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.     

Dynamics of Viral Evolution and Neutralizing Antibody Response after HIV-1 Superinfection

Investigating the incidence and prevalence of HIV-1 superinfection is challenging due to the complex dynamics of two infecting strains. The superinfecting strain can replace the initial strain, be transiently expressed, or persist along with the initial strain in distinct or in recombined forms. Various selective pressures influence these alternative scenarios in different HIV-1 coding regions. We hypothesized that the potency of the neutralizing antibody (NAb) response to autologous viruses would modulate viral dynamics in env following superinfection in a limited set of superinfection cases. HIV-1 env pyrosequencing data were generated from blood plasma collected from 7 individuals with evidence of superinfection. Viral variants within each patient were screened for recombination, and viral dynamics were evaluated using nucleotide diversity. NAb responses to autologous viruses were evaluated before and after superinfection. In 4 individuals, the superinfecting strain replaced the original strain. In 2 individuals, both initial and superinfecting strains continued to cocirculate. In the final individual, the surviving lineage was the product of interstrain recombination. NAb responses to autologous viruses that were detected within the first 2 years of HIV-1 infection were weak or absent for 6 of the 7 recently infected individuals at the time of and shortly following superinfection. These 6 individuals had detectable on-going viral replication of distinct superinfecting virus in the env coding region. In the remaining case, there was an early and strong autologous NAb response, which was associated with extensive recombination in env between initial and superinfecting strains. This extensive recombination made superinfection more difficult to identify and may explain why the detection of superinfection has typically been associated with low autologous NAb titers.     

The breadth and potency of passively acquired human immunodeficiency virus type 1-specific neutralizing antibodies do not correlate with the risk of infant infection

Although a major goal of human immunodeficiency virus type 1 (HIV-1) vaccine efforts is to elicit broad and potent neutralizing antibodies (NAbs), there are no data that directly demonstrate a role for such NAbs in protection from HIV-1 infection in exposed humans. The setting of mother-to-child transmission provides an opportunity to examine whether NAbs provide protection from HIV-1 infection because infants acquire passive antibodies from their mothers prior to exposure to HIV-1 through breastfeeding. We evaluated the characteristics of HIV-1-specific NAbs in 100 breast-fed infants of HIV-1-positive mothers who were HIV-1 negative at birth and monitored them until age 2. A panel of eight viruses that included variants representative of those in the study region as well as more diverse strains was used to determine the breadth of the infant NAbs. From their mothers, infants acquired broad and potent NAbs that were capable of recognizing heterologous circulating HIV-1 variants of diverse subtypes, but the presence of NAbs of broad HIV-1 specificity was not associated with transmission risk. There was also no correlation between responses to any particular virus tested, which included a range of diverse variants that demonstrated different neutralization profiles, including recognition by specific antibodies with known epitope targets. The eight viruses tested exhibited neutralization profiles to a variety of monoclonal antibodies (2F5, PG9, and VRC01) similar to those of viruses present in pregnant women in the cohort. These results suggest that the breadth and potency of the heterologous antibody response in exposed infants, measured against a virus panel comprised of variants typical of those circulating in the population, does not predict protection.     

Evidence for frequent reinfection with human immunodeficiency virus type 1 of a different subtype

A major premise underlying current human immunodeficiency virus type 1 (HIV-1) vaccine approaches is that preexisting HIV-1-specific immunity will block or reduce infection. However, the recent identification of several cases of HIV-1 reinfection suggests that the specific immune response generated for chronic HIV-1 infection may not be adequate to protect against infection by a second HIV-1 strain. It has been unclear, though, whether these individuals are representative of the global epidemic or are rare cases. Here we show that in a population of high-risk women, HIV-1 reinfection occurs almost as commonly as first infections. The study was designed to detect cases of reinfection by HIV-1 of a different subtype and thus captured cases where there was considerable diversity between the first and second strain. In each case, the second virus emerged approximately 1 year after the first infection, and in two cases, it emerged when viral levels were high, suggesting that a well-established HIV-1 infection may provide little benefit in terms of immunizing against reinfection, at least by more-divergent HIV-1 variants. Our findings indicate an urgent need for studies of larger cohorts to determine the incidence and timing of both intersubtype and intrasubtype reinfection.     

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.     

