The Development of a Rabies Virus-Vectored Vaccine against Borrelia burgdorferi, Targeting BBI39.

Lyme disease (LD) is the most common tick-borne illness in the United States (U.S.), Europe, and Asia. Borrelia burgdorferi, a spirochete bacterium transmitted by the tick vector Ixodes scapularis, causes LD in the U.S. If untreated, Lyme arthritis, heart block, and meningitis can occur. Given the absence of a human Lyme disease vaccine, we developed a vaccine using the rabies virus (RABV) vaccine vector BNSP333 and an outer surface borrelial protein, BBI39. BBI39 was previously utilized as a recombinant protein vaccine and was protective in challenge experiments; therefore, we decided to utilize this protective antigen in a rabies virus-vectored vaccine against Borrelia burgdorferi. To incorporate BBI39 into the RABV virion, we generated a chimeric BBI39 antigen, BBI39RVG, by fusing BBI39 with the final amino acids of the RABV glycoprotein by molecular cloning and viral recovery with reverse transcription genetics. Here, we have demonstrated that the BBI39RVG antigen was incorporated into the RABV virion via immunofluorescence and Western blot analysis. Mice vaccinated with our BPL inactivated RABV-BBI39RVG (BNSP333-BBI39RVG) vaccine induced high amounts of BBI39-specific antibodies, which were maintained long-term, up to eight months post-vaccination. The BBI39 antibodies neutralized Borrelia in vaccinated mice when challenged with Borrelia burgdorferi by either syringe injection or infected ticks and they reduced the Lyme disease pathology of arthritis in infected mouse joints. Overall, the RABV-based LD vaccine induced more and longer-term antibodies compared to the recombinant protein vaccine. This resulted in lower borrelial RNA in RABV-based vaccinated mice compared to recombinant protein vaccinated mice. The results of this study indicate the successful use of BBI39 as a vaccine antigen and RABV as a vaccine vector for LD.

GP38 as a vaccine target for Crimean-Congo hemorrhagic fever virus.

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a tick-borne virus that causes severe hemorrhagic disease in humans. There is a great need for effective vaccines and therapeutics against CCHFV for humans, as none are currently internationally approved. Recently, a monoclonal antibody against the GP38 glycoprotein protected mice against lethal CCHFV challenge. To show that GP38 is required and sufficient for protection against CCHFV, we used three inactivated rhabdoviral-based CCHFV-M vaccines, with or without GP38 in the presence or absence of the other CCHFV glycoproteins. All three vaccines elicited strong antibody responses against the respective CCHFV glycoproteins. However, only vaccines containing GP38 showed protection against CCHFV challenge in mice; vaccines without GP38 were not protective. The results of this study establish the need for GP38 in vaccines targeting CCHFV-M and demonstrate the efficacy of a CCHFV vaccine candidate based on an established vector platform.

Effects of adjuvants in a rabies-vectored Ebola virus vaccine on protection from surrogate challenge.

Ebola virus is the primary contributor to the global threat of filovirus severe hemorrhagic fever, and Ebola virus disease has a case fatality rate of 50-90%. An inactivated, bivalent filovirus/rabies virus vaccine, FILORAB1, consists of recombinant rabies virus virions expressing the Ebola virus glycoprotein. FILORAB1 is immunogenic and protective from Ebola virus challenge in mice and non-human primates, and protection is enhanced when formulated with toll-like receptor 4 agonist Glucopyranosyl lipid adjuvant (GLA) in a squalene oil-in-water emulsion (SE). Through an adjuvant comparison in mice, we demonstrate that GLA-SE improves FILORAB1 efficacy by activating the innate immune system and shaping a Th1-biased adaptive immune response. GLA-SE adjuvanted mice and those adjuvanted with the SE component are better protected from surrogate challenge, while Th2 alum adjuvanted mice are not. Additionally, the immune response to FILORAB1 is long-lasting, as exhibited by highly-maintained serum antibody titers and long-lived cells in the spleen and bone marrow.

