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. Author manuscript; available in PMC: 2025 Jun 1.
Published in final edited form as: AIDS. 2023 Dec 1;38(7):963–973. doi: 10.1097/QAD.0000000000003804

The Immunogenicity of an HIV-1 Gag Conserved Element DNA Vaccine in People with HIV and Receiving Antiretroviral Therapy

Jeffrey M JACOBSON a, Barbara K FELBER b, Huichao CHEN c, George N PAVLAKIS d, James I MULLINS e, Stephen C DE ROSA f, Daniel R KURITZKES g, Georgia D TOMARAS h, Jennifer KINSLOW i, Yajing BAO c, Maxine OLEFSKY c, Margherita ROSATI d, Jenifer BEAR b, Drew HANNAMAN j, Gregory M LAIRD k, Joshua C CYKTOR l, Sonya L HEATH m, Ann C COLLIER n, Susan L KOLETAR o, Babafemi O TAIWO p, Pablo TEBAS q, David A WOHL r, Pablo F BELAUNZARAN-ZAMUDIO s, M Juliana MCELRATH f, Alan L LANDAY i; ACTG 5369 Study Team
PMCID: PMC11062837  NIHMSID: NIHMS1948308  PMID: 38051788

Abstract

Objective:

The primary objective of the study was to assess the immunogenicity of an HIV-1 Gag conserved element (CE) DNA vaccine (p24CE DNA) in people with HIV (PWH) receiving suppressive antiretroviral therapy (ART).

Design:

AIDS Clinical Trials Group A5369 was a phase I/IIa, randomized, double-blind, placebo-controlled study of PWH receiving ART with plasma HIV-1 RNA < 50 copies/mL, current CD4+ T-cell counts > 500 cells/mm3, and nadir CD4+ T-cell counts > 350 cells/mm3.

Methods:

The study enrolled 45 participants randomized 2:1:1 to receive p24CE DNA vaccine at weeks 0 and 4, followed by p24CE DNA admixed with full-length p55Gag DNA vaccine at weeks 12 and 24 (Arm A); full-length p55Gag DNA vaccine at weeks 0, 4, 12, and 24 (Arm B); or placebo at weeks 0, 4, 12, and 24 (Arm C). The active and placebo vaccines were administered by intramuscular electroporation.

Results:

There was a modest, but significantly greater increase in the number of CEs recognized by CD4+ and/or CD8+ T-cells in Arm A compared to Arm C (p=0.014). The percentage of participants with an increased number of CEs recognized by T-cells was also highest in Arm A (8/18, 44.4%) vs. Arm C (0/10, 0.0%) (p=0.025). There were no significant differences between treatment groups in the change in magnitude of responses to total CE.

Conclusions:

A DNA-delivered HIV-1 Gag CE vaccine boosted by a combination of this vaccine with a full-length p55Gag DNA vaccine induced a new CE-directed cellular immune response in approximately half the treated PWH on ART.

Keywords: HIV, therapeutic vaccination, DNA vaccine, conserved epitope, HIV-1 Gag, clinical trial, electroporation

INTRODUCTION

In nearly all individuals, the immune response following acquisition of HIV-1 is insufficient to eliminate the virus, resulting in chronic infection[1]. Cellular immune responses to HIV-1 provide partial control of viral replication, establishing a “viral load set point” measurable in plasma and remaining relatively stable during asymptomatic infection[2]. Most notably, Gag-specific CD4+ and CD8+ T-cell responses correlate with control of viremia in people with HIV (PWH)[3–6]. Development of preventive and therapeutic vaccines for HIV-1 has been hindered by the enormous sequence diversity and the constantly evolving virus population within PWH facilitating evasion of host defenses. Dominant immune responses are most often directed against variable epitopes of HIV-1 proteins, whose encoding genes can often mutate to achieve immune escape without compromising viral replication capacity[7].

The DNA immunogen studied here directs immune targeting to seven highly conserved regions (referred to as conserved elements or CEs) of HIV-1 p24Gag, some of which have been associated with virologic control[7–11]. p24CEs exclude variable, immunodominant epitopes of p24Gag, thus avoiding the potential negative effects of directing T-cell responses to epitopes that do not benefit virologic control [7–11]. Preclinical studies in macaques showed that the p24CE DNA vaccine boosted by a DNA expressing the full-length p55Gag protein was the optimal regimen to induce cellular immune responses against CE with the greatest breadth, magnitude, and cytotoxicity[8,12].

AIDS Clinical Trials Group (ACTG) A5639 evaluated the safety and immunogenicity of the HIV-1 p24CE DNA vaccine boosted by the full-length HIV-1 p55Gag DNA vaccine compared to the p55Gag vaccine alone and to placebo in ART-suppressed PWH.

METHODS

Study Design

A5369 was a phase I/IIa, randomized, double-blind, placebo-controlled clinical trial. Participants included PWH receiving ART with plasma HIV-1 RNA <50 copies/mL for at least 2 years (one instance of detectable viremia <400 copies/ml allowed), current CD4+ T-cell counts >500 cells/mm3, and nadir CD4+ T-cell count >350 cells/mm3 (See Supplemental Methods for study entry criteria). Participants were randomized 2:1:1 to 3 arms: Arm A received 4mg p24CE DNA in 1ml administered at weeks 0 and 4, followed by 2mg p24CE DNA in 0.5ml admixed with 2mg p55Gag DNA in 0.5ml at weeks 12 and 24; Arm B received 4mg p55Gag DNA in 1ml at weeks 0, 4, 12, and 24; Arm C received 1ml placebo (Sodium Chloride for Injection, USP 0.9%) at weeks 0, 4, 12, and 24. Previous clinical studies demonstrated that electroporation-mediated intramuscular injection of DNA encoding HIV antigens can dramatically enhance the magnitude, frequency and breadth of immune response compared to conventional methods of injection[13–14]. The vaccines and placebo were administered using the investigational TriGrid Delivery System (TDS-IM v2.0) for electroporation-based intramuscular administration (ICHOR Medical Systems, San Diego, California)[13–16].

