Although antiviral medications can suppress symptomatic disease, asymptomatic shedding and transmission, they neither cure nor alter the natural history of HSV infections. Manipulation of the immune response is one potential method to decrease disease burden. Current research on prophylactic and therapeutic vaccination approaches is discussed in this review, with a focus on compounds that have entered clinical trials or that display novel compositions or proposed mechanisms of action. One such vaccine is an alum and monophosphoryl lipid A-adjuvanted subunit glycoprotein D2 vaccine that has demonstrated activity in the prevention of HSV-2 infection and disease in HSV-uninfected women in a phase III clinical trial. Further confirmatory clinical trials of this vaccine are currently underway. Other vaccine formats also in development include attenuated live or replication-incompetent HSV-2 strains and technologies that target virus-specific CD8 T-cell responses. PMID:16499283

  2. Attenuated Measles Virus as a Vaccine Vector

    PubMed Central

    Zuniga, Armando; Wang, ZiLi; Liniger, Matthias; Hangartner, Lars; Caballero, Michael; Pavlovic, Jovan; Wild, Peter; Viret, Jean Francois; Glueck, Reinhard; Billeter, Martin A.; Naim, Hussein Y.

    2013-01-01

    Live attenuated measles virus (MV) vaccines have an impressive record of safety, efficacy and ability to induce life-long immunity against measles infection. Using reverse genetics technology, such negative-strand RNA viruses can now be rescued from cloned DNA. This technology allows the insertion of exogenous genes encoding foreign antigens into the MV genome in such a way that they can be expressed by the MV vaccine strain, without affecting virus structure, propagation and cell targeting. Recombinant viruses rescued from cloned cDNA induce immune responses against both measles virus and the cloned antigens. The tolerability of MV to gene(s) insertion makes it an attractive flexible vector system, especially if broad immune responses are required. The fact that measles replication strictly occurs in the cytoplasm of infected cells without DNA intermediate has important biosafety implications and adds to the attractiveness of MV as a vector. In this article we report the characteristics of reporter gene expression (GFP, LacZ and CAT) and the biochemical, biophysical and immunological properties of recombinant MV expressing heterologous antigens of simian immunogeficiency virus (SIV). PMID:17303293

  3. Human papilloma virus vaccines: Current scenario.

    PubMed

    Pandhi, Deepika; Sonthalia, Sidharth

    2011-07-01

    Genital human papillomavirus (HPV) infection is the most common sexually transmitted infection with an estimated worldwide prevalence of 9-13% and approximately 6 million people being infected each year. Mostly acquired during adolescence or young adulthood, HPV presents clinically as anogenital warts and may progress to precancerous lesions and cancers of the cervix, vagina, vulva, penis and anus, and oropharynx. HPV infection is considered to contribute to almost 100% cervical cancers and at least 80% of anal and 40-60% of vulvar, vaginal, and penile cancers. At present, two prophylactic HPV vaccines are commercially available and both are prepared from purified L1 structural proteins. These proteins self-assemble to form virus-like particles that induce a protective immunity. Gardasil(®) is a quadrivalent vaccine against HPV types 6, 11, 16, and 18 and is recommended for use in females 9-26 years of age, for the prevention of cervical, vulvar, and vaginal cancers and intraepithelial neoplasia and condyloma acuminata and recently for vaccination in boys and men 9-26 years of age for the prevention of genital warts. Cervarix™ is a bivalent vaccine approved for the prevention of cervical cancer and precancerous lesions caused by HPV 16 and 18, in females 10-25 years. HPV vaccines are safe and efficacious against type-specific HPV-induced anogenital warts, precancerous lesions, and cervical cancer. The vaccines are most effective when given before the onset of sexual activity and provide long-term protection. Effective vaccination coverage in young adolescent females will substantially reduce the incidence of these anogenital malignancy-related morbidity and mortality. There is need to generate India-specific data on HPV epidemiology and HPV vaccination efficacy as well as continue worldwide surveillance and development of newer vaccines. PMID:22021967

  4. Heterologous Immunity between Adenoviruses and Hepatitis C Virus: A New Paradigm in HCV Immunity and Vaccines

    PubMed Central

    Singh, Shakti; Vedi, Satish; Samrat, Subodh Kumar; Li, Wen; Kumar, Rakesh; Agrawal, Babita

    2016-01-01

    Adenoviruses (Ad) are commonly used as vectors for gene therapy and/or vaccine delivery. Recombinant Ad vectors are being tested as vaccines for many pathogens. We have made a surprising observation that peptides derived from various hepatitis C virus (HCV) antigens contain extensive regions of homology with multiple adenovirus proteins, and conclusively demonstrate that adenovirus vector can induce robust, heterologous cellular and humoral immune responses against multiple HCV antigens. Intriguingly, the induction of this cross-reactive immunity leads to significant reduction of viral loads in a recombinant vaccinia-HCV virus infected mouse model, supporting their role in antiviral immunity against HCV. Healthy human subjects with Ad-specific pre-existing immunity demonstrated cross-reactive cellular and humoral immune responses against multiple HCV antigens. These findings reveal the potential of a previously uncharacterized property of natural human adenovirus infection to dictate, modulate and/or alter the course of HCV infection upon exposure. This intrinsic property of adenovirus vectors to cross-prime HCV immunity can also be exploited to develop a prophylactic and/or therapeutic vaccine against HCV. PMID:26751211

  5. Japanese encephalitis: the virus and vaccines.

    PubMed

    Yun, Sang-Im; Lee, Young-Min

    2014-01-01

    Japanese encephalitis (JE) is an infectious disease of the central nervous system caused by Japanese encephalitis virus (JEV), a zoonotic mosquito-borne flavivirus. JEV is prevalent in much of Asia and the Western Pacific, with over 4 billion people living at risk of infection. In the absence of antiviral intervention, vaccination is the only strategy to develop long-term sustainable protection against JEV infection. Over the past half-century, a mouse brain-derived inactivated vaccine has been used internationally for active immunization. To date, however, JEV is still a clinically important, emerging, and re-emerging human pathogen of global significance. In recent years, production of the mouse brain-derived vaccine has been discontinued, but 3 new cell culture-derived vaccines are available in various parts of the world. Here we review current aspects of JEV biology, summarize the 4 types of JEV vaccine, and discuss the potential of an infectious JEV cDNA technology for future vaccine development. PMID:24161909

  6. The Membrane Fusion Step of Vaccinia Virus Entry Is Cooperatively Mediated by Multiple Viral Proteins and Host Cell Components

    PubMed Central

    Laliberte, Jason P.; Weisberg, Andrea S.; Moss, Bernard

    2011-01-01

    For many viruses, one or two proteins allow cell attachment and entry, which occurs through the plasma membrane or following endocytosis at low pH. In contrast, vaccinia virus (VACV) enters cells by both neutral and low pH routes; four proteins mediate cell attachment and twelve that are associated in a membrane complex and conserved in all poxviruses are dedicated to entry. The aim of the present study was to determine the roles of cellular and viral proteins in initial stages of entry, specifically fusion of the membranes of the mature virion and cell. For analysis of the role of cellular components, we used well characterized inhibitors and measured binding of a recombinant VACV virion containing Gaussia luciferase fused to a core protein; viral and cellular membrane lipid mixing with a self-quenching fluorescent probe in the virion membrane; and core entry with a recombinant VACV expressing firefly luciferase and electron microscopy. We determined that inhibitors of tyrosine protein kinases, dynamin GTPase and actin dynamics had little effect on binding of virions to cells but impaired membrane fusion, whereas partial cholesterol depletion and inhibitors of endosomal acidification and membrane blebbing had a severe effect at the later stage of core entry. To determine the role of viral proteins, virions lacking individual membrane components were purified from cells infected with members of a panel of ten conditional-lethal inducible mutants. Each of the entry protein-deficient virions had severely reduced infectivity and except for A28, L1 and L5 greatly impaired membrane fusion. In addition, a potent neutralizing L1 monoclonal antibody blocked entry at a post-membrane lipid-mixing step. Taken together, these results suggested a 2-step entry model and implicated an unprecedented number of viral proteins and cellular components involved in signaling and actin rearrangement for initiation of virus-cell membrane fusion during poxvirus entry. PMID:22194690

  7. Molecular Smallpox Vaccine Delivered by Alphavirus Replicons Elicits Protective Immunity in Mice and Non-human Primates

    PubMed Central

    Hooper, Jay W.; Ferro, Anthony M.; Golden, Joseph W.; Silvera, Peter; Dudek, Jeanne; Alterson, Kim; Custer, Max; Rivers, Bryan; Morris, John; Owens, Gary; Smith, Jonathan F.; Kamrud, Kurt I.

    2009-01-01

    Naturally occurring smallpox was eradicated as a result of successful vaccination campaigns during the 1960s and 70s. Because of its highly contagious nature and high mortality rate, smallpox has significant potential as a biological weapon. Unfortunately, the current vaccine for orthopoxviruses is contraindicated for large portions of the population. Thus, there is a need for new, safe, and effective orthopoxvirus vaccines. Alphavirus replicon vectors, derived from strains of Venezuelan equine encephalitis virus, are being used to develop alternatives to the current smallpox vaccine. Here, we demonstrated that virus-like replicon particles (VRP) expressing the vaccinia virus A33R, B5R, A27L, and L1R genes elicited protective immunity in mice comparable to vaccination with live-vaccinia virus. Furthermore, cynomolgus macaques vaccinated with a combination of the four poxvirus VRPs (4pox-VRP) developed antibody responses to each antigen. These antibody responses were able to neutralize and inhibit the spread of both vaccinia virus and monkeypox virus. Macaques vaccinated with 4pox-VRP, flu HA VRP (negative control), or live-vaccinia virus (positive control) were challenged intravenously with 5 × 106 PFU of monkeypox virus 1 month after the second VRP vaccination. Four of the six negative control animals succumbed to monkeypox and the remaining two animals demonstrated either severe or grave disease. Importantly, all 10 macaques vaccinated with the 4pox-VRP vaccine survived without developing severe disease. These findings revealed that a single-boost VRP smallpox vaccine shows promise as a safe alternative to the currently licensed live-vaccinia virus smallpox vaccine. PMID:19833247

  8. Hendra virus and Nipah virus animal vaccines.

    PubMed

    Broder, Christopher C; Weir, Dawn L; Reid, Peter A

    2016-06-24

    Hendra virus (HeV) and Nipah virus (NiV) are zoonotic viruses that emerged in the mid to late 1990s causing disease outbreaks in livestock and people. HeV appeared in Queensland, Australia in 1994 causing a severe respiratory disease in horses along with a human case fatality. NiV emerged a few years later in Malaysia and Singapore in 1998-1999 causing a large outbreak of encephalitis with high mortality in people and also respiratory disease in pigs which served as amplifying hosts. The key pathological elements of HeV and NiV infection in several species of mammals, and also in people, are a severe systemic and often fatal neurologic and/or respiratory disease. In people, both HeV and NiV are also capable of causing relapsed encephalitis following recovery from an acute infection. The known reservoir hosts of HeV and NiV are several species of pteropid fruit bats. Spillovers of HeV into horses continue to occur in Australia and NiV has caused outbreaks in people in Bangladesh and India nearly annually since 2001, making HeV and NiV important transboundary biological threats. NiV in particular possesses several features that underscore its potential as a pandemic threat, including its ability to infect humans directly from natural reservoirs or indirectly from other susceptible animals, along with a capacity of limited human-to-human transmission. Several HeV and NiV animal challenge models have been developed which have facilitated an understanding of pathogenesis and allowed for the successful development of both active and passive immunization countermeasures. PMID:27154393

