Ongoing evolution of SARS-CoV-2 drives escape from mRNA vaccine-induced humoral immunity (preprint)

Since the COVID-19 pandemic began in 2020, viral sequencing has documented 131 individual mutations in the viral spike protein across 48 named variants. To determine the ability of vaccine-mediated humoral immunity to keep pace with continued SARS-CoV-2 evolution, we assessed the neutralization potency of sera from 76 vaccine recipients collected after 2 to 6 immunizations against a comprehensive panel of mutations observed during the pandemic. Remarkably, while many individual mutations that emerged between 2020 and 2022 exhibit escape from sera following primary vaccination, few escape boosted sera. However, progressive loss of neutralization was observed across newer variants, irrespective of vaccine doses. Importantly, an updated XBB.1.5 booster significantly increased titers against newer variants but not JN.1. These findings demonstrate that seasonal boosters improve titers against contemporaneous strains, but novel variants continue to evade updated mRNA vaccines, demonstrating the need for novel approaches to adequately control SARS-CoV-2 transmission.

Immunogenicity of SARS-CoV-2 vaccines in patients with breast cancer receiving CDK 4/6 inhibitors

Background: CDK 4/6 inhibitors have transformed the landscape of breast oncology. A CDK 4/6 inhibitor in combination with endocrine therapy is recommended as 1st line therapy for patients with metastatic hormone receptor positive breast cancer. CDK 4/6 inhibitors have purported immunomodulatory effects and while effective, myelosuppression is a common adverse effect of CDK 4/6 inhibitor treatment of breast cancer. The impact of CDK 4/6 inhibitor therapy on immunogenicity of vaccines is not known. In this study, we evaluated the spike antibody response to SARS-CoV-2 vaccines among patients with breast cancer receiving endocrine therapy with or without CDK 4/6 inhibitors. Methods: In the Cancer COVID and Vaccine (CANVAX) study eligible patients included patients with breast cancer who had completed all scheduled doses of SARS-CoV-2 vaccines. Chart review was conducted to identify patients who had received endocrine therapy with or without CDK 4/6 inhibitor. We used validated assays to measure anti-SARS-CoV-2 total IgA/M/G spike antibodies and virus neutralization. We evaluated the magnitude of antibody response based on geometric mean concentrations (GMCs) as well as the % of patients with inadequate seroconversion (defined as levels <100 U/ml). Independent T-test based on log-transformed antibody values was utilized to compare the spike antibody levels and p value of ≤ 0.05 Swas considered statistically significant. Results: Between April 2021 and June 2021, 203 patients with breast cancer were enrolled. As of the cut-off date (2nd July 2021), results were available for 73 patients treated with endocrine therapy alone (N = 23), or with CDK 4/6 inhibitor-based therapy (N = 50). Most were females (98.6%), white (83.6%), and had metastatic breast cancer (68.5%). 49.3% had received BNT162b2 (Pfizer), 37% mRNA1273 (Moderna), and 13.7% Ad26.COV2.S (Johnson and Johnson/Janssen) vaccines. Overall, the mean spike antibody levels were similar between patients treated with endocrine therapy alone vs CDK 4/6 inhibitor-based therapy (GMC: 326 vs. 719 U/mL;p=0.704). Mean spike antibody levels were higher in patients with early breast cancer vs. metastatic breast cancer (GMC: 555 vs. 465 U/mL;p=0.031). However, patients who received Ad26.COV2.S had lower levels of mean spike antibody levels (GMC 47 U/ml), compared with patients treated with BNT162b2 (GMC 400 U/ml) or mRNA1273 (GMC 2203 U/mL;P<0.01 for both comparisons). Comparison of neutralization titers in 66 individuals supported the above results. 11 (15.1%) patients had low antibody titers (<100U/ml) of seroconversion and 3 received a booster vaccine, with 1 having available repeat titer results thus far demonstrating a significant improvement. Conclusions: The majority of patients receiving CDK 4/6 inhibitor have adequate antibody response to SARS-CoV-2 vaccines, particularly mRNA vaccines. However, a minority of patients may require booster vaccine to augment immunity. Monitoring spike antibody levels could be helpful to identify patients with inadequate seroconversion and guide mitigation strategies for patients with breast cancer.

