Delineating the functional activity of antibodies with cross-reactivity to SARS-CoV-2, SARS-CoV-1 and related sarbecoviruses.

The recurring spillover of pathogenic coronaviruses and demonstrated capacity of sarbecoviruses, such SARS-CoV-2, to rapidly evolve in humans underscores the need to better understand immune responses to this virus family. For this purpose, we characterized the functional breadth and potency of antibodies targeting the receptor binding domain (RBD) of the spike glycoprotein that exhibited cross-reactivity against SARS-CoV-2 variants, SARS-CoV-1 and sarbecoviruses from diverse clades and animal origins with spillover potential. One neutralizing antibody, C68.61, showed remarkable neutralization breadth against both SARS-CoV-2 variants and viruses from different sarbecovirus clades. C68.61, which targets a conserved RBD class 5 epitope, did not select for escape variants of SARS-CoV-2 or SARS-CoV-1 in culture nor have predicted escape variants among circulating SARS-CoV-2 strains, suggesting this epitope is functionally constrained. We identified 11 additional SARS-CoV-2/SARS-CoV-1 cross-reactive antibodies that target the more sequence conserved class 4 and class 5 epitopes within RBD that show activity against a subset of diverse sarbecoviruses with one antibody binding every single sarbecovirus RBD tested. A subset of these antibodies exhibited Fc-mediated effector functions as potent as antibodies that impact infection outcome in animal models. Thus, our study identified antibodies targeting conserved regions across SARS-CoV-2 variants and sarbecoviruses that may serve as therapeutics for pandemic preparedness as well as blueprints for the design of immunogens capable of eliciting cross-neutralizing responses.

Pro-inflammatory feedback loops define immune responses to pathogenic Lentivirus infection.

BACKGROUND: The Lentivirus human immunodeficiency virus (HIV) causes chronic inflammation and AIDS in humans, with variable rates of disease progression between individuals driven by both host and viral factors. Similarly, simian lentiviruses vary in their pathogenicity based on characteristics of both the host species and the virus strain, yet the immune underpinnings that drive differential Lentivirus pathogenicity remain incompletely understood. METHODS: We profile immune responses in a unique model of differential lentiviral pathogenicity where pig-tailed macaques are infected with highly genetically similar variants of SIV that differ in virulence. We apply longitudinal single-cell transcriptomics to this cohort, along with single-cell resolution cell-cell communication techniques, to understand the immune mechanisms underlying lentiviral pathogenicity. RESULTS: Compared to a minimally pathogenic lentiviral variant, infection with a highly pathogenic variant results in a more delayed, broad, and sustained activation of inflammatory pathways, including an extensive global interferon signature. Conversely, individual cells infected with highly pathogenic Lentivirus upregulated fewer interferon-stimulated genes at a lower magnitude, indicating that highly pathogenic Lentivirus has evolved to partially escape from interferon responses. Further, we identify CXCL10 and CXCL16 as important molecular drivers of inflammatory pathways specifically in response to highly pathogenic Lentivirus infection. Immune responses to highly pathogenic Lentivirus infection are characterized by amplifying regulatory circuits of pro-inflammatory cytokines with dense longitudinal connectivity. CONCLUSIONS: Our work presents a model of lentiviral pathogenicity where failures in early viral control mechanisms lead to delayed, sustained, and amplifying pro-inflammatory circuits, which in turn drives disease progression.

Using viral sequence diversity to estimate time of HIV infection in infants.

Age at HIV acquisition may influence viral pathogenesis in infants, and yet infection timing (i.e. date of infection) is not always known. Adult studies have estimated infection timing using rates of HIV RNA diversification, however, it is unknown whether adult-trained models can provide accurate predictions when used for infants due to possible differences in viral dynamics. While rates of viral diversification have been well defined for adults, there are limited data characterizing these dynamics for infants. Here, we performed Illumina sequencing of gag and pol using longitudinal plasma samples from 22 Kenyan infants with well-characterized infection timing. We used these data to characterize viral diversity changes over time by designing an infant-trained Bayesian hierarchical regression model that predicts time since infection using viral diversity. We show that diversity accumulates with time for most infants (median rate within pol = 0.00079 diversity/month), and diversity accumulates much faster than in adults (compare previously-reported adult rate within pol = 0.00024 diversity/month [1]). We find that the infant rate of viral diversification varies by individual, gene region, and relative timing of infection, but not by set-point viral load or rate of CD4+ T cell decline. We compare the predictive performance of this infant-trained Bayesian hierarchical regression model with simple linear regression models trained using the same infant data, as well as existing adult-trained models [1]. Using an independent dataset from an additional 15 infants with frequent HIV testing to define infection timing, we demonstrate that infant-trained models more accurately estimate time since infection than existing adult-trained models. This work will be useful for timing HIV acquisition for infants with unknown infection timing and for refining our understanding of how viral diversity accumulates in infants, both of which may have broad implications for the future development of infant-specific therapeutic and preventive interventions.

