BACKGROUND:Lassa fever, caused by Lassa virus, is a severe disease, endemic in Western Africa, for which no vaccines or therapeutics are yet approved. Understanding the immune responses elicited by candidate vaccines is key for approval, including characterisation of antibody epitopes recognised and capacity for neutralisation. METHODS:Here we used negative-stain electron microscopy polyclonal antibody epitope mapping (EMPEM), in-vitro pseudovirus neutralisation assays, and biophysical antibody competition assays to uncover components of polyclonal antibody responses elicited in nonhuman primates 26 days after receipt of a single immunisation with a fully protective, recombinant, replication-competent vesicular stomatitis virus-based vaccine bearing the Lassa virus glycoprotein GPC. FINDINGS:Although the vaccinee sera are overall poorly-neutralising, we do directly visualise, within the polyclonal pool, antibodies targeting epitopes on GPC that are consistent with neutralisation, as well as competition with known neutralising mAbs. Nearly every animal, for example, produced antibodies that compete with mAbs against GP1-A and GPC-A neutralising epitopes. The most abundant classes of antibodies, however, are directed against interior interfaces of GPC, while other antibodies recognise post-fusion GPC epitopes not consistent with neutralisation. INTERPRETATION:It may be that some individual antibodies in the pool are neutralising, but that the abundance of non-neutralising epitopes reduces potency as measured at the polyclonal level. The finding, however, neutralisation-consistent sites and competition with known neutralising antibodies are important steps in vaccine design toward eliciting more potent neutralisation. FUNDING:A complete list of funding bodies that supported this study is presented in the Funding section.

Summary: Background: Lassa fever (LF) is a zoonotic haemorrhagic disease caused by Lassa virus (LASV), which is endemic in West African countries. The multimammate rat is the main animal reservoir and its geographic range is expected to expand due to influences like climate change and land usage, and this will place larger parts of Africa at risk. We conducted preclinical development on a promising experimental vaccine that allowed its advancement into human trials. Methods: The LF vaccine is based on a vesicular stomatitis virus (VSV) vector in which the VSV glycoprotein (G) was replaced with the LASV glycoprotein complex (GPC). Earlier studies showed that this vaccine (VSVΔG-LASV-GPC) was efficacious in macaques, thus we regenerated VSVΔG-LASV-GPC using laboratory and documentation practices required to support vaccine manufacturing and human trials. The efficacy of the clinical vaccine candidate was assessed in cynomolgus macaques and more extensive immunologic analysis was performed than previously to investigate immune responses associated with protection. Findings: A single VSVΔG-LASV-GPC vaccination elicited innate, humoural and cellular immune responses, prevented development of substantial LASV viraemia, and protected animals from disease. Vaccinated macaques developed polyfunctional antibodies and serum was shown to neutralize virus expressing GPCs representative of geographically diverse LASV lineages. Interpretation: The VSVΔG-LASV-GPC clinical candidate elicited immunity that protected 10 of 10 vaccinated macaques from disease supporting its use in a clinical development program, which recently progressed to phase 2 clinical trials. Moreover, immunologic analysis showed that virus-neutralizing serum antibodies likely played a role in preventing LASV disease in vaccinated macaques. Funding: This work was supported by the Coalition for Epidemic Preparedness Innovations (CEPI), The National Institute of Allergy and Infectious Diseases (NIAID)/National Institutes of Health (NIH), The Bill and Melinda Gates Global Vaccine Accelerator Program, the Burroughs Wellcome Fund, and financial gifts and support by Nancy Zimmerman, Mark and Lisa Schwartz, and Terry and Susan Ragon.

