Human immunodeficiency virus type 1 (HIV-1) superinfection occurs when an individual chronically infected with one strain of HIV-1 becomes infected with a second strain, indicating that natural immune responses to HIV-1 are not always protective. Since superinfection occurs despite ongoing immune responses to the first HIV-1 strain, it provides an avenue to explore how specific immune deficits allow HIV-1 infection to become established. To date, approximately 30 well-characterized cases of HIV-1 superinfection have been described based on longitudinal follow-up (1
); many other presumed cases have been defined in cross-sectional studies, where there is evidence of dual infection at the time when viral sequences were examined (reviewed in reference 43
). Many of the cases of superinfection identified in longitudinal studies occurred within the first year following initial infection, when immune responses to HIV-1 are often not fully mature. However, HIV-1 superinfections have also been found frequently during chronic infection (38
), when the immune response to HIV-1 should be fully developed.
The frequency of superinfection likely depends on a variety of factors, including the nature of the superinfecting strains, the use of antiretroviral medications, and the immune status of the individual. Several studies, which screened more than 3,000 individuals, found no evidence of HIV-1 superinfection, though many of these individuals were receiving antiretroviral therapy (6
). In contrast, a study of Thai intravenous drug users found two cases of HIV-1 superinfection among 130 chronically infected individuals (41
). More recently, three population-based studies found that HIV-1 superinfection occurred at a rate close to that of initial infection. In a study of high-risk women in Kenya, the incidence of superinfection was approximately 4% per year (7
), approximately half the incidence of primary infection in the same cohort of 8% per year (15
). Among a cohort of men in southern California, the incidence of superinfection was 5% (45
), which was equal to the initial infection rate of 5% per year in a similar cohort (12
). The frequent detection of superinfection in these more recent studies calls into question what role, if any, immunity to the first strain has in protection from the second strain.
The relatively small number of well-characterized cases of superinfection has limited analysis of the role of the immune response in superinfection. Thus, it remains unclear whether only a subset of individuals with particularly poor immune responses succumb to superinfection or whether immune responses during HIV-1 infection are in general inadequate to prevent infection. All six superinfected subjects in whom cellular immune responses have been assessed had cytotoxic T lymphocytes (CTL) directed toward their initial strain, as measured by gamma interferon enzyme-linked immunospot assay (1
). While there were differences between the studies in the number of potential epitopes evaluated, the breadth of the immune responses to the initial HIV-1 strain varied in these superinfected individuals, with four individuals having very broad responses to multiple epitopes (1
) and two individuals having relatively narrow responses predominantly directed to a single epitope (13
). In four of these cases, at least some of the CTL present were cross-reactive with the superinfecting strain prior to reinfection (1
). However, in all six cases at least some of the targeted CTL epitopes were altered in the superinfecting strains, which could have contributed to the ability of these strains to establish infection (1
). Furthermore, as CTL play a critical role in controlling an established infection but are ineffective in preventing initial infection (reviewed in references 5
), it is perhaps not surprising that these cellular responses were insufficient to prevent reinfection.
Unlike CTL, neutralizing antibodies (NAbs) to HIV-1 can prevent infection in animal models (reviewed in reference 46
); NAbs, therefore, might be able to prevent superinfection in humans if sufficiently broad and potent. Among two cases of superinfection described by Ramos et al., binding antibodies to the V3 region were generated to the initial strain though these did not appear to cross-react with the superinfection strain (41
). Moreover, in this study, neutralization was not assessed (41
). In a third case, only weak NAbs were present to the initial virus, and these antibodies did not neutralize the superinfecting strain (1
). In the only study to compare superinfected individuals to those with similar risk factors who did not become superinfected, three individuals who became superinfected had weak NAb responses to their initial infection (44
). These three superinfections all occurred relatively early, within 6 months of initial infection. In addition, this study examined the ability of plasma from the superinfected individuals to neutralize just three viruses, and responses to the superinfecting virus itself were not examined. In order to more rigorously evaluate the potential role of NAb responses in protection from superinfection, we assessed the NAb responses in six cases of superinfection that occurred throughout the course of chronic HIV-1 infection using a larger panel of viruses, including the superinfecting strains.
