Susan E. Witko

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.

Recombinant Vesicular Stomatitis Virus Vectors Expressing Herpes Simplex Virus Type 2 gD Elicit Robust CD4+ Th1 Immune Responses and Are Protective in Mouse and Guinea Pig Models of Vaginal Challenge

ABSTRACT Recombinant vesicular stomatitis virus (rVSV) vectors offer an attractive approach for the induction of robust cellular and humoral immune responses directed against human pathogen target antigens. We evaluated rVSV vectors expressing full-length glycoprotein D (gD) from herpes simplex virus type 2 (HSV-2) in mice and guinea pigs for immunogenicity and protective efficacy against genital challenge with wild-type HSV-2. Robust Th1-polarized anti-gD immune responses were demonstrated in the murine model as measured by induction of gD-specific cytotoxic T lymphocytes and increased gamma interferon expression. The isotype makeup of the serum anti-gD immunoglobulin G (IgG) response was consistent with the presence of a Th1-CD4+ anti-gD response, characterized by a high IgG2a/IgG1 IgG subclass ratio. Functional anti-HSV-2 neutralizing serum antibody responses were readily demonstrated in both guinea pigs and mice that had been immunized with rVSV-gD vaccines. Furthermore, guinea pigs and mice were prophylactically protected from genital challenge with high doses of wild-type HSV-2. In addition, guinea pigs were highly protected against the establishment of latent infection as evidenced by low or absent HSV-2 genome copies in dorsal root ganglia after virus challenge. In summary, rVSV-gD vectors were successfully used to elicit potent anti-gD Th1-like cellular and humoral immune responses that were protective against HSV-2 disease in guinea pigs and mice.

Neurovirulence and Immunogenicity of Attenuated Recombinant Vesicular Stomatitis Viruses in Nonhuman Primates

ABSTRACT 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.

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.