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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC 2012 September 15.
Published in final edited form as:
PMCID: PMC3180898

HIV-DNA priming alters T-cell responses to HIV-adenovirus vaccine even when responses to DNA are undetectable1

Stephen C. De Rosa,* Evan P. Thomas, John Bui, Yunda Huang, Allan deCamp, Cecilia Morgan, Spyros Kalams,§ Georgia D. Tomaras, Rama Akondy, Rafi Ahmed, Chuen-Yen Lau,# Barney S. Graham,** Gary J. Nabel,** M. Juliana McElrath,*†† and the NIAID HIV Vaccine Trials Network


Many candidate HIV vaccines are designed to primarily elicit T-cell responses. Although repeated immunization with the same vaccine boosts antibody responses, the benefit for T-cell responses is ill-defined. We compared two immunization regimens that include the same recombinant adenoviral serotype 5 (rAd5) boost. Repeated homologous rAd5 immunization fails to increase T-cell responses, but increases gp140 antibody responses ten-fold. DNA prime, as compared with rAd5 prime, directs long-term memory CD8+ T cells toward a terminally differentiated effector memory phenotype with cytotoxic potential. Based on the kinetics of activated cells measured directly ex vivo, the DNA vaccination primes for both CD4+ and CD8+ T cells, despite the lack of detection of the latter until after the boost. These results suggest that heterologous prime-boost combinations have distinct immunological advantages over homologous prime-boosts, and suggest that the effect of DNA on subsequent boosting may not be easily detectable directly after the DNA vaccination.


The goal of HIV vaccine development is to prevent or control HIV infection by inducing effective long-term immune memory. Vaccine-mediated induction of broadly neutralizing antibodies has been unsuccessful due to extensive HIV envelope protein variability(1). Therefore, most vaccines in development primarily induce cellular immune responses.

Based upon experience with licensed vaccines, multiple vaccinations, known as a prime-boost regimen, will likely be needed for efficacy. Of interest is the effect on the magnitude and quality of long-term memory induced by priming and boosting with the same (homologous) or different (heterologous) vaccines. Heterologous vaccination could improve the quality of T-cell responses(2).

To test this hypothesis, we designed a study to compare homologous priming and boosting with a recombinant adenoviral serotype 5 (rAd5) vaccine versus priming with DNA and boosting with rAd5 in healthy HIV-uninfected Ad5-seronegative adults (HVTN protocol 068). This is of particular interest considering the results of recent HIV vaccine efficacy trials. Repeated homologous boosting with rAd5 expressing Gag, Pol, and Nef was associated with increased rate of infection in some subgroups(3), while a recombinant canarypox vector prime expressing Env, Gag, and protease followed by recombinant gp120 protein boost was associated with a modest reduction in infection rates(4).

The vaccines used in this study each encode Env, Gag and Pol. One treatment group received two rAd5 vaccinations (Ad5-Ad5), while the other group received two DNA primes and an rAd5 boost (DNA-Ad5). To evaluate the quality of the immune response, we assessed polyfunctionality by measuring cytokine production, evaluated cytotoxic potential, and quantified the types of memory cells elicited. We charted the temporal evolution of the response over multiple timepoints after each vaccination and at a longer-term timepoint one year after the first vaccination. DNA priming significantly affected the magnitude and character of the memory response to the rAd5 boost, even for CD8+ T cells that were not detectable following DNA.

Materials and Methods

Clinical Trial

We conducted a phase I multicenter double-blind, randomized, placebo-controlled study ( NCT00270218) comparing two different primes: recombinant adenoviral serotype 5 (rAd5) vector vaccine (group 1) or a DNA vaccine (group 2), each followed by an rAd5 boost. The DNA prime consisted of two injections one month apart, rather than a single injection, because a prior study had shown that one dose rarely resulted in measurable T-cell responses (5). This prior study included three doses of DNA, but we chose only to use two doses since the third dose minimally increased the response beyond the response following the second dose. The rAd5 prime consisted of a single injection since prior studies have demonstrated a high response rate following a single dose (6, 7). The 66 (33 in each group) participants were healthy HIV-1-uninfected adults (ages 18-50) lacking detectable preexisting Ad5 neutralizing antibodies (titers <1:12)(8). The vaccines, developed by the National Institute of Allergy and Infectious Diseases Vaccine Research Center, encoded HIV-1 Env glycoprotein (clades A/B/C) and clade B Gag/Pol fusion gene; the 4-plasmid DNA prime also encoded clade B Nef (included with Gag/Pol as a Gag/Pol/Nef fusion gene)(5, 6). Group 1 received 1010 particle units (PU) of rAd5 IM on day 0 and week 24; group 2 received 4mg of the DNA vaccine IM via biojector on day 0 and week 4, and 1010 PU rAd5 IM at week 24. Placebo recipients (three in each group) received PBS for DNA and final formulation buffer for rAd5. Blood was collected by venipuncture on the day of each vaccination, weekly for the first four weeks, and then six weeks following the second DNA injection and each rAd5 injection. A final sample was obtained 365 days following the first injection.

