Potent,persistent induction and modulation of cellular immune responses in rhesus macaques primed with Ad5hr-simian immunodeficiency virus (SIV) env/rev,gag, and/or nef vaccines and boosted with SIV gp120

Immunity elicited by multicomponent vaccines delivered by replication-competent Ad5hr-simian immunodeficiency virus (SIV) recombinants was systematically investigated. Rhesus macaques were immunized mucosally at weeks 0 and 12 with Ad5hr-SIV(smH4) env/rev, with or without Ad5hr-SIV(mac239) gag or Ad5hr-SIV(mac239) nef, or with all three recombinants. The total Ad5hr dosage was comparably adjusted among all animals with empty Ad5hr-DeltaE3 vector. The macaques were boosted with SIV gp120 in monophosphoryl A-stable emulsion adjuvant at 24 and 36 weeks. Controls received Ad5hr-DeltaE3 vector or adjuvant only. By ELISPOT analysis, all four SIV gene products elicited potent cellular immune responses that persisted 42 weeks post-initial immunization. Unexpectedly, modulation of this cellular immune response was observed among macaques receiving one, two, or three Ad5hr-SIV recombinants. Env responses were significantly enhanced throughout the immunization period in macaques immunized with Ad5hr-SIV env/rev plus Ad5hr-SIV gag and tended to be higher in macaques that also received Ad5hr-SIV nef. Macaques primed with all three recombinants displayed significant down-modulation in numbers of gamma interferon (IFN-gamma)-secreting cells specific for SIV Nef, and the Env- and Gag-specific responses were also diminished. Modulation of antibody responses was not observed. Down-modulation was seen only during the period of Ad5hr-recombinant priming, not during subunit boosting, although SIV-specific IFN-gamma-secreting cells persisted. The effect was not attributable to Ad5hr replication differences among immunization groups. Vaccine delivery via replication-competent live vectors, which can persistently infect new cells and continuously present low-level antigen, may be advantageous in overcoming competition among complex immunogens for immune recognition. Effects of current multicomponent vaccines on individual immune responses should be evaluated with regard to future vaccine design.     

Potent,persistent cellular immune responses elicited by sequential immunization of rhesus macaques with Ad5 host range mutant recombinants encoding SIV Rev and SIV Nef

Vaccines incorporating multiple HIV components should elicit broad immunity and protection against a spectrum of HIV strains. Early regulatory and accessory gene products are attractive candidates for such vaccines. Here, immunogenicity studies on SIV Rev and Nef expressed in replication competent Adenovirus type 5 host range mutant vectors (Ad5hr) are summarized. Interferon-gamma (IFN-gamma)-secreting cells in response to Env and Rev peptides were enumerated by ELISPOT after two sequential immunizations of 55 macaques with Ad5hr-SIVenv/rev. Responses to SIV Nef were assessed in 16 macaques also immunized with Ad5hr-SIVnef. Potent cellular immunity to both Rev and Nef was induced following the second Ad-recombinant immunization and persisted for at least 30 weeks. Persistent cellular immunity to SIV Env was also seen, with a mean of 700 IFN-gamma-secreting cells per million PBMC. Rev and Env responses were positively correlated. While greater responses to early gene products occur in natural infection, as immunogens Rev and Nef elicited the same number of IFN-gamma secreting cells as Env, after adjusting for differences in protein size. The same percentage of macaques also responded to Rev, Nef, and Env: 59, 63, and 64%, respectively. Overall, Ad5hrSIVenv/rev and -SIVnef were highly effective immunogens. Their contribution to protective efficacy will be addressed in future studies.     

An adenovirus-simian immunodeficiency virus env vaccine elicits humoral, cellular, and mucosal immune responses in rhesus macaques and decreases viral burden following vaginal challenge.