  9. Recombinant vaccinia virus GLV-1h68 is a promising oncolytic vector in the treatment of cholangiocarcinoma.

    PubMed

    Pugalenthi, Amudhan; Mojica, Kelly; Ady, Justin W; Johnsen, Clark; Love, Damon; Chen, Nanhai G; Aguilar, Richard J; Szalay, Aladar A; Fong, Yuman

    2015-12-01

    Although early stage cholangiocarcinoma (CC) can be cured by surgical extirpation, the options for treatment of advanced stage CC are very few and suboptimal. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) is a promising new strategy to treat human cancers. The ability of oncolytic VACV GLV-1h68 to infect, replicate in, and lyse three human CC cell lines was assayed in vitro and in subcutaneous flank xenografts in athymic nude mice. In this study, we have demonstrated that GLV-1h68 effectively infects and lyses three CC cell lines (KMC-1, KMBC, and KMCH-1) in vitro. Expression of the viral marker gene ruc-gfp facilitated real-time monitoring of infection and replication. Furthermore in athymic nude mice, a single dose of GLV-1h68 significantly suppressed tumor growth. The treatment was well tolerated in all animals. Recombinant VACV GLV-1h68 has significant oncolytic ability against CC both in vitro and in vivo. GLV-1h68 has the potential to be used clinically as a therapeutic agent against CC. PMID:26584530

  10. NK Cell-extrinsic IL-18 Signaling Is Required for Efficient NK Cell Activation to Vaccinia Virus

    PubMed Central

    Brandstadter, Joshua D.; Huang, Xiaopei; Yang, Yiping

    2014-01-01

    Summary NK cells are important for the control of vaccinia virus (VV) in vivo. Recent studies have shown that multiple pathways are required for effective activation of NK cells. These include both TLR-dependent and -independent pathways, as well as the NKG2D activating receptor that recognizes host stress-induced NKG2D ligands. However, it remains largely unknown what controls the upregulation of NKG2D ligands in response to VV infection. In this study, we first showed that IL-18 is critical for NK cell activation and viral clearance. We then demonstrated that IL-18 signaling on both NK cells and DCs is required for efficient NK cell activation upon VV infection in vitro. We further showed in vivo that efficient NK cell activation to VV is dependent on DCs and IL-18 signaling in non-NK cells, suggesting an essential role for NK cell-extrinsic IL-18 signaling in NK cell activation. Mechanistically, IL-18 signaling in DCs promotes expression of Rae-1, an NKG2D ligand. Collectively, our data reveal a previously unrecognized role for NK cell-extrinsic IL-18 signaling in NK cell activation through upregulation of NKG2D ligands. These observations may provide insights into the design of effective NK cell-based therapies for viral infections and cancer. PMID:24846540

  11. Interaction between the G3 and L5 proteins of the vaccinia virus entry-fusion complex

    SciTech Connect

    Wolfe, Cindy L.; Moss, Bernard

    2011-04-10

    The vaccinia virus entry-fusion complex (EFC) consists of 10 to 12 proteins that are embedded in the viral membrane and individually required for fusion with the cell and entry of the core into the cytoplasm. The architecture of the EFC is unknown except for information regarding two pair-wise interactions: A28 with H2 and A16 with G9. Here we used a technique to destabilize the EFC by repressing the expression of individual components and identified a third pair-wise interaction: G3 with L5. These two proteins remained associated under several different EFC destabilization conditions and in each case were immunopurified together as demonstrated by Western blotting. Further evidence for the specific interaction of G3 and L5 was obtained by mass spectrometry. This interaction also occurred when G3 and L5 were expressed in uninfected cells, indicating that no other viral proteins were required. Thus, the present study extends our knowledge of the protein interactions important for EFC assembly and stability.

  12. Vaccination against bovine herpes mammillitis virus infections in guinea pigs.

    PubMed

    Smee, D F; Leonhardt, J A

    1994-01-01

    Bovine herpes mammillitis virus or bovine herpesvirus type 2 (BHV-2) causes ulcerative lesions on the teats and udders of infected cows. Since no commercial vaccine is available for this disease, we investigated certain experimental BHV-2 vaccines against this virus in infected guinea pigs. Vaginally infected guinea pigs get severe, self-limiting vaginal infections characterized by erythema and swelling and the production of measurable vaginal virus titers. Two vaccine approaches were investigated: vaccination with wild-type (WT) virus by the subcutaneous route, and vaccination either subcutaneously or intravaginally with a thymidine kinase (TK) deficient (TK-) virus. The TK- strain was prepared by passage of BHV-2 in the presence of the potent TK-dependent antiviral agent 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU). The antiviral activity of FMAU against the virus in plaque reduction assays changed from 0.05 to 2 microM at the same time that the TK activity of the mutant virus decrease to 7% of WT virus TK activity. Subcutaneous vaccination of guinea pigs with WT and TK- viruses did not induce vaginal infection. Primary vaginal infection (vaccination) with the TK- virus led to greatly reduced lesion severity compared to vaginal infection with the WT virus. However, the amount of vaginal virus titers recovered during these primary infections was similar for both TK- and WT viruses, indicating that both viruses had equal infecting potential. Thirty days after vaccination the animals were re-infected intravaginally with WT virus. The vaccinated animals showed dramatically reduced lesion severity and low recoverable virus titers compared to age-matched nonvaccinated animals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7928285

  13. Smallpox Vaccination

    MedlinePlus

    ... Newsletters Events Also Known As Smallpox = Vaccinia Smallpox Vaccination Recommend on Facebook Tweet Share Compartir The smallpox ... like many other vaccines. For that reason, the vaccination site must be cared for carefully to prevent ...

  14. 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.

  15. Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence.

    PubMed

    Stack, Julianne; Haga, Ismar R; Schröder, Martina; Bartlett, Nathan W; Maloney, Geraldine; Reading, Patrick C; Fitzgerald, Katherine A; Smith, Geoffrey L; Bowie, Andrew G

    2005-03-21

    Viral immune evasion strategies target key aspects of the host antiviral response. Recently, it has been recognized that Toll-like receptors (TLRs) have a role in innate defense against viruses. Here, we define the function of the vaccinia virus (VV) protein A46R and show it inhibits intracellular signalling by a range of TLRs. TLR signalling is triggered by homotypic interactions between the Toll-like-interleukin-1 resistance (TIR) domains of the receptors and adaptor molecules. A46R contains a TIR domain and is the only viral TIR domain-containing protein identified to date. We demonstrate that A46R targets the host TIR adaptors myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like, TIR domain-containing adaptor inducing IFN-beta (TRIF), and the TRIF-related adaptor molecule and thereby interferes with downstream activation of mitogen-activated protein kinases and nuclear factor kappaB. TRIF mediates activation of interferon (IFN) regulatory factor 3 (IRF3) and induction of IFN-beta by TLR3 and TLR4 and suppresses VV replication in macrophages. Here, A46R disrupted TRIF-induced IRF3 activation and induction of the TRIF-dependent gene regulated on activation, normal T cell expressed and secreted. Furthermore, we show that A46R is functionally distinct from another described VV TLR inhibitor, A52R. Importantly, VV lacking the A46R gene was attenuated in a murine intranasal model, demonstrating the importance of A46R for VV virulence. PMID:15767367

  16. Envelope formation is blocked by mutation of a sequence related to the HKD phospholipid metabolism motif in the vaccinia virus F13L protein.

    PubMed

    Roper, R L; Moss, B

    1999-02-01

    The outer envelope of the extracellular form of vaccinia virus is derived from Golgi membranes that have been modified by the insertion of specific viral proteins, of which the major component is the 37-kDa, palmitylated, nonglycosylated product of the F13L gene. The F13L protein contains a variant of the HKD (His-Lys-Asp) motif, which is conserved in numerous enzymes of phospholipid metabolism. Vaccinia virus mutants with a conservative substitution of either the K (K314R) or the D (D319E) residue of the F13L protein formed only tiny plaques similar to those produced by an F13L deletion mutant, were unable to produce extracellular enveloped virions, and failed to mediate low-pH-induced fusion of infected cells. Membrane-wrapped forms of intracellular virus were rarely detected in electron microscopic images of cells infected with either of the mutants. Western blotting and pulse-chase experiments demonstrated that the D319E protein was less stable than either the K314R or wild-type F13L protein. Most striking, however, was the failure of either of the two mutated proteins to concentrate in the Golgi compartment. Palmitylation, oleation, and partitioning of the F13L protein in Triton X-114 detergent were unaffected by the K314R substitution. These results indicated that the F13L protein must retain the K314 and D319 for it to localize in the Golgi compartment and function in membrane envelopment of vaccinia virus. PMID:9882312

  17. The Vaccinia Virus O1 Protein Is Required for Sustained Activation of Extracellular Signal-Regulated Kinase 1/2 and Promotes Viral Virulence

    PubMed Central

    Lukassen, Susanne; Späth, Michaela; Wolferstätter, Michael; Babel, Eveline; Brinkmann, Kay; Wielert, Ursula; Chaplin, Paul; Suter, Mark

    2012-01-01

    Sustained activation of the Raf/MEK/extracellular signal-regulated kinase (ERK) pathway in infected cells has been shown to be crucial for full replication efficiency of orthopoxviruses in cell culture. In infected cells, this pathway is mainly activated by the vaccinia virus growth factor (VGF), an epidermal growth factor (EGF)-like protein. We show here that chorioallantois vaccinia virus Ankara (CVA), but not modified vaccinia virus Ankara (MVA), induced sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in infected human 293 cells, although both viruses direct secretion of functional VGF. A CVA mutant lacking the O1L gene (CVA-ΔO1L) demonstrated that the O1 protein was required for sustained upregulation of the ERK1/2 pathway in 293 cells as well as in other mammalian cell lines. The highly conserved orthopoxvirus O1L gene encodes a predicted 78-kDa protein with a hitherto-unknown function. CVA-ΔO1L showed reduced plaque size and an attenuated cytopathic effect (CPE) in infected cell cultures and reduced virulence and spread from lungs to ovaries in intranasally infected BALB/c mice. Reinsertion of an intact O1L gene into MVA, which in its original form harbors a fragmented O1L open reading frame (ORF), restored ERK1/2 activation in 293 cells but did not increase replication and spread of MVA in human or other mammalian cell lines. Thus, the O1 protein was crucial for sustained ERK1/2 activation in CVA- and MVA-infected human cells, complementing the autocrine function of VGF, and enhanced virulence in vivo. PMID:22171261

  18. Plant Viruses as Nanoparticle-Based Vaccines and Adjuvants

    PubMed Central

    Lebel, Marie-Ève; Chartrand, Karine; Leclerc, Denis; Lamarre, Alain

    2015-01-01

    Vaccines are considered one of the greatest medical achievements in the battle against infectious diseases. However, the intractability of various diseases such as hepatitis C, HIV/AIDS, malaria, tuberculosis, and cancer poses persistent hurdles given that traditional vaccine-development methods have proven to be ineffective; as such, these challenges have driven the emergence of novel vaccine design approaches. In this regard, much effort has been put into the development of new safe adjuvants and vaccine platforms. Of particular interest, the utilization of plant virus-like nanoparticles and recombinant plant viruses has gained increasing significance as an effective tool in the development of novel vaccines against infectious diseases and cancer. The present review summarizes recent advances in the use of plant viruses as nanoparticle-based vaccines and adjuvants and their mechanism of action. Harnessing plant-virus immunogenic properties will enable the design of novel, safe, and efficacious prophylactic and therapeutic vaccines against disease. PMID:26350598

  19. Newcastle disease virus as a vaccine vector for infectious laryngotracheitis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Effective, safe, and incapable of reverting to virulence are characteristics desirable for infectious laryngotracheitis virus (ILTV) vaccines. Recombinant Newcastle disease virus (NDV) expressing foreign antigens of avian and mammalian pathogens have been demonstrated to elicit protective immunity....

