Validation of a triple-color pseudovirion-based neutralization assay for immunogenicity assessment of a 14-valent recombinant human papillomavirus vaccine.

Validation of bioanalytical methods is crucial, especially in the pharmaceutical industry, to determine their suitability for specific purposes and the accuracy of analytical results. The pseudovirion-based neutralization assay (PBNA) is considered the gold standard for detecting and quantifying neutralizing antibodies against human papillomavirus in vaccine development for disease prevention. This paper introduces an improved triple-color PBNA method, capable of simultaneous detection of two or three human papillomavirus (HPV types for use in the development of a 14-valent HPV vaccine candidate. The primary objective was to comprehensively validate the triple-color PBNA method for general vaccine immunogenicity assays. Results show that the method has good specificity, accuracy, precision, linearity, robustness, and applicability. This innovative triple-color PBNA offers an improved approach for large-scale immunogenicity assessment in vaccine development. This study lays a solid foundation that can serve as a guiding paradigm for assessing vaccine responses in preclinical and clinical phases, providing valuable insights to the field.

Virus neutralization assays for human respiratory syncytial virus using airway organoids.

Neutralizing antibodies are considered a correlate of protection against severe human respiratory syncytial virus (HRSV) disease. Currently, HRSV neutralization assays are performed on immortalized cell lines like Vero or A549 cells. It is known that assays on these cell lines exclusively detect neutralizing antibodies (nAbs) directed to the fusion (F) protein. For the detection of nAbs directed to the glycoprotein (G), ciliated epithelial cells expressing the cellular receptor CX3CR1 are required, but generation of primary cell cultures is expensive and labor-intensive. Here, we developed a high-throughput neutralization assay based on the interaction between clinically relevant HRSV grown on primary cells with ciliated epithelial cells, and validated this assay using a panel of infant sera. To develop the high-throughput neutralization assay, we established a culture of differentiated apical-out airway organoids (Ap-O AO). CX3CR1 expression was confirmed, and both F- and G-specific monoclonal antibodies neutralized HRSV in the Ap-O AO. In a side-by-side neutralization assay on Vero cells and Ap-O AO, neutralizing antibody levels in sera from 125 infants correlated well, although titers on Ap-O AO were consistently lower. We speculate that these lower titers might be an actual reflection of the neutralizing antibody capacity in vivo. The organoid-based neutralization assay described here holds promise for further characterization of correlates of protection against HRSV disease.

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.

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.

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).

Precision and correlation of ED50 and endpoint titer method in measuring HPV vaccine immunogenicity.

Cervical cancer, the second leading cause of cancer-related deaths among women, is caused by human papillomavirus (HPV), a sexually transmitted virus. Vaccination is an effective preventive measure against viral infections and subsequent development of cervical cancer. Enzyme-linked immunosorbent assay (ELISA) is commonly used to measure specific binding antibody titers and assess the immunogenicity of test vaccines in preclinical models or clinical volunteers. Two methods of deriving titers, the endpoint titer (ET) and the effective dilution producing a median maximal effective fold of dilution (ED50) with a cut-off value, are widely used. For HPV, a pseudovirion-based neutralization assay (PBNA) is used to measure functional antibody titers. The ELISA binding titers and functional PBNA titers were found to be well-correlated for all nine HPV types tested in the vaccine, consistent with previous studies on HPV 16/18. Comparing the PBNA results with the two titration methods, the ED50 method showed higher precision and a closer correlation with PBNA results, both for individual types and pooled data analysis for all nine types. When comparing the titration results of the ET method based on a cut-off value with the ED50 method using all the data points across the dilution series, the ED50 method demonstrated better precision and a stronger correlation with PBNA results.

High-titer neutralization of Mu and C.1.2 SARS-CoV-2 variants by vaccine-elicited antibodies of previously infected individuals.

