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The demonstrated clinical efficacy of a recombinant vesicular stomatitis virus (rVSV) vaccine vector has stimulated the investigation of additional serologically distinct Vesiculovirus vectors as therapeutic and/or prophylactic vaccine vectors to combat emerging viral diseases. Among these viral threats are the encephalitic alphaviruses Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV), which have demonstrated potential for natural disease outbreaks, yet no licensed vaccines are available in the event of an epidemic. Here we report the rescue of recombinant Isfahan virus (rISFV) from genomic cDNA as a potential new vaccine vector platform. The rISFV genome was modified to attenuate virulence and express the VEEV and EEEV E2/E1 surface glycoproteins as vaccine antigens. A single dose of the rISFV vaccine vectors elicited neutralizing antibody responses and protected mice from lethal VEEV and EEEV challenges at 1 month postvaccination as well as lethal VEEV challenge at 8 months postvaccination. A mixture of rISFV vectors expressing the VEEV and EEEV E2/E1 glycoproteins also provided durable, single-dose protection from lethal VEEV and EEEV challenges, demonstrating the potential for a multivalent vaccine formulation. These findings were paralleled in studies with an attenuated form of rVSV expressing the VEEV E2/E1 glycoproteins. Both the rVSV and rISFV vectors were attenuated by using an approach that has demonstrated safety in human trials of an rVSV/HIV-1 vaccine. Vaccines based on either of these vaccine vector platforms may present a safe and effective approach to prevent alphavirus-induced disease in humans. IMPORTANCE This work introduces rISFV as a novel vaccine vector platform that is serologically distinct and phylogenetically distant from VSV. The rISFV vector has been attenuated by an approach used for an rVSV vector that has demonstrated safety in clinical studies. The vaccine potential of the rISFV vector was investigated in a well-established alphavirus disease model. The findings indicate the feasibility of producing a safe, efficacious, multivalent vaccine against the encephalitic alphaviruses VEEV and EEEV, both of which can cause fatal disease. This work also demonstrates the efficacy of an attenuated rVSV vector that has already demonstrated safety and immunogenicity in multiple HIV-1 phase I clinical studies. The absence of serological cross-reactivity between rVSV and rISFV and their phylogenetic divergence within the Vesiculovirus genus indicate potential for two stand-alone vaccine vector platforms that could be used to target multiple bacterial and/or viral agents in successive immunization campaigns or as heterologous prime-boost agents.

Attenuated, replication-competent viral vector vaccines are highly immunogenic in that they mimic natural infection. They offer the promise of eliciting a more integrated immune response and more abundant and sustained expression of the encoded viral antigens. Although a vaccine to prevent human immunodeficiency virus (HIV) infection remains an urgent global health priority, concerns about the safety of using live-attenuated HIV itself as a vaccine [1] has led the field to focus heavily on the exploration of replication-defective viral vectors to deliver HIV-1 antigens. There are limited data to suggest these vectors are able to induce an optimal, integrated immune response, which may include CD4+ T-cell help for B-cell/antibody and polyfunctional cytotoxic T-cell responses together with long-term immunologic memory [2]. To date, only 1 replication-defective viral vector, based on canarypox, administered sequentially with a gp120 subunit boost vaccine, has demonstrated partial protection from HIV acquisition in an efficacy trial [3]. Currently, only a few replicating viruses, including vaccinia (clinical trial ID NCT01705223), Sendai virus (NCT01705990), measles (NCT01320176), and adenovirus subtype 4 (NCT01989533), have advanced to clinical trials as HIV-1 vaccine vectors. Vesicular stomatitis virus (VSV) is a member of the genus Vesiculovirus in the family Rhabdoviridae. The 2 major serotypes are New Jersey and Indiana. Vesicular stomatitis virus has a single-strand, negative-sense, nonsegmented RNA genome. Similar to Sendai and measles viruses, the virus genomic organization of VSV (Figure ​(Figure1A)1A) and life cycle make it particularly attractive as a candidate vaccine vector because the genome can accommodate multiple gene inserts, is stable over many generations, and does not undergo recombination. In addition, the VSV genome replicates in the cytoplasm and is incapable of integrating within the genomes of infected host cells. Finally, VSV is a zoonosis, cycling between biting insects and livestock (cattle, horses, and swine). Human infection with VSV is rare in most regions of the world except in certain regions of Central and South America where VSV is endemic. Where VSV infection does occur, it is typically asymptomatic or is associated with an acute influenza-like illness with symptoms such as fever, muscle aches, and malaise [4, 5]. Thus, the general population is largely free of pre-existing, virus-neutralizing immunity—a factor that has limited the clinical utility of HIV-1 vaccines based on adenovirus subtype 5 [6, 7]. Figure 1. (A) Wild-type vesicular stomatitis virus (VSV) genome organization and virion structure. Viral transcription is polar, initiating at the single 3′ promoter and proceeding to the 5′ end of the genome, producing a steep 3′ to 5′ ... Recombinant VSV (rVSV) has been extensively studied as a potential HIV vaccine vector in preclinical, nonhuman primate (NHP) models [8–11]. After 2 sequential immunizations, rhesus macaques that received rVSV expressing HIV-1 env and simian immunodeficiency virus (SIV) gag were all infected but were protected from disease progression for 4 years or more after infection with SHIV 89.6P, maintaining low or undetectable viral load set points and preserved CD4+ T-cell counts [11]. This encouraging level of postchallenge protection from disease suggested that rVSV vectors expressing HIV genes might be an effective HIV vaccine in humans. To maximize safety for first-in-human clinical evaluation, a highly attenuated form of rVSV Indiana was developed. This involved downregulating VSV N protein expression by translocating the N gene further away from the 3′ transcription promoter and truncating the VSV G protein cytoplasmic tail from 29 amino acids found in wild-type virus to a single amino acid [12]. In addition, HIV-1 gag expression was achieved by insertion of the gag gene adjacent to the 3′ transcription promoter (Figure ​(Figure1B).1B). The resulting vector (rVSVN4CT1gag1) was shown to be safe, well tolerated, and immunogenic in several murine and NHP studies [13, 14]. In the work described here, the HIV Vaccine Trials Network (HVTN) evaluated the safety and immunogenicity of this novel vaccine vector in a phase 1a dose-escalation, first-in-human trial, HVTN 090.

