Letter to the Editor

Serological markers of SARS-CoV-2 infection; anti-nucleocapsid antibody positivity may not be the ideal marker of natural infection in vaccinated individuals

The prevalence and strength of serological responses mounted toward SARS-CoV-2 proteins other than nucleocapsid (N) and spike (S), which may be of use as additional serological markers, remains underexplored. Using high-content microscopy to assess antibody responses against full-length StrepTagged SARS-CoV-2 proteins, we found that 85% (166/196) of unvaccinated individuals with RT-PCR confirmed SARS-CoV-2 infections and 74% (31/42) of individuals infected after being vaccinated developed detectable IgG against the structural protein M, which is higher than previous estimates. Compared with N antibodies, M IgG displayed a shallower time-dependent decay and greater specificity. Sensitivity for SARS-CoV-2 seroprevalence was enhanced when N and M IgG detection was combined. These findings indicate that screening for M seroconversion may be a good approach for detecting additional vaccine breakthrough infections and highlight the potential to use HCM as a rapidly deployable method to identify the most immunogenic targets of newly emergent pathogens.

Introduction of the Omicron variant caused a steep rise in SARS-CoV-2 infections despite high vaccination coverage in the Danish population. We used blood donor serosurveillance to estimate the percentage of recently infected residents in the similarly aged background population with no known comorbidity.

To detect SARS-CoV-2 antibodies induced due to recent infection, and not vaccination, we assessed anti-nucleocapsid (anti-N) immunoglobulin G (IgG) in blood donor samples. Individual level data on SARS-CoV-2 RT-PCR results and vaccination status were available. Anti-N IgG was measured fortnightly from January 18 to April 3, 2022. Samples from November 2021 were analysed to assess seroprevalence before introduction of the Omicron variant in Denmark.

A total of 43 088 donations from 35 309 Danish blood donors aged 17–72 years were screened. In November 2021, 1·2% (103/8 701) of donors had detectable anti-N IgG antibodies. Adjusting for test sensitivity (estimates ranging from 74%–81%) and November seroprevalence, we estimate that 66% (95% confidence intervals (CI): 63%–70%) of the healthy, similarly aged Danish population had been infected between November 1, 2021, and March 15, 2022. One third of infections were not captured by SARS-CoV-2 RT-PCR testing. The infection fatality rate (IFR) was 6·2 (CI: 5·1–7·5) per 100 000 infections.

Screening for anti-N IgG and linkage to national registers allowed us to detect recent infections and accurately assess assay sensitivity in vaccinated or previously infected individuals during the Omicron outbreak. The IFR was lower than during previous waves.

The Danish Ministry of Health.

Older adults in nursing homes are at greatest risk of morbidity and mortality from SARS-CoV-2 infection. Nursing home residents constituted one-third to more than half of all deaths during the early waves of the COVID-19 pandemic. Following this, widespread adaptation of infection prevention and control measures and the supply and use of personal protective equipment resulted in a significant decrease in nursing home infections and deaths. For nursing homes, the most important determinant of experiencing a SARS-CoV-2 outbreak in the first instance appears to be community-transmission levels (particularly with variants of concern), although nursing home size and quality, for-profit status, and sociodemographic characteristics are also important. Use of visitation bans, imposed to reduce the impact of COVID-19 on residents, must be delicately balanced against their impact on resident, friend or family, and staff well-being. The successful rollout of primary vaccination has resulted in a sharp decrease in morbidity and mortality from SARS-CoV-2 in nursing homes. However, emerging evidence suggests that vaccine efficacy may wane over time, and the use of a third or additional vaccine “booster” doses in nursing home residents restores protection afforded by primary vaccination. Ongoing monitoring of vaccine efficacy in terms of infection, morbidity, and mortality is crucial in this vulnerable group in informing ongoing SARS-CoV-2 vaccine boosting strategies. Here, we detail the impact of SARS-CoV-2 on nursing home residents and discuss important considerations in the management of nursing home SARS-CoV-2 outbreaks. We additionally examine the use of testing strategies, nonpharmacologic outbreak control measures and vaccination strategies in this cohort. Finally, the impact of SARS-CoV-2 on the sector is reflected on as we emphasize the need for adoption of universal standards of medical care and integration with wider public health infrastructure in nursing homes in order to provide a safe and effective long-term care sector.

Background Serosurveillance can be used to investigate the extent and distribution of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a population. Characterisation of humoral immune responses gives insight into whether immunity is infection- or vaccine-derived.

Methods A longitudinal study of health care workers (HCWs) in Dili, Timor-Leste, was conducted during vaccine rollout (ChAdOx1) and a concurrent SARS-CoV-2 outbreak.

Results A total of 324 HCWs were included at baseline (April-May 2021). Out of those, 32 (9.9%) were seropositive for anti-nucleocapsid protein (anti-N) IgG antibodies, indicating a significant sub-clinical infection among HCWs early in the local outbreak. Follow-up was conducted in 157 (48.5%) participants (July-September 2021), by which time there had been high uptake of vaccination (91.7%), and 86.0% were seropositive for anti-spike protein antibodies. Acquisition of anti-N antibodies was observed in partially vaccinated HCWs (30/76, 39.5%), indicating some post-dose-1 infections.

Discussion Serosurveillance of HCWs may provide early warning of SARS-CoV-2 outbreaks and should be considered in non-endemic settings, particularly where there is limited availability/uptake of testing for acute infection. Characterisation of humoral immune responses may be used to assess vaccine impact and coverage. Such studies should be considered in national and international efforts to investigate and mitigate against future emerging pathogens.

With COVID-19 vaccine roll-out ongoing in many countries globally, monitoring of breakthrough infections is of great importance. Antibodies persist in the blood after a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Since COVID-19 vaccines induce immune response to the Spike protein of the virus, which is the main serosurveillance target to date, alternative targets should be explored to distinguish infection from vaccination.

Multiplex immunoassay data from 1,513 SARS-CoV-2 RT-qPCR-tested individuals (352 positive and 1,161 negative) without COVID-19 vaccination history were used to determine the accuracy of Nucleoprotein-specific immunoglobulin G (IgG) in detecting past SARS-CoV-2 infection. We also described Spike S1 and Nucleoprotein-specific IgG responses in 230 COVID-19 vaccinated individuals (Pfizer/BioNTech).

The sensitivity of Nucleoprotein seropositivity was 85% (95% confidence interval: 80–90%) for mild COVID-19 in the first two months following symptom onset. Sensitivity was lower in asymptomatic individuals (67%, 50–81%). Participants who had experienced a SARS-CoV-2 infection up to 11 months preceding vaccination, as assessed by Spike S1 seropositivity or RT-qPCR, produced 2.7-fold higher median levels of IgG to Spike S1 ≥ 14 days after the first dose as compared to those unexposed to SARS-CoV-2 at ≥ 7 days after the second dose (p = 0.011). Nucleoprotein-specific IgG concentrations were not affected by vaccination in infection-naïve participants.

Serological responses to Nucleoprotein may prove helpful in identifying SARS-CoV-2 infections after vaccination. Furthermore, it can help interpret IgG to Spike S1 after COVID-19 vaccination as particularly high responses shortly after vaccination could be explained by prior exposure history.