H5 influenza is considered a potential pandemic threat. Recently, H5 viruses belonging to clade 2.3.4.4b have caused large outbreaks in avian and multiple nonhuman mammalian species. Previous studies have identified molecular phenotypes of the viral hemagglutinin (HA) protein that contribute to pandemic potential in humans, including cell entry, receptor preference, HA stability, and reduced neutralization by polyclonal sera. However, prior experimental work has only measured how these phenotypes are affected by a handful of the >10,000 different possible amino-acid mutations to HA. Here, we use pseudovirus deep mutational scanning to measure how all mutations to a 2.3.4.4b H5 HA affect each phenotype. We identify mutations that allow HA to better bind α2-6-linked sialic acids and show that some viruses already carry mutations that stabilize HA. We also measure how all HA mutations affect neutralization by sera from mice and ferrets vaccinated against or infected with 2.3.4.4b H5 viruses. These antigenic maps enable rapid assessment of when new viral strains have acquired mutations that may create mismatches with candidate vaccine virus, and we show that a mutation present in some recent H5 HAs causes a large antigenic change. Overall, the systematic nature of deep mutational scanning combined with the safety of pseudoviruses enables comprehensive measurements of the phenotypic effects of mutations that can inform real-time interpretation of viral variation observed during surveillance of H5 influenza.
Lassa virus is estimated to cause thousands of human deaths per year, primarily due to spillovers from its natural host, Mastomys rodents. Efforts to create vaccines and antibody therapeutics must account for the evolutionary variability of the Lassa virus's glycoprotein complex (GPC), which mediates viral entry into cells and is the target of neutralizing antibodies. To map the evolutionary space accessible to GPC, we used pseudovirus deep mutational scanning to measure how nearly all GPC amino-acid mutations affected cell entry and antibody neutralization. Our experiments defined functional constraints throughout GPC. We quantified how GPC mutations affected neutralization with a panel of monoclonal antibodies. All antibodies tested were escaped by mutations that existed among natural Lassa virus lineages. Overall, our work describes a biosafety-level-2 method to elucidate the mutational space accessible to GPC and shows how prospective characterization of antigenic variation could aid the design of therapeutics and vaccines.
Continuously evolving influenza viruses cause seasonal epidemics and pose global pandemic threats. Although viral neuraminidase (NA) is an effective drug and vaccine target, our understanding of the NA antigenic landscape still remains incomplete. Here, we describe NA-specific human antibodies that target the underside of the NA globular head domain, inhibit viral propagation of a wide range of human H3N2, swine-origin variant H3N2, and H2N2 viruses, and confer both pre- and post-exposure protection against lethal H3N2 infection in mice. Cryo-EM structures of two such antibodies in complex with NA reveal non-overlapping epitopes covering the underside of the NA head. These sites are highly conserved among N2 NAs yet inaccessible unless the NA head tilts or dissociates. Our findings help guide the development of effective countermeasures against ever-changing influenza viruses by identifying hidden conserved sites of vulnerability on the NA underside.
Humoral immunity to SARS-CoV-2 can be supplemented with polyclonal sera from convalescent donors or an engineered monoclonal antibody (mAb) product. While pentameric IgM antibodies are responsible for much of convalescent sera's neutralizing capacity, all available mAbs are based on the monomeric IgG antibody subtype. We now show that IgM mAbs derived from immune memory B cell receptors are potent neutralizers of SARS-CoV-2. IgM mAbs outperformed clonally identical IgG antibodies across a range of affinities and SARS-CoV-2 receptor-binding domain epitopes. Strikingly, efficacy against SARS-CoV-2 viral variants was retained for IgM but not for clonally identical IgG. To investigate the biological role for IgM memory in SARS-CoV-2, we also generated IgM mAbs from antigen-experienced IgM+ memory B cells in convalescent donors, identifying a potent neutralizing antibody. Our results highlight the therapeutic potential of IgM mAbs and inform our understanding of the role for IgM memory against a rapidly mutating pathogen.
The isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. We determined the structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and showed that its domain 0 recognizes sialosides. We identified that the CCoV-HuPn-2018 spike binds canine, feline, and porcine aminopeptidase N (APN) orthologs, which serve as entry receptors, and determined the structure of the receptor-binding B domain in complex with canine APN. The introduction of an oligosaccharide at position N739 of human APN renders cells susceptible to CCoV-HuPn-2018 spike-mediated entry, suggesting that single-nucleotide polymorphisms might account for viral detection in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 spike-mediated entry, underscoring the cross-neutralizing activity among ɑ-coronaviruses. These data pave the way for vaccine and therapeutic development targeting this zoonotic pathogen representing the eighth human-infecting coronavirus.
Understanding the presence and durability of antibodies against SARS-CoV-2 in the airways is required to provide insights into the ability of individuals to neutralize the virus locally and prevent viral spread. Here, we longitudinally assessed both systemic and airway immune responses upon SARS-CoV-2 infection in a clinically well-characterized cohort of 147 infected individuals representing the full spectrum of COVID-19 severity, from asymptomatic infection to fatal disease. In addition, we evaluated how SARS-CoV-2 vaccination influenced the antibody responses in a subset of these individuals during convalescence as compared with naive individuals. Not only systemic but also airway antibody responses correlated with the degree of COVID-19 disease severity. However, although systemic IgG levels were durable for up to 8 months, airway IgG and IgA declined significantly within 3 months. After vaccination, there was an increase in both systemic and airway antibodies, in particular IgG, often exceeding the levels found during acute disease. In contrast, naive individuals showed low airway antibodies after vaccination. In the former COVID-19 patients, airway antibody levels were significantly elevated after the boost vaccination, highlighting the importance of prime and boost vaccinations for previously infected individuals to obtain optimal mucosal protection.
