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.

A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles.

Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viral vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1.

Angiogenic, lymphangiogenic and adipogenic effects of HIV-1 matrix protein p17.

Lymphangiogenesis and concurrent angiogenesis are essential in supporting proliferation and survival of AIDS-related lymphomas, which are often metastatic. In vitro studies suggest a candidate angiogienic and lymphangiogenic factor encoded by HIV: the matrix protein p17. p17 accumulates in lymph nodes of patients even when they are undergoing highly active antiretroviral therapy. p17 has been found to affect immune cells, and recent data showed that a variant p17, called S75X, induces cell growth by triggering MAPK/ERK and PI3K/AKT pathways. We tested the in vivo angiogenic activity of p17 by injecting it in Matrigel plugs in nude mice. Plugs were retrieved 7 days after injection, and assessed macroscopically, and by light and confocal microscopy. Our data revealed that both reference and S75X variant p17 promote angiogenesis and lymphangiogenesis in vivo. Our results suggest that the induction of angiogenesis and lymphangiogenesis by HIV-1 p17 may generate a favorable microenvironment that could trigger tumor growth and maintenance. Moreover, the presence of adipocytes infiltration observed at the histological level suggests a possible interplay between angiogenesis, lymphangiogenesis and adipogenesis. These findings offer new opportunities for the development of treatment strategies to combat HIV-related cancers.

Evaluation of humoral, mucosal, and cellular immune responses following co-immunization of HIV-1 Gag and Env proteins expressed by Newcastle disease virus.

The combination of multiple HIV antigens in a vaccine can broaden antiviral immune responses. In this study, we used NDV vaccine strain LaSota to generate rNDV (rLaSota/optGag) expressing human codon optimized p55 Gag protein of HIV-1. We examined the effect of co-immunization of rLaSota/optGag with rNDVs expressing different forms of Env protein gp160, gp120, gp140L [a version of gp140 that lacked cytoplasmic tail and contained complete membrane-proximal external region (MPER)] and gp140S (a version of gp140 that lacked cytoplasmic tail and distal half of MPER) on magnitude and breadth of humoral, mucosal and cellular immune responses in guinea pigs and mice. Our results showed that inclusion of rLaSota/optGag with rNDVs expressing different forms of Env HIV Gag did not affect the Env-specific humoral and mucosal immune responses in guinea pigs and that the potent immune responses generated against Env persisted for at least 13 weeks post immunization. The highest Env-specific humoral and mucosal immune responses were observed with gp140S+optGag group. The neutralizing antibody responses against HIV strains BaL.26 and MN.3 induced by gp140S+optGag and gp160+optGag were higher than those elicited by other groups. Inclusion of Gag with gp160, gp140S and gp140L enhanced the level of Env-specific IFN-γ-producing CD8(+) T cells in mice. Inclusion of Gag with gp160 and gp140L also resulted in increased Env-specific CD4(+) T cells. The level of Gag-specific CD8(+) and CD4(+) T cells was also enhanced in mice immunized with Gag along with gp140S and gp120. These results indicate lack of antigen interference in a vaccine containing rNDVs expressing Env and Gag proteins.

Short conserved sequences of HIV-1 are highly immunogenic and shift immunodominance.

