The HIV epidemic severely affects the Caribbean and Latin America, with an estimated 30,000 new infections in the Caribbean and 200,000 in Latin America in 2005.1 In the Caribbean, HIV transmission is largely through heterosexual intercourse, whereas in Latin America the epidemic is fueled by combinations of men who have sex with men, heterosexual transmission, and injection drug use.
Recognizing the scientific need and ethical mandate to conduct HIV vaccine trials in Caribbean and Latin American countries heavily affected by the AIDS epidemic, the HIV Network for Prevention Trials (HIVNET) began planning in November 1997 for an HIV vaccine trial in Brazil, Haiti, and Trinidad and Tobago. Peru was later added as a site. The primary objective of HIVNET 026, only the second National Institutes of Health (NIH)-sponsored vaccine clinical trial in the developing world,* was to evaluate the immunogenicity and safety of ALVAC-HIV vCP1452 with or without a MN rgp120 boost. The long-term objective was to develop capacity in sites in this region to evaluate vaccines developed to prevent clade B HIV-1.
Given the lack of knowledge of protective immunity against HIV, such a vaccine would ideally elicit both memory cytotoxic T lymphocyte (CTL) responses and HIV-neutralizing antibodies. Because of difficulties in eliciting neutralizing antibodies against primary HIV strains,2-4 the approach taken in this trial was a vaccine combination designed to primarily elicit CTLs.5-10 HIV-specific CTLs play an important role in the control of lentivirus replication in primate models.11-15
Recombinant poxviruses vectors have successfully delivered HIV proteins to the host immune system.16-18 Canarypox is host-range restricted19,20 and does not fully replicate in cells of human origin. Canarypox-based vectors have had an excellent safety profile in over 10,000 individuals, including HIV-infected persons.
ALVAC-HIV (vCP205), a recombinant canarypox vector that encodes the clade B envelope, gag, and protease of HIV-1, elicited HIV-specific CTL in up to 35% of participants when measured 2 weeks after final vaccination.5,6,8-10 Prior vaccinia exposure did not abate the HIV-specific CTL response. In an effort to make the vaccine more immunogenic, another recombinant, ALVAC-HIV vCP1452, was engineered so that in addition to the HIV-1 gene inserts encoded by vCP205, it contains genes encoding for CTL epitopes in nef and pol. Furthermore, two vaccinia virus genes, E3L and K3L, were inserted into the new construct. E3L and K3L had been previously shown to inhibit apoptosis of infected cells,22 thus theoretically prolonging antigen production and potentiating antigen presentation. At the time of initiation of HIVNET 026, ALVAC constructs were considered the leading HIV vaccine candidates and a parallel, large phase 2 study was being conducted in the United States (HIV Vaccine Trials Network [HVTN] 203).
Recombinant canarypox ALVAC-HIV vCP1452 (developed by Virogenetics, Troy, NY, and manufactured by Sanofi Pasteur, Marcy L'Etoile, France) is a modified recombinant canarypox virus expressing products of the HIV-1 env, gag, and pol genes encoding the protease, a synthetic polypeptide encompassing known human CTL epitopes from nef and pol, and sequences encoding the E3L and K3L vaccinia virus proteins.23 The lot of vCP1452 used in Brazil, Haiti, and Trinidad contained 107.15 TCID50 and that used in Peru contained 107.26 TCID50 per 1.0 mL dose. The subunit vaccine, MN rgp120, is a recombinant HIV-1 envelope glycoprotein of the HIV-1 MN strain formulated as 300 μmg/mL MN rgp120/HIV-1 adsorbed in 0.6 mg alum adjuvant (VaxGen, Brisbane, CA). Placebo ALVAC contained virus stabilizer and freeze-drying medium reconstituted in saline. The rgp120 placebo was aluminum hydroxide adjuvant.
