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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.

Postexposure immunization can prevent disease and reduce transmission following pathogen exposure. The rapid immunostimulatory properties of recombinant vesicular stomatitis virus (rVSV)-based vaccines make them suitable postexposure treatments against the filoviruses Ebola virus and Marburg virus (MARV); however, the mechanisms that drive this protection are undefined. Previously, we reported 60–75% survival of rhesus macaques treated with rVSV vectors expressing MARV glycoprotein (GP) 20–30 minutes after a low dose exposure to the most pathogenic variant of MARV, Angola. Survival in this model was linked to production of GP-specific antibodies and lower viral load. To confirm these results and potentially identify novel correlates of postexposure protection, we performed a similar experiment, but analyzed plasma cytokine levels, frequencies of immune cell subsets, and the transcriptional response to infection in peripheral blood. In surviving macaques (80–89%), we observed induction of genes mapping to antiviral and interferon-related pathways early after treatment and a higher percentage of T helper 1 (Th1) and NK cells. In contrast, the response of non-surviving macaques was characterized by hypercytokinemia; a T helper 2 signature; recruitment of low HLA-DR expressing monocytes and regulatory T-cells; and transcription of immune checkpoint (e.g., PD-1, LAG3) genes. These results suggest dysregulated immunoregulation is associated with poor prognosis, whereas early innate signaling and Th1-skewed immunity are important for survival.

A recombinant vesicular stomatitis virus (rVSV) expressing the Marburg virus (MARV) Musoke variant glycoprotein fully protects macaques against 2 MARV variants and Ravn virus as a preventive vaccine and MARV variant Musoke as a postexposure treatment. To evaluate postexposure efficacy against the most pathogenic MARV variant, Angola, we engineered rVSVs expressing homologous Angola glycoprotein. Macaques were challenged with high or low doses of variant Angola and treated 20–30 minutes after exposure. A total of 25% and 60%–75% of treated macaques survived the high-dose and low-dose challenges, respectively. The more rapid disease progression of variant Angola versus variant Musoke may account for the incomplete protection observed.

The HIV Vaccine Trials Network (HVTN) 087 vaccine trial assessed the effect of increasing doses of pIL-12 (interleukin-12 delivered as plasmid DNA) adjuvant on the immunogenicity of an HIV-1 multiantigen (MAG) DNA vaccine delivered by electroporation and boosted with a vaccine comprising an attenuated vesicular stomatitis virus expressing HIV-1 Gag (VSV-Gag). We randomized 100 healthy adults to receive placebo or 3 mg HIV-MAG DNA vaccine (ProfectusVax HIV-1 gag / pol or ProfectusVax nef / tat / vif , env ) coadministered with pIL-12 at 0, 250, 1,000, or 1,500 μg intramuscularly by electroporation at 0, 1, and 3 months followed by intramuscular inoculation with 3.4 × 10 7 PFU VSV-Gag vaccine at 6 months. Immune responses were assessed after the prime and boost and 6 months after the last vaccination. High-dose pIL-12 increased the magnitude of CD8 + T-cell responses postboost compared to no pIL-12 ( P = 0.02), while CD4 + T-cell responses after the prime were higher in the absence of pIL-12 than with low- and medium-dose pIL-12 ( P ≤ 0.05). The VSV boost increased Gag-specific CD4 + and CD8 + T-cell responses in all groups ( P < 0.001 for CD4 + T cells), inducing a median of four Gag epitopes in responders. Six to 9 months after the boost, responses decreased in magnitude, but CD8 + T-cell response rates were maintained. The addition of a DNA prime dramatically improved responses to the VSV vaccine tested previously in the HVTN 090 trial, leading to broad epitope targeting and maintained CD8 + T-cell response rates at early memory. The addition of high-dose pIL-12 given with a DNA prime by electroporation and boosted with VSV-Gag increased the CD8 + T-cell responses but decreased the CD4 + responses. This approach may be advantageous in reshaping the T-cell responses to a variety of chronic infections or tumors. (This study has been registered at ClinicalTrials.gov under registration no. NCT01578889.)

The family Filoviridae contains three genera, Ebolavirus (EBOV), Marburg virus, and Cuevavirus1. Some members of the EBOV genus, including Zaire ebolavirus (ZEBOV), can cause lethal hemorrhagic fever in humans. During 2014 an unprecedented ZEBOV outbreak occurred in West Africa and is still ongoing, resulting in nearly 10,000 deaths, and causing global concern of uncontrolled disease. To meet this challenge a rapid acting vaccine is needed. Many vaccine approaches have shown promise in being able to protect nonhuman primate (NHPs) against ZEBOV2. In response to the current ZEBOV outbreak several of these vaccines have been fast tracked for human use. However, it is not known whether any of these vaccines can provide protection against the new outbreak Makona strain of ZEBOV. One of these approaches is a first generation recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing the ZEBOV glycoprotein (GP) (rVSV/ZEBOV). To address safety concerns associated with this vector, we developed two candidate, further attenuated rVSV/ZEBOV vaccines. Both attenuated vaccines produced an approximately ten-fold lower vaccine-associated viremia compared to the first generation vaccine and both provided complete, single dose protection of macaques from lethal challenge with the Makona outbreak strain of ZEBOV.

Previous studies demonstrated that a single intramuscular (i.m.) dose of an attenuated recombinant vesicular stomatitis virus (rVSV) vector (VesiculoVax vector platform; rVSV-N4CT1) expressing the glycoprotein (GP) from the Mayinga strain of Zaire ebolavirus (EBOV) protected nonhuman primates (NHPs) from lethal challenge with EBOV strains Kikwit and Makona. Here, we studied the immunogenicities of an expanded range of attenuated rVSV vectors expressing filovirus GP in mice. Based on data from those studies, an optimal attenuated trivalent rVSV vector formulation was identified that included rVSV vectors expressing EBOV, Sudan ebolavirus (SUDV), and the Angola strain of Marburg marburgvirus (MARV) GPs. NHPs were vaccinated with a single dose of the trivalent formulation, followed by lethal challenge 28 days later with each of the three corresponding filoviruses. At day 14 postvaccination, a serum IgG response specific for all three GPs was detected in all the vaccinated macaques. A modest and balanced cell-mediated immune response specific for each GP was also detected in a majority of the vaccinated macaques. No matter the level of total GP-specific immune response detected postvaccination, all the vaccinated macaques were protected from disease and death following lethal challenge with each of the three filoviruses. These findings indicate that vaccination with a single dose of attenuated rVSV-N4CT1 vectors each expressing a single filovirus GP may provide protection against the filoviruses most commonly responsible for outbreaks of hemorrhagic fever in sub-Saharan Africa. IMPORTANCE The West African Ebola virus Zaire outbreak in 2013 showed that the disease was not only a regional concern, but a worldwide problem, and highlighted the need for a safe and efficacious vaccine to be administered to the populace. However, other endemic pathogens, like Ebola virus Sudan and Marburg, also pose an important health risk to the public and therefore require development of a vaccine prior to the occurrence of an outbreak. The significance of our research was the development of a blended trivalent filovirus vaccine that elicited a balanced immune response when administered as a single dose and provided complete protection against a lethal challenge with all three filovirus pathogens.

