Long-term control of simian immunodeficiency virus mac251 viremia to undetectable levels in half of infected female rhesus macaques nasally vaccinated with simian immunodeficiency virus DNA/recombinant modified vaccinia virus Ankara

The efficacy of two SIV DNA plus recombinant modified vaccinia virus Ankara nasal vaccine regimens, one combined with plasmids expressing IL-2 and IL-15, the other with plasmids expressing GM-CSF, IL-12, and TNF-α, which may better stimulate humoral responses, was evaluated in two female rhesus macaque groups. Vaccination stimulated significant SIV-specific mucosal and systemic cell-mediated immunity in both groups, whereas SIV-specific IgA titers were sporadic and IgG titers negative. All vaccinated animals, except one, became infected after intravaginal SIV(mac251) low-dose challenge. Half of the vaccinated, infected animals (7/13) promptly controlled virus replication to undetectable viremia for the duration of the trial (130 wk) and displayed virological and immunological phenotypes similar to those of exposed, uninfected individuals. When all vaccinated animals were considered, a 3-log viremia reduction was observed, compared with controls. The excellent viral replication containment achieved in vaccinated animals translated into significant preservation of circulating α4β7(high+)/CD4(+) T cells and of circulating and mucosal CD4(+)/C(M) T cells and in reduced immune activation. A more significant long-term survival was also observed in these animals. Median survival was 72 wk for the control group, whereas >50% of the vaccinated animals were still disease free 130 wk postchallenge, when the trial was closed. There was a statistically significant correlation between levels of CD4(+)/IFN-γ(+) and CD8(+)/IFN-γ(+) T cell percentages on the day of challenge and the control of viremia at week 60 postchallenge or survival. Postchallenge immunological correlates of protection were systemic anti-SIV Gag + Env CD4(+)/IL-2(+), CD4(+)/IFN-γ(+), and CD8(+)/TNF-α(+) T cells and vaginal anti-SIV Gag + Env CD8(+) T cell total monofunctional responses.     

Control of viremia and prevention of simian-human immunodeficiency virus-induced disease in rhesus macaques immunized with recombinant vaccinia viruses plus inactivated simian immunodeficiency virus and human immunodeficiency virus type 1 particles

An effective vaccine against the human immunodeficiency virus type 1 (HIV-1) will very likely have to elicit both cellular and humoral immune responses to control HIV-1 strains of diverse geographic and genetic origins. We have utilized a pathogenic chimeric simian-human immunodeficiency virus (SHIV) rhesus macaque animal model system to evaluate the protective efficacy of a vaccine regimen that uses recombinant vaccinia viruses expressing simian immunodeficiency virus (SIV) and HIV-1 structural proteins in combination with intact inactivated SIV and HIV-1 particles. Following virus challenge, control animals experienced a rapid and complete loss of CD4(+) T cells, sustained high viral loads, and developed clinical disease by 17 to 21 weeks. Although all of the vaccinated monkeys became infected, they displayed reduced postpeak viremia, had no significant loss of CD4(+) T cells, and have remained healthy for more than 15 months postinfection. CD8(+) T-cell and neutralizing antibody responses in vaccinated animals following challenge were demonstrable. Despite the control of disease, virus was readily isolated from the circulating peripheral blood mononuclear cells of all vaccinees at 22 weeks postchallenge, indicating that immunologic control was incomplete. Virus recovered from the animal with the lowest postchallenge viremia generated high virus loads and an irreversible loss of CD4(+) T-cell loss following its inoculation into a na?ve animal. These results indicate that despite the protection from SHIV-induced disease, the vaccinated animals still harbored replication-competent and pathogenic virus.     