Higher HIV-1 Env gp120-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) Activity Is Associated with Lower Levels of Defective HIV-1 Provirus.

A cure for HIV-1 (HIV) remains unrealized due to a reservoir of latently infected cells that persist during antiretroviral therapy (ART), with reservoir size associated with adverse health outcomes and inversely with time to viral rebound upon ART cessation. Once established during ART, the HIV reservoir decays minimally over time; thus, understanding factors that impact the size of the HIV reservoir near its establishment is key to improving the health of people living with HIV and for the development of novel cure strategies. Yet, to date, few correlates of HIV reservoir size have been identified, particularly in pediatric populations. Here, we employed a cross-subtype intact proviral DNA assay (CS-IPDA) to quantify HIV provirus between one- and two-years post-ART initiation in a cohort of Kenyan children (n = 72), which had a median of 99 intact (range: 0-2469), 1340 defective (range: 172-3.84 × 104), and 1729 total (range: 178-5.11 × 104) HIV proviral copies per one million T cells. Additionally, pre-ART plasma was tested for HIV Env-specific antibody-dependent cellular cytotoxicity (ADCC) activity. We found that pre-ART gp120-specific ADCC activity inversely correlated with defective provirus levels (n = 68, r = -0.285, p = 0.0214) but not the intact reservoir (n = 68, r = -0.0321, p-value = 0.800). Pre-ART gp41-specific ADCC did not significantly correlate with either proviral population (n = 68; intact: r = -0.0512, p-value = 0.686; defective: r = -0.109, p-value = 0.389). This suggests specific host immune factors prior to ART initiation can impact proviruses that persist during ART.

Host cell glycosylation selects for infection with CCR5- versus CXCR4-tropic HIV-1.

HIV-1 infection involves a selection bottleneck that leads to transmission of one or a few HIV variants, which nearly always use CCR5 as the coreceptor (R5 viruses) for viral entry as opposed to CXCR4 (X4 viruses). The host properties that drive this selection are not well understood and may hold keys to factors that govern HIV susceptibility. In this report, we identified SLC35A2, a transporter of UDP-galactose, as a candidate X4-specific restriction factor in CRISPR-knockout screens in primary target CD4+ T cells. SLC35A2 inactivation in CD4+ T cells, which resulted in truncation of glycans due to the absence of galactose, not only increased X4 infection levels, but also consistently decreased infection levels of R5 HIV strains. Single cycle infections demonstrated that the effect is host cell dependent. SLC35A2 is expressed in CD4+ T cells at different tissue sites, with high levels in the genital tract - the site of most HIV infections. These data support a role for a host cell protein that regulates glycan structure on HIV infection, with enhanced R5 infection but reduced X4 infection associated with SLC35A2-mediated glycosylation. Host cell glycosylation may therefore contribute to R5 selection and host susceptibility during HIV transmission.

phippery: a software suite for PhIP-Seq data analysis.

SUMMARY: We present the phippery software suite for analyzing data from phage display methods that use immunoprecipitation and deep sequencing to capture antibody binding to peptides, often referred to as PhIP-Seq. It has three main components that can be used separately or in conjunction: (i) a Nextflow pipeline, phip-flow, to process raw sequencing data into a compact, multidimensional dataset format and allows for end-to-end automation of reproducible workflows. (ii) a Python API, phippery, which provides interfaces for tasks such as count normalization, enrichment calculation, multidimensional scaling, and more, and (iii) a Streamlit application, phip-viz, as an interactive interface for visualizing the data as a heatmap in a flexible manner. AVAILABILITY AND IMPLEMENTATION: All software packages are publicly available under the MIT License. The phip-flow pipeline: https://github.com/matsengrp/phip-flow. The phippery library: https://github.com/matsengrp/phippery. The phip-viz Streamlit application: https://github.com/matsengrp/phip-viz.