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.

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.

Background The addition of plasmid cytokine adjuvants, electroporation, and live attenuated viral vectors may further optimize immune responses to DNA vaccines in heterologous prime-boost combinations. The objective of this study was to test the safety and tolerability of a novel prime-boost vaccine regimen incorporating these strategies with different doses of IL-12 plasmid DNA adjuvant. Methods In a phase 1 study, 88 participants received an HIV-1 multiantigen (gag/pol, env, nef/tat/vif) DNA vaccine (HIV-MAG, 3000 μg) co-administered with IL-12 plasmid DNA adjuvant at 0, 250, 1000, or 1500 μg (N = 22/group) given intramuscularly with electroporation (Ichor TriGrid™ Delivery System device) at 0, 1 and 3 months; followed by attenuated recombinant vesicular stomatitis virus, serotype Indiana, expressing HIV-1 Gag (VSV-Gag), 3.4 ⊆ 107 plaque-forming units (PFU), at 6 months; 12 others received placebo. Injections were in both deltoids at each timepoint. Participants were monitored for safety and tolerability for 15 months. Results The dose of IL-12 pDNA did not increase pain scores, reactogenicity, or adverse events with the co-administered DNA vaccine, or following the VSV-Gag boost. Injection site pain and reactogenicity were common with intramuscular injections with electroporation, but acceptable to most participants. VSV-Gag vaccine often caused systemic reactogenicity symptoms, including a viral syndrome (in 41%) of fever, chills, malaise/fatigue, myalgia, and headache; and decreased lymphocyte counts 1 day after vaccination. Conclusions HIV-MAG DNA vaccine given by intramuscular injection with electroporation was safe at all doses of IL-12 pDNA. The VSV-Gag vaccine at this dose was associated with fever and viral symptoms in some participants, but the vaccine regimens were safe and generally well-tolerated. Trial registration Clinical Trials.gov NCT01578889.

Intramuscular inoculation of rhesus macaques with one or more doses of recombinant vesicular stomatitis virus (rVSV) expressing human immunodeficiency virus type 1 (HIV-1) Gag (rVSVgag) typically elicits peak cellular immune responses of 500 to 1,000 gamma interferon (IFN-γ) enzyme-linked immunospots (ELISPOTS)/10 6 peripheral blood lymphocytes (PBL). Here, we describe the generation of a novel recombinant mumps virus (rMuV) expressing HIV-1 Gag (rMuVgag) and measure the Gag-specific cellular immune responses detected in rhesus macaques following vaccination with a highly attenuated form of rVSV expressing HIV-1 Gag (rVSVN4CT1gag1) and rMuVgag in various prime-boost combinations. Notably, peak Gag-specific cellular immune responses of 3,000 to 3,500 ELISPOTS/10 6 PBL were detected in macaques that were primed with rMuVgag and boosted with rVSVN4CT1gag1. Lower peak cellular immune responses were detected in macaques that were primed with rVSVN4CT1gag1 and boosted with rMuVgag, although longer-term gag-specific responses appeared to remain higher in this group of macaques. These findings indicate that rMuVgag may significantly enhance Gag-specific cellular immune responses when administered with rVSVN4CT1gag1 in heterologous prime-boost regimens.

The highly sensitive gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay permits the investigation of the role of cell-mediated immunity (CMI) in the protection of young children against influenza. Preliminary studies of young children confirmed that the IFN-γ ELISPOT assay was a more sensitive measure of influenza memory immune responses than serum antibody and that among seronegative children aged 6 to 7fluorescent focus units (FFU) of a live attenuated influenza virus vaccine (CAIV-T) elicited substantial CMI responses. A commercial inactivated influenza virus vaccine elicited CMI responses only in children with some previous exposure to related influenza viruses as determined by detectable antibody levels prevaccination. The role of CMI in actual protection against community-acquired, culture-confirmed clinical influenza by CAIV-T was investigated in a large randomized, double-blind, placebo-controlled dose-ranging efficacy trial with 2,172 children aged 6 to 6peripheral blood mononuclear cells were protected against clinical influenza, establishing a possible target level of CMI for future influenza vaccine development. The ELISPOT assay for IFN-γ is a sensitive and reproducible measure of CMI and memory immune responses and contributes to establishing requirements for the future development of vaccines against influenza, especially those used for children.

The cell-mediated immune profile induced by a recombinant DNA vaccine was assessed in the simian/HIV (SHIV) and macaque model. The vaccine strategy included coimmunization of a DNA-based vaccine alone or in combination with an optimized plasmid encoding macaque IL-15 (pmacIL-15). We observed strong induction of vaccine-specific IFN-γ-producing CD8+and CD4+effector T cells in the vaccination groups. Animals were subsequently challenged with 89.6p. The vaccine groups were protected from ongoing infection, and the IL-15 covaccinated group showed a more rapidly controlled infection than the group treated with DNA vaccine alone. Lymphocytes isolated from the group covaccinated with pmacIL-15 had higher cellular proliferative responses than lymphocytes isolated from the macaques that received SHIV DNA alone. Vaccine antigen activation of lymphocytes was also studied for a series of immunological molecules. Although mRNA for IFN-γ was up-regulated after antigen stimulation, the inflammatory molecules IL-8 and MMP-9 were down-regulated. These observed immune profiles are potentially reflective of the ability of the different groups to control SHIV replication. This study demonstrates that an optimized IL-15 immune adjuvant delivered with a DNA vaccine can impact the cellular immune profile in nonhuman primates and lead to enhanced suppression of viral replication.

Plasmid-based IL-12 has been demonstrated to successfully enhance the immunogenicity of DNA vaccines, thus enabling a reduction of the amount of DNA required for immunization. IL-15 is thought to affect the maintenance and enhance effector function of CD8(+) memory T cells. Since the ability to elicit a long-term memory response is a desirable attribute of a prophylactic vaccine, we sought to evaluate the ability of these plasmid-based cytokines to serve as vaccine adjuvants in rhesus macaques. Macaques were immunized with plasmid DNA encoding SIVgag in combination with plasmid IL-12, IL-15, or a combination of IL-12 and IL-15. The plasmid-based cytokines were monitored for their ability to augment SIVgag-specific cellular and humoral immune responses and to alter the clinical outcome following pathogenic SHIV(89.6P) challenge. Macaques receiving SIVgag pDNA in combination with plasmid IL-12 alone, or in combination with plasmid IL-12 and IL-15, demonstrated significantly elevated cell-mediated and humoral immune responses resulting in an improved clinical outcome following virus challenge compared to macaques receiving SIVgag pDNA alone. Macaques receiving SIVgag pDNA in combination with plasmid IL-15 alone demonstrated minor increases in cell-mediated and humoral immune responses, however, the clinical outcome following virus challenge was not improved. These results have important implications for the continued development of plasmid DNA vaccines for the prevention of HIV-1 infection.