Inactivated rabies-vectored SARS-CoV-2 vaccine provides long-term immune response unaffected by vector immunity.

The objective of this study is to further analyze recombinant rabies virus-vectored SARS-CoV-2 vaccine, CORAVAX, as an effective COVID-19 vaccine strategy. CORAVAX has proven immunogenic and protective against SARS-CoV-2 in animal models. Here, we have screened adjuvants for the highest quality antibody titers, negated the concern of pre-existing rabies-vector immunity, and established its potential as a long-term COVID-19 vaccine. We have tested toll-like receptor 4 (TLR4) agonists, inflammasome activators, and alum adjuvants in CORAVAX and found TLR4-activating MPLA-AddaVax to have the greatest potential. We followed the humoral immune response to CORAVAX in mice with pre-existing rabies virus immunity and saw no significant differences compared to naive mice. We then followed the immune response to CORAVAX over several months and 1-year post-immunization. Mice maintained high antigen-specific serum antibody titers as well as long-lived antibody-secreting cells in the spleen and bone marrow. We believe this rabies-vector strategy combats the problem of waning immunity of other COVID-19 vaccines. These results together support CORAVAX's potential during the ongoing COVID-19 pandemic.

A Single Dose of the Deactivated Rabies-Virus Vectored COVID-19 Vaccine, CORAVAX, Is Highly Efficacious and Alleviates Lung Inflammation in the Hamster Model.

Without sufficient herd immunity through either vaccination or natural infection, the coronavirus disease 2019 pandemic is unlikely to be controlled. Waning immunity with the currently approved vaccines suggests the need to evaluate vaccines causing the induction of long-term responses. Here, we report the immunogenicity and efficacy of our adjuvanted single-dose Rabies-vectored SARS-CoV-2 S1 vaccine, CORAVAX, in hamsters. CORAVAX induces high SARS-CoV-2 S1-specific and virus-neutralizing antibodies (VNAs) that prevent weight loss, viral loads, disease, lung inflammation, and the cytokine storm in hamsters. We also observed high Rabies VNA titers. In summary, CORAVAX is a promising dual-antigen vaccine candidate for clinical evaluation against SARS-CoV-2 and Rabies virus.

Current vaccine strategies against SARS-CoV-2: Promises and challenges.

In the years since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic began and spread across the globe, lessons have been learned about the challenges and opportunities that a pandemic brings to humankind. Researchers have produced many vaccines at unprecedented speed to protect people, but they have also been cognizant of the challenges presented by a new and unexpected infectious disease. The scope of this review is to examine the path of vaccine discovery so far and identify potential targets. Here, we provide insight into the leading vaccines and their advantages and challenges. We discuss the emerging mutations within the SARS-CoV-2 spike protein and other issues that need to be addressed to overcome coronavirus disease 2019 (COVID-19) completely. Future research is needed to develop a cheap, temperature-stable vaccine providing long-term immunity that protects the upper respiratory tract.

Measles-based Zika vaccine induces long-term immunity and requires NS1 antibodies to protect the female reproductive tract.

Zika virus (ZIKV) can cause devastating effects in the unborn fetus of pregnant women. To develop a candidate vaccine that can protect human fetuses, we generated a panel of live measles vaccine (MV) vectors expressing ZIKV-E and -NS1. Our MV-based ZIKV-E vaccine, MV-E2, protected mice from the non-lethal Zika Asian strain (PRVABC59) and the lethal African strain (MR766) challenge. Despite 100% survival of the MV-E2 mice, however, complete viral clearance was not achieved in the brain and reproductive tract of the lethally challenged mice. We then tested MV-based vaccines that expressed E and NS1 together or separately in two different vaccines. We observed complete clearance of ZIKV from the female reproductive tract and complete fetal protection in the lethal African challenge model in animals that received the dual antigen vaccines. Additionally, MV-E2 and MV-NS1, when administered together, induced durable plasma cell responses. Our findings suggest that NS1 antibodies are required to enhance the protection of ZIKV-E antibodies in the female reproductive tract.