Six female rhesus macaques were immunized orally and intranasally at 0 weeks and intratracheally at 12 weeks with an adenovirus type 5 host range mutant (Ad5hr)-simian immunodeficiency virus SIVsm env recombinant and at 24 and 36 weeks with native SIVmac251 gp120 in Syntex adjuvant. Four macaques received the Ad5hr vector and adjuvant alone; two additional controls were naive. In vivo replication of the Ad5hr wild-type and recombinant vectors occurred with detection of Ad5 DNA in stool samples and/or nasal secretions in all macaques and increases in Ad5 neutralizing antibody in 9 of 10 macaques following Ad administrations. SIV-specific neutralizing antibodies appeared after the second recombinant immunization and rose to titers > 10,000 following the second subunit boost. Immunoglobulin G (IgG) and IgA antibodies able to bind gp120 developed in nasal and rectal secretions, and SIV-specific IgGs were also observed in vaginal secretions and saliva. T-cell proliferative responses to SIV gp140 and T-helper epitopes were sporadically detected in all immunized macaques. Following vaginal challenge with SIVmac251, transient or persistent infection resulted in both immunized and control monkeys. The mean viral burden in persistently infected immunized macaques was significantly decreased in the primary infection period compared to that of control macaques. These results establish in vivo use of the Ad5hr vector, which overcomes the host range restriction of human Ads for rhesus macaques, thereby providing a new model for evaluation of Ad-based vaccines. In addition, they show that a vaccine regimen using the Ad5hr-SIV env recombinant and gp120 subunit induces strong humoral, cellular, and mucosal immunity in rhesus macaques. The reduced viral burden achieved solely with an env-based vaccine supports further development of Ad-based vaccines comprising additional viral components for immune therapy and AIDS vaccine development.     

A replication competent adenovirus 5 host range mutant-simian immunodeficiency virus (SIV) recombinant priming/subunit protein boosting vaccine regimen induces broad,persistent SIV-specific cellular immunity to dominant and subdominant epitopes in Mamu-A*01 rhesus macaques

CTL are important in controlling HIV and SIV infection. To quantify cellular immune responses induced by immunization, CD8(+) T cells specific for the subdominant Env p15m and p54m epitopes and/or the dominant Gag p11C epitope were evaluated by tetramer staining in nine macaques immunized with an adenovirus (Ad) 5 host range mutant (Ad5hr)-SIVenv/rev recombinant and in four of nine which also received an Ad5hr-SIVgag recombinant. Two Ad5hr-SIV recombinant priming immunizations were followed by two boosts with gp120 protein or an envelope polypeptide representing the CD4 binding domain. Two mock-immunized macaques served as controls. IFN-gamma-secreting cells were also assessed by ELISPOT assay using p11C, p15m, and p54m peptide stimuli and overlapping pooled Gag and Env peptides. As shown by tetramer staining, Ad-recombinant priming elicited a high frequency of persistent CD8(+) T cells able to recognize p11C, p15m, and p54m epitopes. The presence of memory cells 38 wk postinitial immunization was confirmed by expansion of tetramer-positive CD8(+) T cells following in vitro stimulation. The SIV-specific CD8(+) T cells elicited were functional and secreted IFN-gamma in response to SIV peptide stimuli. Although the level and frequency of response of peripheral blood CD8(+) T cells to the subdominant Env epitopes were not as great as those to the dominant p11C epitope, elevated responses were observed when lymph node CD8(+) T cells were evaluated. Our data confirm the potency and persistence of functional cellular immune responses elicited by replication competent Ad-recombinant priming. The cellular immunity elicited is broad and extends to subdominant epitopes.     

Comparison of Systemic and Mucosal Immunization with Helper-Dependent Adenoviruses for Vaccination against Mucosal Challenge with SHIV

Most HIV-1 infections are thought to occur at mucosal surfaces during sexual contact. It has been hypothesized that vaccines delivered at mucosal surfaces may mediate better protection against HIV-1 than vaccines that are delivered systemically. To test this, rhesus macaques were vaccinated by intramuscular (i.m.) or intravaginal (ivag.) routes with helper-dependent adenoviral (HD-Ad) vectors expressing HIV-1 envelope. Macaques were first immunized intranasally with species C Ad serotype 5 (Ad5) prior to serotype-switching with species C HD-Ad6, Ad1, Ad5, and Ad2 vectors expressing env followed by rectal challenge with CCR5-tropic SHIV-SF162P3. Vaccination by the systemic route generated stronger systemic CD8 T cell responses in PBMC, but weaker mucosal responses. Conversely, mucosal immunization generated stronger CD4 T cell central memory (Tcm) responses in the colon. Intramuscular immunization generated higher levels of env-binding antibodies, but neither produced neutralizing or cytotoxic antibodies. After mucosal SHIV challenge, both groups controlled SHIV better than control animals. However, more animals in the ivag. group had lower viral set points than in in the i.m. group. These data suggest mucosal vaccination may have improve protection against sexually-transmitted HIV. These data also demonstrate that helper-dependent Ad vaccines can mediate robust vaccine responses in the face of prior immunity to Ad5 and during four rounds of adenovirus vaccination.     