Participants were HIV-negative individuals, aged 18 to 60 years. Initially volunteers were judged at low risk of acquisition of HIV infection by virtue of having ≤1 low-risk sexual partner; no injection drug or crack cocaine use; no newly acquired sexually transmitted diseases; and no exchange of sex for money, drugs, or services in the 6 months before joining the trial. Having an HIV-infected sexual partner was not exclusionary if the couple were abstinent or consistently used latex condoms. Based on subsequent recruitment difficulty, the criterion for number of sexual partners was liberalized to ≤2 in the past 6 months. Eligibility criteria included general good health and normal hematologic, hepatic, and renal screening tests. Participants had negative assays for hepatitis B surface antigen, HTLV-1, syphilis, and sickle cell disease. Exclusion criteria included histories of anaphylaxis or serious allergic reaction to any substance; allergy to egg products, neomycin, or thimerosal; prior participation in an HIV vaccine trial; and for women, current pregnancy or lactation. Women were required to utilize an effective contraception method during the period of immunization. For the Haiti, Brazil, and Trinidad sites, a viable autologous Epstein-Barr virus-transformed B lymphocyte (EBV) cell line was required before vaccination.
Informed consent was obtained from each volunteer in accordance with guidelines of the individual countries, US Department of Health and Human Services, and the sites' collaborating US institutions.
HIVNET 026 was a multicenter, randomized, double-blind, placebo-controlled clinical trial sponsored by the National Institutes of Allergy and Infectious Diseases (NIAID) and conducted at 4 research sites: the Institut National de Laboratoire et de Recherches (GHESKIO), Port-au-Prince, Haiti; Medical Research Foundation of Trinidad, Port of Spain, Trinidad and Tobago; Projeto Praça Onze, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; and, added 2 years later, Asociacion Civil Impacta Salud y Educacion, Lima, Peru. Each site randomized 40 participants in a 15:15:10 ratio to ALVAC-HIV vCP1452 boosted with MN rgp120; ALVAC with alum placebo boost; or placebo ALVAC and alum placebo (Table 1). An intramuscular injection in the left deltoid of 1 mL vCP1452 or placebo ALVAC was given at months 0, 1, 3, and 6. An intramuscular injection of 1 mL MN rgp120 or alum placebo was given in the right deltoid at months 3 and 6. All participants, clinic staff performing clinical evaluations, and laboratory personnel were blinded to immunization received.
Participants remained at the clinic for 30 minutes after each injection and returned the next day for assessment of erythema and induration at the injection site, lymphadenopathy, oral temperature, and self-reported local pain and tenderness, as well as systemic symptoms of malaise, myalgia, headache, nausea, and feeling feverish. Self-reported symptoms were collected for 7 days following each vaccination.
Participants with moderate reactions that required medication or with severe reactions were seen in the clinic within 24 hours of onset. Adverse experiences were assessed at all participant contacts for 18 months. Hematologies, chemistries, urinalyses, and HIV testing and counseling were performed throughout the study. Blinded clinical and laboratory safety data were reviewed weekly by a clinical group. The NIAID Data and Safety Monitoring Board (DSMB) reviewed unblinded data on trial conduct and safety every 6 months during the vaccination phase of the trial.
Fresh blood was shipped from the Brazil, Haiti, and Trinidad sites to the California Department of Health Services in Richmond, CA (CA-DHS), for establishment of autologous B lymphocyte cell lines (B-LCL) for chromium release and lymphocyte proliferation assays (LPA). Bloods for immunogenicity assays were drawn within 10 to 21 days of the third (day 98) and fourth (day 182) vaccinations and at day 364. The Peru site cryopreserved peripheral blood mononuclear cells (PBMCs) on site for shipment to CA-DHS for day 182 enzyme-linked immunosorbent spot (ELISpot) assays. Cell viability was similar to that of fresh specimens from the other sites that were processed and cryopreserved by CA-DHS if received within 36 hours of blood draw. Humoral assays were performed at the HVTN Duke Central Laboratory using stored frozen serum samples.
CD8+ cytotoxic T-cell responses were evaluated after in vitro stimulation of freshly isolated PBMC with recombinant vaccinia viruses as previously described.5,8,9,24-26 Assessments of specific CTL activity were carried out using a 51Cr release assay.21,25 Percent of specific lysis and positivity were calculated using published methods.21,25
The interferon-gamma (IFN-γ) ELISpot assay, using cryopreserved PBMC, was performed as previously described using 3gag peptide pools.26 This analysis of positivity has been used in all recent HVTN trials. Spots were counted using a CTL Analyzer and version 2.8 of the software (CTL Analyzers, Cleveland, OH). A statistical distribution free resampling method was used to determine positivity.27
3H Lymphoproliferative responses were measured on freshly isolated peripheral blood mononuclear cells using previously described methods.7,28 Antigens used were baculovirus-expressed recombinant HIV-1LAI p24, HIV-1MN gp160, and baculovirus control protein (Protein Sciences, Meriden, CT). The stimulation index (SI) was defined by median counts per minute (cpm) in the stimulated wells divided by the median cpm of control wells. An SI ≥3 was considered to be a positive response.