Elevated binding and functional antibody responses to SARS-CoV-2 in infants versus mothers.

Infant antibody responses to viral infection can differ from those in adults. However, data on the specificity and function of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in infants, and direct comparisons between infants and adults are limited. Here, we characterize antibody binding and functionality against Wuhan-Hu-1 (B lineage) strain SARS-CoV-2 in convalescent plasma from 36 postpartum women and 14 of their infants infected with SARS-CoV-2 from a vaccine-naïve prospective cohort in Nairobi, Kenya. We find significantly higher antibody titers against SARS-CoV-2 Spike, receptor binding domain and N-terminal domain, and Spike-expressing cell-surface staining levels in infants versus mothers. Plasma antibodies from mothers and infants bind to similar regions of the Spike S2 subunit, including the fusion peptide (FP) and stem helix-heptad repeat 2. However, infants display higher antibody levels and more consistent antibody escape pathways in the FP region compared to mothers. Finally, infants have significantly higher levels of antibody-dependent cellular cytotoxicity (ADCC), though, surprisingly, Spike pseudovirus neutralization titers between infants and mothers are similar. These results suggest infants develop distinct SARS-CoV-2 binding and functional antibody activities and reveal age-related differences in humoral immunity to SARS-CoV-2 infection that could be relevant to protection and COVID-19 disease outcomes.

Passively Acquired Constant Region 5-Specific Antibodies Associated With Improved Survival in Infants Who Acquire Human Immunodeficiency Virus.

Studying vertical human immunodeficiency virus (HIV) transmission enables the impact of passively transferred antibodies on HIV transmission and pathogenesis to be examined. Using phage display of HIV envelope peptides and peptide enzyme-linked immunosorbent assay (ELISA), we found that, in infants who acquired HIV, passive antibody responses to constant region 5 (C5) were associated with improved survival in 2 cohorts. In a combined analysis, C5 peptide ELISA activity was correlated directly with survival and estimated infection time and inversely with set point viral load. These results suggest that preexisting C5-specific antibodies may be correlated with the survival of infants living with HIV, motivating additional research into their protective potential.

Mapping the neutralizing specificity of human anti-HIV serum by deep mutational scanning.

Understanding the specificities of human serum antibodies that broadly neutralize HIV can inform prevention and treatment strategies. Here, we describe a deep mutational scanning system that can measure the effects of combinations of mutations to HIV envelope (Env) on neutralization by antibodies and polyclonal serum. We first show that this system can accurately map how all functionally tolerated mutations to Env affect neutralization by monoclonal antibodies. We then comprehensively map Env mutations that affect neutralization by a set of human polyclonal sera that neutralize diverse strains of HIV and target the site engaging the host receptor CD4. The neutralizing activities of these sera target different epitopes, with most sera having specificities reminiscent of individual characterized monoclonal antibodies, but one serum targeting two epitopes within the CD4-binding site. Mapping the specificity of the neutralizing activity in polyclonal human serum will aid in assessing anti-HIV immune responses to inform prevention strategies.

Several cell-intrinsic effectors drive type I interferon-mediated restriction of HIV-1 in primary CD4+ T cells.

Type I interferon (IFN) upregulates proteins that inhibit HIV within infected cells. Prior studies have identified IFN-stimulated genes (ISGs) that impede lab-adapted HIV in cell lines, yet the ISG(s) that mediate IFN restriction in HIV target cells, primary CD4+ T cells, are unknown. Here, we interrogate ISG restriction of primary HIV in CD4+ T cells by performing CRISPR-knockout screens with a custom library that specifically targets ISGs expressed in CD4+ T cells. Our investigation identifies previously undescribed HIV-restricting ISGs (HM13, IGFBP2, LAP3) and finds that two factors characterized in other HIV infection models (IFI16 and UBE2L6) mediate IFN restriction in T cells. Inactivation of these five ISGs in combination further diminishes IFN's protective effect against diverse HIV strains. This work demonstrates that IFN restriction of HIV is multifaceted, resulting from several effectors functioning collectively, and establishes a primary cell ISG screening model to identify both single and combinations of HIV-restricting ISGs.

Identification of broad, potent antibodies to functionally constrained regions of SARS-CoV-2 spike following a breakthrough infection.

