Surrogate Neutralization Assay Research Articles

The temporal course of T- and B-cell responses to vaccination with BNT162b2 and mRNA-1273

ObjectivesTo investigate the response of the immune system (and its influencing factors) to vaccination with BNT162b2 or mRNA-1273. Methods531 vaccinees, recruited from healthcare professionals, donated samples before, in between, and after the administration of the two doses of the vaccine. T- and B-cell responses were examined via interferon-γ (IFN-γ) release assay, and antibodies against different epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (S1 and NCP) were detected via ELISA and surrogate neutralization assay. Results were correlated with influencing factors such as age, sex, prior infection, vaccine received (BNT162b2 or mRNA-1273), and immunosuppression. Furthermore, antinuclear antibodies (ANAs) were measured to screen for autoimmune responses following vaccination with an mRNA vaccine. ResultsNo markers of immunity against SARS-CoV-2 were found before the first vaccination. Two weeks after it, specific responses against SARS-CoV-2 were already measurable (median ± median absolute deviation (MAD): anti-S1 IgG 195.5 ± 172.7 BAU/mL; IgA 6.7 ± 4.9 OD; surrogate neutralization 39 ± 23.7%), and were significantly increased two weeks after the second dose (anti-S1 IgG 3744 ± 2571.4 BAU/mL; IgA 12 ± 0 OD; surrogate neutralization 100 ± 0%, IFN-γ 1897.2 ± 886.7 mIU/mL). Responses were stronger for younger participants (this difference decreasing after the second dose). Further influences were previous infection with SARS-CoV-2 (causing significantly stronger responses after the first dose compared to unexposed individuals (p ≤ 0.0001)) and the vaccine received (significantly stronger reactions for recipients of mRNA-1273 after both doses, p < 0.05–0.0001). Some forms of immunosuppression significantly impeded the immune response to the vaccination (with no observable immune response in three immunosuppressed participants). There was no significant induction of ANAs by the vaccination (no change in qualitative ANA results (p 0.2592) nor ANA titres (p 0.08) from pre-to post-vaccination. ConclusionsBoth vaccines elicit strong and specific immune responses against SARS-CoV-2 which become detectable one week (T-cell response) or two weeks (B-cell response) after the first dose.

Early IgG / IgA response in hospitalized COVID-19 patients is associated with a less severe disease

We determined the kinetics of anti-SARS-CoV-2 antibody response in fifteen hospitalized COVID-19 patients. Patients were divided into mild/moderate (mild, n = 1; moderate, n = 4) or severe (n = 10) and virus-specific anti-Nucleocapsid IgM, anti-Spike IgG and anti-Spike IgA were measured in serial serum samples collected 0 to 15 days after hospital admission. Surrogate neutralization assays were performed by testing inhibition of ACE-2 binding to Spike. In 3 patients (2 severe and 1 moderate case), serum antibodies and T-cell memory were monitored 6 months after baseline. Although IgM response tended to appear first, patients affected by less severe disease were more prone to an early IgG/IgA response. Neutralization of Spike binding to ACE2 correlated with anti-Spike IgG and IgA. IgG and IgA antibody response persisted at the 6 months follow-up. A recall T-cell response to the Spike antigen was observed in 2 out of 3 patients, not related to disease severity.

Novel Surrogate Neutralizing Assay Supports Parvovirus B19 Vaccine Development for Children with Sickle Cell Disease.

Children with sickle cell disease (SCD) suffer life-threatening transient aplastic crisis (TAC) when infected with parvovirus B19. In utero, infection of healthy fetuses may result in anemia, hydrops, and death. Unfortunately, although promising vaccine candidates exist, no product has yet been licensed. One barrier to vaccine development has been the lack of a cost-effective, standardized parvovirus B19 neutralization assay. To fill this void, we evaluated the unique region of VP1 (VP1u), which contains prominent targets of neutralizing antibodies. We discovered an antigenic cross-reactivity between VP1 and VP2 that, at first, thwarted the development of a surrogate neutralization assay. We overcame the cross-reactivity by designing a mutated VP1u (VP1uAT) fragment. A new VP1uAT ELISA yielded results well correlated with neutralization (Spearman’s correlation coefficient = 0.581; p = 0.001), superior to results from a standard clinical diagnostic ELISA or an ELISA with virus-like particles. Virus-specific antibodies from children with TAC, measured by the VP1uAT and neutralization assays, but not other assays, gradually increased from days 0 to 120 post-hospitalization. We propose that this novel and technically simple VP1uAT ELISA might now serve as a surrogate for the neutralization assay to support rapid development of a parvovirus B19 vaccine.

