Peter Silvera

Phase 1 Trials of rVSV Ebola Vaccine in Africa and Europe.

BACKGROUND The replication-competent recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing a Zaire ebolavirus (ZEBOV) glycoprotein was selected for rapid safety and immunogenicity testing before its use in West Africa. METHODS We performed three open-label, dose-escalation phase 1 trials and one randomized, double-blind, controlled phase 1 trial to assess the safety, side-effect profile, and immunogenicity of rVSV-ZEBOV at various doses in 158 healthy adults in Europe and Africa. All participants were injected with doses of vaccine ranging from 300,000 to 50 million plaque-forming units (PFU) or placebo. RESULTS No serious vaccine-related adverse events were reported. Mild-to-moderate early-onset reactogenicity was frequent but transient (median, 1 day). Fever was observed in up to 30% of vaccinees. Vaccine viremia was detected within 3 days in 123 of the 130 participants (95%) receiving 3 million PFU or more; rVSV was not detected in saliva or urine. In the second week after injection, arthritis affecting one to four joints developed in 11 of 51 participants (22%) in Geneva, with pain lasting a median of 8 days (interquartile range, 4 to 87); 2 self-limited cases occurred in 60 participants (3%) in Hamburg, Germany, and Kilifi, Kenya. The virus was identified in one synovial-fluid aspirate and in skin vesicles of 2 other vaccinees, showing peripheral viral replication in the second week after immunization. ZEBOV-glycoprotein-specific antibody responses were detected in all the participants, with similar glycoprotein-binding antibody titers but significantly higher neutralizing antibody titers at higher doses. Glycoprotein-binding antibody titers were sustained through 180 days in all participants. CONCLUSIONS In these studies, rVSV-ZEBOV was reactogenic but immunogenic after a single dose and warrants further evaluation for safety and efficacy. (Funded by the Wellcome Trust and others; ClinicalTrials.gov numbers, NCT02283099, NCT02287480, and NCT02296983; Pan African Clinical Trials Registry number, PACTR201411000919191.).

A Recombinant Vesicular Stomatitis Virus Ebola Vaccine - Preliminary Report.

Background The worst Ebola virus disease (EVD) outbreak in history has resulted in more than 28,000 cases and 11,000 deaths. We present the final results of two phase 1 trials of an attenuated, replication‐competent, recombinant vesicular stomatitis virus (rVSV)–based vaccine candidate designed to prevent EVD. Methods We conducted two phase 1, placebo‐controlled, double‐blind, dose‐escalation trials of an rVSV‐based vaccine candidate expressing the glycoprotein of a Zaire strain of Ebola virus (ZEBOV). A total of 39 adults at each site (78 participants in all) were consecutively enrolled into groups of 13. At each site, volunteers received one of three doses of the rVSV‐ZEBOV vaccine (3 million plaque‐forming units [PFU], 20 million PFU, or 100 million PFU) or placebo. Volunteers at one of the sites received a second dose at day 28. Safety and immunogenicity were assessed. Results The most common adverse events were injection‐site pain, fatigue, myalgia, and headache. Transient rVSV viremia was noted in all the vaccine recipients after dose 1. The rates of adverse events and viremia were lower after the second dose than after the first dose. By day 28, all the vaccine recipients had seroconversion as assessed by an enzyme‐linked immunosorbent assay (ELISA) against the glycoprotein of the ZEBOV‐Kikwit strain. At day 28, geometric mean titers of antibodies against ZEBOV glycoprotein were higher in the groups that received 20 million PFU or 100 million PFU than in the group that received 3 million PFU, as assessed by ELISA and by pseudovirion neutralization assay. A second dose at 28 days after dose 1 significantly increased antibody titers at day 56, but the effect was diminished at 6 months. Conclusions This Ebola vaccine candidate elicited anti‐Ebola antibody responses. After vaccination, rVSV viremia occurred frequently but was transient. These results support further evaluation of the vaccine dose of 20 million PFU for preexposure prophylaxis and suggest that a second dose may boost antibody responses. (Funded by the National Institutes of Health and others; rVSV&Dgr;G‐ZEBOV‐GP ClinicalTrials.gov numbers, NCT02269423 and NCT02280408.)

