Progressive loss of conserved spike protein neutralizing antibody sites in Omicron sublineages is balanced by preserved T-cell recognition epitopes (preprint)

The continued evolution of the SARS-CoV-2 Omicron variant has led to the emergence of numerous sublineages with different patterns of evasion from neutralizing antibodies. We investigated neutralizing activity in immune sera from individuals vaccinated with SARS-CoV-2 wild-type spike (S) glycoprotein-based COVID-19 mRNA vaccines after subsequent breakthrough infection with Omicron BA.1, BA.2, or BA.4/BA.5 to study antibody responses against sublineages of high relevance. We report that exposure of vaccinated individuals to infections with Omicron sublineages, and especially with BA.4/BA.5, results in a boost of Omicron BA.4.6, BF.7, BQ.1.1, and BA.2.75 neutralization, but does not efficiently boost neutralization of sublineages BA.2.75.2 and XBB. Accordingly, we found in in silico analyses that with occurrence of the Omicron lineage a large portion of neutralizing B-cell epitopes were lost, and that in Omicron BA.2.75.2 and XBB less than 12% of the wild-type strain epitopes are conserved. In contrast, HLA class I and class II presented T-cell epitopes in the S glycoprotein were highly conserved across the entire evolution of SARS-CoV-2 including Alpha, Beta, and Delta and Omicron sublineages, suggesting that CD8+ and CD4+ T-cell recognition of Omicron BQ.1.1, BA.2.75.2, and XBB may be largely intact. Our study suggests that while some Omicron sublineages effectively evade B-cell immunity by altering neutralizing antibody epitopes, S protein-specific T-cell immunity, due to the very nature of the polymorphic cell-mediated immune, response is likely to remain unimpacted and may continue to contribute to prevention or limitation of severe COVID-19 manifestation.

Omicron BA.2 breakthrough infection enhances cross-neutralization of BA.2.12.1 and BA.4/BA.5 (preprint)

Recently, we reported that BNT162b2-vaccinated individuals after Omicron BA.1 breakthrough infection have strong serum neutralizing activity against Omicron BA.1, BA.2, and previous SARS-CoV-2 variants of concern (VOCs), yet less against the highly contagious Omicron sublineages BA.4 and BA.5 that have displaced previous variants. As the latter sublineages are derived from Omicron BA.2, we characterized serum neutralizing activity of COVID-19 mRNA vaccine triple-immunized individuals who experienced BA.2 breakthrough infection. We demonstrate that sera of these individuals have broadly neutralizing activity against previous VOCs as well as all tested Omicron sublineages, including BA.2 derived variants BA.2.12.1, BA.4/BA.5. Furthermore, applying antibody depletion we showed that neutralization of BA.2 and BA.4/BA.5 sublineages by BA.2 convalescent sera is driven to a significant extent by antibodies targeting the N-terminal domain (NTD) of the spike glycoprotein, whereas their neutralization by Omicron BA.1 convalescent sera depends exclusively on antibodies targeting the receptor binding domain (RBD). These findings suggest that exposure to Omicron BA.2, in contrast to BA.1 spike glycoprotein, triggers significant NTD specific recall responses in vaccinated individuals and thereby enhances the neutralization of BA.4/BA.5 sublineages. Given the current epidemiology with a predominance of BA.2 derived sublineages like BA.4/BA.5 and rapidly ongoing evolution, these findings are of high relevance for the development of Omicron adapted vaccines.

Omicron breakthrough infection drives cross-variant neutralization and memory B cell formation (preprint)

Omicron is the evolutionarily most distinct SARS-CoV-2 variant (VOC) to date and displays multiple amino acid alterations located in neutralizing antibody sites of the spike (S) protein. We report here that Omicron breakthrough infection in BNT162b2 vaccinated individuals results in strong neutralizing activity not only against Omicron, but also broadly against previous SARS-CoV-2 VOCs and against SARS-CoV-1. We found that Omicron breakthrough infection mediates a robust B cell recall response, and primarily expands preformed memory B cells that recognize epitopes shared broadly by different variants, rather than inducing new B cells against strictly Omicron-specific epitopes. Our data suggest that, despite imprinting of the immune response by previous vaccination, the preformed B cell memory pool has sufficient plasticity for being refocused and quantitatively remodeled by exposure to heterologous S protein, thus allowing effective neutralization of variants that evade a previously established neutralizing antibody response.

