Conceived and designed the experiments: ST BSG JG GJN PF. Performed the experiments: WJ EK KK GSOM BF OA CMM JB TTF PJB PH GS JC. Analyzed the data: BSG RAK RTB CS LD MH MB JC JG PF. Contributed reagents/materials/analysis tools: RAK RTB JG GJN. Wrote the paper: BSG CS LD MB JC CS PF. Director of the Rwanda Zambia HIV Research Group: SAA. Medical monitoring: EA. Data manager: KL. Project Manager: WK. Director of African Laboratory Operations: GS. African Clinical Program Manager: HT. Principal Investigator at KAVI: JJB.
Current address: WHO-UNAIDS HIV Vaccine Initiative (HVI), Initiative for Vaccine Research (IVR), World Health Organization (WHO), Geneva, Switzerland
Current address: Pfizer Inc., New York, New York, United States of America
Current address: Department of Clinical Biology, Faculty of Medicine, National University of Rwanda, Butare, Rwanda
Current address: Division of Biostatistics, Office of Surveillance and Biometrics, Center for Devices and Radiological Health, Food and Drug Administration, Rockville, Maryland, United States of America
Current address: Sanofi Pasteur, Swiftwater, Pennsylvania, United States of America
IAVI is a non-profit organization. None of the co-authors affiliated with IAVI reports any competing interest, that might interfere with the objective assessment of this manuscript or with the ability to adhere to all PloS ONE policies on sharing data and materials. Gary J. Nabel is named on patent applications for the DNA and adenovirus vector components of this vaccine concept (patents # E-173-2004/0-US-01, E-355-2003/0-US-01, and E-267-2004/0). This does not alter the author's ability to adhere to all PloS ONE policies on sharing data and materials. None of the co-authors employed by government agencies declares any competing interest that might interfere with the objective assessment of this manuscript or with the ability to adhere to all PloS ONE policies on sharing data and materials. Affiliations of Carol Smith, Len Dally, Martin Ho, Kelley Loughran, Mark Boaz and Soe Than do not alter the authors' ability to adhere to all the PLoS ONE policies on sharing data and materials. Drs. Than and Boaz report no competing interests.
We conducted a double-blind, randomized, placebo-controlled Phase I study of a recombinant replication-defective adenovirus type 5 (rAd5) vector expressing HIV-1 Gag and Pol from subtype B and Env from subtypes A, B and C, given alone or as boost following a DNA plasmid vaccine expressing the same HIV-1 proteins plus Nef, in 114 healthy HIV-uninfected African adults.
Volunteers were randomized to 4 groups receiving the rAd5 vaccine intramuscularly at dosage levels of 1×1010 or 1×1011 particle units (PU) either alone or as boost following 3 injections of the DNA vaccine given at 4 mg/dose intramuscularly by needle-free injection using Biojector® 2000. Safety and immunogenicity were evaluated for 12 months. Both vaccines were well-tolerated. Overall, 62% and 86% of vaccine recipients in the rAd5 alone and DNA prime - rAd5 boost groups, respectively, responded to the HIV-1 proteins by an interferon-gamma (IFN-γ) ELISPOT. The frequency of immune responses was independent of rAd5 dosage levels. The highest frequency of responses after rAd5 alone was detected at 6 weeks; after DNA prime - rAd5 boost, at 6 months (end of study). At baseline, neutralizing antibodies against Ad5 were present in 81% of volunteers; the distribution was similar across the 4 groups. Pre-existing immunity to Ad5 did not appear to have a significant impact on reactogenicity or immune response rates to HIV antigens by IFN-γ ELISPOT. Binding antibodies against Env were detected in up to 100% recipients of DNA prime - rAd5 boost. One volunteer acquired HIV infection after the study ended, two years after receipt of rAd5 alone.
The HIV-1 rAd5 vaccine, either alone or as a boost following HIV-1 DNA vaccine, was well-tolerated and immunogenic in African adults. DNA priming increased the frequency and magnitude of cellular and humoral immune responses, but there was no effect of rAd5 dosage on immunogenicity endpoints.