BackgroundTo investigate a vaccine technology with potential to protect against coronavirus disease 2019 (COVID-19) and reduce transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with a single vaccine dose, we developed a SARS-CoV-2 candidate vaccine using the live vesicular stomatitis virus (VSV) chimeric virus approach previously used to develop a licensed Ebola virus vaccine.MethodsWe generated a replication-competent chimeric VSV-SARS-CoV-2 vaccine candidate by replacing the VSV glycoprotein (G) gene with coding sequence for the SARS-CoV-2 Spike glycoprotein (S). Immunogenicity of the lead vaccine candidate (VSV∆G-SARS-CoV-2) was evaluated in cotton rats and golden Syrian hamsters, and protection from SARS-CoV-2 infection also was assessed in hamsters.FindingsVSV∆G-SARS-CoV-2 delivered with a single intramuscular (IM) injection was immunogenic in cotton rats and hamsters and protected hamsters from weight loss following SARS-CoV-2 challenge. When mucosal vaccination was evaluated, cotton rats did not respond to the vaccine, whereas mucosal administration of VSV∆G-SARS-CoV-2 was found to be more immunogenic than IM injection in hamsters and induced immunity that significantly reduced SARS-CoV-2 challenge virus loads in both lung and nasal tissues.InterpretationVSV∆G-SARS-CoV-2 delivered by IM injection or mucosal administration was immunogenic in golden Syrian hamsters, and both vaccination methods effectively protected the lung from SARS-CoV-2 infection. Hamsters vaccinated by mucosal application of VSV∆G-SARS-CoV-2 also developed immunity that controlled SARS-CoV-2 replication in nasal tissue.FundingThe study was funded by Merck Sharp & Dohme, Corp., a subsidiary of Merck & Co., Inc., Rahway, NJ, USA, and The International AIDS Vaccine Initiative, Inc. (IAVI), New York, USA. Parts of this research was supported by the Biomedical Advanced Research and Development Authority (BARDA) and the Defense Threat Reduction Agency (DTRA) of the US Department of Defense.

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.

The structure of the HIV-1 envelope membrane-proximal external region (MPER) is influenced by its association with the lipid bilayer on the surface of virus particles and infected cells. To develop a replicating vaccine vector displaying MPER sequences in association with membrane, Env epitopes recognized by the broadly neutralizing antibodies 2F5, 4E10, or both were grafted into the membrane-proximal stem region of the vesicular stomatitis virus (VSV) glycoprotein (G). VSV encoding functional G-MPER chimeras based on G from the Indiana or New Jersey serotype propagated efficiently, although grafting of both epitopes (G-2F5-4E10) modestly reduced replication and resulted in the acquisition of one to two adaptive mutations in the grafted MPER sequence. Monoclonal antibodies 2F5 and 4E10 efficiently neutralized VSV G-MPER vectors and bound to virus particles in solution, indicating that the epitopes were accessible in the preattachment form of the G-MPER chimeras. Overall, our results showed that the HIV Env MPER could functionally substitute for the VSV G-stem region implying that both perform similar functions even though they are from unrelated viruses. Furthermore, we found that the MPER sequence grafts induced low but detectable MPER-specific antibody responses in rabbits vaccinated with live VSV, although additional vector and immunogen modifications or use of a heterologous prime-boost vaccination regimen will be required to increase the magnitude of the immune response.

We are investigating canine distemper virus (CDV) as a vaccine vector for the delivery of HIV envelope (Env) that closely resembles the native trimeric spike. We selected CDV because it will promote vaccine delivery to lymphoid tissues, and because human exposure is infrequent, reducing potential effects of pre-existing immunity. Using SIV Env as a model, we tested a number of vector and gene insert designs. Vectors containing a gene inserted between the CDV H and L genes, which encoded Env lacking most of its cytoplasmic tail, propagated efficiently in Vero cells, expressed the immunogen on the cell surface, and incorporated the SIV glycoprotein into progeny virus particles. When ferrets were vaccinated intranasally, there were no signs of distress, vector replication was observed in the gut-associated lymphoid tissues, and the animals produced anti-SIV Env antibodies. These data show that live CDV-SIV Env vectors can safely induce anti-Env immune responses following intranasal vaccination.

