ABSTRACT
BackgroundA fraction of COVID-19 patients develops severe disease requiring hospitalization, while the majority, including high-risk individuals, experience mild symptoms. Severe disease has been associated with higher levels of antibodies and inflammatory cytokines, but the association has often resulted from comparison of patients with diverse demographics and comorbidity status. This study examined patients with defined demographic risk factors for severe COVID-19 who developed mild vs. severe COVID-19. MethodsThis study evaluated hospitalized vs. ambulatory COVID-19 patients in the James J. Peters VA Medical Center, Bronx, NY. This cohort presented demographic risk factors for severe COVID-19: median age of 63, >80% male, >85% black and/or Hispanic. Sera were collected four to 243 days after symptom onset and evaluated for binding and functional antibodies as well as 48 cytokines/chemokines. FindingsAmbulatory and hospitalized patients showed no difference in SARS-CoV-2-specific antibody levels and functions. However, a strong correlation between anti-S2 antibody levels and the other antibody parameters was observed in hospitalized but not in ambulatory cases. Cytokine/chemokine levels also revealed differences, with notably higher IL-27 levels in hospitalized patients. Hence, among the older, mostly male patients studied here, SARS-CoV-2-specific antibody levels and functions did not distinguish hospitalized and ambulatory cases but a discordance in S2-specific antibody responses was noted in ambulatory patients, and elevated levels of specific cytokines were maintained in convalescent sera of hospitalized cases. InterpretationThe data indicate that antibodies against the relatively conserved S2 spike subunit and immunoregulatory cytokines such as IL-27 are potential immune determinants of COVID-19. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSPrevious studies demonstrated that high levels of SARS-CoV-2 spike binding antibodies and neutralizing antibodies were associated with COVID-19 disease severity. However, the comparisons were often made without considering demographics and comorbidities. Correlation was similarly shown between severe disease and marked elevation of several plasma cytokines but again, most analyses of cytokine responses to COVID-19 were conducted by comparison of patient cohorts with diverse demographic characteristics and risk factors. Added value of this studyWe evaluated here a comprehensive profile of SARS-CoV-2-specific antibodies (total Ig, isotypes/subtypes, Fab- and Fc-mediated functions) and a panel of 48 cytokines and chemokines in serum samples from a cohort of SARS-CoV-2-infected patients with demographic risk factors for severe COVID-19: 81% were male, 79% were >50 years old (median of 63), and 85% belonged to US minority groups (black and/or Hispanic). Comparison of hospitalized vs. ambulatory patients within this cohort revealed two features that differed between severe vs. mild COVID-19 cases: a discordant Ab response to the S2 subunit of the viral spike protein in the mild cases and an elevated response of specific cytokines and chemokines, notably IL-27, in the severe cases. Implications of all the available evidenceData from the study identified key immunologic markers for severe vs. mild COVID-19 that provide a path forward for investigations of their roles in minimizing or augmenting disease severity.
ABSTRACT
Antibodies (Abs) are essential for the host immune response against SARS-CoV-2, and all the vaccines developed so far have been designed to induce Abs targeting the SARS-CoV-2 spike. Many studies have examined Ab responses in the blood from vaccinated and infected individuals. However, since SARS-CoV-2 is a respiratory virus, it is also critical to understand the mucosal Ab responses at the sites of initial virus exposure. Here, we examined plasma versus saliva Ab responses in vaccinated and convalescent patients. Although saliva levels were significantly lower, a strong correlation was observed between plasma and saliva total Ig levels against all SARS-CoV-2 antigens tested. Virus-specific IgG1 responses predominated in both saliva and plasma, while a lower prevalence of IgM and IgA1 Abs was observed in saliva. Antiviral activities of plasma Abs were also studied. Neutralization titers against the initial WA1 (D614G), B.1.1.7 (alpha) and B.1.617.2 (delta) strains were similar but lower against the B.1.351 (beta) strain. Spike-specific antibody-dependent cellular phagocytosis (ADCP) activities were also detected and the levels correlated with spike-binding Ig titers. Interestingly, while neutralization and ADCP potencies of vaccinated and convalescent groups were comparable, enhanced complement deposition to spike-specific Abs was noted in vaccinated versus convalescent groups and corresponded with higher levels of IgG1 plus IgG3 among the vaccinated individuals. Altogether, this study demonstrates the detection of Ab responses after vaccination or infection in plasma and saliva that correlate significantly, although Ig isotypic differences were noted. The induced plasma Abs displayed Fab-mediated and Fc-dependent functions with comparable neutralization and ADCP potencies, but a greater capacity to activate complement was elicited upon vaccination.
