rVSV-based Vaccine Research Articles

Fed-batch strategies for intensified rVSV vector production in high cell density cultures of suspension HEK293 cells.

Vesicular stomatitis virus (VSV) has been increasingly demonstrated as a promising viral vector platform. As the interest over this modality for vaccine and gene therapy applications increases, the need for intensified processes to produce these vectors emerge. In this study, we develop fed-batch-based operations to intensify the production of a recombinant VSV-based vaccine candidate (rVSV-SARS-CoV-2) in suspension cultures of HEK293 cells. A feeding strategy, in which a commercial concentrated medium was added to cultures based on cell growth through a fixed cell specific feeding rate (CSFR), was applied for the development of two different processes using Ambr250 modular bioreactors. Cultures operated in hybrid fed-batch/perfusion (FB/P) or fed-batch (FB) were able to sustain infections performed at 8.0 × 106 cells/mL, respectively resulting in 3.9 and 5.0-fold increase in total yield (YT) and 1.7 and 5.6-fold increase in volumetric productivity (VP) when compared with a batch reference. A maximum viral titer of 4.5 × 1010 TCID50/mL was reached, which is comparable or higher than other processes for VSV production in different cell lines. Overall, our study reports efficient fed-batch options to intensify the production of a rVSV-based vaccine candidate in suspension HEK293 cells.

The rVSV-EBOV vaccine provides limited cross-protection against Sudan virus in guinea pigs

Recombinant vesicular stomatitis viruses (rVSVs) engineered to express heterologous viral glycoproteins have proven to be remarkably effective vaccines. Indeed, rVSV-EBOV, which expresses the Ebola virus (EBOV) glycoprotein, recently received clinical approval in the United States and Europe for its ability to prevent EBOV disease. Analogous rVSV vaccines expressing glycoproteins of different human-pathogenic filoviruses have also demonstrated efficacy in pre-clinical evaluations, yet these vaccines have not progressed far beyond research laboratories. In the wake of the most recent outbreak of Sudan virus (SUDV) in Uganda, the need for proven countermeasures was made even more acute. Here we demonstrate that an rVSV-based vaccine expressing the SUDV glycoprotein (rVSV-SUDV) generates a potent humoral immune response that protects guinea pigs from SUDV disease and death. Although the cross-protection generated by rVSV vaccines for different filoviruses is thought to be limited, we wondered whether rVSV-EBOV might also provide protection against SUDV, which is closely related to EBOV. Surprisingly, nearly 60% of guinea pigs that were vaccinated with rVSV-EBOV and challenged with SUDV survived, suggesting that rVSV-EBOV offers limited protection against SUDV, at least in the guinea pig model. These results were confirmed by a back-challenge experiment in which animals that had been vaccinated with rVSV-EBOV and survived EBOV challenge were inoculated with SUDV and survived. Whether these data are applicable to efficacy in humans is unknown, and they should therefore be interpreted cautiously. Nevertheless, this study confirms the potency of the rVSV-SUDV vaccine and highlights the potential for rVSV-EBOV to elicit a cross-protective immune response.

Comparison of the immunogenicity of nasal-spray rVSV vector, adenovirus vector, and inactivated COVID-19-based vaccines in rodent models.

Intranasal (i.n.) vaccines can induce mucosal and systemic immunity against respiratory pathogens. Previously, we demonstrated that the recombinant vesicular stomatitis virus (rVSV)-based COVID-19 vaccine rVSV-SARS-CoV-2, with poor immunogenicity via the intramuscular route (i.m.), is more suitable for i.n. administration in mice and nonhuman primates. Here, we found that the rVSV-SARS-CoV-2 Beta variant was more immunogenic than the wild-type strain and other variants of concern (VOCs) in golden Syrian hamsters. Furthermore, the immune responses elicited by rVSV-based vaccine candidates via the i.n. route weresignificantly higher than thoseof two licensed vaccines:the inactivated vaccine KCONVAC delivered via the i.m. route and the adenovirus-based Vaxzevria delivered i.n. or i.m. We next assessed the booster efficacy of rVSV following two i.m. doses of KCONVAC. Twenty-eight days after receiving two i.m. doses of KCONVAC, hamsters were boosted with a third dose of KCONVAC (i.m.), Vaxzevria (i.m. or i.n.), or rVSVs (i.n.). Consistent with other heterologous booster studies, Vaxzevria and rVSV elicited significantly higher humoral immunity than the homogenous KCONVAC. In summary, our results confirmed that two i.n. doses of rVSV-Beta elicited significantly higher humoral immune responses than commercial inactivated and adeno-based COVID vaccines in hamsters. As a heterologous booster dose, rVSV-Beta induced potent, persistent, and broad-spectrum humoral and mucosal neutralizing responses against all VOCs, highlighting its potential to be developed into a nasal-spray vaccine.

