HIV-1 subunit vaccines based on recombinant forms of the monomeric gp120 glycoprotein were first developed over 10 years ago. Their creation was a logical consequence of the assumption that neutralizing antibodies were likely to be a correlate of protection against HIV-1 and of experimental observations that most serum HIV-1-neutralizing antibody activity was directed at gp120 (58
). It is still a central tenet of HIV-1 vaccine design that it would be desirable for a vaccine to induce broadly neutralizing antibodies able to prevent HIV-1 transmission or to significantly limit systemic dissemination of the virus. It also remains true that gp120 contains most, but not all, of the neutralizing-antibody epitopes identified to date (71
). Most studies of the gp120 vaccines in chimpanzees have indicated that the apparent correlate of protection against infection is the anti-gp120 antibody response, more specifically the response to the V3 region (7
). Therefore, much of our effort has focused on characterizing the antibody response to gp120 in the infected vaccinees.
Assuming that a preexisting anti-gp120 antibody response alone is sufficient for protection of humans from HIV-1 infection, which is far from certain, the most probable explanation for infection of these vaccine recipients is that the subunit rgp120 immunogens did not induce antibodies of sufficient quality, abundance, or specificity to afford protection. Most of the infected vaccinees who received the complete course of vaccination did develop a reasonable antibody response to the immunogen (C20 was an exception in this regard). However, the anti-gp120 response was relatively type restricted compared to infection-induced responses, in that there was better reactivity with the vaccine rgp120 than with a different rgp120. It is possible that many of the antibodies induced by the rgp120 proteins were directed at the more variable epitopes on gp120, a feature which is not necessarily advantageous for neutralization of heterologous primary viruses under field conditions. However, some cross-reactive rgp120 monomer binding antibodies could clearly be detected in most vaccinees prior to infection. Although the peak antibody titers in many vaccinees approached infection-induced anti-rgp120 titers (Table ), the immune responses to the immunogens were generally transient. This meant that exposure to the infecting HIV-1 strain rarely occurred near the time when the anti-rgp120 response to booster immunizations was maximal, although C16 and C17 were notable exceptions. This could be significant, since successful protection in the HIV-1 LAI chimpanzee model was found when the animals were challenged at the time of their maximal antibody response to the immunogen (7
). However, even under these favorable conditions, using a neutralization-sensitive, vaccine sequence-matched HIV-1 strain, protection was found to occur in only about half the challenged chimpanzees. Conditions in the human trials are less favorable to successful protection in several respects, such as the timing of challenge with respect to immunization and the heterologous nature of the challenge. Furthermore, mucosal antibodies in genital tract secretions induced by systemic immunization were either absent or present at low levels. Assuming that HIV-1 infection in these vaccinees was acquired by the mucosal route, the potential protective effect of local antibodies was not manifest.
What is relevant to protection is not the ability of a vaccine such as rgp120 to induce antibodies reactive with itself but the ability of the immunogens to induce antibodies capable of binding to the infecting strain and neutralizing it. A correlate of virus neutralization is antibody reactivity with the native, oligomeric form of the envelope glycoproteins (48
). We did not test the ability of the rgp120 vaccines to induce oligomer-reactive antibodies; however, using assays based on TCLA viruses, MN and SF-2 rgp120s have been shown to induce in humans significant titers of antibodies able to neutralize the HIV-1 strain from which the immunogen was derived (6
). Information on the neutralizing-antibody titers elicited against the vaccine strain in the individuals we have studied is reported elsewhere (57
). It is now clear, however, that passage of HIV-1 in T-cell lines selects for variants with an abnormal sensitivity to neutralization that is not typical of primary viruses (96
). This may reflect, in part, adaptation of HIV-1 to use the CXCR4 coreceptor after passage through cell lines instead of the CCR5 coreceptor that is used by most primary, NSI strains to enter PBMC (26
). Consequently, it is not now clear whether the induction by the rgp120 vaccines of antibodies able to neutralize HIV-1 MN or HIV-1 SF-2 has any real significance for the protection of humans against primary viruses under in vivo conditions.
We found that the subunit rgp120 vaccines failed to induce, in any individual, antibodies capable of neutralizing (by 90%) the primary HIV-1 strain isolated from that individual during the acute phase of infection or soon thereafter. This finding is in accord with those of others, who observed that a spectrum of primary viruses were strongly resistant to neutralization by serum antibodies from rgp120 vaccinees (96
). We showed, however, that most HIV-1 strains isolated from the infected vaccinees could be neutralized by human MAbs and by a CD4-based reagent with sensitivities that were comparable to those of two sets of control primary HIV-1 strains. This suggests that the preexisting anti-gp120 antibody response has not exerted a selection pressure which permits infection of the vaccine recipients only by especially neutralization-resistant HIV-1 strains.
