A single-cycle vaccine vector based on vesicular stomatitis virus can induce immune responses comparable to those generated by a replication-competent vector

Live attenuated vaccine vectors based on recombinant vesicular stomatitis virus (VSV) are effective in several viral disease models. In this study, we asked if a VSV vector capable of only a single cycle of replication might be an effective alternative to replication-competent VSV vectors. We compared the cellular immune responses to human immunodeficiency virus (HIV) envelope protein (Env) expressed by replication-competent and single-cycle VSV vectors and also examined the antibody response to Env. The single-cycle vector was grown by complementation with VSV G protein and then tested initially for immunogenicity when given by four different routes. When given by the intramuscular route in mice, we found that the single-cycle vector was equivalent to the replication-competent VSV vector in generating high-level primary and memory CD8 T-cell responses as well as antibody responses to Env. Cellular responses were analyzed using major histocompatibility complex class I tetramers and direct measurement of cytotoxic T-lymphocyte activity in vivo. We also found that the recall responses after boosting were equivalent in animals vaccinated with replication-competent or single-cycle vectors. Additionally, we observed recall and heightened memory responses after boosting animals with a single-cycle vector complemented with G protein from a different vesiculovirus. Because expression of HIV Env by G-deleted VSV might allow replication in human cells expressing CD4, we generated a single-cycle VSV recombinant expressing a secreted form of the HIV Env protein. This virus was just as effective as the recombinant expressing the membrane-anchored Env protein at producing CD8 T cells and antibody responses.     

Characterization of nonpathogenic, live, viral vaccine vectors inducing potent cellular immune responses

Experimental vaccines based on recombinant vesicular stomatitis viruses (VSV) expressing foreign viral proteins are protective in several animal disease models. Although these attenuated viruses are nonpathogenic in nonhuman primates when given by nasal, oral, or intramuscular routes, they are pathogenic in mice when given intranasally, and further vector attenuation may be required before human trials with VSV-based vectors can begin. Mutations truncating the VSV glycoprotein (G) cytoplasmic domain from 29 to 9 or 1 amino acid (designated CT9 or CT1, respectively) were shown previously to attenuate VSV growth in cell culture and pathogenesis in mice. Here we show that VSV recombinants carrying either the CT1 or CT9 deletion and expressing the human immunodeficiency virus (HIV) Env protein are nonpathogenic in mice, even when given by the intranasal route. We then carried out a detailed analysis of the CD8+ T-cell responses, including in vivo cytotoxic T-cell activity, induced by these vectors. When given by either the intranasal or intraperitoneal route, the VSV-CT9 vector expressing HIV Env elicited primary and memory CD8+ T-cell responses to Env equivalent to those elicited by recombinant wild-type VSV expressing Env. The VSV-CT1 vector also induced potent CD8+ T-cell responses after intraperitoneal vaccination, but was less effective when given by the intranasal route. The VSV-CT1 vector was also substantially less effective than the VSV-CT9 or wild-type vector at inducing antibody to Env. The VSV-CT9 vector appears ideal because of its lack of pathogenesis, propagation to high titers in vitro, and stimulation of strong cellular and humoral immune responses.     

Attenuation of recombinant vesicular stomatitis virus-human immunodeficiency virus type 1 vaccine vectors by gene translocations and g gene truncation reduces neurovirulence and enhances immunogenicity in mice

Recombinant vesicular stomatitis virus (rVSV) has shown great potential as a new viral vector for vaccination. However, the prototypic rVSV vector described previously was found to be insufficiently attenuated for clinical evaluation when assessed for neurovirulence in nonhuman primates. Here, we describe the attenuation, neurovirulence, and immunogenicity of rVSV vectors expressing human immunodeficiency virus type 1 Gag. These rVSV vectors were attenuated by combinations of the following manipulations: N gene translocations (N4), G gene truncations (CT1 or CT9), noncytopathic M gene mutations (Mncp), and positioning of the gag gene into the first position of the viral genome (gag1). The resulting N4CT1-gag1, N4CT9-gag1, and MncpCT1-gag1 vectors demonstrated dramatically reduced neurovirulence in mice following direct intracranial inoculation. Surprisingly, in spite of a very high level of attenuation, the N4CT1-gag1 and N4CT9-gag1 vectors generated robust Gag-specific immune responses following intramuscular immunization that were equivalent to or greater than immune responses generated by the more virulent prototypic vectors. MncpCT1-gag1 also induced Gag-specific immune responses following intramuscular immunization that were equivalent to immune responses generated by the prototypic rVSV vector. Placement of the gag gene in the first position of the VSV genome was associated with increased in vitro expression of Gag protein, in vivo expression of Gag mRNA, and enhanced immunogenicity of the vector. These findings demonstrate that through directed manipulation of the rVSV genome, vectors that have reduced neurovirulence and enhanced immunogenicity can be made.     

