Induction of anti-Env antibody responses in small animals was one of the first pieces of evidence that established DNA immunization as a novel approach for vaccination [35
]. Although significant progress has been made using DNA immunization to elicit HIV-1-specific CMI in small animals, non-human primates and humans over the past 15 years [12
], there has been no report of using Env DNA immunization to elicit broadly cross-reactive antibodies in humans. Levels of anti-Env antibodies elicited in previous DNA vaccine clinical trials were either low or undetectable [40
], nor there was a clear induction of NAbs against even sensitive viruses [14
]. In the current study, one Env protein immunization following DNA prime was able to elicit human anti-Env antibody responses to a level that has proven difficult to achieve in previous studies that employed multiple injections of recombinant HIV-1 Env proteins [2
]. Therefore, data from this study provides evidence that DNA vaccination can effectively prime the induction of high-level anti-Env antibody responses in humans.
In prior recombinant Env protein-based clinical HIV vaccine studies, up to two Env antigens were included in the vaccine formulation and as many as seven Env protein immunizations had to be administered in order to achieve high antibody responses [42
]. While the overall levels of Env-specific antibodies were low and not broadly cross-reactive, their binding specificity was measured against selected peptides of Env proteins rather than the primary Env antigens themselves [4
]. Our results clearly demonstrate the benefit of delivering polyvalent Env antigens through the DNA prime–protein boost approach. Not only did we elicit Env-specific antibody responses comparable to those seen in persons chronically infected with HIV-1, but the immune sera were able to react with primary Env antigens from every HIV subtype included in the study. Furthermore, the antibody responses appeared to be long lasting, with only a moderate decrease in titers at the end of the 52-week study period.
Given the importance of NAb responses in a prophylactic HIV vaccine and the complexity of measuring such responses, three neutralization studies were conducted, each with a slightly different approach and each using different panels of HIV viruses, to provide a more complete picture of the spectrum of neutralizing activities contained in the immune sera. The DP6-001 formulation elicited neutralizing activities against the sensitive viruses (TCLA and SF162) that were comparable to or better than those elicited by recombinant gp120 alone [30
], and clearly much better than a recently reported DNA vaccine alone approach which did not show neutralizing antibody activities [14
]. In the current studies, neutralization activity, against pseudoviruses expressing the homologous or randomly selected primary Env antigens, was detected in most of the post-immunization sera against approximately half of the viruses tested, independent of subtype. Not all homologous Env viruses showed high sensitivity to neutralization by post-immunization sera, which was not surprising given previous reports showing that infected HIV patient sera did not always have a high neutralizing activity against autologous viruses [44
The neutralizing activities against the standard panel of Tier 2 HIV-1 viruses were low in the current study, even at low serum dilutions. While it is unclear whether this panel represents more resistant viruses than the viruses included in the other two neutralization assays, it is possible that the difference reflects the inclusion of more contemporary viral isolates in the Tier 2 panel while Env antigens included in the DP6-001 formulation were cloned from patient samples collected around or before the early 1990s. While the current DP6-001 formulation, as a proof of concept study, may not have the optimal profile to move to more advanced clinical trials, the results included in this report clearly indicate that the use of a polyvalent, DNA prime–protein boost approach is feasible in order to elicit human neutralizing activities against a wide range of HIV-1 isolates. Future studies should test whether modified formulations including contemporary Env antigens can improve neutralizing activities against Tier 2 viruses.
Recently published studies with optimized DNA or other gene-based HIV vaccines elicited robust CMI responses [14
]. One key question asked in the current trial was whether robust CMI responses would be maintained when DNA immunization was used in conjunction with a protein boost component. The levels of Env-specific CMI responses elicited after three DNA immunizations in this study were similar to those previously reported [12
]. One new finding from the current study is that the Env protein boost further increased the magnitude of Env-specific CMI responses when compared to the Gag-specific CMI, for which there was no protein boost. The Env-specific CMI responses were cross-reactive against at least four different primary Env antigens that were included in this study. In further support for the presence of HIV-1-specific CMI responses in DP6-001 vaccinees, a subset of volunteers developed DTH-like skin reactions at the sites of DNA immunization after receiving an Env protein boost at a distant inoculation (Kennedy et al., submitted for publication).
We also detected a robust Gag-specific CMI response in Group C volunteers, which was higher than previously observed by others [14
]. Given the 1:5 ratio of Gag to Env DNA vaccines in DP6-001, the actual dose of Gag DNA vaccine was 1.2 mg in Group C, suggesting that the minimum immunogenic dose for naked DNA vaccines would be at least 1 mg with the current design of DNA vaccines. There is a clear dose–response relationship for both Env and Gag DNA vaccines. Due to the small sample size in the current study, future trials should study the boost effect of proteins on CMI responses. Furthermore, this study also shows that the ID route of administration was not more effective than IM in eliciting an immune response by DNA vaccines.
In summary, results from this first report of a human study with DNA prime–protein boost HIV-1 vaccine confirms the immunogenicity of this novel approach in eliciting balanced humoral and cell-mediated immune responses in a healthy adult population. These results are significant in that they confirm initial observations on the immunogenicity of DNA vaccines in animal models [35
]. The DNA prime–protein boost approach will not only accelerate the testing of more candidate HIV vaccines that aim to achieve improved neutralizing antibody responses, but will also provide a new platform for the development of future vaccines against a wide range of existing or emerging pathogens. Future studies should include in-depth analysis on the structural basis for both antibody and CMI cross-reactivities observed in the current report. The composition of Env antigens should be further optimized to identify a polyvalent formulation that may expand the breadth of neutralizing activities against viruses that were resistant to immune sera elicited by DP6-001. The immunization schedule including the use of adjuvant should be also optimized to reduce the reactogenicity of DP6-001 before moving this DNA prime and protein boost approach to more advanced human studies.