Surrogate Neutralization Assay Research Articles

No difference in anti-spike antibody and surrogate viral neutralization following SARS-CoV-2 booster vaccination in persons with HIV compared to controls (CO-HIV Study).

Understanding the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination will enable accurate counseling and inform evolving vaccination strategies. Little is known about antibody response following booster vaccination in people living with HIV (PLWH). We enrolled SARS-CoV-2 vaccinated PLWH and controls without HIV in similar proportions based on age and comorbidities. Participants completed surveys on prior SARS-CoV-2 infection, vaccination, and comorbidities, and provided self-collected dried blood spots (DBS). Quantitative anti-spike IgG and surrogate viral neutralization assays targeted wild-type (WT), Delta, and Omicron variants. We also measured quantitative anti-nucleocapsid IgG. The analysis population had received full SARS-CoV-2 vaccination plus one booster dose. Bivariate analyses for continuous outcomes utilized Wilcoxon tests and multivariate analysis used linear models. The analysis population comprised 140 PLWH and 75 controls with median age 58 and 55 years, males 95% and 43%, and DBS collection on 112 and 109 days after the last booster dose, respectively. Median CD4 count among PLWH was 760 cells/mm3 and 91% had an undetectable HIV-1 viral load. Considering WT, Delta, and Omicron variants, there was no significant difference in mean quantitative anti-spike IgG between PLWH (3.3, 2.9, 1.8) and controls (3.3, 2.9, 1.8), respectively (p-values=0. 771, 0.920, 0.708). Surrogate viral neutralization responses were similar in PLWH (1.0, 0.9, and 0.4) and controls (1.0, 0.9, 0.5), respectively (p-values=0.594, 0.436, 0.706). PLWH whose CD4 counts are well preserved and persons without HIV have similar anti-spike IgG antibody levels and viral neutralization responses after a single SARS-CoV-2 booster vaccination.

Validation and Establishment of the SARS-CoV-2 Lentivirus Surrogate Neutralization Assay as a Prescreening Tool for the Plaque Reduction Neutralization Test.

Neutralization assays are important for understanding and quantifying neutralizing antibody responses toward severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 lentivirus surrogate neutralization assay (SCLSNA) can be used in biosafety level 2 (BSL-2) laboratories and has been shown to be a reliable alternative approach to the plaque reduction neutralization test (PRNT). In this study, we optimized and validated the SCLSNA to assess its ability as a comparator and prescreening method to support the PRNT. Comparability between the PRNT and SCLSNA was determined through clinical sensitivity and specificity evaluations. Clinical sensitivity and specificity assays produced acceptable results, with 100% (95% confidence interval [CI], 94% to 100%) specificity and 100% (95% CI, 94% to 100%) sensitivity against ancestral Wuhan spike-pseudotyped lentivirus. The sensitivity and specificity against B.1.1.7 spike-pseudotyped lentivirus were 88.3% (95% CI, 77.8% to 94.2%) and 100% (95% CI, 94% to 100%), respectively. Assay precision measuring intra-assay variability produced acceptable results for high (50% PRNT [PRNT50], 1:≥640), mid (PRNT50, 1:160), and low (PRNT50, 1:40) antibody titer concentration ranges based on the PRNT50, with coefficients of variation (CVs) of 14.21%, 12.47%, and 13.28%, respectively. Intermediate precision indicated acceptable ranges for the high and mid concentrations, with CVs of 15.52% and 16.09%, respectively. However, the low concentration did not meet the acceptance criteria, with a CV of 26.42%. Acceptable ranges were found in the robustness evaluation for both intra-assay and interassay variability. In summary, the validation parameters tested met the acceptance criteria, making the SCLSNA method fit for its intended purpose, which can be used to support the PRNT. IMPORTANCE Neutralization studies play an important role in providing guidance and justification for vaccine administration and helping prevent the spread of diseases. The neutralization data generated in our laboratory have been included in the decision-making process of the National Advisory Committee on Immunization (NACI) in Canada. During the coronavirus 2019 (COVID-19) pandemic, the plaque reduction neutralization test (PRNT) has been the gold standard for determining neutralization of SARS-CoV-2. We validated a SARS-CoV-2 lentivirus surrogate neutralization assay (SCLSNA) as an alternative method to help support the PRNT. The advantages of using the SCLSNA is that it can process more samples, is less tedious to perform, and can be used in laboratories with a lower biosafety level. The use of the SCLSNA can further expand our capabilities to help fulfill the requirements for NACI and other important collaborations.