The primary efficacy objective of the study was to compare to baseline the additional number of CEs recognized by CD4+ and/or CD8+ T-cells between Arm A and Arm C at week 26. The target sample size of 40 (20 Arm A; 10 Arm B; 10 Arm C) for the randomized arms was selected to detect a 0.54 difference in the probability that a participant had increased CE responses (0.64 for Arm A and 0.10 for Arm C) between arms based on a two-sided Fisher’s exact test with 80% power, 5% alpha level, and 10% inflation for loss-to-follow-up or unusable laboratory specimens. The study also had approximately 80% power to detect a difference of 0.85 versus 0.30 when comparing Arms A and B.

The protocol was approved by the institutional review board at each study site. Written informed consent was obtained from all participants. The study was conducted according to human experimentation guidelines of the US Department of Health and Human Services and was monitored by an independent Study Monitoring Committee of the ACTG.

Study Products

The p24CE DNA vaccine comprises two gene segments (p24CE1 and p24CE2) on one plasmid, expressing portions of HIV-1 p24Gag that are conserved in 98–100% of available HIV-1 M-group sequences[7–10] (Supplemental Fig 1). p24CE sequences represent 54% (127 AA) of p24Gag. They are enriched for amino acids (AA) at p24Gag hexamer interfaces[17] and a mutation to the second-most common AA in the HIV database often result in attenuated or noninfectious virus[17,18]. The 7 CE segments of 13, 18, 24, 20, 20, 14, and 18 AA, each separated by 0–3 AA spacers, comprise p24CE proteins[7]. One “toggled” AA is included in each of the CE segments, resulting in the two protein coding elements, p24CE1 and p24CE2, differing by 7 codons. Coding sequences were optimized to enhance expression in human cells[19–22].

The p55Gag DNA (plasmid 114H) contains an expression-optimized full-length HIV-1 p55gag gene from the molecular clone HXB2 [23] (Supplemental Fig. 1). HXB2 is one of the earliest HIV isolates. As such it is near the ancestral root of HIV and a large portion of its proteome sequence is shared by extant strains. Both vaccines were provided by DAIDS, NIAID, NIH, DHHS (Bethesda, MD, USA).

Evaluation of Participants and Follow-up

Clinical assessments and laboratory safety monitoring were done at weeks 0 (baseline), 4, 12, 24, 26, and 48. After each vaccination, participants completed a tolerability questionnaire that assessed the level of discomfort experienced during the vaccination process. They were also contacted by telephone 2 to 3 days post-vaccination to identify changes in health status and answer questions about vaccination-related signs and/or symptoms and given vaccine report cards to record daily temperatures, injection site reactions, and systemic reactions. Plasma HIV-1 RNA levels were measured at Quest Diagnostics using the Amplicor HIV-1 Monitor test (version 1.5; Roche Diagnostics) at weeks 0, 12, 24, and 48. CD4+ and CD8+ T-cell counts were measured at weeks 0, 12 and 24. Leukaphereses or large volume blood draws were performed at weeks 0 and 26 for immunologic and virologic studies. Bloods were drawn for plasma and peripheral blood mononuclear cells (PBMC) processing and frozen storage to perform subsequent immunologic and virologic assays within 14 days before study entry (pre-entry), and at weeks 0, 6, 26, and 48.

Immunologic Evaluations

Intracellular cytokine staining (ICS) assay

CD4+ and CD8+ T-cell response rates and magnitudes were evaluated by flow cytometry using a validated intracellular cytokine staining (ICS) assay[24] with a 28-color staining panel (Supplemental Table 1) and CE and Gag peptide pools using PBMC obtained at week 0 and week 26 (2 weeks after the 4th vaccination).

T-cell epitope mapping

ICS was used to map to the individual CE peptide pools, and to regions of p55Gag using p17Gag, p24Gag and p15Gag peptide pools using a 17-color staining panel (Supplemental Table 3). Stained cells were examined on a BD FACSymphony instrument and data analyzed using FlowJo (Tree Star) version 9.9.4 (Supplemental Methods).

Binding antibody multiplex assay

HIV-1-specific IgG binding antibody responses to clade B p24Gag protein were measured by a customized HIV-1 specific Binding Antibody Multiplex Assay (BAMA)[25,26].

Flow cytometry and soluble markers of inflammation and immune activation

At pre-entry and weeks 0, 6, 26, and 48, T-cell subsets (CD3/CD4, CD3/CD8), markers of immune activation (CD38/HLA-DR), naïve and memory T-cell subsets (CD45RA/CCR7), senescence (CD57/CD28) and exhaustion (PD-1), monocyte subsets (HLA-DR/CD14/CD16) and markers of activation (CD80) and migration (CCR2/CX3CR1) were measured by flow cytometry. Soluble markers of inflammation and immune activation [sCD14, sCD163, sCRP, HS-IL-6, IP-10, and D-Dimer were measured by ELISA (see Supplemental Methods).

Virologic Evaluations

Intact Proviral DNA Assay

The Accelevir Diagnostics IPDA™ assay was performed on genomic DNA extracted from peripheral CD4+ T-cells[27,28].

Plasma HIV-1 RNA Quantification

Automated HIV-1 RNA single copy assay (autoSCA) was performed[29,30].