Reduction of simian-human immunodeficiency virus 89.6P viremia in rhesus monkeys by recombinant modified vaccinia virus Ankara vaccination

Since cytotoxic T lymphocytes (CTLs) are critical for controlling human immunodeficiency virus type 1 (HIV-1) replication in infected individuals, candidate HIV-1 vaccines should elicit virus-specific CTL responses. In this report, we study the immune responses elicited in rhesus monkeys by a recombinant poxvirus vaccine and the degree of protection afforded against a pathogenic simian-human immunodeficiency virus SHIV-89.6P challenge. Immunization with recombinant modified vaccinia virus Ankara (MVA) vectors expressing SIVmac239 gag-pol and HIV-1 89.6 env elicited potent Gag-specific CTL responses but no detectable SHIV-specific neutralizing antibody (NAb) responses. Following intravenous SHIV-89.6P challenge, sham-vaccinated monkeys developed low-frequency CTL responses, low-titer NAb responses, rapid loss of CD4+ T lymphocytes, high-setpoint viral RNA levels, and significant clinical disease progression and death in half of the animals by day 168 postchallenge. In contrast, the recombinant MVA-vaccinated monkeys demonstrated high-frequency secondary CTL responses, high-titer secondary SHIV-89.6-specific NAb responses, rapid emergence of SHIV-89.6P-specific NAb responses, partial preservation of CD4+ T lymphocytes, reduced setpoint viral RNA levels, and no evidence of clinical disease or mortality by day 168 postchallenge. There was a statistically significant correlation between levels of vaccine-elicited CTL responses prior to challenge and the control of viremia following challenge. These results demonstrate that immune responses elicited by live recombinant vectors, although unable to provide sterilizing immunity, can control viremia and prevent disease progression following a highly pathogenic AIDS virus challenge.     

Effect of CD8+ lymphocyte depletion on virus containment after simian immunodeficiency virus SIVmac251 challenge of live attenuated SIVmac239delta3-vaccinated rhesus macaques

Although live attenuated vaccines can provide potent protection against simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus challenges, the specific immune responses that confer this protection have not been determined. To test whether cellular immune responses mediated by CD8+ lymphocytes contribute to this vaccine-induced protection, we depleted rhesus macaques vaccinated with the live attenuated virus SIVmac239Delta3 of CD8+ lymphocytes and then challenged them with SIVmac251 by the intravenous route. While vaccination did not prevent infection with the pathogenic challenge virus, the postchallenge levels of virus in the plasmas of vaccinated control animals were significantly lower than those for unvaccinated animals. The depletion of CD8+ lymphocytes at the time of challenge resulted in virus levels in the plasma that were intermediate between those of the vaccinated and unvaccinated controls, suggesting that CD8+ cell-mediated immune responses contributed to protection. Interestingly, at the time of challenge, animals expressing the Mamu-A*01 major histocompatibility complex class I allele showed significantly higher frequencies of SIV-specific CD8+ T-cell responses and lower neutralizing antibody titers than those in Mamu-A*01- animals. Consistent with these findings, the depletion of CD8+ lymphocytes abrogated vaccine-induced protection, as judged by the peak postchallenge viremia, to a greater extent in Mamu-A*01+ than in Mamu-A*01- animals. The partial control of postchallenge viremia after CD8+ lymphocyte depletion suggests that both humoral and cellular immune responses induced by live attenuated SIV vaccines can contribute to protection against a pathogenic challenge and that the relative contribution of each of these responses to protection may be genetically determined.     

Different patterns of immune responses but similar control of a simian-human immunodeficiency virus 89.6P mucosal challenge by modified vaccinia virus Ankara (MVA) and DNA/MVA vaccines

Recently we demonstrated the control of a mucosal challenge with a pathogenic chimera of simian and human immunodeficiency virus (SHIV-89.6P) by priming with a Gag-Pol-Env-expressing DNA and boosting with a Gag-Pol-Env-expressing recombinant modified vaccinia virus Ankara (DNA/MVA) vaccine. Here we evaluate the ability of the MVA component of this vaccine to serve as both a prime and a boost for an AIDS vaccine. The same immunization schedule, MVA dose, and challenge conditions were used as in the prior DNA/MVA vaccine trial. Compared to the DNA/MVA vaccine, the MVA-only vaccine raised less than 1/10 the number of vaccine-specific T cells but 10-fold-higher titers of binding antibody for Env. Postchallenge, the animals vaccinated with MVA alone increased their CD8 cell numbers to levels that were similar to those seen in DNA/MVA-vaccinated animals. However, they underwent a slower emergence and contraction of antiviral CD8 T cells and were slower to generate neutralizing antibodies than the DNA/MVA-vaccinated animals. Despite this, by 5 weeks postchallenge, the MVA-only-vaccinated animals had achieved as good control of the viral infection as the DNA/MVA group, a situation that has held up to the present time in the trial (48 weeks postchallenge). Thus, MVA vaccines, as well as DNA/MVA vaccines, merit further evaluation for their ability to control the current AIDS pandemic.     

