HIV-1 DNA Vaccine Research Articles

Enhancement of DNA vaccine potency in rhesus macaques by electroporation

The potency of an HIV DNA vaccine was enhanced in rhesus macaques by in vivo electroporation, as judged by increased onset, magnitude and duration of antibody and cell-mediated immune responses against both components of a combination Gag and Env vaccine. These data demonstrate the utility of the electroporation technology for use in large animals.

Immunogenicity of HIV-1 Env and Gag in baboons using a DNA prime/protein boost regimen

To evaluate the immunogenicity of sequence-modified HIV env and gag in baboons using DNA prime and protein boost strategy. Synthetic sequence-modified HIV gene cassettes were constructed that expressed three different forms of Env proteins, gp140, gp140mut and gp140TM, plus or minus a mutation in the protease-cleavage site. These plasmids were used to immunize baboons (Papio cynocephalus). A group of baboons was also immunized with both env and gag DNA followed by p55Gag virus-like particles (VLP) boost. Modest antibody responses and low or no lymphoproliferative responses were observed following multiple DNA immunizations. In contrast, strong antibodies and substantial antigen-specific lymphoproliferative responses were seen following booster immunizations with oligomeric Env protein (o-gp140US4) in MF59. Neutralizing antibody responses were scored against T cell line adapted HIV-1 strains after the protein boosters, but neutralizing responses were low or absent against homologous and heterologous primary isolate strains. In the group receiving both gag and env vaccines, modest antigen-specific antibody and lymphoproliferative responses were scored after the DNA immunizations; these responses were enhanced several-fold upon boosting with the VLP preparations. The addition of Gag antigen did not interfere with Env-specific antibody responses, but there was a negative effect on the levels of Env-specific lymphoproliferation. These results highlight the importance of improving the potency of HIV DNA vaccines by enhanced DNA delivery and prime-boost vaccine technologies to generate more robust immune responses in larger animal models. In addition, care must be taken when immunizations with Env and Gag antigens are performed together.

Cellular Immunity and DNA Vaccines for the Treatment of HIV / AIDS

Data from the first phase III HIV vaccine trial does not indicate efficacy regarding the production of neutralizing antibodies and prevention of HIV infection. The induction of a potent HIV-specific cellular response through therapeutic vaccination is now thought to be a more attainable goal through HIV DNA vaccines. This review will summarize the immunologic rationale behind the development of vaccines that elicit cellular immune responses, and will then illustrate examples of DNA-based preventive and therapeutic vaccine strategies, which have recently been evaluated in pre-clinical animal studies as well as some human clinical trials; they include: (i) cytokine-augmented DNA vaccines; (ii) CpG enriched plasmid DNAs; (iii) viral vectors and prime-boost strategies; (iv) lipopeptide vaccines; (v) ex vivo dendritic cell-based, and (vii) dendritic cell-targeted vaccines. This set of data will be carefully reviewed in order to understand how this know-how has been or can be used to design meaningful “next-generation” therapeutic vaccines. Keywords: hiv dna vaccine, cellular immune response, cytokines

Chitin Micro-Particles (CMP): a useful adjuvant for inducing viral specific immunity when delivered intranasally with an HIV-DNA vaccine.

