Recombinant Vesicular Stomatitis Virus Vectors Research Articles

Immunological correlates of protection afforded by PHV02 live, attenuated recombinant vesicular stomatitis virus vector vaccine against Nipah virus disease.

Immune correlates of protection afforded by PHV02, a recombinant vesicular stomatitis (rVSV) vector vaccine against Nipah virus (NiV) disease, were investigated in the African green monkey (AGM) model. Neutralizing antibody to NiV has been proposed as the principal mediator of protection against future NiV infection. Two approaches were used to determine the correlation between neutralizing antibody levels and outcomes following a severe (1,000 median lethal doses) intranasal/intratracheal (IN/IT) challenge with NiV (Bangladesh): (1) reduction in vaccine dose given 28 days before challenge and (2) challenge during the early phase of the antibody response to the vaccine. Reduction in vaccine dose to very low levels led to primary vaccine failure rather than a sub-protective level of antibody. All AGMs vaccinated with the nominal clinical dose (2 × 107 pfu) at 21, 14, or 7 days before challenge survived. AGMs vaccinated at 21 days before challenge had neutralizing antibodies (geometric mean titer, 71.3). AGMs vaccinated at 7 or 14 days before challenge had either undetectable or low neutralizing antibody titers pre-challenge but had a rapid rise in titers after challenge that abrogated the NiV infection. A simple logistic regression model of the combined studies was used, in which the sole explanatory variable was pre-challenge neutralizing antibody titers. For a pre-challenge titer of 1:5, the predicted survival probability is 100%. The majority of animals with pre-challenge neutralizing titer of ≥1:20 were protected against pulmonary infiltrates on thoracic radiograms, and a majority of those with titers ≥1:40 were protected against clinical signs of illness and against a ≥fourfold antibody increase following challenge (indicating sterile immunity). Controls receiving rVSV-Ebola vaccine rapidly succumbed to NiV challenge, eliminating the innate immunity stimulated by the rVSV vector as a contributor to survival in monkeys challenged as early as 7 days after vaccination. It was concluded that PHV02 vaccine elicited a rapid onset of protection and that any detectable level of neutralizing antibody was a functional immune correlate of survival.

Two Point Mutations in the Glycoprotein of SFTSV Enhance the Propagation Recombinant Vesicular Stomatitis Virus Vectors at Assembly Step.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen for which approved therapeutic drugs or vaccines are not available. We previously developed a recombinant vesicular stomatitis virus-based vaccine candidate (rVSV-SFTSV) by replacing the original glycoprotein with Gn/Gc from SFTSV, which conferred complete protection in a mouse model. Here, we found that two spontaneous mutations, M749T/C617R, emerged in the Gc glycoprotein during passaging that could significantly increase the titer of rVSV-SFTSV. M749T/C617R enhanced the genetic stability of rVSV-SFTSV, and no further mutations appeared after 10 passages. Using immunofluorescence analysis, we found that M749T/C617R could increase glycoprotein traffic to the plasma membrane, thus facilitating virus assembly. Remarkably, the broad-spectrum immunogenicity of rVSV-SFTSV was not affected by the M749T/C617R mutations. Overall, M749T/C617R could enhance the further development of rVSV-SFTSV into an effective vaccine in the future.

A recombinant VSV-vectored vaccine rapidly protects nonhuman primates against heterologous lethal Lassa fever.

Lassa virus (LASV) is recognized by the World Health Organization as one of the top five pathogens likely to cause a severe outbreak. A recent unprecedented resurgence of LASV in Nigeria caused by genetically diverse strains underscores the need for licensed medical countermeasures. Single-injection vaccines that can rapidly control outbreaks and confer long-term immunity are needed. Vaccination of cynomolgus monkeys with a recombinant vesicular stomatitis virus vector expressing the glycoprotein precursor of LASV lineage IV strain Josiah (rVSVΔG-LASV-GPC) induces fast-acting protection in monkeys challenged 3 or 7days later with a genetically heterologous lineage II isolate of LASV from Nigeria, while nonspecifically vaccinated control animals succumb to challenge. The rVSVΔG-LASV-GPC vaccine induces rapid activation of adaptive immunity and the transcription of natural killer (NK) cell-affiliated mRNAs. This study demonstrates that rVSVΔG-LASV-GPC may provide rapid protection in humans against LASV infections in cases where immediate public-health intervention is required.

