Vaccines inducing pathogen-specific cell-mediated immunity are being developed using attenuated adenoviral (Ad) vectors. We report the results of two independent Phase I trials of similar replication-deficient Ad5 vaccines containing a near-consensus HIV-1 clade B gag transgene. Healthy HIV-uninfected adults were enrolled in two separate, multicenter, dose-escalating, blinded, placebo-controlled studies to assess the safety and immunogenicity of a three-dose homologous regimen of Ad5 and MRKAd5 HIV-1 gag vaccines given on day 1, week 4, and week 26. Adverse events were collected for 29 days following each intradeltoid injection. The primary immunogenicity endpoint was the proportion of subjects with a positive unfractionated Gag-specific IFN-γ ELISPOT response measured 4 weeks after the last dose (week 30). Analyses were performed after combining data for each dose group from both protocols, stratifying by baseline Ad5 titers. Overall, 252 subjects were randomized to receive either vaccine or placebo, including 229 subjects (91%) who completed the study through week 30. Tolerability and immunogenicity did not appear to differ between the Ad5 and MRKAd5 vaccines. The frequency of injection-site reactions was dose dependent. Systemic adverse events were also dose dependent and more frequent in subjects with baseline Ad5 titers <200 versus ≥200, especially after the first dose. The percent of ELISPOT responders and the ELISPOT geometric means overall were significantly higher for all four vaccine doses studied compared to placebo, and were generally higher in vaccine recipients with baseline Ad5 titers <200 versus ≥200. Ad5 titers increased after vaccination in a dose-dependent fashion. Both Ad5-vectored HIV-1 vaccines were generally well tolerated and induced cell-mediated immune responses against HIV Gag-peptides in the majority of healthy adults with baseline Ad5 titers <200. Preexistent and/or vaccine-induced immunity to the Ad5 vector may dampen the CMI response to HIV Gag.
We report the primary analysis of the safety and efficacy of the MRKad5 gag/pol/nef HIV-1 sub-type B vaccine in South Africa (SA), where the major circulating clade is sub-type C.
This phase IIb double-blind, randomized test-of-concept study was conducted in sexually active HIV-1 sero-negative participants in SA. The co-primary endpoints were a vaccine-induced reduction in HIV-1 acquisition or viral-load setpoint. These were assessed independently in the modified intent-to-treat (MITT) cohort with two-tailed significance tests stratified by gender. Immunogenicity was assessed by interferon-gamma (IFNγ) ELISPOT in peripheral blood mononuclear cells. Following the lack of efficacy of the MRKAd5 HIV-1 vaccine in the Step study, enrollment and vaccination in this study was halted, treatment unblinding occurred and follow-up continued. This study is registered with the SA National Health Research Database (DOH-27-0207-1539) and ClinicalTrials.gov (NCT00413725).
801 of a scheduled 3000 participants were enrolled, of whom 360 (44.9%) were women, more than half (55.6%) had Ad5 titres > 200, and almost a third (29.3%) of men were circumcised. 62 MITT participants were diagnosed with HIV-1, 34 in the vaccine arm and 28 in the placebo arm, with infection rates of 4.54 and 3.70 per 100 person-years, respectively. There was no evidence of vaccine efficacy (VE); the hazard ratio adjusted for gender was 1.25 (95% CI: 0.76, 2.05). VE did not differ by Ad5 titre, gender, age, HSV-2 status, or circumcision. The geometric mean viral load setpoint was 20,483 copies/ml (N=33) in vaccinees and 34,032 copies/ml (N=28) in placebo recipients (p=0.39). The vaccine elicited IFNγ-secreting T cells recognizing both clade B (89.2%) and C (77.4%) antigens.
The MRKAd5 HIV-1 vaccine did not prevent HIV-1 infection or lower viral-load setpoint however early stopping likely compromised our ability to draw conclusions. The high incidence rates seen in SA highlight the critical need for intensified efforts to develop an efficacious vaccine.
HIV; HIV vaccine efficacy studies; South Africa
The development of an effective human immunodeficiency virus (HIV) vaccine is a high global priority. Here, we report the safety, tolerability, and immunogenicity of a replication-defective recombinant adenovirus serotype 5 (rAd5) vector HIV-1 candidate vaccine.
The vaccine is a mixture of 4 rAd5 vectors that express HIV-1 subtype B Gag-Pol fusion protein and envelope (Env) from subtypes A, B, and C. Healthy, uninfected adults were randomized to receive 1 intramuscular injection of placebo (n = 6) or vaccine at dose levels of 109 (n = 10), 1010 (n = 10), or 1011 (n = 10) particle units and were followed for 24 weeks to assess immunogenicity and safety.
The vaccine was well tolerated but was associated with more reactogenicity at the highest dose. At week 4, vaccine antigen–specific T cell responses were detected in 28 (93.3%) and 18 (60%) of 30 vaccine recipients for CD4+ and CD8+ T cells, respectively, by intracellular cytokine staining assay and in 22 (73%) of 30 vaccine recipients by enzyme-linked immunospot assay. Env-specific antibody responses were detected in 15 (50%) of 30 vaccine recipients by enzyme-linked immunosorbant assay and in 28 (93.3%) of 30 vaccine recipients by immunoprecipitation followed by Western blotting. No neutralizing antibody was detected.
