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.     

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.     

Coexpression of the simian immunodeficiency virus Env and Rev proteins by a recombinant human adenovirus host range mutant.

Recombinant human adenoviruses (Ads) that replicate in the intestinal tract offer a novel, yet practical, means of immunoprophylaxis against a wide variety of viral and bacterial pathogens. For some infectious agents such as human immunodeficiency virus (HIV), the potential for residual infectious material in vaccine preparations must be eliminated. Therefore, recombinant human Ads that express noninfectious HIV or other microbial proteins are attractive vaccine candidates. To test such an approach for HIV, we chose an experimental model of AIDS based on simian immunodeficiency virus (SIV) infection of macaques. Our data demonstrate that the SIV Env gene products are expressed in cultured cells after infection with a recombinant Ad containing both SIV env and rev genes. An E3 deletion vector derived from a mutant of human Ad serotype 5 that efficiently replicates in both human and monkey cells was used to bypass the usual host range restriction of Ad infection. In addition, we show that the SIV rev gene is properly spliced from a single SIV subgenomic DNA fragment and that the Rev protein is expressed in recombinant Ad-SIV-infected human as well as monkey cells. The expression of SIV gene products in suitable live Ad vectors provides an excellent system for studying the regulation of SIV gene expression in cultured cells and evaluating the immunogenicity and protective efficacy of SIV proteins in macaques.     

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.     

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.     

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.     

Multiprotein genetic vaccine in the SIV-Macaca animal model: a promising approach to generate sterilizing immunity to HIV infection

BACKGROUND: Vaccine combining structural and regulatory proteins is an emerging approach to develop an HIV/AIDS vaccine and therefore, the immunogenicity and efficacy of two regimens of immunization combining structural (Gag/Pol, Env) and regulatory (Rev, Tat, Nef) Simian immunodeficiency virus (SIV) proteins were compared in cynomolgus monkeys. METHODS: Monkeys were immunized with Modified Vaccine Ankara vector (MVA-J5) (protocol 1) or with DNA, Semliki forest virus and MVA vectors (DNA/SFV/MVA) (protocol 2). At week 32, all monkeys were challenge intravenously (protocol 1) or intrarectally (protocol 2) with 50 MID(50) of SIVmac251. Humoral, proliferative responses and in particular in protocol 2, the frequency of IFN-gamma producing cells, were measured in all monkeys before and after the challenge. RESULTS: Both vaccine regimens elicited humoral and proliferative responses but failed to induce neutralizing antibodies. Upon intravenous challenge, two out of three MVA-J5 vaccinated monkeys exhibited a long-term control of the viral replication whereas DNA/SFV/MVA vaccine abrogated the virus replication up to undetectable level in three out of four vaccinated monkeys. A major contribution to this vaccine effect appeared to be the IFN-gamma/ELISPOT responses to vaccine antigens (Gag, Rev Tat and Nef). CONCLUSIONS: These results, indicate that multiprotein heterologous prime-boost vaccination can induce a robust vaccine-induced immunity able to abrogate virus replication.     

Th-1-type cytotoxic CD8+ T-lymphocyte responses to simian immunodeficiency virus (SIV) are a consistent feature of natural SIV infection in sooty mangabeys

Sooty mangabeys are a natural host of simian immunodeficiency virus (SIV) that remain asymptomatic and do not exhibit increased immune activation or increased T-lymphocyte turnover despite sustained high levels of SIV viremia. In this study we asked whether an altered immune response to SIV contributes to the lack of immunopathology in sooty mangabeys as opposed to species with pathogenic lentivirus infection. SIV-specific cellular immune responses were investigated in a cohort of 25 sooty mangabeys with natural SIV infection. Gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay responses targeting a median of four SIV proteins were detected in all 25 mangabeys and were comparable in magnitude to those of 13 rhesus macaques infected with SIVmac251 for more than 6 months. As with rhesus macaques, Th2 ELISPOT responses to SIV were absent or >10-fold lower than the IFN-gamma ELISPOT response to the same SIV protein. The SIV-specific ELISPOT response was predominantly mediated by CD8+ T lymphocytes; the frequency of circulating SIV-specific CD8+ T lymphocytes ranged between 0.11% and 3.26% in 13 mangabeys. Functionally, the SIV-specific CD8+ T lymphocytes were cytotoxic; secreted IFN-gamma, tumor necrosis factor alpha, and macrophage inflammatory protein 1beta; and had an activated effector phenotype. Although there was a trend toward higher frequencies of SIV-specific CD8+ T lymphocytes in mangabeys with lower viral loads, a significant inverse correlation between SIV viremia and SIV-specific cellular immunity was not detected. The consistent detection of Th1-type SIV-specific cellular immune responses in naturally infected sooty mangabeys suggests that immune attenuation is neither a feature of nor a requirement for maintenance of nonpathogenic SIV infection in its natural host.     