ABSTRACT
The Pfizer/BioNTech and Moderna mRNA-based COVID-19 vaccines are licensed under emergency use authorization, with millions of doses already administered globally [1]. No COVID-19 vaccines are yet under investigation for use in infants or young children. As such, the passive immunity of the antibodies (Abs) provided through milk from a vaccinated person may be one of the only ways to protect this population until pediatric COVID-19 vaccines are licensed. Our early work (as well as an expanded study being published concurrently with this report) examining the milk Ab response after SARS-CoV-2 infection demonstrated that Spike-specific IgA in milk after infection is dominant and highly correlated with a secretory Ab response [2]. Determining if secretory Abs are elicited in milk is critical, as this Ab class is highly stable and resistant to enzymatic degradation in all mucosae - not only in the infant oral/nasal cavity and gut, but in the airways and GI tract as well [3, 4]. Presently, we describe our analysis of the milk Ab response 14 days after completion of an mRNA-based COVID-19 vaccine regimen among 10 individuals. It was evident that unlike the post-infection milk Ab profile, IgG dominates after COVID-19 vaccination. One hundred percent of post-vaccine milk contained significant levels of Spike-specific IgG, with 8/10 samples exhibiting high IgG endpoint titers. Conversely, 6/10 (60%) of post-vaccine samples were positive for Spike specific IgA, with only 1 (10%) exhibiting high IgA endpoint titer. Furthermore, 5/10 (50%) post-vaccine milk samples contained Spike-specific secretory Ab, none of which were found to be high-titer. As our analyses of the immune response in milk to COVID-19 vaccination continues, it will provide a critical opportunity to address huge knowledge gaps, inform the field as to which COVID-19 vaccine, if any, is likely to provide the best milk Ab response, and highlight the need to design improved vaccines with protection of the breastfeeding infant in mind.
ABSTRACT
Approximately 10% of infants will experience COVID-19 illness requiring advanced care (1). A potential mechanism to protect this population could be provided by passive immunity through the milk of a previously infected mother. We and others have reported on the presence of SARS-CoV-2-specific antibodies in human milk (2-5). We now report the prevalence of SARS-CoV-2 IgA in the milk of 75 COVID-19-recovered participants, and find that 88% of samples are positive for Spike-specific IgA. In a subset of these samples, 95% exhibited robust IgA activity as determined by endpoint binding titer, with 50% considered high-titer. These IgA positive specimens were also positive for Spike-specific antibodies bearing the secretory component. Levels of IgA antibodies and antibodies bearing secretory component were shown to be strongly positively correlated. The secretory IgA response was dominant among the milk samples tested compared to the IgG response, which was present in 75% of samples and found to be of high-titer in only 13% of cases. Our IgA durability analysis using 28 paired samples, obtained 4-6 weeks and 4-10 months after infection, found that all samples exhibited persistently significant Spike-specific IgA, with 43% of donors exhibiting increasing IgA titers over time. Finally, COVID-19 and pre-pandemic control milk samples were tested for the presence of neutralizing antibodies; 6 of 8 COVID-19 samples exhibited neutralization of Spike-pseudotyped VSV (IC50 range, 2.39 - 89.4ug/mL) compared to 1 of 8 controls. IgA binding and neutralization capacities were found to be strongly positively correlated. These data are highly relevant to public health, not only in terms of the protective capacity of these antibodies for breastfed infants, but also for the potential use of such antibodies as a COVID-19 therapeutic, given that secretory IgA is highly stable not only in milk and the infant mouth and gut, but in all mucosa including the gastrointestinal tract, upper airway, and lungs (6).