Early Biodistribution and Persistence of a Protective Live Attenuated SIV Vaccine Elicits Localised Innate Responses in Multiple Lymphoid Tissues

Vaccination of Mauritian cynomolgus macaques with the attenuated nef-truncated C8 variant of SIVmac251/32H (SIVmacC8) induces early, potent protection against pathogenic, heterologous challenge before the maturation of cognate immunity. To identify processes that contribute to early protection in this model the pathogenesis, anatomical distribution and viral vaccine kinetics were determined in relation to localised innate responses triggered by vaccination. The early biodistribution of SIVmacC8 was defined by rapid, widespread dissemination amongst multiple lymphoid tissues, detectable after 3 days. Cell-associated viral RNA dynamics identified mesenteric lymph nodes (MLN) and spleen, as well as the gut mucosae, as early major contributors of systemic virus burden. Rapid, localised infection was populated by discrete foci of persisting virus-infected cells. Localised productive infection triggered a broad innate response, with type-1 interferon sensitive IRF-7, STAT-1, TRIM5α and ApoBEC3G genes all upregulated during the acute phase but induction did not prevent viral persistence. Profound changes in vaccine-induced cell-surface markers of immune activation were detected on macrophages, B-cells and dendritic cells (DC-SIGN, S-100, CD40, CD11c, CD123 and CD86). Notably, high DC-SIGN and S100 staining for follicular and interdigitating DCs respectively, in MLN and spleen were detected by 3 days, persisting 20 weeks post-vaccination. Although not formally evaluated, the early biodistribution of SIVmacC8 simultaneously targets multiple lymphoid tissues to induce strong innate immune responses coincident at the same sites critical for early protection from wild-type viruses. HIV vaccines which stimulate appropriate innate, as well as adaptive responses, akin to those generated by live attenuated SIV vaccines, may prove the most efficacious.     

Comparative prime-boost vaccinations using Semliki Forest virus, adenovirus, and ALVAC vectors demonstrate differences in the generation of a protective central memory CTL response against the P815 tumor

Tumor-specific Ags are potential target molecules in the therapeutic treatment of cancer. One way to elicit potent immune responses against these Ags is to use recombinant viruses, which activate both the innate and the adaptive arms of the immune system. In this study, we have compared Semliki Forest virus (SFV), adenovirus, and ALVAC (poxvirus) vectors for their capacity to induce CD8(+) T cell responses against the P1A tumor Ag and to elicit protection against subsequent challenge injection of P1A-expressing P815 tumor cells in DBA/2 mice. Both homologous and heterologous prime-boost regimens were studied. In most cases, both higher CD8(+) T cell responses and better tumor protections were observed in mice immunized with heterologous prime-boost regimens, suggesting that the combination of different viral vectors is beneficial for the induction of an effective immune response. However, homologous immunization with SFV provided potent tumor protection despite a rather moderate primary CD8(+) T cell response as compared with mice immunized with recombinant adenovirus. SFV-immunized mice showed a rapid and more extensive expansion of P1A-specific CD8(+) T cells in the tumor-draining lymph node after tumor challenge and had a higher frequency of CD62L(+) P1A-specific T cells in the blood, spleen, and lymph nodes as compared with adenoimmunized mice. Our results indicate that not only the magnitude but in particular the quality of the CD8(+) T cell response correlates with tumor protection.     