Viral ImmunologyVol. 16, No. 4 Brief ReportsChitin Micro-Particles (CMP): A Useful Adjuvant for Inducing Viral Specific Immunity when Delivered Intranasally with an HIV-DNA VaccineKenji Hamajima, Yoshizumi Kojima, Kiyohiko Matsui, Yoshihiko Toda, Nao Jounai, Tomomi Ozaki, Ke-Qin Xin, Peter Strong, and Kenji OkudaKenji HamajimaSearch for more papers by this author, Yoshizumi KojimaSearch for more papers by this author, Kiyohiko MatsuiSearch for more papers by this author, Yoshihiko TodaSearch for more papers by this author, Nao JounaiSearch for more papers by this author, Tomomi OzakiSearch for more papers by this author, Ke-Qin XinSearch for more papers by this author, Peter StrongSearch for more papers by this author, and Kenji OkudaSearch for more papers by this authorPublished Online:9 Jul 2004https://doi.org/10.1089/088282403771926355AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited ByAntiviral Potency of Chitosan, Its Derivatives, and Nanocomposites2 October 2022Chitin and chitosan as tools to combat COVID-19: A triple approachInternational Journal of Biological Macromolecules, Vol. 183Immunomodulatory activities of chitin and chitosan microparticlesTechnologies to Improve ImmunizationBiological Applications of Marine Biopolymers6 July 2017Fabrication of magnetic and fluorescent chitin and dibutyrylchitin sub-micron particles by oil-in-water emulsificationActa Biomaterialia, Vol. 45Chitosan and its derivatives-based nano-formulations in drug deliveryThe beneficial properties of marine polysaccharides in alleviation of allergic responses5 December 2014 | Molecular Nutrition & Food Research, Vol. 59, No. 1Chitin and Its Beneficial Activity as an Immunomodulator in Allergic Reactions11 March 2013Marine-Derived Polysaccharides for Regulation of Allergic ResponsesAlternative vaccine delivery methodsProtection against bovine tuberculosis induced by oral vaccination of cattle with Mycobacterium bovis BCG is not enhanced by co-administration of mycobacterial protein vaccinesVeterinary Immunology and Immunopathology, Vol. 144, No. 3-4Immunomodulatory Activities of Chitin Microparticles on Leishmania major-infected Murine MacrophagesArchives of Medical Research, Vol. 42, No. 7Role of Chitin and Chitinase/Chitinase-Like Proteins in Inflammation, Tissue Remodeling, and InjuryAnnual Review of Physiology, Vol. 73, No. 1Chitins and Chitosans as Immunoadjuvants and Non-Allergenic Drug Carriers21 February 2010 | Marine Drugs, Vol. 8, No. 2Chitin, Chitinases and Chitinase-like Proteins in Allergic Inflammation and Tissue RemodelingYonsei Medical Journal, Vol. 50, No. 1Chitin regulation of immune responses: an old molecule with new rolesCurrent Opinion in Immunology, Vol. 20, No. 6Is intranasal vaccination a feasible solution for tuberculosis?9 January 2014 | Expert Review of Vaccines, Vol. 7, No. 9Alternative vaccine delivery methodsProphylactic effects of chitin microparticles on highly pathogenic H5N1 influenza virus1 January 2007 | Journal of Medical Virology, Vol. 79, No. 6Intranasal delivery of vaccines against HIV28 February 2006 | Expert Opinion on Drug Delivery, Vol. 3, No. 2Phagocytosis of N-acetyl-d-glucosamine particles, a Th1 adjuvant, by RAW 264.7 cells results in MAPK activation and TNF-α, but not IL-10, productionCellular Immunology, Vol. 239, No. 2Current Efforts on Generation of Optimal Immune Responses against HIV through Mucosal ImmunisationsDrugs in R & D, Vol. 7, No. 5Expert Review of Vaccines, Vol. 3, No. 4 suppl 1 Volume 16Issue 4Dec 2003 To cite this article:Kenji Hamajima, Yoshizumi Kojima, Kiyohiko Matsui, Yoshihiko Toda, Nao Jounai, Tomomi Ozaki, Ke-Qin Xin, Peter Strong, and Kenji Okuda.Chitin Micro-Particles (CMP): A Useful Adjuvant for Inducing Viral Specific Immunity when Delivered Intranasally with an HIV-DNA Vaccine.Viral Immunology.Dec 2003.541-547.http://doi.org/10.1089/088282403771926355Published in Volume: 16 Issue 4: July 9, 2004PDF download

Construction and characterisation of a candidate HIV-1 subtype C DNA vaccine for South Africa

A candidate DNA vaccine pTHgagC expressing the immunodeficiency virus-1 (HIV-1) gag gene from South African isolate Du422 was constructed and characterised. The isolate was selected on the basis of being the closest to the South African subtype C consensus sequence. Sequence analysis of cytotoxic T lymphocyte (CTL) epitopes showed that HIV subtype C-infected individuals have CTL responses to a number of epitopes present in the vaccine, but also revealed a more limited presence of subtype A- and any B-derived epitopes. A high level of expression of the immunogen was demonstrated in human cells and a potent, long-lived CTL response to a single inoculation of the DNA vaccine was elicited in BALB/c mice, which could be significantly increased by a boost vaccination at 4 weeks. This is the first candidate HIV-1 DNA vaccine employing the South African subtype C sequences, and constitutes a part of a vaccine scheduled to enter a clinical evaluation in South Africa in 2004.

In vivo kinetics and biodistribution of a HIV-1 DNA vaccine after administration in mice.

