Gag-specific Immune Responses Research Articles

The role of mRNA-galsomes and LNPs in enhancing HIV-specific T cell responses across various lymphoid organs

mRNA nanoparticles have been investigated in the context of prophylactic vaccination against HIV but their effectivity has not been widely investigated in therapeutic vaccination. It has been suggested that a profound CD8+ T-cell response within lymphoid tissues, a primary site for viral reservoirs, is crucial for achieving optimal viral control, potentially correlating with protection. This study aimed to evaluate the effectiveness of mRNA lipid nanoparticles (LNPs), including a modified variant containing α-galactosylceramide as an adjuvant, termed galsomes. C57BL/6 mice were immunized intramuscularly with nucleoside-modified mRNA encoding ovalbumin (li80tOVA), revealing that mRNA-galsomes induced slightly higher proliferation levels of li80tOVA-specific CD8+ T cells in lymphoid tissues across various anatomical sites compared to mRNA-LNPs. In addition, immunization with nucleoside-modified HIV-1 Gag mRNA elicited a notable Gag-specific immune response in both formulations even at low mRNA doses. Remarkably, mRNA-galsomes induced lower polyfunctional responses in CD4+ T cells, but similar polyfunctional responses in CD8+ T cells in the spleen compared to mRNA LNPs. Importantly, the Gag-specific CD8+ T cells demonstrated cytolytic capacity against target cells in both the spleen and lymphoid tissues, including gut-associated lymph nodes. These findings underscore the potential of both mRNA-galsomes and mRNA-LNPs as tools for therapeutic vaccination against HIV.

DNA prime/MVTT boost regimen with HIV-1 mosaic Gag enhances the potency of antigen-specific immune responses

HIV-1 diversity and latent reservoir are the major challenges for the development of an effective AIDS vaccine. It is well indicated that Gag-specific CD8+ T cells serve as the dominant host immune surveillance for HIV-1 control, but it still remains a challenge for vaccine design to induce broader and stronger cytotoxic T cell immunity against the virus. Genetic variation of the HIV-1 gag gene across different clades is one of the reasons for the reduction of antigenic epitope coverage. Here, we report an immunization strategy with heterologous vaccines expressing a mosaic Gag antigen aimed to increase antigenic breadth against a wider spectrum of HIV-1 strains. Priming using a DNA vaccine via in vivo electroporation, followed by boosting with a live replication-competent modified vaccinia TianTan (MVTT) vectored vaccine, elicited greater and broader protective Gag-specific immune responses in mice. Compared to DNA or MVTT homologous immunization, the heterologous DNA/MVTT vaccination resulted in higher frequencies of broadly reactive, Gag-specific, polyfunctional, long-lived cytotoxic CD8+ T cells, as well as increased anti-Gag antibody titer. Importantly, the DNA/MVTT heterologous vaccination induced protection against EcoHIV and mesothelioma AB1-Gag challenges. In summary, the stronger protective Gag-specific immunity induced by the heterologous regimen using two safe vectors shows promise for further development to enhance anti-HIV-1 immunity. Our study has important implications for immunogen design and the development of an effective HIV-1 heterologous vaccination strategy.

Vector Order Determines Protection against Pathogenic Simian Immunodeficiency Virus Infection in a Triple-Component Vaccine by Balancing CD4+ and CD8+ T-Cell Responses.