T-cell-mediated protective efficacy of a systemic vaccine approach in cynomolgus monkeys after SIV mucosal challenge

The immunogenicity and the protective efficacy of a new polyvalent triple vector (DNA/SFV/MVA) based vaccine against mucosal challenge with pathogenic SIVmac251 were investigated. Cynomolgus monkeys (Macaca fascicularis) were primed intradermally with DNA, boosted twice subcutaneously with recombinant Semliki Forest virus (rSFV) and finally intramuscularly with recombinant Modified Vaccinia Virus Ankara strain (rMVA). Both DNA and recombinant viral vectors expressed SIV proteins (Gag, Pol, Tat, Rev, Nef and Env). The vaccinated monkeys developed T helper proliferative responses to viral antigens after the second immunization while interferon (IFN)-gamma enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) specific responses appeared only after the last boost with rMVA. Upon intrarectal challenge with pathogenic SIVmac251, three of four vaccinated monkeys were either fully protected or exhibited a dramatic reduction of virus replication up to undetectable level. A major contribution to this protective effect appeared to be the anamnestic T-cell IFN-gamma ELISPOT responses to vaccine antigens (Gag, Rev, Tat, Nef) that mirrored the viral clearance. These results underline the efficacy of a multiprotein approach in combination with a triple vector system of antigen delivery.     

Rapid dissemination of SIV follows multisite entry after rectal inoculation

Receptive ano-rectal intercourse is a major cause of HIV infection in men having sex with men and in heterosexuals. Current knowledge of the mechanisms of entry and dissemination during HIV rectal transmission is scarce and does not allow the development of preventive strategies. We investigated the early steps of rectal infection in rhesus macaques inoculated with the pathogenic isolate SIVmac251 and necropsied four hours to nine days later. All macaques were positive for SIV. Control macaques inoculated with heat-inactivated virus were consistently negative for SIV. SIV DNA was detected in the rectum as early as four hours post infection by nested PCR for gag in many laser-microdissected samples of lymphoid aggregates and lamina propria but never in follicle-associated epithelium. Scarce SIV antigen positive cells were observed by immunohistofluorescence in the rectum, among intraepithelial and lamina propria cells as well as in clusters in lymphoid aggregates, four hours post infection and onwards. These cells were T cells and non-T cells that were not epithelial cells, CD68(+) macrophages, DC-SIGN(+) cells or fascin(+) dendritic cells. DC-SIGN(+) cells carried infectious virus. Detection of Env singly spliced mRNA in the mucosa by nested RT-PCR indicated ongoing viral replication. Strikingly, four hours post infection colic lymph nodes were also infected in all macaques as either SIV DNA or infectious virus was recovered. Rapid SIV entry and dissemination is consistent with trans-epithelial transport. Virions appear to cross the follicle-associated epithelium, and also the digestive epithelium. Viral replication could however be more efficient in lymphoid aggregates. The initial sequence of events differs from both vaginal and oral infections, which implies that prevention strategies for rectal transmission will have to be specific. Microbicides will need to protect both digestive and follicle-associated epithelia. Vaccines will need to induce immunity in lymph nodes as well as in the rectum.     