T cell reactivity to the SARS-CoV-2 Omicron variant is preserved in most but not all prior infected and vaccinated individuals (preprint)

ABSTRACT The SARS-CoV-2 Omicron variant (B.1.1.529) contains mutations that mediate escape from infection and vaccine-induced antibody responses, although the extent to which these substitutions in spike and non-spike proteins affect T cell recognition is unknown. Here we show that T cell responses in individuals with prior infection, vaccination, both prior infection and vaccination, and boosted vaccination are largely preserved to Omicron spike and non-spike proteins. However, we also identify a subset of individuals (∼21%) with a >50% reduction in T cell reactivity to the Omicron spike. Evaluation of functional CD4 + and CD8 + memory T cell responses confirmed these findings and reveal that reduced recognition to Omicron spike is primarily observed within the CD8 + T cell compartment. Booster vaccination substantially enhanced T cell responses to Omicron spike. In contrast to neutralizing immunity, these findings suggest preservation of T cell responses to the Omicron variant, although with reduced reactivity in some individuals.

Preferential expansion upon boosting of cross-reactive “pre-existing” switched memory B cells that recognize the SARS-CoV-2 Omicron variant Spike protein (preprint)

In previously unvaccinated and uninfected individuals, non-RBD SARS-CoV-2 spike-specific B cells were prominent in two distinct, durable, resting, cross-reactive, “pre-existing” switched memory B cell compartments. While pre-existing RBD-specific B cells were extremely rare in uninfected and unvaccinated individuals, these two pre-existing switched memory B cell compartments were molded by vaccination and infection to become the primary source of RBD-specific B cells that are triggered by vaccine boosting. The frequency of wild-type RBD-binding memory B cells that cross-react with the Omicron variant RBD did not alter with boosting. In contrast, after a boost, B cells recognizing the full-length Omicron variant spike protein expanded, with pre-existing resting memory B cells differentiating almost quantitatively into effector B cell populations. B cells derived from “ancient” pre-existing memory cells and that recognize the full-length wild-type spike with the highest avidity after boosting are the B cells that also bind the Omicron variant spike protein. Abstract Figure

mRNA-based COVID-19 vaccine boosters induce neutralizing immunity against SARS-CoV-2 Omicron variant (preprint)

SUMMARY Recent surveillance has revealed the emergence of the SARS-CoV-2 Omicron variant (BA.1/B.1.1.529) harboring up to 36 mutations in spike protein, the target of vaccine-induced neutralizing antibodies. Given its potential to escape vaccine-induced humoral immunity, we measured neutralization potency of sera from 88 mRNA-1273, 111 BNT162b, and 40 Ad26.COV2.S vaccine recipients against wild type, Delta, and Omicron SARS-CoV-2 pseudoviruses. We included individuals that were vaccinated recently (<3 months), distantly (6-12 months), or recently boosted, and accounted for prior SARS-CoV-2 infection. Remarkably, neutralization of Omicron was undetectable in most vaccinated individuals. However, individuals boosted with mRNA vaccines exhibited potent neutralization of Omicron only 4-6-fold lower than wild type, suggesting that boosters enhance the cross-reactivity of neutralizing antibody responses. In addition, we find Omicron pseudovirus is more infectious than any other variant tested. Overall, this study highlights the importance of boosters to broaden neutralizing antibody responses against highly divergent SARS-CoV-2 variants.

Neutralization breadth of SARS CoV-2 viral variants following primary series and booster SARS CoV-2 vaccines in patients with cancer (preprint)

Patients with cancer are more likely to have impaired immune responses to SARS CoV-2 vaccines. We studied the breadth of responses against SARS CoV-2 variants followingly primary vaccination in 178 patients with a variety of tumor types, and after booster doses in a subset. Neutralization of alpha, beta, gamma and delta SARS-CoV-2 variants was impaired relative to wildtype (Wuhan), regardless of vaccine type. Regardless of viral variant, mRNA1273 was the most immunogenic, followed by BNT162b2 and then Ad26.COV2.S. Neutralization of more variants (breadth) was associated with higher magnitude of wildtype neutralization, and increase with time since vaccination; increased age associated with lower breadth. Anti-spike binding antibody concentrations were a good surrogate for breadth (PPV=90% at >1000U/ml). Booster SARS-CoV-2 vaccines conferred enhanced breadth. These data suggest that achieving a high antibody titer is desirable to achieve broad neutralization; a single booster dose with current vaccines increases breadth of responses against variants.