An effective AIDS vaccine should elicit strong humoral and cellular immune responses while maintaining low levels of CD4+ T-cell activation to avoid the generation of target cells for viral infection. The present study investigated two prime-boost regimens, both starting vaccination with single-cycle immunodeficiency virus, followed by two mucosal boosts with either recombinant adenovirus (rAd) or fowlpox virus (rFWPV) expressing SIVmac239 or SIVmac251 gag/pol and env genes, respectively. Finally, vectors were switched and systemically administered to the reciprocal group of animals. Only mucosal rFWPV immunizations followed by systemic rAd boost significantly protected animals against a repeated low-dose intrarectal challenge with pathogenic SIVmac251, resulting in a vaccine efficacy (i.e., risk reduction per exposure) of 68%. Delayed viral acquisition was associated with higher levels of activated CD8+ T cells and Gag-specific gamma interferon (IFN-γ)-secreting CD8+ cells, low virus-specific CD4+ T-cell responses, and low Env antibody titers. In contrast, the systemic rFWPV boost induced strong virus-specific CD4+ T-cell activity. rAd and rFWPV also induced differential patterns of the innate immune responses, thereby possibly shaping the specific immunity. Plasma CXCL10 levels after final immunization correlated directly with virus-specific CD4+ T-cell responses and inversely with the number of exposures to infection. Also, the percentage of activated CD69+ CD8+ T cells correlated with the number of exposures to infection. Differential stimulation of the immune response likely provided the basis for the diverging levels of protection afforded by the vaccine regimen.IMPORTANCE A failed phase II AIDS vaccine trial led to the hypothesis that CD4+ T-cell activation can abrogate any potentially protective effects delivered by vaccination or promote acquisition of the virus because CD4+ T helper cells, required for an effective immune response, also represent the target cells for viral infection. We compared two vaccination protocols that elicited similar levels of Gag-specific immune responses in rhesus macaques. Only the animal group that had a low level of virus-specific CD4+ T cells in combination with high levels of activated CD8+ T cells was significantly protected from infection. Notably, protection was achieved despite the lack of appreciable Env antibody titers. Moreover, we show that both the vector and the route of immunization affected the level of CD4+ T-cell responses. Thus, mucosal immunization with FWPV-based vaccines should be considered a potent prime in prime-boost vaccination protocols.

Live virus vaccines based on a vesicular stomatitis virus (VSV) backbone: Standardized template with key considerations for a risk/benefit assessment

The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety of live, recombinant viral vaccines incorporating genes from heterologous viral and other microbial pathogens in their genome (so-called “chimeric virus vaccines”). Many such viral vector vaccines are now at various stages of clinical evaluation. Here, we introduce an attenuated form of recombinant vesicular stomatitis virus (rVSV) as a potential chimeric virus vaccine for HIV-1, with implications for use as a vaccine vector for other pathogens. The rVSV/HIV-1 vaccine vector was attenuated by combining two major genome modifications. These modifications acted synergistically to greatly enhance vector attenuation and the resulting rVSV vector demonstrated safety in sensitive mouse and non-human primate neurovirulence models. This vector expressing HIV-1 gag protein has completed evaluation in two Phase I clinical trials. In one trial the rVSV/HIV-1 vector was administered in a homologous two-dose regimen, and in a second trial with pDNA in a heterologous prime boost regimen. No serious adverse events were reported nor was vector detected in blood, urine or saliva post vaccination in either trial. Gag specific immune responses were induced in both trials with highest frequency T cell responses detected in the prime boost regimen. The rVSV/HIV-1 vector also demonstrated safety in an ongoing Phase I trial in HIV-1 positive participants. Additionally, clinical trial material has been produced with the rVSV vector expressing HIV-1 env, and Phase I clinical evaluation will initiate in the beginning of 2016. In this paper, we use a standardized template describing key characteristics of the novel rVSV vaccine vectors, in comparison to wild type VSV. The template facilitates scientific discourse among key stakeholders by increasing transparency and comparability of information. The Brighton Collaboration V3SWG template may also be useful as a guide to the evaluation of other recombinant viral vector vaccines.

Mucosal Immunization with Newcastle Disease Virus Vector Coexpressing HIV-1 Env and Gag Proteins Elicits Potent Serum, Mucosal, and Cellular Immune Responses That Protect against Vaccinia Virus Env and Gag Challenges.

ABSTRACTNewcastle disease virus (NDV) avirulent strain LaSota was used to coexpress gp160 Env and p55 Gag from a single vector to enhance both Env-specific and Gag-specific immune responses. The optimal transcription position for both Env and Gag genes in the NDV genome was determined by generating recombinant NDV (rNDV)-Env-Gag (gp160 located between the P and M genes and Gag between the HN and L genes), rNDV-Gag-Env (Gag located between the P and M genes and gp160 between the HN and L genes), rNDV-Env/Gag (gp160 followed by Gag located between the P and M genes), and rNDV-Gag/Env (Gag followed by gp160 located between the P and M genes). All the recombinant viruses replicated at levels similar to those seen with parental NDV in embryonated chicken eggs and in chicken fibroblast cells. Both gp160 and Gag proteins were expressed at high levels in cell culture, with gp160 found to be incorporated into the envelope of NDV. The Gag and Env proteins expressed by all the recombinants except rNDV-Env-Gag self-assembled into human immunodeficiency virus type 1 (HIV-1) virus-like particles (VLPs). Immunization of guinea pigs by the intranasal route with these rNDVs produced long-lasting Env- and Gag-specific humoral immune responses. The Env-specific humoral and mucosal immune responses and Gag-specific humoral immune responses were higher in rNDV-Gag/Env and rNDV-Env/Gag than in the other recombinants. rNDV-Gag/Env and rNDV-Env/Gag were also more efficient in inducing cellular as well as protective immune responses to challenge with vaccinia viruses expressing HIV-1 Env and Gag in mice. These results suggest that vaccination with a single rNDV coexpressing Env and Gag represents a promising strategy to enhance immunogenicity and protective efficacy against HIV.

