Efficacy of different AV7909 dose regimens in a nonclinical model of pulmonary anthrax.

Pulmonary anthrax caused by exposure to inhaled Bacillus anthracis, the most lethal form of anthrax disease, is a continued military and public health concern for the United States. The vaccine AV7909, consisting of the licensed anthrax drug substance AVA adjuvanted with CpG7909, induces high levels of toxin neutralizing antibodies in healthy adults using fewer doses than AVA. This study compares the ability of one- or two-dose regimens of AV7909 to induce a protective immune response in guinea pigs challenged with a lethal dose of aerosolized B. anthracis spores 6 weeks after the last vaccine dose. The results indicated that AV7909 was less effective when delivered as a single dose compared to the two-dose regimen that resulted in dose-dependent protection against death. The toxin neutralizing assay (TNA) titer and anti-PA IgG responses were proportional to the protective efficacy, with a 50% TNA neutralizing factor (NF50) greater than 0.1 associated with survival in animals receiving two doses of vaccine. The strong protection at relatively low TNA NF50 titers in this guinea pig model supports the exploration of lower doses in clinical trials to determine if these protective levels of neutralizing antibodies can be achieved in humans; however, protection with a single dose may not be feasible.

Protection of rabbits and immunodeficient mice against lethal poxvirus infections by human monoclonal antibodies.

Smallpox (variola virus) is a bioweapon concern. Monkeypox is a growing zoonotic poxvirus threat. These problems have resulted in extensive efforts to develop potential therapeutics that can prevent or treat potentially lethal poxvirus infections in humans. Monoclonal antibodies (mAbs) against smallpox are a conservative approach to this problem, as the licensed human smallpox vaccine (vaccinia virus, VACV) primarily works on the basis of protective antibody responses against smallpox. Fully human mAbs (hmAbs) against vaccinia H3 (H3L) and B5 (B5R), targeting both the mature virion (MV) and extracellular enveloped virion (EV) forms, have been developed as potential therapeutics for use in humans. Post-exposure prophylaxis was assessed in both murine and rabbit animal models. Therapeutic efficacy of the mAbs was assessed in three good laboratory practices (GLP) studies examining severe combined immunodeficiency mice (SCID) given a lethal VACV infection. Pre-exposure combination hmAb therapy provided significantly better protection against disease and death than either single hmAb or vaccinia immune globulin (VIG). Post-exposure combination mAb therapy provided significant protection against disease and death, and appeared to fully cure the VACV infection in ≥50% of SCID mice. Therapeutic efficacy was then assessed in two rabbit studies examining post-exposure hmAb prophylaxis against rabbitpox (RPXV). In the first study, rabbits were infected with RPVX and then provided hmAbs at 48 hrs post-infection, or 1 hr and 72 hrs post-infection. Rabbits in both groups receiving hmAbs were 100% protected from death. In the second rabbitpox study, 100% of animal treated with combination hmAb therapy and 100% of animals treated with anti-B5 hmAb were protected. These findings suggest that combination hmAb treatment may be effective at controlling smallpox disease in immunocompetent or immunodeficient humans.

Attenuated NYCBH vaccinia virus deleted for the E3L gene confers partial protection against lethal monkeypox virus disease in cynomolgus macaques.

The New York City Board of Health (NYCBH) vaccinia virus is the currently licensed vaccine for use in the US against smallpox. The vaccine under investigation in this study has been attenuated by deletion of the innate immune evasion gene, E3L, and shown to be protective in homologous virus mouse challenge and heterologous virus mouse and rabbit challenge models. In this study we compared NYCBH deleted for the E3L gene (NYCBHΔE3L) to NYCBH for the ability to induce phosphorylation of proinflammatory signaling proteins and the ability to protect cynomolgus macaques from heterologous challenge with monkeypox virus (MPXV). NYCBHΔE3L induced phosphorylation of PKR and eIF2α as well as p38, SAPK/JNK, and IRF3 which can lead to induction of proinflammatory gene transcription. Vaccination of macaques with two doses of NYCBHΔE3L resulted in negligible pock formation at the site of scarification in comparison to vaccination using a single dose of NYCBH, but still elicited neutralizing antibodies and protected 75% of the animals from mortality after challenge with MPXV. However, NYCBHΔE3L-vaccinated animals developed a high number of secondary skin lesions and blood viral load similar to that seen in unvaccinated controls. The NYCBHΔE3L-vaccinated animals that survived MPXV challenge were able to show resolution of blood viral load, a decrease in number of skin lesions, and an improved clinical score by three weeks post challenge. These results suggest that although the highly attenuated NYCBHΔE3L allows proinflammatory signal transduction to occur, it does not provide full protection against monkeypox challenge.

