The worldwide HIV-1 vaccine research endeavor is focused increasingly on subtype C, which is now the predominant strain of the present HIV/AIDS epidemic. Expression cassettes of HIV-1 subtype C gag, pol and versions of gagpol fusion cassettes were constructed and evaluated for their relative abilities to induce cellular immune responses in mice. Animals were vaccinated with DNA or alphavirus replicon particle-based vaccines and cellular immune responses were measured by flow cytometry. Five new major histocompatibility complex (MHC) class I-restricted T cell epitopes in subtype C Gag and Pol were identified. Although two CD8+ T cell epitopes within Gag were immunodominant in BALB/c and CB6F1 mice, the overall breadth of the T cell responses in mice immunized with plasmids or recombinant alphavirus replicon particles encoding gagpol fusion genes was improved over single antigen genes (i.e. gag or pol alone). The patterns of epitope dominance were consistent among mice although there were variations observed between different animals in the relative contributions of the various epitopes to the total response. These data are consistent with observations in non-human primates (Otten GR, Schaefer M, Doe B, Liu H, Magede JZ, Donnelly J, et al. Potent immunogenicity of an HIV-1 gag-pol fusion DNA vaccine delivered by in vivo electroporation. Vaccine 2005, in press [1]) and support a subtype C in-frame gagpol fusion gene vaccine.

The urgent need for a vaccine against HIV/AIDS requires that multiple strategies be employed and evaluated in a clinical setting. V2-loop deleted trimeric envelope (Env) immunogens (protein and DNA) from subtypes B and C human immunodeficiency virus type 1 (HIV-1) strains were produced for ongoing and future clinical evaluations with other HIV antigens, adjuvants and deliveries.

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) subtype C infections are on the rise in Sub-Saharan Africa and Asia. Therefore, there is a need to develop an HIV vaccine capable of eliciting broadly reactive immune responses against members of this subtype. We show here that modified HIV envelope (env) DNA vaccines derived from the South African subtype C TV1 strain are able to prime for humoral responses in rabbits and rhesus macaques. Priming rabbits with DNA plasmids encoding V2-deleted TV1 gp140 (gp140TV1ΔV2), followed by boosting with oligomeric protein (o-gp140TV1ΔV2) in MF59 adjuvant, elicited higher titers of env-binding and autologous neutralizing antibodies than priming with DNA vaccines encoding the full-length TV1 env (gp160) or the intact TV1 gp140. Immunization with V2-deleted subtype B SF162 env and V2-deleted TV1 env together using a multivalent vaccine approach induced high titers of oligomeric env-binding antibodies and autologous neutralizing antibodies against both the subtypes B and C vaccine strains, HIV-1 SF162 and TV1, respectively. Low-level neutralizing activity against the heterologous South African subtype C TV2 strain, as well as a small subset of viruses in a panel of 13 heterologous primary isolates, was observed in some rabbits immunized with the V2-deleted vaccines. Immunization of rhesus macaques with the V2-deleted TV1 DNA prime/protein boost also elicited high titers of env-binding antibodies and moderate titers of autologous TV1 neutralizing antibodies. The pilot-scale production of the various TV1 DNA vaccine constructs and env proteins described here should provide an initial platform upon which to improve the immunogenicity of these subtype C HIV envelope vaccines.

Investigation into the mechanism of action of vaccine adjuvants provides opportunities to define basic immune principles underlying the induction of strong immune responses and insights useful for the rational development of subunit vaccines. A novel HIV vaccine composed of plasmid DNA-encoding p55 gag formulated with poly-lactide-co-glycolide microparticles (PLG) and cetyl trimethyl ammonium bromide (CTAB) elicits both serum antibody titers and cytotoxic lymphocyte activity in mice at doses two orders of magnitude lower than those required for comparable response to plasmid DNA in saline. Using this model, we demonstrated the increase in potency requires the DNA to be complexed to the PLG–CTAB microparticles. Furthermore, the PLG–CTAB–DNA formulation increased the persistence of DNA at the injection site, recruited mononuclear phagocytes to the site of injection, and activated a population of antigen presenting cells. Intramuscular immunization with the PLG–CTAB–DNA complex induced antigen expression at both the injection site and the draining lymph node. These findings demonstrate that the PLG–CTAB–DNA formulation exhibits multiple mechanisms of immunopotentiation.

