Early Treatment, Inflammation and Post-COVID Conditions (preprint)

ABSTRACT Background: Post-COVID conditions (PCC) are common and have significant morbidity. Risk factors for PCC include advancing age, female sex, obesity, and diabetes mellitus. Little is known about early treatment, inflammation, and PCC. Methods: Among 883 individuals with confirmed SARS-CoV-2 infection participating in a randomized trial of CCP vs. control plasma with available biospecimens and symptom data, the association between early COVID treatment, cytokine levels and PCC was evaluated. Cytokine and chemokine levels were assessed at baseline, day 14 and day 90 using a multiplexed sandwich immuosassay (Mesoscale Discovery). Presence of any self-reported PCC symptoms was assessed at day 90. Associations between COVID treatment, cytokine levels and PCC were examined using multivariate logistic regression models. Results: One-third of the 882 participants had day 90 PCC symptoms, with fatigue (14.5%) and loss of smell (14.5%) being most common. Cytokine levels decreased from baseline to day 90. In a multivariable analysis including diabetes, body mass index, race, and vaccine status, female sex (adjusted odds ratio[AOR]=2.70[1.93-3.81]), older age (AOR=1.32[1.17-1.50]), and elevated baseline levels of IL-6 (AOR=1.59[1.02-2.47]) were associated with development of PCC. There was a trend for decreased PCC in those with early CCP treatment (<5 days after symptom onset) compared to late CCP treatment. Conclusion: Increased IL-6 levels were associated with the development of PCC and there was a trend for decreased PCC with early CCP treatment in this predominately unvaccinated population. Future treatment studies should evaluate the effect of early treatment and anti-IL-6 therapies on PCC development.

Differential antibody production by symptomatology in SARS-CoV-2 convalescent individuals (preprint)

The association between COVID-19 symptoms and antibody responses against SARS-CoV-2 is poorly characterized. We analyzed antibody levels in individuals with known SARS-CoV-2 infection to identify potential antibody-symptom associations. Convalescent plasma from 216 SARS-CoV-2 RNA+ individuals with symptomatology information were tested for the presence of IgG to the spike S1 subunit (Euroimmun ELISA), IgG to receptor binding domain (RBD, CoronaCHEK rapid test), and for IgG, IgA, and IgM to nucleocapsid (N, Bio-Rad ELISA). Logistic regression was used to estimate the odds of having a COVID-19 symptom from the antibody response, adjusting for sex and age. Cough strongly associated with antibodies against S1 (adjusted odds ratio [aOR]= 5.33; 95% CI from 1.51 to 18.86) and RBD (aOR=4.36; CI 1.49, 12.78). In contrast, sore throat significantly associated with the absence of antibodies to S1 and N (aOR=0.25; CI 0.08, 0.80 and aOR=0.31; 0.11, 0.91). Similarly, lack of symptoms associated with the absence of antibodies to N and RBD (aOR=0.16; CI 0.03, 0.97 and aOR=0.16; CI 0.03, 1.01). Cough appeared to be correlated with a seropositive result, suggesting that SARS-CoV-2 infected individuals exhibiting lower respiratory symptoms generate a robust antibody response. Conversely, those without symptoms or limited to a sore throat while infected with SARS-CoV-2 were likely to lack a detectable antibody response. These findings strongly support the notion that severity of infection correlates with robust antibody response.

A Third Dose of SARS-CoV-2 Vaccine Increases Neutralizing Antibodies Against Variants of Concern in Solid Organ Transplant Recipients (preprint)

Vaccine-induced SARS-CoV-2 antibody responses are attenuated in solid organ transplant recipients (SOTRs) and breakthrough infections are more common. Additional SARS-CoV-2 vaccine doses increase anti-spike IgG in some SOTRs, but it is uncertain whether neutralization of variants of concern (VOCs) is enhanced. We tested 47 SOTRs for clinical and research anti-spike IgG, pseudoneutralization (ACE2 blocking), and live-virus neutralization (nAb) against VOCs before and after a third SARS-CoV-2 vaccine dose (70% mRNA, 30% Ad26.COV2.S) with comparison to 15 healthy controls after two mRNA vaccine doses. We used correlation analysis to compare anti-spike IgG assays and focused on thresholds associated with neutralizing activity. A third SARS-CoV-2 vaccine dose increased median anti-spike (1.6-fold) and receptor-binding domain (1.5-fold) IgG, as well as pseudoneutralization against VOCs (2.5-fold versus Delta). However, IgG and neutralization activity were significantly lower than healthy controls (p<0.001); 32% of SOTRs had zero detectable nAb against Delta after third vaccination. Correlation with nAb was seen at anti-spike IgG >4 AU on the clinical assay and >10^4 AU on the research assay. These findings highlight benefits of a third vaccine dose for some SOTRs and the need for alternative strategies to improve protection in a significant subset of this population.

