A vaccine capable of inducing broadly neutralizing antibodies (bnAbs) is essential for effective prevention against HIV in children and adolescents. Germline-targeting vaccine strategies aim to stimulate bnAb precursor B cells through carefully designed immunogens, such as the stabilized SOSIP trimers, which mimic native HIV envelope (Env) proteins while presenting key neutralizing epitopes to germline B cell receptors. Given the ability of children living with HIV to develop bnAbs earlier and at a higher frequency than adults, we compared the immunogenicity of a CD4 binding site (CD4bs) bnAb germline-targeting SOSIP trimer immunization strategy in infant (n = 5) and juvenile (n = 4) rhesus macaques (RMs). Animals received 3 doses of the germline-targeting BG505 GT1.1 immunogen, followed by 3 boosts of wild-type BG505 SOSIP, each adjuvanted with the TLR7/8 agonist, 3M-052-SE. After 1.5 years, the RMs were further boosted with a mixed clade B Env trimer nanoparticle to enhance heterologous virus neutralization responses. This germline-targeting strategy induced equivalent titers of neutralizing antibodies in both groups of RMs, yet the infants exhibited a higher magnitude of vaccine-specific IgG binding. Notably, after 3 doses of BG505 GT1.1 SOSIP, infants had higher BG505 GT1.1-specific IgD- B cells. Upon completion of the vaccine regimen, 4 of 5 infants developed a CD4bs bnAb precursor response detectable in serum compared to only 1 of 4 juveniles. Finally, administration of the mixed clade B nanoparticle was able to increase the breadth of antibody responses in 3 of 5 infants and 2 of 4 juveniles. These results suggest that immunization in early-life may enhance bnAb induction and highlight the potential for future pediatric HIV-1 vaccine strategies.

Adolescents are a growing population of people living with HIV. The period between weaning and sexual debut presents a low-risk window for HIV acquisition, making early childhood an ideal time for implementing an immunization regimen. Because the elicitation of broadly neutralizing antibodies (bnAbs) is critical for an effective HIV vaccine, our goal was to assess the ability of a bnAb B cell lineage-designed HIV envelope SOSIP (protein stabilized by a disulfide bond between gp120-gp41-named "SOS"-and an isoleucine-to-proline point mutation-named "IP"-at residue 559) to induce precursor CD4 binding site (CD4bs)-targeting bnAbs in early life. Infant rhesus macaques received either a BG505 SOSIP, based on the infant BG505 transmitted/founder virus, or the CD4bs germ line-targeting BG505 SOSIP GT1.1 (n = 5 per group). Although both strategies induced durable, high-magnitude plasma autologous virus neutralization responses, only GT1.1-immunized infants (n = 3 of 5) exhibited VRC01-like CD4bs bnAb precursor development. Thus, a multidose immunization regimen with bnAb lineage-designed SOSIPs shows promise for inducing early B cell responses with the potential to mature into protective HIV bnAbs before sexual debut.

A vaccine that can achieve protective immunity prior to sexual debut is critical to prevent the estimated 410,000 new HIV infections that occur yearly in adolescents. As children living with HIV can make broadly neutralizing antibody (bnAb) responses in plasma at a faster rate than adults, early childhood is an opportune window for implementation of a multi-dose HIV immunization strategy to elicit protective immunity prior to adolescence. Therefore, the goal of our study was to assess the ability of a B cell lineage-designed HIV envelope SOSIP to induce bnAbs in early life. Infant rhesus macaques (RMs) received either BG505 SOSIP or the germline-targeting BG505 GT1.1 SOSIP (n=5/group) with the 3M-052-SE adjuvant at 0, 6, and 12 weeks of age. All infant RMs were then boosted with the BG505 SOSIP at weeks 26, 52 and 78, mimicking a pediatric immunization schedule of multiple vaccine boosts within the first two years of life. Both immunization strategies induced durable, high magnitude binding antibodies and plasma autologous virus neutralization that primarily targeted the CD4-binding site (CD4bs) or C3/465 epitope. Notably, three BG505 GT1.1-immunized infants exhibited a plasma HIV neutralization signature reflective of VRC01-like CD4bs bnAb precursor development and heterologous virus neutralization. Finally, infant RMs developed precursor bnAb responses at a similar frequency to that of adult RMs receiving a similar immunization strategy. Thus, a multi-dose immunization regimen with bnAb lineage designed SOSIPs is a promising strategy for inducing protective HIV bnAb responses in childhood prior to adolescence when sexual HIV exposure risk begins.

