Pre- and Postlicensure Animal Efficacy Studies Comparing Anthrax Antitoxins.

Background: The deliberate use of Bacillus anthracis spores is believed by the US government to be a high bioweapons threat. The first line of defense following potential exposure to B. anthracis spores would be postexposure prophylaxis with antimicrobials that have activity against B.

Natural History of to Support Medical Countermeasure Development.

(SUDV) is one of four members of the genus known to cause Ebola Virus Disease (EVD) in humans, which is characterized by hemorrhagic fever and a high case fatality rate. While licensed therapeutics and vaccines are available in limited number to treat infections of Zaire ebolavirus, there are currently no effective licensed vaccines or therapeutics for SUDV. A well-characterized animal model of this disease is needed for the further development and testing of vaccines and therapeutics.

Use of JYNNEOS (Smallpox and Monkeypox Vaccine, Live, Nonreplicating) for Preexposure Vaccination of Persons at Risk for Occupational Exposure to Orthopoxviruses: Recommendations of the Advisory Committee on Immunization Practices - United States, 2022.

Certain laboratorians and health care personnel can be exposed to orthopoxviruses through occupational activities. Because orthopoxvirus infections resulting from occupational exposures can be serious, the Advisory Committee on Immunization Practices (ACIP) has continued to recommend preexposure vaccination for these persons since 1980 (1), when smallpox was eradicated (2). In 2015, ACIP made recommendations for the use of ACAM2000, the only orthopoxvirus vaccine available in the United States at that time (3).

Selection of Filovirus Isolates for Vaccine Development Programs.

The continuing outbreaks of ebola virus disease highlight the ongoing threat posed by filoviruses. Fortunately, licensed vaccines and therapeutics are now available for . However, effective medical countermeasures, such as vaccines for other filoviruses such as and the Marburg virus, are presently in early stages of development and, in the absence of a large outbreak, would require regulatory approval via the U.

The development and approval of tecoviromat (TPOXX), the first antiviral against smallpox.

The classification of smallpox by the U.S. Centers for Disease Control and Prevention (CDC) as a Category A Bioterrorism threat agent has resulted in the U.

Rabbitpox in New Zealand White Rabbits: A Therapeutic Model for Evaluation of Poxvirus Medical Countermeasures Under the FDA Animal Rule.

The elimination of smallpox as an endemic disease and the obvious ethical problems with clinical challenge requires the efficacy evaluation of medical countermeasures against smallpox using the FDA Animal Rule. This approach requires the evaluation of antiviral efficacy in an animal model whose infection recapitulates the human disease sufficiently well enough to provide predictive value of countermeasure effectiveness. The narrow host range of variola virus meant that no other animal species was sufficiently susceptible to variola to manifest a disease with predictive value.

Ectromelia Virus Disease Characterization in the BALB/c Mouse: A Surrogate Model for Assessment of Smallpox Medical Countermeasures.

In 2007, the United States- Food and Drug Administration (FDA) issued guidance concerning animal models for testing the efficacy of medical countermeasures against variola virus (VARV), the etiologic agent for smallpox. Ectromelia virus (ECTV) is naturally-occurring and responsible for severe mortality and morbidity as a result of mousepox disease in the murine model, displaying similarities to variola infection in humans. Due to the increased need of acceptable surrogate animal models for poxvirus disease, we have characterized ECTV infection in the BALB/c mouse.

Effect of the deletion of genes encoding proteins of the extracellular virion form of vaccinia virus on vaccine immunogenicity and protective effectiveness in the mouse model.

Antibodies to both infectious forms of vaccinia virus, the mature virion (MV) and the enveloped virion (EV), as well as cell-mediated immune response appear to be important for protection against smallpox. EV virus particles, although more labile and less numerous than MV, are important for dissemination and spread of virus in infected hosts and thus important in virus pathogenesis. The importance of the EV A33 and B5 proteins for vaccine induced immunity and protection in a murine intranasal challenge model was evaluated by deletion of both the A33R and B5R genes in a vaccine-derived strain of vaccinia virus.

New neutralizing antibody epitopes in hepatitis C virus envelope glycoproteins are revealed by dissecting peptide recognition profiles.

One of the greatest challenges to HCV vaccine development is the induction of effective immune responses using recombinant proteins or vectors. In order to better understand which vaccine-induced antibodies contribute to neutralization of HCV the quality of polyclonal anti-E1E2 antibody responses in immunized mice and chimpanzees was assessed at the level of epitope recognition using peptide scanning and neutralization of chimeric 1a/2a, 1b/2a and 2a HCVcc after blocking or affinity elution of specific antibodies. Mice and chimpanzees were immunized with genotype 1a (H77) HCV gpE1E2; all samples contained cross-neutralizing antibody against HCVcc.

