A major challenge for the next generation of human immunodeficiency virus (HIV) vaccines is the induction of potent, broad, and durable cellular immune responses. The structural protein Gag is highly conserved among the HIV type 1 (HIV-1) gene products and is believed to be an important target for the host cellmediated immune control of the virus during natural infection. Expression of Gag proteins for vaccines has been hampered by the fact that its expression is dependent on the HIV Rev protein and the Rev-responsive element, the latter located on the env transcript. Moreover, the HIV genome employs suboptimal codon usage, which further contributes to the low expression efficiency of viral proteins. In order to achieve high-level Rev-independent expression of the Gag protein, the sequences encoding HIV-1 SF2 p55Gag were modified extensively. First, the viral codons were changed to conform to the codon usage of highly expressed human genes, and second, the residual inhibitory sequences were removed. The resulting modified gag gene showed increases in p55Gag protein expression to levels that ranged from 322-to 966-fold greater than that for the native gene after transient expression of 293 cells. Additional constructs that contained the modified gag in combination with modified protease coding sequences were made, and these showed high-level Rev-independent expression of p55Gag and its cleavage products. Density gradient analysis and electron microscopy further demonstrated that the modified gag and gagprotease genes efficiently expressed particles with the density and morphology expected for HIV virus-like particles. Mice immunized with DNA plasmids containing the modified gag showed Gag-specific antibody and CD8 ؉ cytotoxic T-lymphocyte (CTL) responses that were inducible at doses of input DNA 100-fold lower than those associated with plasmids containing the native gag gene. Most importantly, four of four rhesus monkeys that received two or three immunizations with modified gag plasmid DNA demonstrated substantial Gag-specific CTL responses. These results highlight the useful application of modified gag expression cassettes for increasing the potency of DNA and other gene delivery vaccine approaches against HIV.

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After more than 25 years of human immunodeficiency virus (HIV) research, a prophylactic vaccine able to control or prevent the worldwide spread of HIV/AIDS remains an elusive goal. Recent results in Thailand with the recombinant canary pox (ALVAC-HIV, vCP1521; Sanofi-Pasteur) prime-gp120 (AIDSVAX B/E) protein boost vaccine approach give us hope that such a vaccine is achievable (45). Nevertheless, the results from this trial as well as the disappointing outcome of the Step Study trial (7, 29, 46) vividly highlight the need to better understand the immune correlates of protection and the immune responses engendered by the diverse new vaccine technologies currently under evaluation (13,18,20,49). In the case of viral vectors, this is particularly critical, as the spectrum of immune responses elicited in animal models does not necessarily predict those eventually observed in human clinical trials and will require more thorough evaluations in order to identify the most predictive models. At the moment, nonhuman primate models, such as simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) infection of macaques appear to be the most informative for guiding vaccine development (3,24,47,55), and more rigorous application of these models has begun to yield new and encouraging insights into protective immunity (5,19,27,56). Moreover, as most HIV transmissions occur through mucosal membranes, understanding the correlates of protection, following successful vaccinations, against mucosal challenge is of strong interest.Alphaviruses are positive-sense single-stranded 11.5-kb RNA viruses in the Togaviridae family. They are relatively simple enveloped viruses of approximately 60-nm diameter that have a cytoplasmic RNA-based life cycle and mature at the plasma membranes of infected cells. Recombinant alphavirus replicon particles used for vaccine applications are composed of a replicon vector that encodes the viral replicases (nonstructural proteins [NSPs]) and the vaccine antigen of interest and two packaging vectors that encode the major viral structural proteins (capsid and glycoproteins E1 and E2) required for particle formation. The chimeric (VEE/SIN) alpha-* Corresponding author. Mailing address: Novartis Vaccines and Diagnostics,

The ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. Toward this goal, we derived variants of the prototype alphavirus, Sindbis virus (SIN), with differential abilities to infect human dendritic cells. Cloning and sequencing of the SIN variant genomes revealed that the genetic determinant for human dendritic cell (DC) tropism mapped to a single amino acid substitution at residue 160 of the envelope glycoprotein E2. Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant conferred a similar ability to efficiently infect immature human DC, whereupon those DC were observed to undergo rapid activation and maturation. The SIN replicon particles infected skin-resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of major histocompatibility complex and costimulatory molecules. Furthermore, SIN replicon particles encoding human immunodeficiency virus type 1 p55Gag elicited robust Gag-specific T-cell responses in vitro and in vivo, demonstrating that infected DC maintained their ability to process and present replicon-encoded antigen. Interestingly, human and mouse DC were differentially infected by selected SIN variants, suggesting differences in receptor expression between human and murine DC. Taken together, these data illustrate the tremendous potential of using a directed approach in generating alphavirus vaccine vectors that target and activate antigen-presenting cells, resulting in robust antigen-specific immune responses.Dendritic cells (DC) are the most potent antigen-presenting cell population and play a major role in the activation of both memory and naïve T cells. Immature DC capture antigen in the periphery and migrate to the draining lymph nodes, where they undergo maturation. Presentation of acquired antigen by mature DC is critical for induction of antigen-specific immune responses (1, 9, 13, 36) and stimulation of protective T-cell responses (3, 10). Transduction of autologous cultured DC ex vivo with gene delivery vectors encoding a desired antigen, followed by adoptive transfer, has been shown to stimulate antigen-specific T-cell responses in vivo (45,46). Unfortunately, the ability to target the DC cell population in vivo has been quite limited or has been shown to interfere with DC function or development (5,17,20,23,32,39). We rationalized that enhanced delivery of antigen to immature DC may provide an opportunity for improvement of vaccines, particularly for gene-based vaccination approaches.Toward a goal of improving DC-targeting approaches, we have focused on alphavirus-based vectors. The use of alphavirus vectors for vaccine and gene therapy applications is a rapidly emerging field (15, 42, 44). These RNA-based vectors, known as "replicons" because they retain the replicase functions necessary for RNA self-amplification and high-level expression, can be launched in vi...

During the asymptomatic phase of human immunodeficiency virus 1 (HIV-1) infection the lymphatic tissues seem to function as a major reservoir of HIV. We have examined the viral load in peripheral blood mononuclear cells (PBMC) and lymph node mononuclear cells (LNMC) of 12 naturally and 4 experimentally long-term simian Immunodeficiency virus (SIV)-infected African green monkeys (AGM) to help explain the apathogenicity of the AGM isolates of SIV (SIVagm) in their natural host. The mean number of SIVagm producing cells determined by limiting dilution assay was found to be 1.7 +/- 2.2 and 2.1 +/- 3.3 per 10(5) PBMC or LNMC, respectively. Similarly, polymerase chain reaction analysis of serially diluted cells showed the mean provirus carrying cell number to be 2.8 +/- 3.7 per 10(5) PBMC and 4.0 +/- 5.5 per 10(5) LNMC. When normalized for CD4+ cells the provirus and infectious virus loads in the LNMC and PBMC were also similar. No trapping of virus particles could be detected by in situ hybridization or immunohistochemistry. The data demonstrate that in contrast to HIV-1-infected humans, the viral burden in the lymph nodes of long-term SIV(agm)-infected AGMs is comparable to that in the PBMC.

Naturally occurring polymorphisms in the protease of human immunodeficiency virus type 1 (HIV-1) subtype C would be expected to lead to adaptive (compensatory) changes in protease cleavage sites. To test this hypothesis, we examined the prevalences and patterns of cleavage site polymorphisms in the Gag, Gag-Pol, and Nef cleavage sites of C compared to those in non-C subtypes. Codon-based maximum-likelihood methods were used to assess the natural selection and evolutionary history of individual cleavage sites. Seven cleavage sites (p17/p24, p24/p2, NC/p1, NC/TFP, PR/RT, RT/p66, and p66/IN) were well conserved over time and in all HIV-1 subtypes. One site (p1/p6 gag ) exhibited moderate variation, and four sites (p2/NC, TFP/p6 pol , p6 pol /PR, and Nef) were highly variable, both within and between subtypes. Three of the variable sites are known to be major determinants of polyprotein processing and virion production. P2/NC controls the rate and order of cleavage, p6gag is an important phosphoprotein required for virion release, and TFP/p6 pol , a novel cleavage site in the transframe domain, influences the specificity of Gag-Pol processing and the activation of protease. Overall, 58.3% of the 12 HIV-1 cleavage sites were significantly more diverse in C than in B viruses. When analyzed as a single concatenated fragment of 360 bp, 96.0% of group M cleavage site sequences fell into subtype-specific phylogenetic clusters, suggesting that they coevolved with the virus. Natural variation at C cleavage sites may play an important role, not only in regulation of the viral cycle but also in disease progression and response to therapy.

