HPV-specific antibodies in female genital tract secretions captured via first-void urine retain their neutralizing capacity.

Human papillomavirus (HPV) vaccines, primarily relying on neutralizing antibodies, have proven highly effective. Recently, HPV-specific antibodies have been detected in the female genital tract secretions captured by first-void urine (FVU), offering a minimally invasive diagnostic approach. In this study, we investigated whether HPV16-specific antibodies present in FVU samples retain their neutralizing capacity by using pseudovirion-based neutralization assays. Paired FVU and serum samples (vaccinated n = 25, unvaccinated n = 25, aged 18-25) were analyzed using two orthogonal pseudovirion-based neutralization assays, one using fluorescence microscopy and the other using luminescence-based spectrophotometry. Results were compared with HPV16-specific IgG concentrations and correlations between neutralizing antibodies in FVU and serum were explored. The study demonstrated the presence of neutralizing antibodies in FVU using both pseudovirion-based neutralization assays, with the luminescence-based assay showing higher sensitivity for FVU samples, while the fluorescence microscopy-based assay exhibited better specificity for serum and overall higher reproducibility. High Spearman correlation values were calculated between HPV16-IgG and HPV16-neutralizing antibodies for both protocols (rs: 0.54-0.94, p < .001). Significant Spearman correlations between FVU and serum concentrations were also established for all assays (rs: 0.44-0.91, p < .01). This study demonstrates the continued neutralizing ability of antibodies captured with FVU, supporting the hypothesis that HPV vaccination may reduce autoinoculation and transmission risk to the sexual partner. Although further protocol optimizations are warranted, these findings provide a foundation for future research and larger cohort studies that could have implications for the optimal design, evaluation, and implementation of HPV vaccination programs.

Multicentric evaluation of sensitivity of eight commercial anti-SARS-CoV-2 antibody assays and their correlation to virus neutralization titers in seropositive subjects.

Diagnosis of SARS-CoV-2 virus is mainly based on direct detection. Determination of specific antibodies has been used mostly for epidemiological reasons. However, select immunoassays showed good correlation to plaque reduction virus neutralization test (PRNT) in smaller patient cohorts, which suggests their potential as predictors of virus neutralization titer. A total of 3,699 samples from Covid-19 patients were included in the multicentric study performed in the Czech Republic. Anti-SARS-CoV-2 antibody levels were evaluated by 8 commercial antibody assays. Simultaneously, PRNT evaluations were performed with the SARS-CoV-2 B.1.258 variant. All immunoassays showed an overall high true positive diagnostic value ranging from 79.17 to 98.04%. Several commercial EIA methods showed highly positive correlation between the assay results and PRNT levels, e.g., Liaison CoV-2 TrimericS IgG DiaSorin (Spearman r = 0.8833; Architect SASRS-CoV-2 IgG Abbott (r = 0.7298); NovaLisa SARS-CoV-2 IgG NovaTec (r = 0.7103) and Anti-SARS-CoV-2 ELISA IgG Euroimmun (r = 0.7094). While this correlation was less positive for other assays, those, conversely, presented higher true positive values. For most immunoassays, the positive percent agreement of the results was ≥ 95% in sera exhibiting PRNT levels of 1:80 and higher. The assays tested have shown variable correlation to PRNT. Those possessing high positive predictive values serve well as qualitative tests, while others can be utilised as quantitative tests highly predictive of neutralization antibody levels.

Development of a proficiency testing program for HPV serology assays used to evaluate antibody responses in vaccine trials.