The effect of dose on the safety and immunogenicity of the VSV Ebola candidate vaccine: a randomised double-blind, placebo-controlled phase 1/2 trial

BACKGROUND Safe and effective vaccines against Ebola could prevent or control outbreaks. The safe use of replication-competent vaccines requires a careful dose-selection process. We report the first safety and immunogenicity results in volunteers receiving 3 × 10(5) plaque-forming units (pfu) of the recombinant vesicular stomatitis virus-based candidate vaccine expressing the Zaire Ebola virus glycoprotein (rVSV-ZEBOV; low-dose vaccinees) compared with 59 volunteers who had received 1 ×10(7) pfu (n=35) or 5 × 10(7) pfu (n=16) of rVSV-ZEBOV (high-dose vaccinees) or placebo (n=8) before a safety-driven study hold. METHODS The Geneva rVSV-ZEBOV study, an investigator-initiated phase 1/2, dose-finding, placebo-controlled, double-blind trial conducted at the University Hospitals of Geneva, Switzerland, enrolled non-pregnant, immunocompetent, and otherwise healthy adults aged 18-65 years. Participants from the low-dose group with no plans to deploy to Ebola-aff5cted regions (non-deployable) were randomised 9:1 in a double-blind fashion using randomly permuted blocks of varying sizes to a single injection of 3 × 10(5) pfu or placebo, whereas deployable participants received single-injection 3 × 10(5) pfu open-label. Primary safety and immunogenicity outcomes were the incidence of adverse events within 14 days of vaccination and day-28 antibody titres, respectively, analysed by intention to treat. After viral oligoarthritis was observed in 11 of the first 51 vaccinees (22%) receiving 10(7) or 5 × 10(7) pfu, 56 participants were given a lower dose (3 × 10(5) pfu, n=51) or placebo (n=5) to assess the effect of dose reduction on safety and immunogenicity. This trial is ongoing with a follow-up period of 12 months; all reported results are from interim databases. This study is registered with ClinicalTrials.gov, number NCT02287480. FINDINGS Between Jan 5 and Jan 26, 2015, 43 non-deployable participants received low-dose rVSV-ZEBOV (3 × 10(5) pfu) or placebo in a double-blind fashion, whereas 13 deployable participants received 3 × 10(5) pfu open-label. Altogether, in the low-dose group, 51 participants received rVSV-ZEBOV and five received placebo. No serious adverse events occurred. At 3 × 10(5) pfu, early-onset reactogenicity remained frequent (45 [88%] of 51 compared with 50 [98%] of 51 high dose and two [15%] of 13 placebo recipients), but mild. Objective fever was present in one (2%) of 51 low-dose versus 13 (25%) of 51 high-dose vaccinees receiving at least 1 ×10(7) pfu (p<0·0001). Subjective fever (p<0·0001), myalgia (p=0·036), and chills (p=0·026) were significantly reduced and their time of onset delayed, reflecting significantly lower viraemia (p<0·0001) and blood monocyte-activation patterns (p=0·0233). Although seropositivity rates remained similarly high (48 [94%] of 51), day-28 EBOV-glycoprotein-binding and neutralising antibody titres were lower in low-dose versus high-dose vaccinees (geometric mean titres 344·5 [95% CI 229·7-516·4] vs 1064·2 [757·6-1495·1]; p<0·0001; and 35·1 [24·7-50·7] vs 127·0 [86·0-187·6]; p<0·0001, respectively). Furthermore, oligoarthritis again occurred on day 10 (median; IQR 9-14) in 13 (25%) of 51 low-dose vaccinees, with maculopapular, vesicular dermatitis, or both in seven (54%) of 13; arthritis was associated with increasing age in low-dose but not high-dose vaccinees. Two vaccinees presented with purpura of the lower legs; histological findings indicated cutaneous vasculitis. The presence of rVSV in synovial fluid and skin lesions confirmed causality. INTERPRETATION Reducing the dose of rVSV-ZEBOV improved its early tolerability but lowered antibody responses and did not prevent vaccine-induced arthritis, dermatitis, or vasculitis. Like its efficacy, the safety of rVSV-ZEBOV requires further definition in the target populations of Africa. FUNDING Wellcome Trust through WHO.

Subunit Recombinant Vaccine Protects against Monkeypox

The smallpox vaccine Dryvax, a live vaccinia virus (VACV), protects against smallpox and monkeypox, but is contraindicated in immunocompromised individuals. Because Abs to VACV mediate protection, a live virus vaccine could be substituted by a safe subunit protein-based vaccine able to induce a protective Ab response. We immunized rhesus macaques with plasmid DNA encoding the monkeypox orthologs of the VACV L1R, A27L, A33R, and B5R proteins by the intradermal and i.m. routes, either alone or in combination with the equivalent recombinant proteins produced in Escherichia coli. Animals that received only DNA failed to produce high titer Abs, developed innumerable skin lesions after challenge, and died in a manner similar to placebo controls. By contrast, the animals vaccinated with proteins developed moderate to severe disease (20–155 skin lesions) but survived. Importantly, those immunized with DNA and boosted with proteins had mild disease with 15 or fewer lesions that resolved within days. DNA/protein immunization elicited Th responses and binding Ab titers to all four proteins that correlated negatively with the total lesion number. The sera of the immunized macaques recognized a limited number of linear B cell epitopes that are highly conserved among orthopoxviruses. Their identification may guide future efforts to develop simpler, safer, and more effective vaccines for monkeypox and smallpox.