Immunogenicity of recombinant adenovirus serotype 35 vaccine in the presence of pre-existing anti-Ad5 immunity

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.     

Neutralizing antibodies and CD8+ T lymphocytes both contribute to immunity to adenovirus serotype 5 vaccine vectors

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.     

Sequential priming with simian immunodeficiency virus (SIV) DNA vaccines, with or without encoded cytokines, and a replicating adenovirus-SIV recombinant followed by protein boosting does not control a pathogenic SIVmac251 mucosal challenge

Previously, combination DNA/nonreplicating adenovirus (Ad)- or poxvirus-vectored vaccines have strongly protected against SHIV(89.6P), DNAs expressing cytokines have modulated immunity elicited by DNA vaccines, and replication-competent Ad-recombinant priming and protein boosting has strongly protected against simian immunodeficiency virus (SIV) challenge. Here we evaluated a vaccine strategy composed of these promising components. Seven rhesus macaques per group were primed twice with multigenic SIV plasmid DNA with or without interleukin-12 (IL-12) DNA or IL-15 DNA. After a multigenic replicating Ad-SIV immunization, all groups received two booster immunizations with SIV gp140 and SIV Nef protein. Four control macaques received control DNA plasmids, empty Ad vector, and adjuvant. All vaccine components were immunogenic, but the cytokine DNAs had little effect. Macaques that received IL-15-DNA exhibited higher peak anti-Nef titers, a more rapid anti-Nef anamnestic response postchallenge, and expanded CD8(CM) T cells 2 weeks postchallenge compared to the DNA-only group. Other immune responses were indistinguishable between groups. Overall, no protection against intrarectal challenge with SIV(mac251) was observed, although immunized non-Mamu-A*01 macaques as a group exhibited a statistically significant 1-log decline in acute viremia compared to non-Mamu-A*01 controls. Possible factors contributing to the poor outcome include administration of cytokine DNAs to sites different from the Ad recombinants (intramuscular and intratracheal, respectively), too few DNA priming immunizations, a suboptimal DNA delivery method, failure to ensure delivery of SIV and cytokine plasmids to the same cell, and instability and short half-life of the IL-15 component. Future experiments should address these issues to determine if this combination approach is able to control a virulent SIV challenge.     

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His₆/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His₆/pH5S, which had His₆ tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His₆/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/∆H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His₆ was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His₆. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His₆ tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His₆. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.     

A replication-competent adenovirus-human immunodeficiency virus (Ad-HIV) tat and Ad-HIV env priming/Tat and envelope protein boosting regimen elicits enhanced protective efficacy against simian/human immunodeficiency virus SHIV89.6P challenge in rhesus macaques