In this study, a proficiency panel was created for evaluation of assay performance and inter- and intra-laboratory assay comparisons, especially the ability to accurately measure negative, low, intermediate, and high levels of HPV type-specific antibodies. Comprised of 80 deidentified samples, this panel is designed for individual labs to evaluate assay performance characteristics on a biennial basis, to promote standardization of methodology and harmonization of data from human papillomavirus (HPV) serology tests in vaccine trials. The proficiency panel was qualified using 2 types of assays (singleplex Enzyme-Linked Immunosorbent Assays [ELISAs] or Multiplex antibody-binding assays and Pseudovirion-based neutralization assays [PBNAs]) in 10 laboratories from 7 countries, monitoring HPV antibody responses for up to 9 HPV types and using 3 different analysis methods. Sensitivity, specificity, and correlations (concordance, accuracy, and precision) were evaluated for each HPV type. In laboratories that tested all 80 samples, results from most (74/80) samples were reported with 100% accuracy across all 9 HPV types. The average sensitivity and specificity for singleplex and multiplex antibody binding assays ranged from 86.7% to 98.3% (sensitivity) and 84.2% to 94.3% (specificity), while the average sensitivity and specificity for the Pseudovirion (PsV)-based neutralization assays (PBNA) ranged from 87.6% to 99.4% (sensitivity) and 52.4% to 94.4% (specificity). This proficiency panel will help with assessing performance characteristics of HPV serology assays used in clinical trial studies and assure the data generated from these assays is harmonized.

Development of a high-throughput flow cytometric neutralization assay to screen for human enterovirus A71 (EVA71) neutralizing antibodies.

The conventional method for screening neutralizing antibodies to human enterovirus A71 (EVA71) (microneutralization assay) is time consuming and requires an expert to perform manual evaluation. An automated neutralization assay could shorten the testing time, improve reproducibility, and provide automatic analysis. This study aimed to develop a high-throughput flow cytometric neutralization assay to screen for EVA71 neutralizing antibodies, and to develop quality control materials to ensure accurate testing. The results of this study demonstrate the high potential viability of the proposed flow cytometric method. Compared to the standard method, the flow cytometric method was shown to require a smaller sample volume, provide a much faster turnaround time, provide a rapid result for interpreting the neutralizing antibody level, and allow for possible quantification of results. The observed drawbacks of the proposed method include higher cost per test, longer hands-on time, and lower sensitivity in low titer conditions, which could lead to false negative results. The developed quality control materials were demonstrated to be effective and storable for 1 month. These results pave the way for the optimization and implementation of an automated neutralization assay to screen for neutralizing antibodies not only against EVA71, but also against other viruses in the enterovirus genus.

Development of an automated, high-throughput SARS-CoV-2 neutralization assay based on a pseudotyped virus using a vesicular stomatitis virus (VSV) vector.

ABSTRACTThe global outbreak of COVID-19 has caused a severe threat to human health; therefore, simple, high-throughput neutralization assays are desirable for developing vaccines and drugs against COVID-19. In this study, a high-titre SARS-CoV-2 pseudovirus was successfully packaged by truncating the C-terminus of the SARS-CoV-2 spike protein by 21 amino acids and infecting 293 T cells that had been stably transfected with the angiotensin-converting enzyme 2 (ACE2) receptor and furin (named AF cells), to establish a simple, high-throughput, and automated 384-well plate neutralization assay. The method was optimized for cell amount, virus inoculation, incubation time, and detection time. The automated assay showed good sensitivity, accuracy, reproducibility, Z' factor, and a good correlation with the live virus neutralization assay. The high-throughput approach would make it available for the SARS-CoV-2 neutralization test in large-scale clinical trials and seroepidemiological surveys which would aid the accelerated vaccine development and evaluation.

Standardization, validation, and comparative evaluation of a faster and high-performance test for quantification of yellow fever neutralizing antibodies.

