Sudan ebolavirus (SUDV) is one of four members of the Ebolavirus 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. In this study, twelve cynomolgus macaques (Macaca fascicularis) were challenged intramuscularly with 1000 PFUs of SUDV and were followed under continuous telemetric surveillance. Clinical observations, body weights, temperature, viremia, hematology, clinical chemistry, and coagulation were analyzed at timepoints throughout the study. Death from SUDV disease occurred between five and ten days after challenge at the point that each animal met the criteria for euthanasia. All animals were observed to exhibit clinical signs and lesions similar to those observed in human cases which included: viremia, fever, dehydration, reduced physical activity, macular skin rash, systemic inflammation, coagulopathy, lymphoid depletion, renal tubular necrosis, hepatocellular degeneration and necrosis. The results from this study will facilitate the future preclinical development and evaluation of vaccines and therapeutics for SUDV.

BACKGROUNDThe 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. anthracis. Additional therapies to address the effects of toxins may be needed in systemically ill individuals. Over the last 2 decades, the United States government (USG) collaborated with the private sector to develop, test, and stockpile 3 antitoxins: anthrax immunoglobulin intravenous (AIGIV), raxibacumab, and obiltoxaximab. All 3 products target protective antigen, a protein factor common to the 2 exotoxins released by B. anthracis, and hamper or block the toxins' effects and prevent or reduce pathogenesis. These antitoxins were approved for licensure by the United States Food and Drug Administration based on animal efficacy studies compared to placebo.METHODSWe describe USG-sponsored pre- and postlicensure studies that compared efficacy of 3 antitoxins in a New Zealand White rabbit model of inhalation anthrax; survival following a lethal aerosolized dose of B. anthracis spores was the key measure of effectiveness. To model therapeutic intervention, intravenous treatments were started following onset of antigenemia.RESULTSIn pre- and postlicensure studies, all 3 antitoxins were superior to placebo; in the postlicensure study, raxibacumab and obiltoxaximab were superior to AIGIV, but neither was superior to the other.CONCLUSIONSThese data illustrate the relative therapeutic benefit of the 3 antitoxins and provide a rationale to prioritize their deployment.

The Biomedical Advanced Research and Development Authority, part of the Administration for Strategic Preparedness and Response within the U.S. Department of Health and Human Services, recognizes that the evaluation of medical countermeasures under the Animal Rule requires well-characterized and reproducible animal models that are likely to be predictive of clinical benefit. Marburg virus (MARV), one of two members of the genus Marburgvirus, is characterized by a hemorrhagic fever and a high case fatality rate for which there are no licensed vaccines or therapeutics available. This natural history study consisted of twelve cynomolgus macaques challenged with 1000 PFU of MARV Angola and observed for body weight, temperature, viremia, hematology, clinical chemistry, and coagulation at multiple time points. All animals succumbed to disease within 8 days and exhibited signs consistent with those observed in human cases, including viremia, fever, systemic inflammation, coagulopathy, and lymphocytolysis, among others. Additionally, this study determined the time from exposure to onset of disease manifestations and the time course, frequency, and magnitude of the manifestations. This study will be instrumental in the design and development of medical countermeasures to Marburg virus disease.

The continuing outbreaks of ebola virus disease highlight the ongoing threat posed by filoviruses. Fortunately, licensed vaccines and therapeutics are now available for Zaire ebolavirus. However, effective medical countermeasures, such as vaccines for other filoviruses such as Sudan ebolavirus 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.S. Food and Drug Administration (FDA) Animal Rule. The selection of an appropriate animal model and virus challenge isolates for nonclinical studies are critical aspects of the development program. Here, we have focused on the recommendation of challenge isolates for Sudan ebolavirus and Marburg virus. Based on analyses led by the Filovirus Animal and Nonclinical Group (FANG) and considerations for strain selection under the FDA Guidance for the Animal Rule, we propose prototype virus isolates for use in nonclinical challenge studies.