Induction of HIV-1 gag specific immune responses by cationic micelles mediated delivery of gag mRNA

In recent years, mRNA-based vaccines have emerged to be a great alternative to DNA-based vaccines due to the safety of not inserting into host genome. However, mRNA molecules are single-stranded nucleic acids that are vulnerable under RNase existing in human skin and tissues. In this study, a self-assembled cationic nanomicelles based on polyethyleneimine-stearic acid (PSA) copolymer were developed to delivery HIV-1 gag encoding mRNA to dendritic cells and BALB/c mice. We evaluated the transfection efficiency and cell uptake efficiency of naked EGFP mRNA, PSA, PEI-2k and PEI-25k nanoparticles format on DC2.4 cell lines. Immune responses after sub-cutaneous administration of gag mRNA to BALB/c mice were notably induced by PSA as compared with naked gag mRNA. We found the PSA/mRNA nanomicelles were potent systems that can effectively deliver mRNA and induce antigen-specific immune response, stimulating various new vaccine strategies using mRNA.

Regulation of HIV-Gag expression and targeting to the endolysosomal/secretory pathway by the luminal domain of lysosomal-associated membrane protein (LAMP-1) enhance Gag-specific immune response.

We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.

Attenuation of immune activation in an open-label clinical trial for HIV-AIDS using a polyherbal formulation.

To explain a stable clinical outcome observed in a previous pilot clinical trial using a polyherbal formulation (PHF) for HIV-AIDS, we, in the present study, evaluated the T cell functions from fresh and stored blood samples. In three clinical groups-the anti-retroviral therapy, PHF and control arms-we compared the circulating levels of lipopolysaccharide, LPS-binding protein, soluble CD14, aspartate transaminase (AST) and alanine transaminase (ALT). Additionally, we evaluated the expression of T cell markers and gag-specific immune responses. The PHF treatment significantly reduced the levels of sCD14, AST and ALT. In a cross-sectional analysis at 30months post-treatment, in comparison to the control group, the PHF arm showed significantly low per-cell expression of PD1, CD95 and HLA-DR. The PHF treatment appears to have attenuated general immune activation and hepatic inflammation in the study participants. Targeting the mediators of immune activation must be pursued as a useful strategy for HIV-AIDS management.

Increased Sequence Coverage through Combined Targeting of Variant and Conserved Epitopes Correlates with Control of HIV Replication

A major challenge in the development of an HIV vaccine is that of contending with the extensive sequence variability found in circulating viruses. Induction of HIV-specific T-cell responses targeting conserved regions and induction of HIV-specific T-cell responses recognizing a high number of epitope variants have both been proposed as strategies to overcome this challenge. We addressed the ability of cytotoxic T lymphocytes from 30 untreated HIV-infected subjects with and without control of virus replication to recognize all clade B Gag sequence variants encoded by at least 5% of the sequences in the Los Alamos National Laboratory HIV database (1,300 peptides) using gamma interferon and interleukin-2 (IFN-γ/IL-2) FluoroSpot analysis. While targeting of conserved regions was similar in the two groups (P = 0.47), we found that subjects with control of virus replication demonstrated marginally lower recognition of Gag epitope variants than subjects with normal progression (P = 0.05). In viremic controllers and progressors, we found variant recognition to be associated with viral load (r = 0.62, P = 0.001). Interestingly, we show that increased overall sequence coverage, defined as the overall proportion of HIV database sequences targeted through the Gag-specific repertoire, is inversely associated with viral load (r = -0.38, P = 0.03). Furthermore, we found that sequence coverage, but not variant recognition, correlated with increased recognition of a panel of clade B HIV founder viruses (r = 0.50, P = 0.004). We propose sequence coverage by HIV Gag-specific immune responses as a possible correlate of protection that may contribute to control of virus replication. Additionally, sequence coverage serves as a valuable measure by which to evaluate the protective potential of future vaccination strategies.