Anti-gag and anti-env binding antibody responses were determined by qualitative validated ELISAs.29 Serum from cryopreserved samples was tested in plates coated with purified gag p24 and gp120 (both Protein Sciences, Meriden, CT). Positivity was scored as duplicate antigen-containing minus nonantigen-containing wells with an optical density (OD) of ≥0.2.
Neutralization of HIV-1MN was measured in MT-2 cells as described previously.30 Heterologous neutralization was assessed in a luciferase reporter gene assay in TZM-bl cells31 using a panel of 12 molecularly cloned, clade B env-pseudotyped reference strains from early seroconverters.32 All serum samples were heat-inactivated at 56°C for 1 hour before assay.
The maximum severity of local and systemic reactions after any vaccination was scored. For immunogenicity responses, net response rates were calculated by subtracting the placebo from the vaccine rates. The net rate was considered statistically significant if the exact 95% confidence interval excluded 0. Pairwise differences in response rates between groups were tested with exact Fisher tests. Pairwise differences between groups in reactogenicity severity and neutralizing antibody titers were tested with nonparametric exact Wilcoxon rank sum tests. P values ≤0.05 were considered statistically significant.
Participant Accrual and Demographics
Each site enrolled 40 participants between March 2001 and February 2003. Enrollment at each site took between 9 and 15 months. Participants represented dominant race/ethnicity groups of their respective countries (Table 2). Vaccine and placebo groups were balanced for all demographic factors.
All 160 participants received their first vaccination and 146 (91%) received all 4 (Table 2). All but 6 (96%) participants completed the 18-month follow-up visit.
Both systemic and local vaccine reactions were mostly mild. Maximum pain or tenderness, and erythema or induration at the ALVAC/placebo injection site were significantly higher in both vaccine groups than placebo. As an example, 12% of ALVAC and 20% of ALVAC + MN rgp120 recipients experienced moderate pain, as compared to 5% of placebo recipients. There were no statistically significant differences between the vaccine and placebo recipients in local reactions at the rgp120/placebo injection site or in any of the elicited systemic reactions. Only minor differences in reaction rates by country were observed (data not shown).
There were no statistically significant differences in safety laboratories by group. Three participants had elevated ALT (≥5 times the laboratory upper limit of normal), all unrelated to vaccine. Other serious unrelated events were: death following an elective abortion, life-threatening injuries from an automobile accident, and cat scratch disease.
Net cellular response rates as measured by chromium release, ELISpot, or LPA assays were <14% and generally not statistically significant from placebo at either day 98 or 182 (Table 3). The only exception was LPA at day 182, for which the net response rate to gp120 in the combination group was statistically significant, 26% (95% confidence interval [CI]: 11% to 42%).
Anti-gag p24 and antigp120 antibody responses were detected by ELISA in all vaccine groups at day 98, peaked at day 182, and were minimal by day 364 (Table 4). Anti-gag p24 response rates between the 2 vaccine groups were not significantly different. The combination group had statistically significant higher response rates for antigp120 compared with ALVAC alone at days 98 and 182 (P < 0.001 for both).
NAb titers were significantly higher in both vaccine groups compared with placebo at days 98, 182, and 364 (all P values < 0.001). The boosted vaccine group had higher titers than ALVAC alone at day 98 (P < 0.001) and day 182 (P = 0.02) (Fig. 1). In the boosted group, nAb titers were higher after 1 protein boost than after 2 boosts (P < 0.001), and boosting had no effect on nAb persistence by day 364.
Blinded serum samples from day 182 for the 14 vaccine recipients with the highest titers on the vital dye neutralization assay (4 ALVAC alone, 10 combination) and 5 randomly selected placebo recipients were screened at a 1:10 dilution for neutralizing activity against 12 heterologous reference strains. One strain, SS1196.1, was neutralized by serum from 12 of 14 vaccine and 0 of 5 placebo recipients. This strain is unusually sensitive to neutralization by V3-specific antibodies. The other 11 strains are relatively insensitive to V3-specifc antibodies and possess a neutralization phenotype that resembles most primary isolates.32 For 2 strains, QH0692.42 and THRO.18, the combination group had significantly higher neutralization percentages than placebo (P = 0.007 and 0.01, respectively). The groups did not differ in the number of isolates with ≥50% neutralization (range of 0 to 5 isolates). In addition to this low frequency of positive neutralization, neutralization potencies against viruses other than SS1196.1 never exceeded an 80% reduction in infectivity, indicating a very weak heterologous neutralizing antibody response overall.