ClinicalTrials.gov
In a Phase IIb/III community-based clinical trial in Thailand, prevention from HIV infection was demonstrated for the first time with a combination of ALVAC-HIV (canarypox vectored HIV vaccine) and AIDSVAX B/E (recombinant protein-based HIV vaccine)
A recombinant multiclade adenovirus type 5 (rAd5) vector-based vaccine expressing HIV-1 subtype B Gag and Pol and subtypes A, B and C Env (VRC HIV-1 rAd5), and a recombinant DNA vaccine encoding the same proteins plus subtype B Nef (VRC HIV-1 DNA), developed by the Vaccine Research Center (VRC) at the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH), have been evaluated previously either alone or in a DNA prime - rAd5 boost combination in healthy, HIV-uninfected volunteers; both vaccines were well-tolerated and immunogenic
Healthy HIV-uninfected adults aged 18–50 years were recruited at the Kenya AIDS Vaccine Initiative (KAVI), Nairobi, Kenya and at Project San Francisco (PSF), Kigali, Rwanda. Volunteers reported no increased risk for HIV (i.e., unprotected vaginal or anal sex with known HIV infected person; sex in exchange for money or drugs; a sexually transmitted infection within 6 months before vaccination). They were willing to undergo HIV testing and receive results. They agreed to use effective contraceptive methods for the duration of their participation if sexually active and not to become pregnant. Baseline serum neutralizing antibodies against Ad5 were measured, but not used as an eligibility criterion. All participants provided written informed consent.
This study was a double-blind, randomized, placebo-controlled Phase I clinical trial (
Group (N/n) |
Vaccines/Dosage Group |
W0 | W2 | W4 | W6 | W8 | W10 | W12 | W24 | W28 | W30 | W36 | W48 |
rAd5 1010 | S |
S |
S |
S |
S |
S |
|||||||
rAd5 1011 | S |
S |
S |
S |
S |
S |
|||||||
3DNA + rAd5 1010 | S |
S |
S |
S |
S |
S |
S |
S |
|||||
3DNA + rAd5 1011 | S |
S |
S |
S |
S |
S |
S |
S |
(N/n) Number of vaccine recipients/number of placebo recipients per group.
Vaccines/Dosage Groups: - rAd5: VRC HIV-1 rAd5 at 1×1010 or 1×1011 PU - 3DNA: 3 doses of VRC HIV-1 DNA at 4 mg/dose given intramuscularly by needle-free injection using Biojector ® 2000.
↓indicates vaccination visit.
S: Sample collection and assessment time points (S):
Serum neutralizing antibodies against Ad5.
Vaccine-induced HIV-1 specific cellular immune responses.
Vaccine-induced HIV-1 specific humoral immune responses.
The study vaccines were designed and constructed at the Vaccine Research Center (VRC), Bethesda, MD, USA and were described previously
The rAd5 vaccine was given intramuscularly by needle injection at dosage levels of 1×1010 particle units (PU; low dosage, LD) or 1×1011 PU (high dosage, HD). The DNA vaccine was given at 4 mg/dose intramuscularly by needle-free injection (Biojector® 2000) (
The primary objective of the V001 study was to evaluate the safety and tolerability of a rAd5 vector expressing multiple HIV-1 proteins at two different dosage levels, given alone or as a boost following DNA plasmid vaccine in healthy HIV-uninfected adults in East Africa. The secondary objective was to evaluate the humoral and cellular immunogenicity of the vaccine at each dosage level.
The protocol for this trial and supporting CONSORT checklist are available as supporting information; see
Study participants were monitored by interim medical history, and by physical and laboratory assessments. Local (pain, tenderness, erythema, induration, edema, papule, blister/vesicle, scab) and systemic signs and symptoms (headache, fever, chills, malaise and/or fatigue, myalgia, arthralgia, nausea, vomiting) were solicited for 3 days. Unsolicited adverse events (AEs) were recorded throughout the study, graded for severity (Grade 1 = mild, Grade 2 = moderate, Grade 3 = severe, Grade 4 = potentially life threatening) and classified by MedDRA (Medical Dictionary for Regulatory Activities). The AEs were assessed for relationship to study vaccines. There were 5 categories of relatedness: definitely, probably, possibly, probably not and not related. All safety data were reviewed quarterly by an independent Data and Safety Monitoring Board (DSMB). Protocol deviations were monitored throughout the trial.
At screening, HIV testing was performed according to the respective national algorithms. Only HIV-seronegative individuals were enrolled. During the study, a protocol-specific algorithm was followed, using two different HIV-1 ELISA tests selected from Abbott Murex HIV Ag/Ab Combination Assay (Manufactured by Murex Biotech Limited, United Kingdom), Vironostika Uni-Form II Plus O (Manufactured by BioMerieux bv, Boxtel, The Netherlands) or Adaltis Detect HIV v2 (ADALTIS Inc. Montreal, Quebec Canada). If an ELISA test result was positive, a nucleic acid test (HIV RNA PCR, Roche Amplicor Standard Assay V1.5 manufactured by Roche Diagnostics GmbH, Mannheim, Germany) was performed to confirm or refute incident HIV infection in the presence of vaccine-induced antibodies. During the study, clinic staff was blinded to HIV test results. Volunteers who had a positive HIV ELISA test results due to vaccine-induced antibodies at the final study visit are being followed in a long-term follow up study until serological test results return to negative.