Simian immunodeficiency virus (SIV) gag DNA-vaccinated rhesus monkeys develop secondary cytotoxic T-lymphocyte responses and control viral replication after pathogenic SIV infection

The potential contribution of a plasmid DNA construct to vaccine-elicited protective immunity was explored in the simian immunodeficiency virus (SIV)/macaque model of AIDS. Making use of soluble major histocompatibility class I/peptide tetramers and peptide-specific killing assays to monitor CD8(+) T-lymphocyte responses to a dominant SIV Gag epitope in genetically selected rhesus monkeys, a codon-optimized SIV gag DNA vaccine construct was shown to elicit a high-frequency SIV-specific cytotoxic T-lymphocyte (CTL) response. This CTL response was demonstrable in both peripheral blood and lymph node lymphocytes. Following an intravenous challenge with the highly pathogenic viral isolate SIVsm E660, these vaccinated monkeys developed a secondary CTL response that arose with more rapid kinetics and reached a higher frequency than did the postchallenge CTL response in control plasmid-vaccinated monkeys. While peak plasma SIV RNA levels were comparable in the experimentally and control-vaccinated monkeys during the period of primary infection, the gag plasmid DNA-vaccinated monkeys demonstrated better containment of viral replication by 50 days following SIV challenge. These findings indicate that a plasmid DNA vaccine can elicit SIV-specific CTL responses in rhesus monkeys, and this vaccine-elicited immunity can facilitate the generation of secondary CTL responses and control of viral replication following a pathogenic SIV challenge. These observations suggest that plasmid DNA may prove a useful component of a human immunodeficiency virus type 1 vaccine.     

Effective Induction of Simian Immunodeficiency Virus-Specific Cytotoxic T Lymphocytes in Macaques by Using a Multiepitope Gene and DNA Prime-Modified Vaccinia Virus Ankara Boost Vaccination Regimen

DNA and modified vaccinia virus Ankara (MVA) are vaccine vehicles suitable and safe for use in humans. Here, by using a multicytotoxic T-lymphocyte (CTL) epitope gene and a DNA prime-MVA boost vaccination regimen, high levels of CTLs specific for a single simian immunodeficiency virus (SIV) gag-derived epitope were elicited in rhesus macaques. These vaccine-induced CTLs were capable of killing SIV-infected cells in vitro. Fluorescence-activated cell sorter analysis using soluble tetrameric major histocompatibility complex-peptide complexes showed that the vaccinated animals had 1 to 5% circulating CD8(+) lymphocytes specific for the vaccine epitope, frequencies comparable to those in SIV-infected monkeys. Upon intrarectal challenge with pathogenic SIVmac251, no evidence for protection was observed in at least two of the three vaccinated animals. This study does not attempt to define correlates of protective immunity nor design a protective vaccine against immunodeficiency viruses, but it demonstrates clearly that the DNA prime-MVA boost regimen is an effective protocol for induction of CTLs in macaques. It also shows that powerful tools for studying the role of CTLs in the control of SIV and human immunodeficiency virus infections are now available: epitope-based vaccines, a protocol for an effective induction of CTLs in primates, and a simple and sensitive method for quantitation of epitope-specific T cells. The advantages of the DNA prime-MVA boost regimen as well as the correlations of tetramer staining of peripheral blood lymphocytes with CTL killing in vitro and postchallenge control of viremia are discussed.     

Enhancement of CD8+ T cell immunity in the lung by CpG oligodeoxynucleotides increases protective efficacy of a modified vaccinia Ankara vaccine against lethal poxvirus infection even in a CD4-deficient host