Immunization with Single-Cycle SIV Significantly Reduces Viral Loads After an Intravenous Challenge with SIVmac239

Strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection were evaluated for the ability to elicit protective immunity against wild-type SIV_mac239 infection of rhesus macaques by two different vaccine regimens. Six animals were inoculated at 8-week intervals with 6 identical doses consisting of a mixture of three different envelope variants of single-cycle SIV (scSIV). Six additional animals were primed with a mixture of cytoplasmic domain-truncated envelope variants of scSIV and boosted with two doses of vesicular stomatitis virus glycoprotein (VSV G) trans-complemented scSIV. While both regimens elicited detectable virus-specific T cell responses, SIV-specific T cell frequencies were more than 10-fold higher after boosting with VSV G trans-complemented scSIV (VSV G scSIV). Broad T cell recognition of multiple viral antigens and Gag-specific CD4⁺ T cell responses were also observed after boosting with VSV G scSIV. With the exception of a single animal in the repeated immunization group, all of the animals became infected following an intravenous challenge with SIV_mac239. However, significantly lower viral loads and higher memory CD4⁺ T cell counts were observed in both immunized groups relative to an unvaccinated control group. Indeed, both scSIV immunization regimens resulted in containment of SIV_mac239 replication after challenge that was as good as, if not better than, what has been achieved by other non-persisting vaccine vectors that have been evaluated in this challenge model. Nevertheless, the extent of protection afforded by scSIV was not as good as typically conferred by persistent infection with live, attenuated SIV. These observations have potentially important implications to the design of an effective AIDS vaccine, since they suggest that ongoing stimulation of virus-specific immune responses may be essential to achieving the degree of protection afforded by live, attenuated SIV.     

Vesicular stomatitis virus expressing tumor suppressor p53 is a highly attenuated, potent oncolytic agent

Vesicular stomatitis virus (VSV), a negative-strand RNA rhabdovirus, preferentially replicates in and eradicates transformed versus nontransformed cells and is thus being considered for use as a potential anticancer treatment. The genetic malleability of VSV also affords an opportunity to develop more potent agents that exhibit increased therapeutic activity. The tumor suppressor p53 has been shown to exert potent antitumor properties, which may in part involve stimulating host innate immune responses to malignancies. To evaluate whether VSV expressing p53 exhibited enhanced oncolytic action, the murine p53 (mp53) gene was incorporated into recombinant VSVs with or without a functional viral M gene-encoded protein that could either block (VSV-mp53) or enable [VSV-M(mut)-mp53] host mRNA export following infection of susceptible cells. Our results indicated that VSV-mp53 and VSV-M(mut)-mp53 expressed high levels of functional p53 and retained the ability to lyse transformed versus normal cells. In addition, we observed that VSV-ΔM-mp53 was extremely attenuated in vivo due to p53 activating innate immune genes, such as type I interferon (IFN). Significantly, immunocompetent animals with metastatic mammary adenocarcinoma exhibited increased survival following treatment with a single inoculation of VSV-ΔM-mp53, the mechanisms of which involved enhanced CD49b+ NK and tumor-specific CD8+ T cell responses. Our data indicate that VSV incorporating p53 could provide a safe, effective strategy for the design of VSV oncolytic therapeutics and VSV-based vaccines.     

Immune Mechanisms Responsible for Vaccination against and Clearance of Mucosal and Lymphatic Norovirus Infection

Two cardinal manifestations of viral immunity are efficient clearance of acute infection and the capacity to vaccinate against secondary viral exposure. For noroviruses, the contributions of T cells to viral clearance and vaccination have not been elucidated. We report here that both CD4 and CD8 T cells are required for efficient clearance of primary murine norovirus (MNV) infection from the intestine and intestinal lymph nodes. Further, long-lasting protective immunity was generated by oral live virus vaccination. Systemic vaccination with the MNV capsid protein also effectively protected against mucosal challenge, while vaccination with the capsid protein of the distantly related human Lordsdale virus provided partial protection. Fully effective vaccination required a broad immune response including CD4 T cells, CD8 T cells, and B cells, but the importance of specific immune cell types varied between the intestine and intestinal lymph nodes. Perforin, but not interferon gamma, was required for clearance of MNV infection by adoptively transferred T lymphocytes from vaccinated hosts. These studies prove the feasibility of both mucosal and systemic vaccination against mucosal norovirus infection, demonstrate tissue specificity of norovirus immune cells, and indicate that efficient vaccination strategies should induce potent CD4 and CD8 T cell responses.     

