The high prevalence of pre-existing immunity to adenovirus serotype 5 (Ad5) in human populations may substantially limit the immunogenicity and clinical utility of recombinant Ad5 vector-based vaccines for HIV-1 and other pathogens. A potential solution to this problem is to use vaccine vectors derived from adenovirus (Ad) serotypes that are rare in humans, such as Ad35. However, cross-reactive immune responses between heterologous Ad serotypes have been described and could prove a major limitation of this strategy. In particular, the extent of immunologic cross-reactivity between Ad5 and Ad35 has not previously been determined. In this study we investigate the impact of pre-existing anti-Ad5 immunity on the immunogenicity of candidate rAd5 and rAd35 vaccines expressing SIV Gag in mice. Anti-Ad5 immunity at levels typically found in humans dramatically blunted the immunogenicity of rAd5-Gag. In contrast, even high levels of anti-Ad5 immunity did not substantially suppress Gag-specific cellular immune responses elicited by rAd35-Gag. Low levels of cross-reactive Ad5/Ad35-specific CD4+ T lymphocyte responses were observed, but were insufficient to suppress vaccine immunogenicity. These data demonstrate the potential utility of Ad35 as a candidate vaccine vector that is minimally suppressed by anti-Ad5 immunity. Moreover, these studies suggest that using Ad vectors derived from immunologically distinct serotypes may be an effective and general strategy to overcome the suppressive effects of pre-existing anti-Ad immunity.

The utility of recombinant adenovirus serotype 5 (rAd5) vector-based vaccines for HIV-1 and other pathogens will likely be limited by the high prevalence of pre-existing Ad5-specific neutralizing Abs (NAbs) in human populations. However, the immunodominant targets of Ad5-specific NAbs in humans remain poorly characterized. In this study, we assess the titers and primary determinants of Ad5-specific NAbs in individuals from both the United States and the developing world. Importantly, median Ad5-specific NAb titers were >10-fold higher in sub-Saharan Africa compared with the United States. Moreover, hexon-specific NAb titers were 4- to 10-fold higher than fiber-specific NAb titers in these cohorts by virus neutralization assays using capsid chimeric viruses. We next performed adoptive transfer studies in mice to evaluate the functional capacity of hexon- and fiber-specific NAbs to suppress the immunogenicity of a prototype rAd5-Env vaccine. Hexon-specific NAbs were remarkably efficient at suppressing Env-specific immune responses elicited by the rAd5 vaccine. In contrast, fiber-specific NAbs exerted only minimal suppressive effects on rAd5 vaccine immunogenicity. These data demonstrate that functionally significant Ad5-specific NAbs are directed primarily against the Ad5 hexon protein in both humans and mice. These studies suggest a potential strategy for engineering novel Ad5 vectors to evade dominant Ad5-specific NAbs.