ABSTRACT
Patients with immune-mediated inflammatory diseases (IMIDs) and acquired and genetic immunodeficiencies receiving therapeutic infusions are considered high risk for SARS-CoV-2 infection. However, the seroprevalance in this group and the safety of routine administrations at outpatient infusion centers are unknown. To determine the infection rate and clinical-social factors related to SARS-CoV-2 in asymptomatic patients with IMIDs and immunodeficiencies receiving routine non-cancer therapeutic infusions, we conducted a seroprevalence study at our outpatient infusion center. We report the first prospective SARS-CoV-2 sero-surveillance of 444 IBD/IMID, immunodeficiency, and immune competent patients at an outpatient infusion center in the U.S. showing lower seroprevalence in patients compared with the general population and provide clinical and social characteristics associated with seroprevalence in this group. These data suggest that patients can safely continue infusions at outpatient centers.
ABSTRACT
SARS-CoV-2 has infected millions of people and is on a trajectory to kill more than one million globally. Virus entry depends on the receptor-binding domain (RBD) of the spike protein. Although previous studies demonstrated anti-spike and -RBD antibodies as essential for protection and convalescent plasma as a promising therapeutic option, little is known about the immunoglobulin (Ig) isotypes capable of blocking virus entry. Here, we studied spike- and RBD-specific Ig isotypes in plasma/sera from two acutely infected and 29 convalescent individuals. Spike- and RBD-specific IgM, IgG1, and IgA1 antibodies were produced by all or nearly all subjects at varying levels and detected at 7-8 days post-disease onset. IgG2, IgG3, IgG4, and IgA2 were also present but at much lower levels. All samples also displayed neutralizing activity. IgM, IgG, and IgA were capable of mediating neutralization, but neutralization titers correlated better with binding levels of IgM and IgA1 than IgG.
ABSTRACT
The global COVID-19 pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the SARS-CoV-2 spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous BSL3 conditions which limits high throughput screening of patient and vaccine sera. Myriad BSL-2 compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVAG-based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale and generate accurate neutralizing titers within 18 hours post-infection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in confirmed convalescent patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n>120). Our data show that absolute (abs) IC50, IC80, and IC90 values can be legitimately compared across diverse cohorts, highlight the substantial but consistent variability in neutralization potency across these cohorts, and support the use of absIC80 as a more meaningful metric for assessing the neutralization potency of vaccine or convalescent sera. Lastly, we used our CoV2pp in a screen to identify ultra-permissive 293T clones that stably express ACE2 or ACE2+TMPRSS2. When used in combination with our CoV2pp, we can now produce CoV2pp sufficient for 150,000 standardized VNA/week. ImportanceVaccines and antibody-based therapeutics like convalescent plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) is an essential part of determining vaccine or therapeutic efficacy. However, such efficacy testing is limited by the inherent dangers of working with the live virus, which requires specialized high-level biocontainment facilities. We therefore developed a standardized replication-defective pseudotyped particle system that mimics entry of live SARS-CoV-2. This tool allows for the safe and efficient measurement of NATs, determination of other forms of entry inhibition, and thorough investigation of virus entry mechanisms. Four independent labs across the globe validated our standardized VNA using diverse cohorts. We argue that a standardized and scalable assay is necessary for meaningful comparisons of the myriad of vaccines and antibody-based therapeutics becoming available. Our data provide generalizable metrics for assessing their efficacy.