In this study we have investigated the pharmacokinetics and tissue distribution of GX-12, a multiple plasmid DNA vaccine for the treatment of HIV-1 infection. Plasmid DNA was rapidly degraded in blood with a half-life of 1.34 min and was no longer detectable at 90 min after intravenous injection in mice. After intramuscular injection, plasmid DNA concentration in the injection site rapidly declined to less than 1% of the initial concentration by 90 min post-injection. However, sub-picogram levels (per mg tissue) were occasionally detected for several days after injection. The relative proportions of the individual plasmids of GX-12 remained relatively constant at the injection site until 90 min post-injection. The concentration of plasmid DNA in tissues other than the injection site peaked at 90 min post-injection and decreased to undetectable levels at 8 h post-injection. The rapid in vivo degradation of GX-12 and absence of persistence in non-target tissues suggest that the risk of potential gene-related toxicities by GX-12 administration, such as expression in non-target tissues, insertional mutagenesis and germline transmission, is minimal.

Progress toward the development of a bacterial vaccine vector that induces high-titer long-lived broadly neutralizing antibodies against HIV-1

Conformationally constrained HIV-1 Env and gp120 immunogens induce broadly cross-reactive neutralizing antibodies. Thus, it is now feasible to rationally design an HIV-1 vaccine that affords protection through humoral mechanisms. This paper reviews our progress toward the development of an oral bacterial vaccine vector that is capable of delivering an HIV-1 DNA vaccine to host lymphoid tissues and inducing broadly neutralizing antibodies to HIV-1 in the mucosal and systemic immune compartments.

Quantitative assessment of antigen-specific CD8+ T cells in the mouse: application to vaccine research

An effective HIV vaccine will likely need to induce potent and broad-based immunity, including CD8+ T cell responses. Hence, a quantitative assay to measure such responses in animal models will be important in the evaluation of candidate HIV vaccines. We show here that a single immunization with HIV DNA vaccines, followed by challenge with recombinant vaccinia virus expressing the relevant HIV antigen, allows quantitative assessment of CD8+ T cell responses. These responses can be profound (>30% of total CD8+ T cells) and directly reflect the level of memory CD8+ T cells at the time of challenge. Therefore, this assay will facilitate the selection of promising HIV vaccine candidates for further evaluation.

Muscle specific versus ubiquitous expression of Gag based HIV-1 DNA vaccines: a comparative analysis

Most studies on DNA-based immunization have used viral promoters to drive antigen expression. In this study, we analyzed the properties of the commonly used CMV promoter, the tissue specific murine muscle creatine kinase (MCK) promoter as well as a hybrid MCK/CMV promoter regarding promoter activity and tissue specificity in vitro. Furthermore, the efficiency of inducing HIV-1 Gag specific immune responses in vivo following intramuscular immunization of nacked DNA containing a codon optimized synthetic gene was compared. Although antibody titers and cellular immune responses using the MCK construct were slightly reduced as compared to convential CMV based vector modules, the utilization of nonviral promoters may add significantly to the safety of future DNA vaccines.

Potent CD4+ T cell responses elicited by a bicistronic HIV-1 DNA vaccine expressing gp120 and GM-CSF.

Virus-specific CD4(+) T cell responses have been shown to play a critical role in controlling HIV-1 replication. Candidate HIV-1 vaccines should therefore elicit potent CD4(+) as well as CD8(+) T cell responses. In this report we investigate the ability of plasmid GM-CSF to augment CD4(+) T cell responses elicited by an HIV-1 gp120 DNA vaccine in mice. Coadministration of a plasmid expressing GM-CSF with the gp120 DNA vaccine led to only a marginal increase in gp120-specific splenocyte CD4(+) T cell responses. However, immunization with a bicistronic plasmid that coexpressed gp120 and GM-CSF under control of a single promoter led to a dramatic augmentation of vaccine-elicited CD4(+) T cell responses, as measured by both cellular proliferation and ELISPOT assays. This augmentation of CD4(+) T cell responses was selective, since vaccine-elicited Ab and CD8(+) T cell responses were not significantly changed by the addition of GM-CSF. A 100-fold lower dose of the gp120/GM-CSF bicistronic DNA vaccine was required to elicit detectable gp120-specific splenocyte proliferative responses compared with the monocistronic gp120 DNA vaccine. Consistent with these findings, i.m. injection of the gp120/GM-CSF bicistronic DNA vaccine evoked a more extensive cellular infiltrate at the site of inoculation than the monocistronic gp120 DNA vaccine. These results demonstrate that bicistronic DNA vaccines containing GM-CSF elicit remarkably potent CD4(+) T cell responses and suggest that optimal Th cell priming requires the precise temporal and spatial codelivery of Ag and GM-CSF.