Statistical Methods

Immunogenicity analyses were per protocol, limited to participants who received all four study vaccination/placebo administrations and who did not take medication prohibited by the study protocol. Wilcoxon rank sum test compared the number of CEs recognized in participants between study arms from baseline to week 26. The percentage of participants in each study arm with a CD4+ and/or CD8+ T-cell response to an increased number of CEs at week 26 was estimated along with an exact binomial 95% confidence interval. Pairwise comparisons between study arms were conducted using Fisher’s exact test. Wilcoxon rank sum test assessed changes in immune responses and markers between study arms. These included magnitude of T-cell responses to: total p24CE, each individual CE, p55Gag proteolytic products, and full-length p55Gag; the 26-week absolute and relative changes in antibody titers; the 26- and 48-week changes in cellular and soluble markers of immune activation, exhaustion, and checkpoint inhibition; and changes in the HIV-1 latent T-cell reservoir (IPDA) and persistence (SCA). Undetectable SCA results were imputed as LOD of 0.4 copies/mL during analysis. All statistical tests were two-sided and interpreted at the 5% nominal level of significance without adjustment for multiple comparisons. Analyses were conducted using SAS version 9.4 (SAS Institute) and R version 3.6.3 (2020–02-29, ggplot2, ggh4x, ggpubr packages).

RESULTS

Study Population

A5369 accrued 45 participants from 15 study sites between March and October 2019. Two of the participants were replaced. One participant in Arm B reported taking exclusionary medication prior to the first vaccination and was replaced and not part of safety or efficacy analyses. The baseline samples of one participant in Arm C were lost due to a shipping error; this participant was replaced before receiving the second dose of study treatment, and then withdrew from study follow-up at week 4. The latter participant contributed to safety analyses, but not to efficacy analyses. Among 44 participants in the safety analysis set, 41 completed 4 doses of study treatment. Three participants did not have test results, which led to 38 participants for the primary efficacy analysis (Figure 1).

Figure 1. A5369 Consort Diagram.

Figure 1.

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Table 1 provides a summary of participant characteristics at baseline for those who received 4 doses of study product (N=41). There were no baseline differences across study arms by sex, gender, age, race, ethnicity, or current or nadir CD4+ T-cell counts. Six (15%) of the participants were women. The median (Q1, Q3) age was 49 (37, 55). The median (Q1, Q3) CD4+ T-cell count (cells/mm3) was 823 (710, 1030). All participants had plasma HIV-1 RNA <40 copies/mL at baseline.

Table 1.

Baseline participant characteristics.

Characteristic Statistic A (N=20) B (N=10) C (N=11) Total (N=41)
Sex Women 2 (10%) 1 (10%) 3 (27%) 6 (15%)
Men 18 (90%) 9 (90%) 8 (73%) 35 (85%)
Gender Identity Women 2 (10%) 1 (10%) 3 (27%) 6 (15%)
Men 17 (85%) 8 (80%) 7 (64%) 32 (78%)
Other 1 (5%) 1 (10%) 1 (9%) 3 (7%)
Age (years) N 20 10 11 41
Mean (s.d.) 47.7 (10.3) 47.4 (10.6) 44.0 (11.0) 46.6 (10.4)
Median (Q1, Q3) 52 (39, 56) 52 (37, 56) 42 (34, 53) 49 (37, 55)
Min, Max 30, 60 30, 60 30, 61 30, 61
Race unknown 1 (5%) 0 (0%) 0 (0%) 1 (2%)
Black or African American 6 (30%) 4 (40%) 5 (45%) 15 (37%)
Multiple 0 (0%) 1 (10%) 0 (0%) 1 (2%)
White 13 (65%) 5 (50%) 6 (55%) 24 (59%)
Ethnicity Hispanic or Latino 3 (15%) 0 (0%) 2 (18%) 5 (12%)
Not Hispanic or latino 17 (85%) 10 (100%) 9 (82%) 36 (88%)
CD4 (cells/mm3) N 20 10 11 41
Mean (s.d.) 841 (251) 969 (352) 907 (268) 890 (280)
Median (Q1, Q3) 802 (633, 1017) 819 (790, 1034) 891 (715, 1030) 823 (710, 1030)
Min, Max 561, 1525 693, 1863 444, 1483 444, 1863
Nadir CD4 (cells/mm3) N 20 10 11 41
Mean (s.d.) 527 (189) 471 (117) 501 (118) 506 (155)
Median (Q1, Q3) 451 (403, 622) 438 (386, 501) 446 (410, 565) 440 (407, 565)
Min, Max 355, 1065 366, 700 375, 750 355, 1065
HIV-1 RNA level (copies/mL) <40 cp/mL 20 (100%) 10 (100%) 11 (100%) 41 (100%)
Weight (kg) N 20 10 11 41
Mean (s.d.) 87.9 (13.4) 80.4 (11.8) 90.7 (15.9) 86.8 (14.0)
Median (Q1, Q3) 86.8 (79.6, 93.2) 78.8 (76.7, 83.7) 86.2 (76.6, 108) 83.7 (77.8, 94.3)
Min, Max 65.2, 118 60.0, 103 70.3, 112 60.0, 118
IV drug use No 18 (90%) 10 (100%) 9 (82%) 37 (90%)
Yes 2 (10%) 0 (0%) 2 (18%) 4 (10%)
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Safety

Most of the adverse events (AEs) reported that were related to study treatment were local injection site reactions, without difference across study arms. Seventy-three AEs were reported for 20 participants. Of these, 62 were grade 1, 9 were grade 2, 1 was non-gradable, and 1 was grade 4. The majority (94.5%) of AEs occurred on the same day as study treatment administration, with the other 4 AEs occurring 1, 2, 3, and 21 days after treatment; all resolved within 4 days of onset, most within 1 day. A grade 4 AE, ‘non-ST elevated myocardial infarction’, was reported for one participant (in Arm A) 21 days after this participant’s second treatment dose and resolved one day after onset. This participant had previously experienced grade 1 injection site tenderness lasting 24 hours. By study team decision and supported by an ad hoc review by the Study Monitoring Committee, this participant discontinued study treatment (one of the two participants in Arm A prematurely discontinued treatment) and remained on study for safety follow-up with no additional AEs reported.