Recently identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants Mu and C.1.2 have spike proteins with mutations that may confer resistance to natural and vaccine-elicited antibodies. Analysis of neutralizing antibody titers in the sera of vaccinated individuals without previous history of infection and from convalescent individuals show partial resistance of the viruses. In contrast, sera from individuals with a previous history of SARS-CoV-2 infection who were subsequently vaccinated neutralize variants with titers 4- to 11-fold higher, providing a rationale for vaccination of individuals with previous infection. The heavily mutated C.1.2 spike is the most antibody neutralization-resistant spike to date; however, the avidity of C.1.2 spike protein for angiotensin-converting enzyme 2 (ACE2) is low. This finding suggests that the virus evolved to escape the humoral response but has a decrease in fitness, suggesting that it may cause milder disease or be less transmissible. It may be difficult for the spike protein to evolve to escape neutralizing antibodies while maintaining high affinity for ACE2.

An Antigenically Diverse, Representative Panel of Envelope Glycoproteins for Hepatitis C Virus Vaccine Development.

BACKGROUND & AIMS: Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpps) for neutralization assays. METHODS: We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpps. Neutralization sensitivity of these HCVpps varied widely. HCVpps clustered into 15 distinct groups based on patterns of relative sensitivity to 7 broadly neutralizing monoclonal antibodies. We used these data to select a final panel of 15 antigenically representative HCVpps. RESULTS: Both the 65 and 15 HCVpp panels span 4 tiers of neutralization sensitivity, and neutralizing breadth measurements for 7 broadly neutralizing monoclonal antibodies were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpps were independent of genetic distances between E1E2 clones. CONCLUSIONS: Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity rather than panels selected solely for genetic diversity. We propose that this multitier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories.

Methods to Measure Antibody Neutralization of Live Human Coronavirus OC43.

The human Betacoronavirus OC43 is a common cause of respiratory viral infections in adults and children. Lung infections with OC43 are associated with mortality, especially in hematopoietic stem cell transplant recipients. Neutralizing antibodies play a major role in protection against many respiratory viral infections, but to date a live viral neutralization assay for OC43 has not been described. We isolated a human monoclonal antibody (OC2) that binds to the spike protein of OC43 and neutralizes the live virus derived from the original isolate of OC43. We used this monoclonal antibody to develop and test the performance of two readily accessible in vitro assays for measuring antibody neutralization, one utilizing cytopathic effect and another utilizing an ELISA of infected cells. We used both methods to measure the neutralizing activity of the OC2 monoclonal antibody and of human plasma. These assays could prove useful for studying humoral responses to OC43 and cross-neutralization with other medically important betacoronaviruses.

Tiempo de permanencia de anticuerpos y reinfección en pacientes con antecedente de infección por SARS-CoV-2 / Length of stay of antibodies and reinfection in patients with a history of SARS-CoV-2 infection