To develop vaccines for certain key global pathogens such as HIV, it is crucial to elicit both neutralizing and non-neutralizing Fc-mediated effector antibody functions. Clinical evidence indicates that non-neutralizing antibody functions including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) contribute to protection against several pathogens. In this study, we demonstrated that conjugation of HIV Envelope (Env) antigen gp120 to a self-assembling nanofiber material named Q11 induced antibodies with higher breadth and functionality when compared to soluble gp120. Immunization with Q11-conjugated gp120 vaccine (gp120-Q11) demonstrated higher tier 1 neutralization, ADCP, and ADCC as compared to soluble gp120. Moreover, Q11 conjugation altered the Fc N-glycosylation profile of antigen-specific antibodies, leading to a phenotype associated with increased ADCC in animals immunized with gp120-Q11. Thus, this nanomaterial vaccine strategy can enhance non-neutralizing antibody functions possibly through modulation of immunoglobulin G Fc N-glycosylation.

Congenital Zika virus (ZIKV) infection results in neurodevelopmental deficits in up to 14% of infants born to ZIKV-infected mothers. Neutralizing antibodies are a critical component of protective immunity. Here, we demonstrate that plasma IgM contributes to ZIKV immunity in pregnancy, mediating neutralization up to 3 months post-symptoms. From a ZIKV-infected pregnant woman, we isolated a pentameric ZIKV-specific IgM (DH1017.IgM) that exhibited ultrapotent ZIKV neutralization dependent on the IgM isotype. DH1017.IgM targets an envelope dimer epitope within domain II. The epitope arrangement on the virion is compatible with concurrent engagement of all ten antigen-binding sites of DH1017.IgM, a solution not avail-able to IgG. DH1017.IgM protected mice against viremia upon lethal ZIKV challenge more efficiently than when expressed as an IgG. Our findings identify a role for antibodies of the IgM isotype in protection against ZIKV and posit DH1017.IgM as a safe and effective candidate immunotherapeutic, particularly during pregnancy.

Despite the worldwide availability of antiretroviral therapy (ART), approximately 150,000 pediatric HIV infections continue to occur annually. ART can dramatically reduce HIV mother-to-child transmission (MTCT), but inconsistent drug access and adherence, as well as primary maternal HIV infection during pregnancy and lactation are major barriers to eliminating vertical HIV transmission. Thus, immunologic strategies to prevent MTCT, such as an HIV vaccine, will be required to attain an HIV-free generation. A primary goal of HIV vaccine research has been to elicit broadly neutralizing antibodies (bnAbs) given the ability of passive bnAb immunization to protect against sensitive strains, yet we previously observed that HIV-transmitting mothers have more plasma neutralization breadth than non-transmitting mothers. Additionally, we have identified infant transmitted/founder (T/F) viruses that escape maternal bnAb responses. In this study, we examine a cohort of postpartum HIV-transmitting women with neutralization breadth to determine if certain maternal bnAb specificities drive the selection of infant T/F viruses. Using HIV pseudoviruses that are resistant to neutralizing antibodies targeting common bnAb epitopes, we mapped the plasma bnAb specificities of this cohort. Significantly more transmitting women with plasma bnAb activity had a mappable plasma bnAb specificity (six of seven, or 85.7%), compared to that of non-transmitting women with plasma bnAb activity (seven of twenty-one, or 33.3%, p=0.029 by 2-sided Fisher exact test). Our study suggests that having multispecific broad activity and/or uncommon epitope-specific bnAbs in plasma may be associated with protection against the vertical HIV transmission in the setting of maternal bnAb responses. Importance As mother to child transmission (MTCT) of HIV plays a major part in the persistence of the HIV/AIDS epidemic and bnAb-based passive and active vaccines are a primary strategy for HIV prevention, research in this field is of great importance. While previous MTCT research has investigated the neutralizing antibody activity of HIV-infected women, this is to our knowledge the largest study identifying differences in bnAb specificity of maternal plasma between transmitting and non-transmitting women. Here, we show that among HIV-infected women with broad and potent neutralization activity, more postpartum-transmitting women had a mappable plasma broadly neutralizing antibody (bnAb) specificity, compared to that of non-transmitting women, suggesting that the non-transmitting women more often have multi-specific bnAb responses or bnAb responses that target uncommon epitopes. Such responses may be required for protection against vertical HIV transmission in the setting of maternal bnAb responses.