The antiviral benefit of antibodies can be compromised by viral escape especially for rapidly evolving viruses. Therefore, durable, effective antibodies must be both broad and potent to counter newly emerging, diverse strains. Discovery of such antibodies is critically important for SARS-CoV-2 as the global emergence of new variants of concern (VOC) has compromised the efficacy of therapeutic antibodies and vaccines. We describe a collection of broad and potent neutralizing monoclonal antibodies (mAbs) isolated from an individual who experienced a breakthrough infection with the Delta VOC. Four mAbs potently neutralize the Wuhan-Hu-1 vaccine strain, the Delta VOC, and also retain potency against the Omicron VOCs through BA.4/BA.5 in both pseudovirus-based and authentic virus assays. Three mAbs also retain potency to recently circulating VOCs XBB.1.5 and BQ.1.1 and one also potently neutralizes SARS-CoV-1. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs that had been approved for therapeutic applications. The mAbs target distinct epitopes on the spike glycoprotein, three in the receptor-binding domain (RBD) and one in an invariant region downstream of the RBD in subdomain 1 (SD1). The escape pathways we defined at single amino acid resolution with deep mutational scanning show they target conserved, functionally constrained regions of the glycoprotein, suggesting escape could incur a fitness cost. Overall, these mAbs are unique in their breadth across VOCs, their epitope specificity, and include a highly potent mAb targeting a rare epitope outside of the RBD in SD1.

Reconstruction of a polyclonal ADCC antibody repertoire from an HIV-1 non-transmitting mother.

Human natural history and vaccine studies support a protective role of antibody dependent cellular cytotoxicity (ADCC) activity against many infectious diseases. One setting where this has consistently been observed is in HIV-1 vertical transmission, where passively acquired ADCC activity in HIV-exposed infants has correlated with reduced acquisition risk and reduced pathogenesis in HIV+ infants. However, the characteristics of HIV-specific antibodies comprising a maternal plasma ADCC response are not well understood. Here, we reconstructed monoclonal antibodies (mAbs) from memory B cells from late pregnancy in mother MG540, who did not transmit HIV to her infant despite several high-risk factors. Twenty mAbs representing 14 clonal families were reconstructed, which mediated ADCC and recognized multiple HIV Envelope epitopes. In experiments using Fc-defective variants, only combinations of several mAbs accounted for the majority of plasma ADCC of MG540 and her infant. We present these mAbs as evidence of a polyclonal repertoire with potent HIV-directed ADCC activity.

Mapping the neutralizing specificity of human anti-HIV serum by deep mutational scanning.

Understanding the specificities of human serum antibodies that broadly neutralize HIV can inform prevention and treatment strategies. Here we describe a deep mutational scanning system that can measure the effects of combinations of mutations to HIV envelope (Env) on neutralization by antibodies and polyclonal serum. We first show that this system can accurately map how all functionally tolerated mutations to Env affect neutralization by monoclonal antibodies. We then comprehensively map Env mutations that affect neutralization by a set of human polyclonal sera known to target the CD4-binding site that neutralize diverse strains of HIV. The neutralizing activities of these sera target different epitopes, with most sera having specificities reminiscent of individual characterized monoclonal antibodies, but one sera targeting two epitopes within the CD4 binding site. Mapping the specificity of the neutralizing activity in polyclonal human serum will aid in assessing anti-HIV immune responses to inform prevention strategies.

Several cell-intrinsic effectors drive type I interferon-mediated restriction of HIV-1 in primary CD4 + T cells.

Type I interferon (IFN) upregulates proteins that inhibit HIV within infected cells. Prior studies have identified IFN-stimulated genes (ISGs) that impede lab-adapted HIV in cell lines, yet the ISG(s) that mediate IFN restriction in HIV target cells, primary CD4 + T cells, are unknown. Here, we interrogate ISG restriction of primary HIV in CD4 + T cells. We performed CRISPR-knockout screens using a custom library that specifically targets ISGs expressed in CD4 + T cells and validated top hits. Our investigation identified new HIV-restricting ISGs (HM13, IGFBP2, LAP3) and found that two previously studied factors (IFI16, UBE2L6) are IFN effectors in T cells. Inactivation of these five ISGs in combination further diminished IFN’s protective effect against six diverse HIV strains. This work demonstrates that IFN restriction of HIV is multifaceted, resulting from several effectors functioning collectively, and establishes a primary cell ISG screening model to identify both single and combinations of HIV-restricting ISGs.