Seroprevalence of anti-SARS-CoV-2 antibodies and cross-variant neutralization capacity after the Omicron BA.2 wave in Geneva, Switzerland: a population-based study.

More than two years into the COVID-19 pandemic, most of the population has developed anti-SARS-CoV-2 antibodies from infection and/or vaccination. However, public health decision-making is hindered by the lack of up-to-date and precise characterization of the immune landscape in the population. Here, we estimated anti-SARS-CoV-2 antibodies seroprevalence and cross-variant neutralization capacity after Omicron became dominant in Geneva, Switzerland. We conducted a population-based serosurvey between April 29 and June 9, 2022, recruiting children and adults of all ages from age-stratified random samples of the general population of Geneva, Switzerland. We tested for anti-SARS-CoV-2 antibodies using commercial immunoassays targeting either the spike (S) or nucleocapsid (N) protein, and for antibody neutralization capacity against different SARS-CoV-2 variants using a cell-free Spike trimer-ACE2 binding-based surrogate neutralization assay. We estimated seroprevalence and neutralization capacity using a Bayesian modeling framework accounting for the demographics, vaccination, and infection statuses of the Geneva population. Among the 2521 individuals included in the analysis, the estimated total antibodies seroprevalence was 93.8% (95% CrI 93.1-94.5), including 72.4% (70.0-74.7) for infection-induced antibodies. Estimates of neutralizing antibodies in a representative subsample (N=1160) ranged from 79.5% (77.1-81.8) against the Alpha variant to 46.7% (43.0-50.4) against the Omicron BA.4/BA.5 subvariants. Despite having high seroprevalence of infection-induced antibodies (76.7% [69.7-83.0] for ages 0-5 years, 90.5% [86.5-94.1] for ages 6-11 years), children aged <12 years had substantially lower neutralizing activity than older participants, particularly against Omicron subvariants. Overall, vaccination was associated with higher neutralizing activity against pre-Omicron variants. Vaccine booster alongside recent infection was associated with higher neutralizing activity against Omicron subvariants. While most of the Geneva population has developed anti-SARS-CoV-2 antibodies through vaccination and/or infection, less than half has neutralizing activity against the currently circulating Omicron BA.5 subvariant. Hybrid immunity obtained through booster vaccination and infection confers the greatest neutralization capacity, including against Omicron. General Directorate of Health in Geneva canton, Private Foundation of the Geneva University Hospitals, European Commission ("CoVICIS" grant), and a private foundation advised by CARIGEST SA.

Antibody Avidity Maturation Following Recovery From Infection or the Booster Vaccination Grants Breadth of SARS-CoV-2 Neutralizing Capacity.

