Infants born to HIV-1-infected mothers in resource-limited areas where replacement feeding is unsafe and impractical are repeatedly exposed to HIV-1 throughout breastfeeding. Despite this, the majority of infants do not contract HIV-1 postnatally, even in the absence of maternal antiretroviral therapy. This suggests that immune factors in breast milk of HIV-1-infected mothers help to limit vertical transmission. We compared the HIV-1 envelope-specific breast milk and plasma antibody responses of clade C HIV-1-infected postnatally transmitting and nontransmitting mothers in the control arm of the Malawi-based Breastfeeding Antiretrovirals and Nutrition Study using multivariable logistic regression modeling. We found no association between milk or plasma neutralization activity, antibody-dependent cell-mediated cytotoxicity, or HIV-1 envelope-specific IgG responses and postnatal transmission risk. While the envelope-specific breast milk and plasma IgA responses also did not reach significance in predicting postnatal transmission risk in the primary model after correction for multiple comparisons, subsequent exploratory analysis using two distinct assay methodologies demonstrated that the magnitudes of breast milk total and secretory IgA responses against a consensus HIV-1 envelope gp140 (B.con env03) were associated with reduced postnatal transmission risk. These results suggest a protective role for mucosal HIV-1 envelope-specific IgA responses in the context of postnatal virus transmission. This finding supports further investigations into the mechanisms by which mucosal IgA reduces risk of HIV-1 transmission via breast milk and into immune interventions aimed at enhancing this response. IMPORTANCEInfants born to HIV-1-infected mothers are repeatedly exposed to the virus in breast milk. Remarkably, the transmission rate is low, suggesting that immune factors in the breast milk of HIV-1-infected mothers help to limit transmission. We compared the antibody responses in plasma and breast milk of HIV-1-transmitting and -nontransmitting mothers to identify responses that correlated with reduced risk of postnatal HIV-1 transmission. We found that neither plasma nor breast milk IgG antibody responses were associated with risk of HIV-1 transmission. In contrast, the magnitudes of the breast milk IgA and secretory IgA responses against HIV-1 envelope proteins were associated with reduced risk of postnatal HIV-1 transmission. The results of this study support further investigations of the mechanisms by which mucosal IgA may reduce the risk of HIV-1 transmission via breastfeeding and the development of strategies to enhance milk envelope-specific IgA responses to reduce mother-to-child HIV transmission and promote an HIV-free generation.

A successful HIV-1 vaccine must elicit immune responses that impede mucosal virus transmission, though functional roles of protective HIV-1 Envelope (Env)-specific mucosal antibodies remain unclear. Colostrum is a rich source of readily accessible mucosal B cells that may help define the mucosal antibody response contributing to prevention of postnatal HIV-1 transmission. To examine the HIV-1 Env-specific colostrum B cell repertoire, single B cells were isolated from 17 chronically HIV-infected, lactating women, producing 51 blood and 39 colostrum HIV-1 Env-specific B cell antibodies. All HIV-1 Env-specific colostrum-derived antibodies were IgG1 isotype and had mean heavy chain complementarity-determining region 3 (CDR3) lengths and mutation frequencies similar to those isolated from blood. However, variable heavy chain (VH) gene subfamily 1~69 usage was higher among colostrum than blood HIV-1 Env-reactive antibodies (49% versus 20%, p = 0.006, Fisher’s exact test). Additionally, more HIV-1 Env-specific colostrum antibodies were gp120-specific than those isolated from blood (44% versus 16%, p = 0.005, Fisher’s exact test). One cross-compartment HIV-1 Env-specific clonal B cell lineage was identified. These unique characteristics of colostrum B cell antibodies suggest selective homing of HIV-1-specific IgG1-secreting memory B cells to the mammary gland and have implications for targeting mucosal B cell populations by vaccination.

