Immunology

Tarke et al. shows a broad T cell repertoire suggesting that viral escape of T-cell
immunity is unlikely. CD4 immunodominant regions correlate with HLA binding and not
with high common cold coronavirus homology.

In an exciting, state-of-the-art study currently available on the medRxiv* preprint server, US researchers created a multi-coronavirus protein microarray containing full-length proteins, peptide libraries, and overlapping protein fragments from the severe acute respiratory syndrome coronavirus 2...

The rapid spread of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory
syndrome–coronavirus 2 (SARS-CoV-2), has resulted in an unprecedented public health
crisis worldwide. Recent studies indicate that a hyperinflammatory syndrome induced by SARS-CoV-2 contributes to disease...

Abstract Tremendous progress has been made in understanding the role of T cell immunity in acute and convalescent COVID-19 infection. Here we shed light on the “known unknowns” of pre-existing and acquired T cell responses in relation to acute and convalescent SARS-CoV-2 infection. INTRODUCTION The broad clinical spectrum of COVID-19 indicates widespread intraindividual differences in the host immune defense against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The underlying cause of disease heterogeneity is probably multifactorial. However, a rapid early host response is likely critical to generate control of SARS-CoV-2 viremia before spread to the lower respiratory tract and onset of damaging hyperinflammation. In this regard, the literature is full of examples where functional T cell responses can provide early control of acute viral infections, including SARS-CoV and MERS-CoV (1, 2). Although multiple studies have indicated that T cells play a role in the early immune response to SARS-CoV-2 and can generate a functional memory pool, there are still multiple unanswered questions in the field (Box 1). Here, we summarize and speculate on a specific set of questions related to T cell immunity against respiratory viral infections, with a focus on COVID-19 severity, immunity, long-term consequences, and vaccination (Fig. 1). Box 1 Open questions about T cell immunity to SARS-CoV-2. T cells in acute SARS-CoV-2 infection • What do acute SARS-CoV-2-specific T cell responses in the blood tell us about contemporaneous T cell responses in the lung? • Which host and viral factors regulate the strength and efficacy of the early antiviral T cell response? Longevity and memory T cell formation • Do CD4+ T cell responses to the virus predominate over CD8+ responses in the lung as well as the blood? • Do poor CD4+ TFH responses to the virus correlate with reduced longevity of antibody responses? • Is severe COVID 19 linked to an impaired development of SARS-CoV-2-specific memory T cells? Cross-reactive T cells • Do pre-existing memory T cells to seasonal CoV epitopes help or hinder the host response to SARS-CoV-2? SARS-CoV-2-specific TRM • Will establishment of SARS-CoV-2-specific CD4+ and CD8+ TRM in the upper respiratory tract provide durable protection from lower pulmonary disease upon reinfection? Long COVID • Are virus-induced or bystander T cells major inducers of persistent symptoms of inflammation associated with long COVID? T cells in vaccines • What type of T cell response elicited by vaccines will be the best predictor of protection from disease following exposure to the virus? T CELLS IN ACUTE SARS-COV-2 INFECTION T cells are critical to generate early control and clearance of many viral infections of the respiratory system (3). Recent studies in transgenic mouse models provided evidence that T cells are also important for viral clearance and disease resolution after SARS-CoV-2 infection (4). As such, it is not surprising that T cell activation has emerged as a hallmark of acute COVID-19; probably as a consequence of an early SARS-CoV-2-specific cellular immune response (5–9). Although early T cell responses may play a critical role in dampening disease severity, there are also reports describing a dysregulated and unchecked T cell activation pattern in severe cases (10–12). Increased T cell activation in severe cases likely reflects increased antigen levels in the respiratory system, but whether the early T cell response reaches a state of exhaustion in subjects with severe hyperinflammation remains to be determined. Furthermore, given that COVID-19 is a disease of the respiratory tract it will be important to define if early detection of T cell activation in blood correlates with tissue-specific events. For instance, will delayed detection of SARS-CoV-2-specific T cells in blood reflect the later onset of cellular immunity in the respiratory tract or are these two compartments independent of each other in relation to disease severity? If elicitation of an early T cell response would be beneficial to dampen COVID-19 severity, what might be the underlying causes and correlates of an early versus late onset of SARS-CoV-2-specific T cell activity? Old age and male sex are both associated with increased risk of COVID-19 complications. Interestingly, females seem to mount a somewhat stronger T cell activation following SARS-CoV-2 infection (13) and disruption of T and B cell coordination has been implicated in elderly patients with severe COVID-19 (14). On the other end of the age spectrum, decreased frequencies of IFN-γ+CD4+ and CD25+CD4+ T cells have been