Rhesus macaques chronically infected with highly pathogenic simian immunodeficiency virus (SIV) SIVmac251 were treated with antiretroviral drugs and vaccinated with combinations of DNA vectors expressing SIV antigens. Vaccination during therapy increased cellular immune responses. After the animals were released from therapy, the virus levels of 12 immunized animals were significantly lower ( P = 0.001) compared to those of 11 animals treated with only antiretroviral drugs. Vaccinated animals showed a persistent increase in immune responses, thus indicating both a virological and an immunological benefit following DNA therapeutic vaccination. Several animals show a long-lasting decrease in viremia, suggesting that therapeutic vaccination may provide an additional benefit to antiretroviral therapy.

Previously, recombinant vesicular stomatitis virus (rVSV) pseudotypes expressing Ebolavirus glycoproteins (GPs) in place of the VSV G protein demonstrated protection of nonhuman primates from lethal homologous Ebolavirus challenge. Those pseudotype vectors contained no additional attenuating mutations in the rVSV genome. Here we describe rVSV vectors containing a full complement of VSV genes and expressing the Ebola virus (EBOV) GP from an additional transcription unit. These rVSV vectors contain the same combination of attenuating mutations used previously in the clinical development pathway of an rVSV/human immunodeficiency virus type 1 vaccine. One of these rVSV vectors (N4CT1-EBOVGP1), which expresses membrane-anchored EBOV GP from the first position in the genome (GP1), elicited a balanced cellular and humoral GP-specific immune response in mice. Guinea pigs immunized with a single dose of this vector were protected from any signs of disease following lethal EBOV challenge, while control animals died in 7-9 days. Subsequently, N4CT1-EBOVGP1 demonstrated complete, single-dose protection of 2 macaques following lethal EBOV challenge. A single sham-vaccinated macaque died from disease due to EBOV infection. These results demonstrate that highly attenuated rVSV vectors expressing EBOV GP may provide safer alternatives to current EBOV vaccines.

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.

Cynomolgus macaques were immunized by either the intramuscular (i.m.) or intranasal (i.n.) route with a HIV-1 peptide-based immunogen (C4-V3 89.6P) alone, or formulated with novel adjuvants to evaluate the ability of the adjuvants to augment peptide-specific systemic and mucosal immune responses. A mutant cholera toxin, CT-E29H, or the combination of recombinant human IL-1alpha (rhIL-1alpha) protein and recombinant human GM-CSF (rhGM-CSF) protein were tested as adjuvants for i.n. immunization, while a stable emulsion of a synthetic monophosphoryl lipid A (MPL) analogue (RC529-SE) plus rhGM-CSF protein was tested as an adjuvant for i.m. immunization. Macaques immunized i.n. with peptide alone failed to elicit an anti-C4-V3 89.6P antibody response in serum. In contrast, all the tested peptide/adjuvant formulations elicited peptide-specific immune responses. RC529-SE/rhGM-CSF elicited the highest peak anti-peptide IgG geometric mean titer in serum (1:32,768 at week 25) followed by rhIL-1alpha/rhGM-CSF (1:1217 at week 10) and CT-E29H (1:256 at week 25). Measurable SHIV neutralizing antibody responses were detectable in only one macaque immunized i.m. with peptide formulated with RC529-SE/rhGM-CSF. Macaques immunized by the i.n. route with peptide in combination with CT-E29H failed to elicit measurable antibody responses at nasal or genital mucosal surfaces. In contrast, antibody responses at the nasal and genital mucosa were detected in macaques immunized by the i.n. route with peptide in combination with rhIL-1alpha/rhGM-CSF. However, antibody responses at the nasal and genital mucosa were highest in macaques immunized parenterally with peptide in combination with the adjuvants RC529-SE/rhGM-CSF. These results suggest that parenteral vaccine administration in combination with the appropriate adjuvant formulation can elicit vaccine-specific humoral immune responses in both systemic and mucosal compartments.

An experimental AIDS vaccine based on attenuated, recombinant vesicular stomatitis virus (rVSV), when administered by a combination of parenteral and mucosal routes, has proven effective at preventing AIDS in a rhesus macaque model (Rose NF, et al.: Cell 2001;106:539-549). In an effort to determine the optimal route of vaccine administration we evaluated the ability of rVSV-based vaccine vectors expressing HIV-1 Env and SIV Gag proteins, when given either intramuscularly (i.m.) or intranasally (i.n.), to elicit antigen-specific cellular and humoral immune responses, and to protect from a subsequent vaginal challenge with simian-human immunodeficiency virus (SHIV89.6P). Our results demonstrate that macaques vaccinated by the i.n. route developed significantly higher antigen-specific cellular immune responses as determined by MHC class I tetramer staining, IFN-gamma ELISPOT, and cytotoxic T cell assays. However, systemic and mucosal humoral immune responses did not vary significantly with the route of vaccine administration. Given the importance of cell-mediated immune responses in slowing AIDS progression, intranasal delivery of a VSV-based AIDS vaccine may be an optimal as well as practical route for vaccination and should be considered in design of clinical trials.

In this study, we evaluated the potential of a genetically modified cholera toxin, CT-E29H as an adjuvant for recombinant Norwalk virus like particle (NV-VLP) vaccine. This detoxified mutant, containing E to H substitution at amino acid 29 of the CT-A1 subunit, was administered with a recombinant Norwalk virus like particle vaccine to Balb/c mice by mucosal routes to monitor the induction of mucosal, humoral and cellular responses. We observed that a low dose of NV-VLP (5 μg) with the adjuvant delivered by the intranasal route (IN) was more effective than the highest dose (200 μg) delivered by oral route at inducing both cellular and NV-VLP specific IgG and IgA responses. Higher counts of antigen specific IgA secreting cells were observed in the Peyer’s Patches (PP) following delivery of the vaccine with CT-E29H as compared to delivery of vaccine by mucosal routes without CT-E29H. Furthermore, there was an increase in antigen specific cells producing IL-4 from animals that received the vaccine with the adjuvant. Delivery of the vaccine by the oral route results in antigen specific CD4 + and CD8 + T cells in PP and spleen. Addition of CT-E29H results in an increase of antigen specific CD4 + cell population in PP and both CD4 + and CD8 + populations in the spleen. These cellular and cytokine responses suggest that combining the vaccine with CT-E29H results in a stronger Th2 type response. Collectively, these results indicate that immune responses to NV-VLP vaccine are qualitatively and quantitatively improved when the vaccine is delivered along with CT-E29H, and thus merits its further consideration as a mucosal adjuvant.