BACKGROUND Recent studies have indicated that broadly neutralizing antibodies (bnAbs) in children may develop earlier after HIV infection compared to adults. METHODS We evaluated plasma from 212 ART-naïve, children living with HIV (1-3 years-old). Neutralization breadth and potency was assessed using a panel of 10 viruses and compared to adults with chronic HIV. The magnitude, epitope specificity and IgG subclass distribution of Env-specific antibodies were assessed using a binding antibody multiplex assay. RESULTS 1-year-old children demonstrated neutralization breadth comparable to chronically-infected adults, while 2 and 3-year olds exhibited significantly greater neutralization breadth (p=0.014). Similarly, binding antibody responses increased with age, with levels in 2 and 3 year-old children comparable to adults. Overall, there was no significant difference in antibody specificities or IgG subclass distribution between the pediatric and adult cohorts. Interestingly, the neutralization activity was mapped to a single epitope (CD4 binding site, V2 or V3 glycans) in only 5 of 38 pediatric broadly neutralizing samples, suggesting most children may develop a polyclonal neutralization response. CONCLUSIONS These results contribute to a growing body of evidence suggesting that initiating HIV immunization early in life may present advantages for the development of broadly neutralizing antibody responses.

Despite considerable reduction of mother-to-child transmission (MTCT) of HIV through use of maternal and infant antiretroviral therapy (ART), over 150,000 infants continue to become infected with HIV annually, falling far short of the World Health Organization goal of reaching <20,000 annual pediatric HIV cases worldwide by 2020. Prior to the widespread use of ART in the setting of pregnancy, over half of infants born to HIV-infected mothers were protected against HIV acquisition. Yet, the role of maternal immune factors in this protection against vertical transmission is still unclear, hampering the development of synergistic strategies to further reduce MTCT. It has been established that infant transmitted/founder (T/F) viruses are often resistant to maternal plasma, yet it is unknown if the neutralization resistance profile of circulating viruses predicts the maternal risk of transmission to her infant. In this study, we amplified HIV-1 envelope genes (env) by single genome amplification and produced representative Env variants from plasma of 19 non-transmitting mothers from the U.S. Women Infant Transmission Study (WITS), enrolled in the pre-ART era. Maternal HIV Env variants from non-transmitting mothers had similar sensitivity to autologous plasma as observed for non-transmitting variants from transmitting mothers. In contrast, infant variants were on average 30% less sensitive to paired plasma neutralization compared to non-transmitted maternal variants from both transmitting and non-transmitting mothers (p = 0.015). Importantly, a signature sequence analysis revealed that motifs enriched in env sequences from transmitting mothers were associated with broadly neutralizing antibody (bnAb) resistance. Altogether, our findings suggest that circulating maternal virus resistance to bnAb-mediated neutralization, but not autologous plasma neutralization, near the time of delivery, predicts increased MTCT risk. These results caution that enhancement of maternal plasma neutralization through passive or active vaccination during pregnancy may potentially drive the evolution of variants fit for vertical transmission.

