Human immunodeficiency virus type 1 superinfection occurs despite relatively robust neutralizing antibody responses

Superinfection by a second human immunodeficiency virus type 1 (HIV-1) strain indicates that gaps in protective immunity occur during natural infection. To define the role of HIV-1-specific neutralizing antibodies (NAbs) in this setting, we examined NAb responses in 6 women who became superinfected between ~1 to 5 years following initial infection compared to 18 women with similar risk factors who did not. Although superinfected individuals had less NAb breadth than matched controls at ~1 year postinfection, no significant differences in the breadth or potency of NAb responses were observed just prior to the second infection. In fact, four of the six subjects had relatively broad and potent NAb responses prior to infection by the second strain. To more specifically examine the specificity of the NAbs against the superinfecting virus, these variants were cloned from five of the six individuals. The superinfecting variants did not appear to be inherently neutralization resistant, as measured against a pool of plasma from unrelated HIV-infected individuals. Moreover, the superinfected individuals were able to mount autologous NAb responses to these variants following reinfection. In addition, most superinfected individuals had NAbs that could neutralize their second viral strains prior to their reinfection, suggesting that the level of NAbs elicited during natural infection was not sufficient to block infection. These data indicate that preventing infection by vaccination will likely require broader and more potent NAb responses than those found in HIV-1-infected individuals.     

Chronic HIV-1 infection frequently fails to protect against superinfection

Reports of HIV-1 superinfection (re-infection) have demonstrated that the immune response generated against one strain of HIV-1 does not always protect against other strains. However, studies to determine the incidence of HIV-1 superinfection have yielded conflicting results. Furthermore, few studies have attempted to identify superinfection cases occurring more than a year after initial infection, a time when HIV-1-specific immune responses would be most likely to have developed. We screened a cohort of high-risk Kenyan women for HIV-1 superinfection by comparing partial gag and envelope sequences over a 5-y period beginning at primary infection. Among 36 individuals, we detected seven cases of superinfection, including cases in which both viruses belonged to the same HIV-1 subtype, subtype A. In five of these cases, the superinfecting strain was detected in only one of the two genome regions examined, suggesting that recombination frequently occurs following HIV-1 superinfection. In addition, we found that superinfection occurred throughout the course of the first infection: during acute infection in two cases, between 1-2 y after infection in three cases, and as late as 5 y after infection in two cases. Our results indicate that superinfection commonly occurs after the immune response against the initial infection has had time to develop and mature. Implications from HIV-1 superinfection cases, in which natural re-exposure leads to re-infection, will need to be considered in developing strategies for eliciting protective immunity to HIV-1.     

In-depth analysis of a heterosexually acquired human immunodeficiency virus type 1 superinfection: evolution, temporal fluctuation, and intercompartment dynamics from the seronegative window period through 30 months postinfection

Human immunodeficiency virus type 1 (HIV-1) superinfection refers to the acquisition of another strain by an already infected individual. Here we report a comprehensive genetic analysis of an HIV-1 superinfection acquired heterosexually. The infected individual was in a high-risk cohort in Tanzania, was exposed to multiple subtypes, and was systematically evaluated every 3 months with a fluorescent multi-region genotyping assay. The subject was identified in the window period and was first infected with a complex ACD recombinant strain, became superinfected 6 to 9 months later with an AC recombinant, and was monitored for >2.5 years. The plasma viral load exceeded 400,000 copies/ml during the first 9 months of infection but resolved to the set point of 67,000 copies/ml by 3 months after superinfection; the CD4 cell count was 377 cells/mul at 30 months. Viral diversity was evaluated with techniques designed to fully sample the quasi-species, permitting direct observation of the evolution, temporal fluctuation, and intercompartment dynamics of the initial and superinfecting strains and recombinants derived from them. Within 3 months of superinfection, seven different molecular forms were detected in gag and six were detected in env. The proportions of forms fluctuated widely over time in plasma and peripheral blood mononuclear cells, illustrating how challenging the detection of dually infected individuals can be. Strain-specific nested PCR confirmed that the superinfecting strain was not present until the 9 month follow-up. This study further defines the parameters and dynamics of superinfection and will foster appropriate studies and approaches to gain a more complete understanding of risk factors for superinfection and its impact on clinical progression, epidemiology, and vaccine design.     

HIV-1 Superinfection in Women Broadens and Strengthens the Neutralizing Antibody Response

Identifying naturally-occurring neutralizing antibodies (NAb) that are cross-reactive against all global subtypes of HIV-1 is an important step toward the development of a vaccine. Establishing the host and viral determinants for eliciting such broadly NAbs is also critical for immunogen design. NAb breadth has previously been shown to be positively associated with viral diversity. Therefore, we hypothesized that superinfected individuals develop a broad NAb response as a result of increased antigenic stimulation by two distinct viruses. To test this hypothesis, plasma samples from 12 superinfected women each assigned to three singly infected women were tested against a panel of eight viruses representing four different HIV-1 subtypes at matched time points post-superinfection (∼5 years post-initial infection). Here we show superinfected individuals develop significantly broader NAb responses post-superinfection when compared to singly infected individuals (RR = 1.68, CI: 1.23–2.30, p = 0.001). This was true even after controlling for NAb breadth developed prior to superinfection, contemporaneous CD4+ T cell count and viral load. Similarly, both unadjusted and adjusted analyses showed significantly greater potency in superinfected cases compared to controls. Notably, two superinfected individuals were able to neutralize variants from four different subtypes at plasma dilutions >1∶300, suggesting that their NAbs exhibit elite activity. Cross-subtype breadth was detected within a year of superinfection in both of these individuals, which was within 1.5 years of their initial infection. These data suggest that sequential infections lead to augmentation of the NAb response, a process that may provide insight into potential mechanisms that contribute to the development of antibody breadth. Therefore, a successful vaccination strategy that mimics superinfection may lead to the development of broad NAbs in immunized individuals.     