The primary immunogenicity timepoints were protocol-defined as four weeks following each rAd5 vaccination and four weeks following the second DNA injection, which was chosen because prior trials rarely found measurable responses to one dose of this DNA vaccine(5). Most analyses use data from these pre-specified timepoints, although they might not have been at the peak response for each individual.

The institutional review committee at each clinical site approved the protocol prior to study initiation, and participants completed a thorough informed consent process.

PBMC Processing

Peripheral blood mononuclear cells (PBMC) were isolated and cryopreserved from heparin-anticoagulated whole blood within eight hours of venipuncture, as described previously(9). PBMC were thawed and, except for T-cell activation analysis, cultured overnight at 37°C/5%CO2 in R10 [RPMI 1640 (GibcoBRL) containing 10% FCS (Gemini Bioproducts), 2mM L-glutamine (GibcoBRL), 100U/ml penicillin G, 100μg/ml streptomycin sulfate] prior to stimulation.

Ex vivo Stimulations

PBMC were assessed for ex vivo responses to pools of HIV-1 15-mer peptides (synthesized by Bio-Synthesis) overlapping by 11 a.a. covering global potential T-cell epitopes (PTE) within Env, Gag, Pol, and Nef (10). All PTE epitopes present at a frequency of ≥15% were identified and pooled by protein based on frequency. For this study only one pool (166 peptides) for each protein was used, which was sufficient for complete coverage of PTEs for Nef, but only 70%, 87% and 71% of PTEs for Env, Gag and Pol, respectively. The final concentration for each peptide was 1μg/ml during stimulations. Staphylococcal enterotoxin B (SEB; Sigma) stimulation was the positive control, while peptide diluent (DMSO at a final concentration of 1%) was the negative control. The six-hour stimulation included Brefeldin A (10 μg/ml, Sigma) and αCD28/αCD49d (each at 1μg/ml; BD Biosciences).

Flow cytometric assays

8-color ICS assay

We used the validated 8-color intracellular cytokine staining (ICS) protocol as described previously(11). Reagents used in this and other panels are described briefly below and listed in Supplemental Table I. Cells were first stained with Violet Live/Dead Fixable Dead Cell Stain(12), then fixed, permeabilized, and stained intracellularly with fluorescent-labeled antibody reagents detecting CD3, CD4, CD8, IFN-γ, IL-2, TNF-α and IL-4. For some assays, anti-IL-4 was replaced with anti-perforin that was conjugated to Alexa 647 in the laboratory.

10-color ICS assay

The 10-color ICS assay(13) included an evaluation of granzyme B and CD57 expression using the same reagents as the modified 8-color assay (including perforin) except for TNF-α–FITC, CD4–APC-H7, GzB–Alexa 700, CD57–Alexa 405, and Aqua Live/Dead Fixable Dead Cell Stain. IFN-γ and IL-2 expression was validated in bridging studies with the 8-color assay; for these cytokines, we report combined data from both assays. The TNF-α reagent was not comparable between the assays, and data for TNF-α are not included in the analyses presented here.

11-color ICS/memory marker assay

This assay was used to determine expression of four memory-defining markers (CCR7, CD45RA, CD27, CD57), IFN-γ and IL-2. After incubation with dead cell stain, cells were surface-labeled with antibody reagents detecting the memory markers. Cells were fixed, permeabilized(11), and stained intracellularly with the remaining antibody reagents (Supplemental Table I). The frequency of cells producing IFN-γ and IL-2 was lower for this assay versus the validated 8-color assay (average of 20% lower, but variable). Therefore, the 8-color assay was performed on all samples as the primary endpoint assay, while this assay was performed as a secondary assay on selected samples.