ABSTRACT Control of the worldwide AIDS pandemic may require not only preventive but also therapeutic immunization strategies. To meet this challenge, the next generation of human immunodeficiency virus type 1 (HIV-1) vaccines must stimulate broad and durable cellular immune responses to multiple HIV antigens. Results of both natural history studies and virus challenge studies with macaques indicate that responses to both Gag and Pol antigens are important for the control of viremia. Previously, we reported increased Rev-independent expression and improved immunogenicity of DNA vaccines encoding sequence-modified Gag derived from the HIV-1SF2 strain (J. zur Megede, M. C. Chen, B. Doe, M. Schaefer, C. E. Greer, M. Selby, G. R. Otten, and S. W. Barnett, J. Virol. 74: 2628-2635, 2000). Here we describe results of expression and immunogenicity studies conducted with novel sequence-modified HIV-1SF2 GagPol and Pol vaccine antigens. These Pol antigens contain deletions in the integrase coding region and were mutated in the reverse transcriptase (RT) coding region to remove potentially deleterious enzymatic activities. The resulting Pol sequences were used alone or in combination with sequence-modified Gag. In the latter, the natural translational frameshift between the Gag and Pol coding sequences was either retained or removed. Smaller, in-frame fusion gene cassettes expressing Gag plus RT or protease plus RT also were evaluated. Expression of Gag and Pol from GagPol fusion gene cassettes appeared to be reduced when the HIV protease was active. Therefore, additional constructs were evaluated in which mutations were introduced to attenuate or inactivate the protease activity. Nevertheless, when these constructs were delivered to mice as DNA vaccines, similar levels of CD8+ T-cell responses to Gag and Pol epitopes were observed regardless of the level of protease activity. Overall, the cellular immune responses against Gag induced in mice immunized with multigenic gagpol plasmids were similar to those observed in mice immunized with the plasmid encoding Gag alone. Furthermore, all of the sequence-modified pol and gagpol plasmids expressed high levels of Pol-specific antigens in a Rev-independent fashion and were able to induce potent Pol-specific T- and B-cell responses in mice. These results support the inclusion of a gagpol in-frame fusion gene in future HIV vaccine approaches.