Neutralizing antibodies do not mediate suppression of human immunodeficiency virus type 1 in elite suppressors or selection of plasma virus variants in patients on highly active antiretroviral therapy

Neutralizing antibodies (NAb) against autologous virus can reach high titers in human immunodeficiency virus type 1 (HIV-1)-infected patients with progressive disease. Less is known about the role of NAb in HIV-1-infected patients with viral loads of <50 copies/ml of plasma, including patients on effective highly active antiretroviral therapy (HAART) and elite suppressors, who control HIV-1 replication without antiretroviral therapy. In this study, we analyzed full-length env sequences from plasma viruses and proviruses in resting CD4(+) T cells of HAART-treated patients, elite suppressors, and untreated HIV-1-infected patients with progressive disease. For each patient group, we assessed plasma virus neutralization by autologous, contemporaneous plasma. The degree of env diversity, the number of N-linked glycosylation sites, and the lengths of variable loops were all lower in elite suppressors than in HAART-treated and untreated viremic patients. Both elite suppressors and HAART-treated patients had lower titers of NAb against HIV-1 lab strains than those of untreated viremic patients. Surprisingly, titers of NAb against autologous, contemporaneous plasma viruses were similarly low in chronic progressors, elite suppressors, and HAART-treated patients. In elite suppressors and HAART-treated patients, titers of NAb against autologous plasma viruses also did not differ significantly from titers against autologous proviruses from resting CD4(+) T cells. These results suggest that high-titer NAb are not required for maintenance of viral suppression in elite suppressors and that NAb do not select plasma virus variants in most HAART-treated patients. Both drug-mediated and natural suppression of HIV-1 replication to levels below 50 copies/ml may limit the stimulation and maintenance of effective NAb responses.     

Generation of Neutralizing Antibodies and Divergence of SIVmac239 in Cynomolgus Macaques Following Short-Term Early Antiretroviral Therapy

Neutralizing antibodies (NAb) able to react to heterologous viruses are generated during natural HIV-1 infection in some individuals. Further knowledge is required in order to understand the factors contributing to induction of cross-reactive NAb responses. Here a well-established model of experimental pathogenic infection in cynomolgus macaques, which reproduces long-lasting HIV-1 infection, was used to study the NAb response as well as the viral evolution of the highly neutralization-resistant SIVmac239. Twelve animals were infected intravenously with SIVmac239. Antiretroviral therapy (ART) was initiated ten days post-inoculation and administered daily for four months. Viral load, CD4⁺ T-cell counts, total IgG levels, and breadth as well as strength of NAb in plasma were compared simultaneously over 14 months. In addition, envs from plasma samples were sequenced at three time points in all animals in order to assess viral evolution. We report here that seven of the 12 animals controlled viremia to below 10⁴ copies/ml of plasma after discontinuation of ART and that this control was associated with a low level of evolutionary divergence. Macaques that controlled viral load developed broader NAb responses early on. Furthermore, escape mutations, such as V67M and R751G, were identified in virus sequenced from all animals with uncontrolled viremia. Bayesian estimation of ancestral population genetic diversity (PGD) showed an increase in this value in non-controlling or transient-controlling animals during the first 5.5 months of infection, in contrast to virus-controlling animals. Similarly, non- or transient controllers displayed more positively-selected amino-acid substitutions. An early increase in PGD, resulting in the generation of positively-selected amino-acid substitutions, greater divergence and relative high viral load after ART withdrawal, may have contributed to the generation of potent NAb in several animals after SIVmac239 infection. However, early broad NAb responses correlated with relatively preserved CD4⁺ T-cell numbers, low viral load and limited viral divergence.     

HIV-1 Superinfection in the Antiretroviral Therapy Era: Are Seroconcordant Sexual Partners at Risk?

Background

Acquisition of more than one strain of human immunodeficiency virus type 1 (HIV-1) has been reported to occur both during and after primary infection, but the risks and repercussions of dual and superinfection are incompletely understood. In this study, we evaluated a longitudinal cohort of chronically HIV-infected men who were sexual partners to determine if individuals acquired their partners'' viral strains.

Methodology

Our cohort of HIV-positive men consisted of 8 couples that identified themselves as long-term sexual partners. Viral sequences were isolated from each subject and analyzed using phylogenetic methods. In addition, strain-specific PCR allowed us to search for partners'' viruses present at low levels. Finally, we used computational algorithms to evaluate for recombination between partners'' viral strains.