described in hospitalized pediatric patients, who have shorter lengths of stay compared with their adult counterparts (15). In conjunction with age and sex, host and viral factors probably also play a role in the early immune defense and coordination of the early SARS-CoV-2-specific T cell response. For instance, SARS-CoV-2 has mechanisms to antagonize proinflammatory signals, particularly type I IFN (IFN-I) signaling (16, 17). IFN-I proteins are key inflammatory mediators to initiate antiviral defense, from which viral evasion might lead to a delayed clearance of SARS-CoV-2 (4). This is supported by the observation that inborn errors of immunity and autoantibodies that diminish IFN-I activity are more commonly detected in patients with severe COVID-19 (18, 19). Concordantly, the early expansion and differentiation of antiviral T cells are dependent on the direct action of IFN-I. Given that activated T cells from older individuals exhibit reduced responses to IFN-I, it is tempting to speculate that higher risk elderly persons experience delayed activation of SARS-CoV-2-specific T cells that may lead to reduced clearance of the virus and exacerbated COVID-19 severity. Collectively, more data are needed from mechanistic studies in animal models as well as large cohort studies on males and females in different age groups to identify beneficial and detrimental viral and host factors that have an impact on the early T cell response against SARS-CoV-2. LONGEVITY AND MEMORY T CELL FORMATION Generation of memory T cells can provide lifelong protection against pathogens (20). Previous studies have demonstrated that SARS-CoV- and MERS-CoV-specific T cells can be detected many years after infection (21–23). Likewise, SARS-CoV-2-specific CD4+ and CD8+ T cells are distinguished in a vast majority of convalescent donors (7, 9, 21, 24–27). Studies using peripheral blood have reported stronger SARS-CoV-2-specific CD4+ than CD8+ T cell responses in most subjects. However, it is well established that CD4+ T cells experience a higher propensity to recirculate between tissues and blood than CD8+ T cells. As such, whether SARS-CoV-2-specific CD4+ T cell responses also predominate in tissues, and particularly at barrier sites close to the epithelium, needs to be confirmed through studies on the upper and lower respiratory tract. Similar to the CD4+ T cell polarized response to many other viral infections, SARS-CoV-2-specific CD4+ T cells mainly possess a Th1 or circulating T follicular helper (TFH) cell phenotype (7–9, 14, 28). Circulating TFH differentiation seems to be impaired in certain patients with severe COVID-19 (11, 29) and recent analysis of postmortem lymph nodes and spleen samples showed an absence of germinal centers along with a defect in Bcl6+ TFH differentiation in deceased COVID-19 patients (30). Whether these consequences are due to sampling from postmortem patients remains unknown, but further studies are needed to clarify whether TFH cell formation is impaired by SARS-CoV-2 and could have an impact on declining antibody responses in specific convalescent donors. Furthermore, more mechanistic studies are needed to understand if memory T cells can generate protective immunity to lethal challenge with SARS-CoV-2, as previously demonstrated in SARS-CoV and MERS-CoV models (1, 2), in the presence or absence of high titers of neutralizing antibodies. Likewise, longitudinal human studies will also inform us of whether functional memory T cell responses are present many years after SARS-CoV-2 infection and correlate with protection from reinfection. CROSS-REACTIVE T CELLS Several studies have demonstrated the presence of CD4+ and to a lesser extent CD8+ T cells recognizing SARS-CoV-2 peptides in a significant proportion of unexposed individuals (7, 21, 24, 26, 31). Mapping of SARS-CoV-2 epitopes in unexposed blood donors revealed pre-existing T cell immunity, potentially induced by seasonal human coronaviruses (HCoVs) causing common colds (27, 32). This is supported by a relatively high amino acid similarity between recognized SARS-CoV-2 epitopes and seasonal HCoVs such as HCoV-OC43, -HKU1, -229E and -NL63. The presence of cross-reactive cellular immune responses in the population generates an obstacle to the use of T cell-based assays to track SARS-CoV-2 infection rates in blood donors. Given that antibodies do not result in the same degree of cross-reactivity as T cells and are consequently easier to use in clinical diagnostic settings, serology will likely be a better readout for tracing the infection rate in the society. Nevertheless, more thorough studies are needed to better understand the full spectrum of cross-reactive versus newly-induced SARS-CoV-2-specific CD4+ and CD8+ T cell responses. A key question in the field is whether pre-existing T cell responses influence the severity of COVID-19. Pre-existing SARS-CoV-2-specific T cells are unlikely to provide sterilizing or herd immunity but may allow the host to bypass immune evasion mechanisms, for instance evasion from IFN-I, and generate early pressure on the virus. This concept is supported by studies in mice showing that airway memory CD4+ T cells recognizing a conserved SARS-CoV epitope provided protection from related CoVs (1). Similar scenarios in which pre-existing T cells may provide earlier viral clearance and thus less severe symptoms have been proposed elsewhere (33). Here, the level of