  20. Vaccination of patients with metastatic renal cancer with modified vaccinia Ankara encoding the tumor antigen 5T4 (TroVax) given alongside interferon-alpha.

    PubMed

    Hawkins, Robert E; Macdermott, Catriona; Shablak, Alaaeldin; Hamer, Caroline; Thistlethwaite, Fiona; Drury, Noel L; Chikoti, Priscilla; Shingler, William; Naylor, Stuart; Harrop, Richard

    2009-05-01

    Approximately 90% of renal cell tumors overexpress the tumor antigen 5T4. The attenuated strain of vaccinia virus, modified vaccinia Ankara, has been engineered to express 5T4 (TroVax). We conducted an open-label phase 1/2 trial in which TroVax was administered alongside interferon-alpha (IFNalpha) to 11 patients with metastatic renal cell carcinoma. Antigen-specific cellular and humoral responses were monitored throughout the study, and clinical responses were assessed by measuring the changes in tumor burden by computed tomography scan (Response Evaluation Criteria In Solid Tumors). The primary objective was to assess the safety, immunogenicity, and efficacy of TroVax when given alongside IFNalpha. Treatment with TroVax plus IFNalpha was well tolerated with no serious adverse events attributed to TroVax. All 11 patients mounted 5T4-specific antibody responses and 5 (45%) mounted cellular responses. No objective tumor responses were seen, but the overall median time to progression (TTP) of 9 months (range: 2.1 to 26+ mo) was longer than expected for IFNalpha alone. For the 10 clear cell patients the TTP ranged from 3.9 to 26+ months, with a median TTP of 10.4 months. The high frequency of 5T4-specific immune responses and prolonged median TTP for clear cell patients compared with that expected for IFNalpha alone is encouraging and warrants further investigation. PMID:19342962

  1. Vaccination of chickens decreased Newcastle disease virus contamination in eggs.

    PubMed

    Sá E Silva, Mariana; Susta, Leonardo; Moresco, Kira; Swayne, David E

    2016-02-01

    Newcastle disease is an important health issue of poultry causing major economic losses and inhibits trade worldwide. Vaccination is used as a control measure, but it is unknown whether vaccination will prevent virus contamination of eggs. In this study, hens were sham-vaccinated or received one or two doses of inactivated LaSota vaccine, followed three weeks later by virulent Newcastle disease virus (NDV) challenge. Eggs were collected daily and shell, albumen and yolk were subjected to virus isolation, as were oral and cloacal swabs at 2 and 4 days post-challenge (dpc). A second experiment evaluated the distribution of the virus in the reproductive tract of non-vaccinates. All vaccinated chickens survived challenge, and the levels of virus shed from cloacal swabs were decreased significantly when compared to shams. In non-vaccinated hens, virus was detected in the ovary and all segments of the oviduct. Yolk, albumen and eggshell surface from eggs laid at day 4 and 5 post-infection by sham-vaccinated hens were positive for NDV, but eggs from LaSota vaccinated hens lacked virus in internal egg components (i.e. yolk and albumen) and had reduction in the number of positive eggshell surfaces. These results indicate virulent NDV can replicate in the reproductive tract of hens and contaminate internal components of eggs and eggshell surface, but vaccination was able to prevent internal egg contamination, reducing eggshell surface contamination, and reducing shedding from digestive and respiratory tracts in virulent NDV challenged hens. PMID:26503831

  2. Percutaneous Vaccination as an Effective Method of Delivery of MVA and MVA-Vectored Vaccines.

    PubMed

    Meseda, Clement A; Atukorale, Vajini; Kuhn, Jordan; Schmeisser, Falko; Weir, Jerry P

    2016-01-01

    The robustness of immune responses to an antigen could be dictated by the route of vaccine inoculation. Traditional smallpox vaccines, essentially vaccinia virus strains, that were used in the eradication of smallpox were administered by percutaneous inoculation (skin scarification). The modified vaccinia virus Ankara is licensed as a smallpox vaccine in Europe and Canada and currently undergoing clinical development in the United States. MVA is also being investigated as a vector for the delivery of heterologous genes for prophylactic or therapeutic immunization. Since MVA is replication-deficient, MVA and MVA-vectored vaccines are often inoculated through the intramuscular, intradermal or subcutaneous routes. Vaccine inoculation via the intramuscular, intradermal or subcutaneous routes requires the use of injection needles, and an estimated 10 to 20% of the population of the United States has needle phobia. Following an observation in our laboratory that a replication-deficient recombinant vaccinia virus derived from the New York City Board of Health strain elicited protective immune responses in a mouse model upon inoculation by tail scarification, we investigated whether MVA and MVA recombinants can elicit protective responses following percutaneous administration in mouse models. Our data suggest that MVA administered by percutaneous inoculation, elicited vaccinia-specific antibody responses, and protected mice from lethal vaccinia virus challenge, at levels comparable to or better than subcutaneous or intramuscular inoculation. High titers of specific neutralizing antibodies were elicited in mice inoculated with a recombinant MVA expressing the herpes simplex type 2 glycoprotein D after scarification. Similarly, a recombinant MVA expressing the hemagglutinin of attenuated influenza virus rgA/Viet Nam/1203/2004 (H5N1) elicited protective immune responses when administered at low doses by scarification. Taken together, our data suggest that MVA and MVA

  3. Percutaneous Vaccination as an Effective Method of Delivery of MVA and MVA-Vectored Vaccines

    PubMed Central

    Meseda, Clement A.; Atukorale, Vajini; Kuhn, Jordan; Schmeisser, Falko; Weir, Jerry P.

    2016-01-01

    The robustness of immune responses to an antigen could be dictated by the route of vaccine inoculation. Traditional smallpox vaccines, essentially vaccinia virus strains, that were used in the eradication of smallpox were administered by percutaneous inoculation (skin scarification). The modified vaccinia virus Ankara is licensed as a smallpox vaccine in Europe and Canada and currently undergoing clinical development in the United States. MVA is also being investigated as a vector for the delivery of heterologous genes for prophylactic or therapeutic immunization. Since MVA is replication-deficient, MVA and MVA-vectored vaccines are often inoculated through the intramuscular, intradermal or subcutaneous routes. Vaccine inoculation via the intramuscular, intradermal or subcutaneous routes requires the use of injection needles, and an estimated 10 to 20% of the population of the United States has needle phobia. Following an observation in our laboratory that a replication-deficient recombinant vaccinia virus derived from the New York City Board of Health strain elicited protective immune responses in a mouse model upon inoculation by tail scarification, we investigated whether MVA and MVA recombinants can elicit protective responses following percutaneous administration in mouse models. Our data suggest that MVA administered by percutaneous inoculation, elicited vaccinia-specific antibody responses, and protected mice from lethal vaccinia virus challenge, at levels comparable to or better than subcutaneous or intramuscular inoculation. High titers of specific neutralizing antibodies were elicited in mice inoculated with a recombinant MVA expressing the herpes simplex type 2 glycoprotein D after scarification. Similarly, a recombinant MVA expressing the hemagglutinin of attenuated influenza virus rgA/Viet Nam/1203/2004 (H5N1) elicited protective immune responses when administered at low doses by scarification. Taken together, our data suggest that MVA and MVA

  4. Different Patterns of Expansion, Contraction and Memory Differentiation of HIV-1 Gag-Specific CD8 T Cells Elicited by Adenovirus Type 5 and Modified Vaccinia Ankara Vaccines

    PubMed Central

    Pillai, Vinod Kumar Bhaskara; Kannanganat, Sunil; Penaloza-MacMaster, Pablo; Chennareddi, Lakshmi; Robinson, Harriet L.; Blackwell, Jerry; Amara, Rama Rao

    2011-01-01

    The magnitude and functional quality of antiviral CD8 T cell responses are critical for the efficacy of T cell based vaccines. Here, we investigate the influence of two popular viral vectors, adenovirus type 5 (Ad5) and modified vaccinia Ankara (MVA), on expansion, contraction and memory differentiation of HIV-1 Gag insert-specific CD8 T cell responses following immunization and show different patterns for the two recombinant viral vectors. The Ad5 vector primed 6-fold higher levels of insert-specific CD8 effector T cells than the MVA vector. The Ad5-primed effector cells also underwent less contraction (< 2-fold) than the MVA-primed cells (>5-fold). The Ad5-primed memory cells were predominantly CD62L negative (effector memory) whereas the MVA-primed memory cells were predominantly CD62L positive (central memory). Consistent with their memory phenotype, MVA-primed CD8 T cells underwent higher fold expansion than Ad5-primed CD8 T cells following a homologous or heterologous boost. Impressively, the Ad5 boost changed the quality of MVA-primed memory response such that they undergo less contraction with effector memory phenotype. However, the MVA boost did not influence the contraction and memory phenotype of Ad5-primed response. In conclusion, our results demonstrate that vaccine vector strongly influences the expansion, contraction and the functional quality of insert-specific CD8 T cell responses and have implications for vaccine development against infectious diseases. PMID:21651938

  5. Evaluating anti-Orthopoxvirus antibodies in individuals from Brazilian rural areas prior to the bovine vaccinia era.

    PubMed

    Figueiredo, Poliana de Oliveira; Silva-Fernandes, André Tavares da; Mota, Bruno Eduardo Fernandes; Costa, Galileu Barbosa; Borges, Iara Apolinário; Ferreira, Paulo César Peregrino; Abrahão, Jônatas Santos; Braga, Erika Martins; Kroon, Erna Geessien; Trindade, Giliane de Souza

    2015-09-01

    Vaccinia virus naturally circulates in Brazil and is the causative agent of a zoonotic disease known as bovine vaccinia (BV). We retrospectively evaluated two populations from the Amazon and Southeast Regions. BV outbreaks had not been reported in these regions before sample collection. Neutralising antibodies were found in 13 individuals (n = 132) with titres ranging from 100 ≥ 6,400 neutralising units/mL. Univariate analysis identified age and vaccination as statistically significant risk factors in individuals from the Southeast Region. The absence of detectable antibodies in vaccinated individuals raises questions about the protection of smallpox vaccine years after vaccination and reinforces the need for surveillance of Orthopoxvirus in Brazilian populations without evidence of previous outbreaks. PMID:26517662

  6. Evaluating anti-Orthopoxvirus antibodies in individuals from Brazilian rural areas prior to the bovine vaccinia era

    PubMed Central

    Figueiredo, Poliana de Oliveira; da Silva-Fernandes, André Tavares; Mota, Bruno Eduardo Fernandes; Costa, Galileu Barbosa; Borges, Iara Apolinário; Ferreira, Paulo César Peregrino; Abrahão, Jônatas Santos; Braga, Erika Martins; Kroon, Erna Geessien; Trindade, Giliane de Souza

    2015-01-01

    Vaccinia virus naturally circulates in Brazil and is the causative agent of a zoonotic disease known as bovine vaccinia (BV). We retrospectively evaluated two populations from the Amazon and Southeast Regions. BV outbreaks had not been reported in these regions before sample collection. Neutralising antibodies were found in 13 individuals (n = 132) with titres ranging from 100 ≥ 6,400 neutralising units/mL. Univariate analysis identified age and vaccination as statistically significant risk factors in individuals from the Southeast Region. The absence of detectable antibodies in vaccinated individuals raises questions about the protection of smallpox vaccine years after vaccination and reinforces the need for surveillance of Orthopoxvirus in Brazilian populations without evidence of previous outbreaks. PMID:26517662

  7. Mucosal Immunization with Newcastle Disease Virus Vector Coexpressing HIV-1 Env and Gag Proteins Elicits Potent Serum, Mucosal, and Cellular Immune Responses That Protect against Vaccinia Virus Env and Gag Challenges

    PubMed Central

    Khattar, Sunil K.; Manoharan, Vinoth; Bhattarai, Bikash; LaBranche, Celia C.; Montefiori, David C.