Principal Findings/Conclusions

All couples had at least one factor associated with increased risk for acquisition of new HIV strains during the study, including detectable plasma viral load, sexually transmitted infections, and unprotected sex. One subject was dually HIV-1 infected, but neither strain corresponded to that of his partner. Three couples'' sequences formed monophyletic clusters at the entry visit, with phylogenetic analysis suggesting that one member of the couple had acquired an HIV strain from his identified partner or that both had acquired it from the same source outside their partnership. The 5 remaining couples initially displayed no evidence of dual infection, using phylogenetic analysis and strain-specific PCR. However, in 1 of these couples, further analysis revealed recombinant viral strains with segments of viral genomes in one subject that may have derived from the enrolled partner. Thus, chronically HIV-1 infected individuals may become superinfected with additional HIV strains from their seroconcordant sexual partners. In some cases, HIV-1 superinfection may become apparent when recombinant viral strains are detected.     

Characterization of human immunodeficiency virus type 1 CRF01_AE env genes derived from recently infected Thai individuals

Transmitted/founder virus is responsible for the establishment of human immunodeficiency virus type 1 (HIV-1) infection and induces primary anti-HIV-1 immune responses; therefore, it is important to study the viral population to understand the early events of HIV-1 infection. We amplified HIV-1 env genes from sera derived from recently infected Thai individuals, and established envelope glycoproteins (Env)-recombinant viruses. Generated Env-recombinant viruses were tested for their neutralization susceptibility to neutralizing human monoclonal antibodies (NHMAbs) and entry inhibitors, as well as being subjected to genotypic analysis. Most recombinant viruses were susceptible to neutralization by NHMAbs to Env gp41, whereas approximately one-third of the recombinant viruses were susceptible to a NHMAb against the CD4 binding site of gp120. In addition, all env genes were classified into CRF01_AE genes and showed low genetic divergence. Taken together with our previous studies on CRF01_AE env genes derived from chronically infected Thai individuals, these results suggested that the immunological and genetic characteristics of CRF01_AE Env derived from recently infected Thai individuals were different from those derived from chronically infected individuals.     

Extensive Diversification of Human Immunodeficiency Virus Type 1 Subtype B Strains during Dual Infection of a Chimpanzee That Progressed to AIDS

A chimpanzee (C-499) infected for more than 9 years with two subtype B isolates of human immunodeficiency virus type 1 (HIV-1), one (HIV-1_SF2) that replicates poorly and one (HIV-1_LAV-1b) that replicates efficiently in chimpanzees, died of AIDS 11 years after initial infection (F. J. Novembre et al., J. Virol. 71:4086–4091, 1997). Nucleotide sequence and phylogenetic analyses of the C2 to V5 region of env (C2-V5^env) in proviral DNA from peripheral blood lymphocytes obtained 22 months before death revealed two distinct virus populations. One of these populations appeared to be a recombinant in env, having the V3 loop from HIV-1_SF2 and the V4-V5 region from HIV-1_LAV-1b; the other population had evolved from HIV-1_LAV-1b. In addition to C2-V5^env, the entire p17^gag and nef genes were sequenced; however, based on nucleotide sequences and phlyogeny, whether the progenitor of the p17^gag and nef genes was SF2 or LAV-1b could not be determined. Compared to the two original viruses, the divergence of all clones of C2-V5^env ranged from 9.37 to 20.2%, that of p17^gag ranged from 3.11 to 9.29%, and that of nef ranged from 4.02 to 7.9%. In contrast, compared to the maximum variation of 20.2% in C2-V5^env for C-499, the maximum diversities in C2-V5^env in proviruses from two chimpanzees infected with HIV-1_LAV-1b for 9 and 10 years were 9.65 and 2.48%, respectively. These results demonstrate that (i) two distinct HIV-1 populations can coexist and undergo extensive diversification in chimpanzees with progressive HIV-1-induced disease and (ii) recombination between two subtype B strains occurred even though the second strain was inoculated 15 months after the first one. Furthermore, evaluation of env genes from three chimpanzees infected with the same strain suggests that the magnitude of HIV-1 diversification could be related to higher viral burdens, manifestations of disease, and/or dual infection.     

HIV-1 Autologous Antibody Neutralization Associates with Mother to Child Transmission

The HIV-1 characteristics associated with mother to child transmission (MTCT) are still poorly understood and if known would indicate where intervention strategies should be targeted. In contrast to horizontally infected individuals, exposed infants possess inherited antibodies (Abs) from their mother with the potential to protect against infection. We investigated the HIV-1 gp160 envelope proteins from seven transmitting mothers (TM) whose children were infected either during gestation or soon after delivery and from four non-transmitting mothers (NTM) with similar viral loads and CD4 counts. Using pseudo-typed viruses we tested gp160 envelope glycoproteins for TZM-bl infectivity, CD4 and CCR5 interactions, DC-SIGN capture and transfer and neutralization with an array of common neutralizing Abs (NAbs) (2F5, 2G12, 4E10 and b12) as well as mother and infant plasma. We found no viral correlates associated with HIV-1 MTCT nor did we find differences in neutralization with the panel of NAbs. We did, however, find that TM possessed significantly higher plasma neutralization capacities than NTM (P  = 0.002). Furthermore, we found that in utero (IU) TM had a higher neutralization capacity than mothers transmitting either peri - partum (PP) or via breastfeeding (BF) (P  = 0.002). Plasma from children infected IU neutralized viruses carrying autologous gp160 viral envelopes as well as those from their corresponding mothers whilst plasma from children infected PP and/or BF demonstrated poor neutralizing capacity. Our results demonstrate heightened autologous NAb responses against gp120/gp41 can associate with a greater risk of HIV-1 MTCT and more specifically in those infants infected IU. Although the number of HIV-1 transmitting pairs is low our results indicate that autologous NAb responses in mothers and infants do not protect against MTCT and may in fact be detrimental when considering IU HIV-1 transmissions.     