Immunostimulatory CpG oligodeoxynucleotides (ODN) have proven effective as adjuvants for protein-based vaccines, but their impact on immune responses induced by live viral vectors is not known. We found that addition of CpG ODN to modified vaccinia Ankara (MVA) markedly improved the induction of longer-lasting adaptive protective immunity in BALB/c mice against intranasal pathogenic vaccinia virus (Western Reserve; WR). Protection was mediated primarily by CD8(+) T cells in the lung, as determined by CD8-depletion studies, protection in B cell-deficient mice, and greater protection correlating with CD8(+) IFN-gamma-producing cells in the lung but not with those in the spleen. Intranasal immunization was more effective at inducing CD8(+) T cell immunity in the lung, and protection, than i.m. immunization. Addition of CpG ODN increased the CD8(+) response but not the Ab response. Depletion of CD4 T cells before vaccination with MVA significantly diminished protection against pathogenic WR virus. However, CpG ODN delivered with MVA was able to substitute for CD4 help and protected CD4-depleted mice against WR vaccinia challenge. This study demonstrates for the first time a protective adjuvant effect of CpG ODN for a live viral vector vaccine that may overcome CD4 deficiency in the induction of protective CD8(+) T cell-mediated immunity.     

Comparative efficacy of recombinant modified vaccinia virus Ankara expressing simian immunodeficiency virus (SIV) Gag-Pol and/or Env in macaques challenged with pathogenic SIV

Prior studies demonstrated that immunization of macaques with simian immunodeficiency virus (SIV) Gag-Pol and Env recombinants of the attenuated poxvirus modified vaccinia virus Ankara (MVA) provided protection from high levels of viremia and AIDS following challenge with a pathogenic strain of SIV (V. M. Hirsch et al., J. Virol. 70:3741-3752, 1996). This MVA-SIV recombinant expressed relatively low levels of the Gag-Pol portion of the vaccine. To optimize protection, second-generation recombinant MVAs that expressed high levels of either Gag-Pol (MVA-gag-pol) or Env (MVA-env), alone or in combination (MVA-gag-pol-env), were generated. A cohort of 24 macaques was immunized with recombinant or nonrecombinant MVA (four groups of six animals) and was challenged with 50 times the dose at which 50% of macaques are infected with uncloned pathogenic SIVsmE660. Although all animals became infected postchallenge, plasma viremia was significantly reduced in animals that received the MVA-SIV recombinant vaccines as compared with animals that received nonrecombinant MVA (P = 0.0011 by repeated-measures analysis of variance). The differences in the degree of virus suppression achieved by the three MVA-SIV vaccines were not significant. Most importantly, the reduction in levels of viremia resulted in a significant increase in median (P < 0.05 by Student's t test) and cumulative (P = 0.010 by log rank test) survival. These results suggest that recombinant MVA has considerable potential as a vaccine vector for human AIDS.     

A consecutive priming-boosting vaccination of mice with simian immunodeficiency virus (SIV) gag/pol DNA and recombinant vaccinia virus strain DIs elicits effective anti-SIV immunity

To evaluate immunity induced by a novel DNA prime-boost regimen, we constructed a DNA plasmid encoding the gag and pol genes from simian immunodeficiency virus (SIV) (SIVgag/pol DNA), in addition to a replication-deficient vaccinia virus strain DIs recombinant expressing SIV gag and pol genes (rDIsSIVgag/pol). In mice, priming with SIVgag/pol DNA, followed by rDIsSIVgag/pol induced an SIV-specific lymphoproliferative response that was mediated by a CD4+-T-lymphocyte subset. Immunization with either vaccine alone was insufficient to induce high levels of proliferation or Th1 responses in the animals. The prime-boost regimen also induced SIV Gag-specific cellular responses based on gamma interferon secretion, as well as cytotoxic-T-lymphocyte responses. Thus, the regimen of DNA priming and recombinant DIs boosting induced Th1-type cell-mediated immunity, which was associated with resistance to viral challenge with wild-type vaccinia virus expressing SIVgag/pol, suggesting that this new regimen may hold promise as a safe and effective vaccine against human immunodeficiency virus type 1.     