Systemic and mucosal immune responses in mice after rectal and vaginal immunization with HIV-DNA vaccine.

We examined the feasibility of inducing local and systemic human immunodeficiency virus (HIV)-specific immune responses by rectal and vaginal application of an HIV-DNA vaccine. Mice were immunized with an HIV-DNA vaccine preparation via a rectal or vaginal route. After several applications, HIV-specific antibodies were detected in sera, fecal extract solutions, and vaginal washes, and these antibodies were potent in inhibiting the syncytium formation of a CD4-positive human T cell line by a cell line capable of inducing HIV-1 infection. Spleen cells from rectally and vaginally immunized mice showed antigen-mediated IFN-gamma-inducing activity. In addition, with rectal immunization, mononuclear cells from both the spleen and the regional lymph nodes of the rectal region were found to be potent at inducing a cytotoxic T lymphocyte response. These humoral and cell-mediated immune responses were enhanced by augmenting the vaccine with granulocyte-macrophage colony-stimulating factor-expressing plasmids or IL-12-expressing plasmid. Our results demonstrated that both rectal and vaginal immunization could induce systemic and mucosal immunity and that these responses were enhanced by the addition of the above cytokine-expressing plasmids.

Adjuvant effect of Ubenimex on a DNA vaccine for HIV-1.

Enhancement of DNA vaccine immunogenicity is a current topic of high priority in the field of applied immunology, especially as a means of controlling HIV infection. The adjuvant effect of Ubenimex (UBX), an anti-cancer immunomodulator, on a DNA AIDS vaccine which we developed was examined in a murine model. UBX was formulated into a preparation containing DNA plasmids encoding env and rev genes of HIV-1 strain III(B), and was inoculated intramuscularly into BALB/c mice. The sera obtained with this mixture had 2(3)-2(5) times higher specific IgG titres than those obtained without the use of the adjuvant. UBX also elicited both a stronger HIV-1-specific DTH reaction, as measured by the footpad swelling test, and stronger cytotoxic T lymphocyte activity, as assayed by the 51Cr-release method, compared with responses using DNA alone. The cytokine secretion profile of restimulated immune lymphoid cells showed that UBX raised IL-2 and interferon-gamma levels and decreased IL-4 production. HIV-1-specific immunoglobulin subtype analysis demonstrated that UBX stimulated IgG2a production but suppressed synthesis of IgG1 and IgE. These results indicate that activation of the T-helper type 1 subset was induced by UBX, suggesting a mechanism of immunomodulation mediated by this agent. We conclude that UBX acts as an immunologic adjuvant for DNA vaccination against HIV-1. UBX may be a suitable adjuvant for clinical use because of its lack of antigenicity and low toxicity.

Immunologic characterization of HIV-specific DNA vaccine.

We developed a method for applying HIV-1 DNA vaccine topically in mice. Topical application of DNA vaccine to the skin is useful against infections. To find a less expensive and less cumbersome vaccination method, we administered HIV-1 DNA vaccine to the skin of mice after elimination of keratinocytes using a fast-acting adhesive. HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. A high level of HIV-1-specific cytotoxic T lymphocyte response was also observed, and a high level of IFN-gamma and IL-4 production was induced by the improved skin application of DNA vaccine. High levels of both HIV-specific cytotoxic T lymphocyte and delayed type hypersensitivity in topical application were induced by coadministration of the DNA vaccine with IL-12 expression plasmids and granulocyte-macrophage colony-stimulating factor expression plasmids. These immune responses were inhibited by intradermal injection of anti-CD11c or anti-I-A/I-E antibody. Therefore, topical administration of DNA vaccine is an effective route, and may be very useful for the prevention of infectious diseases.