    2015-01-01

    ABSTRACT Newcastle disease virus (NDV) avirulent strain LaSota was used to coexpress gp160 Env and p55 Gag from a single vector to enhance both Env-specific and Gag-specific immune responses. The optimal transcription position for both Env and Gag genes in the NDV genome was determined by generating recombinant NDV (rNDV)-Env-Gag (gp160 located between the P and M genes and Gag between the HN and L genes), rNDV-Gag-Env (Gag located between the P and M genes and gp160 between the HN and L genes), rNDV-Env/Gag (gp160 followed by Gag located between the P and M genes), and rNDV-Gag/Env (Gag followed by gp160 located between the P and M genes). All the recombinant viruses replicated at levels similar to those seen with parental NDV in embryonated chicken eggs and in chicken fibroblast cells. Both gp160 and Gag proteins were expressed at high levels in cell culture, with gp160 found to be incorporated into the envelope of NDV. The Gag and Env proteins expressed by all the recombinants except rNDV-Env-Gag self-assembled into human immunodeficiency virus type 1 (HIV-1) virus-like particles (VLPs). Immunization of guinea pigs by the intranasal route with these rNDVs produced long-lasting Env- and Gag-specific humoral immune responses. The Env-specific humoral and mucosal immune responses and Gag-specific humoral immune responses were higher in rNDV-Gag/Env and rNDV-Env/Gag than in the other recombinants. rNDV-Gag/Env and rNDV-Env/Gag were also more efficient in inducing cellular as well as protective immune responses to challenge with vaccinia viruses expressing HIV-1 Env and Gag in mice. These results suggest that vaccination with a single rNDV coexpressing Env and Gag represents a promising strategy to enhance immunogenicity and protective efficacy against HIV. PMID:26199332

  8. [Prevention of virus-related neurological diseases by vaccines].

    PubMed

    Takahashi, M

    1997-04-01

    Prevention of virus-related neurological diseases are surveyed. Patients of poliomyelitis has recently been drastically reduced by world-wide administrating live vaccines. In view of rare incidence of paralysis after giving live vaccine, adoption of inactivated vaccine has recently been reconsidered. A live varicella vaccine was developed and has been world-wide used for normal and high-risk children. Incidence of zoster in vaccinated acute leukemic children is several times higher in those who with rash after vaccination as compared with those without rash, and as no or few rash appears after vaccination of normal children, it is expected that vaccination of normal children would lead to reduction of zoster after their aging. Measles encephalitis has rapidly been reduced by world-wide use of live vaccines. Mouse-brain derived vaccine against Japanese encephalitis(JE) has been used in Asian countries. Development of tissue-culture derived JE vaccine is under way. PMID:9103901

  9. Efficacy Evaluation of an Inactivated Duck Tembusu Virus Vaccine.

    PubMed

    Lin, Jian; Liu, Yuehuan; Wang, Xiuqing; Yang, Baoshou; He, Pingyou; Yang, Zhiyuan; Duan, Huijuan; Xie, Jia; Zou, Lihong; Zhao, Jicheng; Pan, Jie

    2015-06-01

    To evaluate the potential use of an inactivated virus-based vaccine for the control and prevention of the newly emerged duck Tembusu virus infection in China, a duck Tembusu virus isolate, Tembusu-HB, was propagated in 12-day-old duck embryos and inactivated by treatment with formaldehyde. The inactivated viral antigen was emulsified with mineral oil, and five batches of the vaccine were manufactured. The immunogenicity and protection efficacy of the vaccine were evaluated in Beijing ducks and Beijing white geese. Results showed that more than 80% of immunized ducks were protected against virulent virus challenge after two intramuscular or subcutaneous injections of the inactivated vaccine, as evidenced by the negative virus isolation results. The protection is also correlated with a positive virus-specific antibody response as detected by ELISA. In contrast, none of the control ducks and geese had any detectable antibody response. Virus was isolated from all control ducks and geese after virulent virus challenge. Interestingly, a variable level of protection (20%-80%) was observed in Beijing white geese immunized twice with the same batches of vaccine, suggesting a species-specific effect of the vaccine. Overall, the results clearly suggest that the inactivated duck Tembusu virus vaccine is immunogenic and provides protection against virulent virus challenge. PMID:26473674

  10. Enhancement of feline immunodeficiency virus infection after immunization with envelope glycoprotein subunit vaccines.

    PubMed Central

    Siebelink, K H; Tijhaar, E; Huisman, R C; Huisman, W; de Ronde, A; Darby, I H; Francis, M J; Rimmelzwaan, G F; Osterhaus, A D

    1995-01-01

    Cats were immunized three times with different recombinant feline immunodeficiency virus (FIV) candidate vaccines. Recombinant vaccinia virus (rVV)-expressed envelope glycoprotein with (vGR657) or without (vGR657 x 15) the cleavage site and an FIV envelope bacterial fusion protein (beta-Galactosidase-Env) were incorporated into immune-stimulating complexes or adjuvanted with Quil A. Although all immunized cats developed antibodies against the envelope protein, only the cats vaccinated with the rVV-expressed envelope glycoproteins developed antibodies which neutralized FIV infection of Crandell feline kidney cells. These antibodies failed to neutralize infection of thymocytes with a molecularly cloned homologous FIV. After the third immunization the cats were challenged with homologous FIV. Two weeks after challenge the cell-associated viral load proved to be significantly higher in the cats immunized with vGR657 and vGR657 x 15 than in the other cats. The cats immunized with vGR657 and vGR657 x 15 also developed antibodies against the Gag proteins more rapidly than the cats immunized with beta-Galactosidase-Env or the control cats. This suggested that immunization with rVV-expressed glycoprotein of FIV results in enhanced infectivity of FIV. It was shown that the observed enhancement could be transferred to naive cats with plasma collected at the day of challenge. PMID:7745719

  11. Active treatment of murine tumors with a highly attenuated vaccinia virus expressing the tumor associated antigen 5T4 (TroVax) is CD4+ T cell dependent and antibody mediated.

    PubMed

    Harrop, Richard; Ryan, Matthew G; Myers, Kevin A; Redchenko, Irina; Kingsman, Susan M; Carroll, Miles W

    2006-09-01

    5T4 is a tumor associated antigen that is expressed on the surface of a wide spectrum of human adenocarcinomas. The highly attenuated virus, modified vaccinia Ankara, has been engineered to express human 5T4 (h5T4). In a pre-clinical murine model, the recombinant virus (TroVax) induces protection against challenge with CT26-h5T4 (a syngeneic tumor line expressing h5T4). Anti-tumor activity is long lived, with protection still evident 6 months after the final vaccination. In a therapeutic setting, injection of mice with TroVax results in a reduction in tumor burden of >90%. Depletion of CD8+ T cells has no effect upon therapy in the active treatment model, whereas depletion of CD4+ T cells completely abrogates anti-tumor activity. In a prophylactic setting, depletion of CD4+ and CD8+ T cells after the induction of a h5T4 immune response has no deleterious effect on protection following challenge with CT26-h5T4. In light of these studies, the role of antibodies in protection against tumor challenge was investigated. 5T4 specific polyclonal serum decreased tumor burden by approximately 70%. Thus, we conclude that CD4+ T cells are essential for the induction of a protective immune response and that antibodies are the likely effector moiety in this xenogeneic murine tumor model. PMID:16311730

  12. The affect of infectious bursal disease virus on avian influenza virus vaccine efficacy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunosuppressive viruses are known to affect vaccinal immunity, however the impact of virally induced immunosuppression on avian influenza vaccine efficacy has not been quantified. In order to determine the effect of exposure to infectious bursal disease virus (IBDV) on vaccinal immunity to highly ...

  13. Vaccination against canine distemper virus infection in infant ferrets with and without maternal antibody protection, using recombinant attenuated poxvirus vaccines.

    PubMed

    Welter, J; Taylor, J; Tartaglia, J; Paoletti, E; Stephensen, C B

    2000-07-01

    Canine distemper virus (CDV) infection of ferrets is clinically and immunologically similar to measles, making this a useful model for the human disease. The model was used to determine if parenteral or mucosal immunization of infant ferrets at 3 and 6 weeks of age with attenuated vaccinia virus (NYVAC) or canarypox virus (ALVAC) vaccine strains expressing the CDV hemagglutinin (H) and fusion (F) protein genes (NYVAC-HF and ALVAC-HF) would induce serum neutralizing antibody and protect against challenge infection at 12 weeks of age. Ferrets without maternal antibody that were vaccinated parenterally with NYVAC-HF (n = 5) or ALVAC-HF (n = 4) developed significant neutralizing titers (log(10) inverse mean titer +/- standard deviation of 2.30 +/- 0.12 and 2.20 +/- 0.34, respectively) by the day of challenge, and all survived with no clinical or virologic evidence of infection. Ferrets without maternal antibody that were vaccinated intranasally (i.n.) developed lower neutralizing titers, with NYVAC-HF producing higher titers at challenge (1.11 +/- 0.57 versus 0.40 +/- 0.37, P = 0.02) and a better survival rate (6/7 versus 0/5, P = 0.008) than ALVAC-HF. Ferrets with maternal antibody that were vaccinated parenterally with NYVAC-HF (n = 7) and ALVAC-HF (n = 7) developed significantly higher antibody titers (1.64 +/- 0. 54 and 1.28 +/- 0.40, respectively) than did ferrets immunized with an attenuated CDV vaccine (0.46 +/- 0.59; n = 7) or the recombinant vectors expressing rabies glycoprotein (RG) (0.19 +/- 0.32; n = 8, P = 7 x 10(-6)). The NYVAC vaccine also protected against weight loss, and both the NYVAC and attenuated CDV vaccines protected against the development of some clinical signs of infection, although survival in each of the three vaccine groups was low (one of seven) and not significantly different from the RG controls (none of eight). Combined i.n.-parenteral immunization of ferrets with maternal antibody using NYVAC-HF (n = 9) produced higher titers (1

  14. Vaccination against Canine Distemper Virus Infection in Infant Ferrets with and without Maternal Antibody Protection, Using Recombinant Attenuated Poxvirus Vaccines

    PubMed Central

    Welter, Janet; Taylor, Jill; Tartaglia, James; Paoletti, Enzo; Stephensen, Charles B.