HIV researchers everywhere are grateful to Science for featuring HIV in their 28 June issue to coincide with this year's AIDS Congress in Barcelona. However, there are two points in Jon Cohen's article “Monkey puzzles” (News Focus, p. [2325][1]) that require further consideration. First, the statement that “monkey studies with AIDS vaccines have completely failed to elicit antibodies that can neutralize the virus” (p. [2325][1]) is not consistent with the published data. Many papers have shown the ability of envelope-based HIV vaccines to induce antibodies that neutralize T cell line-adapted virus isolates, although neutralization of primary CCR5-dependent HIV-1 isolates was rarely observed. In contrast, our papers clearly show that immunization of Rhesus macaques with a plasmid DNA vaccine prime followed by a recombinant oligomeric V2 loop-deleted SF162 envelope protein boost is capable of inducing serum antibodies that neutralize multiple primary isolates of HIV-1 that are both antigenically distinct and CCR5-dependent ([1][2], [2][3]). To our knowledge, this was the first time that a vaccine-induced immune response was shown to be capable of broad primary isolate neutralization, and we are dismayed that this significant milestone in HIV vaccine research is overlooked by a review in a widely read journal. Second, the table on p. [2326][1] (“AIDS Vaccine Pipeline”) does not include vaccines in preclinical testing sponsored by the National Institute of Allergy and Infectious Diseases (NIAID) in collaboration with Wyeth Laboratories and Chiron Corporation (a version of the DNA prime-protein boost vaccine mentioned above). These IND-enabling preclinical studies are supported by the NIH HIV Vaccine Design and Development Team Contracts, which have been well publicized. We believe that underreporting the breadth and scope of NIAID's commitment to research and development of HIV vaccines does this important agency a great disservice. 1. [↵][4]1. S. Cherpelis 2. et al. , J. Virol. 75, 1547 (2001). [OpenUrl][5][Abstract/FREE Full Text][6] 2. [↵][7]1. S. W. Barnett 2. et al. , J. Virol. 75, 5526 (2001). [OpenUrl][8][Abstract/FREE Full Text][9] # Response {#article-title-2} The pipeline table should have made clear that the list was not all-inclusive. The Chiron and Wyeth studies appear on a more comprehensive pipeline table that accompanied an article that I wrote for the 2 March 2001 issue (“AIDS vaccines show promise after years of frustration,” News Focus, p. [1686][10]). That article includes a prediction from Chiron that its vaccine would be in human trials in 2002 (which does not look likely now), and it describes the work in some detail. The assertion that antibodies have “completely failed” to neutralize the virus in monkey studies may be a bit of an overstatement, but not much, as is clear simply by looking closely at the counterevidence Donnelly et al. present. First, they cite in vitro studies of antibodies from human HIV vaccine tests—which, if anything, underscore the difficulties antibodies have in stopping real-world isolates of the virus. Their own monkey data, from two animals, did not reach statistical significance, and, as far as I can tell, have not been independently replicated. [1]: /lookup/doi/10.1126/science.296.5577.2325 [2]: #ref-1 [3]: #ref-2 [4]: #xref-ref-1-1 View reference 1 in text [5]: {openurl}?query=rft.jtitle%253DJournal%2Bof%2BVirology%26rft.stitle%253DJ.%2BVirol.%26rft.issn%253D0022-538X%26rft.aulast%253DCherpelis%26rft.auinit1%253DS.%26rft.volume%253D75%26rft.issue%253D3%26rft.spage%253D1547%26rft.epage%253D1550%26rft.atitle%253DDNA%2BVaccination%2Bwith%2Bthe%2BHuman%2BImmunodeficiency%2BVirus%2BType%2B1%2BSF162%257BDelta%257DV2%2BEnvelope%2BElicits%2BImmune%2BResponses%2BThat%2BOffer%2BPartial%2BProtection%2Bfrom%2BSimian%252FHuman%2BImmunodeficiency%2BVirus%2BInfection%2Bto%2BCD8%252B%2BT-Cell-Depleted%2BRhesus%2BMacaques%26rft_id%253Dinfo%253Adoi%252F10.1128%252FJVI.75.3.1547-1550.2001%26rft_id%253Dinfo%253Apmid%252F11152527%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [6]: /lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MzoianZpIjtzOjU6InJlc2lkIjtzOjk6Ijc1LzMvMTU0NyI7czo0OiJhdG9tIjtzOjIzOiIvc2NpLzI5Ny81NTg1LzEyNzcuYXRvbSI7fXM6ODoiZnJhZ21lbnQiO3M6MDoiIjt9 [7]: #xref-ref-2-1 View reference 2 in text [8]: {openurl}?query=rft.jtitle%253DJournal%2Bof%2BVirology%26rft.stitle%253DJ.%2BVirol.%26rft.issn%253D0022-538X%26rft.aulast%253DBarnett%26rft.auinit1%253DS.%2BW.%26rft.volume%253D75%26rft.issue%253D12%26rft.spage%253D5526%26rft.epage%253D5540%26rft.atitle%253DThe%2BAbility%2Bof%2Ban%2BOligomeric%2BHuman%2BImmunodeficiency%2BVirus%2BType%2B1%2B%2528HIV-1%2529%2BEnvelope%2BAntigen%2BTo%2BElicit%2BNeutralizing%2BAntibodies%2Bagainst%2BPrimary%2BHIV-1%2BIsolates%2BIs%2BImproved%2Bfollowing%2BPartial%2BDeletion%2Bof%2Bthe%2BSecond%2BHypervariable%2BRegion%26rft_id%253Dinfo%253Adoi%252F10.1128%252FJVI.75.12.5526-5540.2001%26rft_id%253Dinfo%253Apmid%252F11356960%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [9]: /lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MzoianZpIjtzOjU6InJlc2lkIjtzOjEwOiI3NS8xMi81NTI2IjtzOjQ6ImF0b20iO3M6MjM6Ii9zY2kvMjk3LzU1ODUvMTI3Ny5hdG9tIjt9czo4OiJmcmFnbWVudCI7czowOiIiO30= [10]: /lookup/doi/10.1126/science.291.5509.1686