Evaluation in rhesus macaques of Tat and rev-targeted immunization as a preventive vaccine against mucosal challenge with SHIV-BX08

Recent evidence suggests that a CD8-mediated cytotoxic T-cell response against the regulatory proteins of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) may control infection after pathogenic virus challenge. Here, we evaluated whether vaccination with Tat or Tat and Rev could significantly reduce viral load in nonhuman primates. Rhesus macaques were primed with Semliki forest Virus (SFV) expressing HIV-1 tat (SFV-tat) and HIV-1 rev (SFV-rev) and boosted with modified vaccinia virus Ankara (MVA) expressing tat and rev. A second group of monkey was primed with SFV-tat only and boosted with MVA-tat. A third group received a tat and rev DNA/MVA prime-boost vaccine regimen. Monitoring of anti-Tat and anti-Rev antibody responses or antigen-specific IFN-gamma production, as measured by enzyme-linked immunospot assays revealed no clear differences between the three groups. These results suggest that priming with either DNA or SFV seemed to be equivalent, but the additive or synergistic effect of a rev vaccine could not be clearly established. The animals were challenged by the rectal route 9 weeks after the last booster immunization, using 10 MID(50) of a SHIV-BX08 stock. Postchallenge follow-up of the monkeys included testing seroconversion to Gag and Env antigens, measuring virus infectivity in PBMC by cocultivation with noninfected human cells, and monitoring of plasma viral load. None of the animals was protected from infection as assessed by PCR, but peak viremia was reduced more than 200-fold compared to sham controls in one third (6/18) of vaccinated macaques, whatever the vaccine regimen they received. Interestingly, among these six protected animals four did not seroconvert. Altogether, these results clearly indicated that the addition of early HIV proteins like Tat and Rev in a multicomponent preventive vaccine including structural proteins like Env or Gag may be beneficial in preventive vaccinal strategies.     

Tenofovir treatment augments anti-viral immunity against drug-resistant SIV challenge in chronically infected rhesus macaques

CTL based vaccine strategies in the macaque model of AIDS have shown promise in slowing the progression to disease. However, rapid CTL escape viruses can emerge rendering such vaccination useless. We hypothesized that such escape is made more difficult if the immunizing CTL epitope falls within a region of the virus that has a high density of overlapping reading frames which encode several viral proteins. To test this hypothesis, we immunized macaques using a peptide-loaded dendritic cell approach employing epitopes in the second coding exon of SIV Tat which spans reading frames for both Env and Rev. We report here that autologous dendritic cells, loaded with SIV peptides from Tat, Rev, and Env, induced a distinct cellular immune response measurable ex vivo. However, conclusive in vivo control of a challenge inoculation of SIVmac239 was not observed suggesting that CTL epitopes within densely overlapping reading frames are also subject to escape mutations.     

Immunogenicity of a DNA prime and recombinant adenovirus boost regime significantly varies between rhesus macaques of Chinese and Indian origins

BACKGROUND: Due to an ever increasing shortage of rhesus macaques of Indian origin (InR) that have been generally used for preclinical AIDS vaccine trials in non-human primates, demand is rising for Chinese rhesus macaques (ChR). However, the immunogenicity of an AIDS vaccine candidate has not been compared in parallel in both rhesus macaque subspecies. METHODS: ChR and InR were immunized with SIV/HIV DNA and adenovirus vaccine and their immune responses to SIV and HIV evaluated. RESULTS: SIV Gag- and Env-specific T-cell responses and SIV-specific lymphoproliferative responses measured in ChR were significantly weaker than those in InR (P < 0.05). By contrast, antibody responses to SIV Env, Tat, and Nef in ChR were stronger than those in InR (P < 0.05). CONCLUSIONS: Immunogenicity of an AIDS vaccine can vary significantly depending on the geographic origin implying genetic differences of macaques. This must be considered when describing and interpreting results of such vaccine studies.     

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.     

Effects of Antigen and Genetic Adjuvants on Immune Responses to Human Immunodeficiency Virus DNA Vaccines in Mice

The effects of genetic adjuvants on humoral and cell-mediated immunity to two human immunodeficiency virus antigens, Env and Nef, have been examined in mice. Despite similar levels of gene expression and the same gene delivery vector, the immune responses to these two gene products differed following DNA immunization. Intramuscular immunization with a Nef expression vector plasmid generated a humoral response and antigen-specific gamma interferon (IFN-gamma) production but little cytotoxic-T-lymphocyte (CTL) immunity. In contrast, immunization with an Env vector stimulated CTL activity but did not induce a high-titer antibody response. The ability to modify these antigen-specific immune responses was investigated by coinjection of DNA plasmids encoding cytokine and/or hematopoietic growth factors, interleukin-2 (IL-2), IL-12, IL-15, Flt3 ligand (FL), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Coadministration of these genes largely altered the immune responses quantitatively but not qualitatively. IL-12 induced the greatest increase in IFN-gamma and immunoglobulin G responses to Nef, and GM-CSF induced the strongest IFN-gamma and CTL responses to Env. A dual approach of expanding innate immunity by administering the FL gene, together with a cytokine that enhances adaptive immune responses, IL-2, IL-12, or IL-15, generated the most potent immune response at the lowest doses of Nef antigen. These findings suggest that intrinsic properties of the antigen determine the character of immune reactivity for this method of immunization and that specific combination of innate and adaptive immune cytokine genes can increase the magnitude of the response to DNA vaccines.     