    2000-01-01

    Canine distemper virus (CDV) infection of ferrets is clinically and immunologically similar to measles, making this a useful model for the human disease. The model was used to determine if parenteral or mucosal immunization of infant ferrets at 3 and 6 weeks of age with attenuated vaccinia virus (NYVAC) or canarypox virus (ALVAC) vaccine strains expressing the CDV hemagglutinin (H) and fusion (F) protein genes (NYVAC-HF and ALVAC-HF) would induce serum neutralizing antibody and protect against challenge infection at 12 weeks of age. Ferrets without maternal antibody that were vaccinated parenterally with NYVAC-HF (n = 5) or ALVAC-HF (n = 4) developed significant neutralizing titers (log10 inverse mean titer ± standard deviation of 2.30 ± 0.12 and 2.20 ± 0.34, respectively) by the day of challenge, and all survived with no clinical or virologic evidence of infection. Ferrets without maternal antibody that were vaccinated intranasally (i.n.) developed lower neutralizing titers, with NYVAC-HF producing higher titers at challenge (1.11 ± 0.57 versus 0.40 ± 0.37, P = 0.02) and a better survival rate (6/7 versus 0/5, P = 0.008) than ALVAC-HF. Ferrets with maternal antibody that were vaccinated parenterally with NYVAC-HF (n = 7) and ALVAC-HF (n = 7) developed significantly higher antibody titers (1.64 ± 0.54 and 1.28 ± 0.40, respectively) than did ferrets immunized with an attenuated CDV vaccine (0.46 ± 0.59; n = 7) or the recombinant vectors expressing rabies glycoprotein (RG) (0.19 ± 0.32; n = 8, P = 7 × 10−6). The NYVAC vaccine also protected against weight loss, and both the NYVAC and attenuated CDV vaccines protected against the development of some clinical signs of infection, although survival in each of the three vaccine groups was low (one of seven) and not significantly different from the RG controls (none of eight). Combined i.n.-parenteral immunization of ferrets with maternal antibody using NYVAC-HF (n = 9) produced higher titers (1.63 ± 0

  15. Adenovirus vectored vaccines against influenza a virus do not result in vaccine associated enhanced respiratory disease following heterologous challenge in contrast to whole inactivated virus vaccine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heterologous influenza A virus (IAV) challenge following vaccination with an intramuscular (IM) whole inactivated vaccine (WIV) can result in vaccine-associated enhanced respiratory disease (VAERD). The objective of this study was to use an adenovirus (Ad5) vector vaccine platform that expressed IAV...

  16. Improvement of BCG protective efficacy with a novel chimpanzee adenovirus and a modified vaccinia Ankara virus both expressing Ag85A

    PubMed Central

    Stylianou, E.; Griffiths, K.L.; Poyntz, H.C.; Harrington-Kandt, R.; Dicks, M.D.; Stockdale, L.; Betts, G.; McShane, H.

    2015-01-01

    A replication-deficient chimpanzee adenovirus expressing Ag85A (ChAdOx1.85A) was assessed, both alone and in combination with modified vaccinia Ankara also expressing Ag85A (MVA85A), for its immunogenicity and protective efficacy against a Mycobacterium tuberculosis (M.tb) challenge in mice. Naïve and BCG-primed mice were vaccinated or boosted with ChAdOx1.85A and MVA85A in different combinations. Although intranasally administered ChAdOx1.85A induced strong immune responses in the lungs, it failed to consistently protect against aerosol M.tb challenge. In contrast, ChAdOx1.85A followed by MVA85A administered either mucosally or systemically, induced strong immune responses and was able to improve the protective efficacy of BCG. This vaccination regime has consistently shown superior protection over BCG alone and should be evaluated further. PMID:26478198

  17. Chikungunya virus and prospects for a vaccine

    PubMed Central

    Weaver, Scott C; Osorio, Jorge E; Livengood, Jill A; Chen, Rubing; Stinchcomb, Dan T

    2013-01-01

    In 2004, chikungunya virus (CHIKV) re-emerged from East Africa to cause devastating epidemics of debilitating and often chronic arthralgia that have affected millions of people in the Indian Ocean Basin and Asia. More limited epidemics initiated by travelers subsequently occurred in Italy and France, as well as human cases exported to most regions of the world, including the Americas where CHIKV could become endemic. Because CHIKV circulates during epidemics in an urban mosquito–human cycle, control of transmission relies on mosquito abatement, which is rarely effective. Furthermore, there is no antiviral treatment for CHIKV infection and no licensed vaccine to prevent disease. Here, we discuss the challenges to the development of a safe, effective and affordable chikungunya vaccine and recent progress toward this goal. PMID:23151166

  18. The detection of Vaccinia virus confirms the high circulation of Orthopoxvirus in buffaloes living in geographical isolation, Marajó Island, Brazilian Amazon.

    PubMed

    Franco-Luiz, Ana Paula Moreira; Fagundes Pereira, Alexandre; de Oliveira, Cairo Henrique Sousa; Barbosa, José Diomedes; Oliveira, Danilo Bretas; Bonjardim, Cláudio Antônio; Ferreira, Paulo César Peregrino; de Souza Trindade, Giliane; Abrahão, Jônatas Santos; Kroon, Erna Geessien

    2016-06-01

    In Brazil, serologic evidence of Orthopoxvirus (OPV) circulation showed positivity around 20% in cattle, humans, monkeys and rodents. Although OPV seropositivity has been described in buffalo herds in southeastern Brazil, no Vaccinia virus (VACV) (member of genus OPV) outbreaks in buffalo herds have been described in this country. This study aimed to investigate the detection of anti-OPV antibodies and to study the OPV genome in Brazilian buffalo herds. Our results demonstrated a high OPV seropositivity in buffalo herds on Marajó Island and molecular data confirmed the circulation of VACV. The geographical isolation conditionmight be a sine qua non condition to explain our results. PMID:27260805

  19. Isolated limb perfusion with melphalan, tumour necrosis factor-alpha and oncolytic vaccinia virus improves tumour targeting and prolongs survival in a rat model of advanced extremity sarcoma.

    PubMed

    Pencavel, Tim D; Wilkinson, Michelle J; Mansfield, David C; Khan, Aadil A; Seth, Rohit; Karapanagiotou, Eleni M; Roulstone, Victoria; Aguilar, Richard J; Chen, Nanhai G; Szalay, Aladar A; Hayes, Andrew J; Harrington, Kevin J

    2015-02-15

    Isolated limb perfusion (ILP) is a treatment for advanced extremity sarcoma and in-transit melanoma. Advancing this procedure by investigating the addition of novel agents, such as cancer-selective oncolytic viruses, may improve both the therapeutic efficacy of ILP and the tumour-targeted delivery of oncolytic virotherapy. Standard in vitro assays were used to characterise single agent and combinatorial activities of melphalan, tumour necrosis factor-alpha (TNF-α) and Lister strain vaccinia virus (GLV-1h68) against BN175 rat sarcoma cells. An orthotopic model of advanced extremity sarcoma was used to evaluate survival of animals after ILP with combinations of TNF-α, melphalan and GLV-1h68. We investigated the efficiency of viral tumour delivery by ILP compared to intravenous therapy, the locoregional and systemic biodistribution of virus after ILP, and the effect of mode of administration on antibody response. The combination of melphalan and GLV-1h68 was synergistic in vitro. The addition of virus to standard ILP regimens was well tolerated and demonstrated superior tumour targeting compared to intravenous administration. Triple therapy (melphalan/TNF-α/GLV-1h68) resulted in increased tumour growth delay and enhanced survival compared to other treatment regimens. Live virus was recovered in large amounts from perfused regions, but in smaller amounts from systemic organs. The addition of oncolytic vaccinia virus to existing TNF-α/melphalan-based ILP strategies results in survival advantage in an immunocompetent rat model of advanced extremity sarcoma. Virus administered by ILP has superior tumour targeting compared to intravenous delivery. Further evaluation and clinical translation of this approach is warranted. PMID:24978211

  20. Control of Influenza and Poliomyelitis with Killed Virus Vaccines

    ERIC Educational Resources Information Center

    Salk, Jonas; Salk, Darrell

    1977-01-01

    Discusses control of poliomyelitis and influenza by live and killed virus vaccines. Considered are the etiological agents, pathogenic mechanisms and epidemiology of each disease. Reviews recent scientific studies of the diseases. Recommends use of killed virus vaccines in controlling both diseases. (CS)

  1. Vaccination of chickens decreased Newcastle disease virus contamination in eggs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Newcastle disease is an important health issue of poultry causing major economic losses and inhibits trade worldwide. Vaccination is used as a control measure, but it is unknown whether vaccination will prevent virus contamination of eggs. In this study, hens were sham-vaccinated or received one or ...

  2. Practical aspects of vaccination of poultry against avian influenza virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although little has changed in vaccine technology for avian influenza virus (AIV) in the past 20 years, the approach to vaccination of poultry (chickens, turkeys and ducks) for avian influenza has evolved as highly pathogenic (HP) AIV has become endemic in several regions of the world. Vaccination f...

  3. Development of an AIDS vaccine using Sendai virus vectors.

    PubMed

    Ishii, Hiroshi; Matano, Tetsuro

    2015-11-01

    Development of an effective AIDS vaccine is crucial for the control of global human immunodeficiency virus type 1 (HIV-1) prevalence. We have developed a novel AIDS vaccine using a Sendai virus (SeV) vector and investigated its efficacy in a macaque AIDS model of simian immunodeficiency virus (SIV) infection. Its immunogenicity and protective efficacy have been shown, indicating that the SeV vector is a promising delivery tool for AIDS vaccines. Here, we describe the potential of SeV vector as a vaccine antigen delivery tool to induce effective immune responses against HIV-1 infection. PMID:26232346

  4. Expression of a biotinylated human thyrotropin receptor in HeLa cells using recombinant vaccinia virus and its application for the detection of Graves' autoantibodies.

    PubMed

    Minich, W B; Weymayer, J D; Loos, U

    1998-01-01

    We have prepared a biotinylated thyrotropin receptor (TSHR-BIO), and characterized its activity in cells and when bound to solid phase (streptavidin agarose). TSHR-BIO consists of the N-terminal 725 amino acids of the human thyrotropin (TSH) receptor linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The C-terminal domain directs the efficient post-translational biotinylation of the protein. TSHR-BIO was expressed using a vaccinia virus expression system. HeLa cells infected with recombinant virus produced large amounts of TSH receptor of approximately 120,000 molecules per cell. Vaccinia virus produced TSHR-BIO was fully functional interacting with TSH (Kd of 2.3+/-0.1 x 10(-10) M) and coupling to cyclic adenosine monophosphate (cAMP) second messenger system. The expressed protein was biotinylated with high efficiency; more than 90% of TSHR-BIO was bound to streptavidin. We have shown the application of streptavidin agarose immobilized TSHR-BIO for the detection of thyroid-binding inhibiting immunoglobulines in unfractionated sera. There was a good positive correlation between the results obtained in this assay and the commercially available TRAK assay performed with solubilized porcine TSH receptor (r = 0.71; p < 0.001, in 45 sera of patients with Graves' disease and 17 normal sera). PMID:9492146

  5. Gene Therapy Using Therapeutic and Diagnostic Recombinant Oncolytic Vaccinia Virus GLV-1h153 for Management of Colorectal Peritoneal Carcinomatosis

    PubMed Central

    Eveno, Clarisse; Mojica, Kelly; Ady, Justin W.; Thorek, Daniel L.J.; Longo, Valerie; Belin, Laurence J.; Gholami, Sepideh; Johnsen, Clark; Zanzonico, Pat; Chen, Nanhai; Yu, Tony; Szalay, Aladar A.; Fong, Yuman

    2015-01-01

    Background Peritoneal carcinomatosis (PC) is a terminal progression of colorectal cancer (CRC). Poor response to cytoreductive surgery and chemotherapy, coupled with the inability to reliably track disease progression using established diagnostic methods make this a deadly disease. This paper examines the effectiveness of the oncolytic vaccinia virus GLV-1h153 as a therapeutic and diagnostic vehicle. We believe that viral expression of the human sodium iodide transporter (hNIS) can provide both real-time monitoring of viral therapy and effective treatment of colorectal peritoneal carcinomatosis (CRPC). Methods Infectivity and cytotoxic effect of GLV-1h153 on CRC cell lines was assayed in-vitro. Viral replication was examined by standard viral plaque assays. Orthotopic CRPC xenografts were generated in athymic nude mice, and subsequently administered GLV-1h153 intraperitoneally. Reduction of tumor burden was assessed by mass. Orthotopic tumors were visualized by SPECT/CT after Iodine (131I) administration and by fluorescence optical imaging. Results GLV-1h153 infected and killed CRC cells in a time and concentration dependent manner. Viral replication demonstrated greater than a 2.35 log increase in titer over 4 days. Intraperitoneal treatment of orthotopic CRPC xenografts resulted in a significant reduction of tumor burden. Infection of orthotopic xenografts was both therapeutic and facilitated monitoring by 131I-SPECT/CT via expression of hNIS in infected tissue. Conclusions GLV-1h153 effectively kills CRC in-vitro and dramatically reduces tumor burden in-vivo. We demonstrate that GLV-1h153 can be used as an agent to provide accurate delineation of tumor burden in-vivo. These findings indicate that GLV-1h153 has significant potential for use as theragnostic agent in the treatment of CRPC. PMID:25616946