A recombinant VSV-vectored vaccine rapidly protects nonhuman primates against lethal Nipah virus disease

SignificanceConcern has increased about the pandemic potential of Nipah virus (NiV). Similar to SARS-CoV-2, NiV is an RNA virus that is transmitted by respiratory droplets. There are currently no NiV vaccines licensed for human use. While several preventive vaccines have shown promise in protecting animals against lethal NiV disease, most studies have assessed protection 1 mo after vaccination. However, in order to contain and control outbreaks, vaccines that can rapidly confer protection in days rather than months are needed. Here, we show that a recombinant vesicular stomatitis virus vector expressing the NiV glycoprotein can completely protect monkeys vaccinated 7 d prior to NiV exposure and 67% of animals vaccinated 3 d before NiV challenge.

Transcriptomic signatures induced by the Ebola virus vaccine rVSVΔG-ZEBOV-GP in adult cohorts in Europe, Africa, and North America: a molecular biomarker study

SummaryBackgroundA recombinant vesicular stomatitis virus vector expressing the Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP) vaccine has been reported as safe, immunogenic, and highly protective in a ring vaccination trial. We aimed to identify transcriptomic immune response biomarker signatures induced by vaccination and associated signatures with its immunogenicity and reactogenicity to better understand the potential mechanisms of action of the vaccine.Methods354 healthy adult volunteers were vaccinated in randomised, double-blind, placebo-controlled trials in Europe (Geneva, Switzerland [November, 2014, to January, 2015]) and North America (USA [Dec 5, 2014, to June 23, 2015]), and dose-escalation trials in Africa (Lambaréné, Gabon [November, 2014, to January, 2015], and Kilifi, Kenya [December, 2014, to January, 2015]) using different doses of the recombinant vesicular stomatitis virus vector expressing the Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP; 3 × 105 to 1 × 108 plaque-forming units [pfu]). Longitudinal transcriptomic responses (days 0, 1, 2, 3, 7, 14, and 28) were measured in whole blood using a targeted gene expression profiling platform (dual-colour reverse-transcriptase multiplex ligation-dependent probe amplification) focusing on 144 immune-related genes. The effect of time and dose on transcriptomic response was also assessed. Logistic regression with lasso regularisation was applied to identify host signatures with optimal discriminatory capability of vaccination at day 1 or day 7 versus baseline, whereas random-effects models and recursive feature elimination combined with regularised logistic regression were used to associate signatures with immunogenicity and reactogenicity.FindingsOur results indicated that perturbation of gene expression peaked on day 1 and returned to baseline levels between day 7 and day 28. The magnitude of the response was dose-dependent, with vaccinees receiving a high dose (≥9 × 106 pfu) of rVSVΔG-ZEBOV-GP exhibiting the largest amplitude. The most differentially expressed genes that were significantly upregulated following vaccination consisted of type I and II interferon-related genes and myeloid cell-associated markers, whereas T cell, natural killer cell, and cytotoxicity-associated genes were downregulated. A gene signature associated with immunogenicity (common to all four cohorts) was identified correlating gene expression profiles with ZEBOV-GP antibody titres and a gene signatures associated with reactogenicity (Geneva cohort) was identified correlating gene expression profiles with an adverse event (ie, arthritis).InterpretationCollectively, our results identify and cross-validate immune-related transcriptomic signatures induced by rVSVΔG-ZEBOV-GP vaccination in four cohorts of adult participants from different genetic and geographical backgrounds. These signatures will aid in the rational development, testing, and evaluation of novel vaccines and will allow evaluation of the effect of host factors such as age, co-infection, and comorbidity on responses to vaccines.FundingInnovative Medicines Initiative 2 Joint Undertaking.

Human Transcriptomic Response to the VSV-Vectored Ebola Vaccine.