Awake Breast Conservative Surgery: A Strategy to Shorten Surgical Waiting Lists During and Post COVID-19 Emergency.

SARS-CoV-2 with a dramatical worldwide spread, impacted greatly daily life and healthcare. In order to avoid delay in cancer treatment, many strategies and measures were implemented. The Awake breast surgery was a strategy implemented in our Unit during the pandemic, aimed to reduce operatory room occupancy and increase the number of procedures performed during the daily surgical session. The aim of the study was to evaluate how the use of this strategy has changed before and after the advent of the COVID-19 pandemic, and its relative benefits. This was a retrospective study analysing all patients subjected to breast conservative surgery for oncological disease from July 2018 to December 2021. Out of 498 patients enrolled in the study, 253 (50.8%) cases were treated before the pandemic and were designated as "pre-COVID-19" group. The remaining 245 (49.1%) cases were considered the "COVID-19" group. Cases of awake surgery in COVID-19 group were 141 (54.7%) vs. 84 (33.2%), p<0.001. Length of hospitalization and surgical time were comparable between the groups: relative p=0.188 and 0.264, respectively. Differently, operation room occupation was significantly shorter in the COVID-19 group, p<0.001; and number of outpatient surgical procedures was higher, p=0.0304. Multivariate analysis identified the period of surgery (OR=1.47) as a statistically significant factor, p=0.011, predictive of prolonged operatory room occupancy. Awake surgery was one of the strategies which made more operating rooms available and allowed avoiding further delays.

A Recombinant VSV-Based Bivalent Vaccine Effectively Protects against Both SARS-CoV-2 and Influenza A Virus Infection.

ABSTRACTCOVID-19 and influenza are both highly contagious respiratory diseases that have been serious threats to global public health. It is necessary to develop a bivalent vaccine to control these two infectious diseases simultaneously. In this study, we generated three attenuated replicating recombinant vesicular stomatitis virus (rVSV)-based vaccine candidates against both SARS-CoV-2 and influenza viruses. These rVSV-based vaccines coexpress SARS-CoV-2 Delta spike protein (SP) bearing the C-terminal 17 amino acid (aa) deletion (SPΔC) and I742A point mutation, or the SPΔC with a deletion of S2 domain, or the RBD domain, and a tandem repeat harboring four copies of the highly conserved influenza M2 ectodomain (M2e) that fused with the Ebola glycoprotein DC-targeting/activation domain. Animal immunization studies have shown that these rVSV bivalent vaccines induced efficient humoral and cellular immune responses against both SARS-CoV-2 SP and influenza M2 protein, including high levels of neutralizing antibodies against SARS-CoV-2 Delta and other variant SP-pseudovirus infections. Importantly, immunization of the rVSV bivalent vaccines effectively protected hamsters or mice against the challenges of SARS-CoV-2 Delta variant and lethal H1N1 and H3N2 influenza viruses and significantly reduced respiratory viral loads. Overall, this study provides convincing evidence for the high efficacy of this bivalent vaccine platform to be used and/or easily adapted to produce new vaccines against new or reemerging SARS-CoV-2 variants and influenza A virus infections.IMPORTANCE Given that both COVID-19 and influenza are preferably transmitted through respiratory droplets during the same seasons, it is highly advantageous to develop a bivalent vaccine that could simultaneously protect against both COVID-19 and influenza. In this study, we generated the attenuated replicating recombinant vesicular stomatitis virus (rVSV)-based vaccine candidates that target both spike protein of SARS-Cov-2 Delta variant and the conserved influenza M2 domain. Importantly, these vaccine candidates effectively protected hamsters or mice against the challenges of SARS-CoV-2 Delta variant and lethal H1N1 and H3N2 influenza viruses and significantly reduced respiratory viral loads.