Cross-neutralizing capacity of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is important in mitigating (re-)exposures. Role of antibody maturation, the process whereby selection of higher affinity antibodies augments host immunity, to determine SARS-CoV-2 neutralizing capacity was investigated. Sera from SARS-CoV-2 convalescents at 2, 6, or 10 months postrecovery, and BNT162b2 vaccine recipients at 3 or 25 weeks postvaccination, were analyzed. Anti-spike IgG avidity was measured in urea-treated ELISAs. Neutralizing capacity was assessed by surrogate neutralization assays. Fold change between variant and wild-type neutralization inferred the breadth of neutralizing capacity. Compared with early-convalescent, avidity indices of late-convalescent sera were significantly higher (median, 37.7 [interquartile range 28.4-45.1] vs 64.9 [57.5-71.5], P < .0001). Urea-resistant, high-avidity IgG best predicted neutralizing capacity (Spearman r = 0.49 vs 0.67 [wild-type]; 0.18-0.52 vs 0.48-0.83 [variants]). Higher-avidity convalescent sera better cross-neutralized SARS-CoV-2 variants (P < .001 [Alpha]; P < .01 [Delta and Omicron]). Vaccinees only experienced meaningful avidity maturation following the booster dose, exhibiting rather limited cross-neutralizing capacity at week 25. Avidity maturation was progressive beyond acute recovery from infection, or became apparent after the booster vaccine dose, granting broader anti-SARS-CoV-2 neutralizing capacity. Understanding the maturation kinetics of the 2 building blocks of anti-SARS-CoV-2 humoral immunity is crucial.

SARS-CoV-2 Neutralizing Responses in Various Populations, at the Time of SARS-CoV-2 Variant Virus Emergence: Evaluation of Two Surrogate Neutralization Assays in Front of Whole Virus Neutralization Test.

The SARS-CoV-2 neutralizing antibodies response is the best indicator of effective protection after infection and/or vaccination, but its evaluation requires tedious cell-based experiments using an infectious virus. We analyzed, in 105 patients with various histories of SARS-CoV-2 infection and/or vaccination, the neutralizing response using a virus neutralization test (VNT) against B.1, Alpha, Beta and Omicron variants, and compared the results with two surrogate assays based on antibody-mediated blockage of the ACE2-RBD interaction (Lateral Flow Boditech and ELISA Genscript). The strongest response was observed for recovered COVID-19 patients receiving one vaccine dose. Naïve patients receiving 2 doses of mRNA vaccine also demonstrate high neutralization titers against B.1, Alpha and Beta variants, but only 34.3% displayed a neutralization activity against the Omicron variant. On the other hand, non-infected patients with half vaccination schedules displayed a weak and inconstant activity against all isolates. Non-vaccinated COVID-19 patients kept a neutralizing activity against B.1 and Alpha up to 12 months after recovery but a decreased activity against Beta and Omicron. Both surrogate assays displayed a good correlation with the VNT. However, an adaptation of the cut-off positivity was necessary, especially for the most resistant Beta and Omicron variants. We validated two simple and reliable surrogate neutralization assays, which may favorably replace cell-based methods, allowing functional analysis on a larger scale.

Design and performance characteristics of the Elecsys anti-SARS-CoV-2 S assay.

Automated, high throughput assays are required to quantify the immune response after infection with or vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study on the Roche Elecsys® Anti-SARS-CoV-2 S (ACOV2S) assay provides insights on the assay design and performance. The ACOV2S assay quantifies antibodies to the receptor-binding domain of the SARS-CoV-2 spike protein. The assigned units and the underlying standardization were compared to the international reference standard in BAU/mL. Assay specificity was assessed in samples (n=5981) collected prior to the COVID-19 pandemic and in samples from patients with non-COVID-19 respiratory infections (n=697) or other infectious diseases (n=771). Sensitivity was measured in 1313 samples from patients with mild COVID-19 and 297 samples from patients hospitalized with COVID-19. Comparison of results was performed to a comparator semi-quantitative anti-S1 assay of indirect detection format as well as a commercially available and an in-house version of a surrogate neutralization assay (ACE2-RBD). The originally assigned units for the ACOV2S assay were shown to be congruent to the units of the First International WHO Standard for anti-SARS-CoV-2 immunoglobulins. Overall specificity was 99.98% with no geographical differences noted and no loss of specificity in samples containing potentially cross-reacting antibodies. High sensitivity was observed, with 98.8% of samples reported to be reactive >14 days after infection and sustained detection of antibodies over time. For all samples, ACOV2S titers and neutralization capacities developed with comparable dynamics. Robust standardization and assay setup enable excellent reproducibility of results, independent of lot or analyzer used. The results from this study confirmed that ACOV2S is a highly sensitive and specific assay and correlates well with surrogate neutralization assays. The units established for ACOV2S are also interchangeable with the units of the First International WHO Standard for anti-SARS-CoV-2 immunoglobulins. Worldwide availability of the assay and analyzers render ACOV2S a highly practical tool for population-wide assessment and monitoring of the humoral response to SARS-CoV-2 infection or vaccination.