A hemagglutination-based, semi-quantitative test for point-of-care determination of SARS-CoV-2 antibody levels (preprint)

Serologic, point-of-care tests to detect antibodies against SARS-CoV-2 are an important tool in the COVID-19 pandemic. The majority of current point-of-care antibody tests developed for SARS-CoV-2 rely on lateral flow assays, but these do not offer quantitative information. To address this, we developed a new method of COVID-19 antibody testing employing hemagglutination tested on a dry card, similar to that which is already available for rapid typing of ABO blood groups. A fusion protein linking red blood cells (RBCs) to the receptor-binding domain (RBD) of SARS-CoV-2 spike protein was placed on the card. 200 COVID-19 patient and 200 control plasma samples were reconstituted with O-negative RBCs to form whole blood and added to the dried protein, followed by a stirring step and a tilting step, 3-minute incubation, and a second tilting step. The sensitivity for the hemagglutination test, Euroimmun IgG ELISA test and RBD-based CoronaChek lateral flow assay was 87.0%, 86.5%, and 84.5%, respectively, using samples obtained from recovered COVID-19 individuals. Testing pre-pandemic samples, the hemagglutination test had a specificity of 95.5%, compared to 97.3% and 98.9% for the ELISA and CoronaChek, respectively. A distribution of agglutination strengths was observed in COVID-19 convalescent plasma samples, with the highest agglutination score (4) exhibiting significantly higher neutralizing antibody titers than weak positives (2) (p<0.0001). Strong agglutinations were observed within 1 minute of testing, and this shorter assay time also increased specificity to 98.5%. In conclusion, we developed a novel rapid, point-of-care RBC agglutination test for the detection of SARS-CoV-2 antibodies that can yield semi-quantitative information on neutralizing antibody titer in patients. The five-minute test may find use in determination of serostatus prior to vaccination, post-vaccination surveillance and travel screening.

Comparative performance of multiplex salivary and commercially available serologic assays to detect SARS-CoV-2 IgG and neutralization titers (preprint)

ABSTRACT Oral fluid (hereafter saliva) offers a non-invasive sampling method for the detection of SARS-CoV-2 antibodies. However, data comparing performance of salivary tests against commercially-available serologic and neutralizing antibody (nAb) assays are lacking. This study compared the performance of a multiplex salivary SARS-CoV-2 IgG assay targeting antibodies to nucleocapsid (N), receptor binding domain (RBD) and spike (S) antigens to three commercially-available SARS-CoV-2 serology enzyme immunoassays (EIAs) (Ortho Vitros, Euroimmun, and BioRad) and nAb. Paired saliva and plasma samples were collected from 101 eligible COVID-19 convalescent plasma (CCP) donors >14 days since PCR+ confirmed diagnosis. Concordance was evaluated using positive (PPA) and negative (NPA) percent agreement, overall percent agreement (PA), and Cohen’s kappa coefficient. The range between salivary and plasma EIAs for SARS-CoV-2-specific N was PPA: 54.4-92.1% and NPA: 69.2-91.7%, for RBD was PPA: 89.9-100% and NPA: 50.0-84.6%, and for S was PPA: 50.6-96.6% and NPA: 50.0-100%. Compared to a plasma nAb assay, the multiplex salivary assay PPA ranged from 62.3% (N) and 98.6% (RBD) and NPA ranged from 18.8% (RBD) to 96.9% (S). Combinations of N, RBD, and S and a summary algorithmic index of all three (N/RBD/S) in saliva produced ranges of PPA: 87.6-98.9% and NPA: 50-91.7% with the three EIAs and ranges of PPA: 88.4-98.6% and NPA: 21.9-34.4% with the nAb assay. A multiplex salivary SARS-CoV-2 IgG assay demonstrated comparable performance to three commercially-available plasma EIAs and a nAb assay, and may be a viable alternative to assist in screening CCP donors and monitoring population-based seroprevalence and vaccine antibody response.