Correction: Priming with Recombinant Auxotrophic BCG Expressing HIV-1 Gag, RT and Gp120 and Boosting with Recombinant MVA Induces a Robust T Cell Response in Mice.

[This corrects the article DOI: 10.1371/journal.pone.0071601.].

Priming with recombinant auxotrophic BCG expressing HIV-1 Gag, RT and Gp120 and boosting with recombinant MVA induces a robust T cell response in mice.

In previous studies we have shown that a pantothenate auxotroph of Myocbacterium bovis BCG (BCGΔpanCD) expressing HIV-1 subtype C Gag induced Gag-specific immune responses in mice and Chacma baboons after prime-boost immunization in combination with matched rMVA and VLP vaccines respectively. In this study recombinant BCG (rBCG) expressing HIV-1 subtype C reverse transcriptase and a truncated envelope were constructed using both the wild type BCG Pasteur strain as a vector and the pantothenate auxotroph. Mice were primed with rBCG expressing Gag and RT and boosted with a recombinant MVA, expressing a polyprotein of Gag, RT, Tat and Nef (SAAVI MVA-C). Priming with rBCGΔpanCD expressing Gag or RT rather than the wild type rBCG expressing Gag or RT resulted in higher frequencies of total HIV-specific CD8+ T cells and increased numbers of T cells specific to the subdominant Gag and RT epitopes. Increasing the dose of rBCG from 105 cfu to 107 cfu also led to an increase in the frequency of responses to subdominant HIV epitopes. A mix of the individual rBCGΔpanCD vaccines expressing either Gag, RT or the truncated Env primed the immune system for a boost with SAAVI MVA-C and generated five-fold higher numbers of HIV-specific IFN-γ-spot forming cells than mice primed with rBCGΔpanCD containing an empty vector control. Priming with the individual rBCGΔpanCD vaccines or the mix and boosting with SAAVI MVA-C also resulted in the generation of HIV-specific CD4+ and CD8+ T cells producing IFN-γ and TNF-α and CD4+ cells producing IL-2. The rBCG vaccines tested in this study were able to prime the immune system for a boost with rMVA expressing matching antigens, inducing robust, HIV-specific T cell responses to both dominant and subdominant epitopes in the individual proteins when used as individual vaccines or in a mix.

A pantothenate suxotroph of BCG rxpressing Gag confers enhanced HIV-specific immunogenicity compared to wildtype and perfingolysin expressing strains

Background In tuberculosis vaccine studies, perfingolysin expressing strains (pfo) of recombinant Mycobacterium bovis (rBCG) have been shown to enhance immunogenicity as compared to wildtype strains whilst pantothenate auxotrophic strains (ΔpanCD) have been shown to be safer and more immunogenic. Our group has recently shown that rBCGΔpanCD expressing HIV-1 Gag is more immunogenic than the wildtype Pasteur strain of BCG in the murine model. In this study, a wild type strain, a ΔpanCDstrain, a pfo strain and a ΔpanCD strain expressing perfringolysin (ΔpanCDpfo) of Danish BCG were used as vectors to express HIV-1 subtype C Gag. Gag specific immune responses induced by a prime with each rBCG-Gag vaccine and boost with modified vaccinia Ankara (MVA) were compared. Methods rBCG vaccines were given intraperitoneally to BALB/c mice followed by an intramuscular MVA boost after 28 days. Twelve days after the boost, mice were sacrificed and single cell suspensions of splenocytes were stimulated with HIV peptides. Immunogenicity was assessed using an IFN-g ELISPOT assay, cytokine bead array and multi-parameter flow cytometry. Results All the rBCG-Gag vaccines primed the immune response to a boost with MVA. Cytokine kinetic measurements and flow cytometry indicated a more rapid and robust release of pro-inflammatory cytokines, in response to ex vivo HIV peptides, from the splenocytes of mice vaccinated with rBCGΔpanCD(gag) as compared to rBCGpfo(gag)(p<0.001- p<0.05) and rBCG(gag) (p<0.01- p<0.05). A rBCGΔpanCD(gag) prime induced increased polyfunctional HIV specific CD4+ and CD8+ T cells as compared to the other strains of BCG. We observed no enhancement of immunogenicity in the rBCGΔpanCDpfo(gag) group as compared to the rBCGΔpanCD(gag) group. Conclusion As this study indicated the Danish ΔpanCD vector induced a more robust and rapid Gag-specific immune response than the wild type and perfringolysin expressing BCG Danish vectors, the Danish ΔpanCD vector maybe an attractive option for HIV vaccines.