Replicating rather than nonreplicating adenovirus-human immunodeficiency virus recombinant vaccines are better at eliciting potent cellular immunity and priming high-titer antibodies

A major challenge in combating the human immunodeficiency virus (HIV) epidemic is the development of vaccines capable of inducing potent, persistent cellular immunity and broadly reactive neutralizing antibody responses to HIV type 1 (HIV-1). We report here the results of a preclinical trial using the chimpanzee model to investigate a combination vaccine strategy involving sequential priming immunizations with different serotypes of adenovirus (Ad)/HIV-1(MN)env/rev recombinants and boosting with an HIV envelope subunit protein, oligomeric HIV(SF162) gp140deltaV2. The immunogenicities of replicating and nonreplicating Ad/HIV-1(MN)env/rev recombinants were compared. Replicating Ad/HIV recombinants were better at eliciting HIV-specific cellular immune responses and better at priming humoral immunity against HIV than nonreplicating Ad-HIV recombinants carrying the same gene insert. Enhanced cellular immunity was manifested by a greater frequency of HIV envelope-specific gamma interferon-secreting peripheral blood lymphocytes and better priming of T-cell proliferative responses. Enhanced humoral immunity was seen in higher anti-envelope binding and neutralizing antibody titers and better induction of antibody-dependent cellular cytotoxicity. More animals primed with replicating Ad recombinants mounted neutralizing antibodies against heterologous R5 viruses after one or two booster immunizations with the mismatched oligomeric HIV-1(SF162) gp140deltaV2 protein. These results support continued development of the replicating Ad-HIV recombinant vaccine approach and suggest that the use of replicating vectors for other vaccines may prove fruitful.     

Expression of simian immunodeficiency virus (SIV) nef in astrocytes during acute and terminal infection and requirement of nef for optimal replication of neurovirulent SIV in vitro

As the most numerous cells in the brain, astrocytes play a critical role in maintaining central nervous system homeostasis, and therefore, infection of astrocytes by human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) in vivo could have important consequences for the development of HIV encephalitis. In this study, we establish that astrocytes are infected in macaques during acute SIV infection (10 days postinoculation) and during terminal infection when there is evidence of SIV-induced encephalitis. Additionally, with primary adult rhesus macaque astrocytes in vitro, we demonstrate that the macrophage-tropic, neurovirulent viruses SIV/17E-Br and SIV/17E-Fr replicate efficiently in astrocytes, while the lymphocyte-tropic, nonneurovirulent virus SIV(mac)239 open-nef does not establish productive infection. Furthermore, aminoxypentane-RANTES abolishes virus replication, suggesting that these SIV strains utilize the chemokine receptor CCR5 for entry into astrocytes. Importantly, we show that SIV Nef is required for optimal replication in primary rhesus macaque astrocytes and that normalizing input virus by particle number rather than by infectivity reveals a disparity between the ability of a Nef-deficient virus and a virus encoding a nonmyristoylated form of Nef to replicate in these central nervous system cells. Since the myristoylated form of Nef has been implicated in functions such as CD4 and major histocompatibility complex I downregulation, kinase association, and enhancement of virion infectivity, these data suggest that an as yet unidentified function of Nef may exist to facilitate SIV replication in astrocytes that may have important implications for in vivo pathogenesis.     

