Comprehensive characterization of the antibody responses to SARS-CoV-2 Spike protein finds additional vaccine-induced epitopes beyond those for mild infection.

Background: Control of the COVID-19 pandemic will rely on SARS-CoV-2 vaccine-elicited antibodies to protect against emerging and future variants; an understanding of the unique features of the humoral responses to infection and vaccination, including different vaccine platforms, is needed to achieve this goal. Methods: The epitopes and pathways of escape for Spike-specific antibodies in individuals with diverse infection and vaccination history were profiled using Phage-DMS. Principal component analysis was performed to identify regions of antibody binding along the Spike protein that differentiate the samples from one another. Within these epitope regions, we determined potential sites of escape by comparing antibody binding of peptides containing wild-type residues versus peptides containing a mutant residue. Results: Individuals with mild infection had antibodies that bound to epitopes in the S2 subunit within the fusion peptide and heptad-repeat regions, whereas vaccinated individuals had antibodies that additionally bound to epitopes in the N- and C-terminal domains of the S1 subunit, a pattern that was also observed in individuals with severe disease due to infection. Epitope binding appeared to change over time after vaccination, but other covariates such as mRNA vaccine dose, mRNA vaccine type, and age did not affect antibody binding to these epitopes. Vaccination induced a relatively uniform escape profile across individuals for some epitopes, whereas there was much more variation in escape pathways in mildly infected individuals. In the case of antibodies targeting the fusion peptide region, which was a common response to both infection and vaccination, the escape profile after infection was not altered by subsequent vaccination. Conclusions: The finding that SARS-CoV-2 mRNA vaccination resulted in binding to additional epitopes beyond what was seen after infection suggests that protection could vary depending on the route of exposure to Spike antigen. The relatively conserved escape pathways to vaccine-induced antibodies relative to infection-induced antibodies suggests that if escape variants emerge they may be readily selected for across vaccinated individuals. Given that the majority of people will be first exposed to Spike via vaccination and not infection, this work has implications for predicting the selection of immune escape variants at a population level. Funding: This work was supported by NIH grants AI138709 (PI JMO) and AI146028 (PI FAM). JMO received support as the Endowed Chair for Graduate Education (FHCRC). The research of FAM was supported in part by a Faculty Scholar grant from the Howard Hughes Medical Institute and the Simons Foundation. Scientific Computing Infrastructure at Fred Hutch was funded by ORIP grant S10OD028685.

When SARS-CoV-2 ­ the virus that causes COVID-19 ­ infects our bodies, our immune system reacts by producing small molecules called antibodies that stick to a part of the virus called the spike protein. Vaccines are thought to work by triggering the production of similar antibodies without causing disease. Some of the most effective antibodies against SARS-CoV-2 bind a specific area of the spike protein called the 'receptor binding domain' or RBD. When SARS-CoV-2 evolves it creates a challenge for our immune system: mutations, which are changes in the virus's genetic code, can alter the shape of its spike protein, meaning that existing antibodies may no longer bind to it as effectively. This lowers the protection offered by past infection or vaccination, which makes it harder to tackle the pandemic. As it stands, it is not clear which mutations to the virus's genetic code can affect antibody binding, especially to portions outside the RBD. To complicate things further, the antibodies people produce in response to mild infection, severe infection, and vaccination, while somewhat overlapping, exhibit some differences. Studying these differences could help minimize emergence of mutations that allow the virus to 'escape' the antibody response. A phage display library is a laboratory technique in which phages (viruses that infect bacteria) are used as a 'repository' for DNA fragments that code for a specific protein. The phages can then produce the protein (or fragments of it), and if the protein fragments bind to a target, it can be easily detected. Garrett, Galloway et al. exploited this technique to study how different portions of the SARS-CoV-2 spike protein were bound by antibodies. They made a phage library in which each phage encoded a portion of the spike protein with different mutations, and then exposed the different versions of the protein to antibodies from people who had experienced prior infection, vaccination, or both. The experiment showed that antibodies produced during severe infection or after vaccination bound to similar parts of the spike protein, while antibodies from people who had experienced mild infection targeted fewer areas. Garrett, Galloway et al. also found that mutations that affected the binding of antibodies produced after vaccination were more consistent than mutations that interfered with antibodies produced during infection. While these results show which mutations are most likely to help the virus escape existing antibodies, this does not mean that the virus will necessarily evolve in that direction. Indeed, some of the mutations may be impossible for the virus to acquire because they interfere with the virus's ability to spread. Further studies could focus on revealing which of the mutations detected by Garrett, Galloway et al. are most likely to occur, to guide vaccine development in that direction. To help with this, Garrett, Galloway et al. have made the data accessible to other scientists and the public using a web tool.