Vaccination of Macaques with Long-Standing SIVmac251 Infection Lowers the Viral Set Point After Cessation of Antiretroviral Therapy

A cohort of rhesus macaques with long-standing SIVmac251 infection (≥5 mo) was treated with continuous antiretroviral therapy (ART). A group of eight macaques was vaccinated with or without simultaneous administration of low dose IL-2 with the highly attenuated poxvirus vector (NYVAC) vaccine candidate expressing the SIVmac structural gag-pol-env (gpe) genes and a novel chimeric fusion protein derived from the rev-tat-nef (rtn) regulatory genes. Control groups consisted of mock-vaccinated macaques or animals treated only with IL-2. Vaccination significantly expanded both virus-specific CD4+ and CD8+ T cell responses, and IL-2 further increased the vaccine-induced response to an immunodominant Gag epitope. Following antiretroviral treatment interruption, the viral set point was significantly lower in vaccinated than in control macaques for at least 4 consecutive mo, and viral containment was inversely correlated with vaccine-induced, virus-specific CD4+ and CD8+ T cell responses. These data provide the proof of concept that therapeutic vaccination before cessation of ART may be a feasible approach in the clinical management of HIV-1 infection.

Protection against simian/human immunodeficiency virus (SHIV) 89.6P in macaques after coimmunization with SHIV antigen and IL-15 plasmid

The cell-mediated immune profile induced by a recombinant DNA vaccine was assessed in the simian/HIV (SHIV) and macaque model. The vaccine strategy included coimmunization of a DNA-based vaccine alone or in combination with an optimized plasmid encoding macaque IL-15 (pmacIL-15). We observed strong induction of vaccine-specific IFN-γ-producing CD8+ and CD4+ effector T cells in the vaccination groups. Animals were subsequently challenged with 89.6p. The vaccine groups were protected from ongoing infection, and the IL-15 covaccinated group showed a more rapidly controlled infection than the group treated with DNA vaccine alone. Lymphocytes isolated from the group covaccinated with pmacIL-15 had higher cellular proliferative responses than lymphocytes isolated from the macaques that received SHIV DNA alone. Vaccine antigen activation of lymphocytes was also studied for a series of immunological molecules. Although mRNA for IFN-γ was up-regulated after antigen stimulation, the inflammatory molecules IL-8 and MMP-9 were down-regulated. These observed immune profiles are potentially reflective of the ability of the different groups to control SHIV replication. This study demonstrates that an optimized IL-15 immune adjuvant delivered with a DNA vaccine can impact the cellular immune profile in nonhuman primates and lead to enhanced suppression of viral replication.

Rapid protection in a monkeypox model by a single injection of a replication-deficient vaccinia virus

The success of the World Health Organization smallpox eradication program three decades ago resulted in termination of routine vaccination and consequent decline in population immunity. Despite concerns regarding the reintroduction of smallpox, there is little enthusiasm for large-scale redeployment of licensed live vaccinia virus vaccines because of medical contraindications and anticipated serious side effects. Therefore, highly attenuated strains such as modified vaccinia virus Ankara (MVA) are under evaluation in humans and animal models. Previous studies showed that priming and boosting with MVA provided protection for >2 years in a monkeypox virus challenge model. If variola virus were used as a biological weapon, however, the ability of a vaccine to quickly induce immunity would be essential. Here, we demonstrate more rapid immune responses after a single vaccination with MVA compared to the licensed Dryvax vaccine. To determine the kinetics of protection of the two vaccines, macaques were challenged intravenously with monkeypox virus at 4, 6, 10, and 30 days after immunization. At 6 or more days after vaccination with MVA or Dryvax, the monkeys were clinically protected (except for 1 of 16 animals vaccinated with MVA), although viral loads and number of skin lesions were generally higher in the MVA vaccinated group. With only 4 days between immunization and intravenous challenge, however, MVA still protected whereas Dryvax failed. Protection correlated with the more rapid immune response to MVA compared to Dryvax, which may be related to the higher dose of MVA that can be tolerated safely.

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.

Molecular smallpox vaccine delivered by alphavirus replicons elicits protective immunity in mice and non-human primates

Naturally occurring smallpox was eradicated as a result of successful vaccination campaigns during the 1960s and 1970s. 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 (VRPs) 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 x 10(6)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.

A protein-based smallpox vaccine protects non-human primates from a lethal monkeypox virus challenge

Concerns about infections caused by orthopoxviruses, such as variola and monkeypox viruses, drive ongoing efforts to develop novel smallpox vaccines that are both effective and safe to use in diverse populations. A subunit smallpox vaccine comprising vaccinia virus membrane proteins A33, B5, L1, A27 and aluminum hydroxide (alum) ± CpG was administered to non-human primates, which were subsequently challenged with a lethal intravenous dose of monkeypox virus. Alum adjuvanted vaccines provided only partial protection but the addition of CpG provided full protection that was associated with a more homogeneous antibody response and stronger IgG1 responses. These results indicate that it is feasible to develop a highly effective subunit vaccine against orthopoxvirus infections as a safer alternative to live vaccinia virus vaccination.