Although it is considered the reference for quantification of neutralizing antibodies, classical method of the plaque reduction neutralization test (PRNT) is labor intensive, requires specific equipment and inputs, besides a long time for its finalization, even in the micro-PRNT version (in 96-well plates). It has a higher sample throughput, however the smaller wells make the reading of plaques more difficult. With an immunoenzymatic revelation step and a semi-automated reading, the µFRN-HRP (micro Focus Reduction Neutralization - Horseradish Peroxidase) is a faster and more efficient test for the quantification of YF neutralizing antibodies. This study aimed to standardize, validate, and compare it with the reference method in 6-well plates (PRNT). Once the execution protocol was standardized, precision, accuracy, selectivity, and robustness were evaluated to validate the µFRN-HRP. In addition, 200 sera of vaccinees were processed by the µFRN-HRP and by the micro-PRNT to compare with the reference test, estimating agreement by Intraclass Correlation Coefficient (ICC). The standardization and validation of the µFRN-HRP was carried out successfully. Weak to moderate agreement was observed between µFRN-HRP and PRNT for titers in reciprocal dilution, while the same comparison between the classical tests resulted in a better ICC. However, titers in milli-international units obtained by µFRN-HRP showed a substantial agreement with PRNT, while the agreement between micro-PRNT and PRNT was inferior. Therefore, µFRN-HRP can be used in the confirmation of natural YF infection and immune response to vaccination, replacing the micro-PRNT, gaining agility, while preserving the specificity of the result.

Development of a Cell-Based SARS-CoV-2 Pseudovirus Neutralization Assay Using Imaging and Flow Cytometry Analysis.

COVID-19 is an ongoing, global pandemic caused by the novel, highly infectious SARS-CoV-2 virus. Efforts to mitigate the effects of SARS-CoV-2, such as mass vaccination and development of monoclonal therapeutics, require precise measurements of correlative, functional neutralizing antibodies that block virus infection. The development of rapid, safe, and easy-to-use neutralization assays is essential for faster diagnosis and treatment. Here, we developed a vesicular stomatitis virus (VSV)-based neutralization assay with two readout methods, imaging and flow cytometry, that were capable of quantifying varying degrees of neutralization in patient serum samples. We tested two different spike-pseudoviruses and conducted a time-course assay at multiple multiplicities of infection (MOIs) to optimize the assay workflow. The results of this assay correlate with the results of previously developed serology and surrogate neutralization assays. The two pseudovirus readout methods produced similar values of 50% neutralization titer values. Harvest-free in situ readouts for live-cell imaging and high-throughput analysis results for flow cytometry can provide unique capabilities for fast evaluation of neutralization, which is critical for the mitigation of future pandemics.

A simplified method for measuring neutralising antibodies against rabies virus.

Rabies virus (RABV) causes a fatal encephalitis that can be prevented through timely vaccination. The levels of virus neutralising antibodies against rabies virus induced by vaccination can be measured using the fluorescent antibody virus neutralisation (FAVN) test. Following incubation of live virus with sera, this method involves the fixation of cell monolayers and staining of rabies virus-specific antigen using fluorescein isothiocyanate (FITC) -conjugated antibody to enable visualisation of rabies virus antigen using a fluorescence microscope. To simplify this procedure, a fluorescent recombinant rabies virus was constructed using reverse genetics by inserting the gene for the mCherry fluorescent protein in front of the ribonucleoprotein gene of the SAD B-19 genome and replacing its glycoprotein with that of the Challenge Virus Standard (CVS)-11 RABV strain to ensure antigenic authenticity with the FAVN. This new recombinant virus (termed mCCCG) expressed the mCherry protein to high levels enabling direct observation of infected cells. In vitro growth kinetics of mCCCG were indistinguishable from that of CVS-11. The stability of the recombinant virus was assessed by sequencing several passages of the rescued virus and only minor changes were detected. Comparative assessment of the virus neutralisation test using mCherry producing virus (NTmCV) against the FAVN demonstrated that test results were equivalent to each other; therefore, mCCCG can be used as an alternative to CVS-11 for measuring antibody titres against the rabies virus. The use of NTmCV removes the need for expensive antibody conjugates and significantly reduces assay time. This would be particularly beneficial for RABV serological assessment in resource limited settings. Moreover, the reading of the plates can be automatically using a cell imaging reader.

Evaluation and validation of a commercial ELISA versus the in vitro toxin neutralization assay for determination of diphtheria anti-toxin in human serum.