Elevated expression levels of inhibitory receptor programmed death 1 on simian immunodeficiency virus-specific CD8 T cells during chronic infection but not after vaccination

Here, we study the temporal expression of the inhibitory receptor programmed death 1 (PD-1) on simian immunodeficiency virus (SIV) Gag-specific T cells following pathogenic SIV infection or following vaccination with a DNA/modified vaccinia virus Ankara (DNA/MVA) vaccine and simian/human immunodeficiency virus (SHIV) challenge in macaques. Following infection, the majority (>95%) of Gag-specific CD8 T cells expressed PD-1, and the level of PD-1 expression per cell increased over time. The level of PD-1 expression in lymph nodes and rectal mucosal tissue, the major sites of virus replication, was higher compared to blood. In vitro blockade of PD-1 resulted in enhanced proliferation of SIV-specific CD8 as well as CD4 T cells. In contrast, following vaccination, the majority of peak effector Gag-specific CD8 T cells expressed low levels of PD-1, and these levels decreased further as the cells differentiated into memory cells. In addition, following SHIV challenge of these vaccinated macaques, the level of PD-1 expression on Gag-specific CD8 T cells correlated positively with plasma viremia. These results demonstrate that SIV-specific CD8 T cells express PD-1 after exposure to antigen but downregulate expression under conditions of antigen clearance and enhance expression under conditions of antigen persistence. They also demonstrate that the level of PD-1 expression per cell rather than the presence or absence of expression plays an important role in regulating CD8 T-cell dysfunction in pathogenic SIV infection. In addition, they demonstrate that similar to HIV infection, the PD-1:PD-1 ligand inhibitory pathway is operational in pathogenic SIV infection, and the macaque/SIV model would be ideal to test the safety and therapeutic benefit of blocking this pathway in vivo.     

Live attenuated lentivirus infection elicits polyfunctional simian immunodeficiency virus Gag-specific CD8+ T cells with reduced apoptotic susceptibility in rhesus macaques that control virus replication after challenge with pathogenic SIVmac239

HIV-specific CD8+ T cells that secrete multiple cytokines in response to Ag stimulation are associated with the control of virus replication during chronic HIV infection. To determine whether the presence of polyfunctional CD8+ T cell responses distinguishes protected and unprotected monkeys in a live attenuated lentivirus model, SIV Gag peptide-specific CD8+ T cell responses of simian HIV (SHIV) 89.6-vaccinated, SIVmac239-challenged rhesus macaques were compared in two monkeys that controlled challenge virus replication and two that did not. The ratio of Bcl-2+ Gag-specific CD8+ T cells to caspase-3+ Gag-specific CD8+ T cells was higher in the vaccinated-protected animals compared with unprotected monkeys. In addition, polyfunctional SIV-specific CD8+ T cells were consistently detected through 12 wk postchallenge in the protected animals but not in the unprotected animals. In the unprotected monkeys, there was an increased frequency of CD8+ T cells expressing markers associated with effector memory T cells. Further, there was increased annexin V expression in central memory T cells of the unprotected animals before challenge. Thus, monkeys that control viral replication after live attenuated SHIV infection have polyfunctional SIV-specific CD8+ T cells with an increased survival potential. Importantly, the differences in the nature of the SIV-specific CD8+ T cell response in the protected and unprotected animals are present during acute stages postchallenge, before different antigenic levels are established. Thus, the polyfunctional capacity and increased survival potential of CD8+ SIV-specific T cells may account for live attenuated, SHIV89.6-mediated protection from uncontrolled SIV replication.     

Immunization of rhesus macaques with a DNA prime/modified vaccinia virus Ankara boost regimen induces broad simian immunodeficiency virus (SIV)-specific T-cell responses and reduces initial viral replication but does not prevent disease progression following challenge with pathogenic SIVmac239

Producing a prophylactic vaccine for human immunodeficiency virus (HIV) has proven to be a challenge. Most biological isolates of HIV are difficult to neutralize, so that conventional subunit-based antibody-inducing vaccines are unlikely to be very effective. In the rhesus macaque model, some protection was afforded by DNA/recombinant viral vector vaccines. However, these studies used as the challenge virus SHIV-89.6P, which is neutralizable, making it difficult to determine whether the observed protection was due to cellular immunity, humoral immunity, or a combination of both. In this study, we used a DNA prime/modified vaccinia virus Ankara boost regimen to immunize rhesus macaques against nearly all simian immunodeficiency virus (SIV) proteins. These animals were challenged intrarectally with pathogenic molecularly cloned SIVmac239, which is resistant to neutralization. The immunization regimen resulted in the induction of virus-specific CD8(+) and CD4(+) responses in all vaccinees. Although anamnestic neutralizing antibody responses against laboratory-adapted SIVmac251 developed after the challenge, no neutralizing antibodies against SIVmac239 were detectable. Vaccinated animals had significantly reduced peak viremia compared with controls (P < 0.01). However, despite the induction of virus-specific cellular immune responses and reduced peak viral loads, most animals still suffered from gradual CD4 depletion and progressed to disease.     