Neutralizing Antibody Escape during HIV-1 Mother-to-Child Transmission Involves Conformational Masking of Distal Epitopes in Envelope

HIV-1 variants transmitted to infants are often resistant to maternal neutralizing antibodies (NAbs), suggesting that they have escaped maternal NAb pressure. To define the molecular basis of NAb escape that contributes to selection of transmitted variants, we analyzed 5 viruses from 2 mother-to-child transmission pairs, in which the infant virus, but not the maternal virus, was resistant to neutralization by maternal plasma near transmission. We generated chimeric viruses between maternal and infant envelope clones obtained near transmission and examined neutralization by maternal plasma. The molecular determinants of NAb escape were distinct, even when comparing two maternal variants to the transmitted infant virus within one pair, in which insertions in V4 of gp120 and substitutions in HR2 of gp41 conferred neutralization resistance. In another pair, deletions and substitutions in V1 to V3 conferred resistance, but neither V1/V2 nor V3 alone was sufficient. Although the sequence determinants of escape were distinct, all of them involved modifications of potential N-linked glycosylation sites. None of the regions that mediated escape were major linear targets of maternal NAbs because corresponding peptides failed to compete for neutralization. Instead, these regions disrupted multiple distal epitopes targeted by HIV-1-specific monoclonal antibodies, suggesting that escape from maternal NAbs occurred through conformational masking of distal epitopes. This strategy likely allows HIV-1 to utilize relatively limited changes in the envelope to preserve the ability to infect a new host while simultaneously evading multiple NAb specificities present in maternal plasma.     

Sequential evolution and escape from neutralization of simian immunodeficiency virus SIVsmE660 clones in rhesus macaques

Simian immunodeficiency virus (SIV) infection of rhesus macaques has become an important surrogate model for evaluating HIV vaccine strategies. The extreme resistance to neutralizing antibody (NAb) of many commonly used strains, such as SIVmac251/239 and SIVsmE543-3, limits their potential relevance for evaluating the role of NAb in vaccine protection. In contrast, SIVsmE660 is an uncloned virus that appears to be more sensitive to neutralizing antibody. To evaluate the role of NAb in this model, we generated full-length neutralization-sensitive molecular clones of SIVsmE660 and evaluated two of these by intravenous inoculation of rhesus macaques. All animals became infected and maintained persistent viremia that was accompanied by a decline in memory CD4(+) T cells in blood and bronchoalveolar lavage fluid. High titers of autologous NAb developed by 4 weeks postinoculation but were not associated with control of viremia, and neutralization escape variants were detected concurrently with the generation of NAb. Neutralization escape was associated with substitutions and insertion/deletion polymorphisms in the V1 and V4 domains of envelope. Analysis of representative variants revealed that escape variants also induced NAbs within a few weeks of their appearance in plasma, in a pattern that is reminiscent of the escape of human immunodeficiency virus type 1 (HIV-1) isolates in humans. Although early variants maintained a neutralization-sensitive phenotype, viruses obtained later in infection were significantly less sensitive to neutralization than the parental viruses. These results indicate that NAbs exert selective pressure that drives the evolution of the SIV envelope and that this model will be useful for evaluating the role of NAb in vaccine-mediated protection.     

Analysis of Neutralization Specificities in Polyclonal Sera Derived from Human Immunodeficiency Virus Type 1-Infected Individuals

During human immunodeficiency virus type 1 (HIV-1) infection, patients develop various levels of neutralizing antibody (NAb) responses. In some cases, patient sera can potently neutralize diverse strains of HIV-1, but the antibody specificities that mediate this broad neutralization are not known, and their elucidation remains a formidable challenge. Due to variable and nonneutralizing determinants on the exterior envelope glycoprotein (Env), nonnative Env protein released from cells, and the glycan shielding that assembles in the context of the quaternary structure of the functional spike, HIV-1 Env elicits a myriad of binding antibodies. However, few of these antibodies can neutralize circulating viruses. We present a systematic analysis of the NAb specificities of a panel of HIV-1-positive sera, using methodologies that identify both conformational and continuous neutralization determinants on the HIV-1 Env protein. Characterization of sera included selective adsorption with native gp120 and specific point mutant variants, chimeric virus analysis, and peptide inhibition of viral neutralization. The gp120 protein was the major neutralizing determinant for most sera, although not all neutralization activity against all viruses could be identified. In some broadly neutralizing sera, the gp120-directed neutralization mapped to the CD4 binding region of gp120. In addition, we found evidence that regions of the gp120 coreceptor binding site may also be a target of neutralizing activity. Sera displaying limited neutralization breadth were mapped to the immunogenic V3 region of gp120. In a subset of sera, we also identified NAbs directed against the conserved, membrane-proximal external region of gp41. These data allow a more detailed understanding of the humoral responses to the HIV-1 Env protein and provide insights regarding the most relevant targets for HIV-1 vaccine design.     