A single injection induced HIV-1 antigen–specific CD4+ T cell, CD8+ T cell, and antibody responses in the majority of vaccine recipients. This multiclade rAd5 HIV-1 vaccine is now being evaluated in combination with a multiclade HIV-1 DNA plasmid vaccine.
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.
Preexisting immunity to adenovirus serotype 5 (Ad5) diminishes immune responses to vaccines using Ad5 as a vector. Alternate Ad serotypes as vaccine vectors might overcome Ad5-specific neutralizing antibodies and enhance immune responses in populations with a high prevalence of Ad5 immunity. To test this hypothesis, healthy human immunodeficiency virus (HIV)-seronegative adults were enrolled in a blinded, randomized, dose-escalating, placebo-controlled study. In part A, subjects with baseline Ad6 titers of ≤18 received the Merck Ad6 (MRKAd6) HIV type 1 (HIV-1) trigene vaccine at weeks 0, 4, and 26. In part B, subjects stratified by Ad5 titers (≤200 or >200) and Ad6 titers (≤18 or >18) received the MRKAd5-plus-MRKAd6 (MRKAd5+6) HIV-1 trigene vaccine at weeks 0, 4, and 26. Immunogenicity was assessed by an enzyme-linked immunospot (ELISPOT) assay at week 30. No serious adverse events occurred. MRKAd6 trigene vaccine recipients responded more often to Nef than to Gag or Pol. In part A, ELISPOT response rates to ≥2 vaccine antigens were 14%, 63%, and 71% at 109, 1010, and 1011 viral genomes (vg)/dose, respectively. All responders had positive Nef-specific ELISPOT results. In part B, Nef-ELISPOT response rates at 1010 vg/dose of the MRKAd5+6 trigene vaccine were 50% in the low-Ad5/low-Ad6 stratum (n = 8), 78% in the low-Ad5/high-Ad6 stratum (n = 9), 75% in the high-Ad5/low-Ad6 stratum (n = 8), and 44% in the high-Ad5/high-Ad6 stratum (n = 9). The MRKAd6 and MRKAd5+6 trigene vaccines elicited dose-dependent responses predominantly to Nef and were generally well tolerated, indicating that Ad6 should be considered a candidate vector for future vaccines. Although small sample sizes limit the conclusions that can be drawn from this exploratory study, combining two Ad vectors may be a useful vaccine strategy for circumventing isolated immunity to a single Ad serotype.
HIV Gag-specific CD4+ and CD8+ T-cell responses are important for HIV immune control. Pulsing overlapping Gag peptides on autologous lymphocytes (OPAL) has proven immunogenic and effective in reducing viral loads in multiple pigtail macaque studies, warranting clinical evaluation.
We performed a phase I, single centre, placebo-controlled, double-blinded and dose-escalating study to evaluate the safety and preliminary immunogenicity of a novel therapeutic vaccine approach ‘OPAL-HIV-Gag(c)’. This vaccine is comprised of 120 15mer peptides, overlapping by 11 amino acids, spanning the HIV Gag C clade sequence proteome, pulsed on white blood cells enriched from whole blood using a closed system, followed by intravenous reinfusion. Patients with undetectable HIV viral loads (<50 copies/ml plasma) on HAART received four administrations at week 0, 4, 8 and 12, and were followed up for 12 weeks post-treatment. Twenty-three people were enrolled in four groups: 12 mg (n = 6), 24 mg (n = 7), 48 mg (n = 2) or matching placebo (n = 8) with 18 immunologically evaluable. T-cell immunogenicity was assessed by IFNγ ELIspot and intracellular cytokine staining (ICS).
The OPAL-HIV-Gag(c) peptides were antigenic in vitro in 17/17 subjects. After vaccination with OPAL-HIV-Gag(c), 1/6 subjects at 12 mg and 1/6 subjects at 24 mg dose groups had a 2- and 3-fold increase in ELIspot magnitudes from baseline, respectively, of Gag-specific CD8+ T-cells at week 14, compared to 0/6 subjects in the placebo group. No Gag-specific CD4+ T-cell responses or overall change in Rev, Nef, Tat and CMV specific responses were detected. Marked, transient and self-limiting lymphopenia was observed immediately post-vaccination (4 hours) in OPAL-HIV-Gag(c) but not in placebo recipients, with median fall from 1.72 to 0.67 million lymphocytes/mL for active groups (P<0.001), compared to post-placebo from 1.70 to 1.56 lymphocytes/ml (P = 0.16).
Despite strong immunogenicity observed in several Macaca nemestrina studies using this approach, OPAL-HIV-Gag(c) was not significantly immunogenic in humans and improved methods of generating high-frequency Gag-specific T-cell responses are required.
Name of Registry
ClinicalTrials.gov, Registry number: NCT01123915, URL trial registry database: http://www.clinicaltrials.gov/ct2/results?term=OPAL-HIV-1001&Search=Search
In the Step Study, the MRKAd5 HIV-1 gag/pol/nef vaccine did not lower post-infection plasma viremia, and HIV-1 incidence was higher in vaccine-treated than placebo-treated males with pre-existing adenovirus serotype 5 (Ad5) immunity. We evaluated vaccine-induced immunity and its potential contributions to infection risk.