Activation marker assay

Samples were stained after overnight culture of thawed PBMC. PBMC were stained with Aqua Live/Dead Fixable Dead Cell Stain(12) and then surface-stained with antibody reagents detecting CCR7, CCR5, CD27, CD38, and HLA-DR. Cells were fixed, permeabilized(11), and stained intracellularly with antibody reagents detecting CD3, CD4, CD8, Ki-67, and BcL-2.

Later experiments demonstrated that the frequency of activated cells was consistently higher when cells were examined immediately after thawing (median of 2.1 times higher, ranging to five times higher). However, we found no difference in the kinetics of the appearance and decline of activated cells whether cells were immediately examined or cultured overnight. Since most samples were examined the day after thawing, data presented here includes only these samples.


Dead cell stains were from Invitrogen/Molecular Probes. Antibodies were from BD, except CD3–ECD (Beckman-Coulter), perforin (Tepnel/Diaclone), CD27 (eBioscience), and HLA-DR (Biolegend). We conjugated the perforin and CD57 antibodies to Alexa 647 and Alexa 405 (Invitrogen), respectively.

Stained samples were collected from 96-well plates using the High Throughput Sample device (BD), and 200,000-300,000 events from each sample were acquired on an LSRII flow cytometer capable of measuring 18 colors (BD). All FACS analyses were performed using either FlowJo (Treestar) or LabKey Flow(14). Positive responses and criteria for evaluable responses were determined as previously described(11), based on background measurements and the number of T cells examined. Since separate criteria are applied to CD4+ and CD8+ cells, the total numbers included in each ICS analysis can differ between the CD4+ and CD8+ T-cell evaluations.

HIV antibody ELISA

Anti-Gag and anti-Env binding antibody responses were determined by validated ELISA as previously described(15, 16). Sera from cryopreserved samples were diluted and tested in duplicate in microtiter plates (NUNC) coated with purified p55 Gag (Protein Sciences), ConS gp140 (Dr. H-X. Liao, Duke University), and gp41 (Immunodiagnostics). Envelope proteins that match the envelopes encoded by the vaccine were not available, but we have found that consensus strain envelopes are extremely sensitive for detecting antibody responses to multiple strains when examining antibody responses in acute HIV infection (16). Plates were washed with an automated calibrated plate washer (Bio-Tek). Optical density was determined (M2 plate reader, Molecular Devices), and non-antigen-containing wells were subtracted from antigen-containing wells. Standard curves were generated from the plot of absorbance (450nm) against the log of serum dilution, and sigmoidal curves were fit using a four-parameter logistic equation (Softmax Pro).

Quantitative Luminex Binding Assay

Serum HIV-1-specific IgG responses to Clade B Consensus (BCon) gp140, Group M Consensus (ConS) gp140, and Con6 gp120 (Drs. H-X. Liao and B. Haynes, Duke University) were measured by a standardized HIV-1 Luminex assay as previously described(16). Antibody measurements were acquired as mean fluorescent intensity on a Bio-Plex instrument (Bio-Rad) under GCLP-compliant conditions. Positive standard curves in each assay included 2G12 monoclonal antibody and purified IgG from HIV-positive sera (HIVIG) that were fit using four-parameter logistic curve fitting to calculate μg/ml equivalence concentration with the serum dilution that fit within the linear range of the curve (1:500 or 1:2500 dilution). Negative controls included HIV-negative human sera and blank beads.

Statistical analysis

Since few responses were detected to Nef and since the rAd5 vaccine did not encode Nef, no Nef data is included here. To reduce the adjustment for multiple comparisons and because fewer Gag and Pol responses were detectable with the DNA prime, statistical comparisons were performed only for the four-week and six-month timepoints post-boost for the overall response and the Env-specific response.

ICS assay positivity was calculated based on comparisons between stimulated and negative control responses via a one-sided Fisher's Exact Test. The resulting multiplicity-adjusted p-values were used to determine positivity, with p≤1×10-5 indicating a positive response. Comparisons of response magnitudes were performed using the non-parametric Wilcoxon rank sums test and adjusted for multiple comparisons using Holm's adjustment(17). Using the score method of Agresti and Coull(18), 95% confidence intervals for response rates were calculated.