The absence of HIV-1-specific IgA antibodies in external secretions of systemically immunized and infected individuals is not surprising; from numerous studies using other microbial antigens, it is well known that systemic immunization is usually not effective in the induction of secretory IgA antibodies (for a review, see reference 103
). Furthermore, it has been the experience of the AVEG that systemically administered HIV-1 vaccines have not been effective inducers of mucosal IgA anti-HIV-1 antibodies (unpublished data). In HIV-1-infected individuals, mucosal antibodies are mainly of the IgG isotype (3
Although the soluble rgp120 immunogens were not designed to induce CTL activity, there is evidence that they can do so to a limited extent (3
). We were unable to assess the ability of the rgp120 vaccines to induce CTL activity prior to infection because of the nature of the biological samples provided to us. However, the envelope CTL responses after infection were infrequent and, when present, were not vigorous among the infected vaccinees. This is not surprising, since the soluble rgp120 proteins would not be expected to enter the class I pathway that allows for presentation to CD8+
CTL. The frequency of Gag responders (55%) was similar to that found for a cohort of HIV-1-infected men from the San Francisco Men’s Health Study (150
). Although the degree of Env responsiveness was low in this group of individuals, this is consistent with previously published reports (129
) and our own unpublished data (150
). The lack of a suitable control group limits speculation on the role that a prior humoral immune response may have had in the generation of Env-specific CD8+
CTL, as has been described for an animal model (157
By definition, the MN and SF-2 rgp120 vaccines did not prevent infection in any of the individuals we studied, but it is also important to evaluate whether vaccination might ameliorate disease in the infected recipients. The individuals in this cohort have not been studied long enough for any clinically beneficial or adverse effects of vaccination to be identified, and there are too few participants for a definitive conclusion to be reached in any case. However, one parameter we could usefully measure was the virus burden. Prechallenge vaccination of chimpanzees with LAI rgp120 was found to reduce postinfection plasma viremia in some HIV-1LAI
-infected animals (143
), and passive administration of the anti-gp41 virus-neutralizing MAb 2F5 to chimpanzees prior to challenge with a primary HIV-1 isolate also reduced the viral burden (24
). Furthermore, there is now compelling evidence linking the level of persistent plasma virion-associated RNA to disease progression (92
). There was a slight trend toward fewer infected vaccinees with very high viral burdens, but there was no significant difference in the overall distribution of viral burdens among the infected vaccinees compared to a control group of symptomatic acutely infected individuals and matched case controls at 9 to 12 months postinfection. The median plasma viral burdens for the infected vaccine recipients and the controls in our study of 8,500 and 9,325 RNA copies/ml, respectively, are comparable to those found by others (52
). The conclusion that rgp120 vaccination did not significantly affect the postinfection viral burden has also been reached by others (57
We determined the phenotypes and growth characteristics of the strains isolated from the infected vaccinees. In all but two cases (C04 and C20), the strains possessed the NSI phenotype, in that they were unable to replicate and form syncytia in the MT-2 T-cell line. The coreceptor usage patterns for several selected isolates were consistent with the phenotype determinations. SI strains were isolated from individuals C04 and C20; it is shown elsewhere that these individuals underwent particularly rapid declines in their CD4 cell counts (57
), a phenomenon associated with the presence of SI virus (70
). The presence of SI strains in individuals C04 and C20 is not necessarily an adverse effect of rgp120 vaccination, since a minority of transmitted strains in unvaccinated individuals have the SI phenotype (46
). Determination of the growth rates of isolates from infected vaccinees in PBMC in vitro revealed that there was nothing unusual about their replication competence; indeed, some of the isolated viruses grew quite vigorously and showed distinct cytopathic effects when cultured in vitro. The phenotype and virus culture data do not, therefore, support the idea that vaccination permitted the transmission only of weakly replicating HIV-1 strains or, conversely, of strains that were especially virulent.
Genetic sequencing and phylogenetic analysis of the HIV-1 strains from infected vaccine recipients indicated that they were not infected with either an unusual lineage or atypically divergent viruses, relative to contemporary clade B viral strains from the United States, the presumed country of infection of all the trial participants. We also systematically examined protein sequence variation, both by scanning gp120 amino acid sequences for signatures and by specifically analyzing linear domains previously identified to be potentially immunogenic. Our goal was to identify distinctive patterns in protein variability that could be considered indicative of vaccine-induced selective pressure among the infected vaccine recipients. However, it should be emphasized that with small sample sizes, moderate effects will not generally provide very low P values. Furthermore, these statistical tests were done in the context of an open-ended exploratory data analysis, and multiple tests confound the interpretation of borderline significance estimates. Thus, the statistical measures used here provide the basis for a ranking system rather than a reliable indication of selection.