  6. Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B sub 1

    SciTech Connect

    Aoyama, Toshifumi; Yamano, Shigeru; Gelboin, H.V.; Gonzalez, F.J. ); Guzelian, P.S. )

    1990-06-01

    Twelve forms of human hepatic cytochrome P450 were expressed in hepatoma cells by means of recombinant vaccinia viruses. The expressed P450s were analyzed for their abilities to activate the potent hepatocarcinogen aflatoxin B{sub 1} to metabolites having mutagenic or DNA-binding properties. Five forms, P450s IA2, IIA3, IIB7, IIIA3, and IIIA4, activated aflatoxin B{sub 1} to mutagenic metabolites as assessed by the production of His revertants of Salmonella typhimurium in the Ames test. The same P450s catalyzed conversion of aflatoxin B{sub 1} to DNA-bound derivatives as judged by an in situ assay in which the radiolabeled carcinogen was incubated with cells expressing the individual P450 forms. Seven other human P450s, IIC8, IIC9, IID6, IIE1, IIF1, and IIIA5, and IVB1, did not significantly activate aflatoxin B{sub 1} as measured by both the Ames test and the DNA-binding assay. Moreover, polyclonal anti-rat liver P450 antibodies that crossreact with individual human P450s IA2, IIA3, IIIA3, and IIIA4 each inhibited aflatoxin B{sub 1} activation catalyzed by human liver S-9 extracts. Inhibition ranged from as low as 10% with antibody against IIA3 to as high as 65% with antibody against IIIA3 and IIIA4. These results establish that metabolic activation of aflatoxin B{sub 1} in human liver involves the contribution of multiple forms of P450.

  7. Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus counterpart inhibits apoptosis independent of Bim

    PubMed Central

    Marshall, B; Puthalakath, H; Caria, S; Chugh, S; Doerflinger, M; Colman, P M; Kvansakul, M

    2015-01-01

    Subversion of host cell apoptosis is an important survival strategy for viruses to ensure their own proliferation and survival. Certain viruses express proteins homologous in sequence, structure and function to mammalian pro-survival B-cell lymphoma 2 (Bcl-2) proteins, which prevent rapid clearance of infected host cells. In vaccinia virus (VV), the virulence factor F1L was shown to be a potent inhibitor of apoptosis that functions primarily be engaging pro-apoptotic Bim. Variola virus (VAR), the causative agent of smallpox, harbors a homolog of F1L of unknown function. We show that VAR F1L is a potent inhibitor of apoptosis, and unlike all other characterized anti-apoptotic Bcl-2 family members lacks affinity for the Bim Bcl-2 homology 3 (BH3) domain. Instead, VAR F1L engages Bid BH3 as well as Bak and Bax BH3 domains. Unlike its VV homolog, variola F1L only protects against Bax-mediated apoptosis in cellular assays. Crystal structures of variola F1L bound to Bid and Bak BH3 domains reveal that variola F1L forms a domain-swapped Bcl-2 fold, which accommodates Bid and Bak BH3 in the canonical Bcl-2-binding groove, in a manner similar to VV F1L. Despite the observed conservation of structure and sequence, variola F1L inhibits apoptosis using a startlingly different mechanism compared with its VV counterpart. Our results suggest that unlike during VV infection, Bim neutralization may not be required during VAR infection. As molecular determinants for the human-specific tropism of VAR remain essentially unknown, identification of a different mechanism of action and utilization of host factors used by a VAR virulence factor compared with its VV homolog suggest that studying VAR directly may be essential to understand its unique tropism. PMID:25766319

  8. A propagation model of computer virus with nonlinear vaccination probability

    NASA Astrophysics Data System (ADS)

    Gan, Chenquan; Yang, Xiaofan; Liu, Wanping; Zhu, Qingyi

    2014-01-01

    This paper is intended to examine the effect of vaccination on the spread of computer viruses. For that purpose, a novel computer virus propagation model, which incorporates a nonlinear vaccination probability, is proposed. A qualitative analysis of this model reveals that, depending on the value of the basic reproduction number, either the virus-free equilibrium or the viral equilibrium is globally asymptotically stable. The results of simulation experiments not only demonstrate the validity of our model, but also show the effectiveness of nonlinear vaccination strategies. Through parameter analysis, some effective strategies for eradicating viruses are suggested.

  9. Infectious laryngotracheitis virus (ILTV) vaccine intake evaluation by detection of virus amplification in feather pulps of vaccinated chickens.

    PubMed

    Davidson, I; Raibshtein, I; Altori, A; Elkin, N

    2016-03-18

    Infectious laryngotracheitis (ILT) is a respiratory disease of poultry caused by an alphaherpesvirus, ILTV. The live vaccine is applied worldwide by drinking water or by the respiratory route, and by the vent application in Israel. No system of direct evaluation of the efficacy of vaccination exists today, except of antibody elicitation, which is an indirect indication of vaccination intake and might happen due to environment exposure. We suggest for the first time an assay for evaluating the accuracy of the vaccination process by spotting the spread of the live vaccine systemically, namely by virus detection in the feather shafts of the vaccinated birds. The feathers are particularly beneficial as they are easy to collect, non-lethal for the bird, therefore advantageous for monitoring purposes. Moreover, the continuous survey of the vaccine virus unveiled the different kinetics of viremia by the different vaccination routes; while after the vent vaccination the systemic viremia peaks during the first week afterwards, after two consecutive vaccine administration by drinking water with 6 day interval, the vireamia peaks only after the second administration. A robust amplification was needed because the vaccine ILTV was present in the bird in minute quantities compared to the wild-type virus. For the vaccine virus identification in feather shafts a nested real-time PCR for the TK ILTV gene was developed. The sensitivity of detection of the nested rtPCR was greater by 1000 compared to conventional nested PCR and 10 times that real-time PCR. PMID:26784685

  10. Advances in Virus-Like Particle Vaccines for Filoviruses

    PubMed Central

    Aman, M. Javad

    2011-01-01

    Ebola virus (EBOV) and Marburg virus (MARV) are among the deadliest human pathogens, with no vaccines or therapeutics available. Multiple vaccine platforms have been tested for efficacy as prophylactic pretreatments or therapeutics for prevention of filovirus hemorrhagic fever. Most successful vaccines are based on a virus-vectored approach expressing the protective glycoprotein (GP); protein-based subunit and DNA vaccines have been tested with moderate success. Virus-like particle (VLP) vaccines have realized promising results when tested in both rodents and nonhuman primates. VLPs rely on the natural properties of the viral matrix protein (VP) 40 to drive budding of filamentous particles that can also incorporate ≥1 other filovirus protein, including GP, VP24, and nucleoprotein (NP). Filovirus VLP vaccines have used particles containing 2 or 3 (GP and VP40, with or without NP) viral proteins generated in either mammalian or insect cells. Early studies successfully demonstrated efficacy of bivalent VLP vaccines in rodents; more recent studies have shown the ability of the VLP vaccines containing GP, NP, and VP40 to confer complete homologous protection against Ebola virus and Marburg virus in a prophylactic setting against in macaques. This review will discuss published work to date regarding development of the VLP vaccines for prevention of lethal filovirus hemorrhagic fever. PMID:21987741

  11. Influenza A virus hemagglutinin protein subunit vaccine elicits vaccine-associated enhanced respiratory disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vaccine-associated enhanced respiratory disease (VAERD) can occur when pigs are challenged with heterologous virus in the presence of non-neutralizing but cross-reactive antibodies elicited by whole inactivated virus (WIV) vaccine. The aim of this study was to compare the effects of heterologous del...

  12. Combination treatment with oncolytic Vaccinia virus and cyclophosphamide results in synergistic antitumor effects in human lung adenocarcinoma bearing mice

    PubMed Central

    2014-01-01

    Background The capacity of the recombinant Vaccinia virus GLV-1h68 as a single agent to efficiently treat different human or canine cancers has been shown in several preclinical studies. Currently, its human safety and efficacy are investigated in phase I/II clinical trials. In this study we set out to evaluate the oncolytic activity of GLV-1h68 in the human lung adenocarcinoma cell line PC14PE6-RFP in cell cultures and analyzed the antitumor potency of a combined treatment strategy consisting of GLV-1h68 and cyclophosphamide (CPA) in a mouse model of PC14PE6-RFP lung adenocarcinoma. Methods PC14PE6-RFP cells were treated in cell culture with GLV-1h68. Viral replication and cell survival were determined by plaque assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. Subcutaneously implanted PC14PE6-RFP xenografts were treated by systemic injection of GLV-1h68, CPA or a combination of both. Tumor growth and viral biodistribution were monitored and immune-related antigen profiling of tumor lysates was performed. Results GLV-1h68 efficiently infected, replicated in and lysed human PC14PE6-RFP cells in cell cultures. PC14PE6-RFP tumors were efficiently colonized by GLV-1h68 leading to much delayed tumor growth in PC14PE6-RFP tumor-bearing nude mice. Combination treatment with GLV-1h68 and CPA significantly improved the antitumor efficacy of GLV-1h68 and led to an increased viral distribution within the tumors. Pro-inflammatory cytokines and chemokines were distinctly elevated in tumors of GLV-1h68-treated mice. Factors expressed by endothelial cells or present in the blood were decreased after combination treatment. A complete loss in the hemorrhagic phenotype of the PC14PE6-RFP tumors and a decrease in the number of blood vessels after combination treatment could be observed. Conclusions CPA and GLV-1h68 have synergistic antitumor effects on PC14PE6-RFP xenografts. We strongly suppose that in the PC14PE6-RFP model the

  13. Probable Congenital Transmission of Reticuloendotheliosis Virus Caused by Vaccination with Contaminated Vaccines

    PubMed Central

    Zhu, Shufen; Guo, Wenlong; Sheng, Pengcheng; Wang, Zunmin; Zhao, Changliang; Zhao, Qingyou; Zhu, Ruiliang

    2012-01-01

    Contaminated vaccine is one unexpected and potential origin of virus infection. In order to investigate the most likely cause of disease in a broiler breeder company of Shandong Province, all 17 batches of live-virus vaccines used in the affected flocks and 478 tissue samples were tested by dot-blot hybridization, nested PCR, and IFA. The results suggested the outbreak of disease was most probably due to the vaccination of REV-contaminated MD-CVI988/Rispens vaccines and ND-LaSota+IB-H120 vaccines. Furthermore, the REV was probably transmitted to the commercial chickens through congenital transmission. PMID:22912872

  14. Influenza A virus hemagglutinin protein subunit vaccine elicits vaccine-associated enhanced respiratory disease in pigs.

