SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, could offer protective immunity, and may affect clinical outcomes of COVID-19 patients. We analyzed 625 serial plasma samples from 40 hospitalized COVID-19 patients and 170 SARS-CoV-2- infected outpatients and asymptomatic individuals. Severely ill patients developed significantly higher SARS-CoV-2-specific antibody responses than outpatients and asymptomatic individuals. The development of plasma antibodies was correlated with decreases in viral RNAemia, consistent with potential humoral immune clearance of virus. Using a novel competition ELISA, we detected antibodies blocking RBD-ACE2 interactions in 68% of inpatients and 40% of outpatients tested. Cross-reactive antibodies recognizing SARS-CoV RBD were found almost exclusively in hospitalized patients. Outpatient and asymptomatic individuals’ serological responses to SARS-CoV-2 decreased within 2 months, suggesting that humoral protection may be short-lived
clinical questions in the ongoing COVID-19 pandemic are the extent to which patient antibody responses may be protective against reinfection, and the duration of individuals’ serological responses. As SARS-CoV-2 vaccine candidates enter clinical trials, comparison of vaccine-induced immune responses to those stimulated by viral infection will be important for understanding immunological correlates of protection. Here, we have analyzed serological responses to SARS-CoV-2 in 210 individuals ranging from ICU patients to asymptomatic individuals, with detailed analysis of antibody responses in 124 patients using a panel of SARS-CoV-2 antigens, and a novel assay testing the ability of RBDspecific antibodies to block binding of human ACE2. The appearance of IgM, IgG, and IgA antibodies, and the development of blocking activity preventing ACE2-RBD interaction were strongly correlated with each other, and were most prominent in severely ill COVID-19 patients. Nucleocapsid-specific responses differed from spike RBD or S1 antigen responses primarily in the low levels of IgM elicited by this antigen. RBD-specific antibody responses were tightly correlated with decreases in viral RNAemia, consistent with the humoral immune response acting to remove virus from circulation, and presumably from other sites in the body. In contrast to some prior studies reporting detection of anti-SARS-CoV-2 IgM at earlier timepoints post-onset of symptoms compared to IgG antibody responses (13, 22), we found no significant difference in the timing of detectable anti-SARS-CoV-2 RBD IgM and IgG in our patient group, as noted in more recent reports (23). The timing of the onset of IgG responses and the finding that severely ill COVID-19 patients who required ICU care developed high anti-RBD antibody titers indicates that delayed or impaired production of virus-specific antibodies relative to the onset of symptoms does not explain differences in disease severity. More robust antibody for this preprint 17 responses in patients with severe as opposed to mild infection has also been reported for other coronavirus infections (16–18). Notably, the IgG responses of inpatients, but not the IgM or IgA responses, were sustained at high levels for at least two months, although not all patients were observed for this length of time. We evaluated patient plasma with a newly developed ACE2 competition ELISA to detect antibodies that can block viral RBD interaction with the host cell receptor. This assay is highly scalable and suitable for population screening as a potential surrogate for cell culture-based virus neutralization testing.
We find that antibodies blocking binding of ACE2 to RBD appear in the majority of inpatients during the course of their infection. Plasma from some patients, including patient 16 (who required ICU care for an extended period of time) and patient 20 (who died of COVID-19 during the study period) contained antibodies with high blocking capacity, indicating that these antibodies, at the time that they developed in these patients, were not sufficient to prevent a deterioration of the patients’ health. Patients 6 and 55, who died of COVID-19 during the study period, did not reach full blocking capacity of ACE2-RBD binding, suggesting that the quality of their humoral responses could have contributed to their deaths, but a counter-example is seen in patient 14, who was discharged from ICU care relatively quickly despite having similarly incomplete ACE2-RBD blocking activity. In contrast to the inpatients in our study, SARS-CoV-2-infected outpatients or asymptomatic individuals showed weaker plasma antibody responses of shorter duration, with a peak of IgG levels at approximately one month after diagnostic rRT-PCR testing, followed by a relatively rapid decline. One implication of these results is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. To further investigate the fine specificity of patient antibody responses we tested patient plasmas for breadth of IgG binding to both SARS-CoV-2 RBD and the RBD from SARS-CoV, the causative agent of the SARS epidemic in 2003. A recent report of COVID-19 patients with unspecified disease severity at up to 22 days post-onset of symptoms found that cross-reactive antibody responses to the SARS-CoV spike ectodomain were more common than responses to the SARS-CoV RBD, and that only one of 15 patients showed cross-neutralization of SARSCoV (24).
Our data demonstrate that it is almost entirely in patients with severe COVID-19 illness that antibodies with breadth of binding including SARS-CoV are found, suggesting that these patients generate antibodies to distinct epitope subsets or a greater diversity of epitopes, in addition to producing higher antibody levels. We observed highly variable time courses of these cross-reactive antibodies, showing a transient appearance in some patients, and a slow increase in others. These responses to specific cross-reactive epitopes may reveal the dynamics of the frequency, extent of plasma cell differentiation, and survival of individual B cell clones amid each patient’s polyclonal response. It is likely that B cell clonal responses to other epitopes may also show variation between individuals. Given that the subgenus Sarbecovirus has already given rise to two coronavirus infection outbreaks in humans in the past two decades, it would be appealing if vaccination efforts could attempt to provide broad protection against this group of viruses. Our data suggest that novel vaccine strategies may be required to stimulate responses to rarely-targeted cross-reactive neutralizing epitopes. Most outpatient plasma samples showed little ACE2 blocking capacity, although there were a few individuals that were exceptions to this pattern. ACE2 blocking activity showed a similar time course to total RBD-specific IgG antibody levels, decreasing at later time points. Recent reports find relatively low titers of neutralizing antibodies for SARS-CoV-2 or pseudotyped viruses expressing SARS-CoV-2 spike in many mildly ill to asymptomatic individuals (25, 26). We find that plasma antibody responses to SARS-CoV-2 are generally of short duration following asymptomatic or mild infection, but this does not necessarily indicate that all immunity will be lost. It is possible that local antibody production in the airways could help prevent or impair SARS-CoV-2-infection upon reexposure (27). Even if serum antibodies have waned to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response in future. Clinical trial results from patients with known reexposure to SARS-CoV-2 after recovery from initial infection will be needed to determine which serological or other immunological assays provide the most accurate correlates of protection from reinfection.
Reference & Source information: https://www.medrxiv.org/
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