Environmental Risk Assessment for rVSVΔG-ZEBOV-GP, a Genetically Modified Live Vaccine for Ebola Virus Disease.

rVSVΔG-ZEBOV-GP is a live, attenuated, recombinant vesicular stomatitis virus (rVSV)-based vaccine for the prevention of Ebola virus disease caused by Zaire ebolavirus. As a replication-competent genetically modified organism, rVSVΔG-ZEBOV-GP underwent various environmental evaluations prior to approval, the most in-depth being the environmental risk assessment (ERA) required by the European Medicines Agency. This ERA, as well as the underlying methodology used to arrive at a sound conclusion about the environmental risks of rVSVΔG-ZEBOV-GP, are described in this review. Clinical data from vaccinated adults demonstrated only infrequent, low-level shedding and transient, low-level viremia, indicating a low person-to-person infection risk. Animal data suggest that it is highly unlikely that vaccinated individuals would infect animals with recombinant virus vaccine or that rVSVΔG-ZEBOV-GP would spread within animal populations. Preclinical studies in various hematophagous insect vectors showed that these species were unable to transmit rVSVΔG-ZEBOV-GP. Pathogenicity risk in humans and animals was found to be low, based on clinical and preclinical data. The overall risk for non-vaccinated individuals and the environment is thus negligible and can be minimized further through defined mitigation strategies. This ERA and the experience gained are relevant to developing other rVSV-based vaccines, including candidates under investigation for prevention of COVID-19.

Titration methods for rVSV-based vaccine manufacturing

The recombinant Vesicular Stomatitis Virus (rVSV) is an emerging platform for viral vector-based vaccines. Promising results have been reported in clinical trials for the rVSV-ZEBOV vaccine for Ebola virus disease prevention. In this study, we describe the titration tools elaborated to assess the titre of rVSV-ZEBOV productions.• A streamlined Median Tissue Culture Infectious Dose (TCID50) assay to determine the infectious titer of this vaccine was established.• A digital polymerase chain reaction (dPCR) assay to assess the total number of viral particles present in cell-free culture supernatants of rVSV productions was developed.• These assays are used to titre rVSV-ZEBOV samples and characterize the ratio of total particles to infectious units for monitoring process robustness and product quality attributes and can be used to titre samples generated in the production of further rVSV vectors.

Single dose of a rVSV-based vaccine elicits complete protection against severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia virus (SFTSV) is an emerging tick-borne phlebovirus that causes lethal human disease, for which there are no licensed antiviral vaccines or therapies. Herein, we developed a live attenuated recombinant vesicular stomatitis virus (rVSV)-based vaccine candidate expressing the SFTSV Gn/Gc glycoproteins (rVSV-SFTSV/AH12-GP). High titers of cross-protective, broadly neutralizing antibodies were elicited by a single dose of rVSV-SFTSV/AH12-GP in both immunocompetent and immunocompromised mice against multiple strains of SFTSV and the related but distinct phlebovirus Heartland virus (HRTV). Remarkably, complete protection against lethal challenge with SFTSV was conferred in young and old immunocompromised mice irrespective of any pre-existing vector-specific immunity. Collectively, these results suggest that a rVSV vector expressing SFTSV glycoproteins is a promising candidate vaccine against two emerging phleboviruses associated with severe human diseases.

Ebola Virus Glycoprotein Induces an Innate Immune Response In vivo via TLR4.

Ebola virus (EBOV), a member of the Filoviridae family, causes the most severe form of viral hemorrhagic fever. Although no FDA licensed vaccine or treatment against Ebola virus disease (EVD) is currently available, Ebola virus glycoprotein (GP) is the major antigen used in all candidate Ebola vaccines. Recent reports of protection as quickly as within 6 days of administration of the rVSV-based vaccine expressing EBOV GP before robust humoral responses were generated suggests that the innate immune responses elicited early after vaccination may contribute to the protection. However, the innate immune responses induced by EBOV GP in the absence of viral vectors or adjuvants have not been fully characterized in vivo. Our recent studies demonstrated that immunization with highly purified recombinant GP in the absence of adjuvants induced a robust IgG response and partial protection against EBOV infection suggesting that GP alone can induce protective immunity. In this study we investigated the early immune response to purified EBOV GP alone in vitro and in vivo. We show that GP was efficiently internalized by antigen presenting cells and subsequently induced production of key inflammatory cytokines. In vivo, immunization of mice with EBOV GP triggered the production of key Th1 and Th2 innate immune cytokines and chemokines, which directly governed the recruitment of CD11b+ macrophages and CD11c+ dendritic cells to the draining lymph nodes (DLNs). Pre-treatment of mice with a TLR4 antagonist inhibited GP-induced cytokine production and recruitment of immune cells to the DLN. EBOV GP also upregulated the expression of costimulatory molecules in bone marrow derived macrophages suggesting its ability to enhance APC stimulatory capacity, which is critical for the induction of effective antigen-specific adaptive immunity. Collectively, these results provide the first in vivo evidence that early innate immune responses to EBOV GP are mediated via the TLR4 pathway and are able to modulate the innate-adaptive interface. These mechanistic insights into the adjuvant-like property of EBOV GP may help to develop a better understanding of how optimal prophylactic efficacy of EBOV vaccines can be achieved as well as further explore the potential post-exposure use of vaccines to prevent filoviral disease.