Broad and potent neutralizing antibody responses elicited in natural HIV-2 infection

Compared with human immunodeficiency virus type 1 (HIV-1), little is known about the susceptibility of HIV-2 to antibody neutralization. We characterized the potency and breadth of neutralizing antibody (NAb) responses in 64 subjects chronically infected with HIV-2 against three primary HIV-2 strains: HIV-2(7312A), HIV-2(ST), and HIV-2(UC1). Surprisingly, we observed in a single-cycle JC53bl-13/TZM-bl virus entry assay median reciprocal 50% inhibitory concentration (IC(50)) NAb titers of 1.7 × 10(5), 2.8 × 10(4), and 3.3 × 10(4), respectively. A subset of 5 patient plasma samples tested against a larger panel of 17 HIV-2 strains where the extracellular gp160 domain was substituted into the HIV-2(7312A) proviral backbone showed potent neutralization of all but 4 viruses. The specificity of antibody neutralization was confirmed using IgG purified from patient plasma, HIV-2 Envs cloned by single-genome amplification, viruses grown in human CD4(+) T cells and tested for neutralization sensitivity on human CD4(+) T target cells, and, as negative controls, env-minus viruses pseudotyped with HIV-1, vesicular stomatitis virus, or murine leukemia virus Env glycoproteins. Human monoclonal antibodies (MAbs) specific for HIV-2 V3 (6.10F), V4 (1.7A), CD4 binding site (CD4bs; 6.10B), CD4 induced (CD4i; 1.4H), and membrane-proximal external region (MPER; 4E10) epitopes potently neutralized the majority of 32 HIV-2 strains bearing Envs from 13 subjects. Patient antibodies competed with V3, V4, and CD4bs MAbs for binding to monomeric HIV-2 gp120 at titers that correlated significantly with NAb titers. HIV-2 MPER antibodies did not contribute to neutralization breadth or potency. These findings indicate that HIV-2 Env is highly immunogenic in natural infection, that high-titer broadly neutralizing antibodies are commonly elicited, and that unlike HIV-1, native HIV-2 Env trimers expose multiple broadly cross-reactive epitopes readily accessible to NAbs.     

HIV-Specific Antibodies Capable of ADCC Are Common in Breastmilk and Are Associated with Reduced Risk of Transmission in Women with High Viral Loads

There are limited data describing the functional characteristics of HIV-1 specific antibodies in breast milk (BM) and their role in breastfeeding transmission. The ability of BM antibodies to bind HIV-1 envelope, neutralize heterologous and autologous viruses and direct antibody-dependent cell cytotoxicity (ADCC) were analyzed in BM and plasma obtained soon after delivery from 10 non-transmitting and 9 transmitting women with high systemic viral loads and plasma neutralizing antibodies (NAbs). Because subtype A is the dominant subtype in this cohort, a subtype A envelope variant that was sensitive to plasma NAbs was used to assess the different antibody activities. We found that NAbs against the subtype A heterologous virus and/or the woman''s autologous viruses were rare in IgG and IgA purified from breast milk supernatant (BMS) – only 4 of 19 women had any detectable NAb activity against either virus. Detected NAbs were of low potency (median IC50 value of 10 versus 647 for the corresponding plasma) and were not associated with infant infection (p = 0.58). The low NAb activity in BMS versus plasma was reflected in binding antibody levels: HIV-1 envelope specific IgG titers were 2.2 log₁₀ lower (compared to 0.59 log₁₀ lower for IgA) in BMS versus plasma. In contrast, antibodies capable of ADCC were common and could be detected in the BMS from all 19 women. BMS envelope-specific IgG titers were associated with both detection of IgG NAbs (p = 0.0001)and BMS ADCC activity (p = 0.014). Importantly, BMS ADCC capacity was inversely associated with infant infection risk (p = 0.039). Our findings indicate that BMS has low levels of envelope specific IgG and IgA with limited neutralizing activity. However, this small study of women with high plasma viral loads suggests that breastmilk ADCC activity is a correlate of transmission that may impact infant infection risk.     

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.     

