The EP HIV-1090 DNA vaccine is an experimental, first-generation product designed to induce responses to highly conserved, HLA class I supertype-restricted, HIV-1 derived CTL epitopes (32
). Our initial preclinical studies showed these conserved epitopes to be antigenic, recognized in vitro using PBMC from a cohort of HIV-1-infected patients, suggesting that they are processed, presented, and immunogenic during natural infection. However, the magnitude of naturally occurring CD8+
T-cell responses targeting these epitopes was low in treated subjects, and the breadth of recognition for a given individual was relatively narrow. Thus, we hypothesized that immunization of HIV-1-infected subjects receiving effective ART with EP HIV-1090 DNA vaccine would induce a stronger and more broadly directed CTL response capable of recognizing autologous HIV-1 variants in vivo.
A major challenge of this type of therapeutic immunization is viral variation, as multiple viral variants, or quasispecies, exist that can evade immune detection through changes in amino acid sequences of CTL epitopes. The evolution of such viral escape mutants, in concert with well-documented HIV-associated T-cell dysfunction, contributes to the loss of immune system control associated with disease progression. With this in mind, the EP HIV-1090 DNA vaccine was designed to induce CTLs that will recognize not only amino acid sequence conserved epitopes but also epitopes with minor changes in sequence (27
). The working hypothesis is that these epitopes cannot be altered significantly without affecting viral fitness, and as such, they are logical targets for therapeutic vaccination.
In this, the first phase 1 trial with HIV-1 infected subjects, the vaccine proved to be safe and very well tolerated but not as immunogenic as was predicted based on animal testing and clinical testing data reported by others. It is difficult to compare the results obtained from the clinical testing of other DNA vaccines because different study designs and types of immune response assays were used. For example, the use of peptide pools rather than individual CTL or HTL epitope peptides complicates comparison, as peptide length can effect rate of detection (10
). The use of ELISPOT assays or intracellular cytokine staining, with and without peptide or other forms of cellular activation or restimulation, also varies between laboratories. Additionally, the variation reported between studies involving the same vaccine products is significant, indicating variability between laboratory capabilities. Finally, preexisting HIV-specific immune responses induced during natural infection may vary between target patient populations and can complicate measurement of vaccine-induced immunity in the therapeutic vaccination setting. However, given all of these variables, the vaccine immunogenicity observed in our studies appears to be comparable to, but certainly not greater than, that reported for other DNA vaccines encoding intact HIV viral gene products or epitopes or for peptide-based products tested clinically.
For example, a vaccine composed of HIV-1 subtype A-derived CTL epitopes fused to the intact Gag p24 gene product and delivered as a DNA vaccine was reported to induce weak and transient CTL responses, measured using the ELISPOT assay, in the PBMC of 14 of 18 healthy uninfected volunteers (78%) (28
). However, a significantly reduced response rate of <15% was reported following subsequent clinical testing, and the immunogenicity of this DNA vaccine could not be demonstrated in HIV-infected volunteers, where preexisting immune responses complicated the analysis (8
). Cellular immune responses measured using an ELISPOT following vaccination of normal volunteers with a similarly designed DNA product encoding Plasmodium falciparum
CTL epitopes fused to the thrombospondin-related adhesion protein appear to be comparable in rate and magnitude and mediated predominantly by CD4+
T lymphocytes (26
). Immune responses were increased significantly following a booster immunization with the P. falciparum
products delivered using an MVA, indicating immune system priming by administration of the DNA vaccine. This is similar to the results we report in this paper, where additional responses could be detected using a culture step to expand and activate responding T lymphocytes to detectable levels.
Higher rates of T-lymphocyte responses were observed using DNA vaccines encoding largely intact but inactivated HIV gene products; response rates of 36 to 40% for CD8+
T lymphocytes and 93 to 97% for CD4+
T lymphocytes were reported (5
). These finding could be interpreted to indicate that DNA vaccines encoding large or intact viral gene products are more immunogenic than epitope-based vaccines such as the product tested in our study. The increased rates of response to the intact Gag p24 protein and TRAP over the defined CTL epitopes may support this interpretation (26
). However, an alternative possibility is that HTL epitopes and the responses induced to them may increase vaccine potency with respect to inducing CTL responses. It should be noted that other experimental vaccines encoding both HTL and CTL epitopes have been developed and are being evaluated clinically; the results of these studies may provide some insight into the results from this first trial.
The use of the primary ELISPOT assay for analysis of PBMC samples from multiple study time points indicated the induction of transient HLA phenotype-restricted CTL responses rather than long-lived circulating and activated CTLs. This has been reported for DNA- and MVA-vectored vaccines by others (16
) and interpreted largely to indicate low-level responses and limitations with assay sensitivity or specificity (18
). Transient CTL responses may also indicate loss in the periphery resulting from homing of lymphocytes to lymphoid tissues, a reduction in cellular function and activation in the absence of continued epitope stimulation or proinflammatory signals, or potentially the progression of vaccine-induced CTLs to memory cells. To investigate these possibilities, an IVS assay was used. The sensitivity of the IVS ELISPOT assay significantly increased our ability to detect responses, resulting in the detection of multiple responses to vaccine-encoded epitopes.
The EP HIV-1090 DNA vaccine was also tested in non-HIV-infected volunteers by the HIV Vaccine Trials Network (HVTN-048) (14
). The product was observed to be safe and well tolerated. Similar to the case for this study, immune responses measured using a primary ELISPOT assay or chromium release assay were only rarely observed; responses were detected in 4 of 35 of vaccine recipients (11%) using PBMC sets from three or four study time points. Further testing using other assays was not completed for the HIV Vaccine Trials Network study, and therefore, the two studies cannot be compared further.
The EP HIV-1090 DNA vaccine proved to be safe and tolerable, and transient new CTL responses were observed in a small subset of vaccinated subjects using prespecified response criteria. However, overall differences in response rates between vaccine and placebo recipients did not achieve statistical significance. The transient nature of the measured CTLs may be a concern; however, additional assays will be needed to better characterize the vaccine-induced T-cell responses to determine if memory CTL responses were effectively primed. As this study was focused on safety and not powered to detect significant differences in vaccine-induced immune responses, additional clinical testing will also be needed to assess vaccine schedules and methods that may augment vaccine potency in HIV-infected subjects. Similarly, larger trials will be necessary to determine whether epitope-based therapeutic vaccine approaches result in better control of HIV-1 replication. Analysis of the combined data from the studies completed with HIV-infected and uninfected volunteers using this vaccine indicates the likely need to increase vaccine immunogenicity through the use of vaccine delivery devices, adjuvants, or potentially the incorporation of HIV-1-derived HTL epitopes. Further studies with second-generation products and vaccine delivery devices are ongoing.