Autologous neutralizing humoral immunity and evolution of the viral envelope in the course of subtype B human immunodeficiency virus type 1 infection

Most human immunodeficiency virus type 1 (HIV-1)-infected individuals develop an HIV-specific neutralizing antibody (NAb) response that selects for escape variants of the virus. Here, we studied autologous NAb responses in five typical CCR5-using progressors in relation to viral NAb escape and molecular changes in the viral envelope (Env) in the period from seroconversion until after AIDS diagnosis. In sera from three patients, high-titer neutralizing activity was observed against the earliest autologous virus variants, followed by declining humoral immune responses against subsequent viral escape variants. Autologous neutralizing activity was undetectable in sera from two patients. Patients with high-titer neutralizing activity in serum showed the strongest positive selection pressure on Env early in infection. In the initial phase of infection, gp160 length and the number of potential N-linked glycosylation sites (PNGS) increased in viruses from all patients. Over the course of infection, positive selection pressure declined as the NAb response subsided, coinciding with reversions of changes in gp160 length and the number of PNGS. A number of identical amino acid changes were observed over the course of infection in the viral quasispecies of different patients. Our results indicate that although neutralizing autologous humoral immunity may have a limited effect on the disease course, it is an important selection pressure in virus evolution early in infection, while declining HIV-specific humoral immunity in later stages may coincide with reversion of NAb-driven changes in Env.     

Human immunodeficiency virus type 1 clade B superinfection: evidence for differential immune containment of distinct clade B strains

Sequential infection with different strains of human immunodeficiency virus type 1 (HIV-1) is a rarely identified phenomenon with important implications for immunopathogenesis and vaccine development. Here, we identify an individual whose good initial control of viremia was lost in association with reduced containment of a superinfecting strain. Subject 2030 presented with acute symptoms of HIV-1 infection with high viremia and an incomplete seroconversion as shown by Western blotting. A low set point of viremia (approximately 1,000 HIV-1 copies/ml) was initially established without drug therapy, but a new higher set point (approximately 40,000 HIV-1 copies/ml) manifested about 5 months after infection. Drug susceptibility testing demonstrated a multidrug-resistant virus initially but a fully sensitive virus after 5 months, and an analysis of pol genotypes showed that these were two phylogenetically distinct strains of virus (strains A and B). Replication capacity assays suggested that the outgrowth of strain B was not due to higher fitness conferred by pol, and env sequences indicated that the two strains had the same R5 coreceptor phenotype. Delineation of CD8+-T-lymphocyte responses against HIV-1 showed a striking pattern of decay of the initial cellular immune responses after superinfection, followed by some adaptation of targeting to new epitopes. An examination of targeted sequences suggested that differences in the recognized epitopes contributed to the poor immune containment of strain B. In conclusion, the rapid overgrowth of a superinfecting strain of HIV-1 of the same subtype raises major concerns for effective vaccine development.     

Intersubtype human immunodeficiency virus type 1 superinfection following seroconversion to primary infection in two injection drug users

In this study, we describe two cases of human immunodeficiency virus type 1 (HIV-1) intersubtype superinfection with CRF01_AE and subtype B strains, which occurred in two injection drug users participating in a prospective cohort study in Bangkok, Thailand. In both cases, the superinfecting strain was detected by molecular and serologic analyses several weeks after complete seroconversion to the primary infection with a strain belonging to a different subtype. Superinfection occurred despite specific T-cell and humoral antibody responses to the primary virus. In both cases, cross-subtype immune responses were limited or absent prior to the second infection. These data show that, in some individuals, the quality and quantity of the immune response elicited by primary HIV-1 infection may not protect against superinfection. This finding has important implications for vaccine design. HIV-1 vaccines, at a minimum, will need to include potent, broadly protective, conserved immunogens derived from several group M subtypes.     

Breadth of Neutralizing Antibody Response to Human Immunodeficiency Virus Type 1 Is Affected by Factors Early in Infection but Does Not Influence Disease Progression