Significant protection against high-dose simian immunodeficiency virus challenge conferred by a new prime-boost vaccine regimen

We constructed vaccine vectors based on live recombinant vesicular stomatitis virus (VSV) and a Semliki Forest virus (SFV) replicon (SFVG) that propagates through expression of the VSV glycoprotein (G). These vectors expressing simian immunodeficiency virus (SIV) Gag and Env proteins were used to vaccinate rhesus macaques with a new heterologous prime-boost regimen designed to optimize induction of antibody. Six vaccinated animals and six controls were then given a high-dose mucosal challenge with the diverse SIVsmE660 quasispecies. All control animals became infected and had peak viral RNA loads of 10(6) to 10(8) copies/ml. In contrast, four of the vaccinees showed significant (P = 0.03) apparent sterilizing immunity and no detectable viral loads. Subsequent CD8(+) T cell depletion confirmed the absence of SIV infection in these animals. The two other vaccinees had peak viral loads of 7 × 10(5) and 8 × 10(3) copies/ml, levels below those of all of the controls, and showed undetectable virus loads by day 42 postchallenge. The vaccine regimen induced high-titer prechallenge serum neutralizing antibodies (nAbs) to some cloned SIVsmE660 Env proteins, but antibodies able to neutralize the challenge virus swarm were not detected. The cellular immune responses induced by the vaccine were generally weak and did not correlate with protection. Although the immune correlates of protection are not yet clear, the heterologous VSV/SFVG prime-boost is clearly a potent vaccine regimen for inducing virus nAbs and protection against a heterogeneous viral swarm.     

PHA activation of encephalitogenic T cells: in vitro line selection overcomes splenic suppression

In this study we compared myelin basic protein (MBP) and phytohemagglutinin (PHA) for their ability to induce proliferation and experimental autoimmune encephalomyelitis (EAE) transfer activity in mixed cell cultures obtained from spleen and lymph nodes versus highly selected MBP-specific T cell lines and clones. Established MBP-specific cells derived initially from immune lymph nodes attained both proliferative and EAE-transfer activities after in vitro activation with either MBP or PHA. In contrast, PHA was unable to induce immune spleen cells to transfer EAE, in spite of its potent mitogenic activity. On the basis of these results, we evaluated the in vitro proliferation and differentiation responses of MBP-specific T cells during the line selection process using cells derived from both immune lymph node and immune spleen. During the initial selection process with MBP, proliferation of MBP-specific T cell precursors from immunized spleen populations was reduced relative to lymph node cells. After antigen-dependent selection the encephalitogenic cells from either organ exhibited identical in vitro response characteristics. Freshly isolated immune spleen cells were potent suppressors of MBP-specific T cell proliferation suggesting that the in vitro differences between the two organs was due to splenic suppression of the encephalitogenic cells.     

Characterization of pulmonary T cell response to helper-dependent adenoviral vectors following intranasal delivery

In spite of the extensive research in the field of gene therapy, host immune responses continue to be the major barrier in translating basic research to clinical practice. Helper-dependent adenoviral (HD-Ad) vectors show great potential for pulmonary gene therapy, but the knowledge of pulmonary immune responses toward these vectors is very limited. In this study, we show that HD-Ad vectors are potent stimulators of dendritic cell (DC) maturation, thus leading to stimulation of T cell proliferation with approximately 6% of naive CD4(+) T cells from pulmonary mediastinal lymph node responding to HD-Ad-treated DCs. In contrast to the belief that HD-Ad vectors are unable to prime adaptive immune response, we show for the first time, through in vivo pulmonary studies in mice, that HD-Ad vectors can prime CD4(+) and CD8(+) T cell responses in the lung at high and substantially low doses. This indicates cross-presentation of HD-Ad-derived epitopes by DCs to prime CD8(+) T cell responses. To assess the basis of pulmonary T cell response against HD-Ad vectors, we examined the response of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) in the lung. In response to HD-Ad delivery, there is induction of maturation in both cDC and pDC subsets, but it is the cDCs, not pDCs, that migrate rapidly to draining lymph nodes within the first 2 days after vector delivery to prime adaptive immune response against these vectors. These findings have implications for development of strategies to prevent adaptive immune responses against gene therapy vectors.     