Vaccine-Induced HIV Antibody Positivity
Among non-HIV-infected vaccinees, 60 of 115 (52%) had a positive enzyme immunoassay (EIA) at their last study visit. Among the 60 who were EIA positive, 54 had an indeterminate Western blot (WB) and 6 a positive WB. Their uninfected status was confirmed by nucleic acid testing.
The percentage of participants reporting more than 2 sexual partners in the previous 6 months increased from a baseline of 2.5% to 11% by month 6 and thereafter remained constant (13% at month 12, 12% at month 18). Rates of other risk behaviors were constant over time, with no significant differences at any post-enrollment visit.
Despite enrollment of a lower risk population and intensive behavioral risk reduction counseling, 4 participants (2 each in the active vaccine groups) became HIV infected between 12 to 18 months post-enrollment. Three of the 4 had completed the vaccination schedule and the other received 3 vaccinations.
Social impact events from trial participation were assessed every 3 months. Thirty-one (19%) participants reported a negative social impact, with most (76%) involving personal relationships. The majority of events (93%) were considered resolved by the participant by the end of the study. Eleven participants reported 12 events that had a major impact on their quality of life (8 involved personal relationships; 2, housing problems; 1, loss of job due to time taken for study visits; 1, distress after testing HIV-antibody-positive on a nonstudy HIV test). Seventy-nine percent of participants also reported benefits from study participation, including the personal satisfaction of helping others, obtaining information on HIV and other health issues, and recognition by family and friends.
The safety and immunogenicity profiles of HIV clade B canarypox virus-vectored HIV vaccines, alone and in combination with recombinant envelope protein boosts, have been studied in the US and France among persons at high and low risk of acquiring HIV.5,6,25,33,34 HIVNET 026 evaluated a prime-boost regimen in clade B-affected developing nations. The safety and immunogenicity in HIVNET 026 paralleled the results obtained in the US and Europe-an important proof of principle for inclusion of international sites in analysis of vaccine responses.
A direct comparison between the reactogenicity profile observed among the 4 ethnically diverse HIVNET 026 international sites can be made with the NIAID-supported US study, HVTN 203. In that trial, ALVAC vCP1452, with or without AIDSVAX B/B boost (300 mcg/mL MN rgp120 and 300 mcg/mL GNE8 rgp120; VaxGen, Brisbane, CA), was administered on a schedule similar to HIVNET 026.34 HIVNET 026 participants reported, on average, less severe local pain and/or tenderness at the ALVAC injection site (P < 0.001) and less severe systemic symptoms (P = 0.01) than the HVTN 203 US population. However, HIVNET 026 participants had significantly more local erythema and/or induration (P = 0.04) and lymph node involvement (P = 0.01), as measured by clinic staff, than measured in HVTN 203 by participant self-reporting.
Vaccine immunogenicity was disappointing in HIVNET 026 (and HVTN 203). Among the 3 measures of CMI (51Cr release; IFN-γ ELISpot assay; and LPA), only an LPA response was elicited by the vaccine. The LPA assay may be less vulnerable to cellular impairments from intercontinental air shipments of fresh cells. Recent studies have shown that processing PBMC within 6 hours of collection influences the ability to detect both CD4 + CD8 + T-cell responses to antigens. Hence, shipping of samples from an international site to a central laboratory is not the optimal way to evaluate responses to candidate vaccines. As such, the T-cell-based results obtained in this trial may underestimate the true immune response to this vaccine.