The determinants of a broad neutralizing antibody (NAb) response and its effect on human immunodeficiency virus type 1 (HIV-1) disease progression are not well defined, partly because most prior studies of a broad NAb response were cross-sectional. We examined correlates of NAb response breadth among 70 HIV-infected, antiretroviral-naïve Kenyan women from a longitudinal seroincident cohort. NAb response breadth was measured 5 years after infection against five subtype A viruses and one subtype B virus. Greater NAb response breadth was associated with a higher viral load set point and greater HIV-1 env diversity early in infection. However, greater NAb response breadth was not associated with a delayed time to a CD4⁺ T-cell count of <200, antiretroviral therapy, or death. Thus, a broad NAb response results from a high level of antigenic stimulation early in infection, which likely accounts for prior observations that greater NAb response breadth is associated with a higher viral load later in infection.Some human immunodeficiency virus (HIV)-infected individuals develop broad neutralizing antibody (NAb) responses, but the factors that lead to NAb response breadth remain elusive. Several cross-sectional studies have found that individuals with greater NAb response breadth have higher contemporaneous viral loads, suggesting that the presence of a greater amount of viral antigen may promote a greater NAb response breadth (9, 10, 25, 30, 32). However, because viral load and NAb response breadth were measured at the same time after HIV type 1 (HIV-1) acquisition in prior studies, it is difficult to discern cause and effect. There is also evidence that NAbs adapt in response to the evolving HIV-1 population throughout infection (11, 29, 35), which may contribute to a greater overall response breadth. Together, these studies support a model in which a greater NAb response breadth is driven by a higher level of antigenic stimulation, in terms of both the absolute level of virus and viral diversity. Confirmation of this model requires an assessment of the temporal relationship of viral load, HIV-1 diversity, and NAb response breadth.In addition to uncertainty regarding the determinants of NAb response breadth, the consequences of a broad NAb response for HIV-1 disease progression remains controversial. Broad NAb responses have been found in long-term nonprogressors (LTNPs) in some studies, suggesting that NAbs may contribute to control of infection in these individuals (6-8, 22, 27, 37). Other studies have found no evidence for NAb control in LTNPs (1, 2, 14, 18), including studies in which NAb response breadth was lower in LTNPs (10) or elite controllers (15, 25) than in viremic individuals. A detailed analysis of NAb response breadth versus clinical outcome has not yet been conducted, particularly for individuals with typical HIV-1 disease progression.To investigate the determinants and consequences of NAb response breadth in HIV-1 infection, we examined NAb responses in women in a seroincident cohort in Mombasa, Kenya, that began in 1993 (19-21). For each woman, the time of infection was defined by both HIV-1 serology and RNA testing (17). Women who had a banked plasma sample ∼5 years after the estimated time of HIV-1 infection were included in this study. This time period was chosen to maximize the chances for the NAb response to broaden while generally testing prior to the beginning of clinical immunodeficiency. We only included samples prior to the initiation of antiretroviral therapy (ART), which in this cohort began in March 2004, according to the WHO and Kenyan National guidelines. Plasma samples meeting these criteria were identified from 70 women and came from a median of 5.0 (range, 4.5 to 6.8) years postinfection (ypi). This subset of women was representative of the entire cohort in terms of their behavioral, clinical, and demographic characteristics (data not shown).HIV-1 subtype A accounts for most of the infections in this cohort (28), including 72% of the 53 women in this study for whom env subtype information was available (Fig. ​(Fig.1).1). Therefore, to test neutralization of viruses relevant to women in this population, we measured NAb response breadth against a panel of five recently transmitted subtype A viruses from other individuals in this cohort, which represented a spectrum of neutralization sensitivities (4). We also included one commonly studied, easy-to-neutralize subtype B virus (SF162) for comparison to other studies. The TZM-bl neutralization assay, using pseudoviruses prepared with these six envelope variants and TZM-bl indicator cells, was performed as described previously (4, 36). The median inhibitory concentration (IC₅₀) was defined as the reciprocal dilution of plasma that resulted in 50% inhibition. Figure ​Figure11 shows the IC₅₀ for each plasma-virus pair, averaged across three independent experiments that included duplicate testing of each pair.Open in a separate windowFIG. 1.Summary of the IC₅₀s and NAb response breadth scores of 70 plasma samples. The first column indicates the subject identifier of each plasma sample, and the next three columns indicate the env V1 to V5 subtype (available for 53/70 women), the set point viral load (available for 64 women), and the viral load at ∼5 ypi, when the NAb response breadth was measured. Data not available are indicated by a period. Each subsequent column shows the results with one panel virus (indicated at the top of the column). Results are the average of three experiments in which each plasma-virus pair was tested in duplicate. In the case of Q769 and Q259, two closely related viruses from the same individual were used in one (Q769.h5, Q259.d217) and two (Q769.b9, Q259.d226) of the three experiments. The IC₅₀ for each plasma-virus pair is the reciprocal dilution of plasma that led to a 50% reduction in infectivity, averaged across the three experiments. IC₅₀s are shown in gray scale to represent increasing neutralization sensitivity, with white for values of <100, light gray for values of >101 and <1,000, and dark gray for values of >1,001. Plasma-virus pairs in which 50% neutralization was not detected at the highest plasma dilution (1:50) are indicated by a pair of dashes. The NAb response breadth score for each plasma sample was calculated as follows. For each experiment, the median IC₅₀ for each virus (across all 70 plasma samples) was determined. Plasma samples were assigned a score of 1 for every virus against which their IC₅₀ was greater than the median IC₅₀, and the score was summed across all six viruses. The NAb response breadth scores that are shown here (and which were used for analysis) were calculated by taking the average response breadth score across the three independent experiments; they were not calculated from the average IC₅₀s shown.In general, we found that the viruses that had been easily neutralized in prior screening with pooled plasma, {"type":"entrez-protein","attrs":{"text":"Q461d1","term_id":"123852094","term_text":"Q461D1"}}Q461d1 and Q168b23 (4), were the most readily neutralized by individual plasma samples from women in this study (Fig. ​(Fig.1).1). Of the 70 plasma samples tested, 68 (97%) showed detectable neutralization activity (IC₅₀, >50) against {"type":"entrez-protein","attrs":{"text":"Q461d1","term_id":"123852094","term_text":"Q461D1"}}Q461d1 and 60 (86%) showed activity against Q168b23. Most (76%) of the plasma samples also neutralized variant Q842d16 at detectable levels, although generally with lower IC₅₀s. By contrast, only about half of the plasma samples neutralized envelope variants Q769b9 and {"type":"entrez-protein","attrs":{"text":"Q259d2","term_id":"122195760","term_text":"Q259D2"}}Q259d2.26 (51% and 46%, respectively). Almost all (93%) of the plasma samples neutralized SF162.Given the different neutralization sensitivities of these viruses, we quantified the NAb responses in these individuals by using a previously described NAb response breadth score that takes into consideration the neutralization sensitivity of each virus (5). Briefly, the NAb response breadth score represents the number of viruses (out of six) that a given plasma sample neutralized at an IC₅₀ that was higher than the median IC₅₀ for that virus (across all 70 plasma samples). The response breadth score was calculated independently for each of three experiments, and the average scores are listed in Fig. ​Fig.1.1. Among all of the individuals, the median response breadth score was 2 and the response breadth scores ranged from 0 to 5.3. A potential limitation of this approach is that response breadth was calculated by using a relatively small number of viruses. However, we found that NAb response breadth measured against this 6-virus panel was highly correlated with the NAb response breadth measured against an expanded 17-virus panel (including these 6 viruses plus an additional 11 viruses representing subtypes A, C, D, A/D, and B; J. Overbaugh et al., unpublished data), for a subset of 29 women whose plasma samples were tested against the expanded panel (Spearman''s rho = 0.62, P < 0.001). Furthermore, the NAb response breadth score measured against this six-virus panel was highly correlated with NAb potency (Spearman''s rho = 0.81, P < 0.001), a measure we have used in prior studies that takes into consideration the magnitude of the IC₅₀ for each plasma-virus pair (5). These findings suggest that the NAb response breadth score measured against the six-virus panel is representative of the overall NAb response breadth.We investigated whether NAb response breadth was associated with the contemporaneous plasma viral load, which was measured at the same time as NAb response breadth (4.5 to 6.8 ypi). Viral loads ranged from 1.7 to 6.7 log₁₀ copies/ml among all of the individuals, with a median of 4.7 log₁₀ copies/ml. As shown in Fig. ​Fig.2a,2a, individuals with higher viral loads had greater NAb response breadth (Spearman''s rho = 0.31, P = 0.009), consistent with prior studies (9, 10, 30, 32). A similar relationship was observed between viral load set point and NAb potency, a measure that takes into account the magnitude of neutralization (data not shown). There was no association between NAb response breadth and CD4⁺ T-cell count (Spearman''s rho = −0.15, P = 0.2) among the 64 women with contemporaneous CD4⁺ T-cell counts available.Open in a separate windowFIG. 2.Associations between NAb response breadth and viral load. In each plot, the NAb response breadth score is indicated on the y axis and the contemporaneous (∼5 ypi) viral load (a) or viral load set point (b) is indicated on the x axis. Each point represents one individual. The results of Spearman correlation analysis are shown above the plots.To further assess whether the viral load may drive NAb response breadth, we examined the relationship between the viral load set point and NAb response breadth. For each individual, the viral load set point was defined as the first available viral load measurement 4 to 24 months after infection (16), and this ranged from 2.1 to 6.2 log₁₀ copies/ml (median, 4.6 log₁₀ copies/ml) among the 64 individuals for whom this measurement was available. As shown in Fig. ​Fig.2b,2b, individuals with higher viral load set points had greater NAb response breadth at ∼5 ypi (Spearman''s rho = 0.35, P = 0.005). The viral load set point was also highly correlated with the viral load measured at ∼5 ypi (Spearman''s rho = 0.42, P = 0.001). Therefore, we investigated whether the relationship between NAb response breadth and the contemporaneous (∼5 ypi) viral load could be explained by the viral load set point. In multivariate linear regression analysis, NAb response breadth was significantly associated with the viral load set point (coefficient of variation = 0.55, P = 0.02) but not with the contemporaneous viral load (coefficient of variation = 0.25, P = 0.3). Thus, the relationship between the contemporaneous viral load and NAb response breadth appeared to be driven by the viral load set point, with each 1-log increase in the viral load set point associated with an increase in the response breadth score of 0.55.Given this association between the viral load set point and NAb response breadth, we wondered whether another factor in early infection—HIV-1 sequence diversity—might influence the development of NAb response breadth. Proviral HIV-1 sequences were available from 26 individuals and had been sampled a median of 87 (range, 17 to 299) days postinfection. For each individual, gag and env V1 to V5 diversity was calculated from a median of seven single-copy sequences per gene as described previously (26). Across all 26 individuals, the median env diversity was 0.28% (range, 0 to 4.0%) and the median gag diversity was 0.19% (range, 0 to 1.28%). Individuals with greater env diversity early in infection had greater NAb response breadth at ∼5 ypi (Spearman''s rho = 0.51, P = 0.008). However, there was no association between early gag diversity and NAb response breadth (Spearman''s rho = 0.10, P = 0.6). Although both early env diversity and the viral load set point were associated with NAb response breadth, there was no association between these factors among the women in this study (Spearman''s rho = 0.21, P = 0.3). However, in a larger study of 156 women in this cohort, women with greater early env heterogeneity (as measured by heteroduplex mobility assay) had higher viral load set points (31). Further work is needed to clarify whether early env diversity and the viral load set point are independent determinants of NAb response breadth or whether early env diversity may drive both the viral load and NAb response breadth.Because the viral load set point and early env diversity have also been shown to be associated with HIV-1 disease progression in this cohort (17, 31), we explored the relationship of NAb response breadth, the viral load set point, and disease progression. We performed Cox proportional hazard analysis by using a composite survival outcome of time to the first occurrence of a CD4⁺ T-cell count of <200, ART initiation, or death. Among all 70 women, 45 reached this composite outcome over a median of 6.8 years of follow-up after HIV-1 infection (range, 1.2 to 14.2 years). In univariate analysis, a greater NAb response breadth was associated with an increased risk of HIV-1 disease progression (Table ​(Table1,1, hazard ratio [HR], 1.27 per unit increase in breadth, P = 0.03). However, this association was attenuated, and no longer statistically significant, in a multivariate analysis adjusting for the viral load set point (HR = 1.06, P = 0.6). In this multivariate model, a higher viral load set point was associated with a greater risk of HIV-1 disease progression (HR = 2.02, P = 0.003), as expected. In a second multivariate analysis considering only those outcome events that occurred after NAb response measurement (n = 25 events among 50 women), there was an association between NAb response breadth and HIV-1 disease outcomes (HR = 1.39, P = 0.03) but again this did not persist after adjustment for the viral load (HR = 1.17, P = 0.4). Thus, we found no evidence that NAb response breadth affected HIV-1 disease progression independently of the viral load set point.