conservation between antigens may have a substantial impact on whether pre-existing T cells are beneficial or detrimental for the host. On the other hand, the concept of “original antigenic sin”, in which earlier induced antibody or T cell responses influence the response against future viral infections, needs further evaluation (34). If pre-existing T cells are less effective in clearing viral infection upon activation but contribute to systemic and permanent increase in inflammatory signals, it might lead to increased hyperinflammation and COVID-19 severity. In a first analysis, comparing T cell responses against SARS-CoV-2 and HCoV sequences did not find any evidence of “original antigenic sin” (32). Again, the level of conservation of targeted epitopes is likely to impact the outcome, and further evaluation of this concept is needed. Collectively, further animal studies and human studies done before and after SARS-CoV-2 infection are needed to define the biological relevance of pre-existing T cell responses and their role as friends or foes in host defense against SARS-CoV-2. SARS-COV-2-SPECIFIC TRM Resident memory T cells (TRM) are a distinct memory T cell lineage. These cells reside within tissues, do not recirculate to peripheral blood, and have been defined as local sentinels mediating rapid protection from reinfection (35). In fact, a vast majority of T cells in nonlymphoid tissues, such as the respiratory tract, are considered to be TRM (36). In terms of respiratory infections, there is a growing body of literature demonstrating that TRM can provide protection against severe pulmonary disease (37, 38). Likewise, airway CD4+ T cells can generate cross-reactive immunity between human and bat coronaviruses (1), emphasizing that cross-reactive T cells in the respiratory tract can provide protection from lethal challenge with pathogenic coronaviruses. Whether cross-reactive TRM, induced by seasonal coronaviruses, can block transmission of SARS-CoV-2 from the upper respiratory tract to the lung and thereby attenuate severe COVID-19 remains unanswered. This scenario, where TRM block the spread of viral disease from upper to lower respiratory tract, has been demonstrated in influenza A infection (37) and might account for partial immunity of secondary infection with heterologous strains (39, 40). Furthermore, whether SARS-CoV-2-specific TRM are induced after COVID-19 and whether these cells will provide protection in the long term also remains unknown (41). Although certain studies in mice have suggested that TRM in the lung are short-lived (42), there is evidence that their counterparts in the upper respiratory tract persist with minimal decay (37) and for more than a year in human lung (43). Altogether, there is currently no evidence supporting the provision of “sterilizing immunity” by TRM, but data presented above suggest that TRM could facilitate rapid control of upper respiratory tract SARS-CoV-2 infection, replication, and spread. In this regard, further work in animal models may provide evidence for whether local immunity mediated by TRM can achieve this type of immunity. LONG COVID A substantial number of COVID-19 patients experience heterogeneous symptoms that persist over a month and onward (44–46). This heterogeneous phenomenon is being referred to as “long COVID” and affects around 10% of all COVID-19 patients (44, 45). Many symptoms can be attributed to persistent tissue damage in severe COVID-19. Nevertheless, the fact that many individuals with milder COVID-19 symptoms also experience chronic lingering symptoms, involving the cardiovascular, nervous, and respiratory systems, indicates that persistent immune activation and/or inflammation may play a role in long COVID. Multiple mechanisms are probably involved in this condition and whether T cells play any role in long COVID is unknown. The higher incidence of long COVID in females than males, similar to autoimmune diseases (47), raises the question of whether T cells orchestrate long COVID through similar mechanisms as in autoimmune or inflammatory conditions (48, 49). One hypothetical underlying mechanism behind autoimmune-related conditions after COVID-19 could be molecular mimicry, given that HCoV-specific T cells can cross-react to myelin in multiple sclerosis patients (50). Whether SARS-CoV-2-specific T cells have the ability to react against self-antigens remains to be determined. In line with a possible effect of HLA type on COVID-19 susceptibility/severity (51, 52), we believe that larger genetic studies are needed to clarify if HLA or other immune-related genes are associated with an increased risk of developing long COVID. T CELLS IN VACCINES Based on the uncertainty of whether cross-reactive T cells or antibodies will provide protective or long-lasting immunity to COVID-19, it will become absolutely critical to administrate a safe and effective vaccine to the population to reach broad immunity and break the negative spiral of new infections. Ongoing vaccine efforts mainly target B cells to promote the induction of neutralizing antibodies (nAbs) against SARS-CoV-2 (53, 54). Although the induction of anti-spike nAbs is the key component for an effective SARS-CoV-2 vaccine, it is well-known that T cells, and in particular TFH cells, are critical to generate antibody-producing plasma cells and long-lived memory B cells. In COVID-19 patients, high nAb titers correlated with strong CD4+ T cell responses, and the lack of functional TFH cells reacting against SARS-CoV-2 was shown to be detrimental (11, 29, 30). Preliminary results from the two major mRNA vaccine trials in humans have demonstrated potent Th1 responses (55, 56). However, previous studies have reported strong TFH responses against certain mRNA vaccines (57), and future trials should therefore include other activation induced markers, such as CD40L and/or CD200, in addition to IFN-γ ELISPOT assays to understand if potent B-helper mechanisms are induced by the current vaccine regimens. Other outstanding questions are whether vaccine-induced TFH responses will be equally induced in all age groups and how long these responses will persist in blood and vaccination site-draining lymph nodes. A final issue to consider is whether high quantities of vaccine-induced CD8+ T cells at local sites need to be elicited by future vaccine candidates. If the initial group of vaccines in clinical trials that are primarily focused on generating an effective nAb response provide recipients with long-standing protection, it may not be necessary to invest in such efforts. However, if problems emerge in the vaccinated population with breakthrough infections, waning antibody levels after vaccination, and/or the emergence of new viral strains, it would be wise to reconsider vaccine approaches specifically designed to induce functional CD8+ TRM responses in the upper respiratory tract. SUMMARY Collective efforts have greatly enhanced our scientific understanding of T cell responses against SARS-CoV-2 but many unknowns remain to be resolved. Although it is clear that T cells play a central role in generating early control and clearance of many viral infections, their role in SARS-CoV-2 infection is only starting to be revealed. Specific T cells may even have a detrimental impact on the clinical outcome and contribute to long COVID symptoms. 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A provocative study suggests that certain colds may leave antibodies against the new coronavirus, perhaps explaining why children are more protected than adults. It’s been a big puzzle of the pandemic: Why are children so much less likely than adults to become infected with the new coronavirus and, if infected, less likely to become ill? A possible reason may be that many children already have antibodies to other coronaviruses, according to researchers at the Francis Crick Institute in London. About one in five of the colds that plague children are caused by viruses in this family. Antibodies to those viruses may also block SARS-CoV-2, the new coronavirus causing the pandemic. In a study published Friday in Science, the group, led by George Kassiotis, who heads the Retroviral Immunology Laboratory at the institute, reports that on average only 5 percent of adults had these antibodies, but 43 percent of children did. 

Summary The factors responsible for the spectrum of coronavirus 19 (COVID‐19) disease severity and the genesis and nature of protective immunity against COVID‐19 remain elusive. Multiple studies ha...

Scientists across the world are closely tracking the spread of mutations in the coronavirus and investigating whether they could render current vaccines less effective. SARS-CoV-2 is no Ferrari among viruses when it comes to mutations. Scientists reckon that its 30,000-base RNA genome acquires around two single-letter mutations a month, a rate around half as fast as influenza and one-quarter the rate of HIV. But allowed to multiply and jump from body to body for more than a year, SARS-CoV-2 has inevitably flourished into a genetically diverse tree branching into countless different variants.  Many variants—defined by a specific assortment of mutations—are relatively unremarkable. But scientists have been keeping a close watch on three rapidly spreading variants—first identified in the UK, South Africa, and Brazil—which harbor an unusual constellation of mutations. They all share a mutation called N501Y that affects the receptor binding domain (RBD) of the spike protein, which the virus uses to clasp onto human cells’ receptors and enter them. That mutation replaces SARS-CoV-2’s 501st amino acid, asparagine, with tyrosine, potentially allowing it to bind more tightly to ACE2 receptors, studies in cells and animal models suggest. By itself, that mutation isn’t unusual, but the variants possess an exceptionally large number of other mutations, some also on the spike protein. Substantive changes to a virus’ behavior, such as heightened transmissibility, are likely the result of multiple mutations rather than individual ones, molecular epidemiologist Emma Hodcroft of the University of Bern tells The Atlantic.  The observation that similar mutations have appeared in three independent variants, and the fact that they are spreading, makes scientists suspect that they may have an evolutionary edge. “They have multiple, eight to ten, mutations in the spike protein all stacked up at once—that suggests that there [is] a lot of evolution and adaption of the protein happening,” Daniel Jones, a molecular pathologist at the Ohio State University, tells The Scientist. “The concern being that since that’s the target of vaccinations and . . . the target for antibody [therapies] like the Regeneron cocktail, for instance, that it might be the beginning of a virus that could evade antibody therapy and/or vaccine coverage.”

