Pentameric complex of viral glycoprotein H is the primary target for potent neutralization by a human cytomegalovirus vaccine.

Human cytomegalovirus (HCMV) can cause serious morbidity/mortality in transplant patients, and congenital HCMV infection can lead to birth defects. Developing an effective HCMV vaccine is a high medical priority. One of the challenges to the efforts has been our limited understanding of the viral antigens important for protective antibodies. Receptor-mediated viral entry to endothelial/epithelial cells requires a glycoprotein H (gH) complex comprising five viral proteins (gH, gL, UL128, UL130, and UL131). This gH complex is notably missing from HCMV laboratory strains as well as HCMV vaccines previously evaluated in the clinic. To support a unique vaccine concept based on the pentameric gH complex, we established a panel of 45 monoclonal antibodies (mAbs) from a rabbit immunized with an experimental vaccine virus in which the expression of the pentameric gH complex was restored. Over one-half (25 of 45) of the mAbs have neutralizing activity. Interestingly, affinity for an antibody to bind virions was not correlated with its ability to neutralize the virus. Genetic analysis of the 45 mAbs based on their heavy- and light-chain sequences identified at least 26 B-cell linage groups characterized by distinct binding or neutralizing properties. Moreover, neutralizing antibodies possessed longer complementarity-determining region 3 for both heavy and light chains than those with no neutralizing activity. Importantly, potent neutralizing mAbs reacted to the pentameric gH complex but not to gB. Thus, the pentameric gH complex is the primary target for antiviral antibodies by vaccination.

Restoration of viral epithelial tropism improves immunogenicity in rabbits and rhesus macaques for a whole virion vaccine of human cytomegalovirus.

Maternal immunity to human cytomegalovirus (HCMV) prior to conception is ~70% protective against congenital transmission and in utero infection of HCMV. Both functional antibodies capable of neutralizing virus and effective T-cells are believed to be important for the protection. Previous HCMV vaccines have rarely been shown able to induce neutralizing antibody titers comparable to those seen in naturally infected HCMV seropositive subjects. Recent studies link a glycoprotein H (gH) complex to receptor-mediated viral entry of endothelial/epithelial cells and leukocytes. This pentameric gH complex, composed of five proteins (gH, gL, UL128, UL130 and UL131 proteins), is notably missing in all HCMV vaccine previously evaluated in clinic. Here we showed that a HCMV virus, with restored expression of the pentameric gH complex, can induce 10-fold higher neutralizing antibody titers than an attenuated AD169 virus or a recombinant glycoprotein B vaccine in multiple animal species in which viral replication is not expected. Encouragingly, the peak neutralizing titers post vaccination in rabbits and monkeys were within 2-4-fold of the levels determined in HCMV seropositive subjects. Functional antibodies by vaccination could further be improved when formulated with a novel adjuvant, and the titers of the antiviral antibodies were sustained in rabbits for over a year after vaccination. These results indicate that the pentameric gH complex is associated with greatly improved functional antibodies following vaccination, and support a vaccine concept based on a nonreplicating whole HCMV with the pentameric gH-associated epithelial tropism restored.

A trivalent recombinant Ad5 gag/pol/nef vaccine fails to protect rhesus macaques from infection or control virus replication after a limiting-dose heterologous SIV challenge.

It has been suggested that poor immunogenicity may explain the lack of vaccine efficacy in preventing or controlling HIV infection in the Step trial. To investigate this issue we vaccinated eight Indian rhesus macaques with a trivalent replication-incompetent adenovirus serotype 5 vaccine expressing SIV Gag, Pol, and Nef using a regimen similar to that employed in the Step trial. We detected broad vaccine-induced CD8(+) (2-7 pool-specific responses) and CD4(+) (5-19 pool-specific responses) T-cell responses in IFN-γ ELISPOT assays at one week post-boost using fresh PBMC. However, using cryopreserved cells at one and four weeks post-boost we observed a reduction in both the number and magnitude of most vaccine-induced responses. This demonstrates that the time points and conditions chosen to perform immune assays may influence the observed breadth and frequency of vaccine-induced T-cell responses. To evaluate protective efficacy, we challenged the immunized macaques, along with naïve controls, with repeated, limiting doses of the heterologous swarm isolate SIVsmE660. Vaccination did not significantly affect acquisition or control of virus replication in vaccinees compared to naïve controls. Post-infection we observed an average of only two anamnestic CD8(+) T-cell responses per animal, which may not have been sufficiently broad to control heterologous virus replication. While the trivalent vaccine regimen induced relatively broad T-cell responses in rhesus macaques, it failed to protect against infection or control viral replication. Our results are consistent with those observed in the Step trial and indicate that SIV immunization and challenge studies in macaque models of HIV infection can be informative in assessing pre-clinical HIV vaccines.