Characterization of antibody and T-cell response after second booster vaccination (preprint)

The recent surge of infections with SARS-CoV-2 Omicron subvariants of prompted countries, such as Israel and Germany, to call for an accelerated booster vaccination program for health care workers and vulnerable groups in order to limit disease and transmission. However, detailed studies analyzing the correlates of protection over time after second booster vaccination are still lacking. Here, we examined the production of Spike receptor binding domain (RBD) -specific antibodies as well as neutralizing antibodies from subjects before, two, and seven weeks after the second booster vaccination against the D614G harboring B.1 variant as well as the variants of concern (VOC) Alpha, Beta, Delta in addition to Omicron BA.1 and BA.2. The second booster vaccination resulted in an increase in anti-RBD IgG antibodies and neutralizing antibodies against B.1 in all individuals tested, then remained nearly constant over the observed period. In addition, a 2nd booster resulted in an increase in neutralizing antibodies against VOCs Alpha, Beta, Delta, and Omicron subvariants BA.1 and BA.2. However, compared to B.1 the neutralizing capacity of both Omicron subvariants remained low. Neutralization of Omicron BA.1 and BA.2 was limited even after the 2nd booster vaccination indicating that an antibody-mediated protection against infection with this VOC is unlikely, as evidenced by the fact that three of the quadruple vaccinated individuals became infected with BA.1 during the course of the study. Moreover, T cell activation measured by interferon gamma release was detected in all subjects after the 2nd booster vaccination. This may offer protection suggesting protection against severe disease. T-cell activation was independent of the age of the subjects, but correlated with the amount of Spike-specific antibodies. Interestingly, in subjects with Omicron BA.1 breakthrough infection, a significant increase in neutralizing antibodies to all tested VOCs studied was observed after the 2nd booster vaccination. Taken together, our data suggest inferior protection from breakthrough infection with the Omicron subvariant BA.1 when compared to other VOCs after four vaccine doses.

Reduced Neutralization of SARS-CoV-2 Omicron Variant by Vaccine Sera and monoclonal antibodies (preprint)

Due to numerous mutations in the spike protein, the SARS-CoV-2 variant of concern Omicron (B.1.1.529) raises serious concerns since it may significantly limit the antibody-mediated neutralization and increase the risk of reinfections. While a rapid increase in the number of cases is being reported worldwide, until now there has been uncertainty about the efficacy of vaccinations and monoclonal antibodies. Our in vitro findings using authentic SARS-CoV-2 variants indicate that in contrast to the currently circulating Delta variant, the neutralization efficacy of vaccine-elicited sera against Omicron was severely reduced highlighting T-cell mediated immunity as essential barrier to prevent severe COVID-19. Since SARS-CoV-2 Omicron was resistant to casirivimab and imdevimab, genotyping of SARS-CoV-2 may be needed before initiating mAb treatment. Variant-specific vaccines and mAb agents may be required to treat COVID-19 due to Omicron and other emerging variants of concern.

Characterization of the humoral immune response to BNT162b2 in elderly residents of long-term care facilities five to seven months after vaccination (preprint)

The elderly residing in long-term care facilities (LTCFs) are a group at high risk for COVID-19. Hence, monitoring of the vaccine-based immunity has a pivotal role in identifying strategies to provide optimal protection in this population. We examined the immune response to the mRNA vaccine BNT162b2 against COVID-19 five to seven months after completing a two-dose regimen. We determined significantly lower anti-SARS-CoV-2 antibody titers in 298 SARS-CoV-2 naive residents who were at least 75 years of age (mean 51.60 BAU/ml) (median age 87 years, range 75 to 101 years) when compared to health care workers (HCWs) aged 18 to 70 years (mean 156.99 BAU/ml, p < 0.001). Of the SARS-CoV-2 naive residents, 29 had detectable neutralizing antibodies against the Delta variant (9.5%), and 14 of those (48.3%) only had a borderline titer of 1:10. Of 114 HCWs, 36 (31.6%) had detectable neutralizing antibodies. In a group of 14 elderly residents who had had a PCR-confirmed breakthrough infection, the mean antibody titer was significantly higher than in the other two groups (3199.65 BAU/mL) (p < 0.001), and 12 (85.7%) had detectable neutralizing antibodies against the Delta variant. Our data demonstrate that 90.5% of elderly residents of LTCFs had no detectable neutralization-competent antibodies against the dominant Delta variant five to seven months after vaccination, and that neutralizing antibody titers were restored following a break-through infection. Our results suggest that both residents and health care workers in LTCFs would benefit from a booster vaccine six months after completing the two-dose schedule or earlier.

