Ratio between IgG against the spike protein of seasonal coronaviruses and SARS- CoV-2 is associated with a fatal outcome of hospitalized COVID-19 patients (preprint)

SARS-CoV-2 infection elicits a cross-reactive and back boosting immune response from prior exposure to any of the human seasonal coronaviruses (HCoVs), but is has yet to be determined whether this response is associated with fatal outcome of severe COVID-19 disease. In a cohort of 58 hospitalized patients, we have shown that heterologous immune responses are associated with increased disease severity. Here, we report that a lower anti-spike IgG ratio of SARS-CoV-2 to HCoVs, in particular to the human coronavirus 229E, correlates with decreased neutralizing antibody titers, and is associated with a fatal disease course. These results support the hypothesis that pre-existing humoral immunity to spike antigens of HCoVs may impair an efficient immune response during COVID-19.

Protection of COVID-19 vaccination and previous infection against Omicron BA.1 and Delta SARS-CoV-2 infections, the Netherlands, 22 November 2021- 19 January 2022 (preprint)

Given the emergence of the SARS-CoV-2 Omicron BA.1 variant and the roll-out of booster COVID-19 vaccination, evidence is needed on protection conferred by primary vaccination, booster vaccination and previous SARS-CoV-2 infection against Omicron BA.1 compared with Delta infection. We employed a test-negative design and used multinomial logistic regression on data from community PCR testing in the Netherlands, from 22 November 2021 to 19 January 2022. S-gene target failure (SGTF) was used as proxy for Omicron BA.1 infection versus Delta. A total of 528,488 tests were included, of which 38,975 SGTF and 41,245 non-SGTF infections. Protection from primary vaccination was 25% (95% confidence interval (CI): 21-29) and from previous infection 33% (95% CI: 31-35) against Omicron BA.1 infection. Protection against Delta infection was higher with 76% (95% CI: 75-76) for primary vaccination and 78% (95% CI: 76-80) for previous infection. Higher protection was observed in individuals with both primary vaccination and earlier infection compared with either one. Waning of vaccine- or infection-induced protection over time was observed against both variants. Booster vaccination considerably increased vaccine effectiveness against Omicron BA.1 to 76% (95% CI: 72-79) and 68% (95% CI: 67-69) with and without previous infection, respectively. Primary vaccination with current COVID-19 vaccines and pre-Omicron SARS-CoV-2 infections offer low protection against Omicron BA.1 infection. Booster vaccination considerably increases protection against Omicron BA.1, although protection remains lower than against Delta.

Shorter serial intervals in SARS-CoV-2 cases with Omicron variant compared to Delta variant in the Netherlands, 13 - 19 December 2021 (preprint)

The SARS-CoV-2 Omicron variant has a growth advantage over the Delta variant, due to higher transmissibility, immune evasion, or a shorter serial interval. Using S-gene target failure (SGTF) as indication for Omicron, we identify 220 SGTF and 869 non-SGTF serial intervals in the same week. Within households, we find a mean serial interval of 3.4 days for SGTF and 3.9 days for non-SGTF cases. This suggests that the growth advantage of Omicron is partly due to a shorter serial interval.

SARS-CoV-2 Omicron efficiently infects human airway, but not alveolar epithelium (preprint)

In late 2021, the highly mutated SARS-CoV-2 Omicron variant emerged, raising concerns about its potential extensive immune evasion, increased transmissibility and pathogenicity. Here, we used organoids of the human airways and alveoli to investigate Omicron's fitness and replicative potential in comparison with earlier SARS-CoV-2 variants. We report that Omicron replicates more rapidly in the airways and has an increased fitness compared to the early 614G variant and Delta. In contrast, Omicron did not replicate productively in human alveolar type 2 cells. Mechanistically, we show that Omicron does not efficiently use TMPRSS2 for entry or spread through cell-cell fusion. Altogether, our data show that Omicron has an altered tropism and protease usage, potentially explaining its higher transmissibility and decreased pathogenicity.