Comparative immunogenicity and effectiveness of mRNA-1273, BNT162b2 and Ad26.COV2.S COVID-19 vaccines (preprint)

Background Understanding immunogenicity and effectiveness of SARS-CoV-2 vaccines is critical to guide rational use. Methods We compared the immunogenicity of mRNA-1273, BNT-162b2 or Ad26.COV2.S in ambulatory adults in Massachusetts, USA. To correlate immunogenicity with effectiveness of the three vaccines, we performed an inverse-variance meta-analysis of population level effectiveness from public health reports in >40 million individuals. Results A single dose of either mRNA vaccine yielded comparable antibody and neutralization titers to convalescent individuals. Ad26.COV2.S yielded lower antibody concentrations and frequently negative neutralization titers. Bulk and cytotoxic T-cell responses were higher in mRNA1273 and BNT162b2 than Ad26.COV2.S recipients, and <50% of vaccinees demonstrate CD8+ T-cell responses to spike peptides. Antibody concentrations and neutralization titers increased comparably after the first dose of either vaccine, and further in recipients of a second dose. Prior infection was associated with high antibody concentrations and neutralization even after a single dose and regardless of vaccine. Neutralization of beta, gamma and delta strains were poorer regardless of vaccine. Relative to mRNA1273, the effectiveness of BNT162b2 was lower against infection and hospitalization; and Ad26COV2.S was lower against infection, hospitalization and death. Conclusions Variation in the immunogenicity correlates with variable effectiveness of the three FDA EUA vaccines deployed in the USA.

Circulating SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity (preprint)

Vaccination elicits immune responses capable of potently neutralizing SARS-CoV-2. However, ongoing surveillance has revealed the emergence of variants harboring mutations in spike, the main target of neutralizing antibodies. To understand the impact of globally circulating variants, we evaluated the neutralization potency of 48 sera from BNT162b2 and mRNA-1273 vaccine recipients against pseudoviruses bearing spike proteins derived from 10 strains of SARS-CoV-2. While multiple strains exhibited vaccine-induced cross-neutralization comparable to wild-type pseudovirus, 5 strains harboring receptor-binding domain mutations, including K417N/T, E484K, and N501Y, were highly resistant to neutralization. Cross-neutralization of B.1.351 variants was weak and comparable to SARS-CoV and bat-derived WIV1-CoV, suggesting that a relatively small number of mutations can mediate potent escape from vaccine responses. While the clinical impact of neutralization resistance remains uncertain, these results highlight the potential for variants to escape from neutralizing humoral immunity and emphasize the need to develop broadly protective interventions against the evolving pandemic.

GPS-estimated foot traffic data and venue selection for COVID-19 serosurveillance studies (preprint)

Tracking the dynamics and spread of COVID-19 is critical to mounting an effective response to the pandemic. In the absence of randomized representative serological surveys, many SARS-CoV-2 serosurveillance studies have relied on convenience sampling to estimate cumulative incidence. One common approach is to recruit at frequently visited community locations ("venue-based" sampling), but the sources of bias and uncertainty associated with this strategy are still poorly understood. Here, we used data from a venue-based community serosurveillance study, GPS-estimated foot traffic data, and data on confirmed COVID-19 cases to report an estimate of cumulative incidence in Somerville, Massachusetts, and a methodological strategy to quantify and reduce uncertainty in serology-based cumulative incidence estimates obtained via convenience sampling. The mismatch between the geographic distribution of participants' home locations (the "participant catchment distribution") and the geographic distribution of infections is an important determinant of uncertainty in venue-based and other convenience sampling strategies. We found that uncertainty in cumulative incidence estimates can vary by a factor of two depending how well the participant catchment distribution matches the known or expected geographic distribution of prior infections. GPS-estimated business foot traffic data provides an important proxy measure for the participant catchment area and can be used to select venue locations that minimize uncertainty in cumulative incidence.