In response to the SARS-CoV-2 pandemic many vaccines have been developed and evaluated in human clinical trials. The humoral immune response magnitude, composition and efficacy of neutralizing SARS-CoV-2 are essential endpoints for these trials. Robust assays that are reproducibly precise, linear, and specific for SARS-CoV-2 antigens would be beneficial for the vaccine pipeline. In this work we describe the methodologies and clinical qualification of three SARS-CoV-2 endpoint assays. We developed and qualified Endpoint titer ELISAs for total IgG, IgG1, IgG3, IgG4, IgM and IgA to evaluate the magnitude of specific responses to the trimeric spike (S) antigen and total IgG specific to the spike receptor binding domain (RBD) of SARS-CoV-2. We also qualified a pseudovirus neutralization assay which evaluates functional antibody titers capable of inhibiting the entry and replication of a lentivirus containing the Spike antigen of SARS-CoV-2. To complete the suite of assays we qualified a plaque reduction neutralization test (PRNT) methodology using the 2019-nCoV/USA-WA1/2020 isolate of SARS-CoV-2 to assess neutralizing titers of antibodies in plasma from normal healthy donors and convalescent COVID-19 individuals.
Most individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop neutralizing antibodies that target the viral spike protein. In this study, we quantified how levels of these antibodies change in the months after SARS-CoV-2 infection by examining longitudinal samples collected approximately 30-152 days after symptom onset from a prospective cohort of 32 recovered individuals with asymptomatic, mild, or moderate-severe disease. Neutralizing antibody titers declined an average of about 4-fold from 1 to 4 months after symptom onset. This decline in neutralizing antibody titers was accompanied by a decline in total antibodies capable of binding the viral spike protein or its receptor-binding domain. Importantly, our data are consistent with the expected early immune response to viral infection, where an initial peak in antibody levels is followed by a decline to a lower plateau. Additional studies of long-lived B cells and antibody titers over longer time frames are necessary to determine the durability of immunity to SARS-CoV-2.
2020
Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19 VirusesAntibodiesCoV
Rodda LB, Netland J, Shehata L, Pruner KB, Morawski PA, Thouvenel CD, Takehara KK, Eggenberger J, Hemann EA, Waterman HR, Fahning ML, Chen Y, Hale M, Rathe J, Stokes C, Wrenn S, Fiala B, Carter L, Hamerman JA, King NP, Gale M, Campbell DJ, Rawlings DJ, Pepper M. (2021) Cell. 184(1)
:169-183.e17 | doi:10.1016/j.cell.2020.11.029Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is causing a global pandemic, and cases continue to rise. Most infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that could contribute to immunity. We performed a longitudinal assessment of individuals recovered from mild COVID-19 to determine whether they develop and sustain multifaceted SARS-CoV-2-specific immunological memory. Recovered individuals developed SARS-CoV-2-specific immunoglobulin (IgG) antibodies, neutralizing plasma, and memory B and memory T cells that persisted for at least 3 months. Our data further reveal that SARS-CoV-2-specific IgG memory B cells increased over time. Additionally, SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral function: memory T cells secreted cytokines and expanded upon antigen re-encounter, whereas memory B cells expressed receptors capable of neutralizing virus when expressed as monoclonal antibodies. Therefore, mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks of antiviral immunity.
Children are strikingly underrepresented in COVID-19 case counts. In the United States, children represent 22% of the population but only 1.7% of confirmed SARS-CoV-2 cases as of April 2, 2020. One possibility is that symptom-based viral testing is less likely to identify infected children, since they often experience milder disease than adults. Here, to better assess the frequency of pediatric SARS-CoV-2 infection, we serologically screen 1,775 residual samples from Seattle Children's Hospital collected from 1,076 children seeking medical care during March and April of 2020. Only one child was seropositive in March, but seven were seropositive in April for a period seroprevalence of ≈1%. Most seropositive children (6/8) were not suspected of having had COVID-19. The sera of seropositive children have neutralizing activity, including one that neutralized at a dilution > 1:18,000. Therefore, an increasing number of children seeking medical care were infected by SARS-CoV-2 during the early Seattle outbreak despite few positive viral tests.
The receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein mediates viral attachment to ACE2 receptor and is a major determinant of host range and a dominant target of neutralizing antibodies. Here, we experimentally measure how all amino acid mutations to the RBD affect expression of folded protein and its affinity for ACE2. Most mutations are deleterious for RBD expression and ACE2 binding, and we identify constrained regions on the RBD's surface that may be desirable targets for vaccines and antibody-based therapeutics. But a substantial number of mutations are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related coronaviruses. However, we find no evidence that these ACE2-affinity-enhancing mutations have been selected in current SARS-CoV-2 pandemic isolates. We present an interactive visualization and open analysis pipeline to facilitate use of our dataset for vaccine design and functional annotation of mutations observed during viral surveillance.
SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.