A guiding principle for HIV vaccine design has been that cellular and humoral immunity work together to provide the strongest degree of efficacy. However, three efficacy trials of Ad5-vectored HIV vaccines showed no protection. Transmission was increased in two of the trials, suggesting that this vaccine strategy elicited CD4+ T-cell responses that provide more targets for infection, attenuating protection or increasing transmission. The degree to which this problem extends to other HIV vaccine candidates is not known. Here, we show that a gp120-CD4 chimeric subunit protein vaccine (full-length single chain) elicits heterologous protection against simian-human immunodeficiency virus (SHIV) or simian immunodeficiency virus (SIV) acquisition in three independent rhesus macaque repeated low-dose rectal challenge studies with SHIV162P3 or SIVmac251. Protection against acquisition was observed with multiple formulations and challenges. In each study, protection correlated with antibody-dependent cellular cytotoxicity specific for CD4-induced epitopes, provided that the concurrent antivaccine T-cell responses were minimal. Protection was lost in instances when T-cell responses were high or when the requisite antibody titers had declined. Our studies suggest that balance between a protective antibody response and antigen-specific T-cell activation is the critical element to vaccine-mediated protection against HIV. Achieving and sustaining such a balance, while enhancing antibody durability, is the major challenge for HIV vaccine development, regardless of the immunogen or vaccine formulation.

There is an urgent need for a vaccine capable of preventing HIV infection or the development of HIV-related disease. A number of approaches designed to stimulate HIV-specific CD8+ cytotoxic T cell responses together with helper responses are presently under evaluation. In this phase 1, multi-center, placebo-controlled trial, we tested the ability of a novel multiepitope peptide vaccine to elicit HIV-specific immunity. To enhance the immunogenicity of the peptide vaccine, half of the vaccine recipients received recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) protein as a coadjuvant. The vaccine was safe; tolerability was moderate, with a number of adverse events related to local injection site reactogenicity. Anti-GM-CSF antibody responses developed in the majority of GM-CSF recipients but were not associated with adverse hematologic events. The vaccine was only minimally immunogenic. Six of 80 volunteers who received vaccine developed HIV-specific responses as measured by interferon-gamma ELISPOT assay, and measurable responses were transient. This study failed to demonstrate that GM-CSF can substantially improve the overall weak immunogenicity of a multiepitope peptide-based HIV vaccine.

Plasmid DNA (pDNA) vaccines are effective at eliciting immune responses in a wide variety of animal model systems, however, pDNA vaccines have generally been incapable of inducing robust immune responses in clinical trials. Therefore, to identify means to improve pDNA vaccine performance, we compared various post-transcriptional and post-translational genetic modifications for their ability to improve antigen-specific CMI responses. Mice vaccinated using a sub-optimal 100 mcg dose of a pDNA encoding an unmodified primary isolate HIV-16101 env gp160 failed to demonstrate measurable env-specific CMI responses. In contrast, significant env-specific CMI responses were seen in mice immunized with pDNA expression vectors encoding env genes modified by RNA optimization or codon optimization. Further modification of the RNA optimized env gp160 gene by the addition of (i) a simian retrovirus type 1 constitutive RNA transport element; (ii) a murine intracisternal A-particle derived RNA transport element; (iii) a tissue plasminogen activator protein signal leader sequences; (iv) a beta-catenin derived ubiquitination target sequence; or (v) a monocyte chemotactic protein-3 derived signal sequence failed to further improve the induction of env-specific CMI responses. Therefore, modification of the env gp160 gene by RNA or codon optimization alone is necessary for high-level rev-independent expression and results in robust env-specific CMI responses in immunized mice. Importantly, further modification(s) of the env gene to alter cellular localization or increase proteolytic processing failed to result in increased env-specific immune responses. These results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.