Longitudinal testing for SARS-CoV-2 RNA in day care centers in Hesse, Germany, during increased local incidence and with VOC Alpha as dominant variant: Results of the SAFE KiDS 2 and SAFE KiDS 3 study (preprint)

In the summer of 2020, we investigated the rate of inapparent shedding of SARS-CoV-2 in a representative sample of day care centers from Hesse, Germany, and found a low positivity rate during a period of low local community spread. To investigate the influence of a high local incidence setting, we conducted the SAFE KiDS 2 study. 577 children and 334 staff members of 47 daycare centers were tested for respiratory and gastrointestinal shedding of SARS-CoV-2, and three infections with SARS-CoV-2 in the infectious period were detected. We conclude that viral shedding occurred infrequently while the original "wild-type" variant was dominant. The more transmissible SARS-CoV-2 variant Alpha (B.1.1.7) became the dominant strain after the SAFE KiDS 2 was concluded. The SAFE KiDS 3 study investigated the impact of the Alpha variant of SARS-Co-2 on inapparent viral shedding in the day care setting. In this study, 756 children and 226 staff members from 46 day care centers provided self-collected saliva swabs, the so-called "Lollipop" swabs, which were tested by RT-PCR. In the four-week study period, none of the participants tested positive for SARS-CoV-2 RNA, demonstrating that inapparent shedding of SARS-CoV-2 in the day care setting was also rare during the dominance of the Alpha variant. The influence of the variant of concern Delta on day care centers has yet to be examined.

Bamlanivimab does not neutralize two SARS-CoV-2 variants carrying E484K in vitro (preprint)

The IgG1 monoclonal antibody (mAb) bamlanivimab (LY-CoV555) prevents viral attachment and entry into human cells by blocking attachment to the ACE2 receptor. However, whether bamlanivimab is equally effective against SARS-CoV-2 emerging variants of concern (VOC) is not fully known. Hence, the aim of this study was to determine whether bamlanivimab is equally effective against SARS-CoV-2 emerging VOC. The ability of bamlanivimab to neutralize five SARS-CoV-2 variants including B.1.1.7 (mutations include N501Y and del69/70), B.1.351 (mutations include E484K and N501Y) and P.2 (mutations include E484K in the absence of a N501Y mutation) was analyzed in infectious cell culture using CaCo2 cells. Additionally, we analyzed vaccine-elicited sera after immunization with BNT162b2, and convalescent sera for its ability to neutralize SARS-CoV-2 variants. We found that the variant B.1.1.7, as well as two isolates from early 2020 (FFM1 and FFM7) could be efficiently neutralized by bamlanivimab (titer 1/1280, respectively), however, no neutralization effect could be detected against either B.1.135 or P.2, both harboring the E484K substitution. Vaccine-elicited sera showed slightly decreased neutralizing activity against B1.1.7, B.1.135 and P.2 Our in vitro findings indicate that, in contrast to vaccine-elicited sera, bamlanivimab may not provide efficacy against SARS-CoV-2 variants harboring the E484K substitution. Confirmation of the SARS-CoV-2 variant, including screening for E484K, may be needed before initiating mAb treatment with bamlanivimab to ensure both efficacious and efficient use of the antibody product. Hence, variant-specific mAb agents may be required to treat emerging VOC.

Evaluation of a SARS-CoV-2 rapid antigen test: potential to help reduce community spread? (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can spread from symptomatic patients with COVID-19, but also from asymptomatic individuals. Therefore, robust surveillance and timely interventions are essential for the control of virus spread within the community. In this regard the frequency of testing and speed of reporting, but not the test sensitivity alone, play a crucial role. In order to reduce the costs and meet the expanding demands in real-time RT-PCR (rRT-PCR) testing for SARS-CoV-2, complementary assays, such as rapid antigen tests, have been developed. Rigorous analysis under varying conditions is required to assess the clinical performance of these tests and to ensure reproducible results. We evaluated the sensitivity and specificity of a recently licensed rapid antigen test using 137 clinical samples in two institutions. Test sensitivity was between 88.2-89.6% when applied to samples with viral loads typically seen in infectious patients. Of 32 rRT-PCR positive samples, 19 demonstrated infectivity in cell culture, and 84% of these samples were reactive with the antigen test. Seven full-genome sequenced SARS-CoV-2 isolates and SARS-CoV-1 were detected with this antigen test, with no cross-reactivity against other common respiratory viruses. Numerous antigen tests are available for SARS-CoV-2 testing and their performance to detect infectious individuals may vary. Head-to-head comparison along with cell culture testing for infectivity may prove useful to identify better performing antigen tests. The antigen test analyzed in this study is easy-to-use, inexpensive, and scalable. It can be helpful in monitoring infection trends and thus has potential to reduce transmission.