Soluble multi-trimeric TNF superfamily ligand adjuvants enhance immune responses to a HIV-1 Gag DNA vaccine

DNA vaccines remain an important component of HIV vaccination strategies, typically as part of a prime/boost vaccination strategy with viral vector or protein boost. A number of DNA prime/viral vector boost vaccines are currently being evaluated for both preclinical studies and in Phase I and Phase II clinical trials. These vaccines would benefit from molecular adjuvants that increase correlates of immunity during the DNA prime. While HIV vaccine immune correlates are still not well defined, there are a number of immune assays that have been shown to correlate with protection from viral challenge including CD8+ T cell avidity, antigen-specific proliferation, and polyfunctional cytokine secretion. Recombinant DNA vaccine adjuvants composed of a fusion between Surfactant Protein D (SP-D) and either CD40 Ligand (CD40L) or GITR Ligand (GITRL) were previously shown to enhance HIV-1 Gag DNA vaccines. Here we show that similar fusion constructs composed of the TNF superfamily ligands (TNFSFL) 4-1BBL, OX40L, RANKL, LIGHT, CD70, and BAFF can also enhanced immune responses to a HIV-1 Gag DNA vaccine. BALB/c mice were vaccinated intramuscularly with plasmids expressing secreted Gag and SP-D-TNFSFL fusions. Initially, mice were analyzed 2 weeks or 7 weeks following vaccination to evaluate the relative efficacy of each SP-D-TNFSFL construct. All SP-D-TNFSFL constructs enhanced at least one Gag-specific immune response compared to the parent vaccine. Importantly, the constructs SP-D-4-1BBL, SP-D-OX40L, and SP-D-LIGHT enhanced CD8+ T cell avidity and CD8+/CD4+ T cell proliferation 7 weeks post vaccination. These avidity and proliferation data suggest that 4-1BBL, OX40L, and LIGHT fusion constructs may be particularly effective as vaccine adjuvants. Constructs SP-D-OX40L, SP-D-LIGHT, and SP-D-BAFF enhanced Gag-specific IL-2 secretion in memory T cells, suggesting these adjuvants can increase the number of self-renewing Gag-specific CD8+ and/or CD4+ T cells. Finally adjuvants SP-D-OX40L and SP-D-CD70 increased T(H)1 (IgG2a) but not T(H)2 (IgG1) antibody responses in the vaccinated animals. Surprisingly, the B cell-activating protein BAFF did not enhance anti-Gag antibody responses when given as an SP-D fusion adjuvant, but nonetheless enhanced CD4+ and CD8+ T cell responses. We present evidence that various SP-D-TNFSFL fusion constructs can enhance immune responses following DNA vaccination with HIV-1 Gag expression plasmid. These data support the continued evaluation of SP-D-TNFSFL fusion proteins as molecular adjuvants for DNA and/or viral vector vaccines. Constructs of particular interest included SP-D-OX40L, SP-D-4-1BBL, SP-D-LIGHT, and SP-D-CD70. SP-D-BAFF was surprisingly effective at enhancing T cell responses, despite its inability to enhance anti-Gag antibody secretion.

HIV-1 Gag-Virus-Like Particles Induce Natural Killer Cell Immune Responses via Activation and Maturation of Dendritic Cells

Despite the extensive efforts that have been made to combat acquired immune deficiency syndrome (AIDS), the number of people infected each year with human immunodeficiency virus type 1 (HIV-1) is still increasing worldwide, and a safe and effective vaccine to control HIV infection is urgently needed. Recently, the natural killer (NK) cell-mediated innate immune response, which represents the first line of defense against infections, has attracted attention for its role in combating HIV infection and disease progression. In the present study, we investigated the immunogenic ability of HIV-1 Gag-virus-like particles (Gag-VLPs) to induce NK cell immune responses in vitro and in vivo. Gag-VLPs efficiently activated human monocyte-derived dendritic cells (MDDCs), eliciting MDDC maturation with an associated increase in the surface expression of CD80, CD86 and MHC classes I and II, MDDC proliferation and proinflammatory cytokine production. Gag-VLP-treated MDDCs subsequently activated autologous NK cells, leading to their proliferation and production of interferon-γ and to the upregulation of NK cell cytotoxicity against YAC-1 cells and HIV-1-infected CD4⁺ T cells. In addition, we introduced a 2-phase immunization strategy in BALB/c mice to assess the role of DCs in the induction of NK cell immune responses by Gag-VLPs in vivo. Our findings reveal that Gag-VLPs efficiently activate DCs, which in turn induce innate and Gag-specific immune responses in NK cells.