Multivalent smallpox DNA vaccine delivered by intradermal electroporation drives protective immunity in nonhuman primates against lethal monkeypox challenge.

The threat of a smallpox-based bioterrorist event or a human monkeypox outbreak has heightened the importance of new, safe vaccine approaches for these pathogens to complement older poxviral vaccine platforms. As poxviruses are large, complex viruses, they present technological challenges for simple recombinant vaccine development where a multicomponent mixtures of vaccine antigens are likely important in protection. We report that a synthetic, multivalent, highly concentrated, DNA vaccine delivered by a minimally invasive, novel skin electroporation microarray can drive polyvalent immunity in macaques, and offers protection from a highly pathogenic monkeypox challenge. Such a diverse, high-titer antibody response produced against 8 different DNA-encoded antigens delivered simultaneously in microvolumes has not been previously described. These studies represent a significant improvement in the efficiency of the DNA vaccine platform, resulting in immune responses that mimic live viral infections, and would likely have relevance for vaccine design against complex human and animal pathogens.

Depo-Provera does not alter disease progression in SIVmac-infected female Chinese rhesus macaques.

Depo-Provera (medroxyprogesterone acetate), a long-acting derivative of progesterone, is utilized during many nonhuman primate microbicide studies to facilitate simian immunodeficiency virus (SIV) infection by thinning the vaginal epithelium. To date, the systemic effects of this steroid hormone in regard to SIV/HIV pathogenesis are not well understood, but an increase in infection rates and lymphoproliferation following progesterone application has been reported. Therefore, a proactive study using 20 Chinese rhesus macaques was designed to investigate the effect of a single Depo-Provera injection on SIV disease progression. Group 1 (n = 10) was treated with 30 mg Depo-Provera intramuscularly 30 days prior to intravenous challenge with 50 TCID(50) SIVmac251, while Group 2 (n = 10) remained untreated, but received the same amount of SIV. Blood samples were taken at predetermined intervals to measure RNA viral loads, CD4(+), CD8(+), and CD20(+) lymphocyte counts and percentages and absolute numbers of naive and memory T lymphocytes. Upon statistical endpoint data analysis, none of the parameters measured were shown to be significantly different between the groups. One animal in the Depo-Provera-treated group and two macaques in the control group were euthanized prior to study end due to the development of clinical signs (in weeks 43 and 51, respectively). All other animals were euthanized between weeks 68 and 71 post-SIV infection. Histopathological evaluations revealed that 5 of 10 animals in each group had developed simian AIDS (SAIDS). In summary, this prospective study demonstrated that a single injection of 30 mg Depo-Provera did not have a significant influence on SIV disease progression.

Structure-antigenicity of the V3 region of SIVmac envelope glycoprotein.

The objective of this study was to analyze the immunogenicity and antigenicity of the V3 domain (Cys313-Cys346) of the external envelope glycoprotein gp125 of SIVmac251. The corresponding peptide was synthesized and characterized as linear and cyclic peptides. Our results showed that this region, as for HIV-1, contained an immunodominant epitope. The antigenicity was similar for the linear and cyclic peptides when tested against a panel of 15 sera from SIV infected macaques. Similarly, both peptide structures presented similar immunogenicity as shown by the characterization of the anti-peptide antibodies produced in rabbits against the cyclic and linear forms. But, unexpectedly, the antibodies produced against linear peptides recognized with a relatively higher intensity the native envelope gp140 than those produced against the cyclic structure. Furthermore, we showed that these antibodies recognized better the deglycosylated form of the glycoprotein. But, in contrast to the neutralizing activity obtained with anti-V3 peptides from HIV-1, no antiviral activity was obtained with antibodies generated against linear or cyclic SIVmac V3 peptides.

Progressive selection for neurovirulent genotypes in the brain of SIV-infected macaques.

OBJECTIVE: To compare the viral genotypes present in RNA from brain and peripheral blood mononuclear cells (PBMC) and DNA from brain during acute, asymptomatic and late stages of SIV infection of macaques. METHODS: Eighteen pigtailed macaques were intravenously inoculated with SIV. At 10, 21 and 56 days postinoculation, six were euthanized and the severity of encephalitis was assessed by microscopic examination. DNA and RNA were isolated from brain and PBMC, and the V1 region of env was amplified by the polymerase chain reaction and sequenced from over 800 different clones. RESULTS: Similar genotypes were detected in RNA from brain and PBMC at 10 days postinoculation, suggesting an unrestricted exchange of virus between the periphery and the brain during acute infection. There was a progressive increase in the percentage of neurovirulent genotypes in brain RNA from acute (14% of all genotypes detected in brain RNA) to early asymptomatic (45%), to late asymptomatic (52%) and to terminal (95%) infection. Fewer different genotypes were found in brain RNA than in PBMC RNA from macaques euthanized during early asymptomatic (2.5 and 5 different genotypes, respectively; P = 0.007), late asymptomatic (2 and 5 different genotypes, respectively; P = 0.003) and terminal (2 and 4 different genotypes, respectively; P < 0.001) infection. CONCLUSION: These data demonstrate that the almost exclusive replication of neurovirulent genotypes in the brain seen at late-stage infection is a progressive process that begins early in infection and continues to late stage disease.