    PubMed

    Rajão, Daniela S; Loving, Crystal L; Gauger, Phillip C; Kitikoon, Pravina; Vincent, Amy L

    2014-09-01

    Vaccine-associated enhanced respiratory disease (VAERD) can occur when pigs are challenged with heterologous virus in the presence of non-neutralizing but cross-reactive antibodies elicited by whole inactivated virus (WIV) vaccine. The aim of this study was to compare the effects of heterologous δ1-H1N2 influenza A virus (IAV) challenge of pigs after vaccination with 2009 pandemic H1N1 virus (H1N1pdm09) recombinant hemagglutinin (HA) subunit vaccine (HA-SV) or temperature-sensitive live attenuated influenza virus (LAIV) vaccine, and to assess the role of immunity to HA in the development of VAERD. Both HA-SV and LAIV vaccines induced high neutralizing antibodies to virus with homologous HA (H1N1pdm09), but not heterologous challenge virus (δ1-H1N2). LAIV partially protected pigs, resulting in reduced virus shedding and faster viral clearance, as no virus was detected in the lungs by 5 days post infection (dpi). HA-SV vaccinated pigs developed more severe lung and tracheal lesions consistent with VAERD following challenge. These results demonstrate that the immune response against the HA protein alone is sufficient to cause VAERD following heterologous challenge. PMID:25077416

  15. Structural analysis and immunogenicity of recombinant major envelope protein (rA27L) of buffalopox virus, a zoonotic Indian vaccinia-like virus.

    PubMed

    Kumar, Amit; Yogisharadhya, Revanaiah; Bhanuprakash, Veerakyathappa; Venkatesan, Gnanavel; Shivachandra, Sathish Bhadravati

    2015-10-01

    Buffalopox virus (BPXV), an Indian variant of vaccinia virus (VACV), is a zoonotic agent and affects buffaloes, cattle and humans. A27L is one of the conserved major immuno-dominant envelope proteins of orthopox viruses (OPVs) involved in viral entry/maturation and elicits neutralizing antibodies. In this study, the A27L gene of BPXV-Vij/96 strain encoding recombinant mature A27L (21S to E110) and C-terminal truncated A27L-LZD (21S to N84aa) proteins were cloned and over-expressed in Escherichia coli as fusion proteins. Structurally, A27L of BPXV was similar to that of VACV and found to contain four regions including a potential coiled-coil motif (CCM) in the centre (43 to 84aa). Oligomerization of recombinant A27L fusion protein (∼30 kDa) leads to the formation of dimer/trimers/tetramers under non-reducing conditions. Further, the purified rA27L protein was used for active immunization of rabbit (250 μg/rabbit) and adult mice (10 μg and 50 μg/mice) with or without adjuvants (FCA, alum and CpG). Immune response measured by using indirect-ELISA and SNT revealed a gradual increase in antigen specific serum IgG as well as neutralization antibody titers. Upon challenge with virulent BPXV strain, a protection of 60% was observed in suckling mice passively administered with anti-rA27L sera. No cross-reactivity of rA27L protein with hyperimmune sera against ORFV, GTPV, SPPV, PPRV, FMDV and BTV was noticed in indirect-ELISA. The study indicated that the rA27L protein is a safe and potential prophylactic as well as diagnostic antigen for buffalopox. PMID:26319070

  16. Oncolytic virotherapy with an armed vaccinia virus in an orthotopic model of renal carcinoma is associated with modification of the tumor microenvironment

    PubMed Central

    Fend, Laetitia; Remy-Ziller, Christelle; Foloppe, Johann; Kempf, Juliette; Cochin, Sandrine; Barraud, Luc; Accart, Nathalie; Erbs, Philippe; Fournel, Sylvie; Préville, Xavier

    2016-01-01

    ABSTRACT Oncolytic virotherapy is an emergent promising therapeutic approach for the treatment of cancer. We have constructed a vaccinia virus (WR strain) deleted for thymidine kinase (TK) and ribonucleotide reductase (RR) genes that expressed the fusion suicide gene FCU1 derived from the yeast cytosine deaminase and uracil phosphoribosyltransferase genes. We evaluated this construct (VV-FCU1) in the orthotopic model of renal carcinoma (RenCa). Systemic administration of VV-FCU1 resulted in orthotopic tumor growth inhibition, despite temporary expression of viral proteins. VV-FCU1 treatment was associated with an infiltration of tumors by CD8+ T lymphocytes and a decrease in the proportion of infiltrating Tregs, thus modifying the ratio of CD8+/CD4+ Treg in favor of CD8+cytotoxic T cells. We demonstrated that VV-FCU1 treatment prolonged survival of animals implanted with RenCa cells in kidney. Depletion of CD8+ T cells abolished the therapeutic effect of VV-FCU1 while depletion of CD4+ T cells enhanced its protective activity. Administration of the prodrug 5-fluorocytosine (5-FC) resulted in a sustained control of tumor growth but did not extend survival. This study shows the importance of CD4+ and CD8+ T cells in vaccinia virus-mediated oncolytic virotherapy and suggests that this approach may be evaluated for the treatment of human renal cell carcinoma. PMID:27057460

  17. Influenza vaccination with live-attenuated and inactivated virus-vaccines during an outbreak of disease.

    PubMed Central

    Rocchi, G.; Ragona, G.; Piga, C.; Pelosio, A.; Volpi, A.; Vella, S.; Legniti, N.; de Felici, A.

    1979-01-01

    Immunization procedures with live attenuated and inactivated vaccines were carried out on a group of young recruits at the beginning of an outbreak of infection due to an A/Victoria/3/75-related virus strain, which occurred in February 1977 in a military camp. A retrospective investigation on protection from clinical influenza was then performed in order to investigate whether immunization with live virus vaccines, administered at the beginning of an epidemic, could provide early protection from the disease. In the course of the two weeks following vaccination, laboratory-confirmed clinical influenza cases occurred in 4 subjects among the 110 volunteers of the control group which received placebo, and in 8, 7 and 4 subjects respectively of the 3 groups of about 125 individuals, each of which received one of the following vaccine preparations: (a), live attenuated A/Victoria/3/75 influenza virus oral vaccine, grown on chick embryo kidney culture; (b), live attenuated nasal vaccine, a recombinant of A/Puerto Rico/8/34 with A/Victoria/3/75 virus; and (c), inactivated A/Victoria/3/75 virus intramuscular vaccine. These data do not support the hypothesis that, during an epidemic of infection, early protection from clinical influenza can be achieved through immunization with live attenuated or inactivated influenza virus vaccines, in spite of the high immunizing capability of the vaccine preparations. PMID:512351

  18. A novel vaccine against Streptococcus equi ssp. zooepidemicus infections: the recombinant swinepox virus expressing M-like protein.

    PubMed

    Lin, Hui-xing; Huang, Dong-yan; Wang, Ye; Lu, Cheng-ping; Fan, Hong-jie

    2011-09-16

    To develop a safer, more immunogenic and efficacious vaccine against Streptococcus equi ssp. zooepidemicus (SEZ) infections, the gene of M-like protein (SzP) was placed under the strong vaccinia virus promoter P28 and then inserted into swinepox virus (SPV) genome. The recombinant swinepox virus (rSPV-szp) was isolated in a non-selective medium by the co-expression of Escherichia coli LacZ gene and verified by PCR, western blotting and immunofluorescence assays. To evaluate the immunogenicity of this rSPV-szp, ICR mice were immunized with the rSPV-szp, inactivated SEZ vaccine (positive control), wild type SPV (negative control), or PBS (challenge control). All mice were intraperitoneally challenged with 5 LD(50) of homogenous ATCC 35246 strain 14 days post-vaccination. The results showed that at least 70% mice in rSPV-szp-vaccinated group were protected against homogenous ATCC 35246 challenge, the survival rate was significantly higher compared with mice in the negative control group and the challenge control group (P<0.001). The antibody titers of the rSPV-szp-vaccinated group were significantly higher (P<0.05) than the other three groups. Passive immune protection assays showed that the hyperimmune sera against M-like protein could provide mice with complete protection against challenge of ATCC 35246. Semi-quantitative RT-PCR analysis showed a marked increased in levels of IL-4 and IFN-γ mRNA in immunized mice. The results suggested that the recombinant rSPV-szp provided mice with significant protection from the SEZ infections. It is a promising candidate for the vaccine development against SEZ infections. PMID:21807055

  19. 9 CFR 113.311 - Bovine Virus Diarrhea Vaccine.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... serum dilution in a varying serum-constant virus neutralization test with less than 500 TCID50 of bovine...-neutralization tests from each of the vaccinates. The test virus shall be less than 500 TCID50 of bovine virus... as prescribed in paragraph (c)(3) of this section, show neutralization in all tubes of the...

  20. 9 CFR 113.311 - Bovine Virus Diarrhea Vaccine.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... serum dilution in a varying serum-constant virus neutralization test with less than 500 TCID50 of bovine...-neutralization tests from each of the vaccinates. The test virus shall be less than 500 TCID50 of bovine virus... as prescribed in paragraph (c)(3) of this section, show neutralization in all tubes of the...

  1. 9 CFR 113.311 - Bovine Virus Diarrhea Vaccine.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... serum dilution in a varying serum-constant virus neutralization test with less than 500 TCID50 of bovine...-neutralization tests from each of the vaccinates. The test virus shall be less than 500 TCID50 of bovine virus... as prescribed in paragraph (c)(3) of this section, show neutralization in all tubes of the...

  2. 9 CFR 113.311 - Bovine Virus Diarrhea Vaccine.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... serum dilution in a varying serum-constant virus neutralization test with less than 500 TCID50 of bovine...-neutralization tests from each of the vaccinates. The test virus shall be less than 500 TCID50 of bovine virus... as prescribed in paragraph (c)(3) of this section, show neutralization in all tubes of the...

  3. 9 CFR 113.311 - Bovine Virus Diarrhea Vaccine.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... serum dilution in a varying serum-constant virus neutralization test with less than 500 TCID50 of bovine...-neutralization tests from each of the vaccinates. The test virus shall be less than 500 TCID50 of bovine virus... as prescribed in paragraph (c)(3) of this section, show neutralization in all tubes of the...

  4. Chitosan nanoparticle encapsulated hemagglutinin-split influenza virus mucosal vaccine.

    PubMed

    Sawaengsak, Chompoonuch; Mori, Yasuko; Yamanishi, Koichi; Mitrevej, Ampol; Sinchaipanid, Nuttanan

    2014-04-01

    Subunit/split influenza vaccines are less reactogenic compared with the whole virus vaccines. However, their immunogenicity is relatively low and thus required proper adjuvant and/or delivery vehicle for immunogenicity enhancement. Influenza vaccines administered intramuscularly induce minimum, if any, mucosal immunity at the respiratory mucosa which is the prime site of the infection. In this study, chitosan (CS) nanoparticles were prepared by ionic cross-linking of the CS with sodium tripolyphosphate (TPP) at the CS/TPP ratio of 1:0.6 using 2 h mixing time. The CS/TPP nanoparticles were used as delivery vehicle of an intranasal influenza vaccine made of hemagglutinin (HA)-split influenza virus product. Innocuousness, immunogenicity, and protective efficacy of the CS/TPP-HA vaccine were tested in influenza mouse model in comparison with the antigen alone vaccine. The CS/TPP-HA nanoparticles had required characteristics including nano-sizes, positive charges, and high antigen encapsulation efficiency. Mice that received two doses of the CS/TPP-HA vaccine intranasally showed no adverse symptoms indicating the vaccine innocuousness. The animals developed higher systemic and mucosal antibody responses than vaccine made of the HA-split influenza virus alone. The CS/TPP-HA vaccine could induce also a cell-mediated immune response shown as high numbers of IFN-γ-secreting cells in spleens while the HA vaccine alone could not. Besides, the CS nanoparticle encapsulated HA-split vaccine reduced markedly the influenza morbidity and also conferred 100% protective rate to the vaccinated mice against lethal influenza virus challenge. Overall results indicated that the CS nanoparticles invented in this study is an effective and safe delivery vehicle/adjuvant for the influenza vaccine. PMID:24343789