To assess immunogenicity, HIV-specific T-cells were characterized ex vivo using validated IFN-γ ELISpot and intracellular cytokine staining (ICS) assays, employing a case-cohort design. To determine effects of vaccine and pre-existing Ad5 immunity on infection risk, flow cytometric studies measured Ad5-specific T-cells and circulating activated (Ki67+/Bcl- 2lo) CD4+ T-cells expressing CCR5.
IFN-γ-secreting HIV-specific T-cells (range, 163–686/106 PBMC) were detected ex vivo by ELISpot in 77% (258/354) of vaccinees; the majority recognized 2–3 HIV proteins. HIV- specific CD4+ T-cells were identified by ICS in 41%; ~85% expressed IL-2, and two-thirds of these co-expressed IFN-γ and/or TNF-α. HIV-specific CD8+ T-cells (range, 0.4–1.0%) were observed in 73%, expressing predominantly either IFN-γ alone or with TNF-α. No major differences were found in vaccine-induced HIV-specific immunity, including response rate, magnitude, and cytokine profile comparing vaccinated male cases (pre-infection) with non-cases. Interestingly, Ad5-specific T-cells were lower in cases than non-cases in several subgroup analyses. The percent circulating Ki67+Bcl-2lo/CCR5+ CD4+ T-cells did not differ between cases and non-cases.
Consistent with previous trials, the MrkAd5/HIV-1 gag/pol/nef vaccine was highly immunogenic for inducing HIV-specific CD8+ T-cells. Comparative analyses did not reveal differences in HIV-specific immunologic responses between cases and non-cases that explain the lack of vaccine efficacy and potential infection enhancement. If T-cell immunity is critical in vaccine-induced HIV protection, our findings suggest that future candidate vaccines must elicit responses that either exceed in magnitude or differ in breadth and/or function from those observed in this trial.
National Institute of Allergy and Infectious Diseases, U.S. National Institute of Health; Merck Research Laboratories
The results of the recent Step Study highlight a need to clarify the effects of pre-existing natural immunity to a vaccine vector on vaccine-induced T-cell responses. To investigate this interaction, we examined the relationship between pre-existing Ad5 immunity and T-cell cytokine response profiles in healthy, HIV-uninfected recipients of MRKAd5 HIV-1 gag vaccine (HVTN 050, ClinicalTrials.gov #NCT00849732). Participants were grouped by baseline Ad5 neutralizing antibody titer as either Ad5-seronegative (titer ≤18; n = 36) or Ad5-seropositive (titer >200; n = 34). Samples from vaccine recipients were analyzed for immune responses to either HIV-1 Gag peptide pools or Ad5 empty vector using an ex vivo assay that measures thirty cytokines in the absence of long-term culture. The overall profiles of cytokine responses to Gag and Ad5 had similar combinations of induced Th1- and Th2-type cytokines, including IFN-γ, IL-2, TNF-α, IP-10, IL-13, and IL-10, although the Ad5-specific responses were uniformly higher than the Gag-specific responses (p<0.0001 for 9 out of 11 significantly expressed analytes). At the peak response time point, PBMC from Ad5-seronegative vaccinees secreted significantly more IP-10 in response to Gag (p = 0.008), and significantly more IP-10 (p = 0.0009), IL-2 (p = 0.006) and IL-10 (p = 0.05) in response to Ad5 empty vector than PBMC from Ad5-seropositive vaccinees. Additionally, similar responses to the Ad5 vector prior to vaccination were observed in almost all subjects, regardless of Ad5 neutralizing antibody status, and the levels of secreted IFN-γ, IL-10, IL-1Ra and GM-CSF were blunted following vaccination. The cytokine response profile of Gag-specific T cells mirrored the Ad5-specific response present in all subjects before vaccination, and included a number of Th1- and Th2-associated cytokines not routinely assessed in current vaccine trials, such as IP-10, IL-10, IL-13, and GM-CSF. Together, these results suggest that vector-specific humoral responses may reduce vaccine-induced T-cell responses by previously undetected mechanisms.
On the basis of positive preclinical data, we evaluated the safety and immunogenicity of an alphavirus replicon HIV-1 subtype C gag vaccine (AVX101), expressing a nonmyristoylated form of Gag, in two double-blind, randomized, placebo-controlled clinical trials in healthy HIV-1-uninfected adults. Escalating doses of AVX101 or placebo were administered subcutaneously to participants in the United States and Southern Africa. Because of vaccine stability issues, the first trial was halted prior to completion of all dose levels and a second trial was implemented. The second trial was also stopped prematurely due to documentation issues with the contract manufacturer. Safety and immunogenicity were evaluated through assessments of reactogenicity, reports of adverse events, and assessment of replication-competent and Venezuelan equine encephalitis (VEE) viremia. Immunogenicity was measured using the following assays: enzyme-linked immunosorbent assay (ELISA), chromium 51 (51Cr)-release cytotoxic T lymphocyte (CTL), gamma interferon (IFN-γ) ELISpot, intracellular cytokine staining (ICS), and lymphoproliferation assay (LPA). Anti-vector antibodies were also measured. AVX101 was well tolerated and exhibited only modest local reactogenicity. There were 5 serious adverse events reported during the trials; none were considered related to the study vaccine. In contrast to the preclinical data, immune responses in humans were limited. Only low levels of binding antibodies and T-cell responses were seen at the highest doses. This trial also highlighted the difficulties in developing a novel vector for HIV.