Serum anti-Ad5 neutralizing titers were measured at baseline and designated time points throughout the study (
An ELISA assay was used to delineate the antibody response to each individual antigen encoded within the vaccine, as previously described
Neutralization was examined in Groups A and B (rAd5 alone) at baseline, at 2 and 4 weeks post vaccination and at final study visit (12 months after injection), and in Groups C and D (DNA prime – rAd5 boost) at baseline, 2 weeks after the 3rd injection of the DNA vaccine, at week 24 immediately prior to the rAd5 boost, 4 weeks and 6 weeks after the rAd5 boost, and at final study visit (6 months after the boost). Neutralizing activity was assessed against HIV isolates representing easy, moderate and hard to neutralize HIV isolates from around the world. The panel contained two primary isolates from each of the following clades: A, B, C, D, circulating recombinant form CRF01/AE and 2 lab adapted clade B isolates.
Neutralization was evaluated using a single-cycle recombinant pseudotype virus assay by Monogram Biosciences, Inc. (San Francisco, CA) as described previously
Vaccine-induced HIV-1 specific T cell responses were assessed at time points shown in
ICS was measured in 7 volunteers each of Groups C and D, and in 6 placebo recipients at only three visits; baseline, 2 weeks post DNA and 4 weeks post rAd5. PBMC were shipped to NVITAL for testing. The method used to detect CD4 and CD8 T cell responsiveness by secretion of Interleukin-2 (IL-2) and/or IFN-γ to HIV peptides via ICS was based upon previously published methods
VIA was performed as described elsewhere
The initial design called for 12 vaccine and 4 placebo recipients in each of the study arms (a total of 64 volunteers). The study was designed so that if there were no serious adverse events (SAE) related to the vaccine in 48 volunteers, the upper bound of 95% confidence interval was 0.06 for the probability that an SAE related to vaccine could have occurred. Following review of initial safety data of the candidate vaccines, Groups C and D were increased to 27 vaccine and 9 placebo recipients to better inform potential subsequent larger phase II studies. Thus, 104 volunteers were planned to provide enhanced precision of the observed point estimates of safety, tolerability and immunogenicity in volunteers who received the VRC DNA prime - rAd5 boost regimen. An over-enrollment of up to 10% was allowed, resulting in 114 volunteers overall.
The study stopping rules were to go into effect if there were one or more grade 4 adverse events that were judged as probably or definitely related to the study vaccine by the site clinician. If a grade 4 adverse event occurred that was possibly related to the study vaccine or a grade 3 adverse event was judged as definitely, probably or possibly related to the vaccine, the independent DSMB would be informed immediately and discuss whether or not the study should pause until more information could be collected.
Annual interim analyses were performed. Blinded summary tables and listings of adverse events, including solicited reactogenicity events, were presented to an independent DSMB.
The randomization schedule was prepared by the statisticians at the Data Coordinating Center, The EMMES Corporation. The randomization list was sent to the site pharmacist of record for dispensing of vaccine and placebo in a double-blind fashion. Study site staff, volunteers, and laboratories remained blinded with respect to the allocation of placebo or vaccine.
Comparisons of reactogenicity and adverse events were made by assigning scores of 0, 1, 2 and 3 to severities of none, mild, moderate and severe. The Wilcoxon Rank sum test was used to compare 2 groups; the Kruskal-Wallis test was used to compare more than 2 groups. Fisher's exact test was used for 2×2 tables. These comparisons were based on the maximum severity per volunteer. Immunogenicity results were tested using Fisher's exact test (2 groups) or the Kruskal-Wallis test. All tests are 2-tailed; statistical significance is defined as a p-value of <0.05. Analyses were performed using SAS version 9.2, Cary, NC.
As shown in
Number of individuals assessed for eligibility, enrolled and randomized to study vaccine(s) and respective placebo, followed-up and analyzed.