The development of an effective maternal HIV-1 vaccine that could synergize with antiretroviral therapy (ART) to eliminate pediatric HIV-1 infection will require the characterization of maternal immune responses capable of blocking transmission of autologous HIV to the infant. We previously determined that maternal plasma antibody binding to linear epitopes within the variable loop 3 (V3) region of HIV envelope (Env) and neutralizing responses against easy-to-neutralize tier 1 viruses were associated with reduced risk of peripartum HIV infection in the historic U.S. Woman and Infant Transmission Study (WITS) cohort. Here, we defined the fine specificity and function of the potentially protective maternal V3-specific IgG antibodies associated with reduced peripartum HIV transmission risk in this cohort. The V3-specific IgG binding that predicted low risk of mother-to-child-transmission (MTCT) was dependent on the C-terminal flank of the V3 crown and particularly on amino acid position 317, a residue that has also been associated with breakthrough transmission in the RV144 vaccine trial. Remarkably, the fine specificity of potentially protective maternal plasma V3-specific tier 1 virus-neutralizing responses was dependent on the same region in the V3 loop. Our findings suggest that MTCT risk is associated with neutralizing maternal IgG that targets amino acid residues in the C-terminal region of the V3 loop crown, suggesting the importance of the region in immunogen design for maternal vaccines to prevent MTCT. IMPORTANCE Efforts to curb HIV-1 transmission in pediatric populations by antiretroviral therapy (ART) have been highly successful in both developed and developing countries. However, more than 150,000 infants continue to be infected each year, likely due to a combination of late maternal HIV diagnosis, lack of ART access or adherence, and drug-resistant viral strains. Defining the fine specificity of maternal humoral responses that partially protect against MTCT of HIV is required to inform the development of a maternal HIV vaccine that will enhance these responses during pregnancy. In this study, we identified amino acid residues targeted by potentially protective maternal V3-specific IgG binding and neutralizing responses, localizing the potentially protective response in the C-terminal region of the V3 loop crown. Our findings have important implications for the design of maternal vaccination strategies that could synergize with ART during pregnancy to achieve the elimination of pediatric HIV infections.KEYWORDS HIV, mother-to-child transmission, V3, antibodies

Despite extensive genetic diversity of HIV-1 in chronic infection, a single or few maternal virus variants become the founders of an infant’s infection. These transmitted/founder (T/F) variants are of particular interest, as a maternal or infant HIV vaccine should raise envelope (Env) specific IgG responses capable of blocking this group of viruses. However, the maternal or infant factors that contribute to selection of infant T/F viruses are not well understood. In this study, we amplified HIV-1 env genes by single genome amplification from 16 mother-infant transmitting pairs from the U.S. pre-antiretroviral era Women Infant Transmission Study (WITS). Infant T/F and representative maternal non-transmitted Env variants from plasma were identified and used to generate pseudoviruses for paired maternal plasma neutralization sensitivity analysis. Eighteen out of 21 (85%) infant T/F Env pseudoviruses were neutralization resistant to paired maternal plasma. Yet, all infant T/F viruses were neutralization sensitive to a panel of HIV-1 broadly neutralizing antibodies and variably sensitive to heterologous plasma neutralizing antibodies. Also, these infant T/F pseudoviruses were overall more neutralization resistant to paired maternal plasma in comparison to pseudoviruses from maternal non-transmitted variants (p = 0.012). Altogether, our findings suggest that autologous neutralization of circulating viruses by maternal plasma antibodies select for neutralization-resistant viruses that initiate peripartum transmission, raising the speculation that enhancement of this response at the end of pregnancy could further reduce infant HIV-1 infection risk.

In the RV144 vaccine trial, IgG responses against the HIV envelope variable loops 1 and 2 (V1V2) were associated with decreased HIV acquisition risk. We previously reported that infants immunized with an MF59-adjuvanted rgp120 vaccine developed higher-magnitude anti-V1V2 IgG responses than adult RV144 vaccinees. To determine whether the robust antibody response in infants is due to differences in vaccine regimens or to inherent differences between the adult and infant immune systems, we compared Env-specific IgG responses in adults and infants immunized with the same MF59- and alum-adjuvanted HIV envelope vaccines. At peak immunogenicity, the magnitudes of the gp120- and V1V2-specific IgG responses were comparable between adults and infants immunized with the alum/MNrgp120 vaccine (gp120 median fluorescence intensities [FIs] in infants = 7,118 and in adults = 11,510, = 0.070; V1V2 median MFIs of 512 [infants] and 804 [adults], = 0.50), whereas infants immunized with the MF59/SF-2 rgp120 vaccine had higher-magnitude antibody levels than adults (gp120 median FIs of 15,509 [infants] and 2,290 [adults], < 0.001; V1V2 median FIs of 23,926 [infants] and 1,538 [adults]; < 0.001). Six months after peak immunogenicity, infants maintained higher levels Env-specific IgG than adults. Anti-V1V2 IgG3 antibodies that were associated with decreased HIV-1 risk in RV144 vaccinees were present in 43% of MF59/rgp120-vaccinated infants but only in 12% of the vaccinated adults ( = 0.0018). Finally, in contrast to the rare vaccine-elicited Env-specific IgA in infants, rgp120 vaccine-elicited Env-specific IgA was frequently detected in adults. Our results suggest that vaccine adjuvants differently modulate gp120-specific antibody responses in adults and infants and that infants can robustly respond to HIV Env immunization. More than 150,000 pediatric HIV infections occur yearly, despite the availability of antiretroviral prophylaxis. A pediatric HIV vaccine could reduce the number of these ongoing infant infections and also prime for long-term immunity prior to sexual debut. We previously reported that immunization of infants with an MF59-adjuvanted recombinant gp120 vaccine induced higher-magnitude, potentially protective anti-V1V2 IgG responses than in adult vaccinees receiving the moderately effective RV144 vaccine. In the present study, we demonstrate that the robust response observed in infants is not due to differences in vaccine regimen or vaccine dose between adults and infants. Our results suggest that HIV vaccine adjuvants may differentially modulate immune responses in adults and infants, highlighting the need to conduct vaccine trials in pediatric populations.

Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants due to beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 Envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. Interestingly, we also identified divergent patterns of colostrum Env-specific B cell lineage evolution with respect to cross-reactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk IgG repertoire. Maternal vaccine strategies to specifically target this breast milk B cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants.

Despite extensive genetic diversity of HIV-1 in chronic infection, infant HIV-1 infection involves selective transmission of a single or few maternal virus variants. These transmitted/founder (T/F) variants are of particular interest, as a maternal or infant HIV vaccine should raise envelope (Env)-specific IgG responses capable of blocking this group of viruses. However, the maternal or infant factors that contribute to selection of infant T/F viruses are not well understood. In this study, we isolated HIV-1envgenes by single genome amplification from 16 mother-infant transmitting pairs from the U.S. pre-antiretroviral era Women Infant Transmission Study (WITS). Infant T/F and representative maternal non-transmitted Env variants from plasma were identified and used to generate pseudoviruses for paired maternal plasma neutralization sensitivity analysis. Eighteen out of 21 (85%) infant T/F Env pseudoviruses were neutralization resistant to paired maternal plasma. Yet, all infant T/F viruses were neutralization sensitive to a panel of HIV-1 broadly neutralizing antibodies and variably sensitive to heterologous plasma neutralizing antibodies. Moreover, infant T/F pseudoviruses were overall more neutralization resistant compared to maternal non-transmitted plasma variants (p=0.012). Altogether, our findings suggest that autologous neutralization of circulating viruses by maternal plasma antibodies select for neutralization-resistant viruses that initiate peripartum transmission, raising the spector that enhancement of this response at the end of pregnancy could further reduce infant HIV infection risk.Author SummaryMother to child transmission (MTCT) of HIV-1 can occur during pregnancy (in utero), at the time of delivery (peripartum) or by breastfeeding (postpartum). With the availability of anti-retroviral therapy (ART), rate of MTCT of HIV-1 have been significantly lowered. However, significant implementation challenges remains in resource-poor areas, making it difficult to eliminate pediatric HIV. An improved understanding of the viral population (escape variants from autologous neutralizing antibodies) that lead to infection of infants at time of transmission will help in designing immune interventions to reduce vertical HIV-1 transmission. Here, we selected 16 HIV-1-infected mother-infant pairs from WITS cohort (from pre anti-retroviral era), where infants became infected peripartum. HIV-1envgene sequences were obtained by the single genome amplification method. The sensitivity of these infant Env pseudoviruses against paired maternal plasma and a panel of broadly neutralizing monoclonal antibodies (bNAbs) was analyzed. We demonstrated that the infant T/F viruses were more resistant against maternal plasma than non-transmitted maternal variants, but sensitive to most (bNAbs). Signature sequence analysis of infant T/F and non-transmitted maternal variants revealed the potential importance of V3 and MPER region for resistance against to paired maternal plasma. These findings provide insights for the design of maternal immunization strategies to enhance neutralizing antibodies that target V3 region of autologous virus populations, which could work synergistically with maternal ARVs to further reduce the rate of peripartum HIV-1 transmission.

Tenascin-C (TNC) is a newly identified innate HIV-1-neutralizing protein present in breast milk, yet its presence and potential HIV-inhibitory function in other mucosal fluids is unknown. In this study, we identified TNC as a component of semen and cervical fluid of HIV-1-infected and uninfected individuals, although it is present at a significantly lower concentration and frequency compared to that of colostrum and mature breast milk, potentially due to genital fluid protease degradation. However, TNC was able to neutralize HIV-1 after exposure to low pH, suggesting that TNC could be active at low pH in the vaginal compartment. As mucosal fluids are complex and contain a number of proteins known to interact with the HIV-1 envelope, we further studied the relationship between the concentration of TNC and neutralizing activity in breast milk. The amount of TNC correlated only weakly with the overall innate HIV-1-neutralizing activity of breast milk of uninfected women and negatively correlated with neutralizing activity in milk of HIV-1 infected women, indicating that the amount of TNC in mucosal fluids is not adequate to impede HIV-1 transmission. Moreover, the presence of polyclonal IgG from milk of HIV-1 infected women, but not other HIV-1 envelope-binding milk proteins or monoclonal antibodies, blocked the neutralizing activity of TNC. Finally, as exogenous administration of TNC would be necessary for it to mediate measurable HIV-1 neutralizing activity in mucosal compartments, we established that recombinantly produced TNC has neutralizing activity against transmitted/founder HIV-1 strains that mimic that of purified TNC. Thus, we conclude that endogenous TNC concentration in mucosal fluids is likely inadequate to block HIV-1 transmission to uninfected individuals.