Comparative efficacy of subtype AE simian-human immunodeficiency virus priming and boosting vaccines in pigtail macaques

Vaccination against AIDS is hampered by great diversity between human immunodeficiency virus (HIV) strains. Heterologous B-subtype-based simian-human immunodeficiency virus (SHIV) DNA prime and poxvirus boost vaccine regimens can induce partial, T-cell-mediated, protective immunity in macaques. We analyzed a set of DNA, recombinant fowlpox viruses (FPV), and vaccinia viruses (VV) expressing subtype AE HIV type 1 (HIV-1) Tat, Rev, and Env proteins and SIV Gag/Pol in 30 pigtail macaques. SIV Gag-specific CD4 and CD8 T-cell responses were induced by sequential DNA/FPV vaccination, although lower FPV doses, VV/FPV vaccination, and DNA vaccines alone were not as consistently immunogenic. The SHIV AE DNA prime, FPV boost regimens were significantly less immunogenic than comparable B-subtype SHIV vaccination. Peak viral load was modestly (0.4 log10 copies/ml) lower among the AE subtype SHIV-immunized animals compared to controls following the virulent B subtype SHIV challenge. Protection from persistent high levels of viremia and CD4 T-cell depletion was less in AE subtype compared to B subtype SHIV-vaccinated macaques. Gag was highly immunodominant over the other AE subtype SHIV vaccine proteins after vaccination, and this immunodominance was exacerbated after challenge. Interestingly, the lower level of priming of immune responses did not blunt postchallenge Gag-specific recall responses, despite more modest protection. These studies suggest priming of T-cell immunity to prevent AIDS in humans is possible, but differences in the immunogenicity of various subtype vaccines and broad cross-subtype protection are substantial hurdles.     

Antibodies to gp120 and PD-1 Expression on Virus-Specific CD8+ T Cells in Protection from Simian AIDS

We compared the relative efficacies against simian immunodeficiency virus (SIV) challenge of three vaccine regimens that elicited similar frequencies of SIV-specific CD4⁺ and CD8⁺ T-cell responses but differed in the level of antibody responses to the gp120 envelope protein. All macaques were primed with DNA plasmids expressing SIV gag, pol, env, and Retanef genes and were boosted with recombinant modified vaccinia Ankara virus (MVA) expressing the same genes, either once (1 × MVA) or twice (2 × MVA), or were boosted once with MVA followed by a single boost with replication-competent adenovirus (Ad) type 5 host range mutant (Ad5 h) expressing SIV gag and nef genes but not Retanef or env (1 × MVA/Ad5). While two of the vaccine regimens (1 × MVA and 1 × MVA/Ad5) protected from high levels of SIV replication only during the acute phase of infection, the 2 × MVA regimen, with the highest anti-SIV gp120 titers, protected during the acute phase and transiently during the chronic phase of infection. Mamu-A*01 macaques of this third group exhibited persistent Gag CD8⁺CM9⁺ effector memory T cells with low expression of surface Programmed death-1 (PD-1) receptor and high levels of expression of genes associated with major histocompatibility complex class I (MHC-I) and MHC-II antigen. The fact that control of SIV replication was associated with both high titers of antibodies to the SIV envelope protein and durable effector SIV-specific CD8⁺ T cells suggests the hypothesis that the presence of antibodies at the time of challenge may increase innate immune recruiting activity by enhancing antigen uptake and may result in improvement of the quality and potency of secondary SIV-specific CD8⁺ T-cell responses.     