UNLABELLED: Cellular immunity is pivotal in HIV-1 pathogenesis but is hampered by viral sequence diversity. An approach to minimize this diversity is to focus immunity on conserved proteome sequences; therefore, we selected four relatively conserved regions (Gag amino acids 148 to 214 and 250 to 335, Env amino acids 521 to 606, and Nef amino acids 106 to 148), each created in three mosaics, to provide better coverage of M-group HIV-1 sequences. A conserved-region vaccine (CRV) delivering genes for these four regions as equal mixtures of three mosaics each (each region at a separate injection site) was compared to a whole-protein vaccine (WPV) delivering equimolar amounts of genes for whole Gag, Env, and Nef as clade B consensus sequences (separate injection sites). Three rhesus macaques were vaccinated via three DNA primes and a recombinant adenovirus type 5 boost (weeks 0, 4, 8, and 24, respectively). Although CRV inserts were about one-fifth that of WPV, the CRV generated comparable-magnitude blood CD4+ and CD8+ T lymphocyte responses against Gag, Env, and Nef. WPV responses preferentially targeted proteome areas outside the selected conserved regions in direct proportion to sequence lengths, indicating similar immunogenicities for the conserved regions and the outside regions. The CRV yielded a conserved-region targeting density that was approximately 5-fold higher than that of the WPV. A similar pattern was seen for bronchoalveolar lymphocytes, but with quadruple the magnitudes seen in blood. Overall, these findings demonstrate that the selected conserved regions are highly immunogenic and that anatomically isolated vaccinations with these regions focus immunodominance compared to the case for full-length protein vaccination. IMPORTANCE: HIV-1 sequence diversity is a major barrier limiting the capability of cellular immunity to contain infection and the ability of vaccines to match circulating viral sequences. To date, vaccines tested in humans have delivered whole proteins or genes for whole proteins, and it is unclear whether including only conserved sequences would yield sufficient cellular immunogenicity. We tested a vaccine delivering genes for four small conserved HIV-1 regions compared to a control vaccine with genes for whole Gag, Env, and Nef. Although the conserved regions ranged from 43 to 86 amino acids and comprised less than one-fifth of the whole Gag/Env/Nef sequence, the vaccines elicited equivalent total magnitudes of both CD4+ and CD8+ T lymphocyte responses. These data demonstrate the immunogenicity of these small conserved regions and the potential for a vaccine to steer immunodominance toward conserved epitopes.

Neurovirulence and immunogenicity of attenuated recombinant vesicular stomatitis viruses in nonhuman primates.

UNLABELLED: 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.

Vaccination with a fusion protein that introduces HIV-1 gag antigen into a multitrimer CD40L construct results in enhanced CD8+ T cell responses and protection from viral challenge by vaccinia-gag.

CD40 ligand (CD40L, CD154) is a membrane protein that is important for the activation of dendritic cells (DCs) and DC-induced CD8(+) T cell responses. To be active, CD40L must cluster CD40 receptors on responding cells. To produce a soluble form of CD40L that clusters CD40 receptors necessitates the use of a multitrimer construct. With this in mind, a tripartite fusion protein was made from surfactant protein D (SPD), HIV-1 Gag as a test antigen, and CD40L, where SPD serves as a scaffold for the multitrimer protein complex. This SPD-Gag-CD40L protein activated CD40-bearing cells and bone marrow-derived DCs in vitro. Compared to a plasmid for Gag antigen alone (pGag), DNA vaccination of mice with pSPD-Gag-CD40L induced an increased number of Gag-specific CD8(+) T cells with increased avidity for major histocompatibility complex class I-restricted Gag peptide and improved vaccine-induced protection from challenge by vaccinia-Gag virus. The importance of the multitrimeric nature of the complex was shown using a plasmid lacking the N terminus of SPD that produced a single trimer fusion protein. This plasmid, pTrimer-Gag-CD40L, was only weakly active on CD40-bearing cells and did not elicit strong CD8(+) T cell responses or improve protection from vaccinia-Gag challenge. An adenovirus 5 (Ad5) vaccine incorporating SPD-Gag-CD40L was much stronger than Ad5 expressing Gag alone (Ad5-Gag) and induced complete protection (i.e., sterilizing immunity) from vaccinia-Gag challenge. Overall, these results show the potential of a new vaccine design in which antigen is introduced into a construct that expresses a multitrimer soluble form of CD40L, leading to strongly protective CD8(+) T cell responses.

Coadministration of polyinosinic:polycytidylic acid and immunostimulatory complexes modifies antigen processing in dendritic cell subsets and enhances HIV gag-specific T cell immunity.