Recent studies conducted in small cohorts of children have indicated that broadly neutralizing antibodies (bnAbs) may develop earlier after HIV infection compared to adults. To define the frequency and kinetics of bnAb responses in a larger pediatric cohort, we evaluated plasma from 212 ART-naïve, children living with HIV aged 1 to 3 years. Neutralization breadth and potency was assessed using a panel of 10 tier-2 viruses and compared to those of adults with chronic HIV. Further, the magnitude, epitope specificity and IgG subclass distribution of Env-specific antibodies were also assessed using a binding antibody multiplex assay. We found that 1-year-old children demonstrated neutralization breadth comparable to that of chronically-infected adults, and breadth continued to increase with age such that the pediatric cohort overall exhibited significantly greater neutralization breadth than adults (p= 0.014). Similarly, binding antibody responses increased with age, and the levels in 2 to 3 year-old children were comparable to those of adults. Overall, there was no significant difference in antibody specificities or IgG subclass distribution between the pediatric and adult cohorts. Interestingly, the neutralization activity was mapped to a single epitope (CD4 binding site, V2 or V3 glycans) in only 5 of 38 pediatric broadly neutralizing samples, suggesting a polyclonal neutralization response may develop in most children. These results contribute to a growing body of evidence suggesting that the early life immune system may present advantages for the development of an effective HIV vaccine. Author summary Understanding the development of broadly neutralizing antibodies during natural HIV infection is critical to provide insights for the development of an effective vaccine. In this study, we used a large cohort (n=212) of HIV infected children aged 1-3 years old to define the frequency and kinetics of broad neutralization in comparison to adults with chronic HIV. We also evaluated the magnitude and epitope specificity of HIV Env-specific antibodies in children and adults. We observed that both the binding and neutralizing antibody responses increased with age. However, by one year of age, children had neutralization breadth comparable to that of chronically infected adults; and overall, 1-3 year-old children demonstrated higher neutralization breadth than adults. Only slight differences in epitope-specificity and IgG subclass distribution were observed between children and adults, suggesting that these factors are not major contributors to the observed age-related difference in neutralization breadth. The epitope specificity of the neutralizing antibodies could not be mapped in the majority of children, suggesting that their response may be polyclonal. Our study contributes to the body of work indicating a potential advantage of the early life immune system for the elicitation of broad neutralization.

A major challenge in developing an effective vaccine against HIV-1 is the genetic diversity of its viral envelope. Because of the broad range of sequences exhibited by HIV-1 strains, protective antibodies must be able to bind and neutralize a widely mutated viral envelope protein. No vaccine has yet been designed which induces broadly neutralizing or protective immune responses against HIV in humans. Nanomaterial-based vaccines have shown the ability to generate antibody and cellular immune responses of increased breadth and neutralization potency. Thus, we have developed supramolecular nanofiber-based immunogens bearing the HIV gp120 envelope glycoprotein. These immunogens generated antibody responses that had increased magnitude and binding breadth compared to soluble gp120. By varying gp120 density on nanofibers, we determined that increased antigen valency was associated with increased antibody magnitude and germinal center responses. This study presents a proof-of-concept for a nanofiber vaccine platform generating broad, high binding antibody responses against the HIV-1 envelope glycoprotein.