Ebolavirus Disease (EVD) is a severe haemorrhagic fever that occurs in epidemic outbreaks, with a high fatality rate and no specific therapies available. rVSVΔG-ZEBOV-GP (Ervebo®), a live-attenuated recombinant vesicular stomatitis virus vector expressing the glycoprotein G of Zaire Ebolavirus, is the first vaccine approved for prevention of EVD. Both innate and adaptive responses are deemed to be involved in vaccine-induced protection, yet the mechanisms are not fully elucidated. A global transcriptomic approach was used to profile the blood host-response in 51 healthy volunteers enrolled in a phase 1/2 clinical trial. Signatures of the host responses were investigated assessing the enrichment in differentially expressed genes (DEGs) of specific “blood transcription modules” (BTM). Comparison of gene-expression levels showed that vaccination produces a peak of 5469 DEGs at day one, representing 38.6% of the expressed genes. Out of 346 BTMs, 144 were significantly affected by vaccination. Innate immunity pathways were induced from day 1 to day 14. At days 2 and 3, neutrophil modules were downregulated and complement-related modules upregulated. T-cell and cell-cycle associated modules were upregulated at days 7 and 14, while at day 28, no modules remained activated. At day 14, a direct correlation was observed between ZEBOV glycoprotein-specific antibody titres and activation of seven BTMs, including two related to B-cell activation and B cell receptor signalling. Transcriptomic analysis identified an rVSVΔG-ZEBOV-GP-induced signature and demonstrated a direct correlation of blood transcriptomic changes with ZEBOV glycoprotein-specific antibody titres.

Evaluation of novel HIV vaccine candidates using recombinant vesicular stomatitis virus vector produced in serum-free Vero cell cultures

Acquired Immune Deficiency Syndrome (AIDS) in humans is a result of the destruction of the immune system caused by Human Immunodeficiency Virus (HIV) infection. This serious epidemic is still progressing world-wide. Despite advances in treatment, a safe and effective preventive HIV vaccine is desired to combat this disease, and to save millions of lives. However, such a vaccine is not available yet although extensive amounts of resources in research and development have been invested over three decades. In light of the recently approved Ebola virus disease vaccine based on a recombinant vesicular stomatitis virus (rVSV-ZEBOV), we present the results of our work on three novel VSV-vectored HIV vaccine candidates. We describe the design, rescue, production and purification method and evaluate their immunogenicity in mice prior to preclinical studies that will be performed in non-human primates. The production of each of the three candidate vaccines (rVSV-B6-NL4.3Env/SIVtm, rVSV-B6-NL4.3Env/Ebtm and rVSV-B6-A74Env(PN6)/SIVtm) was evaluated in small scale in Vero cells and it was found that production kinetics on Vero cells vary depending on the HIV gp surface protein used. Purified virus preparations complied with the WHO restrictions for the residual DNA and host cell protein contents. Finally, when administered to mice, all three rVSV-HIV vaccine candidates induced an HIV gp140-specific antibody response.

Characterization of rVSVΔG-ZEBOV-GP glycoproteins using automated capillary western blotting

Ebolavirus (EBOV) entry to host cells requires membrane-associated glycoprotein (GP). A recombinant vesicular stomatitis virus vector carrying Zaire Ebola virus glycoprotein (rVSV-ZEBOV) was developed as a vaccine against ebolaviruses. The VSV glycoprotein gene was deleted (rVSVΔG) and ZEBOV glycoprotein (GP) was inserted into the deleted VSV glycoprotein open reading frame (ORF) resulting in a live, replication-competent vector (rVSVΔG-ZEBOV-GP). Automated capillary westerns were used to characterize the rVSVΔG-ZEBOV-GP vaccine (ERVEBO®) manufacturing process with regards to glycoprotein (GP) structure and variants. The method shows a unique electropherogram profile for each process step which could be used to monitor process robustness. rVSVΔG-ZEBOV-GP encodes GP (GP1-GP2), secreted GP (sGP), and small secreted GP (ssGP) variants. Furthermore, a TACE-like activity was observed indirectly by detecting soluble GP2Δ after virus precipitation by ultracentrifugation. Capillary western blotting techniques can guide process development by evaluating process steps such as enzyme treatment. In addition, the technique can assess GP stability and process lot-to-lot consistency. Finally, capillary western-based technology was used to identify a unique biochemical profile of the rVSVΔG-ZEBOV-GP vaccine strain in final product. Virion membrane-bound GP1-GP2 is critical to vaccine-elicited protection by providing both neutralizing antibodies and T-cell response.

Advances in Designing and Developing Vaccines, Drugs, and Therapies to Counter Ebola Virus.

