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

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

Maternal Neutralization-Resistant Virus Variants Do Not Predict Infant HIV Infection Risk

Mother-to-child transmission (MTCT) of HIV provides a setting for studying immune correlates of protection. Neutralizing antibodies (NAbs) are suggested to contribute to a viral bottleneck during MTCT, but their role in blocking transmission is unclear, as studies comparing the NAb sensitivities of maternal viruses have yielded disparate results. We sought to determine whether transmitting mothers differ from nontransmitting mothers in the ability to neutralize individual autologous virus variants present at transmission. Ten transmitting and 10 nontransmitting HIV-infected mothers at high risk of MTCT were included in this study. Full-length HIV envelope genes (n = 100) were cloned from peripheral blood mononuclear cells obtained near transmission from transmitting mothers and at similar time points from nontransmitting mothers. Envelope clones were tested as pseudoviruses against contemporaneous, autologous maternal plasma in neutralization assays. The association between transmission and the log2 50% inhibitory concentration (IC50) for multiple virus variants per mother was estimated by using logistic regression with clustered standard errors. t tests were used to compare proportions of neutralization-resistant viruses. Overall, transmitting mothers had a median IC50 of 317 (interquartile range [IQR], 202 to 521), and nontransmitting mothers had a median IC50 of 243 (IQR, 95 to 594). Transmission risk was not significantly associated with autologous NAb activity (odds ratio, 1.25; P = 0.3). Compared to nontransmitting mothers, transmitting mothers had similar numbers of or fewer neutralization-resistant virus variants, depending on the IC50 neutralization resistance cutoff. In conclusion, HIV-infected mothers harbor mostly neutralization-sensitive viruses, although resistant variants were detected in both transmitting and nontransmitting mothers. These results suggest that MTCT during the breastfeeding period is not driven solely by the presence of maternal neutralization escape variants

ADCC activity in relation to HIV-1 Env specific IgG titers in BMS.

<p>The Y-axis shows % ADCC activity in BMS (1∶100) and the X-axis shows the log₁₀ BM HIV-1 env specific IgG titers. Filled and open symbols represent transmitting and non-transmitting women, respectively. The trend line ‘Linear (ALL)’ is the regression line including both transmitting and non-transmitting women.</p

ADCC activity in BMS and plasma.

<p>Percent ADCC activity in BMS(A) and plasma (B). BMS was tested at a 1∶100 dilution and plasma at a 1∶1000 dilution. The subject ID for the corresponding ADCC measure is shown below each bar. The results are from duplicate testing and are an average of at least two independent experiments each done using effector cells from a single donor. nd indicates not done.</p

Neutralization potency of IgG and IgA from two mothers against autologous virus.

<p>Representative graphs showing percent neutralization versus BMS purified IgG or IgA dilution. (a) Neutralization by IgG and IgA fractions from subject MM471 and (b) neutralization by IgG and IgA fractions from subject MA411. IgG (filled square) and IgA (open square) responses against pseudovirus generated with autologous HIV-1 env are shown in black lines and against SIVMneCl8 (SIV) are shown in grey lines. The 50% neutralization level is shown with a dotted line. The results are from duplicate testing and are representative of at least two independent experiments.</p

Neutralization potency of plasma and BMS from four mothers against heterologous virus.

<p>The graphs show percent neutralization versus plasma (A) or BMS (B) dilution. Results using pseudovirus generated with heterologous Q461.d1 env (HIV-1 in black lines) are shown in the left graph and with SIVMneCl8 (SIV in grey lines) are shown in the right graph. The corresponding symbol for the data from each of the four mothers is shown in the upper right corner. The 50% neutralization level is shown with a dotted line. The results are from triplicate testing and are representative of at least two independent experiments. The average IC50s for the two experiments for all 19 women is reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002739#ppat-1002739-t001" target="_blank">Table 1</a>.</p

The characteristics of transmitting and non-transmitting women in the study and the neutralization IC50s of their plasma and BMS.

a<p>Plasma neutralization assays were performed at a starting dilution of 1∶100; an IC50 of 50 was assigned in cases where 50% neutralization was not achieved.</p>b<p>BMS neutralization assays were performed at a starting dilution of 1∶20; an IC50 of 10 was assigned in cases where 50% neutralization was not achieved.</p>c<p>Viral Load.</p>d<p>Indicates week since delivery when infant was first HIV-1 DNA positive.</p>e<p>Indicates time-point after delivery at which BM sample was obtained.</p>f<p>Q461.d1.</p>g<p>Not done.</p>h<p>Not applicable.</p>i<p>Below detection.</p

Neutralization potency of purified IgG and IgA from four mothers against heterologous virus.

<p>The graphs show percent neutralization versus BMS purified IgG (A) or IgA (B) dilution. The corresponding symbol for the data from each of the four mothers is shown in the upper right corner. Neutralization by IgG and IgA is represented by filled and open symbols, respectively. Results using pseudovirus generated with Q461.d1 env (HIV-1 in black lines) are shown in the left graph and with SIVMneCl8 (SIV in grey lines) are shown in the right graph. The 50% neutralization level is shown with a dotted line. The results are from duplicate testing and are representative of at least two independent experiments. The average IC50s for the two experiments for all 19 women is reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002739#ppat.1002739.s002" target="_blank">Table S1</a>.</p