TMPRSS2 and ADAM17 interactions with ACE2 complexed with SARS-CoV-2 and B0AT1 putatively in intestine, cardiomyocytes, and kidney (preprint)

COVID-19 outcomes reflect organ-specific interplay of SARS-CoV-2 and its receptor, ACE2, with TMPRSS2 and ADAM17. Confirmed active tropism of SARS-CoV-2 in epithelial cells of intestine and kidney proximal tubule, and in aging cardiomyocytes, capriciously manifests extra pulmonary organ-related clinical symptoms in about half of COVID-19 patients, occurring by poorly understood mechanisms. We approached this knowledge gap by recognizing a clue that these three particular cell types share a common denominator kindred of uniquely expressing the SLC6A19 neutral amino acid transporter B0AT1 protein (alternatively called NBB, B, B0) serving glutamine and tryptophan uptake. B0AT1 is a cellular trafficking chaperone partner of ACE2, shown by cryo-EM to form a thermodynamically-favored stabilized 2ACE2:2B0AT1 dimer-of-heterodimers. The gut is the body's site of greatest magnitude expression depot of both ACE2 and B0AT1. This starkly contrasts with pulmonary pneumocyte expression of monomeric ACE2 with conspicuously undetectable B0AT1. We hypothesized that B0AT1 steers the organ-related interplay amongst ACE2, TMPRSS2, ADAM17, and SARS-CoV-2 RBD. The present study employed molecular docking modeling that indicated active site catalytic pocket residues of TMPRSS2 and ADAM17 each formed bonds [≤] 2 [A] with monomer ACE2 specific residues within a span R652-D713 involved in cleaving sACE2 soluble ectodomain release. These bonds are consistent with competitive binding interactions of experimental anti-SARS-CoV-2 drug small molecules including Camostat and Nafamostat. Without B0AT1, ACE2 residues K657 and N699 dominated docking bonding with TMPRSS2 or ADAM17 active sites, with ACE2 R710 and R709 contributing electrostatic attractions, but notably ACE2 S708 never closer than 16-44 [A]. However, in the dimer-of-heterodimers arrangement all ACE2 neck region residues were limited to TMPRSS2 or ADAM17 approaches [≥]{approx} 35 [A], with the interference directly attributed to the presence of a neighboring B0AT1 subunit complexed to the partnering ACE2 subunit of 2ACE2:2B0AT1; ADAM17 failed to dock by bumping its active site pocket oriented dysfunctionally outwardly facing 180{degrees} away. Results were the same whether the dimer-of-heterodimers was in either the "closed" or "open" conformation, or whether or not SARS-CoV-2 RBD was complexed to ACE2. The results implicate B0AT1-and in particular the 2ACE2:2B0AT1 complex-as a major player in the landscape of COVID-19 pathophysiology engaging TMPRSS2 and ADAM17, consistent with experimental evidence in the literature and in clinical reports. These findings provide a gateway to understanding the roles of B0AT1 relating to COVID-19 manifestations putatively assigned to intestinal and renal epithelial cells and cardiomyocytes, with underpinnings useful for considerations in public hygiene policy and drug development.

An insight into SARS-CoV-2 Membrane protein interaction with Spike, Envelope, and Nucleocapsid proteins (preprint)

Intraviral protein-protein interactions are crucial for replication, pathogenicity, and viral assembly. Among these, virus assembly is a critical step as it regulates the arrangements of viral structural proteins and helps in the encapsulation of genomic material. SARS-CoV-2 structural proteins play an essential role in the self-rearrangement, RNA encapsulation, and mature virus particle formation. In SARS-CoV, the membrane protein interacts with the envelope and spike protein in Endoplasmic Reticulum Golgi Intermediate Complex (ERGIC) to form an assembly in the lipid bilayer, followed by membrane-ribonucleoprotein (nucleocapsid) interaction. In this study, using protein-protein docking, we tried to understand the interaction of membrane protein's interaction with envelope, spike and nucleocapsid proteins. Further, simulation studies performed up to 100ns agreed that protein complexes M-E, M-S, and M-N were stable. Moreover, the calculated free binding energy and dissociation constant values support the protein complex formation. The interaction identified in the study will be of great importance, as it provides valuable insight into the protein complex, which could be the potential drug targets for future studies.