Introduction. Diphtheria is a potentially life-threatening infection and remains endemic in many low- and middle-income countries (LMICs). A reliable, low-cost method for serosurveys in LMICs is warranted to estimate the accurate population immunity to control diphtheria.Hypothesis/Gap Statement. The correlation between the ELISA results against diphtheria toxoid and the gold standard diphtheria toxin neutralization test (TNT) values is poor when ELISA values are <0.1 IU ml-1, which results in inaccurate estimates of susceptibility in populations when ELISA is used for measuring antibody levels.Aim. To explore methods to accurately predict population immunity and TNT-derived anti-toxin titres from ELISA anti-toxoid results.Methodology. A total of 96 paired serum and dried blood spot (DBS) samples collected in Vietnam were used for comparison of TNT and ELISA. The diagnostic accuracy of ELISA measurement with reference to TNT was assessed by area under the receiver operating characteristic (ROC) curve (AUC) and other parameters. Optimal ELISA cut-off values corresponding to TNT cut-off values of 0.01 and 0.1 IU ml-1 were identified by ROC analysis. A method based on the multiple imputation approach was also applied to estimate TNT measurements in a dataset that only included ELISA results. These two approaches were then applied to ELISA results previously generated from 510 subjects in a serosurvey in Vietnam.Results. The ELISA results on DBS samples showed a good diagnostic performance compared to TNT. The cut-off values for ELISA measurement corresponding to the TNT cut-off values of 0.01 IU ml-1 were 0.060 IU ml-1 in serum samples, and 0.044 IU ml-1 in DBS samples. When a cut-off value of 0.06 IU ml-1 was applied to the 510 subject serosurvey data, 54 % of the population were considered susceptible (<0.01 IU ml-1). The multiple imputation-based approach estimated that 35 % of the population were susceptible. These proportions were much larger than the susceptible proportion estimated by the original ELISA measurements.Conclusion. Testing a subset of sera by TNT combined with ROC analysis or a multiple imputation approach helps to adjust ELISA thresholds or values to assess population susceptibility more accurately. DBS is an effective low-cost alternative to serum for future serological studies for diphtheria.

An ELISA system for tetanus toxoid potency tests: An alternative to lethal challenge.

For a long time, a widely used method for tetanus toxoid (Ttd) potency has been the challenge test, in which animals are immunized and then challenged with tetanus toxin in lethal or non-lethal way. In the context of animal welfare, an alternative is desired because the method causes unsustainable distress to animals. We aimed to replace the system for describing test results, in which scores are assigned to symptoms exhibited by challenged animals, with scores assigned to antibody ELISA titers in immunized mouse sera. The potency values and confidence intervals calculated by the absorbance score system were equivalent to those calculated by the symptom score system. We also attempted to utilize the raw ELISA absorbance instead of the assigned absorbance score and obtained similar results. ELISA may serve as an alternative to the lethal challenge for Ttd potency tests, not only in Japan but also in other countries in which mouse challenge tests are employed.

Comparison of measles IgG enzyme immunoassays (EIA) versus plaque reduction neutralization test (PRNT) for measuring measles serostatus: a systematic review of head-to-head analyses of measles IgG EIA and PRNT.