Enhanced HIV-1 Gag-specific immunity induced by a sPD-1-based vaccine directed to dendritic cells (53.23)

Abstract HIV/AIDS is a serious disease worldwide. The development of a safe, effective and affordable HIV-1 vaccine remains the ultimate solution. This study is based on the discovery of a negative immune regulatory system, the PD-1/PD-L pathway, which exists on both human and other animals. The PD-1 ligands (PD-L1 and PD-L2) are expressed on DCs. So we hypothesize to utilize the soluble PD-1 parts to intervene in the negative signal transmission of the PD-1 pathway and to target the antigen to DCs as a mean to indentify vaccine strategy to enhance the virus specific immune responses. Here we constructed the novel vaccine and controls by inserting soluble PD-1 gene and/or HIV-1 p24 gene into the pVAX1 vector with fusing of rabbit Fc fragment named mspd1-p24-fc, and controls named mspd1-IgVΔ-p24-fc and p24-fc. When compared with control vaccines, we discovered that mspd1-p24fc can significantly enhance HIV-1 Gag-specific immune responses by measuring the number of IFN-γ expressed CD4 and CD8 T cells using Elispot assays and CD8 T cell tetramer staining. Furthermore, the mspd1-p24-fc can significantly enhance humoral immune responses by anti-Gag antibody titer analysis. Importantly, this novel vaccine protects mice against recombinant vaccinia virus-gagpol challenges. This novel vaccine design may be used as DNA vaccine model against other infectious disease and cancer which need eliciting significant antigen specific humoral and cellular immune responses.

Newcastle Disease Virus Expressing a Dendritic Cell-Targeted HIV Gag Protein Induces a Potent Gag-Specific Immune Response in Mice

Viral vaccine vectors have emerged as an attractive strategy for the development of a human immunodeficiency virus (HIV) vaccine. Recombinant Newcastle disease virus (rNDV) stands out as a vaccine vector since it has a proven safety profile in humans, it is a potent inducer of both alpha interferon (IFN-α) and IFN-β) production, and it is a potent inducer of dendritic cell (DC) maturation. Our group has previously generated an rNDV vector expressing a codon-optimized HIV Gag protein and demonstrated its ability to induce a Gag-specific CD8(+) T cell response in mice. In this report we demonstrate that the Gag-specific immune response can be further enhanced by the targeting of the rNDV-encoded HIV Gag antigen to DCs. Targeting of the HIV Gag antigen was achieved by the addition of a single-chain Fv (scFv) antibody specific for the DC-restricted antigen uptake receptor DEC205 such that the DEC205 scFv-Gag molecule was encoded for expression as a fusion protein. The vaccination of mice with rNDV coding for the DC-targeted Gag antigen induced an enhanced Gag-specific CD8(+) T cell response and enhanced numbers of CD4(+) T cells and CD8(+) T cells in the spleen relative to vaccination with rNDV coding for a nontargeted Gag antigen. Importantly, mice vaccinated with the DEC205-targeted vaccine were better protected from challenge with a recombinant vaccinia virus expressing the HIV Gag protein. Here we demonstrate that the targeting of the HIV Gag antigen to DCs via the DEC205 receptor enhances the ability of an rNDV vector to induce a potent antigen-specific immune response.

Dendritic cells infected by recombinant rabies virus vaccine vector expressing HIV-1 Gag are immunogenic even in the presence of vector-specific immunity

Dendritic cells (DC) are the most potent antigen presenting cells whose ability to interact with T cells, B cells and NK cells has led to their extensive use in vaccine design. Here, we designed a DC-based HIV-1 vaccine using an attenuated rabies virus vector expressing HIV-1 Gag (RIDC-Gag). To test this, BALB/c mice were immunized with RIDC-Gag, and the primary, secondary as well as humoral immune responses were monitored. Our results indicate that RIDC-Gag stimulated HIV-1 Gag-specific immune responses in mice. When challenged with vaccinia virus (VV) expressing HIV-1 Gag, they elicited a potent Gag-specific recall response characterized by CD8+ T cells expressing multiple cytokines that were capable of specifically lysing Gag-pulsed target cells. Moreover, RIDC-Gag also enhanced CD8+ T cell responses via a homologous prime-boost regimen. These results show that a DC-based vaccine using live RV is immunogenic and a potential candidate for a therapeutic HIV-1 vaccine.