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.

Comparison of T cell immune responses induced by vectored HIV vaccines in non-human primates and humans.

Following the disappointing outcome of the phase IIb test-of-concept step study in which Merck's adenovirus type 5 (Ad5) HIV-1 clade B gag/pol/nef vaccine failed to demonstrate efficacy in HIV high-risk individuals, an extensive review of the trial and preclinical studies which supported the trial is ongoing. One point of interest is how well preclinical nonhuman primate immunogenicity studies predicted what was observed in humans. Here we compare the HIV-1-specific cellular immune responses elicited in nonhuman primates and human clinical trial subjects to several HIV-1 vaccine candidates. We find that although rhesus macaques are immunologically more responsive to vaccination than humans, the hierarchy in potency of single-modality prime-boost regimens using several vector approaches (adenovirus, DNA, and pox vectors) was well predicted. Vaccine approaches using complex formulations such as novel adjuvants (DNA+CRL1005) or mixed-modality prime-boost (DNA/Ad5; Ad5/ALVAC) did not correlate as well between rhesus macaques and humans. Although the immunogenicity of the vaccines and vaccine regimens evaluated were not all accurately predicted, testing in rhesus macaques generally offers an indispensable tool for ranking the immunological potential of HIV-1 vaccine candidates.

Comparative analysis of immune responses induced by vaccination with SIV antigens by recombinant Ad5 vector or plasmid DNA in rhesus macaques.

DNA vaccines have undergone important enhancements in their design, formulation, and delivery process. Past literature supports that DNA vaccines are not as immunogenic in nonhuman primates as live vector systems. The most potent recombinant vector system for induction of cellular immune responses in macaques and humans is adenovirus serotype 5 (Ad5), an important benchmark for new vaccine development. Here, we performed a head-to-head evaluation of the Merck Ad5 SIV vaccine and an optimized electroporation (EP) delivered SIV DNA vaccine in macaques. Animals receiving the Ad5 vaccine were immunized three times, whereas the DNA-vaccinated animals were immunized up to four times based on optimized protocols. We observed significant differences in the quantity of IFNgamma responses by enzyme-linked immunosorbent spot (ELISpot), greater proliferative capacity of CD8(+) T cells, and increased polyfunctionality of both CD4(+) and CD8(+) T cells in the DNA-vaccinated group. Importantly, Ad5 immunizations failed to boost following the first immunization, whereas DNA responses were continually boosted with all four immunizations demonstrating a major advantage of these improved DNA vaccines. These optimized DNA vaccines induce very different immune phenotypes than traditional Ad5 vaccines, suggesting that they could play an important role in vaccine research and development.

Vaccination with peptide mimetics of the gp41 prehairpin fusion intermediate yields neutralizing antisera against HIV-1 isolates.

Eliciting a broadly neutralizing polyclonal antibody response against HIV-1 remains a major challenge. One approach to vaccine development is prevention of HIV-1 entry into cells by blocking the fusion of viral and cell membranes. More specifically, our goal is to elicit neutralizing antibodies that target a transient viral entry intermediate (the prehairpin intermediate) formed by the HIV-1 gp41 protein. Because this intermediate is transient, a stable mimetic is required to elicit an immune response. Previously, a series of engineered peptides was used to select a mAb (denoted D5) that binds to the surface of the gp41 prehairpin intermediate, as demonstrated by x-ray crystallographic studies. D5 inhibits the replication of HIV-1 clinical isolates, providing proof-of-principle for this vaccine approach. Here, we describe a series of peptide mimetics of the gp41 prehairpin intermediate designed to permit a systematic analysis of the immune response generated in animals. To improve the chances of detecting weak neutralizing polyclonal responses, two strategies were employed in the initial screening: use of a neutralization-hypersensitive virus and concentration of the IgG fraction from immunized animal sera. This allowed incremental improvements through iterative cycles of design, which led to vaccine candidates capable of generating a polyclonal antibody response, detectable in unfractionated sera, that neutralize tier 1 HIV-1 and simian HIV primary isolates in vitro. Our findings serve as a starting point for the design of more potent immunogens to elicit a broadly neutralizing response against the gp41 prehairpin intermediate.

