Neurovirulence and immunogenicity of attenuated recombinant vesicular stomatitis viruses in nonhuman primates.

UNLABELLED: In previous work, a prototypic recombinant vesicular stomatitis virus Indiana serotype (rVSIV) vector expressing simian immunodeficiency virus (SIV) gag and human immunodeficiency virus type 1 (HIV-1) env antigens protected nonhuman primates (NHPs) from disease following challenge with an HIV-1/SIV recombinant (SHIV). However, when tested in a stringent NHP neurovirulence (NV) model, this vector was not adequately attenuated for clinical evaluation. For the work described here, the prototypic rVSIV vector was attenuated by combining specific G protein truncations with either N gene translocations or mutations (M33A and M51A) that ablate expression of subgenic M polypeptides, by incorporation of temperature-sensitive mutations in the N and L genes, and by deletion of the VSIV G gene to generate a replicon that is dependent on trans expression of G protein for in vitro propagation. When evaluated in a series of NHP NV studies, these attenuated rVSIV variants caused no clinical disease and demonstrated a very significant reduction in neuropathology compared to wild-type VSIV and the prototypic rVSIV vaccine vector. In spite of greatly increased in vivo attenuation, some of the rVSIV vectors elicited cell-mediated immune responses that were similar in magnitude to those induced by the much more virulent prototypic vector. These data demonstrate novel approaches to the rational attenuation of VSIV NV while retaining vector immunogenicity and have led to identification of an rVSIV N4CT1gag1 vaccine vector that has now successfully completed phase I clinical evaluation. IMPORTANCE: The work described in this article demonstrates a rational approach to the attenuation of vesicular stomatitis virus neurovirulence. The major attenuation strategy described here will be most likely applicable to other members of the Rhabdoviridae and possibly other families of nonsegmented negative-strand RNA viruses. These studies have also enabled the identification of an attenuated, replication-competent rVSIV vector that has successfully undergone its first clinical evaluation in humans. Therefore, these studies represent a major milestone in the development of attenuated rVSIV, and likely other vesiculoviruses, as a new vaccine platform(s) for use in humans.

Nonreplicating vaccines can protect african green monkeys from the memphis 37 strain of respiratory syncytial virus.

BACKGROUND: We evaluated the immunological responses of African green monkeys immunized with multiple F and G protein-based vaccines and assessed protection against the Memphis 37 strain of respiratory syncytial virus (RSV). METHODS: Monkeys were immunized with F and G proteins adjuvanted with immunostimulatory (CpG) oligodeoxyribonucleotides admixed with either Alhydrogel or ISCOMATRIX adjuvant. Delivery of F and G proteins via replication incompetent recombinant vesicular stomatitis viruses (VSVs) and human adenoviruses was also evaluated. Mucosally or parenterally administered recombinant adenoviruses were used in prime-boost regimens with adjuvanted proteins or recombinant DNA. RESULTS: Animals primed by intranasal delivery of recombinant adenoviruses, and boosted by intramuscular injection of adjuvanted F and G proteins, developed neutralizing antibodies and F/G protein-specific T cells and were protected from RSV infection. Intramuscular injections of Alhydrogel (plus CpG) adjuvanted F and G proteins reduced peak viral loads in the lungs of challenged monkeys. Granulocyte numbers were not significantly elevated, relative to controls, in postchallenge bronchoalveolar lavage samples from vaccinated animals. CONCLUSIONS: This study has validated the use of RSV (Memphis 37) in an African green monkey model of intranasal infection and identified nonreplicating vaccines capable of eliciting protection in this higher species challenge model.

A novel approach to generate a recombinant toxoid vaccine against Clostridium difficile.

The Clostridium difficile toxins A and B are primarily responsible for symptoms of C. difficile associated disease and are prime targets for vaccine development. We describe a plasmid-based system for the production of genetically modified toxins in a non-sporulating strain of C. difficile that lacks the toxin genes tcdA and tcdB. TcdA and TcdB mutations targeting established glucosyltransferase cytotoxicity determinants were introduced into recombinant plasmids and episomally expressed toxin mutants purified from C. difficile transformants. TcdA and TcdB mutants lacking glucosyltransferase and autoproteolytic processing activities were ~10 000-fold less toxic to cultured human IMR-90 cells than corresponding recombinant or native toxins. However, both mutants retained residual cytotoxicity that could be prevented by preincubating the antigens with specific antibodies or by formalin treatment. Such non-toxic formalin-treated mutant antigens were immunogenic and protective in a hamster model of infection. The remaining toxicity of untreated TcdA and TcdB mutant antigens was associated with cellular swelling, a phenotype consistent with pore-induced membrane leakage. TcdB substitution mutations previously shown to block vesicular pore formation and toxin translocation substantially reduced residual toxicity. We discuss the implications of these results for the development of a C. difficile toxoid vaccine.

Quadrivalent VesiculoVax vaccine protects nonhuman primates from viral-induced hemorrhagic fever and death.