Currently approved adjuvants induce protective Ab responses but are more limited for generating cellular immunity. In this study, we assessed the effect of combining two adjuvants with distinct mechanisms of action on their ability to prime T cells: the TLR3 ligand, polyinosinic:polycytidylic acid (poly I:C), and immunostimulatory complexes (ISCOMs). Each adjuvant was administered alone or together with HIV Gag protein (Gag), and the magnitude, quality, and phenotype of Gag-specific T cell responses were assessed. For CD8 T cells, all adjuvants induced a comparable response magnitude, but combining poly I:C with ISCOMs induced a high frequency of CD127(+), IL-2-producing cells with decreased expression of Tbet compared with either adjuvant alone. For CD4 T cells, combining poly I:C and ISCOMs increased the frequency of multifunctional cells, producing IFN-γ, IL-2, and TNF, and the total magnitude of the response compared with either adjuvant alone. CD8 or CD4 T cell responses induced by both adjuvants mediated protection against Gag-expressing Listeria monocytogenes or vaccinia viral infections. Poly I:C and ISCOMs can alter Ag uptake and/or processing, and we therefore used fluorescently labeled HIV Gag and DQ-OVA to assess these mechanisms, respectively, in multiple dendritic cell subsets. Poly I:C promoted uptake and retention of Ag, whereas ISCOMs enhanced Ag degradation. Combining poly I:C and ISCOMs caused substantial death of dendritic cells but persistence of degraded Ag. These data illustrate how combining adjuvants, such as poly I:C and ISCOMs, that modulate Ag processing and have potent innate activity, can enhance the magnitude, quality, and phenotype of T cell immunity.

Robust immunity to an auxotrophic Mycobacterium bovis BCG-VLP prime-boost HIV vaccine candidate in a nonhuman primate model.

We previously reported that a recombinant pantothenate auxotroph of Mycobacterium bovis BCG expressing human immunodeficiency virus type 1 (HIV-1) subtype C Gag (rBCGpan-Gag) efficiently primes the mouse immune system for a boost with a recombinant modified vaccinia virus Ankara (rMVA) vaccine. In this study, we further evaluated the immunogenicity of rBCGpan-Gag in a nonhuman primate model. Two groups of chacma baboons were primed or mock primed twice with either rBCGpan-Gag or a control BCG. Both groups were boosted with HIV-1 Pr55(gag) virus-like particles (Gag VLPs). The magnitude and breadth of HIV-specific cellular responses were measured using a gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay, and the cytokine profiles and memory phenotypes of T cells were evaluated by polychromatic flow cytometry. Gag-specific responses were detected in all animals after the second inoculation with rBCGpan-Gag. Boosting with Gag VLPs significantly increased the magnitude and breadth of the responses in the baboons that were primed with rBCGpan-Gag. These responses targeted an average of 12 Gag peptides per animal, compared to an average of 3 peptides per animal for the mock-primed controls. Robust responses of Gag-specific polyfunctional T cells capable of simultaneously producing IFN-γ, tumor necrosis alpha (TNF-α), and interleukin-2 (IL-2) were detected in the rBCGpan-Gag-primed animals. Gag-specific memory T cells were skewed toward a central memory phenotype in both CD4(+) and CD8(+) T cell populations. These data show that the rBCGpan-Gag prime and Gag VLP boost vaccine regimen is highly immunogenic, inducing a broad and polyfunctional central memory T cell response. This report further indicates the feasibility of developing a BCG-based HIV vaccine that is safe for childhood HIV immunization.

Lipid nanocapsule as vaccine carriers for his-tagged proteins: evaluation of antigen-specific immune responses to HIV I His-Gag p41 and systemic inflammatory responses.

The purpose of this study was to design novel nanocapsules (NCs) with surface-chelated nickel (Ni-NCs) as a vaccine delivery system for histidine (His)-tagged protein antigens. Ni-NCs were characterized for binding His-tagged model proteins through high-affinity non-covalent interactions. The mean diameter and zeta potential of the optimized Ni-NCs were 214.9 nm and -14.8 mV, respectively. The optimal binding ratio of His-tagged Green Fluorescent Protein (His-GFP) and His-tagged HIV-1 Gag p41 (His-Gag p41) to the Ni-NCs was 1:221 and 1:480 w/w, respectively. Treatment of DC2.4 cells with Ni-NCs did not result in significant loss in the cell viability up to 24h (<5%). We further evaluated the antibody response of the Ni-NCs using His-Gag p41 as a model antigen. Formulations were administered subcutaneously to BALB/c mice at day 0 (prime) and 14 (boost) followed by serum collection on day 28. Serum His-Gag p41-specific antibody levels were found to be significantly higher at 1 and 0.5 µg doses of Gag p41-His-Ni-NCs (His-Gag p41 equivalent) compared with His-Gag p41 (1 µg) adjuvanted with aluminum hydroxide (AH). The serum IgG2a levels induced by Gag p41-His-Ni-NCs (1 µg) were significantly higher than AH adjuvanted His-Gag p41. The Ni-NCs alone did not result in the elevation of systemic IL-12/p40 and CCL5/RANTES inflammatory cytokine levels upon subcutaneous administration in BALB/c mice. In conclusion, the proposed Ni-NCs can bind His-tagged proteins and have the potential to be used as antigen delivery system capable of generating strong antigen-specific antibodies at doses much lower than with aluminum-based adjuvant and causing no significant elevation of systemic pro-inflammatory IL-12/p40 and CCL5/RANTES cytokines.