Immune responses to a single dose of the AZD1222/Covishield vaccine in health care workers

Several COVID-19 vaccines have received emergency approval. Here we assess the immunogenicity of a single dose of the AZD1222 vaccine, at one month, in a cohort of health care workers (HCWs) (629 naïve and 26 previously infected). 93.4% of naïve HCWs seroconverted, irrespective of age and gender. Haemagglutination test for antibodies to the receptor binding domain (RBD), surrogate neutralization assay (sVNT) and ex vivo IFNγ ELISpot assays were carried out in a sub-cohort. ACE2 blocking antibodies (measured by sVNT) were detected in 67/69 (97.1%) of naïve HCWs. Antibody levels to the RBD of the wild-type virus were higher than to RBD of B.1.1.7, and titres to B.1.351 were very low. Ex vivo T cell responses were observed in 30.8% to 61.7% in naïve HCWs. Previously infected HCWs, developed significantly higher (p < 0.0001) ACE2 blocking antibodies and antibodies to the RBD for the variants B.1.1.7 and B.1.351. This study shows high seroconversion after one vaccine dose, but also suggests that one vaccine dose may be insufficient to protect against emerging variants.

Anti-SARS-CoV-2 Antibody Levels Measured by the AdviseDx SARS-CoV-2 Assay Are Concordant with Previously Available Serologic Assays but Are Not Fully Predictive of Sterilizing Immunity.

ABSTRACTWith the availability of widespread SARS-CoV-2 vaccination, high-throughput quantitative anti-spike protein serological testing will likely become increasingly important. Here, we investigated the performance characteristics of the recently FDA-authorized semiquantitative anti-spike protein AdviseDx SARS-CoV-2 IgG II assay compared to the FDA-authorized anti-nucleocapsid protein Abbott Architect SARS-CoV-2 IgG, Roche Elecsys anti-SARS-CoV-2-S, EuroImmun anti-SARS-CoV-2 enzyme-linked immunosorbent assay (ELISA), and GenScript surrogate virus neutralization assays and examined the humoral response associated with vaccination, natural protection, and vaccine breakthrough infection. The AdviseDx assay had a clinical sensitivity at 14 days after symptom onset or 10 days after PCR detection of 95.6% (65/68; 95% confidence interval [CI], 87.8 to 98.8%), with two discrepant individuals seroconverting shortly thereafter. The AdviseDx assay demonstrated 100% positive percent agreement with the four other assays examined using the same symptom onset or PCR detection cutoffs. Using a recently available WHO international standard for anti-SARS-CoV-2 antibody, we provide assay unit conversion factors to international units for each of the assays examined. We performed a longitudinal survey of healthy vaccinated individuals, finding that median AdviseDx immunoglobulin levels peaked 7 weeks after first vaccine dose at approximately 4,000 IU/ml. Intriguingly, among the five assays examined, there was no significant difference in antigen binding level or neutralizing activity between two seropositive patients protected against SARS-CoV-2 infection in a previously described fishing vessel outbreak and five health care workers who experienced vaccine breakthrough of SARS-CoV-2 infection, all with variants of concern. These findings suggest that protection against SARS-CoV-2 infection cannot currently be predicted exclusively using in vitro antibody assays against wild-type SARS-CoV-2 spike. Further work is required to establish protective correlates for SARS-CoV-2 infection.

A luciferase-based approach for measuring HBGA blockade antibody titers against human norovirus