We previously demonstrated that replication-competent adenovirus (Ad)-simian immunodeficiency virus (SIV) recombinant prime/protein boost regimens elicit potent immunogenicity and strong, durable protection of rhesus macaques against SIV(mac251). Additionally, native Tat vaccines have conferred strong protection against simian/human immunodeficiency virus SHIV(89.6P) challenge of cynomolgus monkeys, while native, inactivated, or vectored Tat vaccines have failed to elicit similar protective efficacy in rhesus macaques. Here we asked if priming rhesus macaques with replicating Ad-human immunodeficiency virus (HIV) tat and boosting with the Tat protein would elicit protection against SHIV(89.6P). We also evaluated a Tat/Env regimen, adding an Ad-HIV env recombinant and envelope protein boost to test whether envelope antibodies would augment acute-phase protection. Further, expecting cellular immunity to enhance chronic viremia control, we tested a multigenic group: Ad-HIV tat, -HIV env, -SIV gag, and -SIV nef recombinants and Tat, Env, and Nef proteins. All regimens were immunogenic. A hierarchy was observed in enzyme-linked immunospot responses (with the strongest response for Env, followed by Gag, followed by Nef, followed by Tat) and antibody titers (with the highest titer for Env, followed by Tat, followed by Nef, followed by Gag). Following intravenous SHIV(89.6P) challenge, all macaques became infected. Compared to controls, no protection was seen in the Tat-only group, confirming previous reports for rhesus macaques. However, the multigenic group blunted acute viremia by approximately 1 log (P = 0.017), and both the multigenic and Tat/Env groups reduced chronic viremia by 3 and 4 logs, respectively, compared to controls (multigenic, P = 0.0003; Tat/Env, P < 0.0001). The strikingly greater reduction in the Tat/Env group than in the multigenic group (P = 0.014) was correlated with Tat and Env binding antibodies. Since prechallenge anti-Env antibodies lacked SHIV(89.6P)-neutralizing activity, other functional anti-Env and anti-Tat activities are under investigation, as is a possible synergy between the Tat and Env immunogens.     

Modulation of antigen-specific humoral responses in rhesus macaques by using cytokine cDNAs as DNA vaccine adjuvants

An important limitation of DNA immunization in nonhuman primates is the difficulty in generating high levels of antigen-specific antibody responses; strategies to enhance the level of immune responses to DNA immunization may be important in the further development of this vaccine strategy for humans. We approached this issue by testing the ability of molecular adjuvants to enhance the levels of immune responses generated by multicomponent DNA vaccines in rhesus macaques. Rhesus macaques were coimmunized intramuscularly with expression plasmids bearing genes encoding Th1 (interleukin 2 [IL-2] and gamma interferon)- or Th2 (IL-4)-type cytokines and DNA vaccine constructs encoding human immunodeficiency virus Env and Rev and simian immunodeficiency virus Gag and Pol proteins. We observed that the cytokine gene adjuvants (especially IL-2 and IL-4) significantly enhanced antigen-specific humoral immune responses in the rhesus macaque model. These results support the assumption that antigen-specific responses can be engineered to a higher and presumably more desirable level in rhesus macaques by genetic adjuvants.     

Anti-tumor immunotherapy despite immunity to adenovirus using a novel adenoviral vector Ad5 [E1-, E2b-]-CEA

Adenovirus serotype 5 (Ad5) has been widely used in clinical trials because it expresses inserted transgenes robustly and augments the innate immune response. Strategies to improve Ad5 vectors that can circumvent Ad5 immunity have become a critical issue, especially for use as a cancer immunotherapeutic in which repeated immunization is required. In this study, we constructed a novel Ad5 vector with unique deletions of the viral DNA polymerase and the pre-terminal protein region (Ad5 [E1-, E2b-]). This vector contains the carcinoembryonic antigen (CEA) gene insert and is designed to induce cell-mediated immunity (CMI) against the tumor-associated target. The CEA immunogenicity and in vivo anti-tumor effects of repeated immunizations with Ad5 [E1-, E2b-]-CEA compared with those observed with current generation Ad5 [E1-]-CEA were tested in Ad5 pre-immunized mice. We report that Ad5-immune mice immunized multiple times with Ad5 [E1-, E2b-]-CEA induced CEA-specific CMI responses that were significantly increased over those detected in Ad5-immune mice immunized multiple times with a current generation Ad5 [E1-]-CEA. Ad5 immune mice bearing CEA-expressing tumors that were treated with Ad5 [E1-, E2b-]-CEA had increased anti-tumor response as compared with Ad5 [E1-]-CEA treated mice. These results demonstrate that Ad5 [E1-, E2b-]-CEA can induce CMI immune responses which result in tumor growth inhibition despite the presence of pre-existing Ad5 immunity. Multiple re-immunizations using the same vector platform are now possible with the novel Ad5 [E1-, E2b-] platform.     