As new vaccines are developed, novel adjuvants may play an important role in eliciting an effective immune response. We evaluated the safety and adjuvant properties of monophosphoryl lipid A (MPL in 129 healthy toddlers immunized with two doses of nine-valent pneumococcal-CRM(197) protein conjugate vaccine (PCV9) combined with 10, 25, or 50 micro g of MPL with or without alum (AlPO(4)). Vaccine-specific humoral and cell-mediated responses were examined following the second dose of study vaccine. All doses of MPL were well-tolerated and a dose-dependent effect of MPL on specific cellular responses was observed. The 10 micro g MPL dose significantly enhanced CRM(197)-specific T-cell proliferation (P=0.02) and interferon-gamma (INF-gamma) production (P=0.009) compared to responses of controls who received PCV9 with AlPO(4). In contrast, CRM(197)-specific T-cell proliferation and interferon-gamma production of the 50 micro g MPL/AlPO(4) group were decreased when compared to controls although these differences did not reach statistical significance. IL-5 and IL-13 responses after immunization showed a similar pattern with increased production in the 10 micro g MPL group and decreased production in the 50 micro g MPL/AlPO(4) group compared to controls. There were no differences in serum IgG antibody concentrations to the nine vaccine pneumococcal capsular polysaccharides and carrier protein between the MPL-containing and control vaccine groups. These findings demonstrate a dose-dependent effect of MPL on T-helper cell type 1 (TH-1) responses to the carrier protein and also suggest an effect on T-helper cell type 2 (TH-2) responses.

Venezuelan equine encephalomyelitis (VEE) virus, a member of the family Togaviridae, genus Alphavirus, causes disease in humans and equids. The virus is normally transmitted by the bite of an infected mosquito however, it can also be highly infectious by aerosol. The purpose of the present study was to determine the effectiveness of formalin-fixed, 60Co-irradiated VEE virus microencapsulated in poly DL-lactide-co-glycolide in inducing immune responses protective against aerosol challenge with virulent VEE virus. Balb/c mice were primed by subcutaneous injection of microencapsulated VEE virus vaccine, followed 30 days later by a single immunization with the same vaccine given via the oral, intratracheal (i.t.) or subcutaneous (s.c.) route. Mice boosted by the i.t. or s.c. route had higher plasma IgG anti-VEE virus levels than orally immunized animals. The responses in the former groups were similar in magnitude to those seen in mice primed and boosted by the i.t. route. Antibody activity was detected in bronchial-alveolar and intestinal washes, fecal extracts and saliva from immunized animals. The levels of IgG and IgA antibody activity in bronchial-alveolar wash fluids from mice boosted by the i.t. route were higher than those seen in animals immunized by the oral or s.c. route with the microsphere vaccine. Mice immunized with the microencapsulated VEE virus vaccine were protected from lethal VEE virus infection following aerosol challenge at approximately three months after the initial immunization. Mucosal immunization via the i.t. route appeared to be the most effective regimen, since 100% of the mice resisted aerosol challenge.

Six female rhesus macaques were immunized orally and intranasally at 0 weeks and intratracheally at 12 weeks with an adenovirus type 5 host range mutant (Ad5hr)-simian immunodeficiency virus SIVsm env recombinant and at 24 and 36 weeks with native SIVmac251 gp120 in Syntex adjuvant. Four macaques received the Ad5hr vector and adjuvant alone; two additional controls were naive. In vivo replication of the Ad5hr wild-type and recombinant vectors occurred with detection of Ad5 DNA in stool samples and/or nasal secretions in all macaques and increases in Ad5 neutralizing antibody in 9 of 10 macaques following Ad administrations. SIV-specific neutralizing antibodies appeared after the second recombinant immunization and rose to titers > 10,000 following the second subunit boost. Immunoglobulin G (IgG) and IgA antibodies able to bind gp120 developed in nasal and rectal secretions, and SIV-specific IgGs were also observed in vaginal secretions and saliva. T-cell proliferative responses to SIV gp140 and T-helper epitopes were sporadically detected in all immunized macaques. Following vaginal challenge with SIVmac251, transient or persistent infection resulted in both immunized and control monkeys. The mean viral burden in persistently infected immunized macaques was significantly decreased in the primary infection period compared to that of control macaques. These results establish in vivo use of the Ad5hr vector, which overcomes the host range restriction of human Ads for rhesus macaques, thereby providing a new model for evaluation of Ad-based vaccines. In addition, they show that a vaccine regimen using the Ad5hr-SIV env recombinant and gp120 subunit induces strong humoral, cellular, and mucosal immunity in rhesus macaques. The reduced viral burden achieved solely with an env-based vaccine supports further development of Ad-based vaccines comprising additional viral components for immune therapy and AIDS vaccine development.

Intranasal (i.n.) immunization with bacterial protein antigens coupled to cholera toxin B subunit (CTB) effectively induces mucosal, especially salivary immunoglobulin A (IgA), and nonmucosal antibody responses in mice. To examine the regional distribution of antigen-specific B and T cells after i.n. immunization, antibody-secreting cells and antigen-responsive T cells in cervical lymph nodes (CLN) were compared with those found after intraoral or subcutaneous (in the neck) administration of the same antigen and with T cells found in mesenteric lymph nodes (MLN) and spleen after intragastric immunization. The i.n. immunization induced predominantly IgA antibody-secreting cells in salivary glands and IgA and IgG antibody-secreting cells in the superficial and central CLN; these responses were quantitatively enhanced if the antigen was coupled to CTB. Intraoral immunization also induced IgA and IgG antibody-secreting cells in the superficial and central CLN, but only if intact cholera toxin was included as an adjuvant. In contrast, subcutaneous (neck) immunization induced IgG antibody-secreting cells mainly in the draining facial lymph nodes. CLN cell populations resembled those of MLN, except that CLN lymphocytes had higher proportions of T cells and lower proportions of B cells and a slightly higher CD4+/CD8+ ratio among T cells than the MLN lymphocytes did. T cells that proliferated in response to antigen in vitro were found especially in central CLN 2 days after i.n. immunization and persisted for up to 6 months, whereas after intragastric immunization, responsive T cells were not found in the MLN for up to 14 days. After culture with antigen in vitro, T cells from the superficial CLN of i.n. immunized mice secreted both gamma interferon and interleukin-4. Therefore, after i.n. immunization, superficial and central CLN represent sites of regional lymphocyte development, and the central CLN in particular appear to be sites where memory T cells persist.

