In this therapeutic vaccination study, we evaluated the safety and immunogenicity of vCP1452 plus recombinant gp160 in 15 HIV-1-infected patients who were diagnosed and treated within 120 days after the onset of symptoms of newly acquired HIV-1 infection. Fourteen subjects completed the vaccination trial and have been assessed for virologic and immunologic changes pre- and postvaccination. The lack of significant (moderate or severe) adverse events establishes the safety of this vaccine regimen in this cohort of HIV-1-infected and HAART-treated patients. Importantly, these vaccines are also immunogenic. Specifically, all except one patient (93%) had increases in anti-gp120 or anti-p24 antibody titers. Nine out of 14 (64%) patients had an early and transient increase in CD4+ T-cell responses to either Env or Gag, and 11 out of 14 (78%) vaccinated subjects had an augmentation of HIV-1-specific CD8+ T-cell responses postvaccination.
We used a novel FACS-based intracellular cytokine staining assay to assess CD8+
T-cell-specific responses. We believe that this assay represents major advantages over standard assays that were available at the time we initiated this trial. Specifically, we elected to avoid the laborious limiting-dilution assay based on chromium release (26
) and the very restrictive and costly tetramer binding assay (3
). It is important that FACS-based intracellular cytokine staining assays using overlapping HIV-1 peptides and vaccinia virus constructs to more accurately assess immune responses have become commonplace (2
). Importantly, the assay we used was highly reproducible and standardized, and the vaccinia virus vectors contained the same HIV-1 proteins as those in the vaccine being tested in this clinical trial.
Previous therapeutic vaccine studies have been done in the setting of nonsuppressive antiretroviral therapy and used monovalent vaccines. The diversity of immune responses generated by these vaccines was limited (28
). In contrast, the vaccine regimen that we used stimulated both humoral and cellular immunity in a majority of patients. Furthermore, CD8+
T-cell responses to all four major HIV-1 gene products included in the canarypox virus vector, Env, Gag, Pol, and Nef, were boosted. It is remarkable to note that over 50% of the vaccine recipients had CD8+
T-cell responses to more than one HIV-1 antigen.
Previous studies using a similar vaccine regimen, albeit an earlier generation of ALVAC vaccine, have shown this strategy to be capable of eliciting both humoral and cellular immune responses in a proportion of vaccinated seronegative persons (7
). The strategy of priming with ALVAC containing a single HIV env
gene and boosting with rgp120 elicited cumulative envelope-specific CTL responses in over one third of seronegative vaccine recipients (7
). When a multivalent canarypox vector was used, 61% of the seronegative vaccine recipients had a CTL response detected at least one time point postvaccination (12
). The cellular immune responses, however, were usually transient.
In the current study, we have demonstrated a more sustained CD8+
T-cell response to multiple HIV-1 antigens in a larger proportion of vaccine recipients. Our current vaccine regimen enhanced HIV-1-specific CD8+
T-cell responses in 78% of vaccine recipients. Furthermore, these CD8+
T-cell responses persisted over the study period once they became detectable. Similar to the results observed in the uninfected individuals, the most dominant response tended to be specific to the Gag protein, although every antigen included in the vCP1452 elicited a CD8+
T-cell response. In most vaccine recipients, the magnitude of increase in HIV-specific CD8+
T cells was between 0.1 and 0.5%, which is equivalent to a frequency of 1,000 to 5,000 per 106
T cells. This level of antigen-specific CD8+
T cells is similar to levels observed in acutely infected patients by others (2
), but less than the magnitude of HIV-1-specific CD8+
T cells in long-term nonprogressors (25
The size of the cohort limits our ability to dissect factors that would predict a favorable CD8+
T-cell-mediated immune response to vaccination. However, all three subjects without a measurable CD8+
T-cell response had at least one HLA-A2 allele. This is an unexpected finding, considering that the Pol/Nef portion of ALVAC 1452 is based on known HLA-A2-restricted epitopes. However, it has been noted recently that responses to HIV-1 in the acute setting may be quite different from the “immunodominant” epitopes identified in chronically infected individuals (2
). Duration of infection pretreatment, duration of treatment, and HLA class I homozygosity at the A, B, or C locus did not predict a more or less robust CD8+
T-cell-mediated immune response, either quantitative or qualitative, to vaccination. As the size of this pilot phase II study was limited, we do not believe it is possible to draw conclusions regarding positive or negative predictors of response until substantially larger cohorts are similarly studied in controlled randomized trials.
The HIV-1-specific CD4+
T-cell responses, as measured by lymphocyte proliferation to Gag, were transiently elevated in 64% of the vaccine recipients. In one third of the patients who had a response, the initial response was boosted for a second time but not sustained. The preponderance and kinetics of these responses are similar to those observed in seronegative individuals receiving similar vaccines (7
). It remains unclear why these responses do not persist. One explanation may be that these memory HIV-1-specific CD4+
T cells are both limited in repertoire and more prone to activation-induced cell death, or apoptosis. Therefore, subsequent stimuli would be met by a limited CD4+
T-cell-mediated memory response. Whether this is vaccine specific or global for all HIV-1 antigens remains unanswered.
