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Principal Investigator: Thomas Quinn
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2020
Award: $134,338
Funding agency: National Institute of Allergy and Infectious Diseases
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged as a global pandemic in early 2020. By August of 2020, over 21 million cases of SARS-CoV-2 have been confirmed, spanning 188 countries and accounting for over have 750,000 deaths. In the US, over 5.3 million cases of SARS-CoV-2 have been reported with over 167,000 deaths from COVID-19 disease. With widespread community transmission now documented for the past 6 months, and with an urgent need for effective therapeutics and prophylaxis, there is a critical need to perform broad-scale population-based testing to better define population infection dynamics, transmission and immunological and therapeutic responses to SARS-CoV-2 infection. Achieving such comprehensive national testing goals will be challenging by relying only nasopharyngeal swabs for molecular COVID-19 diagnostics. When used effectively and in conjunction with routine molecular testing, accurate serological tests can identify individuals who have already mounted an antibody response to SARS-CoV-2 infection including both symptomatic and asymptomatic infections. These tests can be deployed in large numbers to describe changes in population level immunity at different geographical scales and over time. In addition, SARS-CoV-2 antibody testing can be used to estimate the incidence and prevalence of SARS-CoV-2 infection at the general population level. Precise knowledge of population immunity could allow government bodies to make informed decisions about how and when to relax stay-at-home directives and to reopen the economy.
In order to compare these tests to those who were positive for SARS-CoV-2 infection based on reverse transcriptase polymerase chain reaction (RT-PCR), it is critical to understand how the predictive value of the test varies with time from exposure and symptom onset to avoid being falsely reassured by negative test results. We reviewed 7 previously published studies providing data on RT-PCR performance by time since symptom onset or SARS-CoV-2 exposure using samples from the upper respiratory tract (n = 1330). Over the 4 days of infection before the typical time of symptom onset (day 5), the probability of a false-negative result in an infected person decreases from 100% (95% CI, 100% to 100%) on day 1 to 67% (CI, 27% to 94%) on day 4. On the day of symptom onset, the median false-negative rate was 38% (CI, 18% to 65%). This decreased to 20% (CI, 12% to 30%) on day 8 (3 days after symptom onset) then began to increase again, from 21% (CI, 13% to 31%) on day 9 to 66% (CI, 54% to 77%) on day 21. Care must be taken in interpreting RT-PCR tests for SARS-CoV-2 infection-particularly early in the course of infection-when using these results as a basis for removing precautions intended to prevent onward transmission. If clinical suspicion is high, infection should not be ruled out on the basis of RT-PCR alone, and the clinical and epidemiologic situation should be carefully considered.
We evaluated the performance of fifteen commercial lateral flow POCTs for the detection of SARS-CoV-2-specific antibodies. Using a set of 100 samples, 40 samples were from known SARS-CoV-2-infected, convalescent individuals (average of 45 days post symptom onset) for sensitivity assessment and 60 samples from the pre-pandemic era (negative control). The pre pandemic samples were from individuals known to have been infected with other respiratory viruses (rhinoviruses A, B, C and/or coronavirus 229E, HKU1, NL63 OC43). The timing of seroconversion was assessed on five POCTs on a panel of 272 longitudinal samples from 47 patients of known time since symptom onset. Sensitivity and specificity for any reactive band ranged from 55%-97% and 78%-100%, respectively. When assessing the performance of the IgM and the IgG bands alone, sensitivity and specificity ranged from 0%-88% and 80%-100% for IgM and 25%-95% and 90%-100% for IgG. Longitudinal testing revealed that median time post symptom onset to a positive result was 7 days (IQR 5.4, 9.8) for IgM and 8.2 days (IQR 6.3 to 11.3). The testing performance varied widely among POCTs with most variation related to the sensitivity of the assays. The IgM band was most likely to misclassify pre-pandemic samples. The appearance of IgM and IgG bands occurred almost simultaneously.
For non-invasive sampling, we hypothesized that salivary antibodies to SARS-CoV-2 could serve as an alternative to serological testing. We used Luminex technology and tested saliva and serum samples, including 134 and 118 negative saliva and serum samples, respectively, and 33 saliva and 206 serum samples from participants with RT-PCR-confirmed SARS-CoV-2 infection.. Matched serum and saliva SARS-CoV-2 antigen-specific IgG responses were significantly correlated. Within the 10-plex SARS-CoV-2 panel, the salivary anti-nucleocapsid (N) protein IgG response resulted in the highest sensitivity for detecting prior SARS-CoV-2 infection (100% sensitivity at 10 days post-SARS-CoV-2 symptom onset). The salivary anti-receptor binding domain (RBD) IgG response resulted in 100% specificity. Among individuals with SARS-CoV-2 infection confirmed with RT-PCR, the temporal kinetics of IgG, IgA, and IgM in saliva were consistent with those observed in serum. SARS-CoV-2 appears to trigger a humoral immune response resulting in the almost simultaneous rise of IgG, IgM and IgA levels both in serum and in saliva, mirroring responses consistent with the stimulation of existing, cross-reactive B cells.
Among 126 potential convalescent plasma donors, the humoral immune response was evaluated by a SARS-CoV-2 virus neutralization assay using Vero-E6-50 TMPRSS2 cells, commercial IgG and IgA ELISA to spike (S) protein S1 domain (Euroimmun), IgA, IgG and IgM indirect ELISAs to the full-length S or S-receptor binding domain (S-RBD), and an IgG avidity assay. IgG titers were greater than either IgM or IgA for S1, full length S, and S-RBD in the overall population. Of the 126 plasma samples, 101 (80%) had detectable neutralizing antibody (nAb) titers. Using nAb titers as the reference, the IgG ELISAs confirmed between 95-98% of the nAb positive, but 20-32% of the nAb negative samples were still IgG ELISA positive. Male sex, older age, and hospitalization emerged as factors that can be used to identify individuals with a high likelihood of having strong antiviral antibody responses.
Not all recovered patients develop suitable antibody titers for donation and the link between avidity and neutralizing titers has yet to be established in COVID-19 acute- and convalescent patients. SARS-CoV-2 anti-spike and anti-nucleocapsid IgG antibody titers and avidity were measured in a longitudinal cohort of COVID-19 hospitalized patients (N=16 individuals n=84 samples) and a cross-sectional sample of convalescent plasma donors (n=130). Epidemiologic correlates of avidity were examined in donors by linear regression. The association of avidity and a high neutralizing titer (NT) was also assessed in donors using modified Poisson regression. Antibody avidity increased over duration of infection and remained elevated. In convalescent plasma donors, higher levels of anti-spike avidity was associated with older age, male sex, and hospitalization. Higher NTs significantly correlated with higher anti-spike IgG avidity (spearmans rho=0.386; p<0.001), but not anti-nucleocapsid IgG avidity. Increasing levels of anti-spike IgG avidity were associated with high NT (>160) (adjusted prevalence ratio=1.66 95%CI=1.47-1.89), independent of age, sex, and hospitalization. Antibody avidity is correlated with higher neutralizing titers, suggesting an alternative screening parameter for identifying optimal convalescent plasma donors.
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