SARS-CoV-2 mutations’ effects on transmissibility

It’s often in a virus’ interest to become more transmissible so it can spread and replicate more quickly. Earlier in the pandemic, a spike protein mutation known as D614G—which is widely believed to have made the virus more transmissible—surged to dominance around the world, notes virologist John Moore of Weill Cornell Medical College. Epidemiological data suggest that the B.1.1.7 variant, a descendant of the D614G lineage first identified in the UK that has spread to other parts of the world, also has heightened transmissibility. Eight of the 17 mutations it has recently accumulated are in the spike protein, which could feasibly have an effect on ACE2 binding and virus replication. Hypothetically, if a virus can bind more tightly to the body’s ACE2 receptors, it could be more capable of establishing an infection once it gets into the body and/or of generating more viral particles in the upper respiratory tract, making it easier to transmit to other people, particularly during the presymptomatic stage, explains Theodora Hatziioannou, a virologist at the Rockefeller University in New York. She adds that in her view, it’s hard to definitively ascribe case surges, including the current one in the UK, to single factors such as increased transmissibility, over other driving factors, such as what she sees as ineffective lockdown policies. “I’m not saying that [increased] transmissibility is out of the question. I’m just saying it’s extremely hard to prove.” In South Africa, epidemiologists have estimated that the new variant there, B.1.351 (also known as 501Y.V2), is around 50 percent more contagious compared with dominant lineages, based on its rapid spread, according to The Wall Street Journal. In Brazil, it’s too early to conclude whether a variant now circulating there, called P.1, is inherently more transmissible. First reported on January 12 in the state of Amazonas, it’s been associated with a devastating surge in cases in Manaus, a city where researchers had previously estimated that 75 percent of residents had already been infected with SARS-CoV-2. But it’s still unclear whether properties of the virus itself are contributing to the surge, says virologist Paola Resende of the Oswaldo Cruz Institute in Rio de Janeiro. “In Brazil, we can see a lot of parties, we can see the pubs crowded, and people are on the streets not wearing masks. I think this behavior of the population is the main reason [for] the increase.”

Evading the immune system

Our immune system—and, in particular, antibodies—is a powerful evolutionary force on viruses. Some pathogens such as influenza, and maybe also common cold-causing coronaviruses, mutate their proteins toward new shapes to avoid being targeted by antibodies that would normally prevent them from infecting cells, a process known as antigenic drift. A study recently posted as a preprint to bioRxiv by Hatziioannou and her colleagues suggests that the RBD mutations present in the B.1.351 variant are due to antigenic drift. The team passaged a model virus bearing the dominant SARS-CoV-2 spike protein in the presence of individual neutralizing antibodies extracted from people who had received either the Moderna or Pfizer/BioNTech vaccine. Depending on which antibody they were cultured with, the viruses would gradually adopt a single mutation—either E484K, K417N, and N501Y—which are present in B.1.351. That suggests that “the virus is mutating in these positions to avoid antibodies,” Hatziioannou says. Such antibody-escape mutations don’t necessarily mean that the virus will cause more severe disease or entirely outwit the immune response, she cautions. There are other parts of the immune system to help clear the virus. There’s no evidence so far to suggest that the variants identified in South Africa or Brazil are more lethal. Based on an analysis of several datasets, scientists in the UK suggested last week there’s a “realistic possibility” that B.1.1.7 is deadlier than previous strains, but experts say it’s still too early to draw that conclusion. Another concern is about whether people who have overcome mild infections with older variants could become reinfected with a new one. Nobody that I know has been losing a moment of sleep over the UK variant from a vaccine-efficacy perspective.....

The transmission and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of paramount importance in controlling and combating the coronavirus disease 2019 (COVID-19) pandemic.