At-home self-testing of teachers with a SARS-CoV-2 rapid antigen test to reduce potential transmissions in schools (preprint)

BackgroundRapid antigen tests for SARS-CoV-2 became available recently, offering an opportunity to vastly increase testing capacities. Antigen tests offer lower sensitivity than the gold standard, RT-PCR, but rapid sample-to-answer time. High-frequency testing with an antigen test may offset the lower sensitivity, and testing can be done with at-home collection of samples, offering potential benefit in screening efforts. In this study, we set out to evaluate the practical application of self-performed high-frequency antigen test in a school setting. MethodA total of 711 teachers from 86 schools were enrolled in a seven-week study. After instruction, participants tested themselves every 48 hours at home with a rapid antigen test for SARS-CoV-2 (target: nucleocapsid protein) in a self-collected anterior nasal swab. Positive results in the antigen test were confirmed via RT-PCR from the same sample that had been determined to be positive by the study participant. A questionnaire was given to all participants to evaluate whether the test failed to detect infection. Findings10 836 tests from 602 teachers were recorded and analyzed. A total of five confirmed cases of viral shedding of SARS-CoV-2 was detected by use of the antigen test. One study participant with a SARS-CoV-2 infection was presymptomatic and four were mildly symptomatic at the time of the antigen test. Sixteen false positive antigen tests (0.15% of all tests) were reported, predominantly when the local incidence in the general population was low. In four cases, the study participant reported that a PCR had detected a SARS-CoV-2 infection, but the antigen test was negative, indicating a false negative result. InterpretationHigh-frequency, self-performed rapid antigen tests can detect individuals with a SARS-CoV-2 infection, and therefore potentially reduce transmissions. Testing may be most beneficial when applied during high local incidence of SARS-CoV-2 infections and when mild or atypical symptoms are present. To avoid a high rate of false positive results, a test with optimized specificity should be used. FundingThe study was commissioned and funded by the Hessian Ministry of Education and the Hessian Ministry of Integration and Social Affairs.

Longitudinal Testing of Respiratory and Gastrointestinal Shedding of SARS-CoV-2 in Day Care Centres in Hesse, Germany: Results of the SAFE KiDS-Study (preprint)

Background: With the pandemic of SARS-CoV-2 ongoing in Europe in July of 2020, day care centres were reopened in the state of Hesse, Germany, after the lockdown. The role young children play in the dynamics of the transmission was unknown.Methods: We conducted a longitudinal study over a period of 12 weeks (18th of June 2020 to 10th of September, 2020) to screen attendees and staff from day care centres in the state of Hesse, Germany, for both respiratory and gastrointestinal shedding of SARS-CoV-2. 825 children (age range 3 months to 8 years) and 372 staff members from 50 day-care centres, which were chosen representatively from throughout the state, participated in the study. Parents were asked to perform both a buccal mucosa and an anal swab on their children once a week. Staff were asked to self-administer the swabs. RT-PCRs for SARS-CoV-2 were performed in a multiple-swab pooling protocol.Findings: 7,366 buccal mucosa swabs and 5,907 anal swabs were analysed. No respiratory or gastrointestinal shedding of SARS-CoV-2 was detected in any of the children. Shedding of SARS-CoV-2 could be detected in two staff members from distinct day care centres. Case 1 was a 37 year old female who was asymptomatic at the time of testing. The second case was a 63 year old female who was symptomatic.Interpretation: Respiratory or gastrointestinal shedding of SARS-CoV-2 in children or staff members in day-care centres was very rare in the context of low community activity. The data indicate day care centres do not pose a reservoir for SARS-CoV-2 in a low prevalence setting, no inapparent transmissions were observed.Funding Statement: The study was commissioned by the Hessian Ministry of Social Affairs and Integration and was supported by Roche, Basel, Switzerland.Declaration of Interests: None to declare.Ethics Approval Statement: This study protocol was approved by the ethics board of the University Hospital Frankfurt, Goethe University Frankfurt am Main, Germany.