HIV DNA Vaccination: Advances and Experiences

The world is in dire need of a vaccine against HIV. Several approaches for vaccine development have been taken to eradicate this deadly virus. DNA vaccines in particular are being considered as prime candidates for this task. The ease with which they can be molecularly manipulated, compounded by their immunogenicity, safety, and simple and reproducible manufacture, have put DNA vaccines in the vaccine spotlight. Co-transfection of plasmids encoding HIV antigens, with those having the potential to veer the immune response in the desired direction (cytokines, chemokines, and co-stimulatory molecules), have shown promise. In addition, manipulation of codons of the HIV structural genes in the plasmid, to alter the dependence on rev have shown promising results. DNA vaccines are proving their efficacy in challenge studies in non-human primates. The first generation of DNA vaccines seem to be well tolerated in humans. In addition to being safe, they were also found to be immunogenic. Improving on these initial clinical studies may deliver a safe and effective DNA vaccine for HIV-1.

Topical application of HIV DNA vaccine with cytokine-expression plasmids induces strong antigen-specific immune responses

The topical application of DNA vaccine to the skin is a useful method of immunization because of its simplicity, painlessness and economy. But the immune responses that it elicits are relatively low. In this study, we administered human immunodeficiency virus type-1 (HIV-1) DNA vaccine with cytokine-expressing plasmids to the skin of mice by a new topical application technique involving prior elimination of keratinocytes using fast-acting adhesive. Our results revealed that the topical application of HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. Co-administration of the DNA vaccine with cytokine expression plasmids of IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by this new method raised the levels of both the HIV-specific cytotoxic T lymphocyte (CTL) response and delayed-type hypersensitivity (DTH) and facilitated the induction of substantial immune responses by DNA vaccine. Skin biopsy sections, thus, immunized showed significant increases of S-100 protein-positive dendritic cells (DCs). These results suggest that the topical application method described here is an efficient route of DNA vaccine administration and that the immune response may be induced by DNA plasmids taken in by DCs, Langerhans cells (LCs), or others such as antigen-presenting cells. This new topical application is likely to be of benefit in clinical use.

Modulation of cellular and humoral immune responses to a multiepitopic HIV-1 DNA vaccine by interleukin-18 DNA immunization/viral protein boost

In this study, the impact of Th1-inducing cytokine gene co-delivery and antigen boosting on humoral and cellular responses induced by multiepitopic DNA immunization in mice have been investigated. Intramuscular injection of mixed DNA constructs encoding for HIV-1 Gag, Tat and Nef proteins, co-administered with the DNA encoding for interleukin-18 (IL-18) have been used. The effect of boosting with the recombinant proteins was also evaluated on the outcome of the responses in DNA-primed mice. It was demonstrated that at least two DNA immunizations were necessary to generate virus specific Th-1 responses detected by the presence of cytotoxic T lymphocyte (CTL) and by the secretion of IL-2 and IFN-γ, but not IL-4 and IL-10, in antigen-stimulated splenocyte cultures. Interestingly, co-delivery of Th-1-inducing IL-18 gene was able to shorten by 2 weeks, the CTL induction time, and to increase the antigen-induced secretion of IL-2 and IFN-γ. Furthermore, IL-18 co-delivery enhanced antigen-specific lymphoproliferative responses, and this was most evident in mice that were primed and boosted with plasmid DNA. However, the induction of detectable antibodies in mice required two DNA vaccinations and a protein boost. In contrast to the effects on cell-mediated immunity, co-administration of IL-18-plasmid resulted in decreased antibody titers against viral proteins.

HIV DNA vaccines + cytokine fusion protein show potential

HIV DNA vaccines + cytokine fusion protein show potential

Therapeutic immunization of HIV-infected chimpanzees using HIV-1 plasmid antigens and interleukin-12 expressing plasmids.

To assess HIV-1 DNA vaccination and co-immunization with interleukin (IL)-12 and IL-10 as immunotherapy in the HIV-1 infected chimpanzee model system. Four chimpanzees that were infected with HIV-1-IIIB for longer than 4 years and remained symptom free were immunized with HIV-1 plasmid vaccines. Two chimpanzees were immunized with DNA plasmids that encoded env/rev, gag/pol along with a plasmid that encoded both chains of human IL-12. A third animal was immunized with HIV-1 DNA vaccine constructs and co-immunized with an IL-10 expressing plasmid. Finally a control animal received the HIV-1 DNA vaccine constructs alone. There was no evidence of systemic toxicity associated with the administration of the DNA vaccines or the cytokine-expressing plasmids. We observed that the IL-12/HIV-1 DNA vaccinated animals had enhanced proliferative responses to multiple HIV-1 antigens at multiple time points. The animal that was co-immunized with HIV-1 and IL-10 did not have any changes in the proliferative responses. Finally, the control chimpanzee demonstrated moderate increases in the proliferative responses to HIV-1 antigens. The animal that received HIV-1 vaccines alone and the animals co-immunized with IL-12 all had declines in viral load over the course of the study, however, the decrease in viral loads were transient in all animals. Immunization of HIV-1 infected chimpanzees with DNA based vaccines containing the env, gag and pol genes can transiently boost the env specific proliferative responses. Co-administration of IL-12 expressing plasmids further leads to transient boosting of the proliferative response to the core protein, p24 as well. However, at these doses the impact on viral load is minimal.