BACKGROUND: As countries move towards or achieve measles elimination status, serosurveillance is an important public health tool. However, a major challenge of serosurveillance is finding a feasible, accurate, cost-effective, and high throughput assay to measure measles antibody concentrations and estimate susceptibility in a population. We conducted a systematic review to assess, characterize, and - to the extent possible - quantify the performance of measles IgG enzyme-linked assays (EIAs) compared to the gold standard, plaque reduction neutralization tests (PRNT). METHODS: We followed the PRISMA statement for a systematic literature search and methods for conducting and reporting systematic reviews and meta-analyses recommended by the Cochrane Screening and Diagnostic Tests Methods Group. We identified studies through PubMed and Embase electronic databases and included serologic studies detecting measles virus IgG antibodies among participants of any age from the same source population that reported an index (any EIA or multiple bead-based assays, MBA) and reference test (PRNT) using sera, whole blood, or plasma. Measures of diagnostic accuracy with 95% confidence intervals (CI) were abstracted for each study result, where reported. RESULTS: We identified 550 unique publications and identified 36 eligible studies for analysis. We classified studies as high, medium, or low quality; results from high quality studies are reported. Because most high quality studies used the Siemens Enzygnost EIA kit, we generate individual and pooled diagnostic accuracy estimates for this assay separately. Median sensitivity of the Enzygnost EIA was 92.1% [IQR = 82.3, 95.7]; median specificity was 96.9 [93.0, 100.0]. Pooled sensitivity and specificity from studies using the Enzygnost kit were 91.6 (95%CI: 80.7,96.6) and 96.0 (95%CI: 90.9,98.3), respectively. The sensitivity of all other EIA kits across high quality studies ranged from 0% to 98.9% with median (IQR) = 90.6 [86.6, 95.2]; specificity ranged from 58.8% to 100.0% with median (IQR) = 100.0 [88.7, 100.0]. CONCLUSIONS: Evidence on the diagnostic accuracy of currently available measles IgG EIAs is variable, insufficient, and may not be fit for purpose for serosurveillance goals. Additional studies evaluating the diagnostic accuracy of measles EIAs, including MBAs, should be conducted among diverse populations and settings (e.g., vaccination status, elimination/endemic status, age groups).

Application of Pseudotyped Viruses.

Highly pathogenic emerging and reemerging viruses have serious public health and socioeconomic implications. Although conventional live virus research methods can more reliably investigate disease pathogenicity and evaluate antiviral products, they usually depend on high-level biosafety laboratories and skilled researchers; these requirements hinder in vitro assessments of efficacy, as well as efforts to test vaccines and antibody drugs. In contrast, pseudotyped viruses (i.e., single-round infectious viruses that mimic the membrane structures of various live viruses) are widely used in studies of highly pathogenic viruses because they can be handled in biosafety level 2 facilities. This chapter provides a concise overview of various aspects of pseudotyped virus technologies, including (1) exploration of the mechanisms of viral infection; (2) evaluation of the efficacies of vaccines and monoclonal antibodies based on pseudovirion-based neutralization assay; (3) assessment of antiviral agents (i.e., antibody-based drugs and inhibitors); (4) establishment of animal models of pseudotyped virus infection in vivo; (5) investigation of the evolution, infectivity, and antigenicity of viral variants and viral glycosylation; and (6) prediction of antibody-dependent cell-mediated cytotoxic activity.

Application and comparison of lyophilisation protocols to enhance stable long-term storage of filovirus pseudotypes for use in antibody neutralisation tests.

AIMS: Filoviruses encompass highly pathogenic viruses placing significant public health burden on countries affected. Efforts for improved diagnostics and surveillance are needed. The requirement for high-containment can be circumvented by using pseudotype viruses (PV), which can be handled safely, in tropism, drug screening, vaccine evaluation, and serosurveillance studies. We assessed the stability and functionality after long-term storage of lyophilised filovirus pseudotypes for use in neutralisation assays. METHODS AND RESULTS: We generated a panel of filovirus lentiviral pseudotypes followed by lyophilisation and storage in different conditions. Next, we reconstituted and tested PVs in infection experiments and pseudotype neutralisation assays where possible. Lyophilised Ebola and Marburg PVs retained production titres for at least two years when stored at +4˚C or less. Lyophilised Ebola PVs performed similarly to non-lyophilised PVs in neutralisation assays after reconstitution. When stored at high temperatures (+37˚C), lyophilised PVs did not retain titres after 1-month storage, however, when lyophilised using pilot-scale facilities EBOV PVs retained titres and performed as standard in neutralisation assays after on 1-month storage at 37˚C. CONCLUSIONS: Filovirus PVs are amenable to lyophilisation and can be stored for at least 2 years in a household fridge to be used in antibody assays. Lyophilisation performed in the right conditions would allow transportation at room temperature, even in warmer climates.

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.

Analysis of cross-reactivity among flaviviruses using sera of patients with dengue showed the importance of neutralization tests with paired serum samples for the correct interpretations of serological test results for dengue.