A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants.

Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.

SARS-CoV-2 neutralizing antibody response in vaccinated and non-vaccinated hospital healthcare workers with or without history of infection

Between March 2021 and February 2022, SARS-CoV-2 neutralizing antibodies dynamics was investigated in a prospective observational study in 903 healthcare workers of a hospital in Switzerland. A surrogate neutralization assay measuring the competitive inhibition of the angiotensin converting enzyme 2 (ACE2) binding to the spike protein (S) of the SARS-CoV-2 wild type virus and to five variants of concern (Alpha, Beta, Gamma, Delta, Omicron) was used. We observed a broad distribution of neutralization activity among participants and substantial differences in neutralizing titers against variants. Participants were grouped based on combinations of vaccination status (1, 2 or 3 doses) and/or prior or subsequent SARS-CoV-2 infection/reinfection. Triple vaccination resulted in the highest neutralization response, as did double vaccination with prior or subsequent infection. Double vaccination without infection showed an intermediate neutralization response while SARS-CoV-2 infection in non-vaccinated participants resulted in poor neutralization response. After triple vaccination or double vaccination plus infection, additional vaccination and/or reinfection had no impact on neutralizing antibody titers over the observed period. These results strongly support the booster dose strategy, while additional booster doses within short time intervals might not improve immunization. However, dynamics of neutralizing antibodies titers needs to be monitored individually, over time and include newly emerging variants.

Automated detection of neutralizing SARS-CoV-2 antibodies in minutes using a competitive chemiluminescence immunoassay

The SARS-CoV-2 pandemic has shown the importance of rapid and comprehensive diagnostic tools. While there are numerous rapid antigen tests available, rapid serological assays for the detection of neutralizing antibodies are and will be needed to determine not only the amount of antibodies formed after infection or vaccination but also their neutralizing potential, preventing the cell entry of SARS-CoV-2. Current active-virus neutralization assays require biosafety level 3 facilities, while virus-free surrogate assays are more versatile in applications, but still take typically several hours until results are available. To overcome these disadvantages, we developed a competitive chemiluminescence immunoassay that enables the detection of neutralizing SARS-CoV-2 antibodies within 7 min. The neutralizing antibodies bind to the viral receptor binding domain (RBD) and inhibit the binding to the human angiotensin-converting enzyme 2 (ACE2) receptor. This competitive binding inhibition test was characterized with a set of 80 samples, which could all be classified correctly. The assay results favorably compare to those obtained with a more time-intensive ELISA-based neutralization test and a commercial surrogate neutralization assay. Our test could further be used to detect individuals with a high total IgG antibody titer, but only a low neutralizing titer, as well as for monitoring neutralizing antibodies after vaccinations. This effective performance in SARS-CoV-2 seromonitoring delineates the potential for the test to be adapted to other diseases in the future.Graphical

Priming conditions shape breadth of neutralizing antibody responses to sarbecoviruses