Different HIV vaccine regimens elicit distinct plasma antibody responses in both human and nonhuman primate models. Previous studies in human and non-human primate infants showed that adjuvants influenced the quality of plasma antibody responses induced by pediatric HIV envelope vaccine regimens. We recently reported that use of the 3M052-SE adjuvant and longer intervals between vaccinations are associated with higher magnitude of antibody responses in infant rhesus macaques. However, the impact of different adjuvants in HIV vaccine regimens on the developing infant B cell receptor (BCR) repertoire has not been studied. This study evaluated whether pediatric HIV envelope vaccine regimens with different adjuvants induced distinct antigen-specific memory B cell repertoires and whether specific immunoglobulin (Ig) immunogenetic characteristics are associated with higher magnitude of plasma antibody responses in vaccinated infant rhesus macaques. We utilized archived preclinical pediatric HIV vaccine studies PBMCs and tissue samples from 19 infant rhesus macaques immunized either with (i) HIV Env protein with a squalene adjuvant, (ii) MVA-HIV and Env protein coadministered using a 3-week interval, (iii) MVA-HIV prime/ protein boost with an extended 6-week interval between immunizations, or (iv) with HIV Env administered with 3M-052-SE adjuvant. Frequencies of vaccine-elicited HIV Env-specific memory B cells from PBMCs and tissues were similar across vaccination groups (frequency range of 0.06-1.72%). There was no association between vaccine-elicited antigen-specific memory B cell frequencies and plasma antibody titer or avidity. Moreover, the epitope specificity and Ig immunogenetic features of vaccine-elicited monoclonal antibodies did not differ between the different vaccine regimens. These data suggest that pediatric HIV envelope vaccine candidates with different adjuvants that previously induced higher magnitude and quality of plasma antibody responses in infant rhesus macaques were not driven by distinct antigen-specific memory BCR repertoires.

The majority of new HIV-1 infections occur in young adults, with adolescent women being 3 times more likely to acquire HIV than young men. Implementation of HIV prevention strategies has been less successful in this age group; thus, a vaccine given prior to adolescence remains a high priority. We propose that instead of starting HIV vaccination during adolescence, an HIV vaccine regimen initiated in early infancy, aligned with the well-accepted pediatric vaccine schedule and followed with booster immunizations, will provide an alternative means to reduce HIV acquisition in adolescence. Importantly, the long window of time between the first infant vaccine dose and the adolescence vaccine dose will allow for the maturation of highly functional HIV Env-specific antibody responses. Our study provides evidence that early life vaccination induces durable Env-specific plasma IgG responses that can be boosted to further improve the quality of the antibody response.

Maternal antiretroviral therapy (ART) has effectively reduced but not eliminated the burden of mother-to-child transmission of HIV across the globe, as an estimated 160,000 children were newly infected with HIV in 2018. Thus, additional preventive strategies beyond ART will be required to close the remaining gap and end the pediatric HIV epidemic. A maternal active immunization strategy that synergizes with maternal ART could further reduce infant HIV infections. In this study, we found that two historic HIV Env vaccines did not enhance the ability of HIV-infected pregnant women to neutralize autologous viruses. Therefore, next-generation maternal HIV vaccine candidates must employ alternate approaches to achieve potent neutralizing antibody and perhaps nonneutralizing antibody responses to effectively impede vertical virus transmission. Moreover, these approaches must reflect the broad diversity of HIV strains and widespread availability of ART worldwide.

Each year, >180,000 infants become infected via mother-to-child transmission (MTCT) of HIV despite the availability of effective maternal antiretroviral treatments, underlining the need for a maternal HIV vaccine. We characterized 224 maternal HIV envelope (Env)-specific IgG monoclonal antibodies (MAbs) from seven nontransmitting and transmitting HIV-infected U.S. and Malawian mothers and examined their neutralization activities against nontransmitted autologous circulating viruses and infant-transmitted founder (infant-T/F) viruses. Only a small subset of maternal viruses, 3 of 72 (4%), were weakly neutralized by maternal linear V3 epitope-specific IgG MAbs, whereas 6 out of 6 (100%) infant-T/F viruses were neutralization resistant to these V3-specific IgG MAbs. We also show that maternal-plasma broadly neutralizing antibody (bNAb) responses targeting the V3 glycan supersite in a transmitting woman may have selected for an N332 V3 glycan neutralization-resistant infant-T/F virus. These data have important implications for bNAb-eliciting vaccines and passively administered bNAbs in the setting of MTCT.IMPORTANCE Efforts to eliminate MTCT of HIV with antiretroviral therapy (ART) have met little success, with >180,000 infant infections each year worldwide. It is therefore likely that additional immunologic strategies that can synergize with ART will be required to eliminate MTCT of HIV. To this end, understanding the role of maternal HIV Env-specific IgG antibodies in the setting of MTCT is crucial. In this study, we found that maternal-plasma broadly neutralizing antibody (bNAb) responses can select for T/F viruses that initiate infection in infants. We propose that clinical trials testing the efficacy of single bNAb specificities should not include HIV-infected pregnant women, as a single bNAb might select for neutralization-resistant infant-T/F viruses.