BackgroundNoroviruses are the most common cause of viral gastroenteritis worldwide, yet there is a deficit in the understanding of protective immunity. Surrogate neutralization assays have been widely used that measure the ability of antibodies to block virus-like particle (VLP) binding to histo-blood group antigens (HBGAs). However, screening large sample sets against multiple antigens using the traditional HBGA blocking assay requires significant investment in terms of time, equipment, and technical expertise, largely associated with the generation of purified VLPs. MethodsTo address these issues, a luciferase immunoprecipitation system (LIPS) assay was modified to measure the norovirus-specific HBGA blockade activity of antibodies. The assay (designated LIPS-Blockade) was validated using a panel of well-characterized homotypic and heterotypic hyperimmune sera as well as strain-specific HBGA blocking monoclonal antibodies. ResultsThe LIPS-Blockade assay was comparable in specificity to a standard HBGA blocking protocol performed with VLPs. Using time-ordered patient sera, the luciferase-based approach was also able to detect changes in HBGA blocking titers following viral challenge and natural infection with norovirus. ConclusionIn this study we developed a rapid, robust, and scalable surrogate neutralization assay for noroviruses that circumvented the need for purified VLPs. This LIPS-Blockade assay should streamline the process of large-scale immunological studies, ultimately aiding in the characterization of protective immunity to human noroviruses.

Rapid and accurate agglutination-based testing for SARS-CoV-2 antibodies

SummaryWe have developed a rapid, accurate, and cost-effective serologic test for SARS-CoV-2 virus, which caused the COVID-19 pandemic, on the basis of antibody-dependent agglutination of antigen-coated latex particles. When validated using plasma samples that are positive or negative for SARS-CoV-2, the agglutination assay detected antibodies against the receptor-binding domain of the spike (S-RBD) or the nucleocapsid protein of SARS-CoV-2 with 100% specificity and ∼98% sensitivity. Furthermore, we found that the strength of the S-RBD antibody response measured by the agglutination assay correlated with the efficiency of the plasma in blocking RBD binding to the angiotensin-converting enzyme 2 in a surrogate neutralization assay, suggesting that the agglutination assay might be used to identify individuals with virus-neutralizing antibodies. Intriguingly, we found that >92% of patients had detectable antibodies on the day of a positive viral RNA test, suggesting that the agglutination antibody test might complement RNA testing for the diagnosis of SARS-CoV-2 infection.

Evaluation of a Commercial Culture-Free Neutralization Antibody Detection Kit for Severe Acute Respiratory Syndrome-Related Coronavirus-2 and Comparison With an Antireceptor-Binding Domain Enzyme-Linked Immunosorbent Assay.

BackgroundSevere acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) surrogate neutralization assays that obviate the need for viral culture offer substantial advantages regarding throughput and cost. The cPass SARS-CoV-2 Neutralization Antibody Detection Kit (GenScript) is the first such commercially available assay that detects antibodies that block receptor-binding domain (RBD)/angiotensin-converting enzyme (ACE)-2 interaction. We aimed to evaluate cPass to inform its use and assess its added value compared with anti-RBD enzyme-linked immunosorbent assays (ELISAs).MethodsSerum reference panels comprising 205 specimens were used to compare cPass to plaque-reduction neutralization test (PRNT) and a pseudotyped lentiviral neutralization (PLV) assay for detection of neutralizing antibodies. We assessed the correlation of cPass with an ELISA detecting anti-RBD immunoglobulin (Ig)G, IgM, and IgA antibodies at a single timepoint and across intervals from onset of symptoms of SARS-CoV-2 infection.ResultsCompared with PRNT-50, cPass sensitivity ranged from 77% to 100% and specificity was 95% to 100%. Sensitivity was also high compared with the pseudotyped lentiviral neutralization assay (93%; 95% confidence interval [CI], 85–97), but specificity was lower (58%; 95% CI, 48–67). Highest agreement between cPass and ELISA was for anti-RBD IgG (r = 0.823). Against the pseudotyped lentiviral neutralization assay, anti-RBD IgG sensitivity (99%; 95% CI, 94–100) was very similar to that of cPass, but overall specificity was lower (37%; 95% CI, 28–47). Against PRNT-50, results of cPass and anti-RBD IgG were nearly identical.ConclusionsThe added value of cPass compared with an IgG anti-RBD ELISA was modest.