Dengue is a febrile illness caused by the dengue virus (DENV) that belongs to the genus Flavivirus in the family Flaviviridae. Cross-reactivity between flaviviruses poses a challenge while interpreting serological test results. In the present study, the cross-reactivity of sera of the patients with dengue, who traveled from Japan to DENV-endemic countries, was analyzed by using an enzyme-linked immunosorbent assay (ELISA) and neutralization test (NT). Sixteen serum samples were collected from patients with dengue and were tested for: i) IgM antibodies against Zika virus (ZIKV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) using IgM ELISA, ii) IgG antibody against TBEV using IgG ELISA, and iii) neutralizing antibody against ZIKV, WNV, TBEV, and JEV. Among the 16 samples tested using ELISA, seven samples were IgM-positive for at least one of the other flaviviruses, and nine samples were IgG-positive for TBEV. Neutralizing antibody titers (NATs) against ZIKV, WNV, and TBEV were one-fourth or lower than those against the causative DENV in all samples. The NATs against JEV were one-fourth or lower than those against the causative DENV in six convalescent-phase serum sample among the seven convalescent-phase serum samples. The NAT against DENV of the residual one convalescent-phase serum was similar to that against JEV and that against JEV of its relevant acute-phase serum sample. These results showed that NTs with paired serum samples are important to correctly interpret the serological test results for DENV.

A Lentiviral Pseudotype System to Characterize SARS-CoV-2 Glycoprotein.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2 causes worldwide COVID-19 pandemic and poses a great threat to global public health. Due to its high pathogenicity and infectivity, live SARS-CoV-2 is classified as a BSL-3 agent and has to be handled in BSL-3 condition. Nevertheless, entry of SARS-CoV-2 is mediated by viral spike (S) glycoprotein, and pseudovirus with SARS-CoV-2 S protein can mimic every entry step of SARS-CoV-2 virus and be studied in BSL-2 settings. In this chapter, we describe a detailed protocol of production of lentivirus-based SARS-CoV-2 S pseudovirus and its application in study of virus entry and determination of neutralizing antibody titer of human sera against SARS-CoV-2.

Novel Affibody Molecules Specifically Bind to SARS-CoV-2 Spike Protein and Efficiently Neutralize Delta and Omicron Variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been an unprecedented public health disaster in human history, and its spike (S) protein is the major target for vaccines and antiviral drug development. Although widespread vaccination has been well established, the viral gene is prone to rapid mutation, resulting in multiple global spread waves. Therefore, specific antivirals are needed urgently, especially those against variants. In this study, the domain of the receptor binding motif (RBM) and fusion peptide (FP) (amino acids [aa] 436 to 829; denoted RBMFP) of the SARS-CoV-2 S protein was expressed as a recombinant RBMFP protein in Escherichia coli and identified as being immunogenic and antigenically active. Then, the RBMFP proteins were used for phage display to screen the novel affibody. After prokaryotic expression and selection, four novel affibody molecules (Z14, Z149, Z171, and Z327) were obtained. Through surface plasmon resonance (SPR) and pseudovirus neutralization assay, we showed that affibody molecules specifically bind to the RBMFP protein with high affinity and neutralize against SARS-CoV-2 pseudovirus infection. Especially, Z14 and Z171 displayed strong neutralizing activities against Delta and Omicron variants. Molecular docking predicted that affibody molecule interaction sites with RBM overlapped with ACE2. Thus, the novel affibody molecules could be further developed as specific neutralization agents against SARS-CoV-2 variants. IMPORTANCE SARS-CoV-2 and its variants are threatening the whole world. Although a full dose of vaccine injection showed great preventive effects and monoclonal antibody reagents have also been used for a specific treatment, the global pandemic persists. So, developing new vaccines and specific agents are needed urgently. In this work, we expressed the recombinant RBMFP protein as an antigen, identified its antigenicity, and used it as an antigen for affibody phage-display selection. After the prokaryotic expression, the specific affibody molecules were obtained and tested for pseudovirus neutralization. Results showed that the serum antibody induced by RBMFP neutralized Omicron variants. The screened affibody molecules specifically bound the RBMFP of SARS-CoV-2 with high affinity and neutralized the Delta and Omicron pseudovirus in vitro. So, the RBMFP induced serum provides neutralizing effects against pseudovirus in vitro, and the affibodies have the potential to be developed into specific prophylactic agents for SARS-CoV-2 and its variants.