Category | Placebo Recipients | Vaccine Recipients | ||||
Group A/B |
Group C/D |
Overall N = 30 | Group A/B |
Group C/D |
Overall N = 84 | |
Male | 6 (66.7%) | 15 (71.4%) | 21 (70.0%) | 19 (73.1%) | 31 (53.4%) | 50 (59.5%) |
Female | 3 (33.3%) | 6 (28.6%) |
9 (30.0%) | 7 (26.9%) | 27 (46.6%) |
34 (40.5%) |
Mean | 28.5 | 28.4 | 28.4 | 27.5 | 26.3 | 26.7 |
Range | 20.3–44.6 | 19.7–39.0 | 19.7–44.6 | 19.5–48.8 | 18.3–37.8 | 18.3–48.8 |
# Non-Missing | 7 | 21 | 28 | 26 | 57 | 83 |
Geometric Mean | 489 | 338 | 370 | 341 | 288 | 303 |
Range | 113–1819 | 16–3255 | 16–3255 | 16–4679 | 16–5000 | 16–5000 |
9/9 (100%) | 20/21 (95%) | 29/30 (97%) | 26/26 (100%) | 53/58 (91%) | 79/84 (94%) |
Groups A/B: rAd5 or Placebo alone.
Groups C/D: DNA prime - rAd5 boost or Placebo - Placebo.
In Groups C/D, the number of vaccinated women is not statistically different from female placebo recipients.
There were 59 minor protocol deviations, mainly procedural errors or study visits occurring outside the specified window period. There was one violation of an exclusion criterion: a volunteer, enrolled and randomized to Group C, received one injection of study vaccine prior to availability of the result of hepatitis B surface antigen (HbsAg), which was positive. Further vaccinations were discontinued. Individual unblinding at study end revealed that this volunteer was a placebo recipient. The interpretation of the data presented here is not affected by the protocol deviations.
Figure 2a; Local reactogenicity and systemic signs and symptoms collected over 3 days post vaccination. Maximum severity of local reactions was significantly greater after rAd5 than after placebo (p = 0.006 and p<0.001 for rAd5 alone and rAd5 boost, respectively). For systemic signs and symptoms, the severity was significantly greater after DNA (p = 0.036) and after rAd5 boost (p = 0.028), than after the corresponding placebo. Figure 2b; Local reactogenicity and systemic events post rAd5 by dosage. Combining low dosage (LD) groups and high dosage (HD) groups, the maximum systemic reaction per volunteer post rAd5 was significantly higher (p = 0.025) in the HD group than in the LD group (41% versus 21%, respectively, were moderate or severe). Mild: open bars; Moderate: cross-hatched bars; and Severe: dark bars.
Local reactions (pain, tenderness and vaccination site reactions/lesions) were reported by 92%, 85% and 100% of volunteers following rAd5 alone, rAd5 boost and DNA injections (all 3 combined), respectively, and by 44%, 45% and 86% following the respective placebo injections. The most common local reactions were mild (reported by 55.3% of all volunteers), followed by moderate (33.3%) and severe (2.6%). Two volunteers reported severe tenderness upon touch after the 1st and 2nd DNA vaccinations, respectively; a third volunteer reported severe pain and tenderness after the rAd5 boost. Two volunteers had moderate induration after the 1st DNA vaccination, and two had moderate edema after DNA placebo and rA5 boost, respectively. There were no other moderate or greater local reactions. No papules, blisters/vesicles or scabs were observed.
Overall, the maximum severity per volunteer was significantly greater after rAd5 than after the corresponding placebo (p = 0.006 and p<0.001 in Groups A and B and Groups C and D, respectively). However, there was no significant difference between DNA and corresponding placebo (p = 0.137). The maximum severity per volunteer over all local reactions was not associated with dosage (p = 0.301 and p = 0.111 in Groups A and B and Groups C and D, respectively), and there was no significant difference between rAd5 alone or rAd5 boost (p = 0.716).
Systemic reactions were reported by 69%, 72% and 91% of volunteers following rAd5 alone, rAd5 boost and DNA injections (all 3 combined), respectively, and by 78%, 55% and 81% following the respective placebo injections. The most common systemic reactions were mild (reported by 49.1% of all volunteers), followed by moderate (25.4%) and severe (9.6%). Headache, malaise and/or fatigue were most commonly reported. Eleven vaccine recipients experienced severe events, lasting no more than 1–2 days. One volunteer reported severe headache, one severe malaise following the first DNA injection and 9 volunteers (2 from Group C, 7 from Group D) reported severe reactions following rAd5 boost: headache (n = 2); malaise (n = 1); myalgia (n = 1); elevated temperature (n = 2); arthralgia + malaise (n = 1); malaise + headache (n = 1); and malaise + myalgia + headache + arthralgia (n = 1). No severe or greater systemic symptoms were reported after rAd5 alone or in placebo recipients. No nausea or vomiting was reported.