SARS-CoV-2 S1 NanoBiT: A nanoluciferase complementation-based biosensor to rapidly probe SARS-CoV-2 receptor recognition

As the COVID-19 pandemic continues, there is an imminent need for rapid diagnostic tools and effective antivirals targeting SARS-CoV-2. We have developed a novel bioluminescence-based biosensor to probe a key host-virus interaction during viral entry: the binding of SARS-CoV-2 viral spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2). Derived from Nanoluciferase binary technology (NanoBiT), the biosensor is composed of Nanoluciferase split into two complementary subunits, Large BiT and Small BiT, fused to the Spike S1 domain of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. The ACE2-S1 interaction results in reassembly of functional Nanoluciferase, which catalyzes a bioluminescent reaction that can be assayed in a highly sensitive and specific manner. We demonstrate the biosensor's large dynamic range, enhanced thermostability and pH tolerance. In addition, we show the biosensor's versatility towards the high-throughput screening of drugs which disrupt the ACE2-S1 interaction, as well as its ability to act as a surrogate virus neutralization assay. Results obtained with our biosensor correlate well with those obtained with a Spike-pseudotyped lentivirus assay. This rapid in vitro tool does not require infectious virus and should enable the timely development of antiviral modalities targeting SARS-CoV-2 entry.

Early detection of neutralizing antibodies against SARS-CoV-2 in COVID-19 patients in Thailand.

BackgroundThe presence of neutralizing antibodies (NAbs) is an indicator of protective immunity for most viral infections. A newly developed surrogate viral neutralization assay (sVNT) offers the ability to detect total receptor binding domain-targeting NAbs in an isotype-independent manner, increasing the test sensitivity. Thus, specimens with low IgM/ IgG antibody levels showed strong neutralization activity in sVNT.MethodsThis study aimed to measure the %inhibition of NAbs measured by sVNT in PCR-confirmed COVID-19 patients. The sensitivity of sVNT for the diagnosis of SARS-CoV-2 infection and its kinetics were determined.ResultsNinety-seven patients with PCR-confirmed SARS-CoV-2 infection were included in this study. Majority of the patients were 21–40 years old (67%) and 63% had mild symptoms. The sensitivity of sVNT for the diagnosis of SARS-CoV-2 infection was 99% (95% confidence interval (CI) 94.4–100%) and the specificity was 100% (95% CI 98.3–100%). The negative predictive value of sVNT from the samples collected before and after 7 days of symptom onset was 99.5% (95% CI 97.4–100%) and 100% (95% CI 93.8–100%), respectively. The level of inhibition at days 8–14 were significantly higher than days 0–7 (p<0.001). The median %inhibition values by severity of COVID-19 symptoms were 79.9% (interquartile range (IQR) 49.7–91.8%); 89.0% (IQR 71.2–92.4%); and 86.6% (IQR 69.5–92.8%), for mild, moderate and severe/critical symptoms respectively. The median level of sVNT %inhibition of severe was significantly higher than the mild group (p = 0.05).ConclusionThe sVNT is a practical and robust serological test for SARS-CoV-2 infection and does not require specialized biosafety containment. It can be used clinically to aid diagnosis in both early and late infection especially in cases when the real-time RT-PCR results in weakly negative or weakly positive, and to determine the protective immune response from SARS-CoV-2 infection in patients.

Antibody persistence in the first 6 months following SARS-CoV-2 infection among hospital workers: a prospective longitudinal study

ObjectivesTo evaluate longitudinally the persistence of humoral immunity for up to 6 months in a cohort of hospital employees with mild coronavirus disease 2019 (COVID-19). MethodsWe measured anti-RBD (receptor binding domain of viral spike protein), anti-N (viral nucleoprotein) and neutralizing antibodies at 1, 3 and 6 months after mostly mild COVID-19 in 200 hospital workers using commercial ELISAs and a surrogate virus neutralization assay. ResultsAntibodies specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persisted in all participants for up to 6 months. Anti-RBD geometric mean concentrations (GMCs) progressively increased between months 1 (74.2 U/mL, 95%CI: 62.7–87.8), 3 (103.2 U/mL, 95%CI: 87.9–121.2; p < 0.001), and 6 (123.3 U/mL, 95%CI: 103.4–147.0; p < 0.001) in the whole cohort. Anti-N antibodies were detectable in >97% at all times. Neutralizing antibodies were detectable in 99.5% of participants (195/196) at 6 months post infection. Their GMC progressively decreased between months 1 (20.1 AU/mL, 95%CI: 16.9–24.0), 3 (15.2 AU/mL, 95%CI: 13.2–17.6; p < 0.001) and 6 (9.4 AU/mL, 95%CI: 7.7–11.4; p < 0.001). RBD-ACE2-inhibiting antibody titres and anti-RBD antibody concentrations strongly correlated at each timepoint (all r > 0.86, p < 0.001). Disease severity was associated with higher initial anti-RBD and RBD-ACE2-inhibiting antibody titres, but not with their kinetics. ConclusionsNeutralizing antibodies persisted at 6 months in almost all participants, indicating more durability than initially feared. Anti-RBD antibodies persisted better and even increased over time, possibly related to the preferential detection of progressively higher-affinity antibodies.