CD4-independent entry and replication of simian immunodeficiency virus in primary rhesus macaque astrocytes are regulated by the transmembrane protein.

Previous studies have demonstrated that the genetic determinants of simian immunodeficiency virus (SIV) neurovirulence map to the env and nef genes. Recent studies from our laboratory demonstrated that SIV replication in primary rhesus macaque astrocyte cultures is dependent upon the nef gene. Here, we demonstrate that macrophage tropism is not sufficient for replication in astrocytes and that specific amino acids in the transmembrane (TM) portion of Env are also important for optimal SIV replication in astrocytes. Specifically, a Gly at amino acid position 751 and truncation of the cytoplasmic tail of TM are required for efficient replication in these cells. Studies using soluble CD4 demonstrated that these changes within the TM protein regulate CD4-independent, CCR5-dependent entry of virus into astrocytes. In addition, we observed that two distinct CD4-independent, neuroinvasive strains of SIV/DeltaB670 also replicated efficiently in astrocytes, further supporting the role of CD4 independence as an important determinant of SIV infection of astrocytes in vitro and in vivo.

Role of microglial cells in selective replication of simian immunodeficiency virus genotypes in the brain.

An accelerated, consistent macaque simian immunodeficiency virus (SIV) model in which over 90% of pigtailed macaques (Macaca nemestrina) coinoculated with SIV/17E-Fr and SIV/DeltaB670 developed encephalitis was used to determine whether central nervous system (CNS) lesions are associated with the replication of specific genotypes in the brain and, more specifically, in the microglia. Ten of 11 inoculated macaques had severe (n = 3), moderate (n = 5), or mild (n = 2) encephalitis at 3 months postinoculation. To compare actively replicating viral genotypes in the CNS and in microglia with those in the periphery, the V1 region of the SIV envelope gene was amplified and sequenced from RNA extracted from basal ganglia, from microglial cells isolated from the brain, and from peripheral blood mononuclear cells (PBMC) isolated from blood at the time of death. To distinguish between actively replicating with latent viral genotypes in the CNS, viral genotypes in RNA and DNA from basal ganglia were compared. Two macrophage-tropic, neurovirulent viruses, SIV/17E-Fr and SIV/DeltaB670 Cl-2, predominated in the brain RNA of macaques with encephalitis, comprising 95% of the genotypes detected. The same two viral genotypes were present at the same frequencies in microglial cell RNA, suggesting that microglia are pivotal in the selective replication of neurovirulent viruses. There was a significantly greater number of viral genotypes in DNA than there were in RNA in the brain (P = 0.004), including those of both the macrophage- and lymphocyte-tropic viral strains. Furthermore, significantly fewer viral genotypes were detected in brain RNA than in PBMC RNA at the time of death (P = 0.004) and the viral strain that predominated in the brain frequently was different from that which predominated in the PBMC of the same animal. These data suggest that many viral genotypes enter the brain, but only a limited subset of macrophage-tropic, neurovirulent viruses replicate terminally in the brains of macaques with encephalitis. They further suggest that the selection of macrophage-tropic, neurovirulent viruses occurs not at the level of the blood-brain barrier but at a stage after virus entry and that microglial cells may play an important role in that selection process.

The central nervous system as a reservoir for simian immunodeficiency virus (SIV): steady-state levels of SIV DNA in brain from acute through asymptomatic infection.

Latent reservoirs of human immunodeficiency virus (HIV) present significant challenges for eradicating HIV from infected persons, particularly reservoirs in the brain established during acute infection. A simian immunodeficiency virus (SIV)/macaque model of HIV dementia was used to show that viral DNA levels in the brain remained at constant levels from acute through asymptomatic infection, despite significant down-regulation of viral RNA in the brain after the acute phase of infection. Viral replication in the brain coincided with activation of macrophages and microglia in the central nervous system; down-regulation of viral replication coincided with increased infiltration of cytotoxic lymphocytes and reduced activation of macrophages and microglia in the brain. Comparison of viral genotypes in the central nervous system and peripheral blood mononuclear cells suggests that recrudescence of viral replication in brain occurs by reactivation of latent viral DNA. Latent virus in the brain must be considered in therapeutic strategies to eliminate HIV from infected persons.