Envelope variable region 4 is the first target of neutralizing antibodies in early simian immunodeficiency virus mac251 infection of rhesus monkeys

A major goal of AIDS vaccine development is to design vaccination strategies that can elicit broad and potent protective antibodies. The initial viral targets of neutralizing antibodies (NAbs) early after human or simian immunodeficiency virus (HIV/SIV) infection are not known. The identification of early NAb epitopes that induce protective immunity or retard the progression of disease is important for AIDS vaccine development. The aim of this study was to determine the Env residues targeted by early SIV NAbs and to assess the influence of prior vaccination on neutralizing antibody kinetics and specificity during early infection. We previously described stereotypic env sequence variations in SIVmac251-infected rhesus monkeys that resulted in viral escape from NAbs. Here, we defined the early viral targets of neutralization and determined whether the ability of serum antibody from infected monkeys to neutralize SIV was altered in the setting of prior vaccination. To localize the viral determinants recognized by early NAbs, a panel of mutant pseudoviruses was assessed in a TZM-bl reporter gene neutralization assay to define the precise changes that eliminate recognition by SIV Env-specific NAbs in 16 rhesus monkeys. Changing R420 to G or R424 to Q in V4 of Env resulted in the loss of recognition by NAbs in vaccinated monkeys. In contrast, mutations in the V1 region of Env did not alter the NAb profile. These findings indicate that early NAbs are directed toward SIVmac251 Env V4 but not the V1 region, and that this env vaccination regimen did not alter the kinetics or the breadth of NAbs during early infection.     

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

Polyvalent envelope glycoprotein vaccine elicits a broader neutralizing antibody response but is unable to provide sterilizing protection against heterologous Simian/human immunodeficiency virus infection in pigtailed macaques

The great difficulty in eliciting broadly cross-reactive neutralizing antibodies (NAbs) against human immunodeficiency virus type 1 (HIV-1) isolates has been attributed to several intrinsic properties of their viral envelope glycoprotein, including its complex quaternary structure, extensive glycosylation, and marked genetic variability. Most previously evaluated vaccine candidates have utilized envelope glycoprotein from a single virus isolate. Here we compare the breadth of NAb and protective immune response following vaccination of pigtailed macaques with envelope protein(s) derived from either single or multiple viral isolates. Animals were challenged with Simian/human immunodeficiency virus strain DH12 (SHIV(DH12)) following priming with recombinant vaccinia virus(es) expressing gp160(s) and boosting with gp120 protein(s) from (i) LAI, RF, 89.6, AD8, and Bal (Polyvalent); (ii) LAI, RF, 89.6, AD8, Bal, and DH12 (Polyvalent-DH12); (iii) 89.6 (Monovalent-89.6); and (iv) DH12 (Monovalent-DH12). Animals in the two polyvalent vaccine groups developed NAbs against more HIV-1 isolates than those in the two monovalent vaccine groups (P = 0.0054). However, the increased breadth of response was directed almost entirely against the vaccine strains. Resistance to SHIV(DH12) strongly correlated with the level of NAbs directed against the virus on the day of challenge (P = 0.0008). Accordingly, the animals in the Monovalent-DH12 and Polyvalent-DH12 vaccine groups were more resistant to the SHIV(DH12) challenge than the macaques immunized with preparations lacking a DH12 component (viz. Polyvalent and Monovalent-89.6) (P = 0.039). Despite the absence of any detectable NAb, animals in the Polyvalent vaccine group, but not those immunized with Monovalent-89.6, exhibited markedly lower levels of plasma virus than those in the control group, suggesting a superior cell-mediated immune response induced by the polyvalent vaccine.     

Sequential immunization with a subtype B HIV-1 envelope quasispecies partially mimics the in vivo development of neutralizing antibodies

A major goal of human immunodeficiency virus type 1 (HIV-1) vaccine efforts is the design of Envelope (Env)-based immunogens effective at eliciting heterologous or broad neutralizing antibodies (NAbs). We hypothesized that programming the B-cell response could be achieved by sequentially exposing the host to a collection of env variants representing the viral quasispecies members isolated from an individual that developed broad NAbs over time. This ordered vaccine approach (sequential) was compared to exposure to a cocktail of env clones (mixture) and to a single env variant (clonal). The three strategies induced comparable levels of the autologous and heterologous neutralization of tier 1 pseudoviruses. Sequential and mixture exposure to quasispecies led to epitope targeting similar to that observed in the simian-human immunodeficiency virus (SHIV)-infected animal from which the env variants were cloned, while clonal and sequential exposure led to greater antibody maturation than the mixture. Therefore, the sequential vaccine approach best replicated the features of the NAb response observed in that animal. This study is the first to explore the use of a collection of HIV-1 env quasispecies variants as immunogens and to present evidence that it is possible to educate the B-cell response by sequential exposure to native HIV-1 quasispecies env variants derived from an individual with a broadened NAb response.     