Immunologic Responses

Cellular Immune Responses

A total of 38 participants were included in the primary immunogenicity analysis; of the 41 participants who completed all four vaccine/placebo administrations, one (Arm A) was excluded because the week 26 visit was missed due to COVID, one (Arm C) had a missing week 26 specimen, and one (Arm A) did not have CE mapping done. More than half the study participants had measurable T-cell responses to at least one of the CEs in the vaccine at baseline prior to vaccination (Arm A: 12/18, Arm B: 5/10, Arm C: 6/10). There was a modest but significantly greater increase in the number of CEs recognized by CD4+ and/or CD8+ T-cells in Arm A compared to Arm C (p=0.014, Table 2), but no significant increase in Arm A vs. Arm B (p=0.137) or Arm B vs. Arm C (p=0.100). Across all participants, however, the change in the number of CE responses between baseline and week 26 was either −1, 0, or +1 (Table 2). The increases, mainly observed in Arm A, were limited to one CE per participant across several CE (1, 4, 5, and 6, Table 3). The percentage of participants with an increased number of CEs recognized by T-cell responses was also highest in Arm A (8/18, 44.4%) vs. Arm C (0/10, 0.0%) (p=0.025). When examined separately for CD4+ and CD8+ T-cells, the percentage of participants with increased responses was not significantly different between Arms (Table 2). No significant differences in the total CE magnitude were found between treatment groups (Supplemental Table 4).

Table 2.

CE responses recognized by CD4 and/or CD8 T-cells at week 26 minus the number at baseline.

Difference in CE responses week 26 minus baseline
A B C Wilcoxon rank-sum test P value
(N=18) (N=10) (N=10) A vs. B A vs. C B vs. C
CD4 and/or CD8 -1 1 (6%) 0 (0%) 2 (20%) 0.137 0.014 * 0.100
0 9 (50%) 9 (90%) 8 (80%)
1 8 (44%) 1 (10%) 0 (0%)
Percentage of participants with an increase in number of CEs
Fisher’s exact test P value
CD4 and/or CD8 8/18 (44.4%) 1/10 (10%) 0/10 (0%) 0.098 0.025 1.00
95% CI 22%,69% 0.3%,45% 0%,31%
CD4 3/18 (16.7%) 0/10 (0%) 0/10 (0%) 0.533 0.533
95% CI 3.6%,41% 0.0%,31% 0.0%,31%
CD8 5/18 (27.8%) 2/10 (20%) 0/10 (0%) 1.000 0.128 0.474
95% CI 9.7%,54% 2.5%,56% 0.0%,31%
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*

Boldface is used to indicate antigens with increase response rates from baseline to Week 26.

Table 3.