Overall, the maximum severity per volunteer was not significantly different between rAd5 alone and placebo in Groups A and B (p = 0.566). However, in Groups C and D combined, the maximum severity was higher in the vaccines than the placebo group (p = 0.036 and p = 0.028 after any DNA injections and the rAd5 boost, respectively).
Comparing the two dosage levels of rAd5, the maximum severity per volunteer over all systemic reactions was not associated with dosage (p = 0.092 and p = 0.126 after rAd5 alone {Groups A versus B} and rAd5 boost {Group C versus D}, respectively); nor was there a significant difference between rAd5 alone (Groups A and B combined) or rAd5 boost (groups C and D combined) (p = 0.444). If the LD groups (A and C) are combined and the HD groups (B and D) are combined, then the maximum systemic reaction per volunteer post rAd5 is significantly higher (p = 0.025) in the HD group than in the LD group (41% versus 21%, respectively, were moderate or severe).
The severity of local reactions tended to be highest in subjects with baseline Ad5 neutralizing antibody titers to Ad5<19, lower in those with titers >200, and lowest in volunteers with titers between 19–200, but the differences were not statistically significant. Baseline Ad5 neutralizing antibody titers had no effect on systemic signs and symptoms. (
During the 12-month observation period, 777 unsolicited AEs were reported, of which 344 occurred within 28 days after any injection.
Group | Treatment | Grade 1 Mild | Grade 2 Moderate | Grade 3 Severe | Total AEs |
rAd5 Alone | Low Dosage | 12 (80.0%) | 3 (20.0%) | 0 (0.0%) | 15 |
High Dosage | 9 (90.0%) | 1 (10.0%) | 0 (0.0%) | 10 | |
Any Dose | 21 (84.0%) | 4 (16.0%) | 0 (0.0%) | 25 | |
Placebo | 8 (57.1%) | 6 (42.9%) | 0 (0.0%) | 14 | |
3DNA |
Vaccine | 143 (75.7%) | 41 (21.7%) | 5 (2.6%) | 189 |
Placebo | 45 (88.2%) | 6 (11.8%) | 0 (0.0%) | 51 | |
rAd5 Boost | Low Dosage | 22 (91.7%) | 2 (8.3%) | 0 (0.0%) | 24 |
High Dosage | 22 (78.6%) | 5 (17.9%) | 1 (3.6%) | 28 | |
Any Dose | 44 (84.6%) | 7 (13.5%) | 1 (1.9%) | 52 | |
Placebo | 10 (76.9%) | 2 (15.4%) | 1 (7.7%) | 13 |
*AEs occurring within 28 days of any DNA or placebo injection (given at W 0, 4 and 8) combined.
Laboratory abnormalities were mostly mild, with neutropenia (absolute counts) being the most frequent. Moderate neutropenia (750 to <1000 cells/µL) occurred in 6 (7.1%) of the 84 vaccine recipients and 3 (10%) of 30 placebo recipients. One severe (grade 3) neutropenia (740 cells/µL) was detected 2 weeks after the 2nd DNA injection, and one grade 4 neutropenia (450 cells/µL) was detected in an asymptomatic volunteer 11 months after rAd5 alone. Two volunteers had a moderate decrease of hemoglobin (6 months after the 1st DNA placebo and 3 months after rAd5 boost). There were 2 isolated, moderate ALT elevations, at 6 months after rAd5 alone and 3 months after rAd5 boost. There were no other moderate or greater laboratory abnormalities.
During the study, 6 serious adverse events (SAEs) occurred in 6 vaccine recipients. These events were cesarean section, complete abortion, phlebitis, discal hernia, pelvic inflammatory disease, and fracture of the middle finger. None of these events was considered related to study product.
Three women became pregnant during the study. One had completed her vaccination regimen before getting pregnant and delivered a healthy baby. One woman, who had no history of spontaneous abortion, was found to be pregnant 4 weeks after the 1st DNA injection and had a complete spontaneous abortion one month later. The third woman delivered a term male child by cesarean section due to cephalo-pelvic disproportion 41 weeks after the 3rd DNA injection. No abnormalities were detected either at birth or at 14 weeks of age. However, at the age of 12 months the child was found to have a congenital malformation (a large arachnoidal cyst with agenesis of corpus callosum and a parieto-occipital calvarial defect) and underwent endoscopic fenestration and is now reaching age-appropriate developmental milestones. The abnormality was assessed as probably not related to study vaccine and was defined as “a potential relationship between study agent and the adverse event could exist (i.e., the possibility cannot be excluded), but the adverse event is most likely explained by causes other than the study agent”.