Randomization and Safety

Participants in each group were well-balanced for age, sex and race (Supplemental Table II). All were Ad5 neutralizing antibody seronegative. As in earlier phase I clinical trials that evaluated the safety of rAd5 alone, DNA alone, or DNA prime/rAd5 boost(5, 6), both regimens were well-tolerated with minimal adverse reactions. Of the 30 participants randomized to receive each regimen, 26 Ad5-Ad5 participants and 28 DNA-Ad5 participants completed all vaccinations. All six control participants completed the regimen. Compared with control participants, more vaccinated individuals reported local and systemic reactogenicity, although severity levels were mainly mild (Supplemental Fig. 1).

Immunogenicity as measured by T-cell cytokine production

To determine whether the vaccine induced T-cell responses, we measured IFN-γ and/or IL-2 production by CD4+ or CD8+ T cells following stimulation with Env, Gag or Pol peptide pools in an intracellular cytokine staining (ICS) assay. At each timepoint the percentage of participants with a positive response was calculated (Fig. 1A and Table I). Comparisons were performed at the primary immunogenicity timepoints (four weeks after each rAd5 and four weeks after the second DNA).

Figure 1
Kinetics of percentage of responding individuals and percentage of cytokine-producing cells
Table I
Percentage of responders in each trial group post-prime and post-boost1

Ad5-Ad5 group

The first dose of rAd5 resulted in high CD4+ and CD8+ T-cell response rates (63% at four weeks after vaccination for each, Table I). Responses were relatively evenly distributed between Env, Gag, and Pol (respectively, 61%, 29% and 46% for CD4+ T cells; 30%, 42% and 50% for CD8+ T cells). Overall response rates were lower four weeks after boosting with a second dose of rAd5 (39% for CD4+ and 57% for CD8+ T cells). Similarly, protein-specific response rates were decreased compared to after the prime, except for CD8+ Gag (Fig. 1A and Table I). The magnitude of responses as determined by percentage of cytokine-producing T cells was decreased for CD4+ T cells (overall median of 0.25% decreased to 0.14%), but was similar for CD8+ T cells (0.3%) following the boost as compared with the response to the prime (Figs. 1B and 1C).

DNA-Ad5 group

The DNA prime induced mainly CD4+ T-cell responses (22% CD4+ vs. 0% CD8+ responders at four weeks after the second DNA; Fig. 1A and Table I), similar to the predominant CD4+ T-cell response observed in prior DNA vaccine trials(5). DNA-induced responses were limited to Env. After the rAd5 boost, the percentage of responders increased for both CD4+ and CD8+ T cells (39% and 50%, respectively). Responses to Env predominated, but responses to Gag and Pol were also detected. Comparing the magnitude of Env-specific CD4+ T-cell responses post-prime and post-boost, the median response within the first three weeks post-boost was increased (Fig. 1B), although at the primary immunogenicity timepoint (four weeks), the prime and boost responses were similar in magnitude (Fig. 1C).

Comparison between treatment groups

Following the prime, all response rates were higher for rAd5 than DNA. However, overall response rates were similar for the two treatment groups soon after the boost (four weeks) and at a longer-term timepoint (six months post-boost) (Table I). Env-specific CD8+ T-cell response rates trended higher for DNA-Ad5 at both the four-week and six-month timepoints post-boost (22% for Ad5-Ad5 vs. 45% for DNA-Ad5 at four weeks and 4% vs. 23%, respectively, at six months; p=0.2 for each). For Gag post-boost, the CD8+ T-cell response rate was comparable between DNA-primed and rAd5-primed subjects (25% vs. 39%, respectively), while the CD4+ T-cell response rate was low for both groups. For Pol post-boost, the CD8+ T-cell response rate was higher for the Ad5-Ad5 group (39% vs. 10% for DNA-Ad5, p=0.04); CD4+ T-cell responses to Pol were low for both groups.

Comparing response magnitudes post-boost, the CD4+ T-cell responses overall and for Env were similar between treatment groups (Fig. 1C). However, Env-specific CD8+ T-cell responses were significantly increased in the DNA-primed group compared to the rAd5-primed group at both the four-week and six-month timepoints post-boost (median 0.23% vs. 0.08% at four weeks, and 0.18% vs. 0.04% at six months; adjusted p=0.02 for each). These differences remained significant when absolute cell concentrations were compared (data not shown). Total CD8+ T-cell response magnitudes were not significantly different, but we observed several responses above 1% only in the DNA-Ad5 group (Fig. 1C). Response magnitudes for other proteins were not compared due to the limited number of detectable responses in the DNA-Ad5 group.