Vaccines that are broadly cross-protective against current and future SARS-CoV-2 variants of concern (VoC) or across the sarbecoviruses subgenus remain a priority for public health. Virus neutralization is the best available correlate of protection. To define the magnitude and breadth of cross-neutralization in individuals with different exposure to SARS-CoV-2 infection and vaccination, we here use a multiplex surrogate neutralization assay based on virus spike receptor binding domains of multiple SARS-CoV-2 VoC, as well as related bat and pangolin viruses. We include sera from cohorts of individuals vaccinated with two or three doses of mRNA (BNT162b2) or inactivated SARS-CoV-2 (Coronavac or Sinopharm) vaccines with or without a history of previous SARS-CoV-2 or SARS-CoV-1 infection. SARS-CoV-2 or SARS-CoV-1 infection followed by BNT162b2 vaccine, Omicron BA.2 breakthrough infection following BNT162b2 vaccine or a third dose of BNT162b2 following two doses of BNT162b2 or Coronavac elicit the highest and broadest neutralization across VoCs. For both breadth and magnitude of neutralization across all sarbecoviruses, those infected with SARS-CoV-1 immunized with BNT162b2 outperform all other combinations of infection and/or vaccination. These data may inform vaccine design strategies for generating broadly neutralizing antibodies to SARS-CoV-2 variants or across the sarbecovirus subgenus.

Comparison of SARS-CoV-2 Antibody Levels after a Third Heterologous and Homologous BNT162b2 Booster Dose.

This study aimed to determine the anti-S (receptor binding protein) RBD IgG antibody titers formed against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the neutralizing antibody inhibition percentages (nAb IH%) in blood samples taken after two doses of inactive or mRNA-based vaccine and a booster dose. Volunteers with two doses of inactivated CoronaVac (heterologous group; n = 75) and BioNTech (BNT)162b2 mRNA vaccine (homologous group; n = 75) were included in this study. All participants preferred the BNT162b2 vaccine as a booster dose. First, peripheral blood samples were taken 3 months after the second vaccine dose. Second, peripheral blood samples were taken 1 month after the booster dose. Anti-S-RBD IgG titers were determined by CMIA (SARS-CoV-2 IgG II Quant). Neutralizing antibodies were detected by a surrogate neutralization assay (SARS-CoV-2 NeutraLISA, Euroimmun, Lübeck, Germany). The median age of the volunteers was 40 (IQR 29–47) years old. After the heterologous booster dose, anti-S-RBD IgG levels and neutralizing antibodies increased approximately 50-fold and 9-fold, respectively. Anti-S-RBD IgG titers increased by 9 and 57 times, respectively, while nAb IH% increased by 1.5 and 16 times, respectively, among those with heterologous reminder doses and those with and without a prior history of coronavirus disease (COVID-19). This study showed that after the administration of a heterologous booster dose with BNT162b2 to those whose primary vaccination was with inactivated CoronaVac, the binding and neutralizing antibody levels were similar to those who received a homologous BNT162b2 booster dose. It was observed that the administration of heterologous and homologous booster doses resulted in the development of similar levels of neutralizing antibodies, independently from a prior history of COVID-19.

SARS-CoV-2 Omicron variant emerged under immune selection.

The SARS-CoV-2 Omicron variant (B.1.1.529 lineage) escapes antibodies that neutralize the ancestral virus. We tested human serum panels from participants with differing infection and vaccination status using a multiplex surrogate virus neutralization assay targeting 20 sarbecoviruses. We found that bat and pangolin sarbecoviruses showed significantly less neutralization escape than the Omicron variant. We propose that SARS-CoV-2 variants have emerged under immune selection pressure and are evolving differently from animal sarbecoviruses.

Dynamics of humoral and cellular immune responses after homologous and heterologous SARS-CoV-2 vaccination with ChAdOx1 nCoV-19 and BNT162b2.