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.S. Government investing significant funds to develop and stockpile a suite of medical countermeasures to ameliorate the consequences of a smallpox epidemic. This stockpile includes both vaccines for prophylaxis and antivirals to treat symptomatic patients. In this manuscript, we describe the path to approval for the first therapeutic against smallpox, identified during its development as ST-246, now known as tecovirimat and TPOXX®, a small-molecule antiviral compound sponsored by SIGA Technologies to treat symptomatic smallpox. Because the disease is no longer endemic, the development and approval of TPOXX® was only possible under the U.S. Food and Drug and Administration Animal Rule (FDA 2002). In this article, we describe the combination of animal model studies and clinical trials that were used to satisfy the FDA requirements for the approval of TPOXX ® under the 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. To address this dilemma, the FDA, after a public forum with virologists in December 2011, suggested the development of two animal models infected with the cognate orthopoxvirus, intradermal infection of rabbits and intranasal infection of mice, to supplement the non-human primate models in use. In this manuscript, we describe the development of an intradermal challenge model of New Zealand White rabbits with rabbitpox virus (RPXV) for poxvirus countermeasure evaluation. Lethality of RPXV was demonstrated in both 9 and 16-weeks old rabbits with an LD50 < 10 PFU. The natural history of RPXV infection was documented in both ages of rabbits by monitoring the time to onset of abnormal values in clinical data at a lethal challenge of 300 PFU. All infected animals became viremic, developed a fever, exhibited weight loss, developed secondary lesions, and were euthanized after 7 or 8 days. The 16-weeks RPXV-infected animals exhibiting similar clinical signs with euthanasia applied about a day later than for 9-weeks old rabbits. For all animals, the first two unambiguous indicators of infection were detection of viral copies by quantitative polymerase chain reaction and fever at 2 and 3 days following challenge, respectively. These biomarkers provide clinically-relevant trigger(s) for initiating therapy. The major advantage for using 16-weeks NZW rabbits is that older rabbits were more robust and less subject to stress-induced death allowing more reproducible studies.

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. Mice were inoculated intranasally with a high lethal dose (125 PFU) of ECTV, resulting in complete mortality 10 days after infection. Decreases in weight and temperature from baseline were observed eight to nine days following infection. Viral titers via quantitative polymerase chain reaction (qPCR) and plaque assay were first observed in the blood at 4.5 days post-infection and in tissue (spleen and liver) at 3.5 days post-infection. Adverse clinical signs of disease were first observed four and five days post-infection, with severe signs occurring on day 7. Pathological changes consistent with ECTV infection were first observed five days after infection. Examination of data obtained from these parameters suggests the ECTV BALB/c model is suitable for potential use in medical countermeasures (MCMs) development and efficacy testing.

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. Deletion of either A33R or B5R resulted in viruses with a small plaque phenotype and reduced virus yields, as reported previously, whereas deletion of both EV protein-encoding genes resulted in a virus that formed small infection foci that were detectable and quantifiable only by immunostaining and an even more dramatic decrease in total virus yield in cell culture. Deletion of B5R, either as a single gene knockout or in the double EV gene knockout virus, resulted in a loss of EV neutralizing activity, but all EV gene knockout viruses still induced a robust neutralizing activity against the vaccinia MV form of the virus. The effect of elimination of A33 and/or B5 on the protection afforded by vaccination was evaluated by intranasal challenge with a lethal dose of either vaccinia virus WR or IHD-J, a strain of vaccinia virus that produces relatively higher amounts of EV virus. The results from multiple experiments, using a range of vaccination doses and virus challenge doses, and using mortality, morbidity, and virus dissemination as endpoints, indicate that the absence of A33 and B5 have little effect on the ability of a vaccinia vaccine virus to provide protection against a lethal intranasal challenge in a mouse model.

ABSTRACT 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. Bioluminescence recorded daily showed that pretreatment with VIGIV (30 mg) or with rVIG (100 μg) on day −2 protected mice from death but did not prevent viral replication at the site of inoculation and dissemination to internal organs. Compositions containing HuMAbs against MV or EV proteins were protective in both infection models at 100 μg per animal, but at 30 μg, only anti-EV antibodies conferred protection. Importantly, the t statistic of the mean total fluxes revealed that viral loads in surviving mice were significantly reduced in at least 3 sites for 3 consecutive days (days 3 to 5) postchallenge, while significant reduction for 1 or 2 days in any individual site did not confer protection. Our data suggest that reduction of viral replication at multiple sites, including respiratory tract, spleen, and liver, as monitored by whole-body bioluminescence can be used to predict the effectiveness of passive immunotherapies in mouse models.