Interestingly, except for Env-specific CD8+ T-cell responses, a single dose of rAd5 administered as the prime resulted in higher response rates than a single dose of rAd5 administered after DNA priming (Table I, comparing four weeks post-prime for Ad5-Ad5 versus four weeks post-boost for DNA-Ad5). However, after adjusting for multiple comparisons, the difference was only significant for Pol-specific CD8+ T-cell responses (50% for single rAd5 vs. 10% for DNA/rAd5, adjusted p=0.05).

Cytokine profile

Since polyfunctional responses are thought to be more effective at mounting successful immune responses(19-22), we examined cells co-producing IFN-γ and IL-2 (Fig. 2). Six months post-boost, the DNA-Ad5 and Ad5-Ad5 groups differed in the proportion of dual-cytokine producing CD4+ T cells. Although not statistically significant when adjusted for multiple comparisons (adjusted p=0.07), the large number of individuals in the DNA-Ad5 group with high percentages of these cells (>50%) was notable, suggesting a long-term effect of DNA priming.

Figure 2
The percentages of Env-specific CD4+ and CD8+ T cells co-producing IFN-γ and IL-2

Memory marker expression

To evaluate whether the vaccine regimens induce different types of memory T cells and to determine whether they change over time, we examined expression of two T-cell differentiation markers (CD45RA and CD57) on vaccine-induced T cells. Naïve T cells express CD45RA, memory T cells lose CD45RA expression, and effector T cells re-express CD45RA and often co-express CD57. We were unable to identify central and effector memory T cells due to technical problems with the CCR7 reagent typically used for this classification(23).

The majority of Env-specific CD4+ T cells (>80%) were CD45RA-CD57- (Fig. 3A). A small proportion were CD45RA+CD57-. These proportions were similar after priming and boosting with only a minor decrease in CD45RA- cells post-boost. No significant differences were seen between treatment groups.

Figure 3
Expression of CD45RA and CD57 on Env-specific CD4+ and CD8+ T cells

In contrast, for Env-specific CD8+ T cells the CD45RA-CD57- and CD45RA+CD57- cell types were equally common four weeks post-prime. At later timepoints post-prime and post-boost, the proportion of CD45RA-CD57- cells decreased and most cells expressed CD45RA, with some also co-expressing CD57 (Fig. 3A and Supplemental Fig 2A). At six months post-boost, the DNA-Ad5 group had a greater percentage of CD45RA+CD57+ Env-specific CD8+ T cells than the Ad5-Ad5 group (adjusted p=0.1, Fig. 3A). The presence of the CD57-expressing CD8+ T cells is not simply a result of the rAd5 vaccine component of the DNA-Ad5 regimen, because few CD8+ T cells at 24 weeks following a single dose of rAd5 express CD57 (Fig. 3A, second row). A similar trend was observed for Gag-specific T cells (Supplemental Fig. 2B), but statistical testing was not performed due to the fewer Gag- and Pol-specific T cells.

We previously demonstrated an association between bright staining for CD57, the purported marker of terminally-differentiated cells(24), and perforin, allowing us to identify cells likely to have cytotoxic potential(25). We therefore examined expression of perforin, granzyme B (GzB) and CD57 for a few participants in each trial arm. Env-specific CD8+ T cells expressed intermediate, but not bright, levels of CD57 (Supplemental Fig. 2A). As expected, these cells did not express high levels of perforin. However, they expressed intermediate levels of GzB. Consistent with their higher frequency of CD8+ T cells expressing CD57, DNA-primed individuals had higher percentages of GzB-expressing Env-specific CD8+ T cells (Fig. 3B).

Kinetics of vaccine-induced T-cell responses

To determine the time at which peak responses were attained and to determine the kinetics of response decline post-peak, we measured response rates and magnitudes at multiple timepoints following each vaccination. The cumulative proportion of individuals reaching their peak response to Env, Gag and/or Pol post-prime and post-boost is charted over time in Fig. 4. For the rAd5-primed group, 10% of individuals who had a prime response attained their peak CD4+ response by two weeks after priming; all individuals reached their peak response by four weeks. The CD8+ rAd5-prime response kinetics were similar, except that some individuals peaked after four weeks. For responders from the DNA-primed group all peak CD4+ responses were attained after the second DNA dose, with the majority of peak responses occurring between two and four weeks after that dose.