BackgroundVaccines are an important means to overcome the SARS-CoV-2 pandemic. They induce specific antibody and T-cell responses but it remains open how well vaccine-induced immunity is preserved over time following homologous and heterologous immunization regimens. Here, we compared the dynamics of humoral and cellular immune responses up to 180 days after homologous or heterologous vaccination with either ChAdOx1-nCoV-19 (ChAd) or BNT162b2 (BNT) or both.MethodsVarious tests were used to determine the humoral and cellular immune response. To quantify the antibody levels, we used the surrogate neutralization (sVNT) assay from YHLO, which we augmented with pseudo- and real virus neutralization tests (pVNT and rVNT). Antibody avidity was measured by a modified ELISA. To determine cellular reactivity, we used an IFN-γ Elispot, IFN-γ/IL Flurospot, and intracellular cytokine staining.FindingsAntibody responses significantly waned after vaccination, irrespective of the regimen. The capacity to neutralize SARS-CoV-2 – including variants of concern such as Delta or Omicron – was superior after heterologous compared to homologous BNT vaccination, both of which resulted in longer-lasting humoral immunity than homologous ChAd immunization. All vaccination regimens induced stable, polyfunctional T-cell responses.InterpretationThese findings demonstrate that heterologous vaccination with ChAd and BNT is a potent alternative to induce humoral and cellular immune protection in comparison to the homologous vaccination regimens.FundingThe study was funded by the German Centre for Infection Research (DZIF), the European Union's “Horizon 2020 Research and Innovation Programme" under grant agreement No. 101037867 (VACCELERATE), the “Bayerisches Staatsministerium für Wissenschaft und Kunst” for the CoVaKo-2021 and the For-COVID projects and the Helmholtz Association via the collaborative research program “CoViPa”. Further support was obtained from the Federal Ministry of Education and Science (BMBF) through the “Netzwerk Universitätsmedizin”, project “B-Fast” and “Cov-Immune”. KS is supported by the German Federal Ministry of Education and Research (BMBF, 01KI2013) and the Else Kröner-Stiftung (2020_EKEA.127).

Evaluation of the T cell and B cell response following the administration of COVID-19 vaccines in Korea.

BackgroundThe coronavirus disease (COVID-19) has been a worldwide concern since 2019. Vaccines are predicted to be crucial in preventing further outbreaks. The development and kinetics of immune responses determine the efficacy of COVID-19 vaccines.MethodsWe measured interferon-gamma (IFN-γ) levels upon administering homologous adenovirus vector-based (ChAdOx1-S [AZ], Ad26.COV2.S [JAN]), mRNA-based (BNT162b2 [PF]; mRNA-1273 [MO]), and heterologous (AZ/PF) vaccines in healthy Korean individuals using two IFN-γ release assays: the Covi-FERON ELISA and T-SPOT Discovery SARS-CoV-2 assay. B cell responses were evaluated by assessing the production of neutralizing antibodies by surrogate virus neutralization assay. The immune response among the vaccine groups was compared after adjusting the vaccination dose and interactions between each group.ResultsAZ triggered the highest T cell response after the first dose but showed instability after the second. PF and MO yielded stable and higher increments of T and B cell responses compared to AZ. MO yielded a higher immune response than PF. JAN yielded T and B cell responses at lower levels than the other vaccines. The booster dose triggered significant increases in the T and B cell responses and is therefore needed to protect against SARS-CoV-2 given the possibility of waning immune responses.ConclusionAdministering two doses of mRNA vaccines provides the most effective results among the administered vaccines in triggering the immune response specific to SARS-CoV-2 in healthy Korean individuals. Administration of booster doses demonstrated a significant increase in the immune response and may provide longer protection against SARS-CoV-2.

Vaccine-Induced T-Cell and Antibody Responses at 12 Months after Full Vaccination Differ with Respect to Omicron Recognition.

More than a year after the first vaccines against the novel SARS-CoV-2 were approved, many questions still remain about the long-term protection conferred by each vaccine. How long the effect lasts, how effective it is against variants of concern and whether further vaccinations will confer additional benefits remain part of current and future research. For this purpose, we examined 182 health care employees—some of them with previous SARS-CoV-2 infection—12 months after different primary immunizations. To assess antibody responses, we performed an electrochemiluminescence assay (ECLIA) to determine anti-spike IgGs, followed by a surrogate virus neutralization assay against Wuhan-Hu-1 and B.1.1.529/BA.1 (Omicron). T cell response against wild-type and the Omicron variants of concern were assessed via interferon-gamma ELISpot assays and T-cell surface and intracellular cytokine staining. In summary, our results show that after the third vaccination with an mRNA vaccine, differences in antibody quantity and functionality observed after different primary immunizations were equalized. As for the T cell response, we were able to demonstrate a memory function for CD4+ and CD8+ T cells alike. Importantly, both T and antibody responses against wild-type and omicron differed significantly; however, antibody and T cell responses did not correlate with each other and, thus, may contribute differentially to immunity.