Breastmilk transmission of HIV remains unacceptably high. Vaccine approaches that prime robust virus-specific immune responses in infancy may prevent acquisition during the breastfeeding period. Moreover, vaccine responses that generate robust immunological memory that persists into adolescence could prevent sexual transmission later in life. The current study investigated the immunological benefit of late boosting following vaccination in infancy. Two groups of neonatal rhesus macaques were immunized with a clade C HIV Env protein adjuvanted with 3M-052 stable emulsion (SE) vaccine (Env Only) alone or together with a modified vaccinia Ankara (MVA) vector expressing HIV Env and SIV Gag (MVA/Env) at 0, 6, and 12 weeks of age. Env-specific B cells were present in blood yet absent from lymph nodes in the Env Only group by week 14. At week 32, 18 weeks following the last immunization, we could still detect Env-specific plasma IgG responses capable of autologous and heterologous HIV Env binding, Env-specific ADCC activity and CD4 blocking in both groups. A final booster immunization at week 32 enhanced avidity and function of Env-specific plasma IgG in both groups. These data confirmed previous work by our group that demonstrated HIV Env protein adjuvanted with the TLR7/8-based 3M-052-SE primed greater immune responses than Span85-Tween 80 Squalene. Our data from the Env Only vaccine regimen are consistent with the induction of Env-specific antibody responses in human infants. These results support the idea that early life vaccination is an effective means to induce persistent HIV Env-specific IgG responses that can be boosted in infancy and could be exploited to drive protective immunity against HIV acquisition prior to sexual debut.

Antibodies within the mucosa are part of the first line of defense against mucosal pathogens. Evaluating mucosal antibody isotypes, specificities, and antiviral functions in relationship to the systemic antibody profile can provide insights into whether the antibody response is coordinated in response to mucosal pathogens. In a natural immunity cohort of HIV-infected lactating women, we mapped the fine specificity and function of IgA in breast milk and plasma and compared these with the autologous IgG responses. Antigen specificities and functions differed between IgG and IgA, with antiviral functions (neutralization and phagocytosis) predominantly mediated by the IgG fraction in both milk and plasma. Furthermore, the specificity of milk IgA differed from that of systemic IgA. Our data suggest that milk IgA and systemic IgA should be separately examined as potential correlates of risk. Preventive vaccines may need to employ different strategies to elicit functional antiviral immunity by both antibody isotypes in the mucosa.

Interactions between innate antiviral factors at mucosal surfaces and HIV-1 virions contribute to the natural inefficiency of HIV-1 transmission and are a platform to inform the development of vaccine and nonvaccine strategies to block mucosal HIV-1 transmission. Tenascin-C (TNC) is a large, hexameric extracellular matrix glycoprotein identified in breast milk and genital fluids that broadly neutralizes HIV-1 via interaction with the HIV-1 Envelope (Env) variable 3 (V3) loop. In this report, we characterize the specific determinants of the interaction between TNC and the HIV-1 Env. We observed that TNC binding and neutralization of HIV-1 is dependent on the TNC fibrinogen-like globe (fbg) and fibronectin-type III (fn) domains, oligomerization, and its newly-mapped glycan structure. Moreover, we observed that TNC-mediated neutralization is also dependent on Env V3 residues 321/322 and 326/327, which surround the IGDIR motif of the V3 loop, as well the N332 glycan, which is critical to the broadly neutralizing activity of glycan-dependent V3-specific antibodies such as PGT128. Our results demonstrate a striking parallel between innate and adaptive immune mechanisms of broad HIV neutralization and provide further insight into the host protein-virus interactions responsible for the natural inefficiency of mucosal HIV-1 transmission.