Another host factor for SARS-CoV-2 Virus-host interactions determine cellular entry and spreading in tissues. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the earlier SARS-CoV use angiotensin-converting enzyme 2 (ACE2) as a receptor; however, their tissue tropism differs, raising the possibility that additional host factors are involved. The spike protein of SARS-CoV-2 contains a cleavage site for the protease furin that is absent from SARS-CoV (see the Perspective by Kielian). Cantuti-Castelvetri et al. now show that neuropilin-1 (NRP1), which is known to bind furin-cleaved substrates, potentiates SARS-CoV-2 infectivity. NRP1 is abundantly expressed in the respiratory and olfactory epithelium, with highest expression in endothelial and epithelial cells. Daly et al. found that the furin-cleaved S1 fragment of the spike protein binds directly to cell surface NRP1 and blocking this interaction with a small-molecule inhibitor or monoclonal antibodies reduced viral infection in cell culture. Understanding the role of NRP1 in SARS-CoV-2 infection may suggest potential targets for future antiviral therapeutics. Science, this issue p. 856, p. 861; see also p. 765 Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), uses the viral spike (S) protein for host cell attachment and entry. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2. Cleavage of S generates a polybasic Arg-Arg-Ala-Arg carboxyl-terminal sequence on S1, which conforms to a C-end rule (CendR) motif that binds to cell surface neuropilin-1 (NRP1) and NRP2 receptors. We used x-ray crystallography and biochemical approaches to show that the S1 CendR motif directly bound NRP1. Blocking this interaction by RNA interference or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection and may potentially provide a therapeutic target for COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the coronavirus responsible for the current coronavirus disease 2019 (COVID-19) pandemic (1, 2). A marked difference between the spike (S) protein of SARS-CoV-2 and SARS-CoV is the presence, in the former, of a polybasic sequence motif, Arg-Arg-Ala-Arg (RRAR), at the S1/S2 boundary. It provides a cleavage site for a host proprotein convertase, furin (3–5) (fig. S1A). The resulting two proteins, S1 and S2, remain noncovalently associated, with the serine protease TMPRSS2 further priming S2 (6). Furin-mediated processing increases infectivity and affects the tropism of SARS-CoV-2, whereas furin inhibition diminishes SARS-CoV-2 entry, and deletion of the polybasic site in the S protein reduces syncytia formation in cell culture (3–5, 7). The C terminus of the S1 protein generated by furin cleavage has an amino acid sequence (682RRAR685) that conforms to a [R/K]XX[R/K] motif, termed the “C-end rule” (CendR) (fig. S1B) (8). CendR peptides bind to neuropilin-1 (NRP1) and NRP2, transmembrane receptors that regulate pleiotropic biological processes, including axon guidance, angiogenesis, and vascular permeability (8–10). To explore the possibility that the SARS-CoV-2 S1 protein may associate with neuropilins, we generated a green fluorescent protein (GFP)–tagged S1 construct (GFP-S1) (fig. S1C). When expressed in human embryonic kidney 293T (HEK293T) cells engineered to express the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2), GFP-S1 immunoprecipitated endogenous NRP1 and ACE2 (Fig. 1A). We transiently coexpressed NRP1-mCherry and either GFP-S1 or GFP-S1 ΔRRAR (a deletion of the terminal 682RRAR685 residues) in HEK293T cells. NRP1 immunoprecipitated the S1 protein, and deletion of the CendR motif reduced this association (Fig. 1B). Comparable binding was also observed with mCherry-NRP2, a receptor with high homology to NRP1 (fig. S1, D and E). In both cases, residual binding was observed with the ΔRRAR mutant, indicating an additional CendR-independent association between neuropilins and the S1 protein. To probe the functional relevance of this interaction, we generated HeLa wild-type and NRP1 knockout (KO) cell lines stably expressing ACE2, designated as HeLawt+ACE2 and HeLaNRP1KO+ACE2, respectively (the level of ACE2 expression was comparable between these lines) (fig. S1F). Using a clinical isolate SARS-CoV-2 (SARS-CoV-2/human/Liverpool/REMRQ001/2020), we performed viral infection assays and fixed the cells at 6 and 16 hours postinfection (hpi). SARS-CoV-2 infection was reduced in HeLaNRP1KO+ACE2 relative to HeLawt+ACE2 (Fig. 1C). HeLa cells lacking ACE2 expression were not infected (fig. S1G). In Caco-2 cells, a human colon adenocarcinoma cell line endogenously expressing ACE2 and widely used in COVID-19 studies, the suppression of NRP1 expression by short hairpin RNA (shRNA) greatly reduced SARS-CoV-2 infection at both 7 and 16 hpi, respectively, whereas that of vesicular stomatitis virus (VSV) pseudotyped with VSV-G was unaffected (Fig. 1D and figs. S1H and S2A). To determine if NRP1 was required for early virus infection, we established a sequential staining procedure using antibodies against SARS-CoV-2 S and N proteins to distinguish extracellular and intracellular viral particles (fig. S2B). Although NRP1 depletion did not affect SARS-CoV-2 binding to the Caco-2 cell surface (Fig. 1E), virus uptake was halved in NRP1-depleted cells compared to control cells after 30 min of internalization (Fig. 1F). Thus, NRP1 enhances SARS-CoV-2 entry and infection. We also observed that SARS-CoV-2–infected HeLawt+ACE2 cells displayed a multinucleated syncytia cell pattern, as reported by others (Fig. 1C) (5). Using an image