A Strategy to Detect Emerging Non-Delta SARS-CoV-2 Variants with a Monoclonal Antibody Specific for the N501 Spike Residue.

Efforts to control SARS-CoV-2 have been challenged by the emergence of variant strains that have important implications for clinical and epidemiological decision making. Four variants of concern (VOCs) have been designated by the Centers for Disease Control and Prevention (CDC), namely, B.1.617.2 (delta), B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma), although the last three have been downgraded to variants being monitored (VBMs). VOCs and VBMs have shown increased transmissibility and/or disease severity, resistance to convalescent SARS-CoV-2 immunity and antibody therapeutics, and the potential to evade diagnostic detection. Methods are needed for point-of-care (POC) testing to rapidly identify these variants, protect vulnerable populations, and improve surveillance. Antigen-detection rapid diagnostic tests (Ag-RDTs) are ideal for POC use, but Ag-RDTs that recognize specific variants have not yet been implemented. Here, we describe a mAb (2E8) that is specific for the SARS-CoV-2 spike protein N501 residue. The 2E8 mAb can distinguish the delta VOC from variants with the N501Y meta-signature, which is characterized by convergent mutations that contribute to increased virulence and evasion of host immunity. Among the N501Y-containing mutants formerly designated as VOCs (alpha, beta, and gamma), a previously described mAb, CB6, can distinguish beta from alpha and gamma. When used in a sandwich ELISA, these mAbs sort these important SARS-CoV-2 variants into three diagnostic categories, namely, (1) delta, (2) alpha or gamma, and (3) beta. As delta is currently the predominant variant globally, they will be useful for POC testing to identify N501Y meta-signature variants, protect individuals in high-risk settings, and help detect epidemiological shifts among SARS-CoV-2 variants.

A single dose of replication-competent VSV-vectored vaccine expressing SARS-CoV-2 S1 protects against virus replication in a hamster model of severe COVID-19.

The development of effective countermeasures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent responsible for the COVID-19 pandemic, is a priority. We designed and produced ConVac, a replication-competent vesicular stomatitis virus (VSV) vaccine vector that expresses the S1 subunit of SARS-CoV-2 spike protein. We used golden Syrian hamsters as animal models of severe COVID-19 to test the efficacy of the ConVac vaccine. A single vaccine dose elicited high levels of SARS-CoV-2 specific binding and neutralizing antibodies; following intranasal challenge with SARS-CoV-2, animals were protected from weight loss and viral replication in the lungs. No enhanced pathology was observed in vaccinated animals upon challenge, but some inflammation was still detected. The data indicate rapid control of SARS-CoV-2 replication by the S1-based VSV-vectored SARS-CoV-2 ConVac vaccine.

SARS-CoV-2 vaccines - the biggest medical research project of the 21st century.

SARS-CoV-2 has been detected in more than 141 million people and caused more than 3 million deaths worldwide. To reduce the additional loss of millions of lives until natural immunity is reached, researchers have focused on the only known method to stop the COVID-19 pandemic: vaccines. The pandemic has propelled high-speed vaccine development, some based on novel technology previously not utilized in the vaccine field. The new technology opens new possibilities and comes with challenges because the long-term performance of the new platforms is unknown. Here we review the current leading vaccine candidates against COVID-19 and outline the advantages and disadvantages as well as the unknowns of each candidate.