Response rates for individual CEs and Gag peptide pools

CD4+ T-cells CD8+ T-cells CD4+ and/or CD8+ T-cells
Antigen Week A B C A B C A B C
CE 1 0 0/18 (0%) 0/10 (0%) 0/10 (0%) 1/18 (5.6%) 2/10 (20%) 0/10 (0%) 1/18 (5.6%) 2/10 (20%) 0/10 (0%)
26 0/18 (0%) 0/10 (0%) 0/10 (0%) 3/18 (16.7%) * 2/10 (20%) 0/10 (0%) 3/18 (16.7%) 2/10 (20%) 0/10 (0%)
CE 2 0 1/18 (5.6%) 0/10 (0%) 0/10 (0%) 2/18 (11.1%) 0/10 (0%) 3/10 (30%) 2/18 (11.1%) 0/10 (0%) 3/10 (30%)
26 1/18 (5.6%) 0/10 (0%) 0/10 (0%) 2/18 (11.1%) 0/10 (0%) 2/10 (20%) 2/18 (11.1%) 0/10 (0%) 2/10 (20%)
CE 3 0 1/18 (5.6%) 0/10 (0%) 0/10 (0%) 3/18 (16.7%) 1/10 (10%) 3/10 (30%) 3/18 (16.7%) 1/10 (10%) 3/10 (30%)
26 0/18 (0%) 0/10 (0%) 0/10 (0%) 2/18 (11.1%) 1/10 (10%) 3/10 (30%) 2/18 (11.1%) 1/10 (10%) 3/10 (30%)
CE 4 0 0/18
(0%)		 0/10 (0%) 0/10 (0%) 0/18
(0%)		 1/10 (10%) 1/10 (10%) 0/18
(0%)		 1/10 (10%) 1/10 (10%)
26 0/18
(0%)		 0/10 (0%) 0/10 (0%) 3/18 (16.7%) 3/10 (30%) 1/10 (10%) 3/18 (16.7%) 3/10 (30%) 1/10 (10%)
CE 5 0 2/18 (11.1%) 1/10 (10%) 0/10 (0%) 3/18 (16.7%) 1/10 (10%) 2/10 (20%) 5/18 (27.8%) 2/10 (20%) 2/10 (20%)
26 4/18 (22.2%) 0/10 (0%) 0/10 (0%) 3/18 (16.7%) 1/10 (10%) 2/10 (20%) 6/18 (33.3%) 1/10 (10%) 2/10 (20%)
CE 6 0 3/18 (16.7%) 0/10 (0%) 2/10 (20%) 3/18 (16.7%) 0/10 (0%) 0/10 (0%) 6/18 (33.3%) 0/10 (0%) 2/10 (20%)
26 4/18 (22.2%) 0/10 (0%) 1/10 (10%) 4/18 (22.2%) 0/10 (0%) 0/10 (0%) 8/18 (44.4%) 0/10 (0%) 1/10 (10%)
CE 7 0 0/18
(0%)		 0/10 (0%) 0/10 (0%) 3/18 (16.7%) 0/10 (0%) 0/10 (0%) 3/18 (16.7%) 0/10 (0%) 0/10 (0%)
26 0/18
(0%)		 0/10 (0%) 0/10 (0%) 3/18 (16.7%) 0/10 (0%) 0/10 (0%) 3/18 (16.7%) 0/10 (0%) 0/10 (0%)
CE Total 0 5/18 (27.8%) 0/10 (0%) 3/10 (30%) 11/18 (61.1%) 5/10 (50%) 7/10 (70%) 11/18 (61.1%) 5/10 (50%) 7/10 (70%)
26 8/18 (44.4%) 2/10 (20%) 2/10 (20%) 13/18 (72.2%) 5/10 (50%) 6/10 (60%) 15/18 (83.3%) 5/10 (50%) 6/10 (60%)
p15 Gag 0 0/17
(0%)		 0/10 (0%) 2/9 (22.2%) 2/18 (11.1%) 1/10 (10%) 1/9 (11.1%) 2/18 (11.1%) 1/10 (10%) 3/9 (33.3%)
26 2/18 (11.1%) 2/10 (20%) 1/9 (11.1%) 2/18 (11.1%) 2/10 (20%) 1/9 (11.1%) 4/18 (22.2%) 4/10 (40%) 2/9 (22.2%)
p17 Gag 0 6/18 (33.3%) 4/10 (40%) 3/9 (33.3%) 4/18 (22.2%) 2/10 (20%) 3/9 (33.3%) 7/18 (38.9%) 5/10 (50%) 5/9 (55.6%)
26 7/18 (38.9%) 5/10 (50%) 3/9 (33.3%) 5/18 (27.8%) 2/10 (20%) 3/9 (33.3%) 9/18 (50%) 6/10 (60%) 6/9 (66.7%)
p24 Gag 0 12/18 (66.7%) 5/10 (50%) 6/9 (66.7%) 11/18 (61.1%) 7/10 (70%) 4/9 (44.4%) 15/18 (83.3%) 8/10 (80%) 7/9 (77.8%)
26 15/18 (83.3%) 7/10 (70%) 6/9 (66.7%) 13/18 (72.2%) 7/10 (70%) 4/9 (44.4%) 16/18 (88.9%) 9/10 (90%) 8/9 (88.9%)
p55 Gag 0 11/18 (61.1%) 6/10 (60%) 10/10 (100%) 15/18 (83.3%) 9/10 (90%) 7/10 (70%) 17/18 (94.4%) 9/10 (90%) 10/10 (100%)
26 16/18 (88.9%) 9/10 (90%) 9/10 (90%) 17/18 (94.4%) 10/10 (100%) 7/10 (70%) 18/18 (100%) 10/10 (100%) 10/10 (100%)
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*

Boldface is used to indicate antigens with increase response rates from baseline to Week 26.

Response rates for individual CEs were also analyzed as a pre-specified secondary objective. Although individual participants mounted substantial increases to single CEs, overall response rates were low (Table 3). There were no significant differences in response rates to individual CEs between treatment groups (Supplemental Table 5). Figure 2A shows the percent of CD4+ and CD8+ T-cells expressing IFN-γ and/or IL-2 in response to stimulation with each of the CE peptide pools at baseline and week 26. Most participants showed little change before and after vaccination; three individuals in Arm A and two individuals in Arm B showed CD8+ T-cell responses to CE 4 that increased from undetectable at baseline to positive at week 26.

Figure 2. T-cell responses in vaccine and placebo recipients.

Figure 2.

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CD4+ and CD8+ T-cells expressing IFN-γ and/or IL-2 in response to stimulation with individual CE peptide pools (A) and peptide pools for the total p24CE, p15Gag, p17Gag, p24Gag and total p55Gag (B) at baseline and week 26 (2 weeks post final vaccination or placebo injection). The peptide pools used were: CE total (combined pool of all 7 p24CE peptide pools, including 15-mer peptides overlapping by 11 AA and 10-mer peptides overlapping by 9 AA; Supplemental Table 2); HXB2 p55gag (15mer peptides overlapping by 11 AA); p17Gag, p24Gag and p15Gag peptide pools (15-mer peptides overlapping by 11 AA and 10-mer peptides overlapping by 9 AA), provided by B. Felber, NIH. Blue, pink or orange dots indicate positive responses and gray triangles indicate negative responses as determined by the Fisher’s exact test comparing the response to peptide pool stimulation and the negative control (DMSO, the peptide diluent).

Unlike the responses to individual CE, as expected, CD4+ and CD8+ T-cell responses to the full-length p55Gag were detected in many participants (Table 3 and Figure 2B) at baseline across all arms. By week 26, CD4+ T-cell response rates increased from 11/18 (61%) to 16/18 (89%) in Arm A and from 6/10 (60%) to 9/10 (90%) in Arm B, while in Arm C the response rate was 10/10 at baseline and 9/10 at week 26. When compared to placebo, the increase was significant for Arm A (p=0.037) and tended toward significance for Arm B (p=0.051, Supplemental Table 6). CD8+ T-cell response rates increased from 15/18 (83%) to 17/18 (94%) in Arm A and from 9/10 (90%) to 10/10 (100%) in Arm B, while in Arm C the response rate was 7/10 (70%) at baseline and week 26. There were significant increases in the total magnitude of the CD4+ T-cell responses to p55Gag in both vaccine arms compared to placebo, and there was a nonsignificant increase in total magnitude of the CD8+ T-cell response in Arm B compared to placebo (Supplemental Table 7). No significant changes were observed for response rates to peptide pools for the p15Gag, p17Gag and p24Gag proteolytic products of p55Gag (Supplemental Table 6).

