In this study, the VRC HIV-1 rAd5 vaccine was generally well-tolerated when given alone or as boost following the VRC HIV-1 DNA vaccine to healthy, HIV-seronegative African adults at low risk for HIV infection. The reactogenicity seen in this study was consistent with earlier phase I studies evaluating these products 
The DNA prime - rAd5 boost vaccination schedule was highly immunogenic even in volunteers with neutralizing antibodies against Ad5 at baseline. IFN-γ ELISPOT responses were most frequent to Env and Gag epitopes and were maintained at least 6 months after the rAd5 boost. Overall, there was no dosage effect on the frequency of IFN-γ ELISPOT responses at 6 weeks after rAd5 alone or rAd5 boost.
Compared to rAd5 alone, DNA priming increased the frequency of IFN-γ ELISPOT response after the rAd5 boost, but the difference was not statistically significant 6 weeks after rAd5 administration. DNA priming also increased the magnitude and the durability of the T-cell responses and resulted in immunodominance of Env and Gag over Pol consistent with the results from the RV 172 study 
. In our study, responses to Pol were highest after rAd5 alone. Contrary to our findings, responses to Env in RV 172 were not different between rAd5 alone and DNA prime – rAd5 boost groups 
. These findings may be relevant for future antigen design of T cell based vaccines.
Vaccine-specific antibodies were detected to EnvA, EnvB and EnvC, and to Gag after DNA prime and rAd5 alone; after DNA prime - rAd5 boost, significantly higher magnitude of antibody titers and frequency of responders were detected. However, there was no dosage effect on frequency of antibody responses comparing Groups A and B and Groups C and D.
This study and others suggest that DNA priming may alter the impact of pre-existing Ad5 immunity on vaccine-induced immune responses and alter the quality and/or quantity of elicited T cell and antibody responses 
A limited evaluation of ICS responses indicated that both CD4 and CD8 responses were elicited. Other studies have shown a predominant CD4 response after DNA alone, predominant CD8 response after rAd5 alone and a balanced response after DNA prime - rAd5 boost 
Although it remains to be seen whether VIA activity is a true correlate of HIV protection, VIA activity has been shown to be associated with control of HIV 
. Treatment-naive HIV+ subjects with a plasma viral load of <10 000/mL had median viral inhibition of 3.17 log10
, whereas those with viral load >10000 had median viral inhibition of 1.12 log10 
. In this study, CD8 T cells from individuals vaccinated with DNA prime - rAd5 boost or rAd5 alone had a range of inhibition up to 3.7 log10
units. The difference in VIA activity between rAd5 alone and DNA prime - rAd5 boost was not statistically significant.
At the end of the study, most vaccine recipients tested positive on at least one commercial HIV antibody kit without being HIV-infected. Use of a commercial kit that is least likely to register vaccine-induced antibody as positive is advisable; a purpose-built kit such as HIV-SELECTEST, if eventually approved, may facilitate diagnostic HIV testing in vaccine recipients 
within the study. In an observational follow up study of V001 volunteers, rapid HIV tests that incorporate only HIV-1 envelope proteins did not consistently detect vaccine-induced antibodies 
. Therefore, cautious use of rapid HIV tests may be possible for long term follow up of HIV vaccine recipients, but volunteers must be warned that they may test falsely HIV positive for some time. Specialized testing services will be provided by these clinical sites until the false positive tests fade.
In Africa, the prevalence of pre-existing antibodies from natural exposure to adenovirus type 5 is at least 80% (IAVI unpublished data) compared to 30–60% in the US 
. This may be of concern: vaccine take may be compromised, leading to attenuation of immune responses to the rAd5 vector. In our study, the overall frequency of HIV-specific immune responses to the rAd5 vaccine was somewhat lower than reported in US volunteers, perhaps because of the higher Ad5 seroprevalence seen in the majority of our study participants 
, but there may be other reasons responsible for the differences observed, e.g., race, geographical area, population characteristics, nutritional status. Our findings are also consistent with the findings from the Merck Phase IIb (STEP) study, where pre-existing immunity reduced the immunogenicity of the MRK rAd5 gag-pol-nef vaccine 
. However, it is important to note, that there are differences in properties and vaccination regimens between the VRC and the MRK vaccines and that results from the V001 and STEP study may not be comparable. With respect to the increased HIV incidence in uncircumcised male vaccinees with pre-existing immunity to Ad5 in the STEP study, a recent publication suggests that pre-existing serological immunity to Ad5 in itself does not appear to be associated with increased risk of HIV acquisition 
. Uncircumcised status in males was a stronger predictor of HIV acquisition than pre-existing immunity to Ad5.
The V001 study was completed before the results from the STEP study (HVTN 502) became available; therefore, those results did not influence the conduct of the study 
. However, all former V001 participants were informed about the STEP study results, as were all IRBs/IECs and national regulatory agencies.
Currently, a focused Phase II study is evaluating the VRC HIV-1 DNA prime - rAd5 boost combination for its effect on early control of viral load in those study participants who become HIV-1 infected. The study is enrolling Ad5 seronegative and circumcised men who have sex with men in the USA (HVTN 505; http://clinicaltrials.gov/ct2/show/NCT00865566
Recombinant adenovectors of serotypes other than type 5, such as rAd35 and rAd26, are also in early clinical trials as prophylactic vaccines for HIV and other diseases. The advantage of these vectors may be a lower seroprevalence 
in humans compared to Ad5. The effect of pre-existing immunity to these vectors on the immunogenicity of rAd vectored vaccines remains to be seen.
In addition, enormous efforts are being made to develop HIV vaccines capable of inducing neutralizing HIV antibodies and to design replicating viral vectors. While basic discovery and applied research are crucial for the development of a safe and efficacious HIV vaccine, it is important to continue to perform focused human clinical trials of different vaccine strategies to develop a highly effective and safe preventive HIV vaccine 
. New functional T cell assays that allow determination of correlates of protection and/or predict vaccine efficacy are also urgently needed.