A randomized, double-blind, placebo controlled phase I trial.
The trial was conducted in 32 HIV-uninfected healthy volunteers to assess the safety and immunogenicity of prime-boost vaccination regimens with either 2 doses of ADVAX, a DNA vaccine containing Chinese HIV-1 subtype C env gp160, gag, pol and nef/tat genes, as a prime and 2 doses of TBC-M4, a recombinant MVA encoding Indian HIV-1 subtype C env gp160, gag, RT, rev, tat, and nef genes, as a boost in Group A or 3 doses of TBC-M4 alone in Group B participants. Out of 16 participants in each group, 12 received vaccine candidates and 4 received placebos.
Both vaccine regimens were found to be generally safe and well tolerated. The breadth of anti-HIV binding antibodies and the titres of anti-HIV neutralizing antibodies were significantly higher (p<0.05) in Group B volunteers at 14 days post last vaccination. Neutralizing antibodies were detected mainly against Tier-1 subtype B and C viruses. HIV-specific IFN-γ ELISPOT responses were directed mostly to Env and Gag proteins. Although the IFN-γ ELISPOT responses were infrequent after ADVAX vaccinations, the response rate was significantly higher in group A after 1st and 2nd MVA doses as compared to the responses in group B volunteers. However, the priming effect was short lasting leading to no difference in the frequency, breadth and magnitude of IFN-γELISPOT responses between the groups at 3, 6 and 9 months post-last vaccination.
Although DNA priming resulted in enhancement of immune responses after 1st MVA boosting, the overall DNA prime MVA boost was not found to be immunologically superior to homologous MVA boosting.
Clinical Trial Registry CTRI/2009/091/000051
To evaluate the safety and immunogenicity of a candidate HIV DNA vaccine administered using a needle-free device.
In this phase 1, dose escalation, double-blind, placebo-controlled clinical trial, 21 healthy adults were randomized to receive placebo or 0.5, 1.5, or 4 mg of a single plasmid expressing a Gag/Pol fusion protein. Each participant received repeat immunizations at days 28 and 56 after the first inoculation. Safety and immunogenicity data were collected.
The vaccine was well tolerated, with most adverse events being mild injection site reactions, including pain, tenderness, and erythema. No dose-limiting toxicities occurred. HIV-specific antibody response was not detected in any vaccinee by enzyme-linked immunosorbent assay. HIV-specific T-cell responses to Gag or Pol as measured by enzyme-linked immunospot assay and intracellular cytokine staining were of low frequency and magnitude.
This candidate HIV DNA vaccine was safe and well tolerated. No HIV-specific antibody responses were detected, and only low-magnitude HIV-specific T-cell responses were detected in 8 (53%) of 15 vaccinees. This initial product led to the development of a 4-plasmid multiclade HIV DNA Vaccine Research Center vaccine candidate in which envelope genes expressing Env from clades A, B, and C and a Nef gene from clade B have been added.
CD4+ T-cell immune response; gene delivery; immunization; needle-free device; plasmid vaccine; safety
A novel AS01-adjuvanted HIV-1 vaccine candidate consisting of a recombinant fusion protein (F4) containing 4 HIV-1 clade B antigens (Gag p24, Pol reverse transcriptase [RT], Nef and Gag p17) induced long-lasting, polyfunctional cross-reactive CD4+ T-cell responses in HIV-seronegative volunteers.
Background. This phase I/II partially blinded, randomized, dose-ranging study assessed the safety and immunogenicity of a novel human immunodeficiency virus type 1 (HIV-1) vaccine candidate consisting of a recombinant fusion protein (F4) containing 4 HIV-1 clade B antigens (Gag p24, Pol reverse transcriptase, Nef, and Gag p17) adjuvanted with AS01 in HIV-seronegative volunteers.
Methods. Two doses of the recombinant F4 protein (10, 30, or 90 μg/dose), adjuvanted with AS01 or reconstituted with water for injection, were administered 1 month apart to 180 healthy volunteers aged 18–40 years. F4-specific CD4+ T cell responses were measured using intracellular cytokine staining after in vitro stimulation by overlapping peptide pools covering the 4 individual antigens.
Results. Reactogenicity was higher during the 7-day period after each vaccine dose in the adjuvanted than in the nonadjuvanted groups. In the adjuvanted groups, the overall immune response rate was high after the second vaccine dose, with highest responder rates seen in the 10-μg F4/AS01 group (100% to 3 HIV-1 antigens and 80% to all 4 HIV-1 antigens). High and long-lasting CD4+ T cell frequencies were observed (up to a median value of 1.2% F4-specific CD4+ T cells at day 44), with strongest responses directed against reverse transcriptase. Antigen-specific CD4+ T cells exhibited a polyfunctional phenotype, expressing at least CD40 ligand and interleukin 2, often in combination with tumor necrosis factor α and/or interferon γ. Vaccine-induced CD4+ T cell responses were broadly cross-reactive to all 4 antigens derived from HIV-1 clades A and C.
Conclusions. These results support further clinical investigation of this HIV-1 vaccine candidate both in a prophylactic setting (alone, in conjunction with an envelope-based antigen or in combination with other vaccine approaches in a heterologous prime-boost regimen) and as a potentially disease-modifying therapeutic vaccine in HIV-1–infected subjects.
