VSV-GP: a potent viral vaccine vector that boosts the immune response upon repeated applications.

UNLABELLED: Antivector immunity limits the response to homologous boosting for viral vector vaccines. Here, we describe a new, potent vaccine vector based on replication-competent vesicular stomatitis virus pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (VSV-GP), which we previously showed to be safe in mice. In mice, VSV and VSV-GP encoding ovalbumin (OVA) as a model antigen (VSV-OVA and VSV-GP-OVA) induced equal levels of OVA-specific humoral and cellular immune responses upon a single immunization. However, boosting with the same vector was possible only for VSV-GP-OVA as neutralizing antibodies to VSV limited the immunogenicity of the VSV-OVA boost. OVA-specific cytotoxic T-lymphocyte (CTL) responses induced by VSV-GP-OVA were at least as potent as those induced by an adenoviral state-of-the-art vaccine vector and completely protected mice in a Listeria monocytogenes challenge model. VSV-GP is so far the only replication-competent vaccine vector that does not lose efficacy upon repeated application. IMPORTANCE: Although there has been great progress in treatment and prevention of infectious diseases in the past several years, effective vaccines against some of the most serious infections, e.g., AIDS, malaria, hepatitis C, or tuberculosis, are urgently needed. Here, several approaches based on viral vector vaccines are under development. However, for all viral vaccine vectors currently in clinical testing, repeated application is limited by neutralizing antibodies to the vector itself. Here, we have exploited the potential of vesicular stomatitis virus pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (VSV-GP) as a vaccine platform. VSV-GP is the first replication-competent viral vector vaccine that does not induce vector-specific humoral immunity, i.e., neutralizing antibodies, and therefore can boost immune responses against a foreign antigen by repeated applications. The vector allows introduction of various antigens and therefore can serve as a platform technology for the development of novel vaccines against a broad spectrum of diseases.

C peptides as entry inhibitors for gene therapy.

Peptides derived from the C-terminal heptad repeat 2 region of the HIV-1 gp41 envelope glycoprotein, so-called C peptides, are very potent HIV-1 fusion inhibitors. Antiviral genes encoding either membrane-anchored (ma) or secreted (iSAVE) C peptides have been engineered and allow direct in vivo production of the therapeutic peptides by genetically modified host cells. Membrane-anchored C peptides expressed in the HIV-1 target cells by T-cell or hematopoietic stem cell gene therapy efficiently prevent virus entry into the modified cells. Such gene-protection confers a selective survival advantage and allows accumulation of the genetically modified cells. Membrane-anchored C peptides have been successfully tested in a nonhuman primate model of AIDS and were found to be safe in a phase I clinical trial in AIDS patients transplanted with autologous gene-modified T-cells. Secreted C peptides have the crucial advantage of not only protecting genetically modified cells from HIV-1 infection, but also neighboring cells, thus suppressing virus replication even if only a small fraction of cells is genetically modified. Accordingly, various cell types can be considered as potential in vivo producer cells for iSAVE-based gene therapeutics, which could even be modified by direct in vivo gene delivery in future. In conclusion, C peptide gene therapeutics may provide a strong benefit to AIDS patients and could present an effective alternative to current antiretroviral drug regimens.

A rationally engineered anti-HIV peptide fusion inhibitor with greatly reduced immunogenicity.

Peptides derived from the C-terminal heptad repeat 2 (HR2) region of the HIV-1 gp41 envelope glycoprotein, so-called C peptides, are very efficient HIV-1 fusion inhibitors. We previously developed innovative gene therapeutic approaches aiming at the direct in vivo production of C peptides from genetically modified host cells and found that T cells expressing membrane-anchored or secreted C peptides are protected from HIV-1 infection. However, an unwanted immune response against such antiviral peptides may significantly impair clinical efficacy and pose safety risks to patients. To overcome this problem, we engineered a novel C peptide, V2o, with greatly reduced immunogenicity and excellent antiviral activity. V2o is based on the chimeric C peptide C46-EHO, which is derived from the HR2 regions of HIV-2(EHO) and HIV-1(HxB2) and has broad anti-HIV and anti-simian immunodeficiency virus activity. Antibody and major histocompatibility complex class I epitopes within the C46-EHO peptide sequence were identified by in silico and in vitro analyses. Using rational design, we removed these epitopes by amino acid substitutions and thus minimized antigenicity and immunogenicity considerably. At the same time, the antiviral activity of the deimmunized peptide V2o was preserved or even enhanced compared to that of the parental C46-EHO peptide. Thus, V2o is an ideal candidate, especially for those novel therapeutic approaches for HIV infection that involve direct in vivo production of antiviral C peptides.