ANTECEDENTES: La enfermedad causada por el coronavirus de tipo 2 causante del síndrome respiratorio agudo severo (SARS-CoV-2) denominada como COVID-19 fue reportada inicialmente en la ciudad de Wuhan en China en diciembre de 2019 (1,3). Al poco tiempo, el 11 de marzo de 2020, fue caracterizada como pandemia por la Organización Mundial de la Salud (OMS). El espectro de la enfermedad es amplio e incluye desde cuadros leves y autolimitados hasta neumonía atípica severa y progresiva, falla multiorgánica y muerte (4,5). De acuerdo a la OMS, en el 2020 se subregistraron más de 1.8 millones de muertes a causa de esta enfermedad a nivel mundial. El Perú ha sido uno de los países más afectado en Latinoamérica con, al 30 de julio de 2021, más de 2 millones de casos y 196 214 fallecidos (letalidad de 9.31%) (6). Por lo tanto, conocer el tiempo de duración de esta inmunidad adquirida por la infección causada por SARS-CoV-2 es importante para poder guiar estrategias de vigilancia epidemiológica y de inmunizaciones. OBJETIVO: El objetivo de esta revisión fue identificar y sistematizar la evidencia disponible sobre el tiempo de permanencia de seropositividad y capacidad de neutralización de los Ac contra el SARS-CoV-2, la incidencia de reinfección y el tiempo hasta el evento en personas con antecedente de infección por SARS-CoV-2. METODOLOGÍA: Se realizó una revisión rápida basada en dos preguntas: En pacientes con antecedente de infección por SARS-CoV-2 ¿Cuál es el tiempo de permanencia de seropositividad y capacidad de neutralización de los Ac contra el SARS-CoV-2? ¿Cuál es la incidencia de reinfección y el tiempo hasta este evento? Se incluyeron estudios de cohorte. Para ello, se realizó una búsqueda sistemática en las bases de datos MEDLINE/PubMed, EMBASE/Ovid y LOVE/Epistemonikos. Luego de eliminar duplicados, los autores seleccionaron los ítems que cumplieran con las preguntas establecidas. RESULTADOS: Se incluyeron 32 estudios: 20 evaluaron el tiempo de permanencia de seropositividad de Ac anti SARS-CoV-2: 11 estudios evaluaron únicamente el tiempo de permanencia de seropositividad de los Ac, 9 estudiaron el tiempo de permanencia de seropositividad y capacidad de neutralización de los Ac, y 5 estudiaron únicamente el tiempo de permanencia de la capacidad de neutralización de los Ac 7 estudios evaluaron la incidencia de reinfección y el tiempo hasta este evento. La población incluida correspondió a adultos con antecedente de infección por SARS-CoV-2, 23 estudios incluyeron cohortes en población general, 6 incluyeron a personal de salud y 3 a donantes de plasma convaleciente. Para los estudios que evaluaron el tiempo de permanencia de seropositividad y capacidad de neutralización de los Ac, los periodos de observación fluctuaron entre 6 meses a 1 año. La mayoría (85%, 17/20) de los estudios coinciden en reportar la persistencia de Ac a los 6 meses de seguimiento, con variaciones en las frecuencias de las inmunoglobulinas (Ig) específicas (IgA, IgM, IgG) para las proteínas Spike (S), el complejo de unión al receptor (RBD) o a la proteína de nucleocápside (N). Los estudios reportaron que, hasta al menos 6 meses, la persistencia de la capacidad de neutralización de los Ac varió entre el 30.7 y 100% en los participantes. La incidencia de reinfección varió desde 0 a 4.85% en un tiempo aproximado de seguimiento de 180 días. De los 4 (57.14%) estudios que consideraron como intervalo de tiempo mínimo de 90 días en su definición de reinfección: la menor media de tiempo hasta la reinfección reportada fue de 116 ± 21 días y la mayor fue de 212 ± 25 días. Las personas que tuvieron una infección inicial por SARS-CoV-2 en comparación a aquellos sin infección previa (grupo control) tuvieron un menor riesgo de infección (reinfección), observándose una reducción de la incidencia de 84 a 89% en pacientes con infección previa en relación al grupo control; esta evidencia procede de 2 estudios. Hall et al., (41) realizó un sub-análisis de acuerdo a la presencia o no de síntomas de COVID-19 durante la infección inicial, reportando que el riesgo de reinfección en aquellos que fueron asintomáticos durante la infección inicial fue menor en comparación al grupo control (RTIa: 0.48, IC 95%: 0.37 ­ 0.63; reducción de riesgo relativo (RRR): 52%), aunque la reducción del riesgo fue mayor para el subgrupo con presencia de síntomas de COVID-19 en la infección inicial en comparación al grupo control (RTIa: 0.074, IC 95%: 0.06 ­ 0.10; RRR: 92.6%). La valoración de riesgos de sesgos indicó que los estudios que presentaron serias preocupaciones de riesgo de sesgos fueron el 65.75%: en los estudios sobre el tiempo de permanencia de seropositividad y capacidad de neutralización de los Ac anti-SARS-CoV-2, esta seria preocupación se presentó principalmente en el dominio 2 (sesgo de deserción) debido la pérdida de más del 60% de los participantes al final del seguimiento lo cual podría afectar la validez de los resultados. En los estudios sobre incidencia de reinfección, fue más frecuente (71.4%) la valoración de alguna preocupación de riesgo de sesgo debido a que el 71.4% fueron estudios retrospectivos y, en el 85.7% de los estudios las definiciones de reinfección no pudieron ser respaldadas por la realización de un análisis de secuenciación genómica. Las limitaciones de esta revisión son: que, debido a la heterogeneidad de las pruebas utilizadas para medir Ac, las definiciones de reinfección y la manera de reportar los desenlaces entre los diferentes estudios no fue posible realizar un meta-análisis para ninguno de los desenlaces. Adicionalmente, se incluyeron 8 (25%) estudios en pre-print y estos podrían modificar sus resultados y/o conclusiones en sucesivas versiones hasta su publicación, no siendo posible garantizar que respondan satisfactoriamente la revisión por pares y sean finalmente publicados. CONCLUSIONES: La evidencia disponible mostró que la mayoría (85%) de los estudios coinciden en reportar la persistencia de Ac a los 6 meses de seguimiento, con variaciones en las frecuencias de (Ig) específicas (IgA, IgM, IgG) para las proteínas S, RBD o a la proteína N. Hasta al menos 6 meses, la persistencia de la capacidad de neutralización de los Ac varió entre el 30.7 y 100% en los participantes. La incidencia de reinfección varió entre 0 a 4.85%, en un tiempo aproximado de seguimiento de 180 días. De los 4 (57.14%) estudios que consideraron como intervalo de tiempo mínimo de 90 días en su definición de reinfección: la menor media de tiempo hasta la reinfección reportada fue de 116 ± 21 días y la mayor fue de 212 ± 25 días. El antecedente de infección por SARS-CoV-2 redujo la incidencia de reinfección entre 84 y 89% en comparación a aquellos individuos sin infección previa (grupo control). La reducción de riesgo se mantuvo inclusive para el subgrupo de personas con infección inicial de tipo asintomática (reducción de incidencia de 52% en relación control); aunque, en menor magnitud que en aquellos que desarrollaron síntomas de COVID-19 (reducción incidencia de 93% en relación al control).