Recent occurrences of filoviruses and the arenavirus Lassa virus (LASV) in overlapping endemic areas of Africa highlight the need for a prophylactic vaccine that would confer protection against all of these viruses that cause lethal hemorrhagic fever (HF). We developed a quadrivalent formulation of VesiculoVax that contains recombinant vesicular stomatitis virus (rVSV) vectors expressing filovirus glycoproteins and that also contains a rVSV vector expressing the glycoprotein of a lineage IV strain of LASV. Cynomolgus macaques were vaccinated twice with the quadrivalent formulation, followed by challenge 28 days after the boost vaccination with each of the 3 corresponding filoviruses (Ebola, Sudan, Marburg) or a heterologous contemporary lineage II strain of LASV. Serum IgG and neutralizing antibody responses specific for all 4 glycoproteins were detected in all vaccinated animals. A modest and balanced cell-mediated immune response specific for the glycoproteins was also detected in most of the vaccinated macaques. Regardless of the level of total glycoprotein-specific immune response detected after vaccination, all immunized animals were protected from disease and death following lethal challenges. These findings indicate that vaccination with attenuated rVSV vectors each expressing a single HF virus glycoprotein may provide protection against those filoviruses and LASV most commonly responsible for outbreaks of severe HF in Africa.

Safety and immunogenicity of a highly attenuated rVSVN4CT1-EBOVGP1 Ebola virus vaccine: a randomised, double-blind, placebo-controlled, phase 1 clinical trial.

BACKGROUND: The safety and immunogenicity of a highly attenuated recombinant vesicular stomatitis virus (rVSV) expressing HIV-1 gag (rVSVN4CT1-HIV-1gag1) was shown in previous phase 1 clinical studies. An rVSV vector expressing Ebola virus glycoprotein (EBOV-GP) in place of HIV-1 gag (rVSVN4CT1-EBOVGP1) showed single-dose protection from lethal challenge with low passage Ebola virus in non-human primates. We aimed to evaluate the safety and immunogenicity of the rVSVN4CT1-EBOVGP1 vaccine in healthy adults. METHODS: We did a randomised double-blind, placebo-controlled, phase 1 dose-escalation study at a single clinical site (Optimal Research) in Melbourne, FL, USA. Eligible participants were healthy men and non-pregnant women aged 18-60 years, with a body-mass index (BMI) of less than 40 kg/m2, no history of filovirus infection, VSV infection, or receipt of rVSV in previous studies, and who had not visited regions where Ebola virus outbreaks have occurred. Three cohorts were enrolled to assess a low (2·5 × 104 plaque forming units [PFU]), intermediate (2 × 105 PFU), or high dose (1·8 × 106 PFU) of the vaccine. Participants within each cohort were randomly allocated (10:3) to receive vaccine or placebo by intramuscular injection in a homologous prime and boost regimen, with 4 weeks between doses. All syringes were masked with syringe sleeves; participants and study site staff were not blinded to dose level but were blinded to active vaccine and placebo. The primary outcomes were safety and tolerability; immunogenicity, assessed as GP-specific humoral immune response (at 2 weeks after each dose) and cellular immune response (at 1 and 2 weeks after each dose), was a secondary outcome. All randomised participants were included in primary and safety analyses. This trial is registered with ClinicalTrials.gov, NCT02718469. FINDINGS: Between Dec 22, 2015, and Sept 15, 2016, 39 individuals (18 [46%] men and 21 [54%] women, mean age 51 years [SD 10]) were enrolled, with ten participants receiving the vaccine and three participants receiving placebo in each of three cohorts. One participant in the intermediate dose cohort was withdrawn from the study because of a diagnosis of invasive ductal breast carcinoma 24 days after the first vaccination, which was considered unrelated to the vaccine. No severe adverse events were observed. Solicited local adverse events occurred in ten (26%) of 39 participants after the first dose and nine (24%) of 38 participants after the second dose; the events lasted 3 days or less, were predominantly injection site tenderness (17 events) and injection site pain (ten events), and were either mild (19 events) or moderate (ten events) in intensity. Systemic adverse events occurred in 13 (33%) of 39 participants after the first dose and eight (21%) of 38 participants after the second dose; the events were mild (45 events) or moderate (11 events) in severity, and the most common events were malaise or fatigue (13 events) and headache (12 events). Arthritis and maculopapular, vesicular, or purpuric rash distal to the vaccination site(s) were not reported. A GP-specific IgG response was detected in all vaccine recipients after two doses (and IgG response frequency was 100% after a single high dose), and an Ebola virus neutralising response was detected in 100% of participants in the high-dose cohort. INTERPRETATION: The rVSVN4CT1-EBOVGP1 vaccine was well tolerated at all dose levels tested and was immunogenic despite a high degree of attenuation. The combined safety and immunogenicity profile of the rVSVN4CT1-EBOVGP1 vaccine vector support phase 1-2 clinical evaluation. FUNDING: US Department of Defense Joint Program Executive Office for Chemical, Biological, Radiological and Nuclear Defense: Joint Project Manager for Chemical, Biological, Radiological and Nuclear Medical.

An efficient helper-virus-free method for rescue of recombinant paramyxoviruses and rhadoviruses from a cell line suitable for vaccine development.