In agreement with previous studies using a different formulation of rgp160 (28
), we observed anti-gp120 antibody responses in all except one patient. However, few subjects demonstrated an induced antibody response to Gag (p24). These data suggest that the rgp160 component was capable of eliciting a humoral response to Env and that the viral vector, ALVAC 1452, is a poor inducer of a humoral response. Alternatively, and less likely, is that ALVAC 1452 does induce an Env antibody response but no Gag antibodies due to differences in antigen expression and processing. However, the kinetics of antibody response in our subjects were similar to that seen in seronegative subjects receiving gp120 vaccination, suggesting that we are indeed seeing a response to the recombinant protein.
The binding antibody titers reached peak levels after the third vaccination and remained at plateau levels during subsequent vaccination (12
). Induced anti-gp120 antibody levels exhibited apparently accelerated decay in subject 918, as did prevaccination anti-gp120 levels in subject 921. Neither subject had evidence of conditions associated with accelerated loss of serum proteins (i.e., nephrosis) or rapid extravasation to the extravascular space. The underlying mechanism of this effect in these subjects remains unclear.
In the limited number of subjects studied, we were unable to demonstrate that the antibodies induced by vaccination neutralized CCR5- and CXCR4-using laboratory strains in vitro. Compared to primary isolates, these viruses are generally more neutralizable (33
) and have envelopes more analogous to the exogenous immunogens in the vaccines (32
). That the induced antibodies did not neutralize these strains discouraged further such studies. With further improvement in the formulation of immunogens, such as using trimeric gp140 capable of eliciting HIV-specific neutralizing antibodies in mice (60
), it is possible that neutralizing antibody responses may be induced by vaccination in human subjects. Though antibody-associated cellular cytotoxicity has recently been described as a potential mechanism of virologic control during primary HIV-1 infection (18
), we have not yet assessed whether the antibodies induced by vaccination with ALVAC 1452 and rgp160 have similar properties and believe these experiments are beyond the scope of the original study.
We occasionally observed discordance between vaccine-induced CD4+
T-cell immune responses as measured by lymphocyte proliferation, CD8+
T-cell-mediated immune responses as measured by intracellular IFN-γ staining, and humoral responses to vaccination. We hypothesize that a minority of subjects, such as 313-2, responded to vaccination with a dominant Th2 response, accounting for a robust humoral (Gag) and T-cell proliferative response (Gag and Env) without a CD8+
T-cell response (8
). On the other hand, if we assume that the Env humoral response was due to the rgp160 component of the vaccine regimen, then most of the subjects appeared to respond in a Th1-dominant pattern of various degrees, with a CD8+
T-cell induced immune response, no humoral response to Gag, and a lymphocyte proliferative response, likely mediated by interleukin-2 or other Th1 cytokines (8
In assessing the immunogenicity of the vaccine, it is important to determine whether the immune responses observed were due to occasional bursts of viremia, i.e., autovaccination, instead of induction by the exogenous immunogens. Three subjects had one episode of measurable plasma viremia of 50 to 100 copies/ml during the period of vaccination. A fourth patient (900) was admittedly nonadherent to HAART and appeared to have augmented already induced HIV-1-specific CD4+ and CD8+ T-cell responses after the third and fourth vaccinations. In this subject, the first detectable CD8+ T-cell response appeared immediately after the first injections of vaccines, about 60 days prior to a detectable viremic episode. In subject 908, the initial CD4+ and CD8+ T-cell responses appeared to correlate with the timing of his viremic episode, but the second augmentation of CD8+ T-cell responses was observed in the absence of any detectable plasma viremia. In contrast, subject 3002 never had detectable CD8+ T-cell responses despite also having a viremic episode during vaccination. The isolated viremic episode in subject 1306 had little impact on the kinetics of the immune responses measured in this subject. Furthermore, viremia in these three subjects had little influence on the anti-p24 antibody or the HIV-1 specific CD4+ T-cell mediated immune responses observed.
More importantly, eight patients had augmentation of HIV-1-specific CD8+ T-cell responses in the absence of any detectable viremia. Thus, in most vaccinated individuals, the elevated immune responses postvaccination appear to be induced by the exogenously provided immunogens instead of autologous viruses, and the occasional viremic episodes are unlikely to have been the major determinants of immunogenicity. It is also difficult to determine whether these occasional isolated viremic episodes were directly related to immune stimulation caused by the vaccines or reflect the well-documented occurrence of low-level viremia due to incomplete suppression of viral replication during HAART.
Although we observed significant immunogenicity of the vaccine regimen, the unique features of this cohort of vaccine recipients should not be overlooked. All subjects included in the study were treated early, within 120 days of the onset of symptoms of acute HIV-1 infection. Early treatment may have preserved HIV-1-specific immunity, which allowed boosting by vaccination. Indeed, immunological manipulations, such as vaccination and structured treatment interruption, have been shown to be effective in the setting of acute lentivirus infection (22
). It is yet to be demonstrated whether such immune-based strategies will be successful in chronic HIV-1 infection.
Although the ALVAC vCP1452 plus rgp160 regimen can induce both humoral and cellular immune responses in HIV-1-infected individuals on HAART, the limited magnitude of the CD8+ T-cell response and the lack of persistence of T helper cell responses may limit the utility of this particular immunogen in this clinical setting. Nevertheless, we have demonstrated the feasibility of using multivalent vaccines as therapeutic modalities in HIV-1-infected patients receiving HAART. The promising safety and immunogenicity data generated from this preliminary study support further controlled studies in a variety of HIV-1-infected patient cohorts to better assess the efficacy of this adjunctive vaccine strategy.