Mapping the antigenic diversification of SARS-CoV-2 (preprint)

Large-scale vaccination campaigns have prevented countless SARS-CoV-2 infections, hospitalizations and deaths. However, the emergence of variants that escape from immunity challenges the effectiveness of current vaccines. Given this continuing evolution, an important question is when and how to update SARS-CoV-2 vaccines to antigenically match circulating variants, similar to seasonal influenza viruses where antigenic drift necessitates periodic vaccine updates. Here, we studied SARS-CoV-2 antigenic drift by assessing neutralizing activity against variants-of-concern (VOCs) of a unique set of sera from patients infected with a range of VOCs. Infections with ancestral or Alpha strains induced the broadest immunity, while individuals infected with other VOCs had more strain-specific responses. Omicron was substantially resistant to neutralization by sera elicited by all other variants. Antigenic cartography revealed that all VOCs preceding Omicron belong to one antigenic cluster, while Omicron forms a new antigenic cluster associated with immune escape and likely requiring vaccine updates to ensure vaccine effectiveness.

Incidence rates and symptomatology of community infections with SARS-CoV-2 in children and parents: The CoKids longitudinal household study (preprint)

AIMThe CoKids study aimed to estimate the community incidence of symptomatic and asymptomatic SARS-CoV-2 in children and parents and to assess the symptomatology of SARS-COV-2 infections relative to SARS-CoV-2 negative respiratory episodes. METHODSIn this prospective study, households with at least one child <18 years were recruited from three existing Dutch cohorts. Participation included SARS-CoV-2 screening at 4-6 weeks intervals for all household members during 23 weeks of follow-up and active reporting of new onset respiratory symptoms until July 1st 2021. Follow-up was temporarily intensified following new onset respiratory symptoms in a household member or a SARS-CoV-2 positive screening test and included daily symptom recording, repeated PCR testing (nose-throat, saliva and fecal samples) and SARS-CoV-2 antibody measurement (paired dried blood spots) in all household members. Age-stratified incidence rates for SARS-CoV-2 positive and negative episodes were calculated. Symptomatology and disease burden of respiratory episodes were compared by SARS-CoV-2 status and age. RESULTSIn total 307 households were enrolled including 1209 subjects. We detected 64 SARS-CoV-2 positive and 118 SARS-CoV-2 negative respiratory outbreaks. The highest incidence rate was found in children <12 years for SARS-CoV-2 negative episodes (0.93/ person-year (PY); 95%CI: 0.88-0.96). The SARS-CoV-2 incidence in this age-group was 0.21/PY for confirmed only, and 0.41/PY if probable cases were included. SARS-CoV-2 incidence did not differ by age group (p>0.27). Nasal congestion/runny nose, with or without cough and fatigue were the three most prevalent symptom clusters for both SARS-CoV-2 positive and negative respiratory episodes. Among children, no differences were observed in the symptomatology and severity of SARS-CoV-2 positive versus negative respiratory episodes, whereas among adults, SARS-CoV-2 positive episodes had a higher number and severity of symptoms and with a longer duration p<0.001). CONCLUSIONUsing active, longitudinal household follow up, we detected a high incidence rate of SARS-CoV-2 infections in children that was similar to adults. The findings suggest that after 20 months of COVID-19 pandemic, up to 2/3 of Dutch children < 12 years have been infected with SARS-CoV-2. Symptomatology and disease severity of SARS-CoV-2 in children is similar to respiratory illness from other causes. In adults, SARS-COV-2 positive episodes are characterized by more and prolonged symptoms, and higher severity. These findings may assist decisions on COVID-19 policies targeting children.

Increased risk of infection with SARS-CoV-2 Beta, Gamma, and Delta variant compared to Alpha variant in vaccinated individuals (preprint)

The extent to which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) break through infection- or vaccine-induced immunity is not well understood. Here, we analyze 28,578 sequenced SARS-CoV-2 samples from individuals with known immune status obtained through national community testing in the Netherlands from March to August 2021. We find evidence for an increased risk of infection by the Beta (B.1.351), Gamma (P.1), or Delta (B.1.617.2) variants compared to the Alpha (B.1.1.7) variant after vaccination. No clear differences were found between vaccines. However, the effect was larger in the first 14-59 days after complete vaccination compared to 60 days and longer. In contrast to vaccine-induced immunity, no increased risk for reinfection with Beta, Gamma or Delta variants relative to Alpha variant was found in individuals with infection-induced immunity.