Recovery of recombinant, negative-strand, nonsegmented RNA viruses from a genomic cDNA clone requires a rescue system that promotes de novo assembly of a functional ribonucleoprotein (RNP) complex in the cell cytoplasm. This is accomplished typically by cotransfecting permissive cells with multiple plasmids that encode the positive-sense genomic RNA, the nucleocapsid protein (N or NP), and the two subunits of the viral RNA-dependent RNA polymerase (L and P). The transfected plasmids are transcribed in the cell cytoplasm by phage T7 RNA polymerase (T7 RNAP), which usually is supplied by infection with a recombinant vaccinia virus or through use of a stable cell line that expresses the polymerase. Although both methods of providing T7 RNAP are effective neither is ideal for viral vaccine development for a number of reasons. Therefore, it was necessary to modify existing technology to make it possible to routinely rescue a variety of recombinant viruses when T7 RNAP was provided by a cotransfected expression plasmid. Development of a broadly applicable procedure required optimization of the helper-virus-free methodology, which resulted in several modifications that improved rescue efficiency such as inclusion of plasmids encoding viral glycoproteins and matrix protein, heat shock treatment, and use of electroporation. The combined effect of these enhancements produced several important benefits including: (1) a helper-virus-free methodology capable of rescuing a diverse variety of paramyxoviruses and recombinant vesicular stomatitis virus (rVSV); (2) methodology that functioned effectively when using Vero cells, a suitable substrate for vaccine production; and (3) a method that enabled rescue of highly attenuated recombinant viruses, which had proven refractory to rescue using published procedures.

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.

Refined methods for propagating vesicular stomatitis virus vectors that are defective for G protein expression.

Propagation-defective vesicular stomatitis virus (VSV) vectors that encode a truncated G protein (VSV-Gstem) or lack the G gene entirely (VSV-DeltaG) are attractive vaccine vectors because they are immunogenic, cannot replicate and spread after vaccination, and do not express many of the epitopes that elicit neutralizing anti-VSV immunity. To consider advancing non-propagating VSV vectors towards clinical assessment, scalable technology that is compliant with human vaccine manufacturing must be developed to produce clinical trial material. Accordingly, two propagation methods were developed for VSV-Gstem and VSV-DeltaG vectors encoding HIV gag that have the potential to support large-scale production. One method is based on transient expression of G protein after electroporating plasmid DNA into Vero cells and the second is based on a stable Vero cell line that contains a G gene controlled by a heat shock-inducible transcription unit. Both methods reproducibly supported production of 1 x 10(7) to 1 x 10(8) infectious units (I.U.s) of vaccine vector per milliliter. Results from these studies also showed that optimization of the G gene is necessary for abundant G protein expression from electroporated plasmid DNA or from DNA integrated in the genome of a stable cell line, and that the titers of VSV-Gstem vectors generally exceeded VSV-DeltaG.

Inhibition of measles virus minireplicon-encoded reporter gene expression by V protein.

Measles virus V protein is a Cys-rich polypeptide that is dispensable for virus propagation in continuous cell lines, but necessary for efficient viral replication in animals. Those functions modulating virus propagation in vivo are not understood completely, although V protein is known to interfere with the host interferon response and control of viral gene expression. The ability to modulate gene expression was investigated further with a minireplicon transient expression system in which V protein was found to repress reporter activity. Two regions of the polypeptide contributed to this repressive effect including the carboxy-terminus and a region conserved in morbillivirus V proteins located between amino acids 110-131, whereas domains known to mediate the interaction between V and the nucleocapsid (N) protein were not essential. Accumulation of encapsidated minigenome in transfected cells was inhibited by V protein suggesting that it acted as a repressor of genome replication thereby limiting availability of template for reporter gene mRNA transcription.

Role of V protein RNA binding in inhibition of measles virus minigenome replication.

Measles virus V protein represses genome replication through a poorly understood mechanism, which led us to investigate whether V protein might be an RNA-binding modulatory factor. Recombinant V protein, expressed from transfected HEp-2 cells or E. coli, formed protein-RNA complexes with poly-guanosine (poly-G) or poly-U linked to agarose beads. RNA binding was not exclusive to ribonucleotide homopolymers as complex formation between V protein and an RNA molecule equivalent to the 3' terminal 107 bases of the measles virus genome was observed with an electrophoretic mobility shift assay (EMSA). The interaction with poly-G was used to further examine the RNA binding properties of V demonstrating that protein-RNA complex formation was dependent upon the unique Cys-rich carboxy terminus, a region also required to induce maximal repression of minireplicon-encoded reporter gene expression in transient assays. Surprisingly, two mutant proteins that contained Cys-to-Ala substitutions in the C-terminus were found to retain their ability to bind poly-G binding and repress minireplicon reporter gene expression indicating that neither activity was dependent on the integrity of all 7 C-terminal Cys residues. Additional genetic analysis revealed that amino acids 238-266 were necessary for efficient RNA binding and overlapped with residues (238-278) required for maximal repression induced by the C-terminal domain. In addition, a 10 amino acid deletion was identified (residues 238-247) that blocked RNA binding and repression indicating that these two activities were related.