Efficacy of multivalent adenovirus-based vaccine against simian immunodeficiency virus challenge.

The prophylactic efficacies of several multivalent replication-incompetent adenovirus serotype 5 (Ad5) vaccines were examined in rhesus macaques using an intrarectal high-dose simian immunodeficiency virus SIVmac239 challenge model. Cohorts of Mamu-A*01(+)/B*17(-) Indian rhesus macaques were immunized with one of several combinations of Ad5 vectors expressing Gag, Pol, Nef, and Env gp140; for comparison, a Mamu-A*01(+) cohort was immunized using the Ad5 vector alone. There was no sign of immunological interference between antigens in the immunized animals. In general, expansion of the antigen breadth resulted in more favorable virological outcomes. In particular, the order of efficacy trended as follows: Gag/Pol/Nef/Env approximately Gag/Pol > Gag approximately Gag/Pol/Nef > Nef. However, the precision in ranking the vaccines based on the study results may be limited by the cohort size, and as such, may warrant additional testing. The implications of these results in light of the recent discouraging results of the phase IIb study of the trivalent Ad5 HIV-1 vaccine are discussed.

Comparative cell-mediated immunogenicity of DNA/DNA, DNA/adenovirus type 5 (Ad5), or Ad5/Ad5 HIV-1 clade B gag vaccine prime-boost regimens.

BACKGROUND: We report composite results from the Merck phase I program of near-consensus clade B human immunodeficiency virus (HIV) type 1 gag vaccines. METHODS: Healthy HIV-uninfected adults were enrolled in 6 blinded placebo-controlled studies evaluating the immunogenicity of (1) a 4-dose regimen of a DNA vaccine, (2) a 3-dose priming regimen of the DNA vaccine with a booster dose of an adenovirus type 5 (Ad5)-vectored vaccine, or (3) a 3-dose regimen of the Ad5 vaccine. The DNA plasmid was provided with or without an aluminum phosphate or CRL1005 adjuvant. The primary end point was the unfractionated HIV-1 gag-specific interferon gamma enzyme-linked immunospot (ELISpot) response 4 weeks after the final dose. RESULTS: Overall, 254 (83%) of 307 subjects randomized to the vaccine groups were evaluable. Adjuvants did not enhance immunogenicity of the DNA vaccine. Postboost ELISpot responder frequencies were higher for Ad5-containing regimens than for the DNA/DNA regimen (33%) but were similar for DNA/Ad5 (55%) and Ad5/Ad5 (50%). DNA/DNA elicited mainly a CD4 response, whereas Ad5/Ad5 elicited mainly a CD8 response; DNA/Ad5 generated CD4 and CD8 responses comparable to those of DNA/DNA and Ad5/Ad5, respectively. CONCLUSIONS: The DNA vaccine alone or as a priming regimen for the Ad5 vaccine did not increase unfractionated ELISpot responses compared with the Ad5 vaccine alone. Qualitative T cell responses to different vaccine regimens deserve further study.

International epidemiology of human pre-existing adenovirus (Ad) type-5, type-6, type-26 and type-36 neutralizing antibodies: correlates of high Ad5 titers and implications for potential HIV vaccine trials.

Replication-defective adenoviruses have been utilized as candidate HIV vaccine vectors. Few studies have described the international epidemiology of pre-existing immunity to adenoviruses. We enrolled 1904 participants in a cross-sectional serological survey at seven sites in Africa, Brazil, and Thailand to assess neutralizing antibodies (NA) for adenovirus types Ad5, Ad6, Ad26 and Ad36. Clinical trial samples were used to assess NA titers from the US and Europe. The proportions of participants that were negative were 14.8% (Ad5), 31.5% (Ad6); 41.2% (Ad26) and 53.6% (Ad36). Adenovirus NA titers varied by geographic location and were higher in non-US and non-European settings, especially Thailand. In multivariate logistic regression analysis, geographic setting (non-US and non-European settings) was statistically significantly associated with having higher Ad5 titers; participants from Thailand had the highest odds of having high Ad5 titers (adjusted OR=3.53, 95% CI: 2.24, 5.57). Regardless of location, titers of Ad5NA were the highest and Ad36 NA were the lowest. Coincident Ad5/6 titers were lower than either Ad5 or Ad6 titers alone. Understanding pre-existing immunity to candidate vaccine vectors may contribute to the evaluation of vaccines in international populations.