SARS-CoV-2 RNA and antibody dynamics in a Dutch household study with dense sampling frame (preprint)

This study investigated the dynamics of SARS-CoV-2 infection and diagnostics in household members of different ages and with different symptom severity after SARS-CoV-2 exposure during the early phase of the pandemic. Households with a SARS-CoV-2 confirmed positive case and at least one child in the Netherlands were followed for 6 weeks. Naso (NP)- and oropharyngeal (OP) swabs, oral fluid and feces specimens were analyzed for SARS-CoV-2 RNA and serum for SARS-CoV-2-specific antibodies. The dynamics of the presence of viral RNA and the serological response was modeled to determine the sampling time-frame and sample type with the highest sensitivity to confirm or reject a SARS-CoV-2 diagnosis. Transmission of SARS-CoV-2 between adults and children within a household was correlated with symptom severity of index cases. In children higher viral loads compared to adults were detected at symptom onset. Early in infection, higher viral loads were detected in NP and OP specimens, while RNA in especially feces were longer detectable. SARS-CoV-2-specific antibodies have a 90% probability of detection from 7 days (total Ig) and 18 days (IgG) since symptom onset. In conclusion this study has shown that on average, children carry higher loads of virus as compared to adults early after infection. For highest probability of detection in SARS-CoV-2 diagnostics early in infection, RT-PCR on NP and OP specimens are more sensitive than on oral fluid and feces. For SARS-CoV-2 diagnostics late after infection, RT-PCR on feces specimens and serology are more valuable.

Potential environmental transmission routes of SARS-CoV-2 inside a large meat processing plant experiencing COVID-19 clusters (preprint)

Worldwide exceptionally many COVID-19 clusters were observed in meat processing plants. Many contributing factors, promoting transmission, were suggested, including climate conditions in cooled production rooms favorable for environmental transmission but actual sampling studies are lacking. We aimed to assess SARS-CoV-2 contamination of air and surfaces to gain insight in potential environmental transmission in a large Dutch meat processing plant experiencing COVID-19 clusters. We performed SARS-CoV-2 screening of workers operating in cooled production rooms and intensive environmental sampling during a two-week study period in June 2020. Sampling of air (both stationary and personal), settling dust, ventilation systems, and sewage was performed. Swabs were collected from high-touch surfaces and workers hands. Screening of workers was done using oronasopharyngeal swabs. Samples were tested for presence of SARS-CoV-2 RNA by RT-qPCR. Of the 76 (predominantly asymptomatic) workers tested, 27 (35.5%) were SARS-CoV-2 RNA positive with modest to low viral loads (Ct[≥]29.7). In total, 6 out of 203 surface swabs were positive (Ct [≥]38), being swabs taken from communal touchscreens/handles. One of the 12 personal air samples and one of the 4 sewage samples were positive, RNA levels were low (Ct[≥]38). All other environmental samples tested negative. Although one-third of workers tested SARS-CoV-2 RT-PCR positive, environmental contamination was limited. Hence widespread transmission of SARS-CoV-2 via air and surfaces was considered unlikely within this plant at the time of investigation in the context of strict COVID-19 control measures in place.

High efficacy of therapeutic equine hyperimmune antibodies against SARS CoV-2 variants of concern (preprint)

SARS-CoV-2 variants of concern (VoC) show reduced neutralization by vaccine-induced and therapeutic monoclonal antibodies. We tested therapeutic equine polyclonal antibodies (pAbs) against four VoC (alpha, beta, epsilon and gamma). We show that equine pAbs efficiently neutralize VoC, suggesting they are an effective, broad coverage, low-cost and a scalable COVID-19 treatment.