Ebola virus (EBOV), a member of the family Filoviridae, is responsible for causing Ebola virus disease (EVD) (formerly named Ebola hemorrhagic fever). This is a severe, often fatal illness with mortality rates varying from 50 to 90% in humans. Although the virus and associated disease has been recognized since 1976, it was only when the recent outbreak of EBOV in 2014–2016 highlighted the danger and global impact of this virus, necessitating the need for coming up with the effective vaccines and drugs to counter its pandemic threat. Albeit no commercial vaccine is available so far against EBOV, a few vaccine candidates are under evaluation and clinical trials to assess their prophylactic efficacy. These include recombinant viral vector (recombinant vesicular stomatitis virus vector, chimpanzee adenovirus type 3-vector, and modified vaccinia Ankara virus), Ebola virus-like particles, virus-like replicon particles, DNA, and plant-based vaccines. Due to improvement in the field of genomics and proteomics, epitope-targeted vaccines have gained top priority. Correspondingly, several therapies have also been developed, including immunoglobulins against specific viral structures small cell-penetrating antibody fragments that target intracellular EBOV proteins. Small interfering RNAs and oligomer-mediated inhibition have also been verified for EVD treatment. Other treatment options include viral entry inhibitors, transfusion of convalescent blood/serum, neutralizing antibodies, and gene expression inhibitors. Repurposed drugs, which have proven safety profiles, can be adapted after high-throughput screening for efficacy and potency for EVD treatment. Herbal and other natural products are also being explored for EVD treatment. Further studies to better understand the pathogenesis and antigenic structures of the virus can help in developing an effective vaccine and identifying appropriate antiviral targets. This review presents the recent advances in designing and developing vaccines, drugs, and therapies to counter the EBOV threat.

Postexposure Efficacy of Recombinant Vesicular Stomatitis Virus Vectors Against High and Low Doses of Marburg Virus Variant Angola in Nonhuman Primates.

A recombinant vesicular stomatitis virus (rVSV) expressing the Marburg virus (MARV) Musoke variant glycoprotein fully protects macaques against 2 MARV variants and Ravn virus as a preventive vaccine and MARV variant Musoke as a postexposure treatment. To evaluate postexposure efficacy against the most pathogenic MARV variant, Angola, we engineered rVSVs expressing homologous Angola glycoprotein. Macaques were challenged with high or low doses of variant Angola and treated 20–30 minutes after exposure. A total of 25% and 60%–75% of treated macaques survived the high-dose and low-dose challenges, respectively. The more rapid disease progression of variant Angola versus variant Musoke may account for the incomplete protection observed.

PET Imaging of Oncolytic VSV Expressing the Mutant HSV-1 Thymidine Kinase Transgene in a Preclinical HCC Rat Model

Hepatocellular carcinoma (HCC) is the most predominant form of liver cancer and the third leading cause of cancer-related death worldwide. Due to the relative ineffectiveness of conventional HCC therapies, oncolytic viruses have emerged as novel alternative treatment agents. Our previous studies have demonstrated significant prolongation of survival in advanced HCC in rats after oncolytic vesicular stomatitis virus (VSV) treatment. In this study, we aimed to establish a reporter system to reliably and sensitively image VSV in a clinically relevant model of HCC for clinical translation. To this end, an orthotopic, unifocal HCC model in immune-competent Buffalo rats was employed to test a recombinant VSV vector encoding for an enhanced version of the herpes simplex virus 1 (HSV-1) thymidine kinase (sr39tk) reporter, which would allow the indirect detection of VSV via positron emission tomography (PET). The resulting data revealed specific tracer uptake in VSV-HSV1-sr39tk-treated tumors. Further characterization of the VSV-HSV1-sr39tk vector demonstrated its optimal detection time-point after application and its detection limit via PET. In conclusion, oncolytic VSV expressing the HSV1-sr39tk reporter gene allows for highly sensitive in vivo imaging via PET. Therefore, this imaging system may be directly translatable and beneficial in further clinical applications.