Figure 4
Representation of the time at which peak response is reached

The kinetics of peak responses post-boost were more variable. Although more than 80% of individuals in either treatment group reached a peak CD4+ T-cell response by six weeks post-boost, the time to peak stretched over the full six weeks, unlike the more restrictive two-week period post-prime. Post-boost peak CD8+ T-cell response kinetics were similar, except that more responses peaked beyond six weeks post-boost.

The duration of peak response and rate of decline can be observed visually in Figs. 1A and 1B. The change in overall response rates and total magnitude suggests a two-phase decline, with a rapid decline shortly after the peak, resulting in a response level well above baseline that continued to slowly decrease.

Kinetics of activated T cells following vaccination

To assess induction of activated T cells in response to vaccination, we examined four markers of T-cell activation (CD38, HLA-DR, Ki-67 and BcL-2) on CD4+ and CD8+ T cells. We first compared the frequency of T cells identified as activated by using pairs of these markers in combination (CD38 and HLA-DR vs. Ki-67 and BcL-2). Although activated cells have been proposed to co-express CD38 and HLA-DR(26), many activated cells we identified as Ki-67+BcL-2lo did not co-express CD38 and HLA-DR. In the example shown in Fig. 5A, the percentage of Ki-67+BcL-2lo T cells that co-expressed CD38 and HLA-DR was 42% for CD4+ and 62% for CD8+ T cells. Because of the higher frequency of activated T cells detected and the more distinct staining pattern provided by Ki-67 and BcL-2, only they were used in subsequent analysis.

Figure 5
Activated T cells detected following vaccination

Unlike the kinetics for cytokine-producing cells, activated T cells had minimal variability in peak response timing, generally peaking by two weeks post-prime and one week post-boost (Fig. 5B). An increase in activated cells was commonly observed after rAd5, but rarely after DNA. For both treatment groups, the magnitude of the peak CD8+ T-cell response for activated cells was generally greater than the peak response in detected cytokines (e.g., medians of approximately 0.3% by cytokine and 0.7% by activation for the Ad5-Ad5 group post-boost). The frequency of activated cells rapidly declined following the peak, returning to pre-vaccination levels within two weeks. Considerable variability in magnitude was observed (Fig. 5C), including some especially high CD8+ T-cell responses (several above 2%, one at 7%, one at 13%).

HIV-specific antibody responses

Env- and Gag-specific antibody responses were measured by ELISA four weeks post-prime (first rAd5 or second DNA) and four weeks post-boost (Fig. 6A). The rAd5 prime induced a low-level antibody response significantly greater than baseline for all three antigens tested (adjusted p<0.001). DNA prime induced a low-level antibody response significantly greater than baseline for gp41 and p55 (adjusted p=0.01 for each), but not for gp140. Unlike the T-cell response, homologous immunization greatly enhanced post-boost Env-specific antibody responses (nearly 20-fold for gp140 and 8-fold for gp41). In contrast, the effect of the DNA prime on boosted Env-specific antibody responses was similar to that observed for T-cell responses. Although the gp140-specific antibody response did not differ from baseline following DNA prime, DNA enhanced the subsequent rAd5-boosted response since this was significantly greater than the gp140-specific response to a single dose of rAd5 measured following the prime in the Ad5-Ad5 group (p<0.001, Fig. 6A).

Figure 6
HIV-specific antibody responses

Comparing treatment regimens post-boost, gp140-specific antibody responses were significantly higher in magnitude in the Ad5-Ad5 group compared with the DNA-Ad5 group (adjusted p=0.02, Fig. 6A). Antibody responses to gp41 and p55 were not significantly different, although there were some higher p55-specific responses in the Ad5-Ad5 group not observed in the DNA-Ad5 group. Concentrations of Env-specific antibodies were also higher in the Ad5-Ad5 group when measured in a Luminex assay and expressed as equivalence concentration for 2G12 monoclonal antibody (Fig. 6B). Neutralization of HIV strains represented in the vaccine constructs was not detected for either treatment group (data not shown).


This study evaluated differences between homologous priming and boosting with an rAd5 vaccine and heterologous DNA prime/rAd5 boost vaccination. Homologous boosting greatly enhanced Env-specific antibody responses, but did not increase T-cell responses. Despite limited post-prime immune responses, DNA priming significantly enhanced the magnitude of post-boost Env-specific antibody and CD4+ T-cell responses and influenced the cytokine and memory marker profiles of the boosted T-cell responses.