Emerging SARS-CoV-2 variants of concern evade humoral immune responses from infection and vaccination (preprint)

Emerging SARS-CoV-2 variants pose a threat to human immunity induced by natural infection and vaccination. We assessed the recognition of three variants of concern (B.1.1.7, B.1.351 and P.1) in cohorts of COVID-19 patients ranging in disease severity (n = 69) and recipients of the Pfizer/BioNTech vaccine (n = 50). Spike binding and neutralization against all three VOC was substantially reduced in the majority of samples, with the largest 4-7-fold reduction in neutralization being observed against B.1.351. While hospitalized COVID-19 patients and vaccinees maintained sufficient neutralizing titers against all three VOC, 39% of non-hospitalized patients did not neutralize B.1.351. Moreover, monoclonal neutralizing antibodies (NAbs) show sharp reductions in their binding kinetics and neutralizing potential to B.1.351 and P.1, but not to B.1.1.7. These data have implications for the degree to which pre-existing immunity can protect against subsequent infection with VOC and informs policy makers of susceptibility to globally circulating SARS-CoV-2 VOC.

Serological Evidence for Reinfection with SARS-CoV-2; An Observational Cohort Study (preprint)

Background: Cases of reinfection with SARS-CoV-2 are reported in increasing numbers. Since the required molecular proof of reinfection is challenging to obtain, we aimed to provide serological evidence of reinfection in a cohort of potential reinfection cases.Methods: The study comprises 38 RT-PCR confirmed reinfection cases, with a COVID-19 symptom-free interval of at least 8 weeks (range 57-133 days) since their first RT-PCR confirmed episode. Specific disease symptoms were retrieved from 22 cases by contact tracing and compared between the two disease episodes. The oropharyngeal specimens from 13 cases enabled adequate genomic sequence comparisons. Seventeen cases provided a serum specimen, of which 12 within 7 days after onset of symptoms. Antibody determinations included SARS-CoV-2-specific total Ig, IgM, IgG, avidity, and virus neutralization. Antibody data were compared to that of a control group of primary cases (n=86) in relation to time since onset of disease symptoms.Findings: Reinfection cases generally experienced fewer or milder symptoms. Five of 8 cases which passed genomic comparison between both disease episodes showed reinfection with a different lineage. From 12 reinfection cases that provided a serum sample within 7 days after onset of symptoms, 11/12 (92%) and 12/12 (100%) showed high levels of specific total Ig and IgG antibodies, respectively, compared to 1/23 (4%) and 2/23 (9%) within the control group. Virus neutralizing antibodies were detected in 9/12 (75%) reinfection cases, 5 of which were above a titer of 30. Serological discrimination diminished after 7 days, except for IgG avidity; all 17 reinfection cases had antibodies of higher avidity when compared to control cases.Interpretation: IgG concentration and avidity can be used as an additional diagnostic marker to confirm reinfection with SARS-Cov-2. Reinfection cases that show a rapid and effective secondary immune response are expected to clear the infection more effectively, thereby reducing contagiousness and clinical severity. Understanding this reinfection response is also important for breakthrough infections following vaccination.Funding Statement: This work was supported by the National Institute for Public Health and the Environment, The Netherlands.Declaration of Interests: None of the authors have an association that poses a conflict of interest.Ethics Approval Statement: Approved by the Medical-Ethical Review Committee of the University Medical Center Utrecht.

High infection attack rates of SARS-CoV-2 in Dutch households revealed by dense sampling (preprint)

Background Indoor environments are considered a main setting for transmission of SARS-CoV-2. Households in particular present a close-contact environment with high probability of transmission between persons of different ages and with different roles in society. Methods Complete households with a laboratory-confirmed SARS-CoV-2 positive case in the Netherlands (March-May 2020) were included. At least three home visits were performed during 4-6 week of follow-up, collecting naso- and oropharyngeal swabs, oral fluid, faeces and blood samples for molecular and serological analyses of all household members. Symptoms were recorded from two weeks before the first visit up to the last visit. Secondary attack rates (SAR) were estimated with logistic regression. A transmission model was used to assess transmission routes in the household. Results A total of 55 households with 187 household contacts were included. In 17 households no transmission took place, and in 11 households all persons were infected. Estimated SARs were high, ranging from 35% (95%CI: 24%-46%) in children to 51% (95%CI: 39%-63%) in adults. Estimated transmission rates in the household were high, with reduced susceptibility of children compared to adolescents and adults (0.67; 95%CI: 0.40-1.1). Conclusion Estimated SARs were higher than reported in earlier household studies, presumably owing to a dense sampling protocol. Children were shown to be less susceptible than adults, but the estimated SAR in children was still high. Our results reinforce the role of households as main multiplier of SARS-CoV-2 infection in the population. Key points We analyze data from a SARS-CoV-2 household study and find higher secondary attack rates than reported earlier. We argue that this is due to a dense sampling strategy that includes sampling at multiple time points and of multiple anatomical sites.