We have shown previously both in vitro and in vivo that IL-6 is an important factor for the development of IgA-producing B cells. However, despite the lack of this cytokine in mice with targeted disruption of the interleukin (IL)-6 gene (gene knockout mice), a substantial number of IgA-producing plasma cells occur in their intestinal mucosa. The experiments reported here indicate that there is a population of IgA-producing B cell precursors originating from the peritoneal cavity, distinguished from conventional Peyer's patch-derived precursors by their expression of CD5, and that IgA secretion by these cells is IL-6-independent. Further, there is an increase in CD5 expression among brightly staining IgA-producing cells obtained from the intestinal lamina propria of IL-6 gene-disrupted mice compared to normal controls. These data suggest an explanation for the persistence of IgA-producing plasma cells in the intestinal mucosa of IL-6-depleted mice and indicate the importance of IL-6 for development of conventional precursors of IgA-producing B cells, but not those derived from the peritoneal cavity pool.

One IgG1 and five IgM murine monoclonal antibodies (mAb) specific for rhesus (Rh) IgA were generated. These mAbs bound to Rh IgA but not IgG or IgM when tested by enzyme-linked immunosorbent assay. Immunoblotting revealed that the mAbs reacted with the alpha heavy chain of Rh but not human IgA. The IgG1 anti-Rh IgA mAb detected IgA-producing cells in sections of monkey gut examined by immunofluorescent staining. These mAbs should be useful for characterizing IgA responses in the Rh monkey.

Quantitative analysis for HIV-1-specific antibodies present in IgA and IgG preparations purified from the serum of HIV-seropositive individuals indicated that the proportion of HIV-specific antibodies present within the IgG isotype was seven times greater than the proportion of IgA HIV antibodies present within the IgA isotype. Dilution of IgA HIV-specific antibodies by nonspecific IgA was observed in patients with elevated serum IgA concentrations, whereas proportions of IgG HIV antibodies rose with increases in concentrations of serum IgG. Although proportions of IgA HIV antibodies were not observed to correlate with the CD4 counts of the individuals from whom immunoglobulins were purified, a significant association between the numbers of such cells and proportion of HIV antibodies present in the IgG isotype was found. Equivalent amounts of IgG were also more effective than IgA at inhibiting HIV-1IIIB infection of a susceptible T cell line. This may be due to the presence of higher proportions of IgG antibodies directed toward non-V3 determinants because reactivity against an HIV-1IIIB V3 peptide was low and did not differ significantly between these isotopes. IgA antibodies reacting against a V3 peptide containing the HIV consensus sequence could be detected in the majority of IgA samples purified from infected individuals. Proportions of IgG consensus V3-specific antibodies within the purified IgG samples were, however, much higher. The presence of accompanying increases in serum IgG concentration and proportions of IgG HIV antibodies, higher proportions of both HIV- and consensus V3-specific antibodies within this isotype, and more effective neutralization by IgG suggests that an HIV-driven response is dominated by B cells committed to production of this immunoglobulin isotype. The observed low proportions of HIV antigen-specific IgA antibodies with dilution in many individuals by elevations in non-HIV-specific IgA suggests that IgA B cells may be more susceptible to factors that mediate the polyclonal activation believed to be responsible for many of the B cell disorders characteristic of HIV infection.

This study has assessed T cell subsets according to the expression of CD4 and CD8 and the isotype of surface Ig+ (sIg+) B cells and plasma cells in mononuclear cells (MC) isolated from mucosa-associated tissues of rhesus macaques in comparison to lymphoid cells from systemic tissues, i.e., spleen and peripheral lymph nodes (PLN). Using enzymatic and/or mechanical dissociation methods, mononuclear cells were isolated from lamina propria (LP) of the small (jejunum and ileum) and large (cecum) intestines, parotid glands (PG), and mesenteric lymph nodes (MLN). Approximately equal numbers of CD4+ and CD8+ T cells occurred in mucosa-associated tissues (CD4:CD8 ratios of 0.9-1.2), while slightly higher numbers of CD8+ T cells were seen in spleen and PLN (CD4:CD8 ratios of 0.6-0.9). When the isotypes of sIg+ B cells were assessed in MC isolated from mucosa-associated tissues, the highest frequency of sIgA+ B cells was seen in MLN, which may represent B cells which had recently migrated from IgA inductive sites. However, IgA effector tissues, such as intestinal LP and PG, contained frequencies of sIgA+ B cells lower than those of the MLN. When Ig-producing cells were examined, 50-90% of Ig-secreting cells were of the IgA class. This suggests that sIgA+ B cells differentiate into IgA plasma cells at higher rates in the mucosal effector tissues. Plasma cells of IgM isotype were also found in significant numbers in the LP of the intestine and PG, while IgG plasma cells were most prevalent in spleen and PLN. Taken together, mucosa-associated tissues of rhesus macaques are characterized by higher numbers of CD4+ T cells and Ig-secreting plasma cells of IgA and IgM isotypes than those of systemic lymphoid tissues.

Past work has shown that a subset of effector T cells with unique characteristics could abrogate hapten- or antigen-induced tolerance, and the reconstitution of this immune response has been termed contrasuppression. We have studied contrasuppression in a model of oral tolerance (OT) in which adoptively transferred antigen-specific T contrasuppressor (Tcs) cells reverse OT and result in antibody responses to the eliciting antigen. In the present study, we show that murine intraepithelial lymphocytes (IELs) from mice orally immunized with sheep red blood cells (SRBC) contain T cells that exhibit Tcs cell activity. This effect was mediated by CD3+ gamma/delta T cell receptor-positive (TCR+), but not alpha/beta TCR+ T cells, and gamma/delta TCR+ Tcs cells were associated with both the CD4-,CD8+ and CD4-,CD8- (double-negative) IEL fractions. The CD4-,CD8+ gamma/delta TCR+ IELs were further separated into Vicia villosa-adherent and -nonadherent fractions. Adoptive transfer of V. villosa-adherent gamma/delta TCR+ T cells to mice with OT to SRBC resulted in splenic IgA, IgM, and IgG subclass anti-SRBC responses, while V. villosa-nonadherent gamma/delta TCR+ T cells were without activity. The gamma/delta TCR+ IELs did not support in vitro antibody responses in B cell cultures, while alpha/beta TCR+ IELs were effective T helper cells. Further, cytokine production by the gamma/delta TCR+ IELs was examined, and the gamma/delta TCR+ V. villosa-adherent fraction, which possessed contrasuppressor function, contained low levels of IL-5 mRNA and small numbers of IL-5-producing cells when compared with alpha/beta TCR+ IELs and V. villosa-nonadherent gamma/delta TCR+ IELs. Our results now show that mouse IELs contain two distinct types of T cells that function in the immune response, e.g., alpha/beta TCR+ T cells that produce IL-5 and function as helper cells, and gamma/delta TCR+ T cells that restore antibody responses in mice that had been orally tolerized with antigen.