The venom of the scorpion Centruroides limpidus, which causes the highest number of stings in Mexico, is neutralized by two recombinant antibody fragments.

Phage display and directed evolution have made it possible to generate recombinant antibodies in the format of single chain variable fragments (scFvs) capable of neutralizing different toxins and venoms of Mexican scorpions. Despite having managed to neutralize a significant number of venoms, some others have not yet been completely neutralized, due to the diversity of the toxic components present in them. An example is the venom of the scorpion Centruroides limpidus, which contains three toxins of medical importance, called Cll1, Cll2 and Cl13. The first two are neutralized by scFv 10FG2, while Cl13, due to its sequence divergence, was not even recognized. For this reason, the aim of the present work was the generation of a new scFv capable of neutralizing Cl13 toxin and thereby helping to neutralize the whole venom of this scorpion. By hybridoma technology, a monoclonal antibody (mAb B7) was generated, which was able to recognize and partially neutralize Cl13 toxin. From mAb B7, its scFv format was obtained, named scFv B7 and subjected to three cycles of directed evolution. At the end of these processes, scFv 11F which neutralized Cl13 toxin was obtained. This scFv, administered in conjunction with scFv 10FG2, allowed to fully neutralize the whole venom of Centruroides limpidus scorpion.

Adjuvant effect of saponin in an oil-based monovalent (serotype O) foot-and-mouth disease virus vaccine on the antibody response in guinea pigs and cattle.

To enhance the potency of a foot-and-mouth disease (FMD) vaccine, saponin was included in the vaccine formula. In this study, the combined effect of Montanide ISA 50 and saponin was evaluated. Two experiments were performed in guinea pigs and one in cattle to determine the optimal antigen and saponin doses. Only serotype O of foot-and-mouth disease virus (O/PanAsia-2 of ME-SA topotype) was employed in preparation of the monovalent vaccine. All animals were immunized twice with a four-week interval, except for the negative controls. Blood was collected 10 days after the second booster, and the immune response was evaluated using a serum neutralization test. Oil-based FMD vaccines containing saponin induced higher neutralizing antibody levels than formulations lacking saponin. The addition of saponin to formulations with low antigen payload (2.5 µg of inactivated whole virus particles [146S particles] per dose) gave significantly higher neutralizing antibody levels (p < 0.005) than 5 µg of 146S without saponin, suggesting that it can be used to improve FMD vaccine potency in susceptible animals. No adverse effects were observed in vaccinated cattle or guinea pigs.

Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization.

The human complement system is an important part of the innate immune system. Its effector pathways largely mediate virus neutralization. Vesicular stomatitis virus (VSV) activates the classical pathway of the complement, leading to virus neutralization by lysis. Two host-derived membrane-associated regulators of complement activation (RCA), CD55 and CD46, which are incorporated into the VSV envelope during egress, confer protection by delaying/resisting complement-mediated neutralization. We showed previously that CD55 is more effective than CD46 in the inhibition of neutralization. In this study, we identified that, at the protein level, VSV infection resulted in the down-regulation of CD46 but not CD55. The mRNA of both the RCAs was significantly down-regulated by VSV, but it was delayed in the case of CD55. The immunoblot analysis of the levels of RCAs in the progeny virion harvested at three specific time intervals, points to an equal ratio of its distribution relative to viral proteins. Besides reconfirming the dominant role of CD55 over CD46 in shielding VSV from complement, our results also highlight the importance of the subtle modulation in the expression pattern of RCAs in a system naturally expressing them.

SARS-CoV-2 neutralizing antibodies in patients with varying severity of acute COVID-19 illness.

In order to support vaccine development, and to aid convalescent plasma therapy, it would be important to understand the kinetics, timing and persistence of SARS-CoV-2 neutralizing antibodies (NAbs), and their association with clinical disease severity. Therefore, we used a surrogate viral neutralization test to evaluate their levels in patients with varying severity of illness, in those with prolonged shedding and those with mild/asymptomatic illness at various time points. Patients with severe or moderate COVID-19 illness had earlier appearance of NAbs at higher levels compared to those with mild or asymptomatic illness. Furthermore, those who had prolonged shedding of the virus, had NAbs appearing faster and at higher levels than those who cleared the virus earlier. During the first week of illness the NAb levels of those with mild illness was significantly less (p = 0.01), compared to those with moderate and severe illness. At the end of 4 weeks (28 days), although 89% had NAbs, 38/76 (50%) in those with > 90 days had a negative result for the presence of NAbs. The Ab levels significantly declined during convalescence (> 90 days since onset of illness), compared to 4 to 8 weeks since onset of illness. Our data show that high levels of NAbs during early illness associated with clinical disease severity and that these antibodies declined in 50% of individuals after 3 months since onset of illness.

Neutralization assay with SARS-CoV-1 and SARS-CoV-2 spike pseudotyped murine leukemia virions.

BACKGROUND: Virus neutralization by antibodies is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal murine leukemia virus genome encoding firefly luciferase. This assay design is intended for use in laboratories with biocontainment level 2 and therefore circumvents the need for the biocontainment level 3 that would be required for replication-competent SARS-CoV-2 virus. To validate the pseudovirion assay, we set up comparisons with other available antibody tests including those from Abbott, Euroimmun and Siemens, using archived, known samples. RESULTS: 11 out of 12 SARS-CoV-2-infected patient serum samples showed neutralizing activity against SARS-CoV-2-spike pseudotyped MLV viruses, with neutralizing titers-50 (NT50) that ranged from 1:25 to 1:1,417. Five historical samples from patients hospitalized for severe influenza infection in 2016 tested negative in the neutralization assay (NT50 < 25). Three serum samples with high neutralizing activity against SARS-CoV-2/MLV pseudoviruses showed no detectable neutralizing activity (NT50 < 25) against SARS-CoV-1/MLV pseudovirions. We also compared the semiquantitative Siemens SARS-CoV-2 IgG test, which measures binding of IgG to recombinantly expressed receptor binding domain of SARS-CoV-2 spike glycoprotein with the neutralization titers obtained in the pseudovirion assay and the results show high concordance between the two tests (R2 = 0.9344). CONCLUSIONS: SARS-CoV-2 spike/MLV pseudovirions provide a practical means of assessing neutralizing activity of antibodies in serum or plasma from infected patients under laboratory conditions consistent with biocontainment level 2. This assay offers promise also in evaluating immunogenicity of spike glycoprotein-based candidate vaccines in the near future.