Binding Antibody Responses

The placebo group tended to have lower median anti-p24Gag IgG titers compared to the vaccine groups at baseline (median IgG p24Gag response AUC for Arms A= 12781, B = 14790, C= 5254). The two vaccine arms had comparable baseline values (Supplemental Fig 2).

Arm B showed a higher median absolute and relative change in antibody titer compared to both Arms A and C. Of note, to deliver the same amount of total DNA and volume in both treatment arms, Arm B received twice the amount of p55Gag in the booster vaccinations than Arm A. The median change in AUC between week 26 and baseline was −205.72 (−3.73%), 1661.91 (37.47%), and 112.06 (0.11%) for Arms A, B and C, respectively (Supplemental Figures 2B and C). Pairwise comparisons between groups showed that absolute and relative changes were significantly different for A vs. B =0.022 and 0.025, and B vs. C =0.024 and 0.019, but not A vs. C =0.783 and 0.561 (Wilcoxon rank sum test).

CD4 T lymphocyte Counts and Markers of Immune Activation/Inflammation

There was no significant difference in CD4+ T-cell counts among the treatment arms (measured at weeks 0, 6, 12, 24, and 48). Vaccination had no significant effect on CD4+ or CD8+ T-cell activation (HLA-DR+/CD38+) or monocyte activation (CD14+/CD16+/CD80+) (Supplemental Table 8). No significant changes were observed in soluble markers of activation/inflammation, including sCD14, sCD163, CRP, and CXCL10 (IP-10) or in the frequency of the PD-1 exhaustion marker between arms (Supplemental Table 8).

Virologic Assays

All participants maintained plasma HIV-1 RNA levels <40 cp/mL, except for one (Arm C) who had a week 12 RNA result of 45 cp/mL that returned to <40 cp/mL at week 24. No significant differences between treatment groups between baseline and week 26 were found in changes in IPDA or in single copy HIV-1 RNA assay results between baseline and week 48 (Supplemental Table 9).

DISCUSSION

In this double-blind, randomized, placebo-controlled trial, we studied whether a DNA vaccine encoding highly conserved HIV-1 p24Gag sequences, some of which are associated with virologic control, could re-direct T-cell responses against these normally subdominant epitopes in PWH on suppressive ART. The vaccine regimens were safe; most of the AEs were transient, mild-moderate local injection site reactions.

Vaccination with the p24CE DNA followed by boosting with a combination of the CE DNA mixed with a DNA vaccine encoding full-length p55Gag induced new CE-directed CD4+ and/or CD8+ T-cell responses in approximately half (8/18) of study participants (Arm A) compared to 10% (1/10) in Arm B and none (0/10) receiving placebo (arm C). Arm A also showed a significantly greater increase in the number of CEs recognized by T-cells compared to placebo (Arm C), although the total change in the magnitude of these immune responses was not different. Note that the study was powered for the primary comparison of Arm A and Arm C. The results for other analyses should be interpreted with caution, considering that no adjustment was done for multiple comparisons.

The vaccine regimens for both Arms A and B included administration of a full-length p55Gag DNA vaccine. Both regimens induced significant increases in the number of participants with p55Gag-specific T cell responses and in the total magnitude of the CD4+ T-cell responses compared to placebo. Gag antibody titers increased in Arm B compared to both Arm A (which received overall one-fourth the amount of p55Gag DNA than Arm B) and Arm C.

A limitation of the study is the absence of an analytical treatment interruption (ATI) of ART. The number of new CEs targeted by the induced cellular immune responses in an individual was at most one, and, although there were individual CE response increases of substantial magnitude, the total change in the magnitude of CE responses did not differ from that of the placebo arm. The CE-directed immune responses induced by the study vaccine regimen alone are unlikely to translate into a clinically important effect at controlling viral replication. On the other hand, the number of responders and the total magnitude of the cellular immune responses to p55Gag increased in both vaccine arms compared to placebo, especially CD4+ T-cell responses. Cellular immune responses to Gag have been shown to be associated with viral control[3–6] and CD4+ T-cell responses to Gag have specifically correlated with control of viremia off ART[6].

The vaccine tested focused on highly conserved sequences in Gag, whereas others are developing different design strategies for coverage of HIV T cell targets and using different vaccine delivery platforms (reviewed in [31] and references therein). Few of these concepts have advanced to clinical trials in PWH, and those studied in PWH have had, at best, limited success[32]. In a randomized, controlled trial, a strategy targeting autologous HIV-1 antigens led to control of viremia after ART discontinuation in a subset of PWH[2]. Unique to the study of our CE DNA vaccine regimen was, in addition to placebo, an inclusion of a direct comparison to vaccination with the full-length molecule. Clearly, we found that the CE vaccine regimen was immunogenic and resulted in a significant increase of CE-specific T-cells compared to the placebo arm, although the difference to the full-length Gag arm did not reach significance. As we reported from studies in the non-human primate model, it is possible that inclusion of IL-12 DNA adjuvant[33–36] and boosting with viral vectors i.e. recombinant MVA or mRNA[37,38] could further increase the magnitude of T cell responses in humans[39,40].