Development of delivery, targeting, and imaging strategies to improve oncolytic vesicular stomatitis virus therapy for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a disease with rising incidence, limited treatment options and poor prognosis, representing a major worldwide health concern. Due to the vast deficit of viable treatment options, oncolytic viruses have emerged as promising alternative therapies to specifically target and lyse tumor cells. We have previously demonstrated the safety and efficacy of oncolytic vesicular stomatitis virus (VSV) applied via hepatic arterial infusion to treat orthotopic HCC in immune-competent rats, and based on encouraging data such as these, a recombinant VSV vector has recently entered the clinic in phase I trials for HCC in patients. Although systemic administration of oncolytic viral therapies is ideal for targeting metastatic disease, the efficacy of such an approach is extremely compromised due to rapid clearance from circulation as a result of neutralizing blood components and non-specific uptake by liver and spleen. Furthermore, the complex microenvironment in the liver poses a unique set of challenges to the fate of oncolytic viral therapies directed at hepatic tumors. Although attempts to shield viruses from adverse interactions have led to increased circulation times in vivo, they are also associated with decreased infectivity of the target cells. We have therefore developed several strategies, including novel cell carrier and synthetic shielding approaches, to simultaneously protect VSV from nonspecific interactions and deliver it specifically to the tumor target, where it can exert its oncolytic effect. Because a variety of immune cells possess the inherent ability to take up oncolytic virus and subsequently home to tumor beds, they offer the unique dual benefit of delivering viruses in stealth, while contributing to antitumor immune responses. Alternatively, synthetic polymers can provide highly effective shielding through surface modification of oncolytic viruses and have the advantage of being adaptable to optimize the shielding effect. Through the incorporation of targeting ligands or selectively cleavable linkers, the ability of the virus to infect is then rescued locally and specifically at the tumor site. Furthermore, advancement of imaging technologies allows us the possibility to monitor the biodistribution of these shielded viruses to observe the circulation time and replication kinetics noninvasively and in real-time, which will further facilitate the clinical translation of oncolytic virus therapies. A summary of these novel approaches to enhance oncolytic VSV accumulation within orthotopic HCC tumors and improve the therapy outcome, as well as virus imaging strategies, will be presented.

Single injection recombinant vesicular stomatitis virus vaccines protect ferrets against lethal Nipah virus disease.

BackgroundNipah virus (NiV) is a highly pathogenic zoonotic agent in the family Paramyxoviridae that is maintained in nature by bats. Outbreaks have occurred in Malaysia, Singapore, India, and Bangladesh and have been associated with 40 to 75% case fatality rates. There are currently no vaccines or postexposure treatments licensed for combating human NiV infection.Methods and resultsFour groups of ferrets received a single vaccination with different recombinant vesicular stomatitis virus vectors expressing: Group 1, control with no glycoprotein; Group 2, the NiV fusion protein (F); Group 3, the NiV attachment protein (G); and Group 4, a combination of the NiV F and G proteins. Animals were challenged intranasally with NiV 28 days after vaccination. Control ferrets in Group 1 showed characteristic clinical signs of NiV disease including respiratory distress, neurological disorders, viral load in blood and tissues, and gross lesions and antigen in target tissues; all animals in this group succumbed to infection by day 8. Importantly, all specifically vaccinated ferrets in Groups 2-4 showed no evidence of clinical illness and survived challenged. All animals in these groups developed anti-NiV F and/or G IgG and neutralizing antibody titers. While NiV RNA was detected in blood at day 6 post challenge in animals from Groups 2-4, the levels were orders of magnitude lower than animals from control Group 1.ConclusionsThese data show protective efficacy against NiV in a relevant model of human infection. Further development of this technology has the potential to yield effective single injection vaccines for NiV infection.

Non-propagating, recombinant vesicular stomatitis virus vectors encoding respiratory syncytial virus proteins generate potent humoral and cellular immunity against RSV and are protective in mice

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract illness in infants, the elderly, and other high-risk individuals. Despite years of research in this field, there is no effective licensed vaccine to prevent RSV infection. We have generated candidate RSV vaccines using a recombinant vesicular stomatitis virus (rVSV) replicon in which the attachment and fusion domains of the VSV glycoprotein (G) have been deleted (rVSV-Gstem), rendering the virus propagation-defective except in the presence of complementing VSV G provided in trans. A form of this vector encoding the RSV fusion protein (F) gene expressed high levels of F in vitro and elicited durable neutralizing antibody responses as well as complete protection against RSV challenge in vivo. Mice vaccinated with rVSV-Gstem-RSV-F replicons also developed robust cellular responses characterized by both primary and memory Th1-biased CD8+ and CD4+ T cells. Furthermore, a single high dose of the Gstem-RSV-F replicon was effective against challenge with both RSV A and B subgroup viruses. Finally, addition of an RSV glycoprotein (G)-expressing Gstem vector significantly improved the incomplete protection achieved with a single low dose of Gstem-RSV-F vector alone.