We comprehensively evaluated the role of the breadth and the potency of the HIV-1 NAb response in protection from infection using six cases of superinfection that were identified among a cohort of female sex workers with extensive long-term follow-up (7
). At the time of superinfection, no significant deficits in NAb responses were observed in the superinfected individuals compared to matched controls. Thus, even NAb levels typically found during chronic infection can fail to protect from reinfection with circulating strains of HIV-1. Furthermore, in four of five cases evaluated, superinfection occurred despite preexisting plasma NAbs capable of neutralizing the strains that established the second infection.
The breadth and potency of the NAb responses were heterogeneous among these individuals at the time of exposure to the superinfecting virus. As the variants within the virus panel were chosen on the basis of their neutralization sensitivity to pooled plasma, most individuals were able to neutralize at least one of these heterologous variants. Overall, the breadth and potency of the NAb responses were similar to those found in the matched controls and other chronically infected individuals. In particular, four subjects had relatively robust NAb responses, while two others had comparatively narrow responses. In fact, the four superinfected subjects with the broadest NAb responses could neutralize the ∼70% of the viruses within the panel with average IC50
s of ∼110, while the matched controls neutralized ∼40% of the panel viruses with average IC50
s of ∼90. Admittedly, limitations in the numbers of cases and controls limited the robustness of the statistical analyses. We therefore compared the NAb responses of the superinfected individuals to those of 72 individuals from the same cohort whose NAb responses were assessed at 5 ypi (K. Bosch, D. Panteleeff, and J. Overbaugh, unpublished data). Three superinfected subjects (QA413, QB045, and QB726) had breadth and potency scores within the upper quartile of these 5-year responses. Subject QA013 had breadth and potency scores at the median of the 5-year NAb responses despite having been superinfected relatively early after the first infection (∼1 year), at a time when the breadth and potency of the NAb response has not yet peaked (42
). These 72 women were not selected based on any clinical or immunological findings; thus, they represent typical NAb responses in high-risk African women during chronic infection. Four of the six superinfections therefore occurred in individuals with relatively broad and potent NAb responses at the time of exposure. It is difficult to draw precise comparisons with our data and NAb responses observed in other cohorts because of differences in assays and test strains used. In addition, most of the published studies have focused on selected individuals, particularly long-term nonprogressors, which is not an ideal comparison group (2
). While rare individuals in these populations exhibited apparently broader and more potent NAb responses (e.g., neutralization of ∼90% of viruses tested at average IC50
s of ∼230) (8
) than the women in our study, many other individuals exhibited less breadth and potency in NAb responses against the test strains used. On the basis of all these comparisons—including to matched controls, to chronically infected women in the same population, and to published studies—we conclude that the women who became superinfected did not have deficits in the breadth or potency of their NAb responses relative to other HIV-infected groups when they became superinfected. Overall, NAb responses in superinfected individuals were typical of HIV-1 infection, ranging from narrow to relatively broad.
At ∼1 ypi, we observed relatively narrow NAb responses in superinfected individuals compared to controls. This early lack of breadth in the superinfected women mirrors the findings in three superinfected men who acquired HIV-1 through sex with men (44
). Interestingly, this same association was observed despite the fact that the study of Smith et al. used a small number of primarily laboratory-adapted viruses whereas ours employed a large collection of variants cloned directly from infected individuals near the time of transmission. This early association between breadth and risk of superinfection in both studies suggests that some association between HIV-1-specific antibodies and risk of superinfection may exist early in infection. However, we did not observe a correlation between NAb breadth at the time of superinfection and whether a woman became superinfected. At these later times, which seem to be a more relevant measure of the role of NAbs in protection from infection, there was a broadening of the NAb responses in most individuals prior to their documented superinfection.