Plasmid DNA vaccines elicit potent and protective immune responses in numerous small-animal models of infectious diseases. However, their immunogenicity in primates appears less potent. Here we investigate a novel approach that optimizes regulatory elements in the plasmid backbone to improve the immunogenicity of DNA vaccines. Among various regions analyzed, we found that the addition of a regulatory sequence from the R region of the long terminal repeat from human T-cell leukemia virus type 1 (HTLV-1) to the cytomegalovirus (CMV) enhancer/promoter increased transgene expression 5-to 10-fold and improved cellular immune responses to human immunodeficiency virus type 1 (HIV-1) antigens. In cynomolgus monkeys, DNA vaccines containing the CMV enhancer/promoter with the HTLV-1 R region (CMV/R) induced markedly higher cellular immune responses to HIV-1 Env from clades A, B, and C and to HIV-1 Gag-Pol-Nef compared with the parental DNA vaccines. These data demonstrate that optimization of specific regulatory elements can substantially improve the immunogenicity of DNA vaccines encoding multiple antigens in small animals and in nonhuman primates. This strategy could therefore be explored as a potential method to enhance DNA vaccine immunogenicity in humans.Plasmid DNA vaccines have shown promise as a novel vaccination modality based on their simplicity and versatility (31,32,36). In particular, DNA vaccines can elicit potent and protective cellular and humoral immune responses in a variety of small-animal models (10). However, they have proven substantially less immunogenic in nonhuman primate studies and in clinical trials to date (8,19,33).Several approaches have been explored to improve the immunogenicity of DNA vaccines. Our laboratories and others have demonstrated that the addition of plasmids expressing cytokines and immunomodulatory molecules can substantially augment DNA vaccine-elicited immune responses in both mice and nonhuman primates (3,4,15,16,21,34,37). However, the practical requirements of manufacturing and establishing the safety of the plasmid cytokines prior to the initiation of clinical trials may prove a limitation of this strategy (7, 26). Other approaches involve the addition of polymer adjuvants (29) and the use of in vivo electroporation techniques (24, 35). These strategies have similarly proven effective in animal models, but their practical utility in clinical trials has yet to be demonstrated.In this study, we investigate a novel strategy involving optimization of regulatory elements in the backbone of the plasmid DNA vaccine. DNA vaccines often utilize a cytomegalovirus (CMV) enhancer, promoter, and intron to drive high-level expression of a transgene in mammalian cells (32,38). Here, we explore the effects of adding the regulatory R region from the 5Ј long terminal repeat (LTR) of human T-cell leukemia virus type 1 (HTLV-1), which acts as a transcriptional and posttranscriptional enhancer (30). We find that these CMV/R DNA vaccines elicit substantially higher human immunodeficiency viru...

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations will likely limit the immunogenicity and clinical utility of recombinant Ad5 vector-based vaccines for human immunodeficiency virus type 1 and other pathogens. Ad5-specific neutralizing antibodies (NAbs) are thought to contribute substantially to anti-Ad5 immunity, but the potential importance of Ad5-specific T lymphocytes in this setting has not been fully characterized. Here we assess the relative contributions of Ad5-specific humoral and cellular immune responses in blunting the immunogenicity of a rAd5-Env vaccine in mice. Adoptive transfer of Ad5-specific NAbs resulted in a dramatic abrogation of Env-specific immune responses following immunization with rAd5-Env. Interestingly, adoptive transfer of Ad5-specific CD8؉ T lymphocytes also resulted in a significant and durable suppression of rAd5-Env immunogenicity. These data demonstrate that NAbs and CD8 ؉ T lymphocytes both contribute to immunity to Ad5. Novel adenovirus vectors that are currently being developed to circumvent the problem of preexisting anti-Ad5 immunity should therefore be designed to evade both humoral and cellular Ad5-specific immune responses.Replication-defective recombinant adenovirus serotype 5 (rAd5) vector-based vaccines elicit potent and protective immune responses in a variety of animal models (4,19,22,23). Clinical trials of rAd5 vaccines for human immunodeficiency virus type 1 (HIV-1) and other pathogens are therefore currently in progress (14). A major limitation of this approach, however, is that the majority of the human population has preexisting anti-Ad5 immunity that may substantially reduce the immunogenicity and clinical utility of rAd5 vaccines. In fact, anti-Ad5 immunity has already been demonstrated to suppress the immunogenicity of rAd5 vaccines in studies in mice (3, 31), rhesus monkeys (4), and humans in early phase I clinical trials.It is generally accepted that potent Ad5-specific neutralizing antibodies (NAbs) contribute substantially to the suppressive effects of anti-Ad5 immunity (7,8,12,25,27,32). Far less is known about the biological importance of Ad5-specific T-lymphocyte responses. Ad5-specific CD4 ϩ and CD8 ϩ T lymphocytes have been found in both humans (9,17,18,20) and mice (11,13,21,29,30). However, the importance of Ad5-specific T lymphocytes in suppressing the immunogenicity of rAd5 vaccines has not been fully characterized.In this study, we investigate the relative contributions of Ad5-specific humoral and cellular immune responses in suppressing the immunogenicity of a rAd5-Env vaccine in mice. By adoptive-transfer studies, we demonstrate that NAbs and CD8 ϩ T lymphocytes both contribute to immunity to Ad5. These data suggest that novel Ad vector-based vaccines should overcome both humoral and cellular immune responses to circumvent preexisting anti-Ad5 immunity. MATERIALS AND METHODSMice and immunizations. BALB/c mice, 6 to 8 weeks old, were purchased from Charles River Laboratories (Wilmington, Mass.). ...