CD8+ T-cell-mediated cross-clade protection in the genital tract following intranasal immunization with inactivated human immunodeficiency virus antigen plus CpG oligodeoxynucleotides

Human immunodeficiency virus (HIV) is a mucosally transmitted infection that rapidly targets and depletes CD4+ T cells in mucosal tissues and establishes a major reservoir for viral persistence in gut-associated lymphoid tissues. Therefore, vaccines designed to prevent HIV infections must induce potent and durable mucosal immune responses, especially in the genital tract. Here we investigated whether intranasal (i.n.) immunization with inactivated gp120-depleted HIV-1 antigen (Ag) plus CpG oligodeoxynucleotide (ODN) as an adjuvant induced local immune responses in the genital tract and cross-clade protection against intravaginal (IVAG) challenge. Lymphocytes isolated from the iliac lymph nodes (ILNs) and genital tracts of female mice i.n. immunized with HIV-1 Ag plus CpG showed significant HIV-specific proliferation and produced significantly higher levels of gamma interferon (IFN-gamma) and beta-chemokines than mice immunized with HIV-1 Ag alone or mixed with non-CpG ODN. CD8+ lymphocytes were dramatically increased in the genital tracts of mice immunized with HIV-1 Ag plus CpG, and protection following IVAG challenge with recombinant vaccinia viruses (rVVs) expressing HIV-1 gag was shown to be CD8 dependent. Finally, cross-clade protection was observed between clades A, C, and G but not B following IVAG challenge with rVVs expressing HIV-1 gag from different clades. These studies provide evidence that mucosal (i.n.) immunization induced strong local T-cell-mediated immune responses in the genital tract and cross-clade protection against IVAG challenge.     

Glycoprotein exchange vectors based on vesicular stomatitis virus allow effective boosting and generation of neutralizing antibodies to a primary isolate of human immunodeficiency virus type 1

Live recombinant vesicular stomatitis viruses (VSVs) expressing foreign antigens are highly effective vaccine vectors. However, these vectors induce high-titer neutralizing antibody directed at the single VSV glycoprotein (G), and this antibody alone can prevent reinfection and boosting with the same vector. To determine if efficient boosting could be achieved by changing the G protein of the vector, we have developed two new recombinant VSV vectors based on the VSV Indiana serotype but with the G protein gene replaced with G genes from two other VSV serotypes, New Jersey and Chandipura. These G protein exchange vectors grew to titers equivalent to wild-type VSV and induced similar neutralizing titers to themselves but no cross-neutralizing antibodies to the other two serotypes. The effectiveness of these recombinant VSV vectors was illustrated in experiments in which sequential boosting of mice with the three vectors, all encoding the same primary human immunodeficiency virus (HIV) envelope protein, gave a fourfold increase in antibody titer to an oligomeric HIV envelope compared with the response in animals receiving the same vector three times. In addition, only the animals boosted with the exchange vectors produced antibodies neutralizing the autologous HIV primary isolate. These VSV envelope exchange vectors have potential as vaccines in immunizations when boosting of immune responses may be essential.     

A Chimeric Vesiculo/Alphavirus Is an Effective Alphavirus Vaccine

While a large number of mosquito-transmitted alphaviruses are known to cause serious human diseases, there are no licensed vaccines that protect against alphavirus infections. The alphavirus chikungunya virus (CHIKV) has caused multiple recent outbreaks of chikungunya fever. This virus has the potential to cause a worldwide epidemic and has generated strong interest in development of a prophylactic CHIKV vaccine. We report here on the development of a potent experimental vaccine for CHIKV based on a chimeric vesicular stomatitis virus (VSV) expressing the entire CHIKV envelope polyprotein (E3-E2-6K-E1) in place of the VSV glycoprotein (G). These VSVΔG-CHIKV chimeras incorporated functional CHIKV glycoproteins into the viral envelope in place of VSV G. The chimeric viruses were attenuated for growth in tissue culture but could be propagated to high titers without VSV G complementation. They also generated robust neutralizing antibody and cellular immune responses to CHIKV in mice after a single dose and protected mice against CHIKV infection. VSVΔG-alphavirus chimeras could have general applicability as alphavirus vaccines.     