Inactivated rabies virus vectored SARS-CoV-2 vaccine prevents disease in a Syrian hamster model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent coronavirus that has caused a worldwide pandemic. Although human disease is often asymptomatic, some develop severe illnesses such as pneumonia, respiratory failure, and death. There is an urgent need for a vaccine to prevent its rapid spread as asymptomatic infections accounting for up to 40% of transmission events. Here we further evaluated an inactivated rabies vectored SARS-CoV-2 S1 vaccine CORAVAX in a Syrian hamster model. CORAVAX adjuvanted with MPLA-AddaVax, a TRL4 agonist, induced high levels of neutralizing antibodies and generated a strong Th1-biased immune response. Vaccinated hamsters were protected from weight loss and viral replication in the lungs and nasal turbinates three days after challenge with SARS-CoV-2. CORAVAX also prevented lung disease, as indicated by the significant reduction in lung pathology. This study highlights CORAVAX as a safe, immunogenic, and efficacious vaccine that warrants further assessment in human trials.

Tetravalent Rabies-Vectored Filovirus and Lassa Fever Vaccine Induces Long-term Immunity in Nonhuman Primates.

BACKGROUND: The objective of this study is to evaluate the immunogenicity of adjuvanted monovalent rabies virus (RABV)-based vaccine candidates against Ebola virus (FILORAB1), Sudan virus (FILORAB2), Marburg virus (FILORAB3), Lassa virus (LASSARAB1), and combined trivalent vaccine candidate (FILORAB1-3) and tetravalent vaccine candidate (FILORAB1-3 and LASSARAB) in nonhuman primates. METHODS: Twenty-four Macaca fascicularis were randomly assigned into 6 groups of 4 animals. Each group was vaccinated with either a single adjuvanted vaccine, the trivalent vaccine, or the tetravalent vaccine at days 0 and 28. We followed the humoral immune responses for 1 year by antigen-specific enzyme-linked immunosorbent assays and RABV neutralization assays. RESULTS: High titers of filovirus and/or Lassa virus glycoprotein-specific immunoglobulin G were induced in the vaccinated animals. There were no significant differences between immune responses in animals vaccinated with single vaccines vs trivalent or tetravalent vaccines. In addition, all vaccine groups elicited strong rabies neutralizing antibody titers. The antigen-specific immune responses were detectable for 1 year in all groups. CONCLUSIONS: In summary, this study shows the longevity of the immune responses up to 365 days for a pentavalent vaccine-against Ebola virus, Sudan virus, Marburg virus, Lassa virus, and RABV-using a safe and effective vaccine platform.

Rhabdoviruses as vectors for vaccines and therapeutics.

Appropriate choice of vaccine vector is crucial for effective vaccine development. Rhabdoviral vectors, such as rabies virus and vesicular stomatitis virus, have been used in a variety of vaccine strategies. These viruses have small, easily manipulated genomes that can stably express foreign glycoproteins due to a well-established reverse genetics system for virus recovery. Both viruses have well-described safety profiles and have been demonstrated to be effective vaccine vectors. This review will describe how these Rhabdoviruses can be manipulated for use as vectors, their various applications as vaccines or therapeutics, and the advantages and disadvantages of their use.

Rabies virus-based COVID-19 vaccine CORAVAX™ induces high levels of neutralizing antibodies against SARS-CoV-2.

The recently emerged coronavirus SARS-CoV-2, the causative agent of COVID-19, is rapidly spreading in the world. The exponentially expanding threat of SARS-CoV-2 to global health highlights the urgent need for a vaccine. Herein we show the rapid development of a novel, highly efficient, and safe COVID-19 vaccine using a rabies virus-based vector that has proven to be an efficient vaccine against several emerging infectious diseases. This study reports that both a live and an inactivated rabies virus containing the SARS-CoV-2 spike S1 protein induces potent virus-neutralizing antibodies at much higher levels than seen in the sera of convalescent patients. In summary, the results provided here warrant further development of this safe and established vaccine platform against COVID-19.

Lyssavirus Vaccine with a Chimeric Glycoprotein Protects across Phylogroups.