Comparison of plasmid vaccine immunization schedules using intradermal in vivo electroporation.

In vivo electroporation (EP) has proven to significantly increase plasmid transfection efficiency and to augment immune responses after immunization with plasmids. In this study, we attempted to establish an immunization protocol using intradermal (i.d.) EP. BALB/c mice were immunized with a plasmid encoding HIV-1 p37Gag, either i.d. with the Derma Vax EP device, intramuscularly (i.m.) without EP, or with combinations of both. A novel FluoroSpot assay was used to evaluate the vaccine-specific cellular immune responses. The study showed that i.d. EP immunizations induced stronger immune responses than i.m. immunizations using a larger amount of DNA and that repeated i.d. EP immunizations induced stronger immune responses than i.m. priming followed by i.d. EP boosting. Two and three i.d. EP immunizations induced immune responses of similar magnitude, and a short interval between immunizations was superior to a longer interval in terms of the magnitude of cellular immune responses. The FluoroSpot assay allowed for the quantification of vaccine-specific cells secreting either gamma interferon (IFN-γ), interleukin-2 (IL-2), or both, and the sensitivity of the assay was confirmed with IFN-γ and IL-2 enzyme-linked immunosorbent spot (ELISpot) assays. The data obtained in this study can aid in the design of vaccine protocols using i.d. EP, and the results emphasize the advantages of the FluoroSpot assay over traditional ELISpot assay and intracellular staining for the detection and quantification of bifunctional vaccine-specific immune responses.

Different patterns of expansion, contraction and memory differentiation of HIV-1 Gag-specific CD8 T cells elicited by adenovirus type 5 and modified vaccinia Ankara vaccines.

The magnitude and functional quality of antiviral CD8 T cell responses are critical for the efficacy of T cell based vaccines. Here, we investigate the influence of two popular viral vectors, adenovirus type 5 (Ad5) and modified vaccinia Ankara (MVA), on expansion, contraction and memory differentiation of HIV-1 Gag insert-specific CD8 T cell responses following immunization and show different patterns for the two recombinant viral vectors. The Ad5 vector primed 6-fold higher levels of insert-specific CD8 effector T cells than the MVA vector. The Ad5-primed effector cells also underwent less contraction (<2-fold) than the MVA-primed cells (>5-fold). The Ad5-primed memory cells were predominantly CD62L negative (effector memory) whereas the MVA-primed memory cells were predominantly CD62L positive (central memory). Consistent with their memory phenotype, MVA-primed CD8 T cells underwent higher fold expansion than Ad5-primed CD8 T cells following a homologous or heterologous boost. Impressively, the Ad5 boost changed the quality of MVA-primed memory response such that they undergo less contraction with effector memory phenotype. However, the MVA boost did not influence the contraction and memory phenotype of Ad5-primed response. In conclusion, our results demonstrate that vaccine vector strongly influences the expansion, contraction and the functional quality of insert-specific CD8 T cell responses and have implications for vaccine development against infectious diseases.

HIV fragment gag vaccine induces broader T cell response in mice.

Broad T-cell response is considered critical for HIV-1 vaccines to compensate viral diversity. Usually, a limited number of immunodominant epitopes are recognized in natural infections, as well as in vaccinations. Here, we seek to overcome immunofocusing of CD8 T Cell responses to HIV-1 CN54 gag DNA (delivered as a plasmid) in BalB/C mice by splitting it into fragments for reducing competition of recognition between dominant and sub-dominant epitopes. As expected, mice immunized with mixture of DNA fragments elicited significantly broader T cell responses than whole-length gag. We also further studied the effects when fragments and full-length DNA vaccines are combined for prime-boost vaccination. Interestingly, mice primed with full-length gag and boosted with DNA vaccine fragments induced similar T-cell response breadth as mice both primed and boosted by fragments DNA. In contrast, mice primed with DNA vaccine fragments and boosted with full-length gag failed to broaden T cell responses, once again, only the dominant epitopes were recognized. In summary, our study demonstrated that "fragmentation strategy" can indeed broaden T cell responses. This enhancement is more likely achieved in boosting stage. This study offers a promising way to design a vaccine with higher chance covering the highly diversified circulating strains.