Protection against mucosal simian immunodeficiency virus SIV(mac251) challenge by using replicating adenovirus-SIV multigene vaccine priming and subunit boosting

Whereas several recent AIDS vaccine strategies have protected rhesus macaques against a pathogenic simian/human immunodeficiency virus (SHIV)(89.6P) challenge, similar approaches have provided only modest, transient reductions in viral burden after challenge with virulent, pathogenic SIV, which is more representative of HIV infection of people. We show here that priming with replicating adenovirus recombinants encoding SIV env/rev, gag, and/or nef genes, followed by boosting with SIV gp120 or an SIV polypeptide mimicking the CD4 binding region of the envelope, protects rhesus macaques from intrarectal infection with the highly pathogenic SIV(mac251). Using trend analysis, significant reductions in acute-phase and set point viremia were correlated with anti-gp120 antibody and cellular immune responses, respectively. Within immunization groups exhibiting significant protection, a subset (39%) of macaques have exhibited either no viremia, cleared viremia, or controlled viremia at the threshold of detection, now more than 40 weeks postchallenge. This combination prime-boost strategy, utilizing replication competent adenovirus, is a promising alternative for HIV vaccine development.     

Protection of Macaques with Diverse MHC Genotypes against a Heterologous SIV by Vaccination with a Deglycosylated Live-Attenuated SIV

HIV vaccine development has been hampered by issues such as undefined correlates of protection and extensive diversity of HIV. We addressed these issues using a previously established SIV-macaque model in which SIV mutants with deletions of multiple gp120 N-glycans function as potent live attenuated vaccines to induce near-sterile immunity against the parental pathogenic SIVmac239. In this study, we investigated the protective efficacy of these mutants against a highly pathogenic heterologous SIVsmE543-3 delivered intravenously to rhesus macaques with diverse MHC genotypes. All 11 vaccinated macaques contained the acute-phase infection with blood viral loads below the level of detection between 4 and 10 weeks postchallenge (pc), following a transient but marginal peak of viral replication at 2 weeks in only half of the challenged animals. In the chronic phase, seven vaccinees contained viral replication for over 80 weeks pc, while four did not. Neutralizing antibodies against challenge virus were not detected. Although overall levels of SIV specific T cell responses did not correlate with containment of acute and chronic viral replication, a critical role of cellular responses in the containment of viral replication was suggested. Emergence of viruses with altered fitness due to recombination between the vaccine and challenge viruses and increased gp120 glycosylation was linked to the failure to control SIV. These results demonstrate the induction of effective protective immune responses in a significant number of animals against heterologous virus by infection with deglycosylated attenuated SIV mutants in macaques with highly diverse MHC background. These findings suggest that broad HIV cross clade protection is possible, even in hosts with diverse genetic backgrounds. In summary, results of this study indicate that deglycosylated live-attenuated vaccines may provide a platform for the elucidation of correlates of protection needed for a successful HIV vaccine against diverse isolates.     

Detailed analysis of CD4+ Th responses to envelope and Gag proteins of simian immunodeficiency virus reveals an exclusion of broadly reactive Th epitopes from the glycosylated regions of envelope

Ag-specific CD4(+) Th cells play a key role in the development, maturation, and maintenance of pathogen-specific humoral and cellular immune responses. To define the fine specificity of broadly reactive Th responses associated with mature immunity in a lentiviral system, we analyzed peptide-specific Th responses in eight macaques chronically infected with a reference live attenuated SIV at 12-14 mo postinoculation. All macaques had stable immunocompetent Th cells at the time of analysis, and a unique array of Th responses to 20-mer overlapping peptides from envelope (Env) and Gag was identified for each macaque, which were then used to define a set of 31 broadly reactive peptide epitopes. Only 5 of the 31 broadly reactive Th epitope peptides mapped to the surface (SU) domain of Env. Interestingly, these were all confined to two conserved nonglycosylated regions toward the carboxyl terminus of SU, suggesting a structural influence of glycosylation on development of Th responses. Gag and the Env transmembrane proteins contained the majority of broadly reactive peptide epitopes (12 and 14 peptides, respectively), which were uniformly distributed throughout their sequence. This study defines for the first time broadly reactive Th epitope peptides of SIV Env and Gag proteins that are associated with enduring broadly protective vaccine immunity to attenuated SIV, which may be used for the design and evaluation of experimental vaccines. Moreover, the data suggest that extensive glycosylation of SU may provide yet another immune escape mechanism developed by lentiviruses to restrict the breadth of Th repertoire to SU, a major immunologically exposed protein of the virus.