Dynamics of antibodies to SARS-CoV-2 in convalescent plasma donors (preprint)

The novel SARS-CoV-2 virus emerged in late 2019 and has caused a global health and economic crisis. The characterization of the human antibody response to SARS-CoV-2 infection is vital for serosurveillance purposes as well for treatment options such as transfusion with convalescent plasma or immunoglobin products derived from convalescent plasma. In this study, we measured antibody responses in 844 longitudinal samples from 151 RT-PCR positive SARS-CoV-2 convalescent adults during the first 34 weeks after onset of symptoms. All donors were seropositive at the first sampling moment and only one donor seroreverted during follow-up analysis. Anti-RBD IgG and anti-nucleocapsid IgG levels slowly declined with median half-lifes of 62 and 59 days during 2-5 months after symptom onset, respectively. The rate of decline of antibody levels diminished during extended follow-up. In addition, the magnitude of the IgG response correlated with neutralization capacity measured in a classic plaque reduction assay as well in our in-house developed competition assay. The result of this study gives valuable insight into the longitudinal response of antibodies to SARS-CoV-2.

Validation and clinical evaluation of a SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT) (preprint)

To understand SARS-CoV-2 immunity after natural infection or vaccination, functional assays such as virus neutralizing assays are needed. So far, assays to determine SARS-CoV-2 neutralizing antibodies rely on cell-culture based infection assays either using wild type SARS-CoV-2 or pseudotyped viruses. Such assays are labour-intensive, require appropriate biosafety facilities and are difficult to standardize. Recently, a new surrogate virus neutralisation assay (sVNT) was described that uses the principle of an ELISA to measure the neutralization capacity of anti-SARS-CoV-2 antibodies directed against the receptor binding domain. Here, we performed an independent evaluation of the robustness, specificity and sensitivity on an extensive panel of sera from 269 PCR-confirmed COVID-19 cases and 259 unmatched samples collected before 2020 and compared it to cell-based neutralization assays. We found a high specificity of 99.2 (95%CI: 96.9-99.9) and overall sensitivity of 80.3 (95%CI: 74.9-84.8) for the sVNT. Clinical sensitivity increased between early (<14 days post symptom onset or post diagnosis, dpos/dpd) and late sera (>14 dpos/dpd) from 75.0 (64.7-83.2) to 83.1 (76.5-88.1). Also, higher severity was associated with an increase in clinical sensitivity. Upon comparison with cell-based neutralisation assays we determined an analytical sensitivity of 74.3 (56.4-86.9) and 98.2 (89.4-99.9) for titres [≥]10 to <40 and [≥]40 to <160, respectively. Only samples with a titre [≥]160 were always positive in the sVNT. In conclusion, the sVNT can be used as an additional assay to determine the immune status of COVID-19 infected of vaccinated individuals but its value needs to be assessed for the specific context of use.

Differences in antibody kinetics and functionality between severe and mild SARS-CoV-2 infections. (preprint)

We determined and compared the humoral immune response in severe, hospitalized and mild, non-hospitalized COVID-19 patients. Severe patients (n=38) develop a robust antibody response to SARS-CoV-2, including IgG and IgA antibodies. The geometric mean 50% virus neutralization titer is 1:240. SARS-CoV-2 infected hospital personnel (n=24), who developed mild symptoms necessitating leave of absence, self-isolation, but not hospitalization, 75 % develop antibodies, but with low/absent virus neutralization (60% < 1:20). While severe COVID-19 patients develop a strong antibody response, mild SARS-CoV-2 infections induce a modest antibody response. Long term monitoring will show whether these responses predict protection against future infections.