DNA vaccines have been used widely in experimental primate models of human immunodeficiency virus (HIV), but their effectiveness has been limited. In this study, we evaluated three technologies for increasing the potency of DNA vaccines in rhesus macaques. These included DNA encoding Sindbis virus RNA replicons (pSINCP), cationic poly(lactide-co-glycolide) (PLG) microparticles for DNA delivery, and recombinant protein boosting. The DNA-based pSINCP replicon vaccines encoding HIV Gag and Env were approximately equal in potency to human cytomegalovirus (CMV) promoter-driven conventional DNA vaccines (pCMV). The PLG microparticle DNA delivery system was particularly effective at enhancing antibody responses induced by both pCMV and pSINCP vaccines and had less effect on T cells. Recombinant Gag and Env protein boosting elicited rapid and strong recall responses, in some cases to levels exceeding those seen after DNA or DNA/PLG priming. Of note, Env protein boosting induced serum-neutralizing antibodies and increased frequencies of gamma interferon-producing CD4 T cells severalfold. Thus, PLG microparticles are an effective means of delivering DNA vaccines in nonhuman primates, as demonstrated for two different types of DNA vaccines encoding two different antigens, and are compatible for use with DNA prime-protein boost regimens.Both neutralizing antibodies and cell-mediated immunity (CMI) likely will be required to protect against viruses that can establish chronic infections, such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Moreover, in animal models for HIV, both neutralizing antibodies against Env (9, 35-37) and cytotoxic T lymphocytes (CTL) that target multiple viral antigens (3-5, 17, 18, 21, 26, 30, 32, 38, 39, 45, 48, 51-53, 59) can contribute to protection through prevention of infection and clearance of virus-infected cells, respectively. Vaccines consisting of inactivated pathogens or recombinant proteins generally are not effective at inducing CTL and typically are used to induce protective antibodies. In contrast, viruses and intracellular bacteria can induce CTL responses, in part due to neoexpression of the antigens during infection.Plasmid DNA vaccines were born out of the need for inducing both antibody and CMI responses, including CTL, without the problems associated with live organism-based vaccines, such as potential reversion to virulence and antivector immunity that can limit boosting. Indeed, DNA vaccines that express antigens from strong viral promoters have been used to elicit protective antibodies and CMI in many animal models (14, 23). However, naked DNA vaccines, i.e., plasmid DNA in saline, have proven to be only modestly potent in humans, thereby limiting their utility. Many approaches have been explored to improve DNA vaccine potency, including better expression vectors, DNA formulation and delivery systems, adjuvants, and the use of booster vaccines.We developed an alternative DNA vector that launches a self-amplifying Sindbis virus (alphavirus) RNA replicon (1...

Human immunodeficiency virus type-1 (HIV-1) particles incorporate a trimeric envelope complex (Env) made of gp120 (SU) and gp41 (TM) heterodimers. It has been previously established that soluble CD4 (sCD4) interaction leads to shedding of gp120 from viral particles, and that gp120 may also be easily lost from virions during incubation or particle purification procedures. In the design of HIV particle or pseudovirion-based HIV vaccines, it may be important to develop strategies to maximize the gp120 content of particles. We analyzed the gp120 retention of HIV-1 laboratory-adapted isolates and primary isolates following incubation with sCD4 and variations in temperature. NL4-3 shed gp120 readily in a temperature- and sCD4-dependent manner. Surprisingly, inactivation of the viral protease led to markedly reduced shedding of gp120. Gp120 shedding was shown to vary markedly between HIV-1 strains, and was not strictly determined by whether the isolate was adapted to growth on immortalized T cell lines or was a primary isolate. Pseudovirions produced by expression of codon-optimized gag and env genes also demonstrated enhanced gp120 retention when an immature core structure was maintained. Pseudovirions of optimal stability were produced through a combination of an immature Gag protein core and a primary isolate Env. These results support the feasibility of utilizing pseudovirion particles as immunogens for the induction of humoral responses directed against native envelope structures.