Characterization of a Single-Cycle Rabies Virus-Based Vaccine Vector

Recombinant rabies virus (RV)-based vectors have demonstrated their efficacy in generating long-term, antigen-specific immune responses in murine and monkey models. However, replication-competent viral vectors pose significant safety concerns due to vector pathogenicity. RV pathogenicity is largely attributed to its glycoprotein (RV-G), which facilitates the attachment and entry of RV into host cells. We have developed a live, single-cycle RV by deletion of the G gene from an RV vaccine vector expressing HIV-1 Gag (SPBN-ΔG-Gag). Passage of SPBN-ΔG-Gag on cells stably expressing RV-G allowed efficient propagation of the G-deleted RV. The in vivo immunogenicity data comparing single-cycle RV to a replication-competent control (BNSP-Gag) showed lower RV-specific antibodies; however, the overall isotype profiles (IgG2a/IgG1) were similar for the two vaccine vectors. Despite this difference, mice immunized with SPBN-ΔG-Gag and BNSP-Gag mounted similar levels of Gag-specific CD8⁺ T-cell responses as measured by major histocompatibility complex class I Gag-tetramer staining, gamma interferon-enzyme-linked immunospot assay, and cytotoxic T-cell assay. Moreover, these cellular responses were maintained equally at immunization titers as low as 10³ focus-forming units for both RV vaccine vectors. CD8⁺ T-cell responses were significantly enhanced by a boost with a single-cycle RV complemented with a heterologous vesicular stomatitis virus glycoprotein. These findings demonstrate that single-cycle RV is an effective alternative to replication-competent RV vectors for future development of vaccines for HIV-1 and other infectious diseases.The global spread of HIV-1 represents one of the most significant pandemics to afflict humans (22). Despite tremendous efforts to increase HIV awareness in the general population, UNAIDS reports that fewer than one in five people has access to HIV prevention strategies and many are subject to cultural stigmas thwarting such efforts (43). As such, an HIV vaccine is paramount for preventing disease transmission. It is not yet clear precisely what characteristics are critical for an effective HIV vaccine, yet evidence suggests one would need to induce both antibody and CD8⁺ T-cell-mediated immunity (reviewed in reference 25). Live viruses are at the forefront of HIV vaccine development (7) because they are powerful inducers of both of these arms of immunity. We previously demonstrated that replication-competent rabies virus (RV)-based vectors can induce long-lasting antigen-specific immune responses in both murine and monkey models, as well as protect rhesus macaques from an AIDS-like disease (23, 24, 26-29, 42). However, there are safety concerns with the use of any replication-competent virus for widespread immunization. To address this, we sought to develop and evaluate the immunogenicity of a safer alternative: a single-cycle RV expressing HIV-1 Gag as a model antigen.Single-cycle viral vectors are defective in certain viral components that are required for infectious particle assembly (reviewed in reference 12). As such, the virus undergoes one complete cycle of replication in the primary infected cell and produces progeny virions that are unable to spread to a second round of cells. The progeny are noninfectious and provide inert antigen that may or may not be immunogenic (12). In contrast, so-called replication-deficient viruses do not complete that initial round of replication. These two attenuation strategies have been adopted for use with many different viruses including, but not limited to, adenovirus (Ad), vaccinia virus (VV), canarypox virus (CPV), herpes simplex virus (HSV), vesicular stomatitis virus (VSV), and, more recently, RV (4, 6, 9, 18, 21, 33, 35, 36, 38). However, the results regarding the immunogenicity of such vectors are mixed. For example, both the replication-deficient Ad5 vector and modified vaccinia Ankara (MVA) showed reduced humoral and cellular immunogenicity compared to their replication-competent counterparts, but the use of higher titers and multiple immunizations did increase such responses (18, 33, 35). In the case of CPV, the replication-deficient vector provided poor HIV-specific cellular responses, causing the termination of phase II HIV-1 vaccine trials (38). In contrast, single-cycle VSV, a rhabdovirus closely related to RV, has been shown to induce HIV-1 Env-specific CD8⁺ T-cell responses equivalent to full-length VSV when administered intramuscularly (36). However, protection of rhesus macaques against highly pathogenic simian immunodeficiency virus (SIV) challenge by both replication-competent and single-cycle VSV needs to be shown.In the study described here, we generated a single-cycle RV vector expressing HIV-1 Gag (SPBN-ΔG-Gag) by deletion of the entire RV glycoprotein (RV-G) from the RV genome. RV-G was chosen due to its critical role in the attachment and entry of RV into host cells, which makes RV-G one of the most important determinants of viral pathogenicity (10, 11, 37). RV particles lacking G are unable to spread, as evidenced by intracranial infection with a G-deleted RV that remains restricted to the primary infected neurons (13, 44). It must be noted that in the absence of RV-G, virions are still capable of budding though at a 30-fold lower efficiency (32). These virions, however, are incapable of attachment and entry into a secondary host cell. Because of this, SPBN-ΔG-Gag was propagated on a trans-complementing cell line induced to express RV-G (or VSV-RV-G), effectively facilitating virus spread. To evaluate the immunogenicity of the single-cycle vector, we immunized mice and compared the humoral and cellular responses to responses generated by replication-competent RV. Our results indicate that single-cycle RV generates reduced vector-specific antibody responses but similar HIV-1 Gag-specific CD8⁺ T-cell responses. Moreover, these responses can be significantly enhanced by a heterologous boost with a single-cycle RV complemented with a VSV glycoprotein. Taken together, the results presented here show evidence that single-cycle RV is a promising platform for a safe, live viral vaccine for use against HIV-1 and other applications.     