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 1/2a, 1/2a and 2a HCVcc after blocking or affinity elution of specific antibodies. Mice and chimpanzees were immunized with genotype la (H77) HCV gpE1E2; all samples contained cross-neutralizing antibody against HCVcc. By functionally dissecting the polyclonal immune responses we identified three new regions important for neutralization within E1 (aa264-318) and E2 (aa448-483 and aa496-515) of the HCV glycoproteins, the third of which (aa496-515) is highly conserved (85-95%) amongst genotypes. Antibodies to aa496-515 were isolated by affinity binding and elution from the serum of a vaccinated chimpanzee and found to specifically neutralize chimeric 1/2a, 1/2a and 2a HCVcc. IC50 titres (IgG ng/mL) for the aa496-515 eluate were calculated as 142.1, 239.37 and 487.62 against 1a/2a, 1b/2a and 2a HCVcc, respectively. Further analysis demonstrated that although antibody to this new, conserved neutralization epitope is efficiently induced with recombinant proteins in mice and chimpanzees; it is poorly induced during natural infection in patients and chimpanzees (7 out of 68 samples positive) suggesting the epitope is poorly presented to the immune system in the context of the viral particle. These findings have important implications for the development of HCV vaccines and strategies designed to protect against heterologous viruses. The data also suggest that recombinant or synthetic antigens may be more efficient at inducing neutralizing antibodies to certain epitopes and that screening virally infected patients may not be the best approach for finding new cross-reactive epitopes. Published by Elsevier Ltd.

Whole body bioluminescence imaging was employed to test the effects of IgG-based therapies and Cidofovir on lethality and virus dissemination in BALB/c mice challenged intranasally with 10^5 pfu of recombinant vaccinia viruses (VV) expressing luciferase, WRvFire or IHDJ-Luc. Both viruses induced lethality within 6-8 days, with slightly different kinetics of replication in internal organs. Using statistical analysis of 80 animals/group we determined that replication of IHDJ-Luc (increased Extracellular Enveloped Virion compared to WR) rose faster and peaked earlier than WRvFire (predominantly Intracellular Mature Virion) in the liver and spleen. In the nasal cavity, WRvFire persisted at significantly higher levels than IHDJ-Luc. Vaccinia immunoglobulin for intravenous injection (VIGIV) administered at day -2 protected 100% of animals from WRvFire and IHDJ-Luc infections. Sym002 (26 anti-vaccinia human mAbs) completely protected mice from both challenges at 300-fold lower dose than VIGIV. A single treatment of Cidofovir (100 mg/kg) at day 1, 2, or 3 p.i. fully protected mice from WRvFire challenge. Complete protection conferred by treatments correlated with significant reductions in bioluminescence in nasal cavity or lungs and in both the liver and spleen between days 1-5. Our data demonstrated that despite some differences observed in the kinetics of VV dissemination, infections with these viruses were similarly susceptible to prophylactic and therapeutic treatments.

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. We found that a prime/boost immunization regimen with rA27 protein and MVA, in either sequence order, conferred protection to mice challenged with a lethal dose of vaccinia virus strain Western Reserve (VV-WR), compared to no protection after immunizations with a similar dose of either MVA or rA27 alone. Moreover, protection was achieved in mice primed simultaneously with combination of both MVA and rA27 in different vaccination routes, without any boost, even though MVA or rA27 alone at the same dose gave no protection. These findings show that rA27 can synergize with MVA to elicit robust protection that has a dose-sparing effect on MVA and can accelerate protection by eliminating the need for a booster dose.

Protection from lethal challenge with recombinant WR-Luciferase (TK+) and IHDJ-Luciferase (TK+) vaccinia viruses (VV) was studied by detecting firefly luciferase activity in live Balb/c mice using IVIS technology. High correlation (R2=0.8-0.9) was observed between viral loads in the organs measured by plaque assay and bioluminescence. WR- and IHDJ VV that generate predominantly IMV and EEV forms, respectively, induced lethality within 8-10 days after intranasal challenge with 10^5 pfu yet their dissemination and susceptibility to therapies differed. WR persisted at significantly higher levels than IHDJ at the site of challenge, while IHDJ replicated better in the liver in agreement with the roles of EEV and IMV in host dissemination and inter host transmission. Vaccinia immunoglobulin (VIGIV) at Day -2 completely protected from the WR but not from IHDJ challenge. Sym002, a mixture of 26 human anti-vaccinia mAbs (Symphogen, Denmark), completely protected from WR challenge at 1000 lower dose than VIGIV, but only partially protected from IHDJ. Protection by Sym002 correlated with a significantly reduced flux at the nasopharyngeal area and in the liver. Importantly, our study detected significantly higher bioluminescence at the nasopharyngeal area and in the lungs in non-survivors than in survivors suggesting that bioimaging can substitute for lethality endpoint for evaluation of anti-smallpox treatments.