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations will likely limit the immunogenicity and clinical utility of recombinant Ad5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 and other pathogens. A potential solution to this problem is to utilize rAd vaccine vectors derived from rare Ad serotypes such as Ad35 and Ad11. We have previously reported that rAd35 vectors were immunogenic in the presence of anti-Ad5 immunity, but the immunogenicity of heterologous rAd prime-boost regimens and the extent that cross-reactive anti-vector immunity may limit this approach have not been fully explored. Here we assess the immunogenicity of heterologous vaccine regimens involving rAd5, rAd35, and novel rAd11 vectors expressing simian immunodeficiency virus Gag in mice both with and without anti-Ad5 immunity. Heterologous rAd prime-boost regimens proved significantly more immunogenic than homologous regimens, as expected. Importantly, all regimens that included rAd5 were markedly suppressed by anti-Ad5 immunity. In contrast, rAd35-rAd11 and rAd11-rAd35 regimens elicited high-frequency immune responses both in the presence and in the absence of anti-Ad5 immunity, although we also detected clear cross-reactive Ad35/Ad11-specific humoral and cellular immune responses. Nevertheless, these data suggest the potential utility of heterologous rAd prime-boost vaccine regimens using vectors derived from rare human Ad serotypes.Preexisting anti-vector immunity represents a major hurdle in the development of vector-based vaccines for human immunodeficiency virus type 1 (HIV-1) and other pathogens. Recombinant adenovirus serotype 5 (rAd5) vectors have been shown to elicit high-frequency immune responses and to afford protective efficacy in a variety of animal models (24,30,31). These vectors are therefore being advanced into large-scale clinical trials (14,25). However, the high prevalence of antiAd5 immunity in human populations will likely limit the immunogenicity and clinical utility of rAd5 vector-based vaccines, particularly in the developing world (13,25,32). Anti-Ad5 immunity has already been shown to suppress substantially the immunogenicity of rAd5 vaccines for HIV-1 in studies in mice (2, 3, 6, 29, 34), rhesus monkeys (4), and humans in phase 1 clinical trials (26).A potential solution to this problem is to develop rAd vectors from alternative Ad serotypes. One approach is to develop rAd vectors from species other than humans. For example, ovine (10), porcine (21), bovine (22), and chimpanzee (5, 33) Ads have been constructed. Of these vector systems, chimpanzee rAd vaccine vectors in particular have been shown to be immunogenic and only marginally affected by anti-Ad5 immunity in preclinical studies (6,20). However, a potential hurdle for the use of nonhuman rAd vaccine vectors is their unknown clinical disease associations in humans, which may raise substantial safety and regulatory concerns.Another approach is to develop rAd vectors from rare human Ad serotypes ...

DCs are critical for priming adaptive immune responses to foreign antigens. However, the utility of harnessing these cells in vivo to optimize the immunogenicity of vaccines has not been fully explored. Here we investigate a novel vaccine approach that involves delivering synergistic signals that both recruit and expand DC populations at the site of antigen production. Intramuscular injection of an unadjuvanted HIV-1 envelope (env) DNA vaccine recruited few DCs to the injection site and elicited low-frequency, env-specific immune responses in mice. Coadministration of plasmids encoding the chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) and the DC-specific growth factor fms-like tyrosine kinase 3 ligand with the DNA vaccine resulted in the recruitment, expansion, and activation of large numbers of DCs at the site of inoculation. Consistent with these findings, coadministration of these plasmid cytokines also markedly augmented DNA vaccine---elicited cellular and humoral immune responses and increased protective efficacy against challenge with recombinant vaccinia virus. These data suggest that the availability of mature DCs at the site of inoculation is a critical rate-limiting factor for DNA vaccine immunogenicity. Synergistic recruitment and expansion of DCs in vivo may prove a practical strategy for overcoming this limitation and potentiating immune responses to vaccines as well as other immunotherapeutic strategies.