Norovirus Infection in Young Nicaraguan Children Induces Durable and Genotype-Specific Antibody Immunity.

There are significant challenges to the development of a pediatric norovirus vaccine, mainly due to the antigenic diversity among strains infecting young children. Characterizing human norovirus serotypes and understanding norovirus immunity in naïve children would provide key information for designing rational vaccine platforms. In this study, 26 Nicaraguan children experiencing their first norovirus acute gastroenteritis (AGE) episode during the first 18 months of life were investigated. We used a surrogate neutralization assay that measured antibodies blocking the binding of 13 different norovirus virus-like particles (VLPs) to histo-blood group antigens (HBGAs) in pre- and post-infection sera. To assess for asymptomatic norovirus infections, stools from asymptomatic children were collected monthly, screened for norovirus by RT-qPCR and genotyped by sequencing. Seroconversion of an HBGA-blocking antibody matched the infecting genotype in 25 (96%) of the 26 children. A subset of 13 (50%) and 4 (15%) of the 26 children experienced monotypic GII and GI seroconversion, respectively, strongly suggesting a type-specific response in naïve children, and 9 (35%) showed multitypic seroconversion. The most frequent pairing in multitypic seroconversion (8/12) were GII.4 Sydney and GII.12 noroviruses, both co-circulating at the time. Blocking antibody titers to these two genotypes did not correlate with each other, suggesting multiple exposure rather than cross-reactivity between genotypes. In addition, GII titers remained consistent for at least 19 months post-infection, demonstrating durable immunity. In conclusion, the first natural norovirus gastroenteritis episodes in these young children were dominated by a limited number of genotypes and induced responses of antibodies blocking binding of norovirus VLPs in a genotype-specific manner, suggesting that an effective pediatric norovirus vaccine likely needs to be multivalent and include globally dominant genotypes. The duration of protection from natural infections provides optimism for pediatric norovirus vaccines administered early in life.

Improved SARS-CoV-2 Neutralization of Delta and Omicron BA.1 Variants of Concern after Fourth Vaccination in Hemodialysis Patients.

Hemodialysis patients are exposed to a markedly increased risk when infected with SARS-CoV-2. To date, it is unclear if hemodialysis patients benefit from four vaccinations. A total of 142 hemodialysis patients received four COVID-19 vaccinations until March 2022. RDB binding antibody titers were determined in a competitive surrogate neutralization assay. Vero-E6 cells were infected with SARS-CoV-2 variants of concern (VoC), Delta (B.1.617.2), or Omicron (B.1.1.529, sub-lineage BA.1) to determine serum infection neutralization capacity. Four weeks after the fourth vaccination, serum infection neutralization capacity significantly increased from a 50% inhibitory concentration (IC50, serum dilution factor 1:x) of 247.0 (46.3–1560.8) to 2560.0 (1174.0–2560.0) for the Delta VoC, and from 37.5 (20.0–198.8) to 668.5 (182.2–2560.0) for the Omicron VoC (each p < 0.001) compared to four months after the third vaccination. A significant increase in the neutralization capacity was even observed for patients with high antibody titers after three vaccinations (p < 0.001). Ten patients with SARS-CoV-2 breakthrough infection after the first blood sampling had by trend lower prior neutralization capacity for Omicron (p = 0.051). Our findings suggest that hemodialysis patients benefit from a fourth vaccination in particular in the light of the highly infectious SARS-CoV-2 Omicron-variants. A routinely applied four-time vaccination seems to broaden immunity against variants and would be recommended in hemodialysis patients.