In addition to identifying vaccine-induced T cells based on antigen-specific ex vivo stimulation and cytokine production, we also directly identified activated T cells ex vivo. Recombinant Ad5, but not DNA, induced large percentages of activated CD4+ and CD8+ T cells shortly after immunization, and, consistent with a secondary immune response, the peak response was attained more quickly following the boost (one week) compared with the rAd5 prime (two to three weeks). Although few DNA-Ad5 individuals had detectable post-prime increases in activated cells, most had accelerated post-boost kinetics similar to the Ad5-Ad5 group for both CD4+ and CD8+ T cells, with responses peaking by one week, suggesting a secondary immune response. Thus DNA is likely inducing undetected HIV-specific T-cell responses. In our trial this was especially notable for CD8+ T cells, since no participants had a detectable ICS response at the primary immunogenicity timepoint following DNA vaccination. Evaluation of immunogenicity and potential benefits of DNA vaccination should therefore not be based solely on ELISpot or ICS assays measured directly after DNA vaccination, but should include these or alternate measures following boost.

Homologous priming and boosting is a common strategy for many licensed vaccines, which likely are effective due to induction of humoral immunity that benefits from repeated immunization with the same immunogen. However, the potential benefits of homologous immunization for T-cell responses are not well defined. In our study, the magnitude of antibody responses to gp140 and gp41 were increased significantly after rAd5 boost; conversely, a second vaccination with rAd5 did not enhance T-cell responses, potentially due to anti-vector immunity induced by the first vaccination. However, the divergent effects on cellular and humoral immune responses indicate a more complex mechanism than simply impairing expression of the HIV proteins from the rAd5 boost. Indeed, antigen presentation may be affected by Ad5-specific neutralizing antibodies forming immune complexes with the vector, resulting in an effect on dendritic cell maturation(27).

In contrast to homologous rAd5 vaccination, rAd5 boosting of DNA resulted in an increased response rate and magnitude compared with the response detected after the DNA prime, most notably for Env-specific CD8+ T cells. At six months post-boost, the proportion of Env-specific CD8+ T cells expressing CD57 and granzyme B also tended to be higher in the DNA-Ad5 group. CD57 expression is associated with terminally-differentiated T cells with high cytotoxic effector potential, which are armed for a quick response to pathogens(24, 25, 28). Induction of these cells by a vaccine is likely beneficial to control viral replication at its earliest stage, but the cells must be maintained over time. Although CD57 is proposed to be a marker of replicative senescence(24), these cells are capable of rapid expansion(29). We also detect these cells at six months post-boost, indicating their potential for long-term maintenance.

The CD8+ T cells from both treatment groups had similar CD45RA expression, with nearly all post-boost Env-specific cells expressing CD45RA. Non-naïve CD45RA+ T cells have been classified as effector, rather than memory, T cells(23), but our data and other studies indicate they can still contribute to long-term memory. The majority of CD8+ T cells induced by vaccinia and yellow fever vaccines, known to produce long-lived protection, highly express CD45RA and persist and maintain proliferative ability for many years(22, 30). In our trial, vaccine-induced CD8+ T cells were detected six months post-boost and thus may similarly contribute to long-term memory.

The magnitude of the CD4+ T-cell response at four weeks and six months post-boost was similar for both treatment groups, but the cytokine profile of Env-specific CD4+ T cells differed at the six-month timepoint. A subset of DNA-Ad5 individuals had high percentages (>50%) of Env-specific T cells that co-produced IFN-γ and IL-2. This might be beneficial since polyfunctionality is linked to mounting a more effective host response(19-22). Thus, DNA priming can favorably influence the long-term vaccine-induced cytokine profile, at least in some individuals.