Functional development of a V3/glycan-specific broadly neutralizing antibody isolated from a case of HIV superinfection.

Stimulating broadly neutralizing antibodies (bnAbs) directly from germline remains a barrier for HIV vaccines. HIV superinfection elicits bnAbs more frequently than single infection, providing clues of how to elicit such responses. We used longitudinal antibody sequencing and structural studies to characterize bnAb development from a superinfection case. BnAb QA013.2 bound initial and superinfecting viral Env, despite its probable naive progenitor only recognizing the superinfecting strain, suggesting both viruses influenced this lineage. A 4.15 Å cryo-EM structure of QA013.2 bound to native-like trimer showed recognition of V3 signatures (N301/N332 and GDIR). QA013.2 relies less on CDRH3 and more on framework and CDRH1 for affinity and breadth compared to other V3/glycan-specific bnAbs. Antigenic profiling revealed that viral escape was achieved by changes in the structurally-defined epitope and by mutations in V1. These results highlight shared and novel properties of QA013.2 relative to other V3/glycan-specific bnAbs in the setting of sequential, diverse antigens.

Development of antibody-dependent cell cytotoxicity function in HIV-1 antibodies.

A prerequisite for the design of an HIV vaccine that elicits protective antibodies is understanding the developmental pathways that result in desirable antibody features. The development of antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) is particularly relevant because such antibodies have been associated with HIV protection in humans. We reconstructed the developmental pathways of six human HIV-specific ADCC antibodies using longitudinal antibody sequencing data. Most of the inferred naive antibodies did not mediate detectable ADCC. Gain of antigen binding and ADCC function typically required mutations in complementarity determining regions of one or both chains. Enhancement of ADCC potency often required additional mutations in framework regions. Antigen binding affinity and ADCC activity were correlated, but affinity alone was not sufficient to predict ADCC potency. Thus, elicitation of broadly active ADCC antibodies may require mutations that enable high-affinity antigen recognition along with mutations that optimize factors contributing to functional ADCC activity.

Nearly four decades after the human immunodeficiency virus (HIV for short) was first identified, the search for a vaccine still continues. An effective immunisation would require elements that coax the human immune system into making HIV-specific antibodies ­ the proteins that can recognise, bind to and deactivate the virus. Crucially, antibodies can also help white blood cells to target and destroy cells infected with HIV. This 'antibody-dependent cellular cytotoxicity' could be a key element of a successful vaccine, yet it has received less attention than the ability for antibodies to directly neutralize the virus. In particular, it is still unclear how antibodies develop the ability to flag HIV-infected cells for killing. Indeed, over the course of an HIV infection, an immune cell goes through genetic changes that tweak the 3D structure of the antibodies it manufactures. This process can improve the antibodies' ability to fight off the virus, but it was still unclear how it would shape antibody-dependent cellular cytotoxicity. To investigate this question, Doepker et al. retraced how the genes coding for six antibody families changed over time in an HIV-carrying individual. This revealed that antibodies could not initially trigger antibody-dependent cellular cytotoxicity. The property emerged and improved thanks to two types of alterations in the genetic sequences. One set of changes increased how tightly the antibodies could bind to the virus, targeting sections of the antibodies that can often vary. The second set likely altered the 3D structure in others ways, potentially affecting how antibodies bind the virus or how they interact with components of the immune system that help to kill HIV-infected cells. These alterations took place in segments of the antibodies that undergo less change over time. Ultimately, the findings by Doepker et al. suggest that an efficient HIV vaccine may rely on helping antibodies to evolve so they can bind more tightly to the virus and trigger cellular cytotoxicity more strongly.

Diversity and Function of Maternal HIV-1-Specific Antibodies at the Time of Vertical Transmission.