ABSTRACT
SARS-CoV-2, commonly termed COVID-19 for the illness it causes, has infected >3.2 million people, including >220,000 deaths. Human milk IgG originates mainly from blood, therefore a SARS-CoV-2-reactive antibody (Ab) response in milk would be expected (1). However, IgG comprises only ~2% of milk Ab, with most milk Abs originating from mucosa-associated lymphatic tissue (1). Therefore, the extent of the milk immune response to SARS-CoV-2 is unknown (2). This response is critical for infants and young children, who tend not to suffer greatly from COVID-19 pathology but are likely responsible for significant virus transmission (3-5). Perhaps even more significant is the fact that milk Abs could be purified and used as a COVID-19 therapeutic, given they would likely be of the secretory (s) class and highly resistant to proteolytic degradation in the respiratory tissue (2, 6). In this preliminary report, 15 milk samples obtained from donors previously-infected with SARS-CoV-2 as well as 10 negative control samples obtained prior to December 2019 were tested for reactivity to the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein by ELISAs measuring IgA, IgG, IgM, and secretory Ab. Eighty percent of samples obtained post-COVID-19 exhibited IgA reactivity, and all these samples were also positive for secretory Ab reactivity, suggesting the IgA is predominantly sIgA. COVID-19 group mean OD values of undiluted milk were significantly greater for IgA (p<0.0001), secretory-type Abs (p<0.0001), and IgG (p=0.017), but not for IgM, compared to pre-pandemic group mean values. Overall, these data indicate that there is strong sIgA-dominant SARS-CoV-2 immune response in human milk after infection in the majority of individuals, and that a comprehensive study of this response is highly warranted.
ABSTRACT
BackgroundMore than one million infections with the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) have been confirmed. While PCR-based assays are used for diagnosis, high through-put serologic methods are needed to detect antibodies for seroserveillance and for identification of seroconversion, potential plasma donors, and the nature of the immune response to this pathogen. MethodsA Luminex binding assay was used to assess the presence of antibodies in human sera from COVID-19-infected and -uninfected individuals specific for two recombinant proteins of SARS-CoV-2. FindingsFluorochrome-labeled beads were coated with a recombinant soluble stabilized trimeric SARS-CoV-2 S protein ectodomain or its central portion, the receptor binding domain (RBD). Coated beads were incubated with sera, followed by incubation with biotinylated anti-human total Ig antibodies and phycoerythrin (PE)-labeled streptavidin. Readout using a Luminex analyzer clearly differentiated between sera of the infected and uninfected subjects, delineating a wide range of serum antibody levels in infected subjects. InterpretationAntibody assays of sera can identify individuals who are infected with SARS-CoV-2 and have seroconverted, as well as subjects who have been infected and recovered. The use of the Luminex binding Ab assay has the advantage that it can be run in approximately 2.5 hours, uses very little antigen, and permits a high through-put of samples/day. FundingNIAID contracts and grants, Department of Veterans Affairs grants, the Microbiology Laboratory Clinical Services, Translational Science Hub, and Personalized Virology Initiative, and Department of Medicine of Mount Sinai Health System and Icahn School of Medicine at Mount Sinai. RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSThe outbreak of infections with SARS-CoV-2 began in late 2019. Specimens from nasopharyngeal swabs are being used in PCR-based assays to test for the presence of the virus. Until the first week in April, 2020 there were no licensed tests for the presence of serum antibodies against proteins of the virus. The first approved tests are now becoming available, but none use a format that can be scaled up for mass screening which is now needed for implementing various public health measures. As per a recent Pubmed search, less than 10 studies using serologic assays have been published and none are high through-put. Added value of this studyHigh through-put antibody tests are needed in order to identify seroconversion, to perform serosurveys, identify potential donors for plasma therapy, assess the prevalence of infection in populations, identify healthcare workers who may be immune to SARS-CoV-2, and to study the nature of the immune response to this pathogen. The method described for detecting antibodies in SARS-CoV-2-infected patients can be applied in hospital and reference labs, allowing the assessment of present and past infection in a much higher number of donors per unit of time than assays described heretofore. Implications of all the available evidenceThis study shows that a test in which magnetic beads are coated with soluble forms of the spike protein of SARS-CoV-2 can be used to test for the presence of antibodies targeting this pathogen. The platform allows for the efficient testing of multiple specimens simultaneously using as little as 5 nanograms of antigen per test. This test affords the possibility of large scale, economical and efficient antibody testing.