Nonhuman Primates Are Protected against Marburg Virus Disease by Vaccination with a Vesicular Stomatitis Virus Vector-Based Vaccine Prepared under Conditions to Allow Advancement to Human Clinical Trials

Vaccines are needed to disrupt or prevent continued outbreaks of filoviruses in humans across Western and Central Africa, including outbreaks of Marburg virus (MARV). As part of a filovirus vaccine product development plan, it is important to investigate dose response early in preclinical development to identify the dose range that may be optimal for safety, immunogenicity, and efficacy, and perhaps demonstrate that using lower doses is feasible, which will improve product access. To determine the efficacious dose range for a manufacturing-ready live recombinant vesicular stomatitis virus vaccine vector (rVSV∆G-MARV-GP) encoding the MARV glycoprotein (GP), a dose-range study was conducted in cynomolgus macaques. Results showed that a single intramuscular injection with as little as 200 plaque-forming units (PFUs) was 100% efficacious against lethality and prevented development of viremia and clinical pathologies associated with MARV Angola infection. Across the vaccine doses tested, there was nearly a 2000-fold range of anti-MARV glycoprotein (GP) serum IgG titers with seroconversion detectable even at the lowest doses. Virus-neutralizing serum antibodies also were detected in animals vaccinated with the higher vaccine doses indicating that vaccination induced functional antibodies, but that the assay was a less sensitive indicator of seroconversion. Collectively, the data indicates that a relatively wide range of anti-GP serum IgG titers are observed in animals that are protected from disease implying that seroconversion is positively associated with efficacy, but that more extensive immunologic analyses on samples collected from our study as well as future preclinical studies will be valuable in identifying additional immune responses correlated with protection that can serve as markers to monitor in human trials needed to generate data that can support vaccine licensure in the future

Genetic layout of the rVSV-EnvG₄-G₆ vector, HIV-1 Env, and the EnvG insert.

<p>VSV genes are shown in the 3′ to 5′ orientation as ordered on the recombinant genomic VSV plasmid and are not to scale. Arrows below each VSV gene depict the diminishing 3′-to-5′ mRNA transcription gradient. ss: signal sequence. MPER: membrane proximal external region. TM: transmembrane domain. CT: cytoplasmic tail domain. Star denotes site of intracellular Env(G) cleavage by furin.</p

Fusogenicity and Functionality of EnvG.

<p>(a) 10⁷ 293T cells were transfected with pEnvG or empty vector using Mirus Trans-IT 293 according to manufacturer's protocol. 48 h post-transfection, 293T cells were overlaid with 2×10⁶ CD4+CCR5+ GHOST cells. 48 h after overlay, cells were visualized under light microscope and images were captured. (b) 10⁶ CD4+CCR5+ GHOST cells were infected as above after pre-incubation with anti-VSV-G (Vi10) and/or anti-Env cocktail. After infection, Vi10 and/or anti-Env cocktail was included in the culture media. Eight hours post infection, cells were visualized under light microscope and images were captured.</p

Effect of IN rVSV-EnvG₄-G₆ adminstration.

<p>(a) Mean body weights and temperatures (b) of inoculated mice. (c) Mean copy numbers of VSV N genomic RNA or (d) VSV N mRNA per mg of indicated tissue or mL of blood. N = 4–12, dependent on study day. Fresh tissue specimens were homogenized, clarified by centrifugation and supernatants were subjected to RNA extraction and qPCR. All samples were tested in duplicate. Dotted lines indicate limits of detection. SEM is shown. *<i>p</i><0.05 for comparison of rVSV-EnvG₄-G₆ to rVSV-G₄. All PBS and rVSV-EnvG₄-ΔG values were found to be significantly lower than rVSV-EnvG₄-G₆ and rVSV-G₄ values.</p

rVSV-EnvG₄-G₆ characterization.

<p>(a) After a 24 hr infection, total infected Vero cell lysates were collected and proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE). Blot was probed with anti-VSV-N and anti-VSV-G_IN CT, which does not recognize G_NJ. IN: rVSV-EnvG₄-G₆^IN; NJ: rVSV-EnvG₄-G₆^NJ. (b-d) Sucrose-gradient purified rVSV-EnvG₄-G₆ particles were separated by SDS-PAGE. (b) Blots of 10⁶ pfu of rVSV-EnvG₄-G₆^IN and 2.5×10⁶ pfu rVSV-EnvG₄-G₆^NJ vectors were probed separately for EnvG using anti-gp120. (c) Blot of 10⁶ pfu rVSV-EnvG₄-G₆^IN was probed with anti-VSV-G^IN CT. (d) Blot of 10⁶ pfu rVSV-EnvG₄-G₆^IN and rVSV-EnvG₄-G₆^NJ was probed with anti-VSV-G. (e) Replication kinetics of rVSV-EnvG₄-G₆ and rVSV-G₄ viruses in Vero cells. Vero cells were infected in 6-well plates at an MOI of 0.1. At various intervals post infection, supernatant was collected from duplicate wells and virus was titrated. Plaques were counted manually after cell fixation.</p

Serum anti-Env IgG antibody responses.

<p>(a) Mean anti-Env endpoint titers (4 animals/group) against JR-FL foldon trimer were determined over the course of the 11 week experiment as described previously.(b) Week 6 endpoint titers for mice primed with pEnvG/pIL-12 (group 1/2/3, N = 12), IN rVSV-EnvG₄-G₆ (group 4/5, N = 8), or IM rVSV-EnvG₄-G₆ (group 6/7, N = 8). *p<0.05 compared to group 1/2/3. (c) Week 8 endpoint titers for all groups. (d) Week 11 endpoint titers for all groups. *p<0.05 compared to group 1. ^#p<0.05 compared to group 2. ⁺p<0.05 compared to group 3. ∧p<0.05 compared to group 4. ^∇p<0.05 compared to group 5. (e) Ratio of IgG2a to IgG1endpoint titers at week 11. (f) Neutralization of HIV-1 virus pseudo-typed with SF162.LS Env as measured in a standard TZM-bl neutralization assay using IgG purified from week 11 sera of selected groups. SEM is shown.</p

Frequency of JR-FL Env-specific CD4+ T cell responses in the lung and spleen.

<p>1.5×10⁶ leukocytes isolated from the lungs (a, b) andspleen (c, d) at the end of the study were stimulated <i>ex vivo</i> with JR-FL gp140, anti-CD28 and brefeldin A before being analyzed by flow cytometry for production of IFNg, IL-2 and TNFa. The total frequency of cytokine secreting CD4+ T cells (%) producing IFNγ, IL-2 or TNFα are shown on the left (a, c) and the frequency of multifunctional CD4+ T cells producing any combination of IFN γ +, IL-2+, and/or TNFα+ on the right (b, d). *p<0.05 compared to group 1. ^#p<0.05 compared to group 2. ∧p<0.05 compared to group 4. ^∇p<0.05 compared to group 5. ^@p<0.05 compared to group 6. ^xp<0.05 compared to group 7. Bars represent the median (with SEM), individual animals are shown.</p

Murine Immunization Regimens.

<p>* Groups 1–3 were primed twice, at weeks 0 and 3; groups 4–7 were primed once at week 3.</p><p>∧All groups were boosted at week 6. <b>IN</b>: Intranasal; <b>IM</b>: Intramuscular. <i>Superscript</i> IN/NJ denotes strain of rVSV used.</p><p>Murine Immunization Regimens.</p