There was no intercurrent HIV infection during study follow up. One Group A volunteer acquired HIV about 2 years after receipt of rAd5 alone, i.e., one year after final study visit. At the time of diagnosis – six weeks after the estimated date of infection - the viral load was 929 HIV RNA copies/mL (2.97 logs). The baseline Ad5 neutralizing antibody titer was 830. This volunteer is currently being followed in a long-term follow up study specifically designed for HIV vaccine recipients from any IAVI - sponsored clinical trial who acquire HIV any time during or up to 2 years after the end of a study.
The magnitude, response rate and breadth of HIV-1 specific T-cell responses in each group and the possible impact of pre-existing Ad5 antibodies on these responses were assessed using an IFN-γ ELISPOT assay.
Six weeks after administration of rAd5 1×1010 and rAd5 1×1011, 6/13 (46%) and 7/13 (54%) volunteers, respectively, had one or more positive responses by IFN-γ ELISPOT to any pool (
ELISPOT Responses |
|||||
Group | Dose | Post Vaccination Time Point | Low Dosage | High Dosage | Total |
0/13 0% (0%–25%) | 1/12 8% (0%–38%) | 1/25 4% (0%–20%) | |||
6/13 46% (19%–75%) | 7/13 54% (25%–81%) | 13/26 50% (30%–70%) | |||
4/13 31% (9%–61%) | 4/12 33% (10%–65%) | 8/25 32% (15%–54%) | |||
4/13 31% (9%–61%) | 5/13 38% (14%–68%) | 9/26 35% (17%–56%) | |||
0/29 0% (0%–12%) | 1/28 4% (0%–18%) | 1/57 2% (0%–9%) | |||
11/28 39% (22%–59%) | 13/29 45% (26%–64%) | 24/57 42% (29%–56%) | |||
18/25 72% (51%–88%) | 18/26 69% (48%–86%) | 36/51 71% (56%–83%) | |||
18/27 67% (46%–83%) | 18/27 67% (46%–83%) | 36/54 67% (53%–79%) | |||
22/27 81% (62%–94%) | 20/25 80% (59%–93%) | 42/52 81% (67%–90%) |
Frequency (# Subjects) and percent (exact 95% confidence interval).
Time interval given is post rAd5 boost.
At 4 weeks after the 3rd dose of DNA, and prior to the rAd5 boost, 42% of vaccine recipients from both groups combined had one or more positive responses (
Comparison of percent ELISPOT responders after rAd5 alone (Groups A and B) versus DNA prime - rAd5 boost over time (Groups C and D). Vertical lines represent 95% Confidence Intervals.
The median (range) background-subtracted number of SFC per 106 PBMC from responders 6 weeks after vaccination with rAd5 alone is 85 (52–297) for the low and 78 (39–230) for the high dosage groups, respectively. Similarly, 6 weeks after the rAd5 boost the median (range) SFC counts are 91 (45–1420) and 105 (41–1707) per 106 PBMC, respectively (see also below). The overall differences between dosage groups after rAd5 alone or rAd5 boost were not statistically significant. However, at each visit, the average magnitude of positive responses per volunteer after the rAd5 boost was statistically significantly greater in those who responded to the DNA vaccine compared to those who did not (
Values plotted are the mean background-subtracted ELISPOT counts (Spot Forming Cells, SFC) over all positive peptide responses per volunteer at that visit. Shaded bars are subjects with any positive response at 4 weeks post 3rd DNA, white boxes are those with no positive responses at this time point.
Six weeks after rAd5 alone there were no statistically significant differences between the low and high dosage groups in the proportions of volunteers responding to Pol, EnvA, EnvB or Gag. Similar results were obtained, including responses to Nef, six weeks after the rAd5 boost (
The magnitude of the response was measured by SFC per million peripheral blood mononuclear cells (SFC/m PBMC). The x-axis row one shows the peptide pool, row two the % response rate and row three the vaccine group. Black dots indicate positive responses, defined by background-subtracted values greater than the cutoff, more than 3 times mean background SFC count, and a coefficient of variation of not more than 70% amongst replicate wells. The rAd5 response rates correspond to both dose groups combined. The box plots summarize positive responses only (i.e., median, 1st and 3rd quartiles, minimum/maximum). The cut-offs for EnvA, EnvB, Gag, Nef, PolB1 and PolB2, as determined by the level of non-specific responses from at least 180 samples from unvaccinated individuals, are 40, 51, 54, 68, 51 and 38 respectively. The Pol response is the maximum of PolB1 and PolB2 and positive if either one is positive.