There was little boost of ALVAC-primed HIV-neutralizing antibody titers after inoculation with the subunit envelope glycoprotein gp120. Because neutralizing antibodies to HIV-1MN were measured under the same conditions for HIVNET 026 and HVTN 203, comparisons can be made. For the ALVAC-only groups, participants in HIVNET 026 had significantly higher titers than US participants in HVTN 203 at both days 98 and 182 (both P < 0.001). For participants in the prime-boost groups, the day 182 titers were slightly higher for HIVNET 026 than HVTN 203 (P = 0.02, GMT of 117 versus 70). In HIVNET 026, as in HVTN 203, the magnitude of nAb titers declined by day 365. Higher antibody response rates to gag p24 were also detected by ELISA in the ALVAC-only group of HIVNET 026 compared with HVTN 203 (P = 0.002 for day 98 and 0.007 for day 182), but not for the prime-boost groups. Thus humoral responses were slightly higher in the HIVNET 026 populations than in US populations (HVTN 203).
Of greater eventual importance may be the impact on planning for global HIV vaccine development provided by operational lessons learned from HIVNET 026. HIVNET 026 was the first HIV vaccine trial conducted at each international site, although several had had preparatory funding to build HIV vaccine clinical trial capacity. In each country, differing but major challenges were presented in obtaining approval from national governments, establishing ethical and regulatory review, working with local press, conducting community education, and establishing formal community advisory boards. The UNAIDS Ethical Review Committee approved the trial at the invitation of the Haitian government. The details of some specific issues arising from the trial have been published, including obtaining optimal informed consent in a partially illiterate population35 and provisions for the treatment of patients who become infected with HIV during the trial.36
HIVNET 026 represented the first multinational HIV vaccine trial and utilized a single adverse-event grading system and centralized immunogenicity assessments. The complexities in initiating HIVNET 026 were immense. These included concordance on protocol design among multiple investigators; establishing an infrastructure for good clinical trials practice; obtaining permits for bringing drugs into the countries; satisfying international (UNAIDS), national, and community-specific recruitments for appropriate population screening and consent; informing the medical community, Ministries of Health, and local press of the trial; and, in some countries, dealing with social unrest.
These ethical and operational elements in initiating the trial were further confounded by challenges in shipping specimens to the central US laboratory. Transit time was calculated for 220 samples over a 6-month period: 46 (21%) were received at CA-DHS after 36 hours, and the mean transit time for shipment was 28 hours. As a consequence, a major paradigm shift in the planning of international HIV vaccine trials took place. Assays using cryopreserved specimens are now the norm, with well-standardized local cryopreservation. The requirement for established B lymphocyte cell lines at the CADHS laboratory before enrollment prolonged accrual. However, without this eligibility requirement, the laboratory was unable to establish autologous B-LCL for 4 participants.
That 4 HIV infections out of 160 participants occurred during the 18 months of the trial is a reminder that HIV infections will occur despite counseling, even in trials enrolling “lower risk” populations. The risk per encounter is inherently greater in settings with higher HIV prevalence. A slight increase from the baseline to 6 months in the number of reported sexual partners suggested increasing risk behavior during HIVNET 026, but may equally likely have represented initial underreporting of sexual activity. The enrollment criteria of ≤1 sexual partner in the past 6 months was a significant impediment to enrollment that was resolved by liberalizing the requirements.
Social harms reported in the trial were successfully addressed by investigators and staff at the sites, but stigma and bias will undoubtedly be seen in many international settings. Those with posttrial HIV-1 antibody positivity will continue to be provided HIV testing to differentiate between vaccine response and HIV infection.
In spite of these challenges, HIVNET 026 demonstrated the capability of these sites to enroll, vaccinate, and retain participants in a complicated vaccine protocol. The conduct of 026 was indistinguishable from the US-based HVTN 203 trial in terms of percentages of participants completing vaccination (91% and 92%, respectively) and 18-month follow-up rates (96% and 85%, respectively). Measures of good clinical practice instituted by the HVTN were and have remained comparable between US and non-US sites. Three of the HIVNET 026 sites continue to actively enroll in HVTN phase 1 and 2 vaccine trials. Two sites are actively enrolling in a large phase 2 proof-of-concept of an adenovirus recombinant vaccine, HVTN 502, to which each has contributed well over 200 participants, fulfilling the promise begun with HIVNET 026.
The authors gratefully acknowledge the participation and support of many colleagues and staff at each site. The contributions of the local Institutional Review Boards and Community Advisory Boards, Ministries of Health, UNAIDS, and US Customs and Border Protection officials are acknowledged. We are particularly grateful for the participation of all 160 study volunteers.
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*HIVNET 007, the first safety and immunogenicity study of vaccine in a developing country, was done in Uganda, also with canarypox.21