Clinical trials registration. NCT00434512.
The recently released results of the Merck's Phase IIb "test-of concept" vaccine trials have shown no protection from HIV-1 infection in the vaccinated group compared with a control group vaccinated with placebo. The study was designed to test the Merck's MRKAd5 trivalent candidate vaccine. The vaccine formulation was expected to stimulate a HIV-specific T cell immune response and to either prevent infection, or to reduce the levels of the viral load in vaccinated subjects. Upon the first evaluation of the interim data, the independent Data and Safety Monitoring Board (DSMB) underscored no protection from HIV-1 infection in the vaccine-inoculated volunteers compared with the control group; accordingly, the vaccine trial was stopped. This disappointing outcome warrants a critical analysis of the current vaccine studies and calls for a renewed effort toward a rational design of novel immunogens to be tested in large primate trials.
The objective was to compare the safety and cellular immunogenicity of intradermal versus intramuscular immunization with an HIV-lipopeptide candidate vaccine (LIPO-4) in healthy volunteers.
A randomized, open-label trial with 24 weeks of follow-up was conducted in France at six HIV-vaccine trial sites. Sixty-eight healthy 21– to 55–year-old HIV-uninfected subjects were randomized to receive the LIPO-4 vaccine (four HIV lipopeptides linked to a T-helper–stimulating epitope of tetanus-toxin protein) at weeks 0, 4 and 12, either intradermally (0.1 ml, 100 µg of each peptide) or intramuscularly (0.5 ml, 500 µg of each peptide). Comparative safety of both routes was evaluated. CD8+ T-cell immune responses to HIV epitopes (ELISpot interferon-γ assay) and tetanus toxin-specific CD4+ T-cell responses (lymphoproliferation) were assessed at baseline, two weeks after each injection, and at week 24.
Results and Conclusion
No severe, serious or life-threatening adverse events were observed. Local pain was significantly more frequent after intramuscular injection, but local inflammatory reactions were more frequent after intradermal immunization. At weeks 2, 6, 14 and 24, the respective cumulative percentages of induced CD8+ T-cell responses to at least one HIV peptide were 9, 33, 39 and 52 (intradermal group) or 14, 20, 26 and 37 (intramuscular group), and induced tetanus toxin-specific CD4+ T-cell responses were 6, 27, 33 and 39 (intradermal), or 9, 46, 54 and 63 (intramuscular). In conclusion, intradermal LIPO-4 immunization was well tolerated, required one-fifth of the intramuscular dose, and induced similar HIV-specific CD8+ T-cell responses. Moreover, the immunization route influenced which antigen-specific T-cells (CD4+ or CD8+) were induced.
We conducted a Phase I dose-escalation trial of ADMVA, a Clade-B'/C-based HIV-1 candidate vaccine expressing env, gag, pol, nef, and tat in a modified vaccinia Ankara viral vector. Sequences were derived from a prevalent circulating HIV-1 recombinant form in Yunnan, China, an area of high HIV incidence. The objective was to evaluate the safety and immunogenicity of ADMVA in human volunteers.
ADMVA or placebo was administered intramuscularly at months 0, 1 and 6 to 50 healthy adult volunteers not at high risk for HIV-1. In each dosage group [1×107 (low), 5×107 (mid), or 2.5×108 pfu (high)] volunteers were randomized in a 3∶1 ratio to receive ADMVA or placebo in a double-blinded design. Subjects were followed for local and systemic reactogenicity, adverse events including cardiac adverse events, and clinical laboratory parameters. Study follow up was 18 months. Humoral immunogenicity was evaluated by anti-gp120 binding ELISA, immunoflourescent staining, and HIV-1 neutralization. Cellular immunogenicity was assessed by a validated IFNγ ELISpot assay and intracellular cytokine staining. Anti-vaccinia binding titers were measured by ELISA.
ADMVA was generally well-tolerated, with no vaccine-related serious adverse events or cardiac adverse events. Local or systemic reactogenicity events were reported by 77% and 78% of volunteers, respectively. The majority of events were of mild intensity. The IFNγ ELISpot response rate to any HIV antigen was 0/12 (0%) in the placebo group, 3/12 (25%) in the low dosage group, 6/12 (50%) in the mid dosage group, and 8/13 (62%) in the high dosage group. Responses were often multigenic and occasionally persisted up to one year post vaccination. Antibodies to gp120 were detected in 0/12 (0%), 8/13 (62%), 6/12 (50%) and 10/13 (77%) in the placebo, low, mid, and high dosage groups, respectively. Antibodies persisted up to 12 months after vaccination, with a trend toward agreement with the ability to neutralize HIV-1 SF162 in vitro. Two volunteers mounted antibodies that were able to neutralize clade-matched viruses.
ADMVA was well-tolerated and elicited durable humoral and cellular immune responses.
We conducted a phase I, randomized, double-blind, placebo-controlled trial to assess the safety and immunogenicity of escalating doses of two recombinant replication defective adenovirus serotype 35 (Ad35) vectors containing gag, reverse transcriptase, integrase and nef (Ad35-GRIN) and env (Ad35-ENV), both derived from HIV-1 subtype A isolates. The trial enrolled 56 healthy HIV-uninfected adults.