Since human immunodeficiency virus (HIV)-specific cell-mediated immune (CMI) responses are critical in the early control and resolution of HIV infection and correlate with postchallenge outcomes in rhesus macaque challenge experiments, we sought to identify a plasmid DNA (pDNA) vaccine design capable of eliciting robust and balanced CMI responses to multiple HIV type 1 (HIV-1)-derived antigens for further development. Previously, a number of two-, three-, and four-vector pDNA vaccine designs were identified as capable of eliciting HIV-1 antigen-specific CMI responses in mice (M. A. Egan et al., Vaccine 24:4510-4523, 2006). We then sought to further characterize the relative immunogenicities of these two-, three-, and four-vector pDNA vaccine designs in nonhuman primates and to determine the extent to which in vivo electroporation (EP) could improve the resulting immune responses. The results indicated that a two-vector pDNA vaccine design elicited the most robust and balanced CMI response. In addition, vaccination in combination with in vivo EP led to a more rapid onset and enhanced vaccine-specific immune responses. In macaques immunized in combination with in vivo EP, we observed a 10- to 40-fold increase in HIV-specific enzyme-linked immunospot assay responses compared to those for macaques receiving a 5-fold higher dose of vaccine without in vivo EP. This increase in CMI responses translates to an apparent 50- to 200-fold increase in pDNA vaccine potency. Importantly, in vivo EP enhanced the immune response against the less immunogenic antigens, resulting in a more balanced immune response. In addition, in vivo EP resulted in an approximate 2.5-log 10 increase in antibody responses. The results further indicated that in vivo EP was associated with a significant reduction in pDNA persistence and did not result in an increase in pDNA associated with high-molecular-weight DNA relative to macaques receiving the pDNA without EP. Collectively, these results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.

We describe methods to identify high and low responders in a whole-blood assay of lipopolysaccharide-stimulated cytokine responses. Two multivariate measures of the cytokine responses both captured high and low responses for each of the four individual cytokines that were assayed.

SUMMARY The humoral and genital secretory immune response to chronic SIV infection was compared between female Rhesus macaques inoculated by i.v. or intravaginal routes. Total IgG levels in serum were 10-fold higher in SIV-infected animals when compared with uninfected controls. Vaginal washes from normal macaques contained predominantly IgA and IgG, while those from SIV-infected animals contained high levels of IgG. The SIV-infected animals had high titres of SIV-specific IgG in serum, with lower but detectable IgA and IgM responses. The genital secretory immune response to SIV was similar in intravenously and intravaginally inoculated animals. The anti-SIV response in the vaginal washes consisted mainly of IgG. Within the lamina propria of the reproductive tract of animals chronically infected with SIV there were essentially no IgA or IgG plasma cells and only a small number of IgM plasma cells, while two normal animals had large numbers of IgA plasma cells. These results suggest that the mucosal immune system of the female reproductive tract is impaired inchronic SIV infection.

Peyer's patch (PP) and mesenteric lymph node (MLN) cell cultures from young adult X-linked immunodeficient (xid) CBA/N and (CBA/N X DBA/2) F1 male mice support primary anti-sheep erythrocyte (SRBC) plaque-forming cell (PFC) responses, which suggests that gut-associated lymphoreticular tissue (GALT) contains a normal B lymphocyte subpopulation. Further support for this was provided by the observation that PP cells from xid mice gave responses to both TI-1 and TI-2 antigens that were similar to the responses of PP cell cultures from normal mice. Spleen cell cultures from xid mice were unresponsive to SRBC and TI-2 antigens. Proof that GALT of xid mice contain mature B lymphocytes was provided by the demonstration of PP B cells that bear a low density of surface immunoglobulin M. When these cells were separated by flow cytometry and immunized with trinitrophenyl (TNP)-Ficoll in vitro, good anti-TNP PFC responses were observed. These results suggest that GALT of young adult xid mice contain mature B cells and may represent the origin for the mature B cell responses seen in aged xid mice.

Methods HVTN 090 enrolled sixty healthy, HIV-1-uninfected adults in a randomized, double-blinded, placebo-controlled dose escalation study. Groups of 12 participants received rVSV Indiana HIV Gag vaccine at 5 dose levels (4.6 x 10 to 3.4 x 10 PFU) (N=10/group) or placebo (N=2/group), delivered intramuscularly at 0 and 2 months. Reactogenicity over 7 days, adverse events (AEs), and viral cultures from whole blood, urine, saliva and swabs of oral lesions were collected. HIV-1-specific CD4+ and CD8+ T-cell responses to Gag peptides were measured 1 and 2 weeks post-boost by intracellular cytokine staining.