Abstract Immunization by injection of plasmids encoding foreign proteins has been used successfully as a research tool and to elicit protective immune responses in animal models.

Intestinal infections with enteropathogenic Escherichia coli are potentially devastating and difficult to treat. Outbreaks linked to food-borne spread of the bacteria have occurred repeatedly in the US in recent years. New approaches to neutralizing the bacterial toxins responsible for the worst effects of the disease may provide lifesaving tools for clinicians ( 265–270 ).

There is reasonable evidence that both cross-priming and direct transfection of antigen-presenting cells (APCs) play a role in induction of immune responses by DNA vaccines. It is not known which mode is more important for priming cytotoxic T cell responses, but both are sufficient and neither alone is necessary. Hence, a rational strategy for increasing DNA vaccine potency would be to facilitate both pathways. With regard to cross-priming, a better understanding of the nature of the antigen transferred and the molecules/cells involved may suggest ways to design DNA vaccines to enhance this pathway. With respect to transfection of APCs, certain DNA formulations or delivery systems may be able to target APCs for increased DNA uptake. Other considerations include recruitment of APCs to the site of DNA injection and manipulation of these cells to ensure the proper activation state for priming immune responses. The burgeoning scientific literature in these areas indicates that much effort is currently being directed toward these goals.

Intramuscular injection of DNA vaccines elicits potent humoral and cellular immune responses in mice. However, DNA vaccines are less efficient in larger animal models and humans. To gain a better understanding of the factors limiting the efficacy of DNA vaccines, we used fluorescence-labeled plasmid DNA in mice to 1) define the macroscopic and microscopic distribution of DNA after injection into the tibialis anterior muscle, 2) characterize cellular uptake and expression of DNA in muscle and draining lymph nodes, and 3) determine the effect of modifying DNA distribution and cellular uptake by volume changes or electroporation on the magnitude of the immune response. Injection of a standard 50-μl dose resulted in the rapid dispersion of labeled DNA throughout the muscle. DNA was internalized within 5 min by muscle cells near the injection site and over several hours by cells that were located along muscle fibers and in the draining lymph nodes. Histochemical staining and analysis of mRNA expression in isolated cells by RT-PCR showed that the transgene was detectably expressed only by muscle cells, despite substantial DNA uptake by non-muscle cells. Reduction of the injection volume to 5 μl resulted in substantially less uptake and expression of DNA by muscle cells, and correspondingly lower immune responses against the transgene product. However, expression and immunogenicity were restored when the 5-μl injection was followed by electroporation in vivo. These findings indicate that distribution and cellular uptake significantly affect the immunogenicity of DNA vaccines.

A pneumococcal conjugate vaccine (PCV) has been developed consisting of capsular polysaccharide (Ps) coupled to the outer membrane protein complex of Neiserria meningitidis serogroup B. Experiments were conducted in infant rhesus monkeys to assess the potential to administer multiple Pn types in a single vaccine. A single type conjugate, 6B, was dosed from 0.025 to 25 mu g Ps. Peak anti-6B Ps Ab titers were seen at lower doses of 0.025 and 0.25 mu g Ps, while reduced titers of anti-6B Ps Ab were observed at the highest doses of conjugate administered, 2.5 and 25 mu g Ps. By mixing free Ps, carrier, or another monovalent PCV with this 6B PCV, it was determined that reduced anti-6B Ps titers at high PCV doses were associated only with the quantity of type-specific Ps in the conjugate. Thus, increasing the amount of carrier protein or adding an additional monovalent conjugate did not significantly affect the response to type 6B Ps. These results suggest that, given an appropriately determined dose per individual pneumococcal Ps type, a multivalent PCV that includes many different types should have satisfactory clinical immunogenicity. (C) 2000 Published by Elsevier Science Ltd. All rights reserved.