Immunogenicity of heterologous prime-boost regimens involving recombinant adenovirus serotype 11 (Ad11) and Ad35 vaccine vectors in the presence of anti-ad5 immunity

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.     

Low-dose penile SIVmac251 exposure of rhesus macaques infected with adenovirus type 5 (Ad5) and then immunized with a replication-defective Ad5-based SIV gag/pol/nef vaccine recapitulates the results of the phase IIb step trial of a similar HIV-1 vaccine

The Step Trial showed that the MRKAd5 HIV-1 subtype B Gag/Pol/Nef vaccine did not protect men from HIV infection or reduce setpoint plasma viral RNA (vRNA) levels but, unexpectedly, it did modestly enhance susceptibility to HIV infection in adenovirus type 5 (Ad5)-seropositive, uncircumcised men. As part of the process to understand the results of the Step Trial, we designed a study to determine whether rhesus macaques chronically infected with a host-range mutant Ad5 (Ad5hr) and then immunized with a replication defective Ad5 SIVmac239 Gag/Pol/Nef vaccine were more resistant or susceptible to SIV infection than unimmunized rhesus macaques challenged with a series of escalating dose penile exposures to SIVmac 251. The Ad5 SIV vaccine induced CD8(+) T cell responses in 70% of the monkeys, which is similar to the proportion of humans that responded to the vaccine in the Step Trial. However, the vaccine did not protect vaccinated animals from penile SIV challenge. At the lowest SIV exposure dose (10(3) 50% tissue culture infective doses), 2 of 9 Ad5-seropositive animals immunized with the Ad5 SIV vaccine became infected compared to 0 of 34 animals infected in the other animal groups (naive animals, Ad5-seropositive animals immunized with the empty Ad5 vector, Ad5-seronegative animals immunized with the Ad5 SIV vaccine, and Ad5-seronegative animals immunized with the empty Ad5 vector). Penile exposure to more concentrated virus inocula produced similar rates of infection in all animal groups. Although setpoint viral loads were unaffected in Step vaccinees, the Ad5 SIV-immunized animals had significantly lower acute-phase plasma vRNA levels compared to unimmunized animals. Thus, the results of the nonhuman primate (NHP) study described here recapitulate the lack of protection against HIV acquisition seen in the Step Trial and suggest a greater risk of infection in the Ad5-seropositive animals immunized with the Ad5 SIV vaccine. Further studies are necessary to confirm the enhancement of virus acquisition and to discern associated mechanisms.     

Improved protection of rhesus macaques against intrarectal simian immunodeficiency virus SIV(mac251) challenge by a replication-competent Ad5hr-SIVenv/rev and Ad5hr-SIVgag recombinant priming/gp120 boosting regimen

In this study we investigated the ability of a replication-competent Ad5hr-SIVenv/rev and Ad5hr-SIVgag recombinant priming/gp120 boosting regimen to induce protective immunity in rhesus macaques against pathogenic simian immunodeficiency virus(mac251). Immunization of macaques by two sequential administrations of the same recombinants by the same route resulted in boosting and persistence of SIV-specific cellular immune responses for 42 weeks past the initial immunization. Anti-SIV gp120 immunoglobulin G (IgG) and IgA antibodies were induced in secretory fluids, and all macaques exhibited serum neutralizing antibody activity. After intrarectal SIV(mac251) challenge, all of the macaques became infected. However, relative protection, as assessed by statistically significant lower SIV viral loads in plasma at both acute infection and set point, was observed in 8 out of 12 immunized non-Mamu-A(*)01 animals. Elevated mean cellular immune responses to Gag and Env, neutralizing antibody activity, and IgG and IgA binding antibody levels were observed in the eight protected macaques. Statistically significant correlations with protective outcome were observed for cellular immune responses to SIV Env and Gag and for SIV gp120-specific IgG antibodies in nasal and vaginal fluids. Two macaques that exhibited the greatest and most persistent viremia control also exhibited strong CD8(+) T-cell antiviral activity. The results suggest that a spectrum of immune responses may be necessary for adequate control of viral replication and disease progression and highlight a potential role for nonneutralizing antibodies at mucosal sites.     