The challenges to inducing new effective anti-HIV-1 immunity in PWH are substantial[41]. Persistent abnormalities of immune function, whether from the early damage due to the infection that may not be fully reparable, or from the ongoing, albeit reduced, immune activation under ART, have been reported to contribute to inabilities to mount optimal responses to vaccination[42,43]. However, the recent experience with SARS-CoV-2 vaccination suggests that PWH with normal CD4+ T-cell counts at least develop neutralizing antibody responses to that virus comparable to people without HIV[43,44]. An additional hurdle demonstrated by this study is the pre-existent immune imprinting from previous and probable continuing exposure to HIV-1 antigens in PWH, as most of the study participants already had detectable immune responses to the antigens targeted by the vaccine.

Together, a DNA-delivered HIV-1 p24CE vaccine boosted by a combination of this vaccine with a full-length p55Gag DNA vaccine modestly induced new CE-directed cellular immune responses in approximately half of participants. This vaccine regimen as well as the p55Gag alone DNA vaccine induced significantly increased full-length Gag-targeted cellular immune responses. Further enhancements of this therapeutic vaccine approach are very likely to be needed to enhance virologic control during an ATI.

Supplementary Material

Supplemental Figure 1

Supplemental Figure S1. Vaccine Components. The dual promoter p24CE1/2 plasmid (plasmid code 306H) expresses the two variants CE proteins CE1 and CE2. They each comprise 7 regions representing 54% of p24Gag. CE1 is expressed from the human cytomegalovirus (huCMV). CE2 is expressed from the simian cytomegalovirus (siCMV) promoter. The plasmid contains the bovine growth hormone polyadenylation (BGH polyA, CE1) and the simian vacuolating virus 40 polyadenylation (SV40 polyA, CE2) signals. The p55Gag plasmid (plasmid code 114H) contains the huCMV promoter and the BGH polyA signal and expresses the full-length HIV-1 p55Gag, comprising the p17Gag, p24Gag and p15Gag proteolytic cleavage products. The location of the 7 CE within p24Gag are indicated.

Supplemental Figure 2

Supplemental Figure S2. HIV-1 p24Gag-specific IgG binding antibody responses. Dot plots, with overlaying lines (A) or boxplots (B, C), depicting HIV-1 p24Gag-specific IgG binding antibody response magnitudes as Area Under the Titration Curve (AUC) by the Binding Antibody Multiplex Assay (BAMA). Serum samples were titrated and the AUC was calculated over dilutions 500, 5000, 50000, and 500000 using trapezoidal integration, with base 10 log-transformation of the dilutions. The tested antigen was the clade B p24 Gag protein. Absolute change in AUC was then calculated as (week 26 AUC – baseline AUC) for each participant. Relative change in AUC, was calculated as [(week 26 AUC – baseline AUC)/baseline AUC] for each participant. All assays were conducted according to Good Clinical Laboratory Practice Guidelines, including tracking of controls with Levey-Jennings charts. Each dot represents a participant from the respective treatment arm and time point being plotted. Lines join the same participant over study time points. Treatment groups are color coded: Arm A: p24CE1/2 pDNA + p55Gag pDNA (blue), Arm B: p55Gag pDNA (pink) and Arm C: placebo (orange). A) AUC is shown on the y-axis and the time points are shown on the x-axis. B) Absolute change in AUC, calculated as week 26 AUC – baseline AUC, the latter taken as the average of entry and pre-entry timepoints, shown on the y-axis, and treatment groups on the x-axis. C) Relative change in AUC, calculated as [100*(week 26 AUC – baseline AUC)/ baseline AUC], shown on the y-axis, and treatment groups on the x-axis. The treatment specific descriptive statistics, N=sample size, median and [Q1, Q3] = interquartile range are shown on top of each treatment group for panels B and C.

Supplemental Data File (.doc, .tif, pdf, etc.)

ACKNOWLEDGEMENTS:

We are indebted to the individuals who participated in the study. We also thank Michael Pensiero, Division of AIDS, NIAID, NIH for overseeing the DNA vaccine cGMP preparation.

Other ACTG A5369 protocol team members.

Parita Rathod, DLH Corporation, Bethesda, MD; Christine Scello, Frontier Science & Technology Research, Amherst, NY; Lynette Purdue, PharmD, Division of AIDS, NIAID, NIH, Rockville, MD; Frederick Bone, Frontier Science & Technology Research, Amherst, NY; Jenifer Baer, R.N., University of Cincinnati, Cincinnati, OH; Gerald L. Tegha, M.Sc., Kamuzu Central Hospital/Tidziwe, Lilongwe, Malawi; William R. Hardin, B.S. Penobscot, ME.

Other investigators at participating sites.

Ann Collier, MD and Sher Storey, PA-C – University of Washington Positive Research CRS (Site 1401); Heather Harber, RN and Brian Greenfelder, BA – Ohio State University CRS (Site 2301); Babafemi Taiwo, MBBS and Claudia Hawkins, MD – Northwestern University CRS (Site 2701); Sonya Heath, MD and E. Turner Overton, MD – Alabama CRS (Site 31788); Matthew Newell, RN, CCRN and Mandy Tipton, LPN – Chapel Hill CRS (Site 3201); Pablo Tebas, MD and William Short, MD, MPH - Penn Therapeutics CRS (Site 6201); Deborah McMahon, MD and Jennifer Sullivano, BSN, RN – University of Pittsburgh CRS (Site 1001); Brett Williams, MD and Rebecca Lai, RN – Rush University CRS (Site 2702); Joan Gottesman, RN, CCRP and Fred Nicotera, BS – Vanderbilt Therapeutics (VT) CRS (Site 3652); Jorge L. Santana, MD, FIDSA and Sigrid D. Perez, MD, AAHIVS – Puerto Rico AIDS Clinical Trials Unit CRS (Site 5401); Jay Dwyer, RN and Anne Luetkemeyer, MD – University of California, San Francisco HIV/AIDS CRS (Site 801); Amy Sbrolla, RN and Rajesh Gandhi, MD – Massachusetts General Hospital CRS (MGH CRS) (Site 101); Michael C. Keefer, MD and Peter Mariuz, MD – University of Rochester Adult HIV Therapeutic Strategies Network CRS (Site 31787); Paul Sax, MD and Cheryl Keenan, RN, BC – Brigham and Women’s Hospital Therapeutics (BWH TCRS) CRS (Site 107); Kara Chew, MD, MS and Aleen Khodabakhshian, MD – University of California Los Angeles CARE Center CRS (Site 601).