Passive immunotherapies protect WRvFire and IHD-J-Luc vaccinia virus-infected mice from lethality by reducing viral loads in the upper respiratory tract and internal organs.

Whole-body bioimaging was employed to study the effects of passive immunotherapies on lethality and viral dissemination in BALB/c mice challenged with recombinant vaccinia viruses expressing luciferase. WRvFire and IHD-J-Luc vaccinia viruses induced lethality with similar times to death following intranasal infection, but WRvFire replicated at higher levels than IHD-J-Luc in the upper and lower respiratory tracts. Three types of therapies were tested: licensed human anti-vaccinia virus immunoglobulin intravenous (VIGIV); recombinant anti-vaccinia virus immunoglobulin (rVIG; Symphogen, Denmark), an investigational product containing a mixture of 26 human monoclonal antibodies (HuMAbs) against mature virion (MV) and enveloped virion (EV); and HuMAb compositions targeting subsets of MV or EV proteins.

Recombinant A27 protein synergizes with modified vaccinia Ankara in conferring protection against a lethal vaccinia virus challenge.

Highly attenuated modified vaccinia virus Ankara (MVA) is being considered as a safer alternative to conventional smallpox vaccines such as Dryvax or ACAM 2000, but it requires higher doses or more-frequent boosting than replication-competent Dryvax. Previously, we found that passive transfer of A27 antibodies can enhance protection afforded by vaccinia immune globulin (VIG), which is derived from Dryvax immunized subjects. Here we investigated whether protective immunity elicited by MVA could be augmented by prime-boost or combination immunizations with a recombinant A27 (rA27) protein.

Comparative evaluation of the immune responses and protection engendered by LC16m8 and Dryvax smallpox vaccines in a mouse model.

The immune response elicited by LC16m8, a candidate smallpox vaccine that was developed in Japan by cold selection during serial passage of the Lister vaccine virus in primary rabbit kidney cells, was compared to Dryvax in a mouse model. LC16m8 carries a mutation resulting in the truncation of the B5 protein, an important neutralizing target of the extracellular envelope form of vaccinia virus (EV). LC16m8 elicited a broad-spectrum immunoglobulin G (IgG) response that neutralized both EV and the intracellular mature form of vaccinia virus and provoked cell-mediated immune responses, including the activation of CD4+ and CD8+ cells, similarly to Dryvax.

Smallpox vaccines induce antibodies to the immunomodulatory, secreted vaccinia virus complement control protein.

Vaccination with Dryvax elicits a broad humoral response against many viral proteins. Human vaccinia immune globulin was used to screen the secreted proteins from cells infected with Dryvax or the candidate smallpox vaccine LC16m8 to determine whether the protective humoral response included antibodies against secreted viral proteins. Many proteins were detected, with the primary band corresponding to a band of 28 or 30 kDa in cells infected with Dryvax or LC16m8, respectively.

Antibodies to the A27 protein of vaccinia virus neutralize and protect against infection but represent a minor component of Dryvax vaccine--induced immunity.

The smallpox vaccine Dryvax, which consists of replication-competent vaccinia virus, elicits antibodies that play a major role in protection. Several vaccinia proteins generate neutralizing antibodies, but their importance for protection is unknown. We investigated the potency of antibodies to the A27 protein of the mature virion in neutralization and protection experiments and the contributions of A27 antibodies to Dryvax-induced immunity.

Characterization and use of mammalian-expressed vaccinia virus extracellular membrane proteins for quantification of the humoral immune response to smallpox vaccines.

The licensed smallpox vaccine Dryvax is used as the standard in comparative immunogenicity and protection studies of new smallpox vaccine candidates. Although the correlates of protection against smallpox are unknown, recent studies have shown that a humoral response against the intracellular mature virion and extracellular enveloped virion (EV) forms of vaccinia virus is crucial for protection. Using a recombinant Semliki Forest virus (rSFV) vector system, we expressed a set of full-length EV proteins for the development of EV antigen-specific enzyme-linked immunosorbent assays (ELISAs) and the production of monospecific antisera.

Microarray assay for evaluation of the genetic stability of modified vaccinia virus Ankara B5R gene.

Adverse events associated with the use of live smallpox vaccines have led to the development of a new generation of attenuated smallpox vaccines that are prepared in cultured cells as alternatives. The inability to conduct direct clinical evaluation of their efficacy in humans demands that licensure be based on animal studies and exhaustive evaluation of their in vitro properties. One of the most important characteristics of live viral vaccines is their genetic stability, including reversion of the vaccine strain to more virulent forms, recombination with other viral sequences to produce potentially pathogenic viruses, and genetic drift that can result in decrease of immunogenicity and efficacy.