An effective AIDS vaccine based on live attenuated vesicular stomatitis virus recombinants

We developed an AIDS vaccine based on attenuated VSV vectors expressing env and gag genes and tested it in rhesus monkeys. Boosting was accomplished using vectors with glycoproteins from different VSV serotypes. Animals were challenged with a pathogenic AIDS virus (SHIV89.6P). Control monkeys showed a severe loss of CD4+ T cells and high viral loads, and 7/8 progressed to AIDS with an average time of 148 days. All seven vaccinees were initially infected with SHIV89.6P but have remained healthy for up to 14 months after challenge with low or undetectable viral loads. Protection from AIDS was highly significant (p = 0.001). VSV vectors are promising candidates for human AIDS vaccine trials because they propagate to high titers and can be delivered without injection.     

Cell Cycle Progression or Translation Control Is Not Essential for Vesicular Stomatitis Virus Oncolysis of Hepatocellular Carcinoma

The intrinsic oncolytic specificity of vesicular stomatitis virus (VSV) is currently being exploited to develop alternative therapeutic strategies for hepatocellular carcinoma (HCC). Identifying key regulators in diverse transduction pathways that define VSV oncolysis in cancer cells represents a fundamental prerequisite to engineering more effective oncolytic viral vectors and adjusting combination therapies. After having identified defects in the signalling cascade of type I interferon induction, responsible for attenuated antiviral responses in human HCC cell lines, we have now investigated the role of cell proliferation and translation initiation. Cell cycle progression and translation initiation factors eIF4E and eIF2Bε have been recently identified as key regulators of VSV permissiveness in T-lymphocytes and immortalized mouse embryonic fibroblasts, respectively. Here, we show that in HCC, decrease of cell proliferation by cell cycle inhibitors or siRNA-mediated reduction of G(1) cyclin-dependent kinase activities (CDK4) or cyclin D1 protein expression, do not significantly alter viral growth. Additionally, we demonstrate that translation initiation factors eIF4E and eIF2Bε are negligible in sustaining VSV replication in HCC. Taken together, these results indicate that cellular proliferation and the initiation phase of cellular protein synthesis are not essential for successful VSV oncolysis of HCC. Moreover, our observations indicate the importance of cell-type specificity for VSV oncolysis, an important aspect to be considered in virotherapy applications in the future.     

Persistent antigen presentation after acute vesicular stomatitis virus infection

Long-term antigen expression is believed to play an important role in modulation of T-cell responses to chronic virus infections. However, recent studies suggest that immune responses may occur late after apparently acute infections. We have now analyzed the CD8 T-cell response to vesicular stomatitis virus (VSV), which is thought to cause to an infection characterized by rapid virus clearance by innate and adaptive immune system components. Unexpectedly, virus-encoded antigen was detectable more than 6 weeks after intranasal VSV infection in both draining and nondraining lymph nodes by adoptively transferred CD8 T cells. Infection with Listeria monocytogenes expressing the same antigen did not result in prolonged antigen presentation. Weeks after VSV infection, discrete T-cell clustering with dendritic cells within the lymph node was observed after transfer of antigen-specific CD8 T cells. Moreover, memory CD8 T cells as defined by phenotype and function were generated from na?ve CD8 T cells entering the response late after infection. These findings suggested that protracted antigen presentation after an apparently acute virus infection may contribute to an ongoing antiviral immune response.