Infants of HIV-positive mothers can acquire HIV infection by various routes, but even in the absence of antiviral treatment, the majority of these infants do not become infected. There is evidence that maternal antibodies provide some protection from infection, but gestational maternal antibodies have not yet been characterized in detail. One of the most studied vertically infected infants is BG505, as the virus from this infant yielded an Envelope protein that was successfully developed as a stable trimer. Here, we isolated and characterized 39 HIV-specific neutralizing monoclonal antibodies (nAbs) from MG505, the mother of BG505, at a time point just prior to vertical transmission. These nAbs belonged to 21 clonal families and employed a variety of VH genes. Many were specific for the HIV-1 Env V3 loop, and this V3 specificity correlated with measurable antibody-dependent cellular cytotoxicity (ADCC) activity. The isolated nAbs did not recapitulate the full breadth of heterologous or autologous virus neutralization by contemporaneous plasma. Notably, we found that the V3-targeting nAb families neutralized one particular maternal Env variant, even though all tested variants had low V3 sequence diversity and were measurably bound by these nAbs. None of the nAbs neutralized BG505 transmitted virus. Furthermore, the MG505 nAb families were found at relatively low frequencies within the maternal B cell repertoire; all were less than 0.25% of total IgG sequences. Our findings illustrate an example of the diversity of HIV-1 nAbs within one mother, cumulatively resulting in a collection of antibody specificities that can contribute to the transmission bottleneck.IMPORTANCE Mother-to-child-transmission of HIV-1 offers a unique setting in which maternal antibodies both within the mother and passively transferred to the infant are present at the time of viral exposure. Untreated HIV-exposed human infants are infected at a rate of 30 to 40%, meaning that some infants do not get infected despite continued exposure to virus. Since the potential of HIV-specific immune responses to provide protection against HIV is a central goal of HIV vaccine design, understanding the nature of maternal antibodies may provide insights into immune mechanisms of protection. In this study, we isolated and characterized HIV-specific antibodies from the mother of an infant whose transmitted virus has been well studied.

Maternal Envelope gp41 Ectodomain-Specific Antibodies Are Associated With Increased Mother-to-Child Transmission of Human Immunodeficiency Virus-1.

Mother-to-child transmission of human immunodeficiency virus (HIV) occurs in the setting of maternal and passively acquired antibodies, providing a unique window into immune correlates of HIV risk. We compared plasma antibody binding to HIV antigens between 51 nontransmitting mother-infant pairs and 21 transmitting mother-infant pairs. Plasma antibody binding to a variety of gp41 ectodomain-containing antigens was associated with increased odds of transmission. Understanding the reasons why gp41 ectodomain-targeting antibodies are associated with transmission risk will be important in determining whether they can directly enhance infection or whether their presence reflects a redirecting of the humoral response away from targeting more protective epitopes.

Antibody-dependent cellular cytotoxicity targeting CD4-inducible epitopes predicts mortality in HIV-infected infants.

BACKGROUND: Antibody-dependent cellular cytotoxicity (ADCC) has been associated with improved infant outcome in mother-to-child transmission (MTCT) of HIV-1. Epitopes of these ADCC-mediating antibodies remain unidentified. CD4-inducible (CD4i) epitopes on gp120 are common ADCC targets in natural infection and vaccination. We tested whether CD4i epitope-specific ADCC mediated by maternal antibodies or passively-acquired antibodies in infants is associated with reduced MTCT and improved infant survival. METHODS: We used variants of CD4i cluster A-specific antibodies, A32 and C11, and a cluster C-specific antibody, 17b, with mutations abolishing Fc-Fc receptor interactions as inhibitors in a competition rapid and fluorometric ADCC assay using gp120-coated CEM-nkr target cells with plasma from 51 non-transmitting and 21 transmitting breastfeeding mother-infant pairs. FINDINGS: Cluster A-specific ADCC was common. Individually, neither A32-like nor C11-like ADCC was statistically significantly associated with risk of MTCT or infected infant survival. In combination, total maternal cluster A-specific ADCC was statistically significantly associated with decreased infected infant survival in a log-rank test (p = 0·017). There was a non-significant association for infant passively-acquired total cluster A-specific ADCC and decreased infected infant survival (p = 0·14). Surprisingly, plasma ADCC was enhanced in the presence of the defective Fc 17b competitor. Defective Fc 17b competitor-mediated maternal ADCC enhancement was statistically significantly associated with reduced infected infant survival (p = 0·011). A non-significant association was observed for passively-acquired infant ADCC enhancement and decreased survival (p = 0·19). INTERPRETATIONS: These data suggest that ADCC targeting CD4i epitopes is not associated with protection against breast milk HIV transmission but is associated with decreased survival of infected infants. FUND: This study was funded by NIH grant R01AI076105 and NIH fellowship F30AI136636.