A Recombinant Vesicular Stomatitis Virus Ebola Vaccine

BackgroundThe 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.MethodsWe 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.ResultsThe 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.ConclusionsThis 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∆G-ZEBOV-GP ClinicalTrials.gov numbers, NCT02269423 and NCT02280408.)

Vesicular Stomatitis Virus-Based Vaccines Protect Nonhuman Primates against Bundibugyo ebolavirus

Ebola virus (EBOV) causes severe and often fatal hemorrhagic fever in humans and nonhuman primates (NHPs). Currently, there are no licensed vaccines or therapeutics for human use. Recombinant vesicular stomatitis virus (rVSV)-based vaccine vectors, which encode an EBOV glycoprotein in place of the VSV glycoprotein, have shown 100% efficacy against homologous Sudan ebolavirus (SEBOV) or Zaire ebolavirus (ZEBOV) challenge in NHPs. In addition, a single injection of a blend of three rVSV vectors completely protected NHPs against challenge with SEBOV, ZEBOV, the former Côte d'Ivoire ebolavirus, and Marburg virus. However, recent studies suggest that complete protection against the newly discovered Bundibugyo ebolavirus (BEBOV) using several different heterologous filovirus vaccines is more difficult and presents a new challenge. As BEBOV caused nearly 50% mortality in a recent outbreak any filovirus vaccine advanced for human use must be able to protect against this new species. Here, we evaluated several different strategies against BEBOV using rVSV-based vaccines. Groups of cynomolgus macaques were vaccinated with a single injection of a homologous BEBOV vaccine, a single injection of a blended heterologous vaccine (SEBOV/ZEBOV), or a prime-boost using heterologous SEBOV and ZEBOV vectors. Animals were challenged with BEBOV 29–36 days after initial vaccination. Macaques vaccinated with the homologous BEBOV vaccine or the prime-boost showed no overt signs of illness and survived challenge. In contrast, animals vaccinated with the heterologous blended vaccine and unvaccinated control animals developed severe clinical symptoms consistent with BEBOV infection with 2 of 3 animals in each group succumbing. These data show that complete protection against BEBOV will likely require incorporation of BEBOV glycoprotein into the vaccine or employment of a prime-boost regimen. Fortunately, our results demonstrate that heterologous rVSV-based filovirus vaccine vectors employed in the prime-boost approach can provide protection against BEBOV using an abbreviated regimen, which may have utility in outbreak settings.

Recombinant Vesicular Stomatitis Virus–Based Vaccines Against Ebola and Marburg Virus Infections

The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with a high mortality rate in humans and nonhuman primates. Among the most-promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV) that expresses a single filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). Importantly, a single injection of blended rVSV-based filovirus vaccines was shown to completely protect nonhuman primates against Marburg virus and 3 different species of Ebola virus. These rVSV-based vaccines have also shown utility when administered as a postexposure treatment against filovirus infections, and a rVSV-based Ebola virus vaccine was recently used to treat a potential laboratory exposure. Here, we review the history of rVSV-based vaccines and pivotal animal studies showing their utility in combating Ebola and Marburg virus infections.

Single Immunization With a Monovalent Vesicular Stomatitis Virus–Based Vaccine Protects Nonhuman Primates Against Heterologous Challenge With Bundibugyo ebolavirus

The recombinant vesicular stomatitis virus (rVSV) vector-based monovalent vaccine platform expressing a filovirus glycoprotein has been demonstrated to provide protection from lethal challenge with Ebola (EBOV) and Marburg (MARV) viruses both prophylactically and after exposure. This platform provides protection between heterologous strains within a species; however, protection from lethal challenge between species has been largely unsuccessful. To determine whether the rVSV-EBOV vaccines have the potential to provide protection against a newly emerging, phylogenetically related species, cynomolgus macaques were vaccinated with an rVSV vaccine expressing either the glycoprotein of Zaire ebolavirus (ZEBOV) or Côte d'Ivoire ebolavirus (CIEBOV) and then challenged with Bundibugyo ebolavirus (BEBOV), which was recently proposed as a new EBOV species following an outbreak in Uganda in 2007. A single vaccination with the ZEBOV-specific vaccine provided cross-protection (75% survival) against subsequent BEBOV challenge, whereas vaccination with the CIEBOV-specific vaccine resulted in an outcome similar to mock-immunized animals (33% and 25% survival, respectively). This demonstrates that monovalent rVSV-based vaccines may be useful against a newly emerging species; however, heterologous protection across species remains challenging and may depend on enhancing the immune responses either through booster immunizations or through the inclusion of multiple immunogens.