Safety and immunogenicity of the Merck adenovirus serotype 5 (MRKAd5) and MRKAd6 human immunodeficiency virus type 1 trigene vaccines alone and in combination in healthy adults.

Preexisting immunity to adenovirus serotype 5 (Ad5) diminishes immune responses to vaccines using Ad5 as a vector. Alternate Ad serotypes as vaccine vectors might overcome Ad5-specific neutralizing antibodies and enhance immune responses in populations with a high prevalence of Ad5 immunity. To test this hypothesis, healthy human immunodeficiency virus (HIV)-seronegative adults were enrolled in a blinded, randomized, dose-escalating, placebo-controlled study. In part A, subjects with baseline Ad6 titers of < or = 18 received the Merck Ad6 (MRKAd6) HIV type 1 (HIV-1) trigene vaccine at weeks 0, 4, and 26. In part B, subjects stratified by Ad5 titers (< or = 200 or >200) and Ad6 titers (< or = 18 or >18) received the MRKAd5-plus-MRKAd6 (MRKAd5+6) HIV-1 trigene vaccine at weeks 0, 4, and 26. Immunogenicity was assessed by an enzyme-linked immunospot (ELISPOT) assay at week 30. No serious adverse events occurred. MRKAd6 trigene vaccine recipients responded more often to Nef than to Gag or Pol. In part A, ELISPOT response rates to > or = 2 vaccine antigens were 14%, 63%, and 71% at 10(9), 10(10), and 10(11) viral genomes (vg)/dose, respectively. All responders had positive Nef-specific ELISPOT results. In part B, Nef-ELISPOT response rates at 10(10) vg/dose of the MRKAd5+6 trigene vaccine were 50% in the low-Ad5/low-Ad6 stratum (n = 8), 78% in the low-Ad5/high-Ad6 stratum (n = 9), 75% in the high-Ad5/low-Ad6 stratum (n = 8), and 44% in the high-Ad5/high-Ad6 stratum (n = 9). The MRKAd6 and MRKAd5+6 trigene vaccines elicited dose-dependent responses predominantly to Nef and were generally well tolerated, indicating that Ad6 should be considered a candidate vector for future vaccines. Although small sample sizes limit the conclusions that can be drawn from this exploratory study, combining two Ad vectors may be a useful vaccine strategy for circumventing isolated immunity to a single Ad serotype.

Adenovirus-specific immunity after immunization with an Ad5 HIV-1 vaccine candidate in humans.

The immunologic basis for the potential enhanced HIV-1 acquisition in adenovirus serotype 5 (Ad5)-seropositive individuals who received the Merck recombinant Ad5 HIV-1 vaccine in the STEP study remains unclear. Here we show that baseline Ad5-specific neutralizing antibodies are not correlated with Ad5-specific T lymphocyte responses and that Ad5-seropositive subjects do not develop higher vector-specific cellular immune responses as compared with Ad5-seronegative subjects after vaccination. These findings challenge the hypothesis that activated Ad5-specific T lymphocytes were the cause of the potential enhanced HIV-1 susceptibility in the STEP study.

Vaccine-induced cellular responses control simian immunodeficiency virus replication after heterologous challenge.

All human immunodeficiency virus (HIV) vaccine efficacy trials to date have ended in failure. Structural features of the Env glycoprotein and its enormous variability have frustrated efforts to induce broadly reactive neutralizing antibodies. To explore the extent to which vaccine-induced cellular immune responses, in the absence of neutralizing antibodies, can control replication of a heterologous, mucosal viral challenge, we vaccinated eight macaques with a DNA/Ad5 regimen expressing all of the proteins of SIVmac239 except Env. Vaccinees mounted high-frequency T-cell responses against 11 to 34 epitopes. We challenged the vaccinees and eight naïve animals with the heterologous biological isolate SIVsmE660, using a regimen intended to mimic typical HIV exposures resulting in infection. Viral loads in the vaccinees were significantly less at both the peak (1.9-log reduction; P < 0.03) and at the set point (2.6-log reduction; P < 0.006) than those in control naïve animals. Five of eight vaccinated macaques controlled acute peak viral replication to less than 80,000 viral RNA (vRNA) copy eq/ml and to less than 100 vRNA copy eq/ml in the chronic phase. Our results demonstrate that broad vaccine-induced cellular immune responses can effectively control replication of a pathogenic, heterologous AIDS virus, suggesting that T-cell-based vaccines may have greater potential than previously appreciated.