Immunization of neonatal mice with LAMP/p55 HIV gag DNA elicits robust immune responses that last to adulthood

Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 ( LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects. Neonatal vaccination with chimeric LAMP/gag generated stronger Gag-specific immune responses, as measured by the breadth of the Gag peptide-specific IFN-γ, proliferative responsiveness, cytokine production and antibody production, all of which revealed activation of CD4+ T cells as well as the generation of a more robust CTL response compared to gag vaccine alone. To induce long-lived T and B cell memory responses, it was necessary to immunize neonates with the chimeric LAMP/gag DNA vaccine. The LAMP/gag DNA vaccine strategy could be particularly useful for generating an anti-HIV immune response in the early postnatal period capable of inducing long-term immunological memory.

Comparison of the Expression and Immunogenicity of Wild-Type and Sequence-Modified HIV-1 gag Genes in a Recombinant Sendai Virus Vector

Efficient expression of HIV-1 structural gene involves regulation by viral Rev and Rev-responsive elements (RRE). Messenger RNAs of wild-type HIV-1 structural genes are either retained in the nucleus or degraded rapidly in the absence of Rev/RRE; therefore little protein can be expressed. Modifying HIV-1 genes is an excellent approach to circumvent this problem, but it is a laborious and costly process. Using certain vectors to deliver wild-type genes may be a promising approach. In this study, the wild-type and modified gag genes, derived from Chinese HIV-1 isolates, were separately constructed in both plasmid DNA and recombinant Sendai virus vectors (rSeV). The expression and immunogenicity of the wild-type and modified gag genes were compared. The results showed that efficient expression of the modified gag gene could be achieved by transfection with DNA and infection with rSeV, but the efficient expression of wild-type gag could only be achieved by rSeV. In addition, the rSeV expressing wild-type gag elicited similar Gag-specific immune responses with modified gag in both SeV/SeV and DNA-prime/rSeV-boost schemes. However, SeV/SeV failed to produce Gag-specific responses as robust as DNA/rSeV. Then the SeV-specific humoral and cellular immune responses were evaluated just before the second rSeV vaccine immunization. It was found that anti-SeV neutralizing antibody was very low but the SeV-specific cellular response was strong. Efficient expression of wild-type HIV-1 structural genes may make the SeV vector a useful tool for the HIV-1 vaccine research, but the strong SeV-specific cellular immune responses may impair the efficacy of SeV-SeV vaccination scheme.

HIV-1 Gag-specific immunity induced by a lentivector-based vaccine directed to dendritic cells

Lentivectors (LVs) have attracted considerable interest for their potential as a vaccine delivery vehicle. In this study, we evaluate in mice a dendritic cell (DC)-directed LV system encoding the Gag protein of human immunodeficiency virus (HIV) (LV-Gag) as a potential vaccine for inducing an anti-HIV immune response. The DC-directed specificity is achieved through pseudotyping the vector with an engineered Sindbis virus glycoprotein capable of selectively binding to the DC-SIGN protein. A single immunization by this vector induces a durable HIV Gag-specific immune response. We investigated the antigen-specific immunity and T-cell memory generated by a prime/boost vaccine regimen delivered by either successive LV-Gag injections or a DNA prime/LV-Gag boost protocol. We found that both prime/boost regimens significantly enhance cellular and humoral immune responses. Importantly, a heterologous DNA prime/LV-Gag boost regimen results in superior Gag-specific T-cell responses as compared with a DNA prime/adenovector boost immunization. It induces not only a higher magnitude response, as measured by Gag-specific tetramer analysis and intracellular IFN-gamma staining, but also a better quality of response evidenced by a wider mix of cytokines produced by the Gag-specific CD8(+) and CD4(+) T cells. A boosting immunization with LV-Gag also generates T cells reactive to a broader range of Gag-derived epitopes. These results demonstrate that this DC-directed LV immunization is a potent modality for eliciting anti-HIV immune responses.