These phase I trials of a recombinant VRP vaccine expressing a subtype C gag gene, modified to express nonmyristoylated Gag, demonstrated that the AVX101 vaccine was well tolerated in healthy adults at doses of up to 1 × 108 IU and that despite promising preclinical immunogenicity data, there were only modest immune responses among these trial participants. These trials also highlighted the difficulties in developing a novel vector for HIV. The trials were conducted sequentially over 3 years (2004 to 2006). Each trial was terminated early: one trial due to loss of vaccine titer during storage and the other due to potential issues with the contract manufacturer.
The vaccine was well tolerated and exhibited only modest local reactogenicity, similar to the modest reactogenicity seen in a phase I clinical trial of a VRP vaccine expressing cytomegalovirus (CMV) antigens (5
). Few severe adverse events were reported during the studies. Subjects were enrolled in the two studies, in the United States and Southern Africa, successfully, and safety assessments in the two populations were similar.
The ELISA data revealed consistent dose-dependent antibody responses in participants who received the higher doses of vaccine (1 × 106
to 1 × 108
IU), with 3/10, 8/10 and 10/10 U.S. participants responding; however, the magnitude was much lower than was seen in preclinical studies. Using an assay in which the endpoint titer was the last dilution eliciting an OD of >0.20, the ELISA GMT ranged from 4,200 to 6,450 after two injections of 1 × 105
IU of AVX101 in four studies in mice and was 508, 1,280, and 3,620 after three injections of AVX101 in rabbits at doses of 1 × 106
, 1 × 107
, and 2 × 108
IU, respectively (2
). In contrast, using an assay in which the OD at a 1:50 serum dilution was determined, the ELISA magnitudes at a dose of 1 × 108
IU in the clinical trial were only slightly above the positivity cutoff-adjusted OD of >0.2. In addition, because the two higher doses (1 × 107
and 1 × 108
IU) were not studied in Southern African participants, any conclusions about the anti-Gag antibody response are limited to U.S. participants.
Similarly, despite promising results in preclinical studies, AVX101 was poorly immunogenic in stimulating Gag-specific cellular immune responses in people. Preclinical immunogenicity studies of AVX101 in mice demonstrated robust cellular responses. In mice given two injections of 1 × 105
IU of AVX101 by subcutaneous footpad injection, positive ELISpot responses were detected in 100% of mice, with responses ranging from 75 to 420 SFC per 105
splenocytes, and 51
Cr-release CTL assays showed >30% specific lysis at a 50:1 E:T ratio using splenocytes stimulated with Gag peptides and >30% specific lysis at a 6:1 E:T ratio using splenocytes stimulated with a vaccine-Gag virus (2
). Additionally, a prototype VRP vaccine expressing simian immunodeficiency virus (SIV) gag
genes was associated with high levels of immunogenicity in nonhuman primates (NHP) (8
). In that trial, rhesus macaques received VRP-expressing proteins encoded in the SIVsm H-4i molecular clone. Four inoculations were administered over a period of 21 weeks: two subcutaneous injections both at 1 × 105
IU and two intravenous (i.v.) injections at 1 × 107
and 5 × 108
IU. Humoral and cellular immune responses were measured, and animals were challenged with SIVsm E660 given i.v. Antibody responses to Env were not detected following the two s.c. injections, but binding antibodies were detected after the first i.v. inoculation (the GMT measured 3 weeks following the last vaccination was 17,947). Two of the four vaccinated monkeys showed strong cellular immune responses to both SIV proteins, one had a low-level response to either Gag or Env, and the fourth vaccinated monkey had no detectable cellular response. Four weeks after immunization, animals were challenged with SIVsm E660 administered i.v. All animals were infected; however, two of the four control animals showed signs of illness by 4 weeks postchallenge and were eventually sacrificed. All four of the vaccinated animals were protected from disease progression with a reduction in viral load of 200-fold compared to controls, and there was an inverse relationship between viral load and humoral and cellular immune response.
In contrast to these positive data from a different VRP vaccine in nonhuman primates, and the encouraging preclinical data with AVX101, the data from this clinical trial demonstrated a dose-dependent anti-Gag antibody response but only a marginal T-cell response to Gag in humans. Only low levels of binding antibodies to p55 were achieved at the highest doses (1 × 107 and 1 × 108 IU), notwithstanding the expectation that infection of dendritic cells by the VRP vaccine would result in a strong immunogenic response. T-cell responses as detected by a 7-day 51Cr release CTL assay were seen in only a few participants and only at the 1 × 105 and 1 × 106 IU doses in participants from Southern Africa, and as measured by ICS assay, CD4+ T-cell responses appeared more frequent than CD8+ T-cell responses. By ELISpot assay only one participant, who received the 1 × 107 IU dose, had detectable T-cell responses.