PD-1 blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination.

Programmed Cell Death 1 (PD-1) plays a crucial role in immunomodulation. Binding of PD-1 to its ligand receptors down-regulates immune responses, and published reports suggest that this immune modulation is exploited in cases of tumor progression or chronic viral infection to evade immune surveillance. Thus, blockade of this signal could restore or enhance host immune functions. To test this hypothesis, we generated a panel of mAbs specific to human PD-1 that block PD ligand 1 and tested them for in vitro binding, blocking, and functional T cell responses, and evaluated a lead candidate in two in vivo rhesus macaque (Macaca mulatta) models. In the first therapeutic model, chronically SIV-infected macaques were treated with a single infusion of anti-PD-1 mAb; viral loads increased transiently before returning to, or falling below, pretreatment baselines. In the second prophylactic model, naive macaques were immunized with an SIV-gag adenovirus vector vaccine. Induced PD-1 blockade caused a statistically significant (p<0.05) increase in the peak percentage of T cells specific for the CM9 Gag epitope. These new results on PD-1 blockade in nonhuman primates point to a broader role for PD-1 immunomodulation and to potential applications in humans.

Safety and immunogenicity of adenovirus-vectored near-consensus HIV type 1 clade B gag vaccines in healthy adults.

Vaccines inducing pathogen-specific cell-mediated immunity are being developed using attenuated adenoviral (Ad) vectors. We report the results of two independent Phase I trials of similar replication-deficient Ad5 vaccines containing a near-consensus HIV-1 clade B gag transgene. Healthy HIV-uninfected adults were enrolled in two separate, multicenter, dose-escalating, blinded, placebo-controlled studies to assess the safety and immunogenicity of a three-dose homologous regimen of Ad5 and MRKAd5 HIV-1 gag vaccines given on day 1, week 4, and week 26. Adverse events were collected for 29 days following each intradeltoid injection. The primary immunogenicity endpoint was the proportion of subjects with a positive unfractionated Gag-specific IFN-gamma ELISPOT response measured 4 weeks after the last dose (week 30). Analyses were performed after combining data for each dose group from both protocols, stratifying by baseline Ad5 titers. Overall, 252 subjects were randomized to receive either vaccine or placebo, including 229 subjects (91%) who completed the study through week 30. Tolerability and immunogenicity did not appear to differ between the Ad5 and MRKAd5 vaccines. The frequency of injection-site reactions was dose dependent. Systemic adverse events were also dose dependent and more frequent in subjects with baseline Ad5 titers <200 versus > or =200, especially after the first dose. The percent of ELISPOT responders and the ELISPOT geometric means overall were significantly higher for all four vaccine doses studied compared to placebo, and were generally higher in vaccine recipients with baseline Ad5 titers <200 versus > or = 200. Ad5 titers increased after vaccination in a dose-dependent fashion. Both Ad5-vectored HIV-1 vaccines were generally well tolerated and induced cell-mediated immune responses against HIV Gag-peptides in the majority of healthy adults with baseline Ad5 titers <200. Preexistent and/or vaccine-induced immunity to the Ad5 vector may dampen the CMI response to HIV Gag.

Characterizations of four monoclonal antibodies against M2 protein ectodomain of influenza A virus.

M2 protein of influenza A virus has been implicated as a target for vaccines with broad cross-strain coverage. Studies in small animal models have shown that antibody responses induced by 23-mer M2 peptide vaccines can provide protection against influenza A virus challenge. To study antiviral mechanisms of Merck M2-OMPC conjugate vaccine, we generated and characterized four M2 peptide-specific monoclonal antibodies (mAbs). Here we demonstrated that the protection by our M2 mAbs is independent of NK-mediated effector functions in mice. The protective mAbs preferentially bind to M2 multimers composed of two or more M2 peptides in parallel orientation. Our findings indicate that the protective M2 Ab prefer to bind to epitopes located within the N-terminal 10 amino acids of the M2 peptide, and the epitopes are likely formed by two M2 peptides in parallel orientation. The implications of these results in antiviral mechanisms of immune responses induced by M2 vaccines are discussed.

HIV-1 vaccine-induced immunity in the test-of-concept Step Study: a case-cohort analysis.