The immunogenicity and protective efficacy of DNA vaccines have been amply demonstrated in numerous animal models of infectious disease. However, the feasibility of DNA vaccines for human use is not yet known. In order to investigate potential means of increasing the potency of DNA vaccines, conventional adjuvants such as aluminum salts were tested. Coadministration of these adjuvants with DNA vaccines substantially enhanced the ability of these vaccines to induce antibody responses up to 100-fold in mice and guinea pigs, and 5–10-fold in non-human primates. Effective formulations had no demonstrable effect on the levels of antigen expression in situ and consisted of adjuvants that did not form complexes with the plasmid DNA; rather they exerted their effects on antigen after expression in situ. Therefore, the potency of DNA vaccines both in laboratory rodents and in non-human primates can be substantially increased by simple formulation with conventional aluminum adjuvants.

Intramuscular injection of BALB/c mice with a DNA plasmid encoding nucleoprotein (NP) from influenza virus A/PR/8/34 (H1N1) provides cross-strain protection against lethal challenge with influenza virus A/HK/68 (H3N2). CTL specific for the H-2Kd-restricted epitope NP147-155 are present in these mice and are thought to play a role in the protection. To assess the effectiveness of NP DNA immunization in comparison with influenza virus infection in the induction of CTL responses, we monitored the frequency of CTL precursors (CTLp) in mice following i.m. injection with NP DNA or intranasal infection with influenza virus and showed that the CTLp frequency in NP DNA-immunized mice can reach levels found in mice that had been infected with influenza virus. We also measured the CTLp frequency, anti-NP Ab titers, and T cell proliferative responses in mice that were injected with titrated dosages of NP DNA and documented a correlation of the CTLp frequency and the Ab titers, but not proliferative responses, with the injection dose. Furthermore, we observed a positive correlation between the frequency of NP147-155 epitope-specific CTLp and the extent of protective immunity against cross-strain influenza challenge induced by NP DNA injection. Collectively, these results and our early observations from adoptive transfer experiments of in vitro activated lymphocytes from NP DNA-immunized mice suggest a protective function of NP-specific CTLp in mice against cross-strain influenza virus challenge.

Influenza is a leading cause of morbidity and mortality in older persons. The current influenza vaccine is only modestly successful, in part because of an age-related decline in immunogenicity and also because it induces only type-specified immunity. To overcome this, we evaluated DNA vaccines encoding A/PR8/34 haemagglutinin (HA) and nucleoprotein (NP) in young and aged BALBc mice. Control mice were given formalin-inactivated A/PR8/34, control DNA, or a non-lethal dose of PR8. Aged mice given HA DNA developed slightly lower anti-HA serum antibodies than young mice; however, both young and aged mice were protected from a homotypic PR8 challenge. Following vaccination with NP DNA, both young and aged mice developed anti-NP bulk cytotoxic T-lymphocyte (CTL) activity and pCTL frequency similar to control animals. When challenged with a low dose of A/HK/68 (H3N2) influenza virus, both young mice and aged mice showed significant protection as measured by inhibition of weight loss. When challenged with a relatively high dose of A/HR/68 (H3N2) influenza virus, however, the anti-NP vaccine only partially protected young mice and failed to protect aged mice. These data demonstrate that DNA-based vaccines are immunogenic in aged animals, but suggest that factors other than the age-related decline in CTL activity also contribute to the increased morbidity and mortality of influenza in the elderly.

