This study was the first clinical trial of ADVAX in humans. Three vaccinations with ADVAX were well-tolerated at all three dosage levels, with minimal local and systemic reactogenicity.
Cellular immunogenicity, as measured by IFNγ ELISpot assays, was generally modest, sporadic, and transient, with no apparent dose response, which is in contrast to the stronger responses observed in small animals 
. Responses occurred after the second or third vaccination in all but one volunteer who formed a transient response to Gag after the first vaccination. This relatively modest response is concurrent with other stand-alone intramuscular DNA vaccines, which have proven weakly immunogenic in humans 
Given that DNA vaccines provide synergistic priming of the cellular immune response when boosted by viral vaccines, the IFNγ ELISpot assay does not adequately measure the ability to prime the immune system. The magnitude of the ELISpot response also does not necessarily correlate with a protective immune response either in non-human primates 
or in the recent STEP trial of an adenoviral-based vaccine 
. In our hands we have seen that the 16 hour detection platform of the ELISpot is more sensitive for IFNγ detection than the 6 hour detection platform of the flow cytometry assay, which may account for the paucity of detectable responses on intracellular cytokine staining. The mechanism of priming by DNA vaccines remains to be elucidated. Because the correlates of protection to HIV remain unknown, the relevance of the IFNγ ELISPOT and other assays ultimately remains unknown.
One volunteer in the low dosage group formed a particularly robust response to polymerase after the third vaccination, which was of high magnitude and sustained for at least nine months following vaccination. This was the same volunteer who formed polyfunctional antigen-specific T cell responses after vaccination. After unblinding, it was noted that this volunteer was a homosexual male who had a history of sexual relations with a long-term HIV-infected partner several years prior to enrolling in the trial. This volunteer remains HIV uninfected, and qualified for enrollment into the trial with a negative HIV ELISA, no bands on HIV western blot, and undetectable viral load. One explanation is that the robust response to polymerase after vaccination with ADVAX may reflect a “boosting” effect by ADVAX after “priming” with exposure to HIV in the past, as described previously 
. It is also possible that this response is a non-specific cross reaction to both polymerase peptide pools. However, this finding may have implications for assessment of responses to HIV vaccines in high-risk, uninfected populations who may have prior immunologic exposure to HIV not detected by conventional screening assays. Further immunologic characterization of this volunteer pre- and post-vaccination is ongoing.
The fact that no antibody responses were detected is disappointing, but consistent with the performance of other stand-alone DNA vaccines delivered to date 
. Other Clade C based HIV-1 vaccine candidates have been tested in DNA prime, Viral vector boost combinations 
. To test the priming ability of ADVAX, two clinical trials are now being conducted in the United Kingdom and in India, respectively, where 2 or 3 doses of 4 mg of ADVAX, either administered by Biojector ® 2000 or regular intramuscular needle injection, are given as prime followed by a recombinant multigenic MVA expressing HIV-1 Clade C env, gag, RT, rev, tat and nef genes 
. Attempts to improve the immunogenicity of DNA vaccines alone are also underway through improvements in DNA vaccine delivery or use of adjuvants 
. ADVAX is currently in a Phase I clinical trial to assess safety and immunogenicity when delivered by in vivo
electroporation with the Tri Grid™ Delivery System