Results from recent HIV-1 vaccine studies have indicated that high serum antibody (Ab) titers may not be necessary for Ab-mediated protection, and that Abs localized to mucosal sites might be critical for preventing infection. Enzyme-linked immunosorbent assay (ELISA) has been used for decades as the gold standard for Ab measurement, though recently, highly sensitive microsphere-based assays have become available, with potential utility for improved detection of Abs. In this study, we assessed the Bio-Plex (R) Suspension Array System for the detection of simian immunodeficiency virus (SIV)-specific Abs in rhesus macaques (RMs) chronically infected with SIV, whose serum or mucosal SIV-specific Ab titers were negative by ELISA. We developed a SIVmac239-specific 4-plex bead array for the simultaneous detection of Abs binding to Env, Gag, Pol, and Nef. The 4-plex assay was used to quantify SIV-specific serum IgG and rectal swab IgA titers from control (SIV-naive) and SIVmac239-infected RMs. The Bio-Plex assay specifically detected anti-SIV Abs in specimens from SIV-infected animals for all four analytes when compared to SIV-naive control samples (p <= 0.04). Furthermore, in 70% of Env and 79% of Gag ELISA-negative serum samples, specific Ab was detected using the Bio-Plex assay. Similarly, 71% of Env and 48% of Gag ELISA-negative rectal swab samples were identified as positive using the Bio-Plex assay. Importantly, assay specificity (i.e., probability of true positives) was comparable to ELISA (94%-100%). The results reported here indicate that microsphere-based methods provide a substantial improvement over ELISA for the detection of Ab responses, aid in detecting specific Abs when analyzing samples containing low levels of Abs, such as during the early stages of a vaccine trial, and may be valuable in attempts to link protective efficacy of vaccines with induced Ab responses.

DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent "blips" in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication.

ABSTRACT Mature dendritic cells (DCs) are potent antigen-presenting cells essential for initiating successful antiviral immune responses and would therefore serve as an ideal target for viruses seeking to evade or delay the immune response by disrupting their function. We have previously reported that VZV productively infects immature DCs (A. Abendroth, G. Morrow, A. L. Cunningham, and B. Slobedman, J. Virol. 75:6183-6192, 2001), and in the present study we assessed the ability of VZV to infect mature DCs. Mature DCs were generated from immature monocyte-derived DCs by lipopolysaccharide treatment before being exposed to VZV-infected fibroblasts. On day 4 postexposure, flow cytometry analysis revealed that 15 to 45% of mature DCs were VZV antigen positive, and immunofluorescent staining together with infectious-center assays demonstrated that these cells were fully permissive for the complete VZV replicative cycle. VZV infection of mature DCs resulted in a selective downregulation of cell surface expression of the functionally important immune molecules major histocompatibility complex (MHC) class I, CD80, CD83, and CD86 but did not alter MHC class II expression. Immunofluorescent staining showed that the downregulation of cell surface CD83 was concomitant with a retention of CD83 in cytoplasmic vesicles. Importantly, VZV infection of mature DCs significantly reduced their ability to stimulate the proliferation of allogeneic T lymphocytes. These data demonstrate that mature DCs are permissive for VZV and that infection of these cells reduces their ability to function properly. Thus, VZV has evolved yet another immune evasion strategy that would likely impair immunosurveillance and enhance the chances for lifelong persistence in the human population.

ABSTRACT During primary varicella-zoster virus (VZV) infection, it is presumed that virus is transmitted from mucosal sites to regional lymph nodes, where T cells become infected. The cell type responsible for VZV transport from the mucosa to the lymph nodes has not been defined. In this study, we assessed the susceptibility of human monocyte-derived dendritic cells to infection with VZV. Dendritic cells were inoculated with the VZV strain Schenke and assessed by flow cytometry for VZV and dendritic cell (CD1a) antigen expression. In five replicate experiments, 34.4% ± 6.6% (mean ± SEM) of CD1a+ cells were also VZV antigen positive. Dendritic cells were also shown to be susceptible to VZV infection by the detection of immediate-early (IE62), early (ORF29), and late (gC) gene products in CD1a+dendritic cells. Infectious virus was recovered from infected dendritic cells, and cell-to-cell contact was required for transmission of virus to permissive fibroblasts. VZV-infected dendritic cells showed no significant decrease in cell viability or evidence of apoptosis and did not exhibit altered cell surface levels of major histocompatibility complex (MHC) class I, MHC class II, CD86, CD40, or CD1a. Significantly, when autologous T lymphocytes were incubated with VZV-infected dendritic cells, VZV antigens were readily detected in CD3+ T lymphocytes and infectious virus was recovered from these cells. These data provide the first evidence that dendritic cells are permissive to VZV and that dendritic cell infection can lead to transmission of virus to T lymphocytes. These findings have implications for our understanding of how virus may be disseminated during primary VZV infection.