TABLE 1.

Association between NAb response breadth and risk of HIV-1 disease progression^a

Genetically Related Human Immunodeficiency Virus Type 1 in Three Adults of a Family with No Identified Risk Factor for Intrafamilial Transmission

A small number of cases of human immunodeficiency virus (HIV) infection have been reported in individuals with no identified risk factors for transmission. We report on the seroconversion of the 61-year-old mother and the subsequent finding of HIV seropositivity in the 66-year-old father of a 31-year-old AIDS patient. Extensive investigation failed to identify any risk factor for intrafamilial transmission. We conducted a genetic analysis and determined the amino acid signature patterns of the V3, V4, and V5 hypervariable domains and flanking regions in the HIV-1 gp120 env gene of 26 clones derived from proviral DNA in peripheral blood mononuclear cells of the members of the family. env sequences of the viruses isolated from the patients were compared with sequences of HIV-1 subtype B viruses from Europe and local field isolates. Phylogenetic analysis revealed that the sequences of the viruses isolated from the patients were genetically related and formed an intrafamilial cluster of HIV-1 distinct from other subtype B viruses. Interindividual nucleotide variability in the C2-V3 and V4-C4-V5 domains ranged between 1.2 and 5.0% and between 2.2 and 7.5%, respectively, whereas divergence between HIV strains from the patients and control viral strains ranged from 6.6 to 29.3%. The amino acid signature patterns of viral clones from the three patients were closely related. In the C2-V3 region, two minor clones derived from the son’s virus showed less nucleotide divergence (mean, 3.5 and 3.9%) than did the clones derived from the viruses of both parents or the seven other predominant clones derived from the virus from the son (mean, 5.4%). The top of the V3 loop of the last two clones and of all viral clones from the parents exhibited an unusual GPGG sequence. This is the first report of genotypic relatedness of HIV-1 in three adults of the same family in the absence of identified risk factor for transmission between the members of the family. Our findings suggest that atypical transmission of HIV may occur.     

Potent autologous and heterologous neutralizing antibody responses occur in HIV-2 infection across a broad range of infection outcomes

Few studies have explored the role of neutralizing antibody (NAb) responses in controlling HIV-2 viremia and disease progression. Using a TZM-bl neutralization assay, we assessed heterologous and autologous NAb responses from a community cohort of HIV-2-infected individuals with a broad range of disease outcomes in rural Guinea-Bissau. All subjects (n = 40) displayed exceptionally high heterologous NAb titers (50% inhibitory plasma dilution or 50% inhibitory concentration [IC(50)], 1:7,000 to 1:1,000,000) against 5 novel primary HIV-2 envelopes and HIV-2 7312A, whereas ROD A and 3 primary envelopes were relatively resistant to neutralization. Most individuals also showed high autologous NAb against contemporaneous envelopes (78% of plasma-envelope combinations in 69 envelopes from 21 subjects), with IC(50)s above 1:10,000. No association between heterologous or autologous NAb titer and greater control of HIV-2 was found. A subset of envelopes was found to be more resistant to neutralization (by plasma and HIV-2 monoclonal antibodies). These envelopes were isolated from individuals with greater intrapatient sequence diversity and were associated with changes in potential N-linked glycosylation sites but not CD4 independence or CXCR4 use. Plasma collected from up to 15 years previously was able to potently neutralize recent autologous envelopes, suggesting a lack of escape from NAb and the persistence of neutralization-sensitive variants over time, despite significant NAb pressure. We conclude that despite the presence of broad and potent NAb responses in HIV-2-infected individuals, these are not the primary forces behind the dichotomous outcomes observed but reveal a limited capacity for adaptive selection and escape from host immunity in HIV-2 infection.     