Prospects for immunisation against Marburg and Ebola viruses.

For more than 30 years the filoviruses, Marburg virus and Ebola virus, have been associated with periodic outbreaks of hemorrhagic fever that produce severe and often fatal disease. The filoviruses are endemic primarily in resource-poor regions in Central Africa and are also potential agents of bioterrorism. Although no vaccines or antiviral drugs for Marburg or Ebola are currently available, remarkable progress has been made over the last decade in developing candidate preventive vaccines against filoviruses in nonhuman primate models. Due to the generally remote locations of filovirus outbreaks, a single-injection vaccine is desirable. Among the prospective vaccines that have shown efficacy in nonhuman primate models of filoviral hemorrhagic fever, two candidates, one based on a replication-defective adenovirus serotype 5 and the other on a recombinant VSV (rVSV), were shown to provide complete protection to nonhuman primates when administered as a single injection. The rVSV-based vaccine has also shown utility when administered for postexposure prophylaxis against filovirus infections. A VSV-based Ebola vaccine was recently used to manage a potential laboratory exposure.

Development of pDNA and Recombinant VSV Vectored Vaccines for Immunization Against HIV

Vaccines based on plasmid DNA (pDNA) or recombinant vesicular stomatitis virus (rVSV) vectors elicit HIV-1-specific cellular and humoral immune responses in small animal models. However, hurdles remain with each of these vaccine modalities before they can be considered for widespread clinical use. For pDNA vaccines, early clinical studies indicate that additional measures are needed to improve immunogenicity. For rVSV-based vaccines, issues related to the potential neurovirulence of the prototype vector represent a safety concern.

Immunogenicity of Attenuated Vesicular Stomatitis Virus Vectors Expressing HIV Type 1 Env and SIV Gag Proteins: Comparison of Intranasal and Intramuscular Vaccination Routes

An experimental AIDS vaccine based on attenuated, recombinant vesicular stomatitis virus (rVSV), when administered by a combination of parenteral and mucosal routes, has proven effective at preventing AIDS in a rhesus macaque model (Rose NF, et al.: Cell 2001;106:539-549). In an effort to determine the optimal route of vaccine administration we evaluated the ability of rVSV-based vaccine vectors expressing HIV-1 Env and SIV Gag proteins, when given either intramuscularly (i.m.) or intranasally (i.n.), to elicit antigen-specific cellular and humoral immune responses, and to protect from a subsequent vaginal challenge with simian-human immunodeficiency virus (SHIV89.6P). Our results demonstrate that macaques vaccinated by the i.n. route developed significantly higher antigen-specific cellular immune responses as determined by MHC class I tetramer staining, IFN-gamma ELISPOT, and cytotoxic T cell assays. However, systemic and mucosal humoral immune responses did not vary significantly with the route of vaccine administration. Given the importance of cell-mediated immune responses in slowing AIDS progression, intranasal delivery of a VSV-based AIDS vaccine may be an optimal as well as practical route for vaccination and should be considered in design of clinical trials.

Immunogenicity of Attenuated Vesicular Stomatitis Virus Vectors Expressing HIV Type 1 Env and SIV Gag Proteins: Comparison of Intranasal and Intramuscular Vaccination Routes

An experimental AIDS vaccine based on attenuated, recombinant vesicular stomatitis virus (rVSV), when administered by a combination of parenteral and mucosal routes, has proven effective at preventing AIDS in a rhesus macaque model (Rose NF, et al.: Cell 2001;106:539–549). In an effort to determine the optimal route of vaccine administration we evaluated the ability of rVSV-based vaccine vectors expressing HIV-1 Env and SIV Gag proteins, when given either intramuscularly (i.m.) or intranasally (i.n.), to elicit antigen-specific cellular and humoral immune responses, and to protect from a subsequent vaginal challenge with simian–human immunodeficiency virus (SHIV89.6P). Our results demonstrate that macaques vaccinated by the i.n. route developed significantly higher antigen-specific cellular immune responses as determined by MHC class I tetramer staining, IFN-γ ELISPOT, and cytotoxic T cell assays. However, systemic and mucosal humoral immune responses did not vary significantly with the route of vaccine administration. Given the importance of cell-mediated immune responses in slowing AIDS progression, intranasal delivery of a VSV-based AIDS vaccine may be an optimal as well as practical route for vaccination and should be considered in design of clinical trials.