Group | Env A | Env B | Gag | Nef | Pol | Any | |
23.1 | 23.1 | 7.7 | 0.0 | 38.5 | 46.2 | ||
30.8 | 30.8 | 15.4 | 7.7 | 46.2 | 53.8 | ||
25.0 | 22.2 | 17.9 | 18.5 | 3.6 | 39.3 | ||
64.0 | 54.2 | 40.0 | 8.3 | 20.0 | 72.0 | ||
20.7 | 24.1 | 31.0 | 7.4 | 0.0 | 44.8 | ||
50.0 | 42.3 | 42.3 | 11.5 | 26.9 | 69.2 | ||
DNA priming improved the proportion of vaccine recipients with any positive response at more than one time point after the rAd5 vaccination, with 31, 54, 69 and 64% in Groups A, B, C and D, respectively. The difference was not statistically significant (p = 0.122 between all four groups, and p = 0.054 for Groups A and B versus Groups C and D, i.e., 42% versus 67%, respectively).
The distribution of baseline Ad5 titers is similar across Groups A to D (p = 0.332). There was no significant difference in response rate to vaccination in any group of vaccine recipients depending on baseline anti-Ad5 titers. (
Samples from 13 vaccine and 5 placebo recipients from groups C and D were assessed using an IL-2/IFN-γ ICS assay at 2 weeks post DNA prime and 4 weeks post Ad5 boost. The percent response rates for ICS are shown in
Number of responders/total tested (% responders) | ||||||
Baseline | 2 weeks post 3DNA | 4 weeks post rAd5 | ||||
Peptide Pool | V02 | Placebo | 3DNA | Placebo | Low Dosage | High Dosage |
0/13 (0.0%) | 0/5 (0.0%) | |||||
0/13 (0.0%) | 0/5 (0.0%) | 0/5 (0.0%) | ||||
0/13 (0.0%) | 0/5 (0.0%) | 0/5 (0.0%) | ||||
0/13 (0.0%) | 0/5 (0.0%) | |||||
0/13 (0.0%) | 0/5 (0.0%) | 0/5 (0.0%) | ||||
0/11 (0.0%) | 0/5 (0.0%) | 0/5 (0.0%) | 0/5 (0.0%) | |||
0/13 (0.0%) | 0/5 (0.0%) | 0/13 (0.0%) | 0/5 (0.0%) | 0/6 (0.0%) | 0/7 (0.0%) | |
0/13 (0.0%) | 0/5 (0.0%) | 0/5 (0.0%) | ||||
0/13 (0.0%) | 0/5 (0.0%) | 0/5 (0.0%) | ||||
0/13 (0.0%) | 0/5 (0.0%) | 0/5 (0.0%) | ||||
0/13 (0.0%) | 0/5 (0.0%) | 0/13 (0.0%) | 0/5 (0.0%) | 0/6 (0.0%) | 0/7 (0.0%) | |
0/13 (0.0%) | 0/5 (0.0%) | 0/13 (0.0%) | 0/5 (0.0%) | 0/6 (0.0%) | ||
0/13 (0.0%) | 0/5 (0.0%) | 0/5 (0.0%) | 0/7 (0.0%) | |||
0/13 (0.0%) | 0/5 (0.0%) | 0/5 (0.0%) |
*Two volunteers had CD4 responses to Nef at all 3 visits. These Nef responses have been excluded.
A viral inhibition assay (VIA) has previously been optimized and qualified using PBMC from HIV-infected individuals with a range of viral loads (VL) and uninfected low risk volunteers. To determine the criteria for assay positivity, viral inhibition from 43 HIV–uninfected subjects was assessed. A cut-off of >1.13 log10 viral inhibition was established based on a median of 0.70 log10 inhibition of p24 release from HIV-1IIIB-infected CD4 T cells with >99% confidence. With a qualified viral inhibition assay, HIV seropositive individuals had a range of responses with a trend toward higher viral inhibition in those with lower viral loads. Treatment-naive HIV+ subjects with a plasma VL of <10 000/mL had median viral inhibition of 3.17 log10, whereas those with VL >10000 had median viral inhibition of 1.12 log10
Viral inhibition was assessed in placebo recipients and prior to any vaccination (•); following DNA alone (▪); DNA prime - rAd5 boost at 6 and 12 weeks (▴and ▵respectively) and after rAd5 alone at 6 and between 36 to 48 weeks (♦ and ⋄ respectively). The 1.13 log10 inhibition value above which inhibition is considered HIV-1 specific is indicated by the hashed line. Medians for all groups are indicated (—).