Elevated binding and functional antibody responses to SARS-CoV-2 in infants versus mothers.

Infant antibody responses to viral infection can differ from those in adults. However, data on the specificity and function of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in infants, and direct comparisons between infants and adults are limited. We characterized antibody binding and functionality in convalescent plasma from postpartum women and their infants infected with SARS-CoV-2 from a vaccine-naïve prospective cohort in Nairobi, Kenya. Antibody titers against SARS-CoV-2 Spike, receptor binding domain and N-terminal domain, and Spike-expressing cell-surface staining levels were significantly higher in infants than in mothers. Plasma antibodies from mothers and infants bound to similar regions of the Spike S2 subunit, including the fusion peptide (FP) and stem helix-heptad repeat 2. However, infants displayed higher antibody levels and more consistent antibody escape pathways in the FP region compared to mothers. Finally, infants had significantly higher levels of antibody-dependent cellular cytotoxicity (ADCC), though, surprisingly, neutralization titers between infants and mothers were similar. These results suggest infants develop distinct SARS-CoV-2 binding and functional antibody repertoires and reveal age-related differences in humoral immunity to SARS-CoV-2 infection that could be relevant to protection and COVID-19 disease outcomes.

Comparison of nucleocapsid and spike antibody ELISAs for determining SARS-CoV-2 seropositivity in Kenyan women and infants.

A multitude of enzyme-linked immunosorbent assays (ELISAs) has been developed to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies since the coronavirus disease 2019 pandemic started in late 2019. Assessing the reliability of these assays in diverse global populations is critical. This study compares the use of the commercially available Platelia Total Ab Assay (Bio-Rad) nucleocapsid ELISA to the widely used Mount Sinai spike IgG ELISA in a Kenyan population seroprevalence study. Using longitudinal plasma specimens collected from a mother-infant cohort living in Nairobi, Kenya between May 2019 and December 2020, this study demonstrates that the two assays have a high qualitative agreement (92.7%) and strong correlation of antibody levels (R2 = 0.973) in repeated measures. Within this cohort, seroprevalence detected by either ELISA closely resembled previously published seroprevalence estimates for Kenya during the sampling period and no significant difference in the incidence of SARS-CoV-2 antibody detection by either assay was observed. Assay comparability was not affected by HIV exposure status. These data support the use of the Platelia SARS-CoV-2 Total Ab ELISA as a suitable high-throughput method for seroprevalence studies in Kenya.

Identification of broad, potent antibodies to functionally constrained regions of SARS-CoV-2 spike following a breakthrough infection.

The antiviral benefit of antibodies can be compromised by viral escape especially for rapidly evolving viruses. Therefore, durable, effective antibodies must be both broad and potent to counter newly emerging, diverse strains. Discovery of such antibodies is critically important for SARS-CoV-2 as the global emergence of new variants of concern (VOC) has compromised the efficacy of therapeutic antibodies and vaccines. We describe a collection of broad and potent neutralizing monoclonal antibodies (mAbs) isolated from an individual who experienced a breakthrough infection with the Delta VOC. Four mAbs potently neutralize the Wuhan-Hu-1 vaccine strain, the Delta VOC, and also retain potency against the Omicron VOCs through BA.4/BA.5 in both pseudovirus-based and authentic virus assays. Three mAbs also retain potency to recently circulating VOCs XBB.1.5 and BQ.1.1 and one also potently neutralizes SARS-CoV-1. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs that had been approved for therapeutic applications. The mAbs target distinct epitopes on the spike glycoprotein, three in the receptor binding domain (RBD) and one in an invariant region downstream of the RBD in subdomain 1 (SD1). The escape pathways we defined at single amino acid resolution with deep mutational scanning show they target conserved, functionally constrained regions of the glycoprotein, suggesting escape could incur a fitness cost. Overall, these mAbs are novel in their breadth across VOCs, their epitope specificity, and include a highly potent mAb targeting a rare epitope outside of the RBD in SD1.

Protocol for high-throughput reservoir quantification across global HIV subtypes using a cross-subtype intact proviral DNA assay.