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.     

Selection of a Rare Neutralization-Resistant Variant following Passive Transfer of Convalescent Immune Plasma in Equine Infectious Anemia Virus-Challenged SCID Horses

Vaccines preventing HIV-1 infection will likely elicit antibodies that neutralize diverse strains. However, the capacity for lentiviruses to escape broadly neutralizing antibodies (NAbs) is not completely understood, nor is it known whether NAbs alone can control heterologous infection. Here, we determined that convalescent immune plasma from a horse persistently infected with equine infectious anemia virus (EIAV) neutralized homologous virus and several envelope variants containing heterologous principal neutralizing domains (PND). Plasma was infused into young horses (foals) affected with severe combined immunodeficiency (SCID), followed by challenge with a homologous EIAV stock. Treated SCID foals were protected against clinical disease, with complete prevention of infection occurring in one foal. In three SCID foals, a novel neutralization-resistant variant arose that was found to preexist at a low frequency in the challenge inoculum. In contrast, SCID foals infused with nonimmune plasma developed acute disease associated with high levels of the predominant challenge virus. Following transfer to an immunocompetent horse, the neutralization-resistant variant induced a single febrile episode and was subsequently controlled in the absence of type-specific NAb. Long-term control was associated with the presence of cytotoxic T lymphocytes (CTL). Our results demonstrate that immune plasma with neutralizing activity against heterologous PND variants can prevent lentivirus infection and clinical disease in the complete absence of T cells. Importantly, however, rare neutralization-resistant envelope variants can replicate in vivo under relatively broad selection pressure, highlighting the need for protective lentivirus vaccines to elicit NAb responses with increased breadth and potency and/or CTL that target conserved epitopes.Development of an effective vaccine will be critical in the efforts to control the human immunodeficiency virus type 1 (HIV-1) pandemic. Unfortunately, vaccines evaluated in completed human efficacy trials have shown moderate to no protective effects, and, clearly, much more work is needed to define the correlates of lentivirus immune protection. Although these correlates are still not entirely known, vaccine strategies that elicit antibodies with broad neutralizing activity are currently of considerable interest, and it is widely believed that HIV-1 envelope glycoproteins that induce broadly neutralizing antibodies (NAbs) will be critical components of a protective vaccine (21, 28, 53, 63).Equine infectious anemia virus (EIAV) is a macrophage-tropic lentivirus that causes persistent infection in horses worldwide and serves as an important large-animal translational model in which to dissect basic correlates of protective lentiviral immunity (9, 31, 33, 38, 57). EIAV is a naturally occurring lentivirus, and infection results in a predictable course of recurrent episodes of plasma viremia and clinical disease. As with HIV-1 and simian immunodeficiency virus (SIV), EIAV infection is not cleared. However, infected horses eventually control viral replication and clinical disease to remain persistently infected inapparent carriers. Adaptive immune responses, including NAbs, are required for EIAV control since young horses (foals) with severe combined immunodeficiency (SCID), unlike normal foals, fail to eliminate the initial viremia following challenge (46). Equine SCID is caused by a frameshift mutation in the gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) (55, 60) and has an autosomal recessive mode of inheritance (47). The equine SCID defect is more severe than its murine counterpart in that SCID foals are incapable of forming either coding or signal joints (55). Adoptive transfer of EIAV-specific T and B lymphocytes to a SCID foal results in functional cytotoxic T lymphocytes (CTL) and NAb activity and is protective against homologous EIAV challenge (33).During acute EIAV infection, each recurrent episode coincides with the emergence of an antigenically distinct EIAV variant as defined by type-specific NAb, which neutralizes virus isolated during early disease episodes but not virus isolated during subsequent disease episodes (2, 20, 22, 43, 52). Amino acid variation primarily occurs within hypervariable regions V1 to V8 of the envelope gp90 surface unit (SU) and particularly within the V3/principal neutralizing domain (PND) region (1, 19, 24, 25, 57). Our work with EIAV-infected SCID foals indicates that significant envelope diversification does not occur in the absence of NAbs but that rapid envelope diversification occurs when adaptive immune responses are reconstituted (35). Thus, adaptive immunity, including NAb, drives selection of EIAV envelope variants during acute infection. Amino acid changes occur primarily within the V3 to V7 hypervariable SU regions, and many changes affect potential N-linked glycosylation sites (PNLGS) (35). Importantly, however, CTL also target the SU, and variants that escape CTL recognizing