The potential of recombinant vesicular stomatitis virus-mediated virotherapy against metastatic colon cancer

Colorectal cancer (CRC) is the fourth most frequently diagnosed cancer and the second leading cause of cancer-related mortality in the United States. The liver and lung are the most common sites of distant metastasis of CRC. The approval of newer chemotherapeutic agents such as oxaliplatin, irinotecan, bevacizumab, cetuximab and panitumumab has significantly improved survival, yet the majority of patients still succumb to the disease in less than 2 years. Novel therapeutic agents that can provide significant clinical benefit for metastatic CRC patients are needed. Oncolytic vesicular stomatitis virus (VSV) is a promising tool as a cancer therapeutic agent. In this study, we examined the feasibility of repeated intravenous infusions of rVSV in multiple CRC lung metastases, compared with repeated hepatic arterial administration in multifocal CRC liver metastasis in immune competent rats. We established a multifocal liver metastases model or the multiple lung metastases model using a CRC cell line, RCN-H4, implanted into syngeneic F344/DuCrj rats. 4.0x10(6) plaque-forming units (pfu) of recombinant VSV vectors expressing mutant (L289A) Newcastle disease virus fusion protein [rVSV-NDV/F(L289A)] were administered 3 times for 3 consecutive days locally via the hepatic artery for liver metastases or systemically via the penial vein for lung metastases. In the liver metastasis model, significantly enhanced survival was observed with rVSV-NDV/F(L289A)-treated rats (P=0.0196). Median survival was 110 and 25 days, respectively. In addition, 4 out of 7 of the rVSV-NDV/F(L289A)-treated rats demonstrated long-term survival exceeding 100 days. The long-term surviving rats were sacrificed to evaluate for residual malignancy. Liver tumors were not detected. In the lung metastasis model, median survival was 10 [VSV-NDV/F(L289A)-treated rats] and 7 days (control). Although survival was significantly prolonged (P<0.001), none of the rats achieved long-term survival. VSV virotherapy has potential for CRC liver and lung metastases, although systemic venous delivery is much less effective than locoregional delivery such as hepatic arterial infusion.

O004 Using type III interferon to improve the efficacy of vaccine and oncolytic viral vectors

Introduction Vaccines based on replicating viral vectors that express foreign antigens can generate superior durable antibody and memory CD8 T cell responses compared to inactivated virus or recombinant protein, with the added benefit of potential needle-free intranasal administration. Despite these significant advantages, safety concerns related to viral vectors as vaccine platforms can be an issue with this approach. Replicating viral vectors are perceived as less safe alternatives compared to other immunization modalities, especially in the context of intranasal administration, where the potential exists for systemic virus spread from the lung. The type III interferons (IL-29, IL-28A, IL-28B; also known as IFN-lambda1, 2, and 3) play a critical role in protecting epithelial cells in the lung and gut from virus infection, and can also stimulate adaptive immunity. We therefore examined the ability of this cytokine family to improve the safety and efficacy of a viral vector by limiting its systemic spread and vector-associated pathological effects after intranasal delivery. Methods Vesicular stomatitis virus (VSV) based vectors are promising candidates for use as vaccines and oncolytic agents. We generated recombinant VSV vectors expressing mouse IL-28A either from the fifth position or the more highly expressing first position of the viral genome, and measured virus replication in cell culture and mice. Results We found that IL-28A is properly expressed, secreted, and active when produced by VSV-infected cells. In cell culture, expression of IL-28A from VSV attenuated virus replication in type III IFN-sensitive cells, but not in type III IFN-resistant cells. Following intranasal inoculation in mice, spread of the VSV.IL28A vector from the lung to the lymph nodes, spleen, blood, liver, and brain was significantly reduced compared to recombinant wild-type VSV. Despite this attenuation, the VSV.mIL28A vector generated virus-specific CD8 T cell and antibody responses in mice that were comparable to wild-type VSV. Conclusion These studies demonstrate the ability of the type III interferon cytokine family to improve the safety and efficacy of a clinically promising viral vaccine vector. These results may potentially also inform the enhancement of VSV oncolytic activity by increasing selectivity of the virus for type III IFN-resistant tumor cells.