Remote fingerstick blood collection for SARS-CoV-2 antibody testing (preprint)

The rapid worldwide spread of SARS-CoV-2 infection has propelled the rapid development of serological tests that can detect anti-SARS-CoV-2 antibodies. These have been used for studying the prevalence and spread of infection in different populations, helping establish a diagnosis of COVID-19, and will likely be used to confirm humoral immunity after infection or vaccination. However, nearly all lab-based high-throughput SARS-CoV-2 serological assays require a serum sample from venous blood draw, limiting their applications and scalability. Here, we present a method that enables large scale SARS-CoV-2 serological studies by combining self or office collection of fingerprick blood with an FDA-approved dried blood spot collection device (Neoteryx Mitra(R)) with a high-throughput electrochemiluminescence-based SARS-CoV-2 total antibody assay (Roche Elecsys(R)) that is EUA approved for use on serum samples and widely used by clinical laboratories around the world. We found that the Roche Elecsys(R) assay has a high dynamic range that allows for accurate detection of SARS-CoV-2 antibodies in serum samples diluted 1:20 as well as contrived dried blood extracts. Extracts of dried blood from Neoteryx Mitra(R) devices acquired in a community seroprevalence study showed near identical sensitivity and specificity in detection of SARS-CoV-2 antibodies as compared to neat sera using predefined thresholds for each specimen type. Overall, this study affirms the use of Neoteryx Mitra(R) dried blood collection device with the Roche Elecsys(R) SARS-CoV-2 total antibody assay for remote or at-home testing as well as large-scale community seroprevalence studies.

Boceprevir, calpain inhibitors II and XII, and GC-376 have broad-spectrum antiviral activity against coronaviruses in cell culture (preprint)

As the COVID-19 pandemic continues to fold out, the morbidity and mortality are increasing daily. Effective treatment for SARS-CoV-2 is urgently needed. We recently discovered four SARS-CoV-2 main protease (Mpro) inhibitors including boceprevir, calpain inhibitors II and XII and GC-376 with potent antiviral activity against infectious SARS-CoV-2 in cell culture. Despite the weaker enzymatic inhibition of calpain inhibitors II and XII against Mpro compared to GC-376, calpain inhibitors II and XII had more potent cellular antiviral activity. This observation promoted us to hypothesize that the cellular antiviral activity of calpain inhibitors II and XII might also involve the inhibition of cathepsin L in addition to Mpro. To test this hypothesis, we tested calpain inhibitors II and XII in the SARS-CoV-2 pseudovirus neutralization assay in Vero E6 cells and found that both compounds significantly decreased pseudoviral particle entry into cells, indicating their role in inhibiting cathepsin L. The involvement of cathepsin L was further confirmed in the drug time-of-addition experiment. In addition, we found that these four compounds not only inhibit SARS-CoV-2, but also SARS-CoV, MERS-CoV, as well as human coronaviruses (CoVs) 229E, OC43, and NL63. The mechanism of action is through targeting the viral Mpro, which was supported by the thermal shift binding assay and enzymatic FRET assay. We further showed that these four compounds have additive antiviral effect when combined with remdesivir. Altogether, these results suggest that boceprevir, calpain inhibitors II and XII, and GC-376 are not only promising antiviral drug candidates against existing human coronaviruses, but also might work against future emerging CoVs.

COVID-19 Neutralizing Antibodies Predict Disease Severity and Survival (preprint)