Rabies is nearly 100% lethal in the absence of treatment, killing an estimated 59,000 people annually. Vaccines and biologics are highly efficacious when administered properly. Sixteen rabies-related viruses (lyssaviruses) are similarly lethal, but some are divergent enough to evade protection from current vaccines and biologics, which are based only on the classical rabies virus (RABV). Here we present the development and characterization of LyssaVax, a vaccine featuring a structurally designed, functional chimeric glycoprotein (G) containing immunologically important domains from both RABV G and the highly divergent Mokola virus (MOKV) G. LyssaVax elicits high titers of antibodies specific to both RABV and MOKV Gs in mice. Immune sera also neutralize a range of wild-type lyssaviruses across the major phylogroups. LyssaVax-immunized mice are protected against challenge with recombinant RABV and MOKV. Altogether, LyssaVax demonstrates the utility of structural modeling in vaccine design and constitutes a broadened lyssavirus vaccine candidate.

Identification and Characterization of a Small-Molecule Rabies Virus Entry Inhibitor.

Rabies virus (RABV) causes a severe and fatal neurological disease, but morbidity is vaccine preventable and treatable prior to the onset of clinical symptoms. However, immunoglobulin (IgG)-based rabies postexposure prophylaxis (PEP) is expensive, restricting access to life-saving treatment, especially for patients in low-income countries where the clinical need is greatest, and does not confer cross-protection against newly emerging phylogroup II lyssaviruses. Toward identifying a cost-effective replacement for the IgG component of rabies PEP, we developed and implemented a high-throughput screening protocol utilizing a single-cycle RABV reporter strain. A large-scale screen and subsequent direct and orthogonal counterscreens identified a first-in-class direct-acting RABV inhibitor, GRP-60367, with a specificity index (SI) of >100,000. Mechanistic characterization through time-of-addition studies, transient cell-to-cell fusion assays, and chimeric vesicular stomatitis virus (VSV) recombinants expressing the RABV glycoprotein (G) demonstrated that GRP-60367 inhibits entry of a subset of RABV strains. Resistance profiling of the chemotype revealed hot spots in conserved hydrophobic positions of the RABV G protein fusion loop that were confirmed in transient cell-to-cell fusion assays. Transfer of RABV G genes with signature resistance mutations into a recombinant VSV backbone resulted in the recovery of replication-competent virions with low susceptibility to the inhibitor. This work outlines a tangible strategy for mechanistic characterization and resistance profiling of RABV drug candidates and identified a novel, well-behaved molecular probe chemotype that specifically targets the RABV G protein and prevents G-mediated viral entry.IMPORTANCE Rabies PEP depends on anti-RABV IgG, which is expensive and in limited supply in geographical areas with the highest disease burden. Replacing the IgG component with a cost-effective and shelf-stable small-molecule antiviral could address this unmet clinical need by expanding access to life-saving medication. This study has established a robust protocol for high-throughput anti-RABV drug screens and identified a chemically well-behaved, first-in-class hit with nanomolar anti-RABV potency that blocks RABV G protein-mediated viral entry. Resistance mapping revealed a druggable site formed by the G protein fusion loops that has not previously emerged as a target for neutralizing antibodies. Discovery of this RABV entry inhibitor establishes a new molecular probe to advance further mechanistic and structural characterization of RABV G that may aid in the design of a next-generation clinical candidate against RABV.

Inactivated Rabies Virus-Based Ebola Vaccine Preserved by Vaporization Is Heat-Stable and Immunogenic Against Ebola and Protects Against Rabies Challenge.