VIA activity was found in the presence of moderate to high Ad5 neutralization titers. At 4 weeks post rAd5, Ad5 neutralization titers were available from 13 of the 14 vaccinees, 9 of the 13 individuals had titers >8748 and 4 had titers >200 (1305–2667). No samples were available from individuals with Ad5 titers of <200.
After rAd5 alone, antibodies to EnvA, EnvB and EnvC were detected in, respectively, 25%, 45% and 42% of volunteers in Group A and in 50%, 58% and 58% of volunteers in Group B. After the rAd5 boost, the response to each Env protein was 89% in Group C and 100% in Group D. There was no effect of dosage. Antibodies to Gag were detected after the LD and HD rAd5 boost in 25% and 31% of volunteers, respectively, compared to 8% and 0% after rAd5 alone, whereas antibodies to pol and nef were detected infrequently in all groups (data not shown). None of the placebo group had antibody responses to the HIV antigens. Antibody ELISA titers are shown in
Distribution of HIV-specific antibody titers at 1 month post rAd5 alone (Groups A and B) and rAd5 boost (Groups C and D) in vaccine recipients, by protein and treatment group. The Y-axis shows the antibody titer on a log scale with box plots showing the median, 1st and 3rd quartiles.
Only 2 individuals in Group D had moderate neutralizing activity against SF162 and no detectable neutralizing activity against 12 other HIV-1 isolates (data not shown). Similar HIV-1 binding ELISA and HIV neutralization activity was seen in individuals in a recently published study of the VRC DNA + Ad5 regimen
Twelve months after rAd5 alone and 6 months after the rAd5 boost, 10/13 (76%) and 48/57 (84%) vaccine recipients, respectively, had a positive HIV test result by at least one commercial HIV ELISA kit without being HIV-infected. Four months after 3 DNA injections and immediately prior to the rAd5 boost, only 6/57 (10%) vaccine recipients had a positive HIV test result. The Detect HIV v2 kit did not detect vaccine-induced antibodies in any vaccine recipient. 2/30 (7%) placebo recipients had a false positive HIV test result.
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
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
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
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
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
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;
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 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
Local reactogenicity by Ad5 baseline titers. Ad5 neutralizing titers were stratified by values obtained prior to vaccination; <19, 19–200 and >200 as measured by the Crucell luciferase-based assay. N = numbers of individuals in each group.
(0.33 MB TIF)
Systemic reactogenicity by Ad5 baseline titers. Ad5 neutralizing titers were stratified by values obtained prior to vaccination; <19, 19–200 and >200 as measured by the Crucell luciferase-based assay. N = numbers of individuals in each group.
(0.22 MB TIF)
Percent of volunteers responding to HIV-peptide pools. The table shows the number of subjects per group at each time point that contributed ELISPOT data for the bar graph. Six pools are included in the analysis; Gag, Nef, Env A, Env B and two Pol pools. The numbers inside the bars represent percent of the responder frequencies.
(0.31 MB TIF)
Impact of Ad5 neutralizing antibody on IFN-γ ELISPOT responses. The bars show the cumulative proportion of vaccine recipients with positive ELISPOT responses in those with a baseline Ad5 titer >200 after the rAd5 boost in groups A–D. The p-values shown on the X-axis are based on Fisher's exact 2-tailed test.
(0.21 MB TIF)
A Phase I Randomized, Placebo-Controlled, Double-Blind Trial to Evaluate the Safety and Immunogenicity of a Multiclade HIV-1 DNA Plasmid Vaccine Followed by Recombinant, Multiclade HIV-1 Adenoviral Vector Vaccine or the Multiclade HIV-1 Adenoviral Vector Vaccine Alone in Healthy Adult Volunteers not Infected with HIV
(5.64 MB PDF)
CONSORT Checklist
(0.22 MB DOC)
We express our thanks to all the study volunteers, to the research staff at the collaborating centers for their outstanding work and dedication, to VRC and DAIDS for their constant support and guidance, and to the DSMB for overseeing the study. For more information, see