an EIAV V3/PND epitope have been identified (37, 38). Thus, both NAbs and CTL are capable of contributing to the selection of EIAV SU variants, but the relative contributions of each to such selection are not known.Recently, SU variation was evaluated in an immunocompetent pony experimentally inoculated with the virulent wild-type Wyoming strain of EIAV (57). Seventy-one distinct V3 variants that partitioned into five major nonoverlapping groups were identified and designated PND1 to PND5. Neutralization assays using chimeric infectious molecular clones containing these PNDs suggested a transition from type-specific NAb responses toward more broadly reactive immune responses during the course of infection and indicated that genetic changes conferring resistance to broadly NAbs lead to recrudescence of clinical disease following a lengthy clinically quiescent period (57). Thus, the NAb response broadens significantly during long-term persistent EIAV infection, and broadly NAbs play a critical role in EIAV immune control.Studies of nonhuman primates have provided important information regarding the protective effects of NAbs. Passive immunization of macaques with purified immunoglobulin from chimpanzees infected with several different HIV-1 isolates results in complete protection from homologous chimeric simian/human immunodeficiency virus (SHIV) infection when the immunoglobulin is given 24 h prior to challenge (54). Passive transfer of a triple combination of broadly neutralizing human monoclonal antibodies directed against the envelope of a primary HIV-1 isolate results in complete protection against SHIV infection in some macaques while others become infected but exhibit decreased plasma viremia (29). The contribution of T cells to partial protection in these studies is not clear, and the presence or absence of viral escape variants in the unprotected macaques has not been evaluated. In neonatal macaques, various combinations of broadly neutralizing human monoclonal antibodies directed against conserved HIV envelope epitopes administered before and after SHIV challenge result in protection against persistent systemic infection in some animals, but clinical disease develops in others (12-14). Virus-specific T-cell proliferative responses are detected in some of the protected animals, indicating that cellular immune responses occur and likely contribute to protection by eliminating infected cells (13).Despite the fact that NAbs can block experimental SHIV infection, selection pressure exerted by NAbs plays a critical role in HIV-1 and SIV envelope evolution during infection, and evasion of NAb responses is an important mechanism of HIV-1 and SIV persistence (11, 16, 27, 48, 59). The maturation of a type-specific NAb response in SIV-infected rhesus macaques significantly correlates with diversification in the V1/V2 region of the SIV envelope (50). In HIV-1, NAbs are detectable within the first 2 months postinfection and result in an early and significant selection force on the virus population (49). Escape from NAbs involves many amino acid substitutions with little cross-neutralization between closely related strains, and NAb responses drive the diversification of the HIV-1 envelope during the early stages of infection (16). The early appearance of NAbs in patients with acute HIV-1 infection results in the replacement of neutralization-sensitive virus by successive populations of resistant virus, and virus escape primarily involves changes in N-linked glycosylation (59). Thus, overcoming neutralization escape constitutes a significant barrier to the ultimate efficacy of any NAb-eliciting HIV-1 vaccine.Because the SCID defect occurs naturally in the horse, it provides a powerful and unique opportunity to finely dissect the protective effects of immune interventions against a naturally occurring lentivirus independent of other de novo adaptive immune responses. This level of dissection is not possible in other lentivirus model systems. The goal of the current study was to determine if broadly NAbs could protect against lentivirus challenge in the complete absence of T lymphocytes and other adaptive immune responses. We hypothesized that convalescent immune plasma from a long-term persistently infected inapparent carrier horse containing antibodies capable of neutralizing homologous and several heterologous EIAV SU PND variants would provide complete protection when infused into SCID foals before experimental virus inoculation. This plasma was administered to four SCID foals 24 h prior to challenge, and four control SCID foals received normal horse plasma. Clinical outcome, plasma viral load, and serum neutralization activity were analyzed in all foals. Although complete protection was achieved in one treated foal, infection occurred in the others. In foals that became viremic, the SU sequence and neutralization phenotype of the breakthrough virus were determined. As part of these experiments, blood containing this virus was inoculated into a naive immunocompetent horse, and the adaptive immune responses associated with its control were further evaluated.     

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.     

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.