To date, only two studies have reported the use of a DNA prime and protein boost strategy in humans. In the first study, a DNA vaccine encoding hepatitis B virus surface antigen given i.d., followed by a recombinant protein boost given i.m., elicited an antibody response only after the protein boost (74
). In the second study, which investigated a malaria vaccine using a DNA and protein boost vaccine approach (80
), antibody and T-cell responses were induced. A single function (IFN-γ production) measured by an enzyme-linked immunospot assay was used to assess the T-cell responses in that study. The present study simultaneously characterized several functions of the vaccine-induced T cells seen with a polyvalent HIV-1 DNA prime/glycoprotein boost vaccine strategy that was previously shown to induce cross-clade neutralizing antibody responses in humans (83
). We demonstrated that this vaccine regimen induced predominantly Env-specific CD4 T-cell responses, irrespective of the dose and route of administration. However, the magnitudes and functional kinetics of the responses differed significantly among groups. CD4+
T-cell responses were very weak in the participants receiving the DNA vaccine i.d. but were detected in the majority of participants receiving the same DNA vaccine dose i.m. The high-dose DNA given i.m. was unique, inducing both CD4 T cells with greater function than those induced by the lower dose and Env-specific CD8 T cells. Similarly, Gag-specific CD4 T cells were reliably detected only in the high-dose DNA group.
The high proportion of IL-2-producing CD4 T cells following vaccination is in accordance with published data that protein-based vaccines induce IL-2-secreting cells whereas infections induce more IFN-γ-secreting cells (21
). The effector memory CD4 T cells generated in this study (CD127hi
) were multifunctional, with CD154-expressing CD4 T cells having the capacity to secrete IFN-γ and IL-2. This phenotype has been reported previously for long-term nonprogressors and aviremic patients on highly active antiretroviral therapy (42
). Cells with such a memory phenotype have proliferative potential and can be the source of the rapid generation and maturation of CD4 T cells with an effector function (42
). Stubbe and colleagues (73
) also demonstrated that hepatitis B vaccination results in the generation of a polyfunctional effector memory T-cell phenotype. Although polyfunctional CD4 T cells are better at effector functions than monofunctional cells and are needed for optimal and sustained protection, repeated vaccine administration may be necessary to maintain such responses when immunogens other than live and replicating vectors are used (20
Antigen-specific CD8 T-cell responses were lower in frequency, seen only in the high-dose DNA group, and the T cells in these cases expressed IFN-γ or CD107, suggesting the possibility that CD8 T-cell responses were underestimated by using pools of 20-mers. We therefore tested an A2- and A11-restricted Env-B peptide (TK-10 peptide), TVYYGVPVWK (amino acids 4 to 13), in four subjects. The frequency of CD8 T cells producing IFN-γ in response to this 10-mer was similar to the frequency of such cells in response to the 20-mer peptide encompassing it (data not shown). This skewing toward a CD4 T-cell response is consistent with the findings of two recent studies in which DNA vaccines induced antigen-specific CD4 and CD8 T cells in about 90 and 40% of participants, respectively (13
We previously demonstrated that following the DNA vaccine priming, antibody responses were reliably induced with only the high-dose preparation (83
). While numerous preclinical data have demonstrated a dose effect with DNA vaccines, there are limited clinical trial data on this issue. Part of the explanation for this lack of information is the historically poor immunogenicity of DNA vaccines when given alone to humans (23
). Nevertheless, in trials of DNA vaccines in which immune responses have been induced, a dose-dependent increase of T-cell responses has been shown in one study (79
) but not in others (41
). Our finding that CD4 T cells generated by the higher DNA vaccination dose were greater in magnitude and function (i.e., the ability to secrete three cytokines and upregulate CD154) demonstrates that the dose is an important factor in determining the quality of immune responses. Despite the improved CD4 T-cell responses induced by the high-dose DNA, the subsequent administration of a recombinant gp120 protein did not boost this response, indicating either the ineffectiveness of the protein along with the QS-21 adjuvant to boost CD4 without subsequent priming or a null or even detrimental effect of repeated vaccinations with recombinant Env on T-cell memory. The former possibility may be explained by the observation that CD4 T cells have an inherently lower proliferation potential than CD8 T cells (30
It is interesting that the i.m. route of DNA vaccination was more effective than the i.d. route in eliciting CD4 T cells. This was true even though the doses of the vaccine administered in the i.d. and i.m. groups (1.2 mg) were identical. The protein boost effect, however, tended to be greater in the i.d. than the i.m. groups, possibly due to the longer persistence of DNA given i.d. (62
). Prior studies have compared the i.d. and i.m. vaccination routes in clinical trials, with disparate effects on the magnitudes of immune responses (4
). These trials have generally used particle-based vaccines, but one trial demonstrated that antibody responses in volunteers receiving a hepatitis B vaccine i.d. were diminished compared to those in subjects receiving the vaccine i.m. (76
). However, in that trial, the i.d. dose was 1/10 of the i.m. dose. We are not aware of any previous study that has compared the i.d. and i.m. routes of delivery of DNA vaccines in humans. Our findings suggest that DNA vaccines and particle-based vaccines may behave differently depending on the route of administration and demonstrate that such information cannot simply be extrapolated from prior studies based on other forms of vaccines.
The identification of positive neutralizing antibody responses (titers of >1:20) against laboratory-adapted and pseudotyped viruses expressing Env from primary isolates from clades A, B, C, D, and E, as well as the gp120-specific immunoglobulin G titers (83
), matched the onset and maturation of T-cell responses as measured in this study (data not shown). However, the magnitude of Env-specific CD4 T-cell responses as measured by any of the four functions did not significantly correlate with the magnitude of binding or neutralizing antibodies (data not shown). The induction of antibody responses is likely the result of a complex series of interactions, and our data indicate that they are impacted by factors in addition to CD4 T cells.
In summary, this polyvalent HIV-1 DNA prime/protein boost vaccine regimen induced different qualities of immune response following the DNA priming depending upon the dose and route of administration. This information may be useful for the future application of DNA vaccines to be used alone or in combination with other vaccines. The protein boosts resulted in CD4 T cells that were similar among the low-dose groups, consistent with the antibody responses observed. The responses induced by the DNA, particularly with the high dose, are encouraging and suggest that this vaccine formulation could be used as an effective prime vaccine for a recombinant vectored vaccine boost. This finding is particularly important information when one considers that the vaccine-induced T-cell responses observed in clinical trials to date have been significantly lower than those observed in natural infection or primate models of vaccine protection.