CD4+ immune escape and subsequent T-cell failure following chimpanzee immunization against hepatitis C virus.

Hepatitis C is a major cause of chronic liver disease, with 170 million individuals infected worldwide and no available vaccine. We analyzed the effects of an induced T-cell response in 3 chimpanzees, targeting nonstructural proteins in the absence of neutralizing antibodies. In all animals the specific T-cell response modified the outcome of infection, producing a 10- to 1,000-fold reduction in peak virus titers.

Vaccinia virus nicking-joining enzyme is encoded by K4L (VACWR035).

Vaccinia virus encodes an enzyme with DNA modifying activity that cleaves and inefficiently cross-links cruciformic DNA. This enzyme is contained within the virion, expressed at late times postinfection, and processes DNA in an energy-independent, Mg2+ ion-independent manner. Viral nuclease activity was measured in extracts from cells infected with well-defined viral mutants.

Identification and preliminary characterization of vaccinia virus (Dryvax) antigens recognized by vaccinia immune globulin.

Using vaccinia immune globulin (VIG), a high-titer antibody preparation from immunized subjects, we demonstrate that the humoral immune response in humans is directed against numerous antigens in the Dryvax vaccine strain. Western blot and immunoprecipitation analyses revealed highly antigenic proteins associated with both the extracellular enveloped virus and intracellular mature virus forms. The modified vaccinia virus Ankara (MVA), a new generation smallpox vaccine that is attenuated for replication in humans, expresses most, but not all, of the major vaccinia antigens recognized by antibodies in VIG, lacking the highly antigenic protein corresponding to the A-type inclusion body protein.

Identification of a novel immunodominant cytotoxic T lymphocyte epitope derived from human factor VIII in a murine model of hemophilia A.

Gene therapy of hemophilia A could be complicated by the development of immune responses against the vector as well as the Factor VIII (FVIII) transgene. Previous efforts have been focused on identifying FVIII inhibitor antibody epitopes, whereas the cytotoxic T lymphocyte (CTL) epitopes have not been characterized. CTL would kill cells expressing such epitopes and thus limit the efficacy of gene therapy.

Enhanced immunogenicity and protective effect conferred by vaccination with combinations of modified vaccinia virus Ankara and licensed smallpox vaccine Dryvax in a mouse model.

Significant adverse events are associated with vaccination with the currently licensed smallpox vaccine. Candidate new-generation smallpox vaccines such as the replication-defective modified vaccinia virus Ankara (MVA) produce very few adverse events in experimental animals and in limited human clinical trials conducted near the end of the smallpox eradication campaign. Efficacy evaluation of such new-generation vaccines will be extraordinarily complex, however, since the eradication of smallpox precludes a clinical efficacy trial and the correlates of protection against smallpox are unknown.

Mouse neurotoxicity test for vaccinia-based smallpox vaccines.

The only US FDA licensed smallpox vaccine, Dryvax, was associated with rare but serious neurological adverse events. After smallpox was eradicated in the United States, mass vaccination ceased in 1971. As counter-bioterrorism/biowarfare measures, new smallpox vaccines are now being investigated.

Development of a novel vaccinia-neutralization assay based on reporter-gene expression.

In anticipation of large-scale smallpox vaccination, clinical trials of new vaccine candidates with improved safety profiles, and new vaccinia immune globulin (VIG) products, there is an immediate need to develop new assays to measure vaccinia-specific immune responses. The classical assay to measure vaccinia neutralization, the plaque-reduction neutralization test (PRNT), is slow, labor intensive, and difficult to validate and transfer. Here we describe the development of a novel vaccinia-neutralization assay based on the expression of a reporter gene, beta-galactosidase (beta-Gal).

Prime-boost immunization with DNA and modified vaccinia virus ankara vectors expressing herpes simplex virus-2 glycoprotein D elicits greater specific antibody and cytokine responses than DNA vaccine alone.

Several reports have indicated that prime-boost strategies of vaccination can enhance the level of specific immunity induced by nucleic acid vaccines. The present report describes such a strategy with herpes simplex virus (HSV)-2 glycoprotein D (gD), using combinations of plasmid vector that expresses gD (pgD2) and a recombinant modified vaccinia virus Ankara vector that expresses gD (MVA-gD2). The IgG antibody response to gD and the HSV-2 neutralizing antibody response were greatest when the MVA-gD2 vector was used as the priming immunization and then was boosted with either pgD2 or MVA-gD2.