Humoral immune response characterization of heterologous prime-boost vaccination with CoronaVac and BNT162b2

BackgroundVaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has proven to be a successful strategy for prevent severe infections. CoronaVac and BNT162b2 are the most used vaccines worldwide, but their use in heterologous vaccination schedules is still subjected to evaluation. MethodsFifty healthy individuals who received heterologous prime-boost vaccination with CoronaVac and BNT162b2 were enrolled in a post-vaccination serological follow-up longitudinal prospective study. We evaluated specific serum anti-receptor binding domain (RBD) IgG antibody levels, and their capacity to block RBD-ACE2 interaction with a surrogate neutralization assay. In 20 participants, we assessed antibody binding kinetics by surface plasmon resonance, and Fc-mediated functions by ADCC and ADCP reporter assays. ResultsOur baseline seronegative cohort, displayed seroconversion after two doses of CoronaVac and an important decrease in serum anti-RBD IgG antibodies levels 80 days post-second dose. These levels increased significantly early after the third dose with BNT162b2, but 73 days after the booster we found a new fall. Immunoglobulin functionalities showed a similar behavior. ConclusionsThe heterologous prime-boost vaccination with CoronaVac and BNT162b2 generated an impressive increase in serum anti-RBD specific antibody levels followed by a drop. Nevertheless, these titers remained well above those found in individuals only vaccinated with CoronaVac in the same elapsed time. Serum IgG levels showed high correlation with antibody binding analysis, their capacity to block RBD-ACE2 interaction, and Fc-effectors mechanisms. Our work sheds light on the humoral immune response to heterologous vaccination with CoronaVac and BNT162b2, to define a post-vaccination correlate of protection against SARS-CoV-2 infection and to discuss the scheduling of future vaccine boosters in general population.

COVID-19 symptom severity predicts neutralizing antibody activity in a community-based serological study

Serological testing for SARS-CoV-2 IgG antibodies is used to assess their presence in blood samples from exposed individuals and provides a measure of the magnitude of immune response to infection. The measurement of neutralizing antibodies (NAbs) in particular provides information about the severity of prior infection and level of protective immunity against re-infection. Much of the work investigating the association between prior infection severity and NAb levels has been conducted among clinical populations, and less is known about this relationship in the general population. Accordingly, we utilize data from a large (n = 790) community-based cohort of unvaccinated, seropositive participants. We analyzed the association between NAb response, measured via surrogate virus neutralization assay, with patterns of symptoms and household exposure. Our results indicate no detectable NAb activity in 63.8% of the seropositive participants (n = 504). Those with detectable NAb levels demonstrated a positive relationship between NAb activity and both self-reported previous symptom severity and household exposure. These findings are significant in light of recent concerns about degree of protective immunity conferred by prior infection or vaccination, and we highlight the value of community-based research for investigating variation in immune response.

Homogeneous surrogate virus neutralization assay to rapidly assess neutralization activity of anti-SARS-CoV-2 antibodies

The COVID-19 pandemic triggered the development of numerous diagnostic tools to monitor infection and to determine immune response. Although assays to measure binding antibodies against SARS-CoV-2 are widely available, more specific tests measuring neutralization activities of antibodies are immediately needed to quantify the extent and duration of protection that results from infection or vaccination. We previously developed a ‘Serological Assay based on a Tri-part split-NanoLuc® (SATiN)’ to detect antibodies that bind to the spike (S) protein of SARS-CoV-2. Here, we expand on our previous work and describe a reconfigured version of the SATiN assay, called Neutralization SATiN (Neu-SATiN), which measures neutralization activity of antibodies directly from convalescent or vaccinated sera. The results obtained with our assay and other neutralization assays are comparable but with significantly shorter preparation and run time for Neu-SATiN. As the assay is modular, we further demonstrate that Neu-SATiN enables rapid assessment of the effectiveness of vaccines and level of protection against existing SARS-CoV-2 variants of concern and can therefore be readily adapted for emerging variants.