COVID-19 exhibits variable symptom severity ranging from asymptomatic to life-threatening, yet the relationship between severity and the humoral immune response is poorly understood. We examined antibody responses in 113 COVID-19 patients and found that severe cases resulting in intubation or death exhibited increased inflammatory markers, lymphopenia, and high anti-RBD antibody levels. While anti-RBD IgG levels generally correlated with neutralization titer, quantitation of neutralization potency revealed that high potency was a predictor of survival. In addition to neutralization of wild-type SARS-CoV-2, patient sera were also able to neutralize the recently emerged SARS-CoV-2 mutant D614G, suggesting protection from reinfection by this strain. However, SARS-CoV-2 sera was unable to cross-neutralize a highly-homologous pre-emergent bat coronavirus, WIV1-CoV, that has not yet crossed the species barrier. These results highlight the importance of neutralizing humoral immunity on disease progression and the need to develop broadly protective interventions to prevent future coronavirus pandemics.Funding: K.L.C. is supported by Ruth L. Kirschstein National Research Service Award (NRSA) Individual Postdoctoral Fellowship 1F32AI143480. T.M.C. and B.M.H. were supported by award Number T32GM007753 from the National Institute of General Medical Sciences. J.F. was supported by T32AI007245. A.G.S. was supported by NIH R01 AI146779 and a Massachusetts Consortium on Pathogenesis Readiness (MassCPR) grant. J.A.B. has received research support from Zeus Scientific, bioMerieux, Immunetics, Alere, DiaSorin, the Bay Area Lyme Foundation (BALF), and the National Institute of Allergy and Infectious Diseases (NIAID; Award 1R21AI119457-01) for unrelated projects. J.A.B. has served as a paid consultant to T2 Biosystems, DiaSorin and Roche Diagnostics. A.J.I. is supported by the Lambertus Family Foundation. A.B.B. is supported by the National Institutes for Drug Abuse (NIDA) Avenir New Innovator Award DP2DA040254, the MGH Transformative Scholars Program as well as funding from the Charles H. Hood Foundation. This independent research was supported by the Gilead Sciences Research Scholars Program in HIV. Ethical Approval: Use of patient samples for the development and validation of SARS-CoV-2 diagnostic tests was approved by Partners Institutional Review Board (protocol 2020P000895).

COVID-19 neutralizing antibodies predict disease severity and survival (preprint)

COVID-19 exhibits variable symptom severity ranging from asymptomatic to life-threatening, yet the relationship between severity and the humoral immune response is poorly understood. We examined antibody responses in 113 COVID-19 patients and found that severe cases resulting in intubation or death exhibited increased inflammatory markers, lymphopenia, and high anti-RBD antibody levels. While anti-RBD IgG levels generally correlated with neutralization titer, quantitation of neutralization potency revealed that high potency was a predictor of survival. In addition to neutralization of wild-type SARS-CoV-2, patient sera were also able to neutralize the recently emerged SARS-CoV-2 mutant D614G, suggesting protection from reinfection by this strain. However, SARS-CoV-2 sera was unable to cross-neutralize a highly-homologous pre-emergent bat coronavirus, WIV1-CoV, that has not yet crossed the species barrier. These results highlight the importance of neutralizing humoral immunity on disease progression and the need to develop broadly protective interventions to prevent future coronavirus pandemics.

Dynamics and significance of the antibody response to SARS-CoV-2 infection (preprint)

BACKGROUND Characterizing the humoral immune response to SARS-CoV-2 and developing accurate serologic assays are needed for diagnostic purposes and estimating population-level seroprevalence. METHODS We measured the kinetics of early antibody responses to the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2 in a cohort of 259 symptomatic North American patients infected with SARS-CoV-2 (up to 75 days after symptom onset) compared to antibody levels in 1548 individuals whose blood samples were obtained prior to the pandemic. RESULTS Between 14-28 days from onset of symptoms, IgG, IgA, or IgM antibody responses to RBD were all accurate in identifying recently infected individuals, with 100% specificity and a sensitivity of 97%, 91%, and 81% respectively. Although the estimated median time to becoming seropositive was similar across isotypes, IgA and IgM antibodies against RBD were short-lived with most individuals estimated to become seronegative again by 51 and 47 days after symptom onset, respectively. IgG antibodies against RBD lasted longer and persisted through 75 days post-symptoms. IgG antibodies to SARS-CoV-2 RBD were highly correlated with neutralizing antibodies targeting the S protein. No cross-reactivity of the SARS-CoV-2 RBD-targeted antibodies was observed with several known circulating coronaviruses, HKU1, OC 229 E, OC43, and NL63. CONCLUSIONS Among symptomatic SARS-CoV-2 cases, RBD-targeted antibodies can be indicative of previous and recent infection. IgG antibodies are correlated with neutralizing antibodies and are possibly a correlate of protective immunity.