The mucosal immune system of the gastrointestinal (GI) tract consists of Peyer's patches (PP), which are IgA inductive sites, and more diffuse effector regions which include cells in the intraepithelial lymphocyte (IEL) compartment. Since autoimmune MRL lpr/lpr (MRL/lpr) mice develop a proliferating CD3+, CD4-, CD8- (double negative; DN), B220+ T cell subset in systemic lymphoid tissue, we have initiated studies to determine the distribution of CD3+, DN, B220+ T cells (B220+ T cells or lpr/lpr T cells) in the GI immune system. Specifically, we examined T cell subsets separated according to expression of CD4, CD8, Thy-1, B220, alpha/beta T cell receptor (TcR) and gamma/delta TcR in PP and IEL of MRL/lpr mice at 6, 12 and 21 weeks of age. Increased numbers of CD3+ T cells were noted in both PP and spleen of 12- and 21-week-old mice in which the development of autoimmune disorders were also evident. However, normal numbers of CD3+ IEL T cells were seen in MRL/lpr mice in all three age groups tested. When the presence of T cell lymphadenopathy was examined in both IgA inductive and effector tissues, the PP followed the B220+ T cell pattern seen in the spleen, where approximately 30%-50% of CD3+ T cells in the PP of 12- and 21-week-old MRL/lpr mice expressed the phenotype of lpr/lpr T cells and greater than 90% were alpha/beta TcR+. On the other hand, B220+ T cells had not developed in PP or spleen of 6-week-old MRL/lpr mice. Of interest was the finding that IEL from lpr/lpr homozygous mice did not contain B220+ T cells in any age group tested. In this regard, the IEL of MRL/lpr mice comprised an identical pattern and frequency of CD4-/CD8+, CD4+/CD8-, DN and CD4+/CD8+ (double positive, DP) T cell subsets as their normal counterparts (i.e. MRL +/+, BALB/c and C3H/HeN mice) which consisted of approximately 75%, approximately 7.5%, approximately 7.5% and approximately 10%, respectively. Further, Thy-1, gamma/delta TcR and alpha/beta TcR expression in these four subsets of MRL/lpr IEL were very similar to normal mice. These results suggest that the intestinal IEL compartment is minimally affected by the lpr/lpr mutation which induces T cell abnormalities and indicate that B220+ T cells do not preferentially home to IEL. Further, our results support the concept that IEL T cells develop as a separate T cell lineage from thymus-derived cells.

Microspheres composed of biocompatible, biodegradable poly(DL-lactide-co-glycolide) (DL-PLG) and staphylococcal enterotoxin B (SEB) toxoid were evaluated as a vaccine delivery system when subcutaneously injected into mice. As measured by circulating immunoglobulin G (IgG) antitoxin titers, the delivery of SEB toxoid via DL-PLG microspheres, 1 to 10 microns in diameter, induced an immune response which was approximately 500 times that seen with nonencapsulated toxoid. The kinetics, magnitude, and duration of the antitoxin response induced with microencapsulated toxoid were similar to those obtained when an equal toxoid dose was administered as an emulsion with complete Freund adjuvant. However, the microspheres did not induce the inflammation and granulomata formation seen with complete Freund adjuvant. The adjuvant activity of the microspheres was not dependent on the superantigenicity of SEB toxin and was equally effective at potentiating circulating IgG antitrinitrophenyl levels in response to microencapsulated trinitrophenyl-keyhole limpet hemocyanin. Empty DL-PLG microspheres were not mitogenic, and SEB toxoid injected as a mixture with empty DL-PLG microspheres was no more effective as an immunogen than toxoid alone. Antigen-containing microspheres 1 to 10 microns in diameter exhibited stronger adjuvant activity than those greater than 10 microns, which correlated with the delivery of the 1- to 10-microns, but not the greater than 10-microns, microspheres into the draining lymph nodes within macrophages. The antibody response induced through immunization with microencapsulated SEB toxoid was protective against the weight loss and splenic V beta 8+ T-cell expansion induced by intravenous toxin administration. These results show that DL-PLG microsphere vaccine delivery systems, which are composed of pharmaceutically acceptable components, possess a strong adjuvant activity for their encapsulated antigens.

The utility of biodegradable and biocompatible microspheres as a vaccine delivery system for the induction of systemic and disseminated mucosal antibody responses was investigated. Intraperitoneal (ip) injection into mice of 1-10 microns microspheres, constructed of the copolymer poly(DL-lactide-coglycolide) (DL-PLG) which contained approximately 1% by weight a formalinized toxoid vaccine of staphylococcal enterotoxin B (SEB), dramatically potentiated the circulating IgG anti-toxin antibody response as compared to the free toxoid. The initiation of vaccine release was delayed in larger microspheres, and a mixture of 1-10 and 20-50 microns microspheres stimulated both a primary and an anamnestic secondary anti-toxin response following a single injection. However, neither free nor microencapsulated SEB toxoid induced a detectable mucosal IgA anti-toxin response following systemic injection. In contrast, three peroral immunizations with toxoid-microspheres stimulated circulating IgM, IgG and IgA anti-toxin antibodies and a concurrent mucosal IgA response in saliva, gut washings and lung washings. Systemic immunization with microencapsulated toxoid primed for the induction of disseminated mucosal IgA responses by subsequent oral or intratracheal (it) boosting in microspheres, while soluble toxoid was ineffective at boosting. These results indicate that biodegradable and biocompatible microspheres represent an adjuvant system with potentially widespread application in the induction of both circulating and mucosal immunity.

The role of CD4+ T cells in the gastrointestinal (GI) immune system in vivo was studied in mice selectively depleted of this subset by treatment with monoclonal anti-L3T4 (GK1.5) mAb. Treatment of young BALB/c mice with weekly injections of anti-L3T4 mAb resulted in a selective depletion of CD4+ T cells in both IgA effector (lamina propria regions of the intestine; LP) and inductive (Peyer's patch; PP) sites. However, levels of CD3+CD4-CD8+ and CD4-CD8- (double negative) T cells remained constant or increased. When sections of small intestine were assessed for the isotype of Ig-containing cells, normal mice contained predominantly IgA plasma cells with small numbers of IgM and IgG plasma cells while anti-L3T4 treatment dramatically reduced the numbers of IgA plasma cells. When numbers of IgA-producing cells were assessed by the isotype-specific ELISPOT assay, the LPL of anti-L3T4 mAb-treated mice showed an 80% reduction in the number of IgA spot-forming cells. The effect of anti-L3T4 mAb treatment on IgA inductive sites was also studied and this treatment reduced the overall size of PP as well as the germinal centers in this tissue. Although anti-L3T4 treatment depleted CD3+CD4+ T cells in PP, the relative frequency of surface IgA-positive (slgA+) B cells in this tissue did not change. These results show that repeated injection of anti-L3T4 mAb results in a CD4+ T cell deficiency in both IgA inductive (PP) and effector (LP) sites. The depletion of CD4+ T cells resulted in reductions in the numbers of mature IgA plasma cells present in the LP of gut-associated tissues, and reduced the overall size of PP including germinal centers, but did not affect the frequency of sIgA+ B cells in this IgA inductive site.