  5. Chimpanzee adenovirus and MVA-vectored respiratory syncytial virus vaccine is safe and expands humoral and cellular immunity in adults

    PubMed Central

    Green, CA; Scarselli, E; Sande, CJ; Thompson, AJ; de Lara, CM; Taylor, K; Haworth, K; Del Sorbo, M; Angus, B; Siani, L; Di Marco, S; Traboni, C; Folgori, A; Colloca, S; Capone, S; Vitelli, A; Cortese, R; Klenerman, P; Nicosia, A; Pollard, AJ

    2015-01-01

    Respiratory syncytial virus (RSV) causes respiratory infection in annual epidemics, with infants and the elderly at particular risk of developing severe disease and death. However, despite its importance, no vaccine exists. The chimpanzee adenovirus, PanAd3-RSV, and modified vaccinia virus Ankara, MVA-RSV, are replication defective viral vectors encoding the RSV proteins F, N and M2-1 for the induction of humoral and cellular responses. We performed an open-label, dose-escalation, phase 1 clinical trial in 42 healthy adults in which four different combinations of prime/boost vaccinations were investigated for safety and immunogenicity, including both intra-muscular and intra-nasal administration of the adenoviral vectored vaccine. The vaccines were safe and well tolerated, with the most common reported adverse events being mild injection site reactions. No vaccine-related serious adverse events occurred. RSV neutralising antibody titres rose in response to intramuscular (IM) prime with PanAd3-RSV, and after IM boost for individuals primed by the intra-nasal (IN) route. Circulating anti-F IgG and IgA antibody secreting cells (ASCs) were observed after IM prime and IM boost. RSV-specific T-cell responses were increased after IM PanAd3-RSV prime and were most efficiently boosted by IM MVA-RSV. IFNγ secretion after boost was from both CD4+ and CD8+ T-cells, without detectable Th2 cytokines that have been previously associated with immune pathogenesis following exposure to RSV after formalin inactivated RSV vaccine. In conclusion, PanAd3-RSV and MVA-RSV are safe and immunogenic in healthy adults. These vaccine candidates warrant further clinical evaluation of efficacy to assess their potential to reduce the burden of RSV disease. PMID:26268313

  6. Influenza virus vaccine live intranasal--MedImmune vaccines: CAIV-T, influenza vaccine live intranasal.

    PubMed

    2003-01-01

    MedImmune Vaccines (formerly Aviron) has developed a cold-adapted live influenza virus vaccine [FluMist] that can be administered by nasal spray. FluMist is the first live virus influenza vaccine and also the first nasally administered vaccine to be marketed in the US. The vaccine will be formulated to contain live attenuated (att) influenza virus reassortants of the strains recommended by the US Public Health Service for each 'flu season. The vaccine is termed cold-adapted (ca) because the virus has been adapted to replicate efficiently at 25 degrees C in the nasal passages, which are below normal body temperature. The strains used in the seasonal vaccine will also be made temperature sensitive (ts) so that their replication is restricted at 37 degrees C (Type B strains) and 39 degrees C (Type A strains). The combined effect of the antigenic properties and the att, ca and ts phenotypes of the influenza strains contained in the vaccine enables the viruses to replicate in the nasopharynx to produce protective immunity. The original formulation of FluMist requires freezer storage throughout distribution. Because many international markets do not have distribution channels well suited to the sale of frozen vaccines, Wyeth and MedImmune are collaborating to develop a second generation, refrigerator-stable, liquid trivalent cold-adapted influenza vaccine (CAIV-T), which is in phase III trials. Initially, the frozen formulation will only be available in the US. For the 2003-2004 season, FluMist will contain A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2) (A/Moscow/10/99-like) and B/Hong Kong/330/2001. Aviron was acquired by MedImmune on 15 January 2002. Aviron is now a wholly-owned subsidiary of MedImmune and is called MedImmune Vaccines. Aviron acquired FluMist in March 1995 through a Co-operative Research and Development Agreement (CRADA) with the US NIAID, and a licensing agreement with the University of Michigan, Ann Arbor, USA. In June 2000, the CRADA was

  7. The nucleotide sequence of the chicken thymidine kinase gene and the relationship of its predicted polypeptide to that of the vaccinia virus thymidine kinase.

    PubMed Central

    Kwoh, T J; Engler, J A

    1984-01-01

    The entire DNA nucleotide sequence of a 3.0 kilobase pair Hind III fragment containing the chicken cytoplasmic thymidine kinase gene was determined. Oligonucleotide linker insertion mutations distributed throughout this gene and having known effects upon gene activity ( Kwoh , T.J., Zipser , D., and Wigler , M. 1983. J. Mol. Appl. Genet. 2, 191-200), were used to access regions of the Hind III fragment for sequencing reactions. The complete nucleotide sequence, together with the positions of the linker insertion mutations within the sequence, allows us to propose a structure for the chicken thymidine kinase gene. The protein coding sequence of the gene is divided into seven small segments (each less than 160 base pairs) by six small introns (each less than 230 base pairs). The proposed 244 amino acid polypeptide encoded by this gene bears strong homology to the vaccinia virus thymidine kinase. No homology with the thymidine kinases of the herpes simplex viruses was found. PMID:6328447

  8. Genetic Confirmation that the H5 Protein Is Required for Vaccinia Virus DNA Replication

    PubMed Central

    Boyle, Kathleen A.; Greseth, Matthew D.

    2015-01-01

    ABSTRACT The duplication of the poxvirus double-stranded DNA genome occurs in cytoplasmic membrane-delimited factories. This physical autonomy from the host nucleus suggests that poxvirus genomes encode the full repertoire of proteins committed for genome replication. Biochemical and genetic analyses have confirmed that six viral proteins are required for efficient DNA synthesis; indirect evidence has suggested that the multifunctional H5 protein may also have a role. Here we show that H5 localizes to replication factories, as visualized by immunofluorescence and immunoelectron microscopy, and can be retrieved upon purification of the viral polymerase holoenzyme complex. The temperature-sensitive (ts) mutant Dts57, which was generated by chemical mutagenesis and has a lesion in H5, exhibits defects in DNA replication and morphogenesis under nonpermissive conditions, depending upon the experimental protocol. The H5 variant encoded by the genome of this mutant is ts for function but not stability. For a more precise investigation of how H5 contributes to DNA synthesis, we placed the ts57 H5 allele in an otherwise wild-type viral background and also performed small interfering RNA-mediated depletion of H5. Finally, we generated a complementing cell line, CV-1–H5, which allowed us to generate a viral recombinant in which the H5 open reading frame was deleted and replaced with mCherry (vΔH5). Analysis of vΔH5 allowed us to demonstrate conclusively that viral DNA replication is abrogated in the absence of H5. The loss of H5 does not compromise the accumulation of other early viral replication proteins or the uncoating of the virion core, suggesting that H5 plays a direct and essential role in facilitating DNA synthesis. IMPORTANCE Variola virus, the causative agent of smallpox, is the most notorious member of the Poxviridae family. Poxviruses are unique among DNA viruses that infect mammalian cells, in that their replication is restricted to the cytoplasm of the cell

  9. Distinct Humoral and Cellular Immunity Induced by Alternating Prime-boost Vaccination Using Plasmid DNA and Live Viral Vector Vaccines Expressing the E Protein of Dengue Virus Type 2

    PubMed Central

    George, Junu A.

    2011-01-01

    Background Dengue virus, which belongs to the Flavivirus genus of the Flaviviridae family, causes fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) with infection risk of 2.5 billion people worldwide. However, approved vaccines are still not available. Here, we explored the immune responses induced by alternating prime-boost vaccination using DNA vaccine, adenovirus, and vaccinia virus expressing E protein of dengue virus type 2 (DenV2). Methods Following immunization with DNA vaccine (pDE), adenovirus (rAd-E), and/or vaccinia virus (VV-E) expressing E protein, E protein-specific IgG and its isotypes were determined by conventional ELISA. Intracellular CD154 and cytokine staining was used for enumerating CD4+ T cells specific for E protein. E protein-specific CD8+ T cell responses were evaluated by in vivo CTL killing activity and intracellular IFN-γ staining. Results Among three constructs, VV-E induced the most potent IgG responses, Th1-type cytokine production by stimulated CD4+ T cells, and the CD8+ T cell response. Furthermore, when the three constructs were used for alternating prime-boost vaccination, the results revealed a different pattern of CD4+ and CD8+ T cell responses. i) Priming with VV-E induced higher E-specific IgG level but it was decreased rapidly. ii) Strong CD8+ T cell responses specific for E protein were induced when VV-E was used for the priming step, and such CD8+ T cell responses were significantly boosted with pDE. iii) Priming with rAd-E induced stronger CD4+ T cell responses which subsequently boosted with pDE to a greater extent than VV-E and rAd-E. Conclusion These results indicate that priming with live viral vector vaccines could induce different patterns of E protein- specific CD4+ and CD8+ T cell responses which were significantly enhanced by booster vaccination with the DNA vaccine. Therefore, our observation will provide valuable information for the establishment of optimal prime-boost vaccination against

  10. Construction and testing of a novel host-range defective myxoma virus vaccine with the M063 gene inactivated that is non-permissive for replication in rabbit cells.

    PubMed

    Adams, Mathew M; van Leeuwen, Barbara H; McFadden, Grant; Kerr, Peter J

    2008-01-01

    Deletion of the M063 gene from myxoma virus produces a virus that is unable to replicate in rabbit cells in vitro or in live rabbits but can be propagated in non-rabbit cell lines. A targeted M063 deletion mutant was constructed in the attenuated Uriarra strain of myxoma virus and the ability of this virus to act as a safe, non-transmissible vaccine against myxomatosis was tested in outbred laboratory rabbits. Immunization with the M063 deletion vaccine provided good short-term protection against lethal challenge with virulent myxoma virus. Long-term protection was similar to reported results with heterologous live virus, with some rabbits protected but others succumbing to challenge. Replication-deficient poxvirus vaccines, like the Modified Vaccinia Virus Ankara (MVA) in man and the myxoma virus vaccine described here in rabbits, are very attractive from a safety perspective. Seasonal boosting would be predicted to provide long-term protection. Targeted host-range gene deletions could have potential for rapid development of poxvirus vaccines in general. PMID:18778680

  11. Measles Virus Infection Among Vaccinated and Unvaccinated Children in Nigeria

    PubMed Central

    Adeniji, Johnson A.; Olusola, Babatunde A.; Motayo, Babatunde O.; Akintunde, Grace B.

    2015-01-01

    Abstract This study investigated measles infection in vaccinated and unvaccinated children presenting with fever and maculopapular rash during measles outbreaks in the southern and western states of Nigeria. Measles, an acute viral illness caused by a virus in the family Paramyxoviridae, is a vaccine-preventable disease. Measles outbreak is common in Nigeria, despite the national immunization program. Children presenting with symptoms of measles infection in general hospitals and health centers in the states of southern and western Nigeria were recruited for this study. Vaccination history, clinical details, and 5 mL of blood were obtained from the children. Their sera samples were screened for specific immunoglobulin M antibodies to measles virus. Of 234 children tested (124 [53.2%] female), 133 (56.8%) had previously been vaccinated against measles virus, while 93 (39.7%) had not been vaccinated. Vaccination information for eight children could not be retrieved. One hundred and forty-three (62.4%) had measles IgM antibodies. Of these, 79 (55.3%) had been vaccinated for measles, while 65 (44.7%) had not. Despite the ongoing vaccination program in Nigeria, a high number of children are still being infected with measles, despite their vaccination status. Therefore, there is need to identify the reason for the low level of vaccine protection. PMID:26102341

  12. A recombinant influenza virus vaccine expressing the F protein of respiratory