A pseudovirus-based platform to measure neutralizing antibodies in Mexico using SARS-CoV-2 as proof-of-concept.

The gold-standard method to evaluate a functional antiviral immune response is to titer neutralizing antibodies (NAbs) against a viral pathogen. This is historically performed using an in vitro assay of virus-mediated infection, which requires BSL-3 facilities. As these are insufficient in Latin American countries, including Mexico, scant information is obtained locally about viral pathogens NAb, using a functional assay. An alternative solution to using a BSL-3 assay with live virus is to use a BSL-2-safe assay with a non-replicative pseudovirus. Pseudoviral particles can be engineered to display a selected pathogen's entry protein on their surface, and to deliver a reporter gene into target cells upon transduction. Here we comprehensively describe the first development of a BSL-2 safe NAbs-measuring functional assay in Mexico, based on the production of pseudotyped lentiviral particles. As proof-of-concept, the assay is based on Nanoluc luciferase-mediated luminescence measurements from target cells transduced with SARS-CoV-2 Spike-pseudotyped lentiviral particles. We applied the optimized assay in a BSL-2 facility to measure NAbs in 65 serum samples, which evidenced the assay with 100% sensitivity, 86.6% specificity and 96% accuracy. Overall, this is the first report of a BSL-2 safe pseudovirus-based functional assay developed in Mexico to measure NAbs, and a cornerstone methodology necessary to measure NAbs with a functional assay in limited resources settings.

Nanoluciferase-based cell fusion assay for rapid and high-throughput assessment of SARS-CoV-2-neutralizing antibodies in patient samples.

After more than two years, COVID-19 still represents a global health burden of unprecedented extent and assessing the degree of immunity of individuals against SARS-CoV-2 remains a challenge. Virus neutralization assays represent the gold standard for assessing antibody-mediated protection against SARS-CoV-2 in sera from recovered and/or vaccinated individuals. Neutralizing antibodies block the interaction of viral spike protein with human angiotensin-converting enzyme 2 (ACE2) receptor in vitro and prevent viral entry into host cells. Classical viral neutralization assays using full replication-competent viruses are restricted to specific biosafety level 3-certified laboratories, limiting their utility for routine and large-scale applications. We developed therefore a cell-fusion-based assay building on the interaction between viral spike and ACE2 receptor expressed on two different cell lines, substantially reducing biosafety risks associated with classical viral neutralization assays. This chapter describes this simple, sensitive, safe and cost-effective approach for rapid and high-throughput evaluation of SARS-CoV-2 neutralizing antibodies relying on high-affinity NanoLuc® luciferase complementation technology (HiBiT). When applied to a variety of standards and patient samples, this method yields highly reproducible results in 96-well, as well as in 384-well format. The use of novel NanoLuc® substrates with increased signal stability like Nano-Glo® Endurazine™ furthermore allows for high flexibility in assay set-up and full automatization of all reading processes. Lastly, the assay is suitable to evaluate the neutralizing capacity of sera against the existing spike variants, and potentially variants that will emerge in the future.

Antibody titers and neutralizing activity in cases of COVID-19 after a single dose of vaccination.

Vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have shown high efficacy in preventing the onset of disease. However, the immune response to infection immediately after the first vaccination remains unknown. We examined the anti-SARS-CoV-2-binding-antibody titers and neutralizing activity in patients who developed coronavirus disease 2019 after the first vaccination. The amount of anti-SARS-CoV-2-binding antibodies and neutralizing activity drastically increased from the first to the second collection. Our results may provide important data on the course of immune response following vaccination.