Ad35-GRIN/ENV (Ad35-GRIN and Ad35-ENV mixed in the same vial in equal proportions) or Ad35-GRIN was administered intramuscularly at 0 and 6 months. Participants were randomized to receive either vaccine or placebo (10/4 per group, respectively) within one of four dosage groups: Ad35-GRIN/ENV 2×109 (A), 2×1010 (B), 2×1011 (C), or Ad35-GRIN 1×1010 (D) viral particles.
No vaccine-related serious adverse event was reported. Reactogenicity events reported were dose-dependent, mostly mild or moderate, some severe in Group C volunteers, all transient and resolving spontaneously. IFN-γ ELISPOT responses to any vaccine antigen were detected in 50, 56, 70 and 90% after the first vaccination, and in 75, 100, 88 and 86% of Groups A–D vaccine recipients after the second vaccination, respectively. The median spot forming cells (SFC) per 106 PBMC to any antigen was 78–139 across Groups A–C and 158–174 in Group D, after each of the vaccinations with a maximum of 2991 SFC. Four to five HIV proteins were commonly recognized across all the groups and over multiple timepoints. CD4+ and CD8+ T-cell responses were polyfunctional. Env antibodies were detected in all Group A–C vaccinees and Gag antibodies in most vaccinees after the second immunization. Ad35 neutralizing titers remained low after the second vaccination.
Ad35-GRIN/ENV reactogenicity was dose-related. HIV-specific cellular and humoral responses were seen in the majority of volunteers immunized with Ad35-GRIN/ENV or Ad35-GRIN and increased after the second vaccination. T-cell responses were broad and polyfunctional.
The EuroVacc 02 phase I trial has evaluated the safety and immunogenicity of a prime-boost regimen comprising recombinant DNA and the poxvirus vector NYVAC, both expressing a common immunogen consisting of Env, Gag, Pol, and Nef polypeptide domain from human immunodeficiency virus (HIV)-1 clade C isolate, CN54. 40 volunteers were randomized to receive DNA C or nothing on day 0 and at week 4, followed by NYVAC C at weeks 20 and 24. The primary immunogenicity endpoints were measured at weeks 26 and 28 by the quantification of T cell responses using the interferon γ enzyme-linked immunospot assay. Our results indicate that the DNA C plus NYVAC C vaccine regimen was highly immunogenic, as indicated by the detection of T cell responses in 90% of vaccinees and was superior to responses induced by NYVAC C alone (33% of responders). The vaccine-induced T cell responses were (a) vigorous in the case of the env response (mean 480 spot-forming units/106 mononuclear cells at weeks 26/28), (b) polyfunctional for both CD4 and CD8 T cell responses, (c) broad (the average number of epitopes was 4.2 per responder), and (d) durable (T cell responses were present in 70% of vaccinees at week 72). The vaccine-induced T cell responses were strongest and most frequently directed against Env (91% of vaccines), but smaller responses against Gag-Pol-Nef were also observed in 48% of vaccinees. These results support the development of the poxvirus platform in the HIV vaccine field and the further clinical development of the DNA C plus NYVAC C vaccine regimen.
Preclinical studies of overlapping 15mer peptides, spanning SIV, SHIV or HIV, pulsed on autologous PBMC ex vivo have demonstrated high level, virus-specific T cell responses and viral suppression in non-human primates (NHP). Opal-HIV-Gag(c) consists of 120 synthetic 15mer peptides spanning Clade C, consensus Gag, manufactured to current good manufacturing practice; having been evaluated in a good laboratory practice toxicology study in Macaca mulatta. We evaluated the safety and preliminary immunogenicity of such peptides administered intravenously after short-duration ex vivo incubation, to HIV-positive adults on suppressive antiretroviral therapy.
Methods and Findings
A first-in-human, placebo-controlled, double-blind, dose escalation study was conducted. Twenty-three patients with virus suppressed by antiretroviral therapy were enrolled in four groups 12 mg (n = 6), 24 mg (n = 6), 48 mg (n = 2) or matching placebo (n = 8). Treatment was administered intravenously after bedside enrichment of 120 mL whole blood for white cells using a closed system (Sepax S-100 device), with ex vivo peptide admixture (or diluent alone) and 37°C incubation for one hour prior to reinfusion. Patients received 4 administrations at monthly intervals followed by a 12-week observation post-treatment. Opal-HIV-Gag(c) was reasonably tolerated at doses of 12 and 24 mg. There was an increased incidence of temporally associated pyrexia, chills, and transient/self-limiting lymphopenia in Opal-HIV-Gag(c) recipients compared to placebo. The study was terminated early, after two patients were recruited to the 48 mg cohort; a serious adverse event of hypotension, tachycardia secondary to diarrhoea occurred following a single product administration. An infectious cause for the event could not be identified, leaving the possibility of immunologically mediated product reaction.
A serious, potentially life-threatening event of hypotension led to early, precautionary termination of the study. In the absence of a clearly defined mechanism or ability to predict such occurrence, further development of Opal-HIV-Gag(c) will not be undertaken in the current form.
ClinicalTrials.gov NCT01123915; EudraCT: 2008-005142-23
Observational data and non-human primate challenge studies suggest that cell-mediated immune (CMI) responses may provide control of HIV replication. The Step Study is the first direct assessment of the efficacy of a CMI vaccine to protect against HIV infection or alter early plasma HIV levels in humans.