The reasons for the differences between the preclinical and human phase I data are unclear. Preexisting anti-vector immunity is not the reason, since anti-VEE virus antibodies were present at enrollment in only one subject. SIV Gag has been shown in several studies to be more immunogenic than HIV-1 Gag. In addition, the gag
gene cDNA in AVX101 was modified by site-specific mutagenesis to remove the amino-terminal myristoylation site in order to prevent the formation of Gag-derived virus-like particles (VLP) during production in the Vero packaging cells (6
). It was unclear at the time of the project's initiation whether the downstream purification process would be sufficient to remove the large majority of contaminating VLP from the VRP vaccine harvest. Thus, the modification was employed to simplify the purification and characterization of the AVX101 vaccine particle components. Based in part on the results of the current clinical trial, an HIV Gag VRP vaccine has been designed using a gag
gene with a normal myristoylation site. The immunogenicities of VRP vaccines expressing nonmyristoylated (myr−
) Gag (i.e., AVX101) and myristoylated (myr+
) Gag were compared in nonhuman primates (Chinese rhesus macaques) injected at weeks 0, 4, and 24. Gag-specific T-cell responses as measured by IFN-γ ELISpot were positive in 5 of 5 Gag (myr+
) VRP-vaccinated animals by 4 weeks after the first dose and remained positive at all later time points. In contrast, only 4 of 5 AVX101-vaccinated animals had an inconsistent positive response, at only one or two time points after the second dose. Mean responses were 4- to 5-fold higher in Gag (myr+
) VRP-vaccinated animals than in AVX101-vaccinated animals after the 2nd (514 versus 126 SFC/106
PBMC) and 3rd (514 versus 126 SFC/106
PBMC) injections (Olmsted et al., unpublished data). Irrespective of the vaccine received, all animals mounted anti-Gag antibody responses after two doses of vaccine and in contrast to the vaccine-specific differences in T-cell responses, the Gag-specific ELISA titers induced by either vaccine were of similar magnitude. The results from this comparative immunogenicity study are encouraging and may warrant clinical evaluation of the Gag (myr+
) VRP vaccine to determine in human subjects whether a VRP vaccine expressing Gag (myr+
) would elicit more frequent and higher-magnitude Gag-specific T-cell responses than did AVX101.
This clinical study with AVX101 also demonstrated a dose-dependent antibody response to the VEE virus vector. This response was consistent with results in preclinical studies, and it is possible that the anti-vector responses played a role in the limited anti-Gag immune response. However, anti-VRP antibody responses do not appear to inhibit the ability of repeated injections of VRP vaccines to boost immune responses to other antigens in humans. In a clinical trial with a CMV VRP vaccine, anti-vector antibodies developed in most subjects after a first dose and all subjects after a second dose, but CMV gB-specific antibody titers were boosted after the second and third doses of vaccine (5
). Similarly, in a cancer immunotherapy clinical trial comparing two dose levels of a carcinoembryonic antigen (CEA) VRP vaccine, after a single dose of vaccine the anti-vector antibody titer increased dramatically and remained elevated throughout the period of repeated immunizations. Despite this, there was a significant increase in immune response for at least one postvaccination time point versus prevaccination for all three assays (anti-CEA antibody, IFN-γ ELISpot, and ICS) in the high-dose cohort (13
The HVTN 040 and 059 trials also demonstrated the difficulty inherent in process development for biological products such as virus-vectored vaccines. Given the fact that the vast majority of products that enter clinical trials do not successfully complete phase III testing and become licensed, it is not feasible to complete all process development activities, such as manufacturing scale-up and long-term stability testing, before initiating clinical trials. With the AVX101 vaccine, a single dose-response clinical trial (HVTN 040) was originally planned. Because of issues with stability, the HVTN 040 trial was halted after enrolling only the first two dose level groups. After the first study was started, improvements in scale-up of the manufacturing process provided an opportunity to test a higher dose level in the second study (HVTN 059). However, because of issues with documentation at the contract research organization (CRO) where the vaccine was manufactured, the second trial was also halted prior to completion of dosing participants in Southern Africa. Stability issues, including loss of potency and critical storage conditions, may come to light only once trials are under way, as was the case for HVTN 040. Further, many candidate HIV-1 prophylactic vaccines have their start in small academic or biotech environments. For many, a contract manufacturer is the only option for production of clinical-grade material. Although these manufacturers may have passed audits and have documented procedures in place, the developer of the vaccine does not have complete control over the facility and processes. As such, problems may arise at any point, including once a trial is under way, that can result in halting a study.
Results in animals often are not completely predictive of results in humans, but there is no alternative that is more predictive. There are no in vitro models of immunogenicity that have demonstrated good predictive ability. These hurdles, as well as the challenges inherent in the manufacture of these complicated vaccines, face all vaccine developers. Despite these problems, and the premature stopping of both trials, these studies were able to address the primary and secondary objectives, to evaluate the safety and tolerability and immunogenicity of increasing doses of AVX101 given by s.c. injection. AVX101 was well tolerated, but the cellular and humoral immune responses were disappointingly modest compared with the promising preclinical results.