BACKGROUND: In the Step Study, the MRKAd5 HIV-1 gag/pol/nef vaccine did not reduce plasma viraemia after infection, and HIV-1 incidence was higher in vaccine-treated than in placebo-treated men with pre-existing adenovirus serotype 5 (Ad5) immunity. We assessed vaccine-induced immunity and its potential contributions to infection risk. METHODS: To assess immunogenicity, we characterised HIV-specific T cells ex vivo with validated interferon-gamma ELISPOT and intracellular cytokine staining assays, using a case-cohort design. To establish effects of vaccine and pre-existing Ad5 immunity on infection risk, we undertook flow cytometric studies to measure Ad5-specific T cells and circulating activated (Ki-67+/BcL-2(lo)) CD4+ T cells expressing CCR5. FINDINGS: We detected interferon-gamma-secreting HIV-specific T cells (range 163/10(6) to 686/10(6) peripheral blood mononuclear cells) ex vivo by ELISPOT in 77% (258/354) of people receiving vaccine; 218 of 354 (62%) recognised two to three HIV proteins. We identified HIV-specific CD4+ T cells by intracellular cytokine staining in 58 of 142 (41%) people. In those with reactive CD4+ T cells, the median percentage of CD4+ T cells expressing interleukin 2 was 88%, and the median co-expression of interferon gamma or tumor necrosis factor alpha (TNFalpha), or both, was 72%. We noted HIV-specific CD8+ T cells (range 0.4-1.0%) in 117 of 160 (73%) participants, expressing predominantly either interferon gamma alone or with TNFalpha. Vaccine-induced HIV-specific immunity, including response rate, magnitude, and cytokine profile, did not differ between vaccinated male cases (before infection) and non-cases. Ad5-specific T cells were lower in cases than in non-cases in several subgroup analyses. The percentage of circulating Ki-67+BcL-2(lo)/CCR5+CD4+ T cells did not differ between cases and non-cases. INTERPRETATION: Consistent with previous trials, the MRKAd5 HIV-1 gag/pol/nef vaccine was highly immunogenic for inducing HIV-specific CD8+ T cells. Our findings suggest that future candidate vaccines have to elicit responses that either exceed in magnitude or differ in breadth or function from those recorded in this trial.

Estimating the impact of vaccination on acute simian-human immunodeficiency virus/simian immunodeficiency virus infections.

The dynamics of HIV infection have been studied in humans and in a variety of animal models. The standard model of infection has been used to estimate the basic reproductive ratio of the virus, calculated from the growth rate of virus in acute infection. This method has not been useful in studying the effects of vaccination, since, for the vaccines developed so far, early growth rates of virus do not differ between control and vaccinated animals. Here, we use the standard model of viral dynamics to derive the reproductive ratio from the peak viral load and nadir of target cell numbers in acute infection. We apply this method to data from studies of vaccination in SHIV and SIV infection and demonstrate that vaccination can reduce the reproductive ratio by 2.3- and 2-fold, respectively. This method allows the comparison of vaccination efficacies among different viral strains and animal models in vivo.

The effect of early versus delayed challenge after vaccination in controlling SHIV 89.6P infection.

We sought to determine how effectively a CD8+ T cell inducing vaccine controls SHIV-89.6P infection in rhesus macaques at a range of challenge times post-vaccination. To this end, twenty eight Mamu-A*01+ rhesus macaques were given replication incompetent human serotype 5 adenovirus vector expressing SIVmac239 gag DNA and boosted 24 weeks later. Groups of 4 monkeys were then challenged with SHIV-89.6P at 1, 3, 6, 12, and 24 weeks after the boost. We compared the kinetics of viral load, CD4+ and virus-specific CD8+ T cells in these macaques. Measurements of CD8+ T cells taken before challenge show an exponential decay between 1 and 12 weeks following vaccination (p<0.0001). After week 12, no further decay was observed. Twenty of 24 vaccinated animals maintained more CD4+ T cells and kept their viral load at least one order of magnitude lower than the control animals throughout the chronic phase of the study. All 24 vaccinated animals survived the duration of the study. The viral and T cell kinetics over the first two weeks differed between the vaccinated groups, with more recent vaccination improving the early control of virus (p-value=0.027). The rates of virus specific CD8+ T cell expansion were greater in animals having higher viral loads at one week (r=0.45, p=0.029), suggesting that the kinetics of early viral load may have a role in virus specific CD8+ T cell generation, although these early differences did not lead to different clinical outcomes within the vaccinated animals.