With minimal systemic T-cell expansion, CD8+ T Cells mediate protection of rhesus macaques immunized with attenuated simian-human immunodeficiency virus SHIV89.6 from vaginal challenge with simian immunodeficiency virus

The presence, at the time of challenge, of antiviral effector T cells in the vaginal mucosa of female rhesus macaques immunized with live-attenuated simian-human immunodeficiency virus 89.6 (SHIV89.6) is associated with consistent and reproducible protection from pathogenic simian immunodeficiency virus (SIV) vaginal challenge (18). Here, we definitively demonstrate the protective role of the SIV-specific CD8(+) T-cell response in SHIV-immunized monkeys by CD8(+) lymphocyte depletion, an intervention that abrogated SHIV-mediated control of challenge virus replication and largely eliminated the SIV-specific T-cell responses in blood, lymph nodes, and genital mucosa. While in the T-cell-intact SHIV-immunized animals, polyfunctional and degranulating SIV-specific CD8(+) T cells were present in the genital tract and lymphoid tissues from the day of challenge until day 14 postchallenge, strikingly, expansion of SIV-specific CD8(+) T cells in the immunized monkeys was minimal and limited to the vagina. Thus, protection from uncontrolled SIV replication in animals immunized with attenuated SHIV89.6 is primarily mediated by CD8(+) T cells that do not undergo dramatic systemic expansion after SIV challenge. These findings demonstrate that despite, and perhaps because of, minimal systemic expansion of T cells at the time of challenge, a stable population of effector-cytotoxic CD8(+) T cells can provide significant protection from vaginal SIV challenge.     

Incomplete protection, but suppression of virus burden, elicited by subunit simian immunodeficiency virus vaccines.

We compared the efficacy of immunization with either simian immunodeficiency virus (SIV) Env glycoprotein (Env), Env plus Gag proteins (Gag-Env), or whole inactivated virus (WIV), with or without recombinant live vaccinia vector (VV) priming, in protecting 23 rhesus macaques (six vaccine and two control groups) from challenge with SIVmac251 clone BK28. Vaccination elicited high titers of syncytium-inhibiting and anti-Env (gp120/gp160) antibodies in all vaccinated macaques and anti-Gag (p27) antibodies in groups immunized with WIV or Gag-Env. Only WIV-immunized macaques developed anticell (HuT78) antibodies. After homologous low-dose intravenous virus challenge, we used frequency of virus isolation, provirus burden, and change in antibody titers to define four levels of resistance to SIV infection as follows. (i) No infection ("sterilizing" immunity) was induced only in WIV-immunized animals. (ii) Abortive infection (strong immunity) was defined when virus or provirus were detected early in the postchallenge period but not thereafter and no evidence of virus or provirus was detected in terminal tissues. This response was observed in two animals (one VV-Env and one Gag-Env). (iii) Suppression of infection (incomplete or partial immunity) described a gradient of virus suppression manifested by termination of viremia, declining postchallenge antibody titers, and low levels (composite mean = 9.1 copies per 10(6) cells) of provirus detectable in peripheral blood mononuclear cells or lymphoid tissues at termination (40 weeks postchallenge). This response occurred in the majority (8 of 12) of subunit-vaccinated animals. (iv) Active infection (no immunity) was characterized by persistent virus isolation from blood mononuclear cells, increasing viral antibody titers postchallenge, and high levels (composite mean = 198 copies per 10(6) cells) of provirus in terminal tissues and blood. Active infection developed in all controls and two of three VV-Gag-Env-immunized animals. The results of this study restate the protective effect of inactivated whole virus vaccines produced in heterologous cells but more importantly demonstrate that a gradient of suppression of challenge virus growth, reflecting partial resistance to SIV infection, is induced by subunit vaccination. The latter finding may be pertinent to studies with human immunodeficiency virus vaccines, in which it is plausible that vaccination may elicit significant suppression of virus infection and pathogenicity rather than sterilizing immunity.     

Early induction of polyfunctional simian immunodeficiency virus (SIV)-specific T lymphocytes and rapid disappearance of SIV from lymph nodes of sooty mangabeys during primary infection