Initiation of antiretroviral therapy leads to a rapid decline in cervical and vaginal HIV-1 shedding.

BACKGROUND: Antiretroviral therapy (ART) may decrease HIV-1 infectivity in women by reducing genital HIV-1 shedding. OBJECTIVES: To evaluate the time course and magnitude of decay in cervical and vaginal HIV-1 shedding as women initiate ART. METHODS: This prospective, observational study of 20 antiretroviral-naive women initiating ART with stavudine, lamivudine, and nevirapine measured HIV-1 RNA in plasma, cervical secretions, and vaginal secretions. Qualitative polymerase chain reaction estimated HIV-1 DNA in cervical and vaginal samples. Perelson's two-phase viral decay model and non-linear random effects were used to compare RNA decay rates. Decreases in proviral DNA were evaluated using logistic regression and generalized estimating equations. RESULTS: Significant decreases in the quantity of HIV-1 RNA were observed by day 2 in plasma (P < 0.001), day 2 in cervical secretions (P = 0.001), and day 4 in vaginal secretions (P < 0.001). Modeled initial and subsequent RNA decay rates in plasma, cervical secretions, and vaginal secretions were 0.6, 0.8, and 1.2 log10 virions/day, and 0.04, 0.05, and 0.06 log10 virions/day, respectively. The initial decay rate for vaginal HIV-1 RNA was more rapid than for plasma RNA (P = 0.02). Detection of HIV-1 DNA decreased significantly in vaginal secretions during the first week (P < 0.001). At day 28, 10 women had detectable HIV-1 RNA or proviral DNA in genital secretions. CONCLUSIONS: Genital HIV-1 shedding decreased rapidly after ART initiation, consistent with a rapid decrease in infectivity. However, incomplete viral suppression in half of these women may indicate an ongoing risk of transmission.

CC and CXC chemokines in breastmilk are associated with mother-to-child HIV-1 transmission.

INTRODUCTION: CC and CXC chemokines may play a role in mother-to-child HIV-1 transmission by blocking HIV-1 binding to chemokine receptors and impeding viral entry into cells. METHODS: To define correlates of breastmilk chemokines and associations with infant HIV-1 acquisition, chemokines in breastmilk and infant HIV-1 infection risk were assessed in an observational, longitudinal cohort study. We measured MIP-1alpha, MIP-1beta, RANTES, and SDF-1 in month 1 breastmilk specimens from HIV-1-infected women in Nairobi and HIV-1 viral load was calculated in maternal plasma and breastmilk at delivery and 1 month postpartum. Infant infection status was determined at birth and months 1, 3, 6, 9, and 12. RESULTS: Among 281 breastfeeding women, 60 (21%) of their infants acquired HIV-1 during follow-up, 39 (65%) of whom became infected intrapartum or after birth. MIP-1alpha, MIP-1beta, RANTES, and SDF-1 were all positively correlated with breastmilk HIV-1 RNA (P<0.0005). Women with clinical mastitis had 50% higher MIP-1alpha and MIP-1beta levels (P<0.001 and P=0.006, respectively) and women with subclinical mastitis (breastmilk Na(+)/K(+)>1) had approximately 70% higher MIP-1alpha, MIP-1beta and RANTES (P<0.002 for all) compared to women without mastitis. Independent of breastmilk HIV-1, increased MIP-1beta and SDF-1 were associated with reduced risk of infant HIV-1 (RR=0.4; 95% CI 0.2-0.9; P=0.03 and RR=0.5; 95% CI=0.3-0.9; P=0.02, respectively) and increased RANTES was associated with higher transmission risk (RR=2.3; 95% CI 1.1- 5.3; P=0.04). CONCLUSIONS: These observations suggest a complex interplay between virus levels, breastmilk chemokines, and mother-to-child HIV-1 transmission and may provide insight into developing novel strategies to reduce infection across mucosal surfaces.