Augmentation of immune responses to HIV-1 and simian immunodeficiency virus DNA vaccines by IL-2/Ig plasmid administration in rhesus monkeys.

The potential utility of plasmid DNA as an HIV-1 vaccination modality currently is an area of active investigation. However, recent studies have raised doubts as to whether plasmid DNA alone will elicit immune responses of sufficient magnitude to protect against pathogenic AIDS virus challenges. We therefore investigated whether DNA vaccine-elicited immune responses in rhesus monkeys could be augmented by using either an IL-2/Ig fusion protein or a plasmid expressing IL-2/Ig. Sixteen monkeys, divided into four experimental groups, were immunized with (i) sham plasmid, (ii) HIV-1 Env 89.6P and simian immunodeficiency virus mac239 Gag DNA vaccines alone, (iii) these DNA vaccines and IL-2/Ig protein, or (iv) these DNA vaccines and IL-2/Ig plasmid. The administration of both IL-2/Ig protein and IL-2/Ig plasmid induced a significant and sustained in vivo activation of peripheral T cells in the vaccinated monkeys. The monkeys that received IL-2/Ig plasmid generated 30-fold higher Env-specific antibody titers and 5-fold higher Gag-specific, tetramer-positive CD8+ T cell levels than the monkeys receiving the DNA vaccines alone. IL-2/Ig protein also augmented the vaccine-elicited immune responses, but less effectively than IL-2/Ig plasmid. Augmentation of the immune responses by IL-2/Ig was evident after the primary immunization and increased with subsequent boost immunizations. These results demonstrate that the administration of IL-2/Ig plasmid can substantially augment vaccine-elicited humoral and cellular immune responses in higher primates.

8 Br-cAMP enhances both humoral and cell-mediated immune responses induced by an HIV-1 DNA vaccine.

From a series of preclinical studies and animal experiments, we have been able to demonstrate that DNA vaccines are a promising tool in strategies for protecting hosts from a variety of infectious diseases. Since the promoter activity of the human cytomegalovirus immediate-early promoter/ enhancer (CMV promoter) is known to be responsive to an elevation in the level of intracellular cAMP, we hypothesized that use of cAMP analogue (8-Bromo adenosine 3'5'-cyclic monophosphate, 8 Br-cAMP) would increase the level of transgene expression supported by the CMV, and enhance the ability of DNA vaccines to evoke an immune response against the transgene product in vivo. To evaluate this hypothesis, immune responses against HIV-1 envelope protein, gp160, an immunogenic HIV-1 component expressed under the control of the CMV promoter, were evaluated in BALB/c mice with or without stimulation by 8 Br-cAMP. DNA vaccine with 8 Br-cAMP was intramuscularly (i.m.) or intranasally (i.n.) administered to BALB/c mice twice on days 0 and 14. Regardless of which route was used, the combination increased the serum IgG antibody (Ab) titer, HIV-1-specific cytotoxic T lymphocyte (CTL) activity and the delayed-type hypersensitivity (DTH) response, compared with the effect of using the vaccine alone. When administered via the i.n. route, the combination also remarkably increased the titer of secretory IgA (sIgA). Moreover, it induced increased production of interferon-gamma with reduction in IL-4 synthesis, and decreased the ratio of serum IgG1/IgG2a. However, these enhancements were not observed when 8 Br-cAMP was coadministered with peptide vaccine or protein antigen. These data suggest that 8 Br-cAMP is able to enhance both humoral and cellular immune responses induced by the DNA vaccine. The induction of T helper type 1 (Th1) immunity against HIV-1 was also enhanced by coadministration of 8 Br-cAMP. A CAT assay study demonstrated that the adjuvant effect of 8 Br-cAMP may be due to the activation of the CMV promoter in the DNA vaccine. The virus challenge experiment in a mouse influenza model also proved our hypothesis.