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. This was identified as the vaccinia virus complement protein (VCP), which migrated more slowly in LC16m8-infected cells due to post-translational glycosylation. Vaccinia virus deleted in VCP, vVCPko, protected mice from a lethal intranasal challenge of vaccinia Western Reserve strain. Mice vaccinated with purified VCP demonstrated a strong humoral response, but were not protected against a moderate lethal challenge of vaccinia virus, suggesting that the humoral response against VCP is not critical for protection.

ABSTRACT 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. Mice inoculated with LC16m8 had detectable but low levels of anti-B5 IgG compared to Dryvax, but both Dryvax and LC16m8 sera neutralized vaccinia virus EV in vitro. A truncated B5 protein (∼8 kDa) was expressed abundantly in LC16m8-infected cells, and both murine immune sera and human vaccinia virus immunoglobulin recognized the truncated recombinant B5 protein in antigen-specific enzyme-linked immunosorbent assays. At a high-dose intranasal challenge (100 or 250 50% lethal doses), LC16m8 and Dryvax conferred similar levels of protection against vaccinia virus strain WR postvaccination. Taken together, the results extend our current understanding of the protective immune responses elicited by LC16m8 and indicate that the relative efficacy in a mouse model rivals that of previously licensed smallpox vaccines.

ABSTRACT 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. The EV-specific ELISAs were used to evaluate the EV humoral response elicited by Dryvax and the nonreplicating modified vaccinia virus Ankara (MVA) in mouse vaccination experiments comparing doses and routes of vaccination. Quantitatively similar titers of antibodies against EV antigens A33R, A56R, and B5R were measured in mice vaccinated with Dryvax and MVA when MVA was administered at a dose of 108 plaque-forming units. Further, a substantial increase in the EV-specific antibody response was induced in mice inoculated with MVA by using a prime-boost schedule. Finally, we investigated the abilities of the EV-expressing rSFV vectors to elicit the production of polyclonal monospecific antisera against the corresponding EV proteins in mice. The monospecific serum antibody levels against A33R, A56R, and B5R were measurably higher than the antibody levels induced by Dryvax. The resulting polyclonal antisera were used in Western blot analysis and immunofluorescence assays, indicating that rSFV particles are useful vectors for generating monospecific antisera.

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. Using a recombinant A27 protein (rA27), we confirmed that A27 contains neutralizing determinants and that vaccinia immune globulin (VIG) derived from Dryvax recipients contains reactivity to A27. However, VIG neutralization was not significantly reduced when A27 antibodies were removed, and antibodies elicited by an rA27 enhanced the protection conferred by VIG in passive transfer experiments. These findings demonstrate that A27 antibodies do not represent the major fraction of neutralizing activity in VIG and suggest that immunity may be augmented by vaccines and immune globulins that include strong antibody responses to A27.

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. To study genetic stability of an immunoessential vaccinia virus gene in a new generation smallpox vaccine, an advanced oligonucleotide microchip was developed and used to assay for mutations that could emerge in B5R gene, a vaccinia virus gene encoding for a protein that contains very important neutralizing epitopes. This microarray contained overlapping oligonucleotides covering the B5R gene of modified vaccinia virus Ankara (MVA), a well‐studied candidate smallpox vaccine. The microarray assay was shown to be able to detect even a single point mutation, and to differentiate between vaccinia strains. At the same time, it could detect newly emerged mutations in clones of vaccinia strains. In the work described here, it was shown that MVA B5R gene was stable after 34 passages in Vero and MRC‐5 cells that were proposed for use as cell substrates for vaccine manufacture. Potentially, the proposed method could be used as an identity test and could be extended for the entire viral genome and used to monitor consistency of vaccine production. J. Med. Virol. 79: 791–802, 2007. © 2007 Wiley‐Liss, Inc.