Factors Associated with Slow Disease Progression in Macaques Immunized with an Adenovirus-Simian Immunodeficiency Virus (SIV) Envelope Priming-gp120 Boosting Regimen and Challenged Vaginally with SIVmac251

Rhesus macaques were immunized with a combination vaccine regimen consisting of adenovirus type 5 host range mutant-simian immunodeficiency virus envelope (Ad5hr-SIVenv) recombinant priming and boosting with native SIV gp120. Upon intravaginal challenge with SIVmac251, both persistently and transiently viremic animals were observed (S. L. Buge, E. Richardson, S. Alipanah, P. Markham, S. Cheng, N. Kalyan, C. J. Miller, M. Lubeck, S. Udem, J. Eldridge, and M. Robert-Guroff, J. Virol. 71:8531-8541, 1997). Long-term follow-up of the persistently viremic immunized macaques, which displayed significantly reduced viral burdens during the first 18 weeks postchallenge compared to controls, has now shown that one of four became a slow progressor, clearing virus from plasma and remaining asymptomatic with stable CD4 counts for 134 weeks postchallenge. Reboosting of the transiently viremic macaques did not reactivate latent virus. Rechallenge with two sequential SIVmac251 intravaginal exposures again resulted in partial protection of one of two immunized macaques, manifested by viral clearance and stable CD4 counts. No single immune parameter was associated with partial protection. Development of a strong antibody response capable of neutralizing a primary SIVmac251 isolate together with SIV-specific cytotoxic T lymphocytes were implicated, while CD8(+) T-cell antiviral activity and mucosal immune responses were not associated with delayed disease progression. Our data show that even a third immunization with the same Ad5hr-SIVenv recombinant can elicit significant immune responses to the inserted gene product, suggesting that preexisting Ad antibodies may not preclude effective immunization. Further, the partial protection against a virulent, pathogenic SIV challenge observed in two of six macaques immunized with a vaccine regimen based solely on the viral envelope indicates that this vectored-vaccine approach has promise and that multicomponent vaccines based in the same system merit further investigation.     

Protection Provided by a Herpes Simplex Virus 2 (HSV-2) Glycoprotein C and D Subunit Antigen Vaccine against Genital HSV-2 Infection in HSV-1-Seropositive Guinea Pigs

A prophylactic vaccine for genital herpes disease remains an elusive goal. We report the results of two studies performed collaboratively in different laboratories that assessed immunogenicity and vaccine efficacy in herpes simplex virus 1 (HSV-1)-seropositive guinea pigs immunized and subsequently challenged intravaginally with HSV-2. In study 1, HSV-2 glycoproteins C (gC2) and D (gD2) were produced in baculovirus and administered intramuscularly as monovalent or bivalent vaccines with CpG and alum. In study 2, gD2 was produced in CHO cells and given intramuscularly with monophosphoryl lipid A (MPL) and alum, or gC2 and gD2 were produced in glycoengineered Pichia pastoris and administered intramuscularly as a bivalent vaccine with Iscomatrix and alum to HSV-1-naive or -seropositive guinea pigs. In both studies, immunization boosted neutralizing antibody responses to HSV-1 and HSV-2. In study 1, immunization with gC2, gD2, or both immunogens significantly reduced the frequency of genital lesions, with the bivalent vaccine showing the greatest protection. In study 2, both vaccines were highly protective against genital disease in naive and HSV-1-seropositive animals. Comparisons between gD2 and gC2/gD2 in study 2 must be interpreted cautiously, because different adjuvants, gD2 doses, and antigen production methods were used; however, significant differences invariably favored the bivalent vaccine. Immunization of naive animals with gC2/gD2 significantly reduced the number of days of vaginal shedding of HSV-2 DNA compared with that for mock-immunized animals. Surprisingly, in both studies, immunization of HSV-1-seropositive animals had little effect on recurrent vaginal shedding of HSV-2 DNA, despite significantly reducing genital disease.     