Robust genital gag-specific CD8+ T-cell responses in mice upon intramuscular immunization with simian adenoviral vectors expressing HIV-1-gag.

Most studies on E1-deleted adenovirus (Ad) vectors as vaccine carriers for antigens of HIV-1 have focused on induction of central immune responses, although stimulation of mucosal immunity at the genital tract (GT), the primary port of entry of HIV-1, would also be highly desirable. In this study, different immunization protocols using chimpanzee-derived adenoviral (AdC) vectors expressing Gag of HIV-1 clade B given in heterologous prime-boost regimens were tested for induction of systemic and genital immune responses. Although i.n. immunization stimulated CD8(+) T-cell responses that could be detected in the GT, this route induced only marginal cellular responses in systemic tissues and furthermore numbers of Gag-specific CD8(+) T cells contracted sharply within a few weeks. On the contrary, i.m. immunization induced higher and more sustained frequencies of vaccine-induced cells which could be detected in the GT as well as systemic compartments. Antigen-specific CD8(+) T cells could be detected 1 year after immunization in all compartments analyzed. Genital memory cells secreted IFN-γ, expressed high levels of CD103 and their phenotypes were consistent with a state of activation. Taken together, the results presented here show that i.m. vaccination with chimpanzee-derived (simian) adenovirus vectors is a suitable strategy to induce a long-lived genital CD8(+) T-cell response.

[Comparison of the immnunogenicity of rAAV2/1 and rAd5 rad5 expressing HIV-1 gag].

OBJECTIVE: To compare the immunogenicity of rAAV2/1 and rAd5 expressing HIV-1 gag in BALB/c mice. METHODS: BALB/c mice were immunized with rAAV2/1-gag or rAd5-gag once or twice. HIV-1 specific cellular immune responses were analyzed by in vivo CTL and intracellular cytokine staining assays. HIV-1 Gag specific antibodies were tested by ELISA. RESULTS: Mice immunized with rAd5-gag once induced stronger Gag specific cellular immune responses and similar level of Gag specific antibody compared with rAAV2/1-gag. Mice immunized with rAd5-gag reached the peak immune responses more rapidly than rAAV2/1-gag. However, mice immunized with rAAV2/1-gag twice elicited better Gag specific IgG. CONCLUSION: rAd5-gag induced strong HIV-1 specific cellular and antibody responses, and rAAV2/1-gag induced high level of HIV-1 specific IgG and moderate cellular immune responses.

Safety and immunogenicity, after nasal application of HIV-1 DNA gagp37 plasmid vaccine in young mice.

BACKGROUND: There is a need for safe and potent adjuvants capable of delivering vaccine candidates over the mucosal barrier, with good capacity to stimulate both mucosal and systemic cell-mediated and humoral immunity. An adjuvant aimed for intranasal delivery should preferably deliver the antigen and minimize the transfer into the close proximity of the central nervous system, thus avoiding damage on the olfactory tissues. Advantages with a mucosal delivery route would be to provide mucosal and systemic immunity, requiring lower vaccine doses then when given parentally. The aim of this study was to study if the N3 adjuvant intranasally administered with HIV DNA plasmids would be transferred into the olfactory tissues and cause local inflammation and tissue damage. RESULTS: The N3 adjuvant alone and when combined with HIV-1 DNA gag plasmid and delivered intranasally did not cause detectable damage to the nasal epithelium or the olfactory epithelium or bulb over a period of 3 days after delivery. The intranasal administration of HIV-1 gagp37 DNA induced both a humoral and a cell-mediated immunity against the gag antigen. Significantly higher HIV-1-specific humoral, but not cell-mediated immune responses were seen in DNA/N3-immunized mice in comparison with HIV-1 DNA/saline-immunized animals. CONCLUSIONS: A safe and convenient intranasal mode of HIV-1 DNA plasmid and adjuvant delivery was shown not to interfere with the tissues in close proximity to the central nervous system. The N3 adjuvant combined with HIV-1 plasmids enhances the HIV-1-specific immunogenicity and merits to be clinically tested.