BACKGROUND: Ebola virus (EBOV) is a highly lethal member of the Filoviridae family associated with human hemorrhagic disease. Despite being a sporadic disease, it caused a large outbreak in 2014-2016 in West Africa and another outbreak recently in the Democratic Republic of Congo. Several vaccine candidates are currently in preclinical and clinical studies but none are stable without cold chain storage. METHODS: We used preservation by vaporization (PBV), a novel processing technology to heat-stabilize FiloRab1 (inactivated rabies-based Ebola vaccine), a candidate Ebola vaccine, and stored the vials at temperatures ranging from 4°C to 50°C for 10 days to 12 months. We immunized Syrian hamsters with the best long-term stable FiloRab1 PBV vaccines and challenged them with rabies virus (RABV). RESULTS: Syrian hamsters immunized with FiloRab1 PBV-processed vaccines stored at temperatures of 4°C and 37°C for 6 months, and at 50°C for 2 weeks, seroconverted against both RABV-G and EBOV-GP. Notably, all of the FiloRab1 PBV vaccines proved to be 100% effective in a RABV challenge model. CONCLUSIONS: We successfully demonstrated that the FiloRab1 PBV vaccines are stable and efficacious for up to 6 months when stored at temperatures ranging from 4°C to 37°C and for up to 2 weeks at 50°C.

Erratum: Publisher Correction: Rabies-based vaccine induces potent immune responses against Nipah virus.

[This corrects the article DOI: 10.1038/s41541-019-0109-5.].

Rabies-based vaccine induces potent immune responses against Nipah virus.

Nipah Virus (NiV) is a re-emerging zoonotic pathogen in the genus Henipavirus of the Paramyxoviridae family of viruses. NiV is endemic to Bangladesh and Malaysia and is highly fatal to both livestock and humans (human case fatality rate = 74.5%). Currently, there is no approved vaccine against NiV on the market. The goal of this study was to use a recombinant RABV vector expressing NiV glycoprotein (NiV G) to develop a bivalent candidate vaccine against NiV disease and rabies virus (RABV) disease, which is also a significant health burden in the regions where NiV is endemic. The rabies vector is a well-established vaccine strain that lacks neurovirulence and can stably expresses foreign antigens that are immunogenic in various animal models. Mice inoculated intranasally with the live recombinant RABV/NiV vaccine (NIPARAB) showed no signs of disease. To test the immunogenicity of the vaccine candidate, groups of C57BL/6 mice were immunized intramuscularly with a single dose of live vaccine particles or two doses of chemically inactivated viral particles. Both vaccination groups showed NiV G-specific seroconversion, and the inactivated (INAC) vaccine group yielded higher titers of NiV G-specific antibodies. Furthermore, cross-reactivity of NiV G-specific immune sera against Hendra virus (HeV), was confirmed by immunofluorescence (IF) and indirect ELISA against soluble recombinant HeV glycoprotein (HeV G). Both live and killed vaccines induced neutralizing antibodies. These results indicate that NIPARAB may be used as a killed virus vaccine to protect humans against NiV and RABV, and possibly as a preventative measure against HeV as well.

A new recombinant rabies virus expressing a green fluorescent protein: A novel and fast approach to quantify virus neutralizing antibodies.

The Rapid Fluorescent Focus Inhibition Test (RFFIT) is a standard assay used to detect and assess the titers of rabies virus neutralizing antibodies (RVNA) in blood sera. To simplify the multistep RFFIT procedure by eliminating the immunostaining step, we generated a new recombinant RV expressing a green fluorescent protein (rRV-GFP) and assess its suitability for quantifying RVNA. We rescued the rRV-GFP virus from plasmid DNA carrying a full-length genome of the CVS-N2c strain of RV in which the eGFP gene was inserted between the glycoprotein and RNA-polymerase genes. The recombinant virus was genetically stable and grew efficiently in appropriate cells expressing sufficient GFP fluorescence to detect directly 20 h post infection (hpi). We evaluated the feasibility of using rRV-GFP in RFFIT by comparing RVNA titers in 27 serum samples measured by conventional RFFIT and RFFIT-GFP. A linear regression analysis of the data demonstrated a good agreement between these two methods (r = 0.9776) including results with samples having RVNA titers close to the minimally acceptable vaccine potency threshold (0.5 IU/ml). Study results showed that the rRV-GFP virus could replace the CVS-11 challenge virus currently used in the conventional RFFIT and enabling more rapid, simpler, and less expensive detection and quantitation of RVNA.