CTL-dependent and -independent antitumor immunity is determined by the tumor not the vaccine

Previously, we compared the efficiency of direct injection with an adenovirus (Ad) expressing human gp100 (hgp100) to immunization with dendritic cells (DC) loaded with the same vector ex vivo. The DC vaccine provided the greatest protection against challenge with B16F10 melanoma, and antitumor immunity was found to be CD8(+) T cell-independent. In the current study, we sought to determine whether lack of CD8(+) T cell-mediated antitumor immunity was a function of the vaccine platform or the tumor line. Both Ad and DC/Ad vaccines elicited CD8(+) CTL reactive against hgp100 and provided protection against B16F10 engineered to express hgp100 demonstrating that both vaccination platforms can effectively generate protective CD8(+) T cell-mediated immunity. The hgp100-induced CTL cross-reacted with murine gp100 (mgp100) and lysed B16F10 cells pulsed with mgp100 peptide indicating that the resistance of B16F10 cells to CTL elicited by hgp100 vaccination may be due to a defect in processing of the endogenous mgp100. Indeed, introduction of the TAP-1 cDNA into B16F10 rendered the cells sensitive to lysis by gp100-specific CTL. Furthermore, gp100-immunized mice were protected from challenge with B16F10-TAP1 cells through a mechanism dependent upon CD8(+) T cells. These results demonstrate that tumor phenotype, not the vaccination platform, ultimately determines CD8(+) or CD4(+) T cell-mediated tumor clearance.     

Novel vaccination protocol with two live mucosal vectors elicits strong cell-mediated immunity in the vagina and protects against vaginal virus challenge

Most HIV infections result from heterosexual transmission to women. Because cellular immunity plays a key role in the control of the infection, we sought to strengthen cellular immune responses in vaginal tissue. We explored a novel prime-boost protocol that used two live mucosal agents that trigger different pathways of innate immunity and induce strong cellular immunity. Adenovirus serotype 5 (Ad5) has frequently been used as a boost for DNA vaccines. In this study we used attenuated, recombinant L. monocytogenes-gag (rLm-gag) to prime mice by various mucosal routes-oral, intrarectal, and intravaginally (ivag)-followed by a systemic or mucosal boost with replication-defective rAd5-gag. Mice primed with a single administration of rLm-gag by any route and then boosted with rAd5-gag intramuscularly exhibited abundant Gag-specific CD8 T cells in spleen and vaginal lamina propria. Conversely, when boosted with rAd5-gag ivag, the immune response was reoriented toward the vagina with strikingly higher CD8 T cell responses in that tissue, particularly after ivag immunization by both vectors (ivag/ivag). Five weeks to 5 mo later, ivag/ivag-immunized mice continued to show high levels of effector memory CD8 T cells in vagina, while the pool of memory T cells in spleen assumed a progressively more central memory T cell phenotype. The memory mice showed high in vivo CTL activity in vagina, a strong recall response, and robust protection after ivag vaccinia-gag challenge, suggesting that this prime-boost strategy can induce strong cellular immunity, especially in vaginal tissues, and might be able to block the heterosexual transmission of HIV-1 at the vaginal mucosa.     

Suppression of immune response and protective immunity to a Japanese encephalitis virus DNA vaccine by coadministration of an IL-12-expressing plasmid

IL-12 plays a central role in both innate and acquired immunity and has been demonstrated to potentiate the protective immunity in several experimental vaccines. However, in this study, we show that IL-12 can be detrimental to the immune responses elicited by a plasmid DNA vaccine. Coadministration of the IL-12-expressing plasmid (pIL-12) significantly suppressed the protective immunity elicited by a plasmid DNA vaccine (pE) encoding the envelope protein of Japanese encephalitis virus. This suppressive effect was associated with marked reduction of specific T cell proliferation and Ab responses. A single dose of pIL-12 treatment with plasmid pE in initial priming resulted in significant immune suppression to subsequent pE booster immunization. The pIL-12-mediated immune suppression was dose dependent and evident only when the IL-12 gene was injected either before or coincident with the pE DNA vaccine. Finally, using IFN-gamma gene-disrupted mice, we showed that the suppressive activity of the IL-12 plasmid was dependent upon endogenous production of IFN-gamma. These results demonstrate that coexpression of the IL-12 gene can sometimes produce untoward effects to immune responses, and thus its application as a vaccine adjuvant should be carefully evaluated.