Clinical Sensitivity and Interpretation of PCR and Serological COVID-19 Diagnostics for Patients Presenting to the Hospital (preprint)

Introduction: The diagnosis of COVID-19 requires integration of clinical and laboratory data. SARS-CoV-2 diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. Interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. Our goal was to examine the clinical sensitivity of two most common SARS-CoV-2 diagnostic test modalities, polymerase chain reaction (PCR) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. Methods: A single-center, retrospective study. To derive clinical sensitivity of PCR, we identified 209 PCR-positive SARS-CoV-2 patients with multiple PCR test results (624 total PCR tests) and calculated daily sensitivity from date of symptom onset or first positive test. To calculate daily clinical sensitivity by serology, we utilized 157 PCR-positive patients with a total of 197 specimens tested by enzyme-linked immunosorbent assay for IgM, IgG, and IgA anti-SARS-CoV-2 antibodies. Results: Clinical sensitivity of PCR decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70-71% from days 9-11, and 30% at day 21. In contrast, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after day 7, >80% after day 12, and 100% by day 21. Conclusion: PCR and serology are complimentary modalities that require time-dependent interpretation. Superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection.

Analytical performance of lateral flow immunoassay for SARS-CoV-2 exposure screening on venous and capillary blood samples (preprint)

Objectives: Numerous serologic immunoassays have been launched to detect antibodies to SARS-CoV-2, including rapid tests. Here, we validate use of a lateral flow immunoassay (LFI) intended for rapid screening and qualitative detection of anti-SARS-CoV-2 IgM and IgG in serum, plasma, and whole blood, and compare results with ELISA. We also seek to establish the value of LFI testing on blood obtained from a capillary blood sample. Methods: Samples collected by venous blood draw and capillary finger stick were obtained from patients with SARS-CoV-2 detected by RT-qPCR and control patients negative for SARS-CoV-2. Samples were tested with the 2019-nCoV IgG/IgM Detection Kit (Colloidal Gold) lateral flow immunoassay, and antibody calls were compared with results obtained by ELISA. Results: The Biolidics LFI kit shows clinical sensitivity of 92% at 7 days after PCR diagnosis of SARS-CoV-2 on venous blood. Test specificity was 92% for IgM and 100% for IgG. There was no significant difference in detecting IgM and IgG with Biolidics LFI and ELISA at D0 and D7 (p=1.00), except for detection of IgM at D7 (p=0.04). Finger stick whole blood of SARS-CoV-2 patients showed 93% sensitivity for antibody detection. Conclusions: Clinical performance of Biolidics 2019-nCoV IgG/IgM Detection Kit (Colloidal Gold) is comparable to ELISA and showed consistent results across different sample types. Furthermore, we show that capillary blood obtained by finger stick shows similar sensitivity for detecting anti-SARS-CoV-2 IgM and IgG antibodies as venous blood samples. This provides an opportunity for decentralized rapid testing in the community and may allow point-of-care and longitudinal self-testing for the presence of anti-SARS-CoV-2 antibodies.

Test performance evaluation of SARS-CoV-2 serological assays (preprint)

Background Serological tests are crucial tools for assessments of SARS-CoV-2 exposure, infection and potential immunity. Their appropriate use and interpretation require accurate assay performance data. Method We conducted an evaluation of 10 lateral flow assays (LFAs) and two ELISAs to detect anti-SARS-CoV-2 antibodies. The specimen set comprised 128 plasma or serum samples from 79 symptomatic SARS-CoV-2 RT-PCR-positive individuals; 108 pre-COVID-19 negative controls; and 52 recent samples from individuals who underwent respiratory viral testing but were not diagnosed with Coronavirus Disease 2019 (COVID-19). Samples were blinded and LFA results were interpreted by two independent readers, using a standardized intensity scoring system. Results Among specimens from SARS-CoV-2 RT-PCR-positive individuals, the percent seropositive increased with time interval, peaking at 81.8-100.0% in samples taken >20 days after symptom onset. Test specificity ranged from 84.3-100.0% in pre-COVID-19 specimens. Specificity was higher when weak LFA bands were considered negative, but this decreased sensitivity. IgM detection was more variable than IgG, and detection was highest when IgM and IgG results were combined. Agreement between ELISAs and LFAs ranged from 75.7-94.8%. No consistent cross-reactivity was observed. Conclusion Our evaluation showed heterogeneous assay performance. Reader training is key to reliable LFA performance, and can be tailored for survey goals. Informed use of serology will require evaluations covering the full spectrum of SARS-CoV-2 infections, from asymptomatic and mild infection to severe disease, and later convalescence. Well-designed studies to elucidate the mechanisms and serological correlates of protective immunity will be crucial to guide rational clinical and public health policies.