The cross-subtype intact proviral DNA assay (CS-IPDA) is a high-throughput method to quantify HIV reservoir size in populations infected with any of the dominant global HIV-1 subtypes. Our protocol includes genomic DNA isolation optimized to minimize DNA shearing, a reference droplet digital PCR (ddPCR) assay to quantify T cells and assess DNA shearing, and a multiplex ddPCR targeting three distinct regions across the HIV genome to quantify intact proviruses as an estimate of replication-competent proviruses in the reservoir. For complete details on the use and execution of this protocol, please refer to Cassidy et al. (2022).

Distinct Antibody Responses to Endemic Coronaviruses Pre- and Post-SARS-CoV-2 Infection in Kenyan Infants and Mothers.

Pre-existing antibodies that bind endemic human coronaviruses (eHCoVs) can cross-react with SARS-CoV-2, which is the betacoronavirus that causes COVID-19, but whether these responses influence SARS-CoV-2 infection is still under investigation and is particularly understudied in infants. In this study, we measured eHCoV and SARS-CoV-1 IgG antibody titers before and after SARS-CoV-2 seroconversion in a cohort of Kenyan women and their infants. Pre-existing eHCoV antibody binding titers were not consistently associated with SARS-CoV-2 seroconversion in infants or mothers; however, we observed a very modest association between pre-existing HCoV-229E antibody levels and a lack of SARS-CoV-2 seroconversion in the infants. After seroconversion to SARS-CoV-2, antibody binding titers to the endemic betacoronaviruses HCoV-OC43 and HCoV-HKU1, and the highly pathogenic betacoronavirus SARS-CoV-1, but not the endemic alphacoronaviruses HCoV-229E and HCoV-NL63, increased in the mothers. However, eHCoV antibody levels did not increase following SARS-CoV-2 seroconversion in the infants, suggesting the increase seen in the mothers was not simply due to cross-reactivity to naively generated SARS-CoV-2 antibodies. In contrast, the levels of antibodies that could bind SARS-CoV-1 increased after SARS-CoV-2 seroconversion in both the mothers and infants, both of whom were unlikely to have had a prior SARS-CoV-1 infection, supporting prior findings that SARS-CoV-2 responses cross-react with SARS-CoV-1. In summary, we found evidence of increased eHCoV antibody levels following SARS-CoV-2 seroconversion in the mothers but not the infants, suggesting eHCoV responses can be boosted by SARS-CoV-2 infection when a prior memory response has been established, and that pre-existing cross-reactive antibodies are not strongly associated with SARS-CoV-2 infection risk in mothers or infants.

Distinct antibody responses to endemic coronaviruses pre- and post-SARS-CoV-2 infection in Kenyan infants and mothers.

Pre-existing antibodies that bind endemic human coronaviruses (eHCoVs) can cross-react with SARS-CoV-2, the betacoronavirus that causes COVID-19, but whether these responses influence SARS-CoV-2 infection is still under investigation and is particularly understudied in infants. In this study, we measured eHCoV and SARS-CoV-1 IgG antibody titers before and after SARS-CoV-2 seroconversion in a cohort of Kenyan women and their infants. Pre-existing eHCoV antibody binding titers were not consistently associated with SARS-CoV-2 seroconversion in infants or mothers, though we observed a very modest association between pre-existing HCoV-229E antibody levels and lack of SARS-CoV-2 seroconversion in infants. After seroconversion to SARS-CoV-2, antibody binding titers to endemic betacoronaviruses HCoV-OC43 and HCoV-HKU1, and the highly pathogenic betacoronavirus SARS-CoV-1, but not endemic alphacoronaviruses HCoV-229E and HCoV-NL63, increased in mothers. However, eHCoV antibody levels did not increase following SARS-CoV-2 seroconversion in infants, suggesting the increase seen in mothers was not simply due to cross-reactivity to naively generated SARS-CoV-2 antibodies. In contrast, the levels of antibodies that could bind SARS-CoV-1 increased after SARS-CoV-2 seroconversion in both mothers and infants, both of whom are unlikely to have had a prior SARS-CoV-1 infection, supporting prior findings that SARS-CoV-2 responses cross-react with SARS-CoV-1. In summary, we find evidence for increased eHCoV antibody levels following SARS-CoV-2 seroconversion in mothers but not infants, suggesting eHCoV responses can be boosted by SARS-CoV-2 infection when a prior memory response has been established, and that pre-existing cross-reactive antibodies are not strongly associated with SARS-CoV-2 infection risk in mothers or infants.