The effectiveness of plasmid DNA (pDNA) vaccines can be improved by the co-delivery of plasmid-encoded molecular adjuvants. We evaluated pDNAs encoding GM-CSF, Flt-3L, IL-12 alone, or in combination, for their relative ability to serve as adjuvants to augment humoral and cell-mediated immune responses elicited by prototype pDNA vaccines. In Balb/c mice we found that co-administration of plasmid-based murine GM-CSF (pmGM-CSF), murine Flt-3L (pmFlt-3L) or murine IL-12 (pmIL-12) could markedly enhance the cell-mediated immune response elicited by an HIV-1 env pDNA vaccine. Plasmid mGM-CSF also augmented the immune response elicited by DNA vaccines expressing HIV-1 Gag and Nef-Tat-Vif. In addition, the use of pmGM-CSF as a vaccine adjuvant appeared to markedly increase antigen-specific proliferative responses and improved the quality of the resulting T cell response by increasing the percentage of poly-functional memory CD8(+) T cells. Co-delivery of pmFlt-3L with pmGM-CSF did not result in a further increase in adjuvant activity. However, the co-administration of pmGM-CSF with pmIL-12 did significantly enhanced Env-specific proliferative responses and vaccine efficacy in the murine vaccinia virus challenge model relative to mice immunized with the env pDNA vaccine adjuvanted with either pmGM-CSF or pmIL-12 alone. These data support the testing of pmGM-CSF and pmIL-12, used alone or in combination, as plasmid DNA vaccine adjuvants in future macaque challenge studies.

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.

During the past several years, much interest has been directed towards delineating and characterizing different subsets of T helper (Th) cells in order to understand their roles in immune processes. In this study, we report the generation of antigen-specific rat Th cell clones and their characterization in terms of phenotype, function, and lymphokine production. The clones were derived by culturing purified splenic T cells from rats immunized with the pathogen Bacteroides gingivalis with equivalent numbers of irradiated spleen cells from nonimmune rats and B. gingivalis whole-cell antigen. The clones required antigen stimulation but not exogenously added interleukin-2 for growth and were maintained in culture for approximately 6 months. The cloned T cells proliferated in response to the mitogen concanavalin A and to B. gingivalis whole-cell antigen but not to other microbial antigens. Phenotypic characterization of the cloned T cells for cell surface markers demonstrated that these cells were OX19+ W3/25+ OX8- OX22- and therefore probably represented a mature subpopulation of CD4+ Th cells. These cloned T cells were positive for interleukin-2 receptor expression. Culture supernatants from the Th cell clones which were collected at various times after antigen stimulation exhibited low interleukin-2 activity and high gamma interferon activity. This in vitro study provides evidence of a rat Th cell subset that could represent an important population in regulating immune responses to microbial antigens.

Although several sensitive and specific assays have been developed to quantify murine cytokines, these assays do not allow individual cells to be correlated with the specific cytokines they produce. The purpose of this study was to develop a sensitive and reproducible method for the detection of individual T cells which secrete either interferon-gamma (IFN-gamma) or interleukin-5 (IL-5). We have used an adaptation of the enzyme-linked immunospot (ELISPOT) assay in which monoclonal antibodies to IFN-gamma (R4-6A2) and to IL-5 (TRFK-5) were used to coat 96-well plates with a nitrocellulose base. Mouse splenic T cells, either nonstimulated or activated with concanavalin A (ConA) or phytohemagglutinin (PHA), were cultured in individual wells. Following incubation, the cells were removed, and the bound cytokines probed with either biotinylated mAb anti-IFN-gamma (XMG 1.2) or anti-IL-5 (TRFK-4) followed by avidin-peroxidase. The spots which developed with 3-amino-9-ethylcarbazole were discrete and enumerated with a dissecting microscope. Although unstimulated splenic T cells contained low numbers of cytokine-specific spot-forming cells (SFC), 24-72 h activation with mitogen was required to induce significant numbers of cytokine producing cells. When mitogen-stimulated splenic CD4+ T cells were assessed, approximately equal numbers of IFN-gamma and IL-5 SFC were seen. Approximately 20-30% of all mitogen-activated splenic T cells produced at least one of these two cytokines. Pre-incubation of biotinylated anti-IFN-gamma with recombinant IFN-gamma (rIFN-gamma) or anti-IL-5 mAbs with rIL-5 completely inhibited cytokine-specific SFC. Further, use of nonrelevant antibodies did not result in spot formation, and treatment of mitogen-activated T cells with cycloheximide inhibited both IFN-gamma- and IL-5-specific SFC. A sensitive method has been developed which allows detection of individual T cells that produce either IFN-gamm or IL-5, and should be useful for detection of cytokine secretion at the single cell level.

Efficient expression vectors for interleukin 15 (IL-15) were developed combining RNA/codon optimization and modification of the IL-15 native long signal peptide. These changes resulted in elevated cytoplasmic levels of the optimized mRNA and more than 100-fold improved production of secreted human IL-15 protein. Similar modifications have also led to greatly increased rhesus macaque and murine IL-15 production. Comparison of different heterologous secretory signals showed that the tissue plasminogen activator signal is most efficient for the production of extracellular IL-15. Upon intramuscular injection of the fully optimized expression vectors in mice, IL-15 was readily detected in the serum. Serum levels represented

Background The cell mediated immune profiles following immunization with a recombinant DNA vaccine was assessed in the simian-human immunodeficiency virus (SHIV) and Macaque model. Earlier work demonstrated increased numbers of antigen specific CD8 and CD4 effector cells able to secrete IFN-γ. Method The vaccine strategy included co-immunization of a DNA based vaccine alone or in combination with a macaque IL-12 expressing plasmid (pmacIL12). Antigen activated lymphocytes were studied for activation of a set of immunological molecules. Results The current study demonstrates lymphocytes isolated and activated from the group that was immunized with DNA and pmacIL12 had a higher level of IFN-γ producing cells. We also observed a different immunological profile when comparing the cells isolated from macaques immunized with DNA as compared to those animals that also received pmacIL12. Conclusion The observed immune profiles are reflective of the co-delivery of pmacIL12 and demonstrates that IL-12 can increase the magnitude and polyfunctionality of the cellular immune response.