Use of replication restricted recombinant vesicular stomatitis virus vectors for detection of antigen-specific T cells

Detection of antigen-specific T cells at the single-cell level by ELISpot or flow cytometry techniques employing intracellular cytokine staining (ICS) is now an indispensable tool in many areas of immunology. When precisely mapped, optimal MHC-binding peptide epitopes are unknown, these assays use antigen in a variety of forms, including recombinant proteins, overlapping peptide sets representing one or more target protein sequences, microbial lysates, lysates of microbially-infected cells, or gene delivery vectors such as DNA expression plasmids or recombinant vaccinia or adenoviruses expressing a target protein of interest. Here we introduce replication-restricted, recombinant vesicular stomatitis virus (VSV) vectors as a safe, easy to produce, simple to use, and highly effective vector for genetic antigen delivery for the detection of human antigen-specific helper and cytotoxic T cells. To demonstrate the broad applicability of this approach, we have used these vectors to detect human T cell responses to the immunodominant pp65 antigen of human cytomegalovirus, individual segments of the yellow fever virus polyprotein, and to various influenza proteins.

Vesicular Stomatitis Virus-Based Ebola Vaccine Is Well-Tolerated and Protects Immunocompromised Nonhuman Primates

Ebola virus (EBOV) is a significant human pathogen that presents a public health concern as an emerging/re-emerging virus and as a potential biological weapon. Substantial progress has been made over the last decade in developing candidate preventive vaccines that can protect nonhuman primates against EBOV. Among these prospects, a vaccine based on recombinant vesicular stomatitis virus (VSV) is particularly robust, as it can also confer protection when administered as a postexposure treatment. A concern that has been raised regarding the replication-competent VSV vectors that express EBOV glycoproteins is how these vectors would be tolerated by individuals with altered or compromised immune systems such as patients infected with HIV. This is especially important as all EBOV outbreaks to date have occurred in areas of Central and Western Africa with high HIV incidence rates in the population. In order to address this concern, we evaluated the safety of the recombinant VSV vector expressing the Zaire ebolavirus glycoprotein (VSVΔG/ZEBOVGP) in six rhesus macaques infected with simian-human immunodeficiency virus (SHIV). All six animals showed no evidence of illness associated with the VSVΔG/ZEBOVGP vaccine, suggesting that this vaccine may be safe in immunocompromised populations. While one goal of the study was to evaluate the safety of the candidate vaccine platform, it was also of interest to determine if altered immune status would affect vaccine efficacy. The vaccine protected 4 of 6 SHIV-infected macaques from death following ZEBOV challenge. Evaluation of CD4+ T cells in all animals showed that the animals that succumbed to lethal ZEBOV challenge had the lowest CD4+ counts, suggesting that CD4+ T cells may play a role in mediating protection against ZEBOV.

Foreign Gene Transfer in Termite Cells Using a Recombinant Vesicular Stomatitis Virus

The Formosan subterranean termite, Coptotermes formosanus Shiraki, and the eastern subterranean termite, Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae), are well known for their destruction of human dwellings and flora in the tropics and subtropics. A method to deliver foreign genes into termite cell cultures would provide a controlled environment to facilitate the study of key regulatory functions at the molecular and cellular level. Here a method for the establishment and cryopreservation of primary embryonic termite cell cultures is described. Evidence is presented of viral-mediated gene transfer in these cells and foreign gene expression using a recombinant vesicular stomatitis virus vector.

Recombinant Vesicular Stomatitis Virus Vector Mediates Postexposure Protection against Sudan Ebola Hemorrhagic Fever in Nonhuman Primates

Recombinant vesicular stomatitis virus (VSV) vectors expressing homologous filoviral glycoproteins can completely protect rhesus monkeys against Marburg virus when administered after exposure and can partially protect macaques after challenge with Zaire ebolavirus. Here, we administered a VSV vector expressing the Sudan ebolavirus (SEBOV) glycoprotein to four rhesus macaques shortly after exposure to SEBOV. All four animals survived SEBOV challenge, while a control animal that received a nonspecific vector developed fulminant SEBOV hemorrhagic fever and succumbed. This is the first demonstration of complete postexposure protection against an Ebola virus in nonhuman primates and provides further evidence that postexposure vaccination may have utility in treating exposures to filoviruses.