analysis algorithm and supervised machine learning (fig. S2, C to F) (11), we quantified syncytia of infected HeLawt+ACE2 and HeLaNRP1KO+ACE2 cells. At 16 hpi, the majority of HeLawt+ACE2 cells formed syncytia, whereas in HeLaNRP1KO+ACE2 cells, this phenotype was reduced (fig. S2G). When infected with a SARS-CoV-2 isolate lacking the furin cleavage site (SARS-CoV-2 ΔS1/S2) (fig. S1A), the differences in infection and syncytia formation were less pronounced (fig. S2, H and I). However, a significant decrease in infection of HeLaNRP1KO+ACE2 was still observed at 16 hpi, indicating that NRP1 may additionally influence infection through a CendR-independent mechanism (fig. S2H). The extracellular regions of NRP1 and NRP2 are composed of two CUB domains (a1 and a2), two coagulation factor domains (b1 and b2), and a MAM domain (9). Of these, the b1 domain contains the specific binding site for CendR peptides (fig. S3A) (12). Accordingly, the mCherry-b1 domain of NRP1 immunoprecipitated GFP-S1, and a shortened GFP-S1 construct spanning residues 493 to 685 (figs. S1C and S3B). Isothermal titration calorimetry (ITC) established that the b1 domain of NRP1 directly bound a synthetic S1 CendR peptide (679NSPRRAR685) with an affinity of 20.3 μM at pH 7.5, which was enhanced to 13.0 μM at pH 5.5 (Fig. 2A). Binding was not observed to an S1 CendR peptide in which the C-terminal arginine was mutated to alanine (679NSPRRAA685) (Fig. 2A). We cocrystallized the NRP1 b1 domain in complex with the S1 CendR peptide (Fig. 2B). The resolved 2.35-Å structure revealed four molecules of b1 with electron density of the S1 CendR peptide clearly visible in the asymmetric unit (fig. S3C). S1 CendR peptide binding displayed strong similarity to the previously solved structure of NRP1 b1 domain in complex with its endogenous ligand VEGF-A164 (Fig. 2B and fig. S3D) (12). The key residues responsible for contacting the C-terminal R685 of the CendR peptide —Y297, W301, T316, D320, S346, T349 and Y353—are almost identical between the two structures (Fig. 2B and fig. S3D). The R682 and R685 side chains together engage NRP1 via stacked cation-π interactions with NRP1 side chains of Y297 and Y353. By projecting these findings onto the structure of the NRP1 ectodomain, the b1 CendR binding pocket appears to be freely accessible to the S1 CendR peptide (fig. S3E) (13). Site-directed mutagenesis of the S1 R685 residue to aspartic acid drastically reduced GFP-S1493-685 immunoprecipitation by mCherry-b1, confirming the critical role of the C-terminal arginine (Fig. 2C). Mutagenesis of the T316 residue within the mCherry-b1 domain of NRP1 to arginine also reduced association with GFP-S1493-685, consistent with its inhibitory impact on VEGF-A164 binding (12) (Fig. 2D). Accordingly, incubation of mCherry-b1 with VSV particles pseudotyped with trimeric S resulted in immunoprecipitation of processed forms of S1, which was dependent on the T316 residue (fig. S3F). Next, we transiently expressed either GFP, full-length NRP1 wt-GFP, or full length NRP1-GFP harboring the T316R mutation in HeLaNRP1KO+ACE2 cells. GFP expression and ACE2 expression levels were comparable and both constructs retained similar cell surface localization (fig. S3, G and H). SARS-CoV-2 infection was significantly enhanced in cells expressing NRP1 wt-GFP compared to GFP control, whereas it was not enhanced in cells expressing the T316R mutant (Fig. 2E). Thus, the SARS-CoV-2 S1 CendR and NRP1 interaction promotes infection. To establish the functional relevance of the S1 CendR-NRP1 interaction, we screened monoclonal antibodies (mAb#1, mAb#2, mAb#3) raised against the NRP1 b1b2 ectodomain. All three bound to the NRP1 b1b2 domain, displayed staining by immunofluorescence in NRP1-expressing PPC-1 (human primary prostate cancer) cells but not in M21 (human melanoma) cells that do not express NRP1 (fig. S4A) (8), and stained the extracellular domain of NRP1-GFP expressed in cells (fig. S4B). Of these antibodies, mAb#3, and to a lesser extent mAb#1, bound to the CendR-binding pocket with high specificity, as defined by reduced ability to bind to a b1b2 mutant that targets residues (S346, E348, T349) at the opening of the binding pocket (Fig. 3A) (12). Incubation of Caco-2 cells with mAbs#1 and 3 reduced SARS-CoV-2 infection compared to a control mAb targeting avian influenza A virus (H11N3) hemagglutinin (Fig. 3B). Consistent with this, mAb#3 inhibited binding of GFP-S1493-685 and mCherry-b1 (Fig. 3C). As a comparison, Caco-2 and Calu-3 cells were incubated with soluble ACE2, which inhibited SARS-CoV-2 infection in both cases (fig. S4C). Next, we turned to the small molecule EG00229, a selective NRP1 antagonist that binds the b1 CendR binding pocket and inhibits VEGF-A binding (Fig. 3D) (14). ITC established that EG00229 bound to the NRP1 b1 domain with a dissociation constant (Kd) of 5.1 and 11.0 μM at pH 7.5 and 5.5, respectively (Fig. 3E). EG00229 inhibited the direct binding between b1 and the S1 CendR peptide, and the immunoprecipitation of