Perhaps a more important measure of the role of NAbs in protection from superinfection is the study of the antigenicity of the specific viruses that established the second infection. In this study, we examined such viruses derived from the first time point following documented superinfection. We found that these viruses were not unusually neutralization resistant as there were no differences in the neutralization sensitivities of these variants and other circulating variants to pooled plasma from HIV-1-infected individuals. Moreover, the infected individuals were able to mount autologous NAb responses to the superinfecting variants, suggesting that the strains could themselves elicit NAbs. Importantly, the superinfecting variants were often susceptible to neutralization by plasma from the person who became infected by these strains: in four of five subjects, at least one superinfecting variant was susceptible to the host plasma from the time prior to superinfection. Even if a “sieve” effect weeded out the majority of the neutralization-sensitive viral variants from the donor virus, at least some of these variants apparently established infection despite encountering NAbs capable of neutralizing them. While we cannot rule out that these variants evolved within a narrow window after transmission, it is not clear what would drive them to become more susceptible to neutralization. Overall, these data suggest that the levels of NAbs found in these individuals were insufficient to prevent infection even by variants that showed some susceptibility.
Several caveats to these data need to be considered. First, as with all cases of superinfection, it remains possible that the superinfecting viruses were present at low levels and/or compartmentalized prior to their first detection. We have reasonable confidence that the superinfecting strains were detected soon after they became established in these cases because we used a sensitive subtype-specific PCR assay that gives >92% probability of detecting a strain present with a prevalence of 5% to define the time of superinfection (38
). Moreover, the women in this cohort have relatively few partners (on average 1 to 2 per week) (16
), making superinfection in a short time frame less likely than reported in women who have many more partners (11
). Secondly, we cannot conclude that control subjects were protected from superinfection because superinfections could be missed if the second virus did not persist (56
) or recombined with the initial virus within the regions of the genome analyzed (38
). If such cases were missed within the control group, this would decrease our ability to detect differences between cases and controls. Third, new infections are generally established at mucosal sites, where NAb levels could be lower than those assessed in the plasma, possibly allowing local establishment and spread of infection before immune control could be attained. It is therefore possible that differences could be observed in NAb levels at the mucosal sites, in particular, against the superinfecting strains near the time of infection.
While this study suggests that the presence of any detectable NAbs of the proper specificity may not protect against HIV-1 infection, it does not rule out the possibility that such antibodies would be effective if present at higher levels. These data are consistent with the nonhuman primate (NHP) model, where sterile protection from infection generally required very high NAb levels that produced >99% in vitro neutralization of the challenge simian-human immunodeficiency virus with between 1:8 and 1:200 dilutions of plasma (26
). While differences in the assays used to assess neutralization between the NHP studies and our human study make direct comparisons difficult, the NAb levels in the superinfected individuals were probably not at this potency. Even the unusually neutralization-sensitive variant QA013.J36* was neutralized at 90% by host plasma at a 1:78 dilution but did not achieve 99% neutralization at the lowest plasma dilution tested (1:50). The remaining superinfecting variants were neutralized at levels of <90% with a 1:50 dilution of plasma and were therefore not at the levels required for sterilizing immunity in the NHP model. Thus, it remains unclear whether the higher levels of NAbs achieved by passive transfer within the NHP model would be protective in humans.
These findings in exposed humans, where there is extensive diversity in potential infecting strains, unfortunately suggest a high bar for the levels of antibodies required for eliciting protective immunity. While weak, narrow NAb responses could have contributed to superinfection in a subset of individuals, others became reinfected despite relatively robust NAb responses to their first strain. Since NAbs can clear virus without dependence on the cellular immune system, the levels of antibodies required for protection in these previously infected individuals are likely similar to those required in uninfected, vaccinated individuals. Furthermore, most effective vaccines are thought to provide protection primarily by stimulating neutralizing antibodies to clear cell-free virus (35
); thus, the assays used here should provide a valid measure of viral protection by this mechanism. An effective HIV-1 vaccine will therefore need to elicit more robust NAb responses than found during natural infection. Indeed, this is the case for some other viral vaccines, such as those for hepatitis B and human papillomavirus, which elicit equivalent or higher levels of NAbs than natural infection (4
). HIV-1 presents additional challenges because of the extreme genetic diversity of the virus. Our results suggesting that reinfection occurs even in individuals who have antibodies capable of neutralizing diverse strains further underscore this challenge.