Although infections among vaccinated individuals lead to milder COVID-19 symptoms relative to unvaccinated subjects, the specificity and durability of antibody responses elicited by breakthrough cases remain unknown. Here, we demonstrate that breakthrough infections induce serum binding and neutralizing antibody responses that are markedly more potent, durable and resilient to spike mutations observed in variants than those in subjects who received only two doses of vaccine. However, we show that breakthrough cases, subjects who were vaccinated after infection and individuals vaccinated three times have serum neutralizing activity of comparable magnitude and breadth, indicating that increased number of exposures to SARS-CoV-2 antigen(s) enhance the quality of antibody responses. Neutralization of SARS-CoV was moderate, however, underscoring the importance of developing vaccines eliciting broad sarbecovirus immunity for pandemic preparedness.

Highlights The expected 100 copies/mL LOD reported in the EUA Abbott RealTi m e SARS-CoV-2 assay product insert was exceeded. All clinical samples positive for 24 non‒SARS-CoV-2 respiratory viruses were SARS-CoV-2 negative on the RealTi m e assay. The assay had high sensitivity (93%) and specificity (100%) for detecting SARS-CoV-2 in clinical samples.

While diagnosis of COVID-19 relies on qualitative molecular testing for the absence or presence of SARS-CoV-2 RNA, quantitative viral load determination for SARS-CoV-2 has many potential applications in antiviral therapy and vaccine trials as well as implications for public health and quarantine guidance. To date, no quantitative SARS-CoV-2 viral load tests have been authorized for clinical use by the FDA. In this study, we modified the FDA emergency use authorized qualitative RealTi m e SARS-CoV-2 assay into a quantitative SARS-CoV-2 Laboratory Developed Test (LDT) using newly developed Abbott SARS-CoV-2 calibration standards. Both analytical and clinical performance of this SARS-CoV-2 quantitative LDT was evaluated using nasopharyngeal swabs (NPS). We further assessed the correlation between Ct and the ability to culture virus on Vero CCL81 cells. The SARS-CoV-2 quantitative LDT demonstrated high linearity with R 2 value of 0.992, high inter- and intra-assay reproducibility across the dynamic range (SDs ±0.08-0.14 log 10 copies/mL for inter-assay reproducibility and ±0.09 to 0.19 log 10 copies/mL for intra-assay reproducibility). Lower limit of detection was determined as 1.90 log 10 copies/mL. The highest Ct at which CPE was detected ranged between 28.21-28.49, corresponding to approximately 4.2 log 10 copies/mL. Quantitative tests, validated against viral culture capacity, may allow more accurate identification of individuals with and without infectious viral shedding from the respiratory tract.

The SARS-CoV-2 Delta variant is currently responsible for most infections worldwide, including among vaccinated individuals. Although these latter infections lead to milder COVID-19 disease relative to unvaccinated subjects, the specificity and durability of antibody responses elicited by Delta breakthrough cases remain unknown. Here, we demonstrate that breakthrough infections induce serum binding and neutralizing antibody responses that are markedly more potent, durable and resilient to spike mutations observed in variants than those observed in subjects who were infected only or received only two doses of vaccine. We show that Delta breakthrough cases, subjects who were vaccinated after infection and individuals vaccinated three times (without infection) have serum neutralizing activity of comparable magnitude and breadth, indicating that multiple types of exposure or increased number of exposures to SARS-CoV-2 antigen(s) enhance antibody responses. Neutralization of SARS-CoV, however, was moderate, underscoring the importance of developing vaccines eliciting broad sarbecovirus immunity for pandemic preparedness.

Background Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectious virus isolation in outpatients with coronavirus disease 2019 (COVID-19) has been associated with viral RNA levels and symptom duration, little is known about the host, disease, and viral determinants of infectious virus detection. Methods COVID-19 adult outpatients were enrolled within 7 days of symptom onset. Clinical symptoms were recorded via patient diary. Nasopharyngeal swabs were collected to quantitate SARS-CoV-2 RNA by reverse transcriptase polymerase chain reaction and for infectious virus isolation in Vero E6-cells. SARS-CoV-2 antibodies were measured in serum using a validated ELISA assay. Results Among 204 participants with mild-to-moderate symptomatic COVID-19, the median nasopharyngeal viral RNA was 6.5 (interquartile range [IQR] 4.7–7.6 log10 copies/mL), and 26% had detectable SARS-CoV-2 antibodies (immunoglobulin (Ig)A, IgM, IgG, and/or total Ig) at baseline. Infectious virus was recovered in 7% of participants with SARS-CoV-2 antibodies compared to 58% of participants without antibodies (prevalence ratio [PR] = 0.12, 95% confidence interval [CI]: .04, .36; P = .00016). Infectious virus isolation was also associated with higher levels of viral RNA (mean RNA difference +2.6 log10, 95% CI: 2.2, 3.0; P < .0001) and fewer days since symptom onset (PR = 0.79, 95% CI: .71, .88 per day; P < .0001). Conclusions The presence of SARS-CoV-2 antibodies is strongly associated with clearance of infectious virus. Seropositivity and viral RNA levels are likely more reliable markers of infectious virus clearance than subjective measure of COVID-19 symptom duration. Virus-targeted treatment and prevention strategies should be administered as early as possible and ideally before seroconversion. Clinical Trials Registration NCT04405570.