Immunogenicity and Safety of an Inactivated SARS-CoV-2 Vaccine in People Living with HIV-1

Background: It has been proven that inactivated COVID-19 vaccines are safe and effective in general population with intact immunity. However, their safety and immunogenicity have not been demonstrated in people living with HIV (PLWH).Methods: 42 HIV-1 infected individuals who were stable on cART and 28 healthy individuals were enrolled in this study. Two doses of an inactivated COVID-19 vaccine (BIBP-CorV) were given 4 weeks apart. The safety and reactogenicity of the vaccine were evaluated by observing clinical adverse events and solicited local and systemic reactions. Humoral responses were measured by anti-spike IgG ELISA and surrogate neutralization assays. Cell-mediated immune responses and vaccine induced T cell activation were measured by flow cytometry.Findings: All the HIV-1 infected participants had a CD4+ T cell count of above 200 cells/μL both at baseline and 4 weeks after vaccination. No solicited adverse reaction was observed among all participants. Similar binding antibody, neutralizing antibody and S protein specific T cell responses were elicited in PLWH and healthy individuals. Further analyses showed that PLWH with low baseline CD4+/CD8+ T cell ratios (+/CD8+ T cell ratios (P+, CD4+ and CD8+ T cell counts of PLWH decreased significantly after vaccination, but it did not lead to any adverse clinical manifestation. Moreover, we found that the general burden of HIV-1 among the PLWH cohort decreased significantly (P=0·0192) after vaccination. And the alteration of HIV-1 viral load was not significantly associated with the vaccine induced CD4+ T cell activation.Interpretation: Our data demonstrate that the inactivated COVID-19 vaccine is safe and immunogenic in PLWH who are stable on cART with unsuppressed CD4 counts.Funding: This work was funded by the National Natural Science Foundation of China (Grant No. 81971559, 82041010).Declaration of Interest: None to declare. Ethical Approval: Written informed consent was obtained from all participants,and the study was approved by the Research Ethics Committee of Hubei CDC (approval reference number: HBCDC-AF/SC-08/02.0).

Antibodies to SARS-CoV-2 in dogs and cats, USA

ABSTRACT To provide more complete data on SARS-CoV-2 infections in dogs and cats in the U.S., we conducted a serosurvey on convenience serum samples from dogs (n=1336) and cats (n=956) collected from 48 states of the USA in 2020. An ELISA targeting the antibody against nucleocapsid identified eleven positive and two doubtful samples in cats, and five positive and five doubtful samples in dogs. A surrogate neutralization assay detecting antibodies blocking the attachment of the spike protein to ACE2 was positive with three of the ELISA positive and doubtful samples, and one of 463 randomly selected ELISA negative samples. These four positive samples were confirmed by SARS-CoV-2 virus neutralization testing. All were from cats, in New York, Florida, and New Jersey (n=2). The serosurvey results, one of the largest yet completed on dogs and cats globally, support the OIE and CDC positions that currently there is no evidence that pets play a role in the spread of SARS CoV-2 in humans.

SARS-CoV-2 Antibody Neutralization Assay Platforms Based on Epitopes Sources: Live Virus, Pseudovirus, and Recombinant S Glycoprotein RBD.

The high virulent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus that emerged in China at the end of 2019 has generated novel coronavirus disease, coronavirus disease 2019 (COVID-19), causing a pandemic worldwide. Every country has made great efforts to struggle against SARS-CoV-2 infection, including massive vaccination, immunological patients’ surveillance, and the utilization of convalescence plasma for COVID-19 therapy. These efforts are associated with the attempts to increase the titers of SARS-CoV-2 neutralizing Abs (nAbs) generated either after infection or vaccination that represent the body’s immune status. As there is no standard therapy for COVID-19 yet, virus eradication will mainly depend on these nAbs contents in the body. Therefore, serological nAbs neutralization assays become a requirement for researchers and clinicians to measure nAbs titers. Different platforms have been developed to evaluate nAbs titers utilizing various epitopes sources, including neutralization assays based on the live virus, pseudovirus, and neutralization assays utilizing recombinant SARS-CoV-2 S glycoprotein receptor binding site, receptor-binding domain. As a standard neutralization assay, the plaque reduction neutralization test (PRNT) requires isolation and propagation of live pathogenic SARS-CoV-2 virus conducted in a BSL-3 containment. Hence, other surrogate neutralization assays relevant to the PRNT play important alternatives that offer better safety besides facilitating high throughput analyses. This review discusses the current neutralization assay platforms used to evaluate nAbs, their techniques, advantages, and limitations.