Given these limitations, we identified the most-distinctive amino acid positions and motifs in gp120 from sequences obtained from infected vaccine recipients relative to the controls. Once a region or site was identified as being among the most distinctive, we used other biological and statistical considerations to evaluate the relevance of the site, some of which are summarized below. For example, the most variable motifs were no more unusual than could be found by analysis of randomly selected sequences. Second, sites that appeared to be distinctive among vaccine recipients compared to the control group were not necessarily distinctive when compared to the B subtype sequences from the recent Los Alamos Human Retroviruses and AIDS Database. Third, the potentially interesting sites or motifs identified were specific for viruses obtained from either the set of MN rgp120 vaccinees or the SF2 rgp120 vaccinees; there was no concordance between regions that were distinctive for the two sets. Finally, the sequence analysis results need to be considered in the context of the neutralizing-antibody assays: vaccinee sera collected prior to infection did not neutralize primary isolates, and strains infecting vaccinees were not particularly resistant to the effects of potent neutralizing MAbs. Taken together, these factors argue against specific vaccine-induced selection pressures.
The V3 amino acid sequences of the strains isolated from the MN rgp120 recipients did not precisely match that of MN rgp120, and MN has a motif at the tip of the V3 loop (GPGRAFY) that is similar to the United States’ B subtype consensus sequence (90
). Additionally, when using an amino acid substitution matrix that estimates protein sequence similarities based on the alignments of the antigenic regions in V3, a trend toward greater distance from the vaccine strain among the viral sequences from MN vaccine recipients was observed. These observations should be qualified. Only the viruses obtained from MN rgp120 vaccinees showed greater variability in this region, and in particular the association was found only in patients given three or more vaccinations prior to infection. If one considered the SF2 cases, which share the GPGRAF motif at the tip of the V3 loop with MN, or the full set of MN vaccinees, greater-than-expected variability in the V3 motifs was not evident. The MN rgp120 vaccinees whom had only two vaccinations prior to infection had detectable anti-V3 antibody prior to infection, while three of those who received three or more vaccinations prior to infection did not (57
). Thus, the divergence from the MN sequence in the V3 regions of some vaccinees is quite possibly attributable purely to chance. It should also be noted that MN and SF-2 are TCLA strains isolated in 1983 to 1984, so the sequence divergence from them in a variable region is an inevitable consequence of more than a decade of evolution of HIV-1 in vivo combined with the consequence of selection in cell lines in vitro.
In the context of analyzing potential vaccine-induced selection pressures, it is also important to point out that many recent studies concerning the V3 loop have brought its relevance to vaccine protection into question. First, there is evidence that while antibodies to the V3 region can potently neutralize HIV-1 TCLA strains, they are not of paramount importance for neutralization of primary strains (96
). Second, HIV-1 V3 sequences are continually diversifying, so the proportion of contemporary strains in the United States that have a V3 sequence closely matching the MN V3 sequence is inexorably diminishing, and most individuals, after primary infection, carry a virus with detectable variation in this region (78
). A particularly graphic example of increasing divergence in the V3 loop crown was provided by a recent study of virus strains circulating in Memphis, Tenn. (128
). Of viruses collected early in 1993, 78% of the sequenced strains contained the GPGRAF motif, while approximately 1 year later, this proportion had decreased to 22%, with a wide spectrum of motifs being found instead. This degree of sequence divergence, in the absence of any putative vaccine-induced selection pressure, should be recalled when considering the sequences of the vaccine and control isolates (Fig. ). Third, the GPGRAFY motif is not common globally (13
). Fourth, neutralization serotype studies (97
) all indicate that V3 antibodies do not contribute to cross-neutralization in any dominant sense. Thus, it seems unlikely that the rgp120 vaccines have exerted selection pressure on the mixtures of strains to which the vaccinees may have been exposed such that only strains with a V3 loop significantly divergent from that of the immunogen were transmitted.
The purpose of our study was not to determine the efficacy of the MN and SF-2 rgp120 vaccines, and the number of cases of infected vaccinees that we have studied to date is too small to draw conclusions about the efficacy of these immunogens. We note, however, that the distribution of infected individuals among the MN rgp120, SF-2 rgp120, and placebo groups was similar to that in the test population as a whole (Table ). Furthermore, the total number of infections of study subjects enrolled in the 201 cohort represents an annual infection rate of about 2.0 cases per 100 person-years, which is not unexpected for a high-risk cohort in the United States. We were unable to obtain evidence that rgp120 vaccination has had any significant impact on the in vitro or in vivo characteristics of the infecting HIV-1 strains. More effort needs to be focused on identifying the forms of immunogens that best present neutralizing antibody and CTL epitopes to the human immune system, defining the kinetics and magnitude of responses needed for protection, and determining how immunogens can best be formulated to induce protective immune responses, rather than on further evaluation of the rgp120 and rgp160 vaccines.