Other laboratory investigators: Jack R. Hepinstall, Lu Zhang, Sheetal Sawant - Duke Center for Human Systems Immunology, Departments of Surgery, Immunology, Molecular Genetics and Microbiology, Durham, North Carolina

Sources of Funding:

Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Numbers UM1 AI068634, UM1 AI068636, and UM1 AI106701, and by funding from the Intramural Research Program, National Institutes of Health, National Cancer Institute, Center for Cancer Research to B.K. Felber and G.N. Pavlakis; ACTG Site Grant Numbers: Case Western Reserve University CTU (Site 2501) Grant UM1AI069501, University of Washington Positive Research CRS (Site 1401) Grant UM1AI69481, Ohio State University CRS (Site 2301) Grant UM1AI69494, Northwestern University CRS (Site 2701) Grant UM1AI69471, Alabama CRS (Site 31788) Grant UM1AI69452, Chapel Hill CRS (Site 3201) Grant UL1TR002489, Penn Therapeutics CRS (Site 6201) Grant UM1AI69534, University of Pittsburgh CRS (Site 1001) Grant UM1AI69494, UL1TR001857, Rush University CRS (Site 2702) Grant UM1AI69471, Vanderbilt Therapeutics (VT) CRS (Site 3652) Grant UM1AI69439, UL1 TR002243, Puerto Rico AIDS Clinical Trials Unit CRS (Site 5401) Grant UM1AI69415, University of California, San Francisco HIV/AIDS CRS (Site 801) Grant UM1AI69496, Massachusetts General Hospital CRS (MGH CRS) (Site 101) Grant UM1AI69412, University of Rochester Adult HIV Therapeutic Strategies Network CRS (Site 31787) Grant UM1AI69511, Brigham and Women’s Hospital Therapeutics (BWH TCRS) CRS (Site 107) Grant UM1AI69412, University of California Los Angeles CARE Center CRS (Site 601) Grant UM1AI69424; and NIH/NIAID HIV Vaccine Trials Network (HVTN) UM1 AI068618; UM1 AI068614 and Duke Center for AIDS Research (P30 AI064518). Accelevir (G.M.L.) received funding from the National Institutes of Health under award numbers U24AI143503, R44AI124996, NSF1738428. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Figure 1

Supplemental Figure S1. Vaccine Components. The dual promoter p24CE1/2 plasmid (plasmid code 306H) expresses the two variants CE proteins CE1 and CE2. They each comprise 7 regions representing 54% of p24Gag. CE1 is expressed from the human cytomegalovirus (huCMV). CE2 is expressed from the simian cytomegalovirus (siCMV) promoter. The plasmid contains the bovine growth hormone polyadenylation (BGH polyA, CE1) and the simian vacuolating virus 40 polyadenylation (SV40 polyA, CE2) signals. The p55Gag plasmid (plasmid code 114H) contains the huCMV promoter and the BGH polyA signal and expresses the full-length HIV-1 p55Gag, comprising the p17Gag, p24Gag and p15Gag proteolytic cleavage products. The location of the 7 CE within p24Gag are indicated.

NIHMS1948308-supplement-Supplemental_Figure_1.jpeg (27.4KB, jpeg)
Supplemental Figure 2

Supplemental Figure S2. HIV-1 p24Gag-specific IgG binding antibody responses. Dot plots, with overlaying lines (A) or boxplots (B, C), depicting HIV-1 p24Gag-specific IgG binding antibody response magnitudes as Area Under the Titration Curve (AUC) by the Binding Antibody Multiplex Assay (BAMA). Serum samples were titrated and the AUC was calculated over dilutions 500, 5000, 50000, and 500000 using trapezoidal integration, with base 10 log-transformation of the dilutions. The tested antigen was the clade B p24 Gag protein. Absolute change in AUC was then calculated as (week 26 AUC – baseline AUC) for each participant. Relative change in AUC, was calculated as [(week 26 AUC – baseline AUC)/baseline AUC] for each participant. All assays were conducted according to Good Clinical Laboratory Practice Guidelines, including tracking of controls with Levey-Jennings charts. Each dot represents a participant from the respective treatment arm and time point being plotted. Lines join the same participant over study time points. Treatment groups are color coded: Arm A: p24CE1/2 pDNA + p55Gag pDNA (blue), Arm B: p55Gag pDNA (pink) and Arm C: placebo (orange). A) AUC is shown on the y-axis and the time points are shown on the x-axis. B) Absolute change in AUC, calculated as week 26 AUC – baseline AUC, the latter taken as the average of entry and pre-entry timepoints, shown on the y-axis, and treatment groups on the x-axis. C) Relative change in AUC, calculated as [100*(week 26 AUC – baseline AUC)/ baseline AUC], shown on the y-axis, and treatment groups on the x-axis. The treatment specific descriptive statistics, N=sample size, median and [Q1, Q3] = interquartile range are shown on top of each treatment group for panels B and C.

NIHMS1948308-supplement-Supplemental_Figure_2.jpeg (6.4MB, jpeg)
Supplemental Data File (.doc, .tif, pdf, etc.)
NIHMS1948308-supplement-Supplemental_Data_File___doc___tif__pdf__etc__.docx (256.3KB, docx)

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