Respiratory syncytial virus (RSV) is a major cause of acute respiratory tract disease in humans. Towards development of a prophylactic vaccine, we genetically engineered Venezuelan equine encephalitis virus (VEEV) replicons encoding the fusion (Fa) or attachment (Ga or Gb) proteins of the A or B subgroups of RSV. Intramuscular immunization with a formulation composed of equal amounts of each replicon particle (3vRSV replicon vaccine) generated serum neutralizing antibodies against A and B strains of RSV in BALB/c mice and rhesus macaques. When contrasted with purified natural protein or formalin-inactivated RSV formulated with alum, the 3vRSV replicon vaccine induced balanced Th1/Th2 T cell responses in mice. This was evident in the increased number of RSV-specific IFN-gamma(+) splenocytes following F or G peptide stimulation, diminished quantity of eosinophils and type 2 T cell cytokines in the lungs after challenge, and increased in vivo lysis of RSV peptide-loaded target cells. The immune responses in mice were also protective against intranasal challenge with RSV. Thus, the replicon-based platform represents a promising new strategy for vaccines against RSV.

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.

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.

The glycoprotein D of HSV-2 (gD2) is currently a leading candidate vaccine target for genital herpes vaccines as both cellular and humoral responses can be generated against it. However, little is known about how vaccine composition will affect T cell epitope selection. A panel of 15-mer peptides (with 11 amino acid overlap) spanning full-length gD2 was used to investigate the fine specificity of T cell responses to gD2 as well as the role of vaccine composition on epitope selection. Spleen cells from BALB/c mice (H-2(d)) immunized with gD2, formulated with or without AlPO(4) and/or IL-12, were stimulated in vitro with overlapping gD2 peptides. Cellular responses (lymphoproliferation and IFN-gamma expression) were mapped to four epitopes within the gD2 molecule: gD2(49-63), gD2(105-119), gD2(245-259), and gD2(333-347). CTL analysis of these four epitopes indicated that not all of them could serve as a CTL epitope. Mice immunized with gD2 expressed from a viral vector mounted CTL responses primarily to one epitope located in the extracellular domain of gD2 (gD2(245-259)). More importantly, mice immunized with gD2 co-administered with IL-12 mounted CTL responses to an additional epitope located at the transmembrane-cytoplasmic junction of gD2 (gD2(333-347)). The location of this novel epitope emphasizes the benefit of using full-length versions of glycoproteins when designing vaccine components.

An experimental pDNA vaccine adjuvant expressing IL-12 was evaluated for its ability to augment the humoral and cellular immune responses elicited by a SIVmac239 gag p39 expressing pDNA vaccine. To determine the effect of vaccine dose on the immune response, rhesus macaques were immunized with 1.5 mg or 5.0 mg of SIVmac239 gag pDNA, with or without co-immunization of IL-12 pDNA at 1.5 mg and 5.0 mg, respectively. Serum antibody responses to simian immunodeficiency virus (SIV) gag were increased 10-fold ( p = 0.044, 0.002) in macaques receiving IL-12 pDNA. Cellular immune responses, monitored by SIV gag-specific IFN-γ ELISpot assay, were also significantly higher ( p = 0.007, 0.019) when the pDNA vaccine was co-immunized with IL-12 pDNA at high and low doses. There was no statistical difference between the immune responses elicited by the high and low dose of IL-12 pDNA ( p = 0.221, 0.917), a finding which could allow a dose reduction of vaccine without the concomitant loss of imunogenicity. Furthermore, analysis of the breadth of the T-cell response during the vaccination schedule, using overlapping peptides to SIV gag, demonstrated a significant correlation ( p = 0.0002) between the magnitude and breadth of the immune responses in the vaccines. These results have important implications for the continuing development of an effective, safe low dose pDNA vaccine adjuvant suitable for human use.

Given the importance of the HIV-specific cell-mediated immune response in the early control and resolution of HIV infection and the observed correlation between pre-challenge vaccine elicited CTL responses and post challenge outcome in SHIV/rhesus macaque experiments, we sought to identify several candidate plasmid DNA (pDNA) vaccine designs capable of eliciting robust and balanced cell-mediated immune responses to multiple HIV-1 derived antigens in mice for further vaccine development. To rationally construct candidate vaccines for immunogenicity testing, we determined the relative immunogenicity of the individual HIV-derived vaccine antigens (env, gag, pol, nef, tat and vif) and the relative strength of various transcriptional control elements (HCMV, SCMV, HSV Lap1) in Balb/c mice. Next, a number of 1-, 2-, 3- and 4-vector pDNA vaccine designs were tested for their ability to elicit HIV-1 antigen-specific CMI responses. For these studies, Balb/c mice were immunized with a fixed total pDNA vaccine dose of 100 mcg in combination with 25 mcg plasmid-based murine IL-12 and tested for the induction of HIV-1 antigen-specific CMI responses by IFN-gamma ELISpot analysis. The results of this study indicate that all pDNA vaccine designs were capable of eliciting CMI responses to multiple HIV-1 antigens. As a result of this iterative comparative analysis, we have identified a number of pDNA vaccine candidates capable of eliciting potent, balanced CMI responses to multiple HIV-1 derived antigens. These results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.

Of the various approaches being developed as prophylactic HIV vaccines, those based on a heterologous plasmid DNA prime, live vector boost vaccination regimen appear especially promising in the nonhuman primate/simian-human immunodeficiency virus (SHIV) challenge model. In this study, we sought to determine whether a series of intramuscular priming immunizations with a plasmid DNA vaccine expressing SIVgag p39, in combination with plasmid expressed rhesus IL-12, could effectively enhance the immunogenicity and postchallenge efficacy of two intranasal doses of recombinant vesicular stomatitis virus (rVSV)-based vectors expressing HIV-1 env 89.6P gp160 and SIVmac239 gag p55 in rhesus macaques. In macaques receiving the combination plasmid DNA prime, rVSV boost vaccination regimen we observed significantly increased SIVgag- specific cell-mediated and humoral immune responses and significantly lower viral loads postintravenous SHIV89.6P challenge relative to macaques receiving only the rVSV vectored immunizations. In addition, the plasmid DNA prime, rVSV boost vaccination regimen also tended to increase the preservation of peripheral blood CD4+ cells and reduce the morbidity and mortality associated with SHIV89.6P infection. An analysis of immune correlates of protection after SHIV89.6P challenge revealed that the prechallenge SHIV-specific IFN-gamma ELISpot response elicited by vaccination and the ability of the host to mount a virus-specific neutralizing antibody response postchallenge correlated with postchallenge clinical outcome. The correlation between vaccine-elicited cell-mediated immune responses and an improved clinical outcome after SHIV challenge provides strong justification for the continued development of a cytokine-enhanced plasmid DNA prime, rVSV vector boost immunization regimen for the prevention of HIV 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.