GFP-S1493-685 by mCherry-b1 (Fig. 3E and fig. S4D). Finally, incubation of Caco-2 cells with EG00229 reduced the efficiency of SARS-CoV-2 infection at 7 and 16 hpi (Fig. 3F). Thus, the SARS-CoV-2 interaction with NRP1 can be targeted to reduce viral infectivity in relevant human cell lines (fig. S5). Cell entry of SARS-CoV-2 depends on priming by host cell proteases (5, 6, 15). Our data indicate that a component of SARS-CoV-2 S protein binding to cell surface neuropilins occurs via the S1 CendR motif generated by the furin cleavage of S1/S2. Though not affecting cell surface attachment, this interaction promotes entry and infection by SARS-CoV-2 in physiologically relevant cell lines widely used in the study of COVID-19. The molecular basis for the effect is unclear, but neuropilins are known to mediate the internalization of CendR ligands through an endocytic process resembling macropinocytosis, (8, 16, 17). Notably, gene expression analysis has revealed an up-regulation of NRP1 and NRP2 in lung tissue from COVID-19 patients (18). A SARS-CoV-2 virus with a natural deletion of the S1/S2 furin cleavage site demonstrated attenuated pathogenicity in hamster models (19). NRP1 binding to the CendR peptide in S1 is thus likely to play a role in the increased infectivity of SARS-CoV-2 compared with SARS-CoV. The ability to target this specific interaction may provide a route for COVID-19 therapies. Supplementary Materials science.sciencemag.org/content/370/6518/861/suppl/DC1 Materials and Methods Figs. 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We thank the University of Queensland Remote Operation Crystallisation and X-ray facility (UQ-ROCX) and the staff for their support with the crystallization experiments, and the staff of the Australian Synchrotron for assistance with x-ray diffraction data collection. Funding: J.L.D. was supported by a Wellcome Trust studentship from the Dynamic Molecular Cell Biology Ph.D. program (203959/Z/16/Z), C.A.P. was supported by Beca Fundación Ramón Areces Estudios Postdoctorales en el Extranjero, and M.K.W. was supported by an MRC grant (MR/R020566/1) awarded to A.D.D. This project has received funding from the MRC (MR/P018807/1), Wellcome Trust (104568/Z/14/2), Lister Institute of Preventive Medicine, and Elizabeth Blackwell Institute for Health Research Rapid Response Call (COVID-19) awarded to P.J.C, the European Research Council under the European Union’s Horizon 2020 research and innovation program (No 856581 - CHUbVi), and from MRC-AMED (MR/T028769/1) awarded to Y.Y., the Swiss National Science Foundation and Kanton Zurich awarded to U.F.G. B.M.C. is supported by an Australian National Health and Medical Research Council (NHMRC) Senior Research Fellowship (APP1136021) and Project Grant (APP1156493), and the United States Food and Drug Administration grant no. HHSF223201510104C “Ebola Virus Disease: correlates of protection, determinants of outcome and clinical management” amended to incorporate urgent COVID-19 studies awarded to J.A.H., A.D.D., and D.A.M. R.H. and P.H. acknowledge support from the LENDULET-BIOMAG Grant (2018-342), from H2020-discovAIR (874656), and from Chan Zuckerberg Initiative, Seed Networks for the HCA-DVP. T.T. was supported by the European Regional Development Fund (Project no. 2014-2020.4.01.15-0012), by European Research Council grant GLIOGUIDE and Estonian Research Council (grants PRG230 and EAG79, to T.T.). Author contributions: J.L.D., B.S., A.H., P.J.C., and Y.Y. conceived the study. J.L.D., B.S., K.K, and Y.Y. performed most of the experiments. K.K., M.K.W., D.A.M., and A.D.D. performed all work with infectious SARS-CoV-2 supervised by A.D.D. M.K.W. and A.D.D. isolated SARS-CoV-2 strains used for the work. K.C., C.A.P., M.B., L.S.G., U.F.G., K.K., R.B.S., D.K.S., J.A.H., and T.T. did experimental work and/or provided essential reagents. R.H. and P.H. performed image analysis. B.S., A.D.D., B.M.C., P.J.C., and Y.Y. supervised the research. J.L.D., B.S., A.D.D., P.J.C., and Y.Y. wrote the manuscript and made the figures. All authors read and approved the final manuscript. Competing interests: T.T. is an inventor of patents on CendR peptides and a shareholder of Cend Therapeutics Inc., a company that holds a license for the CendR peptides and is developing the peptides for cancer therapy. J.A.H. is a member of the Department of Health, New and Emerging Respiratory Virus Threats Advisory Group (NERVTAG) and the Department of Health, Testing Advisory Group. U.F.G. is a consultant to F. Hoffmann–La Roche Ltd, Switzerland. All other authors declare no competing interests. Data and materials availability: Coordinates and structure factors for the NRP1 b1-S1 CendR peptide complex have been deposited at the Protein Data Bank (PDB) with accession code 7JJC. All other data are available in the manuscript or the supplementary materials. 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Scientists have found a second doorway, called neuropilin-1, that the novel coronavirus uses to invade human cells.