Longitudinal testing for respiratory and gastrointestinal shedding of SARS-CoV-2 in day care centres in Hesse, Germany. Results of the SAFE KiDS Study (preprint)

Abstract Background With the pandemic of SARS-CoV-2 ongoing in Europe in July of 2020, day care centres were reopened in the state of Hesse, Germany, after the lockdown. The role young children play in the dynamics of the transmission was unknown. Methods We conducted a longitudinal study over a period of 12 weeks (18th of June 2020 to 10th of September, 2020) to screen attendees and staff from day care centres in the state of Hesse, Germany, for both respiratory and gastrointestinal shedding of SARS-CoV-2. 825 children (age range 3 months to 8 years) and 372 staff members from 50 day care centres, which were chosen representatively from throughout the state, participated in the study. Parents were asked to perform both a buccal mucosa and an anal swab on their children once a week. Staff were asked to self-administer the swabs. RT-PCRs for SARS-CoV-2 were performed in a multiple-swab pooling protocol. Findings 7,366 buccal mucosa swabs and 5,907 anal swabs were analysed. No respiratory or gastrointestinal shedding of SARS-CoV-2 was detected in any of the children. Shedding of SARS-CoV-2 could be detected in two staff members from distinct day care centres. One was asymptomatic at the time of testing, and one was symptomatic. Interpretation Respiratory or gastrointestinal shedding of SARS-CoV-2 in children or staff members in day care centres was very rare in the context of low community activity. The data indicate day care centres do not pose a reservoir for SARS-CoV-2 in a low prevalence setting, no inapparent transmissions were observed. Funding The study was commissioned by the Hessian Ministry of Social Affairs and Integration and was supported by Roche Diagnostics, Basel, Switzerland

Evaluation of stability and inactivation methods of SARS-CoV-2 in context of laboratory settings (preprint)

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Laboratory work with SARS-CoV-2 in a laboratory setting was rated to biosafety level 3 (BSL-3) biocontainment level. However, certain research applications in particular in molecular biology require incomplete denaturation of the proteins, which might cause safety issues handling contaminated samples. In particular, it is critical to provide proof of inactivation before samples can be removed from the BSL-3. In this study, we evaluated common lysis buffers that are used in molecular biological laboratories for their ability to inactivate SARS-CoV-2. We have found that guanidine thiocyanate, SDS, and Triton-X containing lysis buffers were effective in inactivation of SARS-CoV-2, however, the M-PER lysis buffer containing a proprietary detergent failed to inactivate SARS-CoV-2. Furthermore, we compared chemical and non-chemical inactivation methods including ethanol, acetone-methanol mixture, PFA, UV-C light, and heat inactivation. In addition, the stability of the virus in cell culture media at 4{degrees}C and on surfaces used in laboratory environment was analyzed. In conclusion, careful evaluation of the used inactivation methods are required and additional inactivation steps are necessary before removal of lysed viral samples from BSL-3.

FACT- Frankfurt adjusted COVID-19 testing- a novel method enables high-throughput SARS-CoV-2 screening without loss of sensitivity (preprint)

Background: In the pandemic, testing for SARS-CoV-2 by RT-PCR in one of the pillars on which countermeasures are based. Factors limiting the output of laboratories interfere with the effectiveness of public health measures. Conserving reagents by pooling samples in low-probability settings is proposed, but may cause dilution and loss of sensitivity. Methods: We tested an alternate approach (FACT) by simultaneously incubating multiple respiratory swabs in a single tube. This protocol was evaluated by serial incubation of a respiratory swab in up to 10 tubes. The analytics validity of this concept was demonstrated in a five-sample mini pool set-up. It was consequently applied in the testing of 50 symptomatic patients (five-sample pools) as well as 100 asymptomatic residents of a nursing home (ten-sample pools). Results: Serial incubation of a respiratory swab in up to 10 tubes did not lead to a significant decline in viral concentration. The novel FACT-protocol did not cause a false negative result in a five-sample mini-pool setup, with non-significantly differing Ct values between single sample and mini-pool NAT. In two routine applications, all mini pools containing positive patient samples were correctly identified. Conclusions: Our proposed FACT- protocol did not cause a significant loss in analytic or diagnostic sensitivity compared to single sample testing in multiple setups. It reduced the amount of reagents needed by up to 40%, and also reduced hands-on time. This method could enhance testing efficiency, especially in groups with a low pretest-probability, such as systemically relevant professional groups.

Optimized qRT-PCR approach for the detection of intra- and extracellular SARS-CoV-2 RNAs (preprint)

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19 which has become a global concern due to its rapid spread. Meanwhile, increased demand in testing has led to shortage of reagents, supplies, and compromised the performance of diagnostic laboratories in many countries. Both the world health organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate interpretation of the test results especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in the diagnostics as well as in research labs using a low cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infection and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.