Secreted antiviral entry inhibitory (SAVE) peptides for gene therapy of HIV infection.

Gene therapeutic strategies for human immunodeficiency virus type 1 (HIV-1) infection could potentially overcome the limitations of standard antiretroviral drug therapy (ART). However, in none of the clinical gene therapy trials published to date, therapeutic levels of genetic protection have been achieved in the target cell population for HIV-1. To improve systemic antiviral efficacy, C peptides, which are efficient inhibitors of HIV-1 entry, were engineered for high-level secretion by genetically modified cells. The size restrictions for efficient peptide export through the secretory pathway were overcome by expressing the C peptides as concatemers, which were processed into monomers by furin protease cleavage. These secreted antiviral entry inhibitory (SAVE) peptides mediated a substantial protective bystander effect on neighboring nonmodified cells, thus suppressing virus replication even if only a small fraction of cells was genetically modified. Accordingly, these SAVE peptides may provide a strong benefit to AIDS patients in future, and, if applied by direct in vivo gene delivery, could present an effective alternative to antiretroviral drug regimen.

Mutations in gp120 contribute to the resistance of human immunodeficiency virus type 1 to membrane-anchored C-peptide maC46.

Binding of the human immunodeficiency virus (HIV) envelope glycoprotein (Env) to the cellular CD4 receptor and a chemokine coreceptor initiates a series of conformational changes in the Env subunits gp120 and gp41. Eventually, the trimeric gp41 folds into a six-helix bundle, thereby inducing fusion of the viral and cellular membranes. C peptides derived from the C-terminal heptad repeat (CHR) of gp41 are efficient entry inhibitors as they block the six-helix bundle formation. Previously, we developed a membrane-anchored C peptide (maC46) expressed from a retroviral vector that also shows high activity against virus strains resistant to enfuvirtide (T-20), an antiviral C peptide approved for clinical use. Here, we present a systematic analysis of mutations in Env that confer resistance of HIV type 1 (HIV-1) to maC46. We selected an HIV-1 BaL strain with 10-fold reduced sensitivity to maC46 (BaL_C46) by passaging virus for nearly 200 days in the presence of gradually increasing concentrations of maC46. In comparison to wild-type BaL, BaL_C46 had five mutations at highly conserved positions in Env, three in gp120, one in the N-terminal heptad-repeat (NHR), and one in the CHR of gp41. No mutations were found in the NHR domain around the GIV motif that are known to cause resistance to enfuvirtide. Instead, maC46 resistance was found to depend on complementary mutations in the NHR and CHR that considerably favor binding of the mutated NHR to the mutated CHR over binding to maC46. In addition, resistance was highly dependent on mutations in gp120 that accelerated entry. Taken together, resistance to maC46 did not develop readily and required multiple cooperating mutations at conserved positions of the viral envelope glycoproteins gp120 and gp41.

Application of interferon modulators to overcome partial resistance of human ovarian cancers to VSV-GP oncolytic viral therapy.

Previously, we described an oncolytic vesicular stomatitis virus variant pseudotyped with the nonneurotropic glycoprotein of the lymphocytic choriomeningitis virus, VSV-GP, which was highly effective in glioblastoma. Here, we tested its potency for the treatment of ovarian cancer, a leading cause of death from gynecological malignancies. Effective oncolytic activity of VSV-GP could be demonstrated in ovarian cancer cell lines and xenografts in mice; however, remission was temporary in most mice. Analysis of the innate immune response revealed that ovarian cancer cell lines were able to respond to and produce type I interferon, inducing an antiviral state upon virus infection. This is in stark contrast to published data for other cancer cell lines, which were mostly found to be interferon incompetent. We showed that in vitro this antiviral state could be reverted by combining VSV-GP with the JAK1/2-inhibitor ruxolitinib. In addition, for the first time, we report the in vivo enhancement of oncolytic virus treatment by ruxolitinib, both in subcutaneous as well as in orthotopic xenograft mouse models, without causing significant additional toxicity. In conclusion, VSV-GP has the potential to be a potent and safe oncolytic virus to treat ovarian cancer, especially when combined with an inhibitor of the interferon response.