Preexisting immunity to adenovirus serotype 5 (Ad5) has been shown to suppress the immunogenicity of recombinant Ad5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 (HIV-1) in both preclinical studies and clinical trials. A potential solution to this problem is to utilize rAd vectors derived from rare Ad serotypes, such as Ad35. However, rAd35 vectors have appeared less immunogenic than rAd5 vectors in preclinical studies to date. In this study, we explore the hypothesis that the differences in immunogenicity between rAd5 and rAd35 vectors may be due in part to differences between the fiber proteins of these viruses. We constructed capsid chimeric rAd35 vectors containing the Ad5 fiber knob (rAd35k5) and compared the immunogenicities of rAd5, rAd35k5, and rAd35 vectors expressing simian immunodeficiency virus Gag and HIV-1 Env in mice and rhesus monkeys. In vitro studies demonstrated that rAd35k5 vectors utilized the Ad5 receptor CAR rather than the Ad35 receptor CD46. In vivo studies showed that rAd35k5 vectors were more immunogenic than rAd35 vectors in both mice and rhesus monkeys. These data suggest that the Ad5 fiber knob contributes substantially to the immunogenicity of rAd vectors. Moreover, these studies demonstrate that capsid chimeric rAd vectors can be constructed to combine beneficial immunologic and serologic properties of different Ad serotypes.Recombinant adenovirus serotype 5 (rAd5) vector-based vaccines have been shown to elicit high-frequency, protective immune responses in a variety of animal models (22,23,26,27). As a result, rAd5 vaccines for human immunodeficiency virus type 1 (HIV-1) and other pathogens are currently being advanced into large-scale clinical trials (5,14,24). However, the high prevalence of preexisting anti-Ad5 immunity in human populations may limit the immunogenicity and clinical utility of rAd5 vector-based vaccines, particularly in the developing world (12,29,30). Importantly, anti-Ad5 immunity has already been shown to suppress the immunogenicity of rAd5 vaccines for HIV-1 in both preclinical studies (2,3,7,8,28,29,33) and clinical trials (24).A potential solution to this problem is to develop rAd vectors from rare human Ad serotypes, such as Ad35 and Ad11 (11,23,30). We have recently reported that rAd35 and rAd11 vectors expressing simian immunodeficiency virus (SIV) Gag were immunogenic and were not suppressed by anti-Ad5 immunity in mice, demonstrating the potential feasibility of this approach (3, 13). However, preclinical studies from our laboratory and others have suggested that rare serotype rAd vectors are intrinsically less immunogenic than rAd5 vectors in both mice and rhesus monkeys (3,13,23,24).In this study, we explore the hypothesis that the differences in immunogenicity between rAd5 and rAd35 vaccines may be due in part to the differences between the Ad5 and Ad35 fiber proteins. The Ad5 fiber knob interacts with the Ad5 receptor CAR (coxsackievirus and adenovirus receptor) on the surfaces of cells and mediates efficient viral attach...

Viral escape from cytotoxic T lymphocytes (CTLs) can undermine immune control of human immunodeficiency virus 1. It is therefore important to assess the stability of viral mutations in CTL epitopes after transmission to naive hosts. Here we demonstrate the persistence of mutations in a dominant CTL epitope after transmission of simian immunodeficiency virus variants to major histocompatibility complex-matched rhesus monkeys. Transient reversions to wild-type sequences occurred and elicited CTLs specific for the wild-type epitope, resulting in immunological pressure that rapidly reselected the mutant viruses. These data suggest that mutations in dominant human immunodeficiency virus 1 CTL epitopes may accumulate in human populations with limited major histocompatibility complex heterogeneity by a mechanism involving dynamic CTL control of transiently reverted wild-type virus.