Development of Serum Antibodies to SARS-CoV-2 and Evidence of Immunity in Older Adults Convalescing from COVID-19

The presence and magnitude of SARS-CoV-2 IgG, IgM, and Neutralizing Antibody (NAbs) response was investigated in study cohort comprised of 58 volunteers who survived COVID-19 in a Virginia LTC facility. All subjects were confirmed positive by PCR nasal swab at least once and blood samples were drawn a minimum of 14 days post symptom onset or first positive COVID-19 test. The cohort was split between LTC residents (n=32, mean age 77.8 yrs, age range 48-97), and the LTC staff (n=26, mean age 41.3 yrs, range 23-61); the age difference between groups was statistically significant (P<0.001). Serum IgG measurement was quantitative over 5 orders of magnitude (0.6-1250µg/mL) and IgM was qualitative, measured with sandwich ELISA; NAbs were measured with surrogate virus neutralization assay (sVNT) competition ELISA (both Genscript). The convalescent older adult LTC patients were fully immuno-competent and showed no significant difference in IgG, IgM, or NAbs compared to the younger staff group. All older adults developed NAbs and were positive for either IgG, IgM, or both. All study participants were then grouped by age range and IgG, IgM, and NAbs compared between the following groups: ≤ 50 yrs old(n=20), 51-60(n=6), 61-70(n=8), 71-80(n=14), 81-90(n=6), and ≥91(n=4). There was no significant difference in immune response parameters between the age groups. Furthermore, repeat testing at 3 months on a subset of participants showed that NAbs, IgG and IgM persist. We conclude that development of competent immune response was age-independent, and that presence of NAbs in serum suggests older adults may develop true immunity.

Comparison of potency assays to assess SARS-CoV-2 neutralizing antibody capacity in COVID-19 convalescent plasma

Convalescent plasma is plasma collected from individuals after resolution of an infection and the development of antibodies. Passive antibody administration by transfusion of convalescent plasma is currently in clinical evaluations to treat COVID-19 patients. The level of neutralizing antibodies vary among convalescent patients and fast and simple methods to identify suitable plasma donations are needed. We compared three methods to determine the SARS-CoV-2 neutralizing activity of human convalescent plasma: life virus neutralization by plaque reduction assay, a lentiviral vector based pseudotype neutralization assay and a competition ELISA-based surrogate virus neutralization assay (sVNT). Neutralization activity correlated among the different assays; however the sVNT assay was overvaluing the low neutralizing plasma. On the other hand, the sVNT assay required the lowest biosafety level, is fast and is sufficient to identify highly neutralizing plasma samples. Though weakly neutralizing samples were more reliable detected by the more challenging lentiviral vector based assays or virus neutralization assays. Spike receptor binding competition assays are suitable to identify highly neutralizing plasma samples under low biosafety requirements. Detailed analysis of in vitro neutralization activity requires more sophisticated methods that have to be performed under higher biosafety levels.

Antibody Persistence in the Six Months Following SARS-CoV-2 Infection Among Hospital Workers