Publisher Summary This chapter describes the various aspects of DNA vaccines. It also discusses the basic materials and methods involved in preparing and administering DNA vaccines. The preparation of Escherichia coli -derived plasmid DNA expression vectors utilizes standard molecular biology reagents. The gene of interest, or fragment, can be generated by polymerase chain reaction (PCR). Wolff demonstrated the expression of proteins in situ after the administration of plasmid DNA containing the genes encoding those proteins. In some cases, it may be desirable to include only the coding region from the ATG to the termination codon. Sequence verify at least three clones using primers 30–50 bp from the restriction site so that bases within the vector can be read, as well as 150–200 bases within the gene. In this way, the orientation and quality of the PCR-generated gene can be assessed. For the characterization of plasmid DNA vectors prior to immunization of animals, expression can be assessed by transient transfection in vitro and immunoblot analysis. A crude estimation of relative vaccine efficacy can be made by comparing relative expression levels of a particular antigen in vitro .

ABSTRACTDNA vaccination is an effective means of eliciting both humoral and cellular immunity, including cytotoxic T lymphocytes (CTL). Using an influenza virus model, we previously demonstrated that injection of DNA encoding influenza virus nucleoprotein (NP) induced major histocompatibility complex class I-restricted CTL and cross-strain protection from lethal virus challenge in mice (J. B. Ulmer et al., Science 259:1745–1749, 1993). In the present study, we have characterized in more detail the cellular immune responses induced by NP DNA, which included robust lymphoproliferation and Th1-type cytokine secretion (high levels of gamma interferon and interleukin-2 [IL-2], with little IL-4 or IL-10) in response to antigen-specific restimulation of splenocytes in vitro. These responses were mediated by CD4+T cells, as shown by in vitro depletion of T-cell subsets. Taken together, these results indicate that immunization with NP DNA primes both cytolytic CD8+T cells and cytokine-secreting CD4+T cells. Further, we demonstrate by adoptive transfer and in vivo depletion of T-cell subsets that both of these types of T cells act as effectors in protective immunity against influenza virus challenge conferred by NP DNA.

DNA immunization offers a novel means to induce cellular immunity in a population with a heterogeneous genetic background. An immunorecessive cytotoxic T-lymphocyte (CTL) epitope in influenza virus nucleoprotein (NP), residues 218 to 226, was identified when mice were immunized with a plasmid DNA encoding a full-length mutant NP in which the anchor residues for the immunodominant NP147-155 epitope were altered. Mice immunized with wild-type or mutant NP DNA were protected from lethal cross-strain virus challenge, and the protection could be adoptively transferred by immune splenocytes, indicating the role of cell-mediated immunity in the protection. DNA immunization is capable of eliciting protective cellular immunity against both immunodominant and immunorecessive CTL epitopes in the hierarchy seen with virus infection.

We have examined in detail the characteristics of the humoral immune response and protective efficacy induced by an influenza hemagglutinin (HA) DNA vaccine. In mice injected intramuscularly with HA DNA, the magnitude of the immune responses generated, as measured by ELISA and hemagglutination inhibiting (HI) antibodies, was directly related to the amount of DNA injected and the number of doses administered. The level of anti-HA antibodies in DNA-vaccinated mice was higher than that in convalescent immune mice and was maintained for at least 1.5 years. The immunoglobulin isotype profile of the antibodies was predominantly IgG2a, similar to that induced by live virus infection but in contrast to the relative abundance of IgG1 antibodies observed after inoculation with formalin-inactivated whole virus. The presence of pre-challenge HI antibodies was found to be a good correlate of protection, in that every animal with a detectable HI titer was protected from a lethal challenge. Complete protection from a lethal dose of influenza virus (A/PR/34), as judged by 100% survival and no weight loss, was conferred by as little as 1 μg of DNA (given twice). Furthermore, mice injected with 10 to 100 μg doses, when subsequently challenged with virus, showed no increase in HI titer and no production of antibodies directed against the challenge virus, suggesting a substantial inhibition of virus replication after challenge.