HIV-seronegative participants (3000) were randomized (1:1) to receive 3 injections of MRKAd5 HIV-1 gag/pol/nef vaccine or placebo. Randomization was pre-stratified by gender, baseline adenovirus type 5 (Ad5) titer, and study site. Participants were tested ~every 6 months for HIV acquisition; early plasma HIV RNA was measured ~3 months post-HIV diagnosis.
The vaccine elicited IFN-γ ELISPOT responses in 75% of vaccinees. In a pre-specified interim analysis among participants with baseline Ad5 ≤200, 24 of 741 vaccinees became HIV infected, versus 21 of 762 placebo recipients. All but one infection occurred in men. The early geometric mean plasma HIV RNA was comparable in infected vaccine and placebo recipients. In exploratory multivariate analyses, HIV incidence was higher in vaccinees versus placebo recipients among Ad5 seropositive men (5.1% versus 2.2% per year, respectively) and uncircumcised men (5.2% versus 1.4% per year, respectively). HIV incidence was similar in vaccinees versus placebo recipients among Ad5 seronegative men and circumcised men.
This CMI vaccine did not prevent HIV infection or lower early viral level. Mechanisms for failure of the vaccine to protect and for the increased HIV infection rates in subgroups of vaccinees are being explored. Additional follow-up will determine if elevated HIV incidence in vaccinee subgroups persists.
HIV vaccine; efficacy; adenovirus; HIV acquisition; viral load; male circumcision; test of concept
It has been suggested that poor immunogenicity may explain the lack of vaccine efficacy in preventing or controlling HIV infection in the Step trial. To investigate this issue we vaccinated eight Indian rhesus macaques with a trivalent replication-incompetent adenovirus serotype 5 vaccine expressing SIV Gag, Pol, and Nef using a regimen similar to that employed in the Step trial. We detected broad vaccine-induced CD8+ (2–7 pool-specific responses) and CD4+ (5–19 pool-specific responses) T-cell responses in IFN-γ ELISPOT assays at one week post-boost using fresh PBMC. However, using cryopreserved cells at one and four weeks post-boost we observed a reduction in both the number and magnitude of most vaccine-induced responses. This demonstrates that the time points and conditions chosen to perform immune assays may influence the observed breadth and frequency of vaccine-induced T-cell responses. To evaluate protective efficacy, we challenged the immunized macaques, along with naïve controls, with repeated, limiting doses of the heterologous swarm isolate SIVsmE660. Vaccination did not significantly affect acquisition or control of virus replication in vaccinees compared to naïve controls. Post-infection we observed an average of only two anamnestic CD8+ T-cell responses per animal, which may not have been sufficiently broad to control heterologous virus replication. While the trivalent vaccine regimen induced relatively broad T-cell responses in rhesus macaques, it failed to protect against infection or control viral replication. Our results are consistent with those observed in the Step trial and indicate that SIV immunization and challenge studies in macaque models of HIV infection can be informative in assessing pre-clinical HIV vaccines.
HIV vaccine; Adenovirus serotype 5; Simian Immunodeficiency Virus; CD8+ T cells; CD4+ T cells; Step trial
Understanding how human genetic variation impacts individual response to immunogens is fundamental for rational vaccine development. To explore host mechanisms involved in cellular immune responses to the MRKAd5 human immunodeficiency virus type 1 (HIV-1) gag/pol/nef vaccine tested in the Step trial, we performed a genome-wide association study of determinants of HIV-specific T cell responses, measured by interferon γ enzyme-linked immunospot assays. No human genetic variant reached genome-wide significance, but polymorphisms located in the major histocompatibility complex (MHC) region showed the strongest association with response to the HIV-1 Gag protein: HLA-B alleles known to be associated with differences in HIV-1 control were responsible for these associations. The implication of the same HLA alleles in vaccine-induced cellular immunity and in natural immune control is of relevance for vaccine design. Furthermore, our results demonstrate the importance of considering the host immunogenetic background in the analysis of immune responses to T cell vaccines.
A randomized, double-blind, placebo-controlled phase I trial was conducted in 32 HIV-uninfected healthy volunteers to assess the safety and immunogenicity of 3 doses of DNA vaccine (Advax) plus 1 dose of recombinant modified vaccinia virus Ankara (MVA) (TBC-M4) or 3 doses of TBC-M4 alone (groups A and B, respectively). Both vaccine regimens were found to be safe and well tolerated. Gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay responses were detected in 1/10 (10%) individuals in group A after three Advax primes and in 9/9 individuals (100%) after the MVA boost. In group B, IFN-γ ELISPOT responses were detected in 6/12 (50%) and 7/11 (64%) individuals after the second and third MVA vaccinations, respectively. Responses to all vaccine components, but predominantly to Env, were seen. The breadth and magnitude of the T cell response and viral inhibition were greater in group A than in group B, indicating that the quality of the T-cell response was enhanced by the DNA prime. Intracellular cytokine staining indicated that the T-cell responses were polyfunctional but were skewed toward Env with a CD4+ phenotype. At 2 weeks after the last vaccination, HIV-specific antibody responses were detected in all (100%) group B and 1/11 (9.1%) group A vaccinees. Vaccinia virus-specific responses were detected in all (100%) group B and 2/11 (18.2%) group A vaccinees. In conclusion, HIV-specific T-cell responses were seen in the majority of volunteers in groups A and B but with a trend toward greater quality of the T-cell response in group A. Antibody responses were better in group B than in group A.