Background: Little is known about the duration and breadth of humoral immunity against SARS-CoV-2 to better inform appropriate pandemic control measures.Methods: We measured anti-RBD, -N and neutralizing antibodies at one, three and six months after mostly mild COVID-19 in 200 hospital workers using commercial ELISAs and a surrogate virus neutralization assay.Findings: Anti-RBD antibodies were detected in all participants at each timepoint. Geometric mean concentrations (GMCs) progressively increased between month one (74 . 2 U/ml [95%CI 62 . 7-87 . 8), three (103 . 2 U/ml [95%CI 87 . 9-121 . 2]; p97% at all times. Neutralizing antibodies were detectable in 92 . 5% (184/199), 94 . 4% (185/196) and 76 . 5% (150/196) participants at one, three and six months, respectively. Their GMC progressively decreased between months one (20 . 1 AU/ml [95%CI 16 . 9-24 . 0]), three (15 . 2 AU/ml [95%CI 13 . 2-17 . 6]; p0 . 86, pInterpretation: Neutralizing antibodies persisted at six months in most participants, indicating more durability than initially feared. Anti-RBD antibodies persisted better and even increased over time, possibly related to the preferential detection of progressively higher-affinity antibodies. Trial Registration: registered (NCT04329546)Funding: Pictet, Ancrage and Schmidheiny Foundations, Geneva University Hospitals’ Private Foundation, PRD, Center for Vaccinology and Centre for Emerging Viral Diseases.Declaration of Interests: The authors have no conflict of interest.Ethics Approval Statement: The study was approved by the local ethics committee (CCER 2020-00516) and registered (NCT04329546) prior to initiation. All hospital workers provided written informed consent before enrolment.

A simple protein-based surrogate neutralization assay for SARS-CoV-2.

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector–based assay.

Virus–Host Interactions Between Nonsecretors and Human Norovirus

Background & AimsHuman norovirus infection is the leading cause of acute gastroenteritis. Genetic polymorphisms, mediated by the FUT2 gene (secretor enzyme), define strain susceptibility. Secretors express a diverse set of fucosylated histoblood group antigen carbohydrates (HBGA) on mucosal cells; nonsecretors (FUT2-/-) express a limited array of HBGAs. Thus, nonsecretors have less diverse norovirus strain infections, including resistance to the epidemiologically dominant GII.4 strains. Because future human norovirus vaccines will comprise GII.4 antigen and because secretor phenotype impacts GII.4 infection and immunity, nonsecretors may mimic young children immunologically in response to GII.4 vaccination, providing a needed model to study cross-protection in the context of limited pre-exposure.MethodsBy using specimens collected from the first characterized nonsecretor cohort naturally infected with GII.2 human norovirus, we evaluated the breadth of serologic immunity by surrogate neutralization assays, and cellular activation and cytokine production by flow cytometry.ResultsGII.2 infection resulted in broad antibody and cellular immunity activation that persisted for at least 30 days for T cells, monocytes, and dendritic cells, and for 180 days for blocking antibody. Multiple cellular lineages expressing interferon-γ and tumor necrosis factor-α dominated the response. Both T-cell and B-cell responses were cross-reactive with other GII strains, but not GI strains. To promote entry mechanisms, inclusion of bile acids was essential for GII.2 binding to nonsecretor HBGAs.ConclusionsThese data support development of within-genogroup, cross-reactive antibody and T-cell immunity, key outcomes that may provide the foundation for eliciting broad immune responses after GII.4 vaccination in individuals with limited GII.4 immunity, including young children.

Immunological Cross-Reactivity of an Ancestral and the Most Recent Pandemic Norovirus GII.4 Variant.

Norovirus (NoV) genotype GII.4 is responsible for the majority of NoV infections causing pandemics every few years. A NoV virus-like particle (VLP)-based vaccine should optimally cover the high antigenic variation within the GII.4 genotype. We compared the immune responses generated by VLPs of the ancestral GII.4 1999 strain (GII.4 1995/96 US variant) and the most recent GII.4 Sydney 2012 pandemic strains in mice. No significant differences were observed in the type-specific responses but GII.4 1999 VLPs were more potent in inducing high-avidity antibodies with better cross-reactivity. GII.4 1999 immune sera blocked binding of GII.4 2006 and GII.4 2012 VLPs to the putative receptors in a surrogate neutralization assay, whereas GII.4 2012 immune sera only had low blocking activity against GII.4 2006 VLPs. Amino acid substitution in the NERK motif (amino acids 310, 316, 484, and 493, respectively), altering the access to conserved blocking epitope F, moderately improved the cross-blocking responses against mutated GII.4 2012 VLPs (D310N). NoV GII.4 1999 VLPs, uptaken and processed by antigen-presenting cells, induced stronger interferon gamma (IFN-γ) production from mice splenocytes than GII.4 2012 VLPs. These results support the use of GII.4 1999 VLPs as a major component of a NoV vaccine.