Except for Env-specific CD8+ T-cell responses, a single dose of rAd5 resulted in the highest response rates not only when compared with a second rAd5 dose, but also when compared with rAd5 administered after DNA prime. The dampening of rAd5-induced responses to some HIV proteins following DNA vaccination is possibly due to a DNA-induced suppressive effect. This effect may not be due to DNA as the method of vaccination, but rather due to the design of the DNA-encoded immunogens. The three Env proteins (clades A, B and C) are encoded on different plasmids, each including a secretory sequence. Gag, Pol and Nef are encoded on one plasmid. The NIH Vaccine Research Center (VRC) has recently developed a new DNA vaccine that encodes Gag, Pol and Nef on separate plasmids(31); trials with this DNA vaccine also include a third DNA vaccination. Those trials do not demonstrate a lower post-boost Gag-specific CD4+ T-cell response rate like we observed(32), suggesting that these changes may have alleviated any potential suppressive effect for Gag, although not for Pol. Finally, our trial was restricted to Ad5-seronegative participants. In contrast to potential suppression, DNA priming of rAd5 is reported to mitigate a potential decreased response to rAd5 vaccination in Ad5-seropositive individuals(32).

The Step Study, a prior efficacy trial of an rAd5-vectored vaccine, failed to reduce HIV infection or reduce HIV viral load(3, 13). Instead, early analyses revealed an increased incidence of HIV infection in Ad5-seropositive vaccinees. Concern was raised as to whether the vaccine increased the number of activated CCR5+ T cells, thus increasing the availability of target cells susceptible to infection. In our study (restricted to Ad5-seronegative participants), we found an increase in activated T cells, and many activated CD4+ T cells expressed CCR5 (data not shown). However, this increase was short-lived, at least as measured in blood, suggesting only a brief period of increased risk, unless vaccine-induced activated T cells persist at mucosal sites. Although kinetics indicate that the majority of activated cells are likely to be HIV-specific, some may be vector-specific. If so, differences in the design of the rAd5 vectors used in the two trials may affect vector persistence and thus persistence of activated cells. In particular, our VRC rAd5 vaccine has additional Ad5 gene deletions that may decrease vector persistence. Supporting this, a recent study demonstrated that the VRC rAd5 vaccine did not induce expansion of vector-specific CD4+ T cells or increase the activation state of Ad5-specific CD4+ T cells(33).

Our data demonstrate no T-cell benefit for repetitive boosting with the same viral vector vaccine. Instead, a single dose of rAd5 induced the highest response rates and magnitudes for many HIV proteins, regardless of DNA priming. However, DNA priming altered the character of the post-boost T-cell response, even without inducing a detectable T-cell response itself. This was most notable for Env, which as noted above has better expression than the other proteins in the DNA vaccine used in this trial(5). The newer DNA vaccine may extend the benefits of DNA priming to the other proteins. It should also be noted that there are other methods for improving the immunogenicity of DNA vaccines. One is through co-administration of DNA that encodes for cytokines that can potentially serve as adjuvants (34, 35). Another is through administration by electroporation. Both of these methods may further enhance the priming potential for DNA vaccination (36, 37).

Unlike the T-cell response, homologous boosting with the same viral vector dramatically increased Env-specific antibody responses. These antibodies bind to Env but do not neutralize HIV. The recent RV144 trial suggests potential benefits to inducing non-neutralizing antibodies, as nearly all vaccinees developed Env-specific binding antibodies with minimal neutralizing activity(4). This trial also highlights the potential utility of adding recombinant protein as one component of a vaccine regimen. The protective effect in RV144 was likely antibody-mediated since vaccination was associated with decreased HIV acquisition but not viral control in those who became infected. Our study, considered in the context of these other trials, suggests that vaccine regimens may need to include both heterologous vaccine modalities to optimize T-cell responses and homologous boosting to increase antibody responses.

Supplementary Material


The authors thank the study participants for their time and effort. We also acknowledge the study site staff and investigators, the HIV Vaccine Trials Network (HVTN) core staff, the HVTN site-affiliated laboratories and repository staff, and the HVTN research and development and endpoints laboratories that made this study possible. We acknowledge the data management and statistical support provided by SCHARP. We thank Stephen Voght, Renee Ireton and Phyllis Stegall for help with editing. We thank Patricia D'Souza and Alan Fix at the NIAID for thoughtful discussions and support.


recombinant Adenovirus serotype 5
Granzyme B
HIV Vaccine Trials Network
intracellular cytokine staining
potential T cell epitope
staphylococcal enterotoxin B


1This work is supported by the HVTN and SCHARP, a cooperative agreement with the National Institutes of Health Division of AIDS (NIAID) (U01 AI068614, U01 AI068618, U01 AI068635). The work is also supported through the University of Washington Center for AIDS Research (CFAR), an NIH funded program (P30 AI027757). We thank the James B. Pendleton Charitable Trust for their generous equipment donation.


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