The safety and immunogenicity of a new candidate tuberculosis (TB) vaccine, FP85A was evaluated alone and in heterologous prime-boost regimes with another candidate TB vaccine, MVA85A. This was an open label, non-controlled, non-randomized Phase I clinical trial. Healthy previously BCG-vaccinated adult subjects were enrolled sequentially into three groups and vaccinated with FP85A alone, or both FP85A and MVA85A, with a four week interval between vaccinations. Passive and active data on adverse events were collected. Immunogenicity was evaluated by Enzyme Linked Immunospot (ELISpot), flow cytometry and Enzyme Linked Immunosorbent assay (ELISA). Most adverse events were mild and there were no vaccine-related serious adverse events. FP85A vaccination did not enhance antigen 85A-specific cellular immunity. When MVA85A vaccination was preceded by FP85A vaccination, cellular immune responses were lower compared with when MVA85A vaccination was the first immunisation. MVA85A vaccination, but not FP85A vaccination, induced anti-MVA IgG antibodies. Both MVA85A and FP85A vaccinations induced anti-FP9 IgG antibodies. In conclusion, FP85A vaccination was well tolerated but did not induce antigen-specific cellular immune responses. We hypothesize that FP85A induced anti-FP9 IgG antibodies with cross-reactivity for MVA85A, which may have mediated inhibition of the immune response to subsequent MVA85A. ClinicalTrials.gov identification number: NCT00653770
phase I clinical trial; tuberculosis vaccines; heterologous prime-boost regimes; poxvirus-vectored subunit vaccines
To investigate the safety and immunogenicity of a booster BCG vaccination delivered intradermally in healthy, BCG vaccinated subjects and to compare with a previous clinical trial where BCG vaccinated subjects were boosted with a new TB vaccine, MVA85A.
Phase I open label observational trial, in the UK. Healthy, HIV-negative, BCG vaccinated adults were recruited and vaccinated with BCG. The primary outcome was safety; the secondary outcome was cellular immune responses to antigen 85, overlapping peptides of antigen 85A and tuberculin purified protein derivative (PPD) detected by ex vivo interferon-gamma (IFN-γ) ELISpot assay and flow cytometry.
Results and Conclusions
BCG revaccination (BCG-BCG) was well tolerated, and boosting of pre-existing PPD-specific T cell responses was observed. However, when these results were compared with data from a previous clinical trial, where BCG was boosted with MVA85A (BCG-MVA85A), MVA85A induced significantly higher levels (>2-fold) of antigen 85-specific CD4+ T cells (both antigen and peptide pool responses) than boosting with BCG, up to 52 weeks post-vaccination (p = 0.009). To identify antigen 85A-specific CD8+ T cells that were not detectable by ex vivo ELISpot and flow cytometry, dendritic cells (DC) were used to amplify CD8+ T cells from PBMC samples. We observed low, but detectable levels of antigen 85A-specific CD8+ T cells producing IFNγ (1.5% of total CD8 population) in the BCG primed subjects after BCG boosting in 1 (20%) of 5 subjects. In contrast, in BCG-MVA85A vaccinated subjects, high levels of antigen 85A-specific CD8+ T cells (up to 14% total CD8 population) were observed after boosting with MVA85A, in 4 (50%) of 8 subjects evaluated.
In conclusion, revaccination with BCG resulted in modest boosting of pre-existing immune responses to PPD and antigen 85, but vaccination with BCG-MVA85A induced a significantly higher response to antigen 85 and generated a higher frequency of antigen 85A-specific CD8+ T cells.
ClinicalTrials.gov NCT00654316 NCT00427830
Recombinant adenovirus vectors have been extensively used in gene therapy clinical studies. More recently, the capability of inducing potent cell-mediated and humoral immunity has made these vectors equally attractive candidates for prophylactic or therapeutic vaccine applications. Merck and Co., Inc., developed HIV-1 vaccine candidates based on adenovirus serotype 5 (Ad5) vectors in which the E1 gene, a critical component for adenovirus replication, was replaced by the cytomegalovirus immediate/early promoter, followed by mutated versions of the HIV-1 gag, pol or nef genes (constructs referred to as MRKAd5gag, MRKAd5pol and MRKAd5nef, respectively). Vaccine performance was evaluated in vitro in a novel assay that measures the level of transgene expression in non-permissive A549 cells. Various combinations of vectors were studied. The results indicate that the vaccine induces a dose-dependent expression of the HIV-1 transgenes in vitro. Furthermore, the gag, pol, and nef transgenes are expressed differentially in A549 cells in an MOI-dependent and formulation-dependent manner, yielding an unexpected enhancement of protein expression in trivalent vs. monovalent formulations. Our data suggest that the presence of additional virus in multivalent formulations increases individual transgene expression in A549 cells, even when the amount of DNA encoding the gene of interest remains constant. This enhancement appears to be controlled at the transcriptional level and related to both the total amount of virus and the combination of transgenes present in the formulation.