Evidence for potent autologous neutralizing antibody titers and compact envelopes in early infection with subtype C human immunodeficiency virus type 1

Information about neutralizing antibody responses in subtype C-infected individuals is limited, even though this viral subtype causes the majority of AIDS cases worldwide. Here we compared the course and magnitude of the autologous neutralizing antibody (NAb) response against viral envelope (Env) glycoproteins present during acute and early infection with subtypes B and C human immunodeficiency virus type 1 (HIV-1). NAb responses were evaluated in 6 subtype B-infected and 11 subtype C-infected subjects over a mean evaluation period of 25 months using a pseudovirus reporter gene assay. All subjects in the C cohort were infected through heterosexual contact, while five of the six subjects in the B cohort were infected via male-to-male contact. The kinetics and magnitude of the NAb responses varied among subjects in the B and C cohorts; however, the median 50% inhibitory concentration (IC(50) titer) reached by antibody in the plasma of subtype C-infected subjects, overall, was 3.5-fold higher than in the subtype B-infected subjects (P = 0.06). The higher titers of NAbs in the C cohort were associated with viruses having significantly shorter amino acid length (P = 0.002) in the V1 to V4 region of the surface Env glycoprotein, gp120, compared to the B cohort. Despite the potency of the autologous subtype C NAb response, it was not directed against cross-neutralizing epitopes. These data demonstrate that subtype C Envs elicit a potent yet restricted NAb response early in infection that frequently reaches IC(50) titers in excess of 1:1,000 and suggest that clade-specific differences may exist in Env immunogenicity or susceptibility to neutralization.     

Immunological and Virological Analyses of Persons Infected by Human Immunodeficiency Virus Type 1 while Participating in Trials of Recombinant gp120 Subunit Vaccines

We have studied 18 participants in phase I/II clinical trials of recombinant gp120 (rgp120) subunit vaccines (MN and SF-2) who became infected with human immunodeficiency virus type 1 (HIV-1) during the course of the trials. Of the 18 individuals, 2 had received a placebo vaccine, 9 had been immunized with MN rgp120, and seven had been immunized with SF-2 rgp120. Thirteen of the 18 infected vaccinees had received three or four immunizations prior to becoming infected. Of these, two were placebo recipients, six had received MN rgp120, and five had received SF-2 rgp120. Only 1 of the 11 rgp120 recipients who had multiple immunizations failed to develop a strong immunoglobulin G antibody response to the immunogen. However, the antibody response to rgp120 was transient, typically having a half-life of 40 to 60 days. No significant neutralizing activity against the infecting strain was detected in any of the infected individuals at any time prior to infection. Antibody titers in subjects infected despite vaccination and in noninfected subjects were not significantly different. Envelope-specific cytotoxic T-lymphocyte responses measured after infection were infrequent and weak in the nine vaccinees who were tested. HIV-1 was isolated successfully from all 18 individuals. Sixteen of these strains had a non-syncytium-inducing (NSI) phenotype, while two had a syncytium-inducing (SI) phenotype. NSI strains used the CCR5 coreceptor to enter CD4⁺ cells, while an SI strain from one of the vaccinees also used CXCR4. Viruses isolated from the blood of rgp120 vaccinees were indistinguishable from viruses isolated from control individuals in terms of their inherent sensitivity to neutralization by specific monoclonal antibodies and their replication rates in vitro. Furthermore, genetic sequencing of the env genes of strains infecting the vaccinees did not reveal any features that clearly distinguished these viruses from contemporary clade B viruses circulating in the United States. Thus, despite rigorous genetic analyses, using various breakdowns of the data sets, we could find no evidence that rgp120 vaccination exerted selection pressure on the infecting HIV-1 strains. The viral burdens in the infected rgp120 vaccine recipients were also determined, and they were found to be not significantly different from those in cohorts of placebo-vaccinated and nonvaccinated individuals. In summary, we conclude that vaccination with rgp120 has had, to date, no obvious beneficial or adverse effects on the individuals we have studied.     

The Unique Envelope Gene of the Subgroup J Avian Leukosis Virus Derives from ev/J Proviruses, a Novel Family of Avian Endogenous Viruses

A new subgroup of avian leukosis virus (ALV), designated subgroup J, was identified recently. Viruses of this subgroup do not cross-interfere with viruses of the avian A, B, C, D, and E subgroups, are not neutralized by antisera raised against the other virus subgroups, and have a broader host range than the A to E subgroups. Sequence comparisons reveal that while the subgroup J envelope gene includes some regions that are related to those found in env genes of the A to E subgroups, the majority of the subgroup J gene is composed of sequences either that are more similar to those of a member (E51) of the ancient endogenous avian virus (EAV) family of proviruses or that appear unique to subgroup J viruses. These data led to the suggestion that the ALV-J env gene might have arisen by multiple recombination events between one or more endogenous and exogenous viruses. We initiated studies to investigate the origin of the subgroup J envelope gene and in particular to determine the identity of endogenous sequences that may have contributed to its generation. Here we report the identification of a novel family of avian endogenous viruses that include env coding sequences that are over 95% identical to both the gp85 and gp37 coding regions of subgroup J viruses. We call these viruses the ev/J family. We also report the isolation of ev/J-encoded cDNAs, indicating that at least some members of this family are expressed. These data support the hypothesis that the subgroup J envelope gene was acquired by recombination with expressed endogenous sequences and are consistent with acquisition of this gene by only one recombination event.