Although the cellular immune response is essential for controlling SIV replication in Asian macaques, its role in maintaining nonpathogenic SIV infection in natural hosts such as sooty mangabeys (SM) remains to be defined. We have previously shown that similar to rhesus macaques (RM), SM are able to mount a T lymphocyte response against SIV infection. To investigate early control of SIV replication in natural hosts, we performed a detailed characterization of SIV-specific cellular immunity and viral control in the first 6 mo following SIV infection in SM. Detection of the initial SIV-specific IFN-γ ELISPOT response in SIVsmE041-infected SM coincided temporally with a decline in peak plasma viremia and was similar in magnitude, specificity, and breadth to SIVsmE041-infected and SIVmac239-infected RM. Despite these similarities, SM showed a greater reduction in postpeak plasma viremia and a more rapid disappearance of productively SIV-infected cells from the lymph node compared with SIVmac239-infected RM. The early Gag-specific CD8(+) T lymphocyte response was significantly more polyfunctional in SM compared with RM, and granzyme B-positive CD8(+) T lymphocytes were present at significantly higher frequencies in SM even prior to SIV infection. These findings suggest that the early SIV-specific T cell response may be an important determinant of lymphoid tissue viral clearance and absence of lymph node immunopathology in natural hosts of SIV infection.     

Role of CD8(+) lymphocytes in control of simian immunodeficiency virus infection and resistance to rechallenge after transient early antiretroviral treatment

Transient antiretroviral treatment with tenofovir, (R)-9-(2-phosphonylmethoxypropyl)adenine, begun shortly after inoculation of rhesus macaques with the highly pathogenic simian immunodeficiency virus (SIV) isolate SIVsmE660, facilitated the development of SIV-specific lymphoproliferative responses and sustained effective control of the infection following drug discontinuation. Animals that controlled plasma viremia following transient postinoculation treatment showed substantial resistance to subsequent intravenous rechallenge with homologous (SIVsmE660) and highly heterologous (SIVmac239) SIV isolates, up to more than 1 year later, despite the absence of measurable neutralizing antibody. In some instances, resistance to rechallenge was observed despite the absence of detectable SIV-specific binding antibody and in the face of SIV lymphoproliferative responses that were low or undetectable at the time of challenge. In vivo monoclonal antibody depletion experiments demonstrated a critical role for CD8(+) lymphocytes in the control of viral replication; plasma viremia rose by as much as five log units after depletion of CD8(+) cells and returned to predepletion levels (as low as <100 copy Eq/ml) as circulating CD8(+) cells were restored. The extent of host control of replication of highly pathogenic SIV strains and the level of resistance to heterologous rechallenge achieved following transient postinoculation treatment compared favorably to the results seen after SIVsmE660 and SIVmac239 challenge with many vaccine strategies. This impressive control of viral replication was observed despite comparatively modest measured immune responses, less than those often achieved with vaccination regimens. The results help establish the underlying feasibility of efforts to develop vaccines for the prevention of AIDS, although the exact nature of the protective host responses involved remains to be elucidated.     

Control of simian/human immunodeficiency virus viremia and disease progression after IL-2-augmented DNA-modified vaccinia virus Ankara nasal vaccination in nonhuman primates

A successful HIV vaccine may need to stimulate antiviral immunity in mucosal and systemic immune compartments, because HIV transmission occurs predominantly at mucosal sites. We report here the results of a combined DNA-modified vaccinia virus Ankara (MVA) vaccine approach that stimulated simian/human immunodeficiency virus (SHIV)-specific immune responses by vaccination at the nasal mucosa. Fifteen male rhesus macaques, divided into three groups, received three nasal vaccinations on day 1, wk 9, and wk 25 with a SHIV DNA plasmid producing noninfectious viral particles (group 1), or SHIV DNA plus IL-2/Ig DNA (group 2), or SHIV DNA plus IL-12 DNA (group 3). On wk 33, all macaques were boosted with rMVA expressing SIV Gag-Pol and HIV Env 89.6P, administered nasally. Humoral responses were evaluated by measuring SHIV-specific IgG and neutralizing Abs in plasma, and SHIV-specific IgA in rectal secretions. Cellular responses were monitored by evaluating blood-derived virus-specific IFN-gamma-secreting cells and TNF-alpha-expressing CD8+ T cells, and blood- and rectally derived p11C tetramer-positive T cells. Many of the vaccinated animals developed both mucosal and systemic humoral and cell-mediated anti-SHIV immune responses, although the responses were not homogenous among animals in the different groups. After rectal challenge of vaccinated and naive animals with SHIV89.6P, all animals became infected. However a subset, including all group 2 animals, were protected from CD4+ T cell loss and AIDS development. Taken together, these data indicate that nasal vaccination with SHIV-DNA plus IL-2/Ig DNA and rMVA can provide significant protection from disease progression.