Antigenicity, stability, and reproducibility of Zika reporter virus particles for long-term applications.

The development of vaccines against flaviviruses, including Zika virus (ZIKV) and dengue virus (DENV), continues to be a major challenge, hindered by the lack of efficient and reliable methods for screening neutralizing activity of sera or antibodies. To address this need, we previously developed a plasmid-based, replication-incompetent DENV reporter virus particle (RVP) production system as an efficient and safe alternative to the Plaque Reduction Neutralization Test (PRNT). As part of the response to the 2015-2016 ZIKV outbreak, we developed pseudo-infectious ZIKV RVPs by modifying our DENV RVP system. The use of ZIKV RVPs as critical reagents in human clinical trials requires their further validation using stability and reproducibility metrics for large-scale applications. In the current study, we validated ZIKV RVPs using infectivity, neutralization, and enhancement assays with monoclonal antibodies (MAbs) and human ZIKV-positive patient serum. ZIKV RVPs are antigenically equivalent to live virus based on binding ELISA and neutralization results and are nonreplicating based on the results of live virus replication assays. We demonstrate reproducible neutralization titer data (NT50 values) across different RVP production lots, volumes, time frames, and laboratories. We also show RVP stability across experimentally relevant time intervals and temperatures. Our results demonstrate that ZIKV RVPs provide a safe, high-throughput, and reproducible reagent for large-scale, long-term studies of neutralizing antibodies and sera, which can facilitate large-scale screening and epidemiological studies to help expedite ZIKV vaccine development.

[Immunity against SARS-CoV-2: walking to the vaccination]. / Inmunidad frente a SARS-CoV-2: caminando hacia la vacunación.

The coronavirus are a wide group of viruses among that the SARS-CoV-2 is included (family Coronaviridae, subfamily Coronavirinae, genus Betacoronavirus and subgenus Sarbecovirus). Its main structural proteins are the membrane (M), the envelope (E), the nucleocapsid (N) and spike (S). The immune response to SARS-CoV-2 involves the cellular and the humoral sides, with neutralizing antibodies fundamentally directed against the S antigen. Although the seroprevalence data are frequently assumed as protection markers, no necessarily they are. In Spain, it is estimated that, to assure the herd immunity, at least four-fifths of the population should be immunoprotected. Due the high fatality rate of COVID-19, the acquisition of the protection only by the natural infection it not assumable and other measures as the mass immunization are required. Currently, there are several vaccine prototypes (including life virus, viral vectors, peptides and proteins and nucleic acid) in different phase of clinical evaluation. Foreseeably, some of these news vaccines would be soon commercially available. In this text, aspects related to these issues are reviewed.

Potential Antiviral Options against SARS-CoV-2 Infection.

As of June 2020, the number of people infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) continues to skyrocket, with more than 6.7 million cases worldwide. Both the World Health Organization (WHO) and United Nations (UN) has highlighted the need for better control of SARS-CoV-2 infections. However, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against SARS-CoV-2 can be time-consuming and costly. Convalescent sera and safe-in-man broad-spectrum antivirals (BSAAs) are readily available treatment options. Here, we developed a neutralization assay using SARS-CoV-2 strain and Vero-E6 cells. We identified the most potent sera from recovered patients for the treatment of SARS-CoV-2-infected patients. We also screened 136 safe-in-man broad-spectrum antivirals against the SARS-CoV-2 infection in Vero-E6 cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. We found that a combination of orally available virus-directed nelfinavir and host-directed amodiaquine exhibited the highest synergy. Finally, we developed a website to disseminate the knowledge on available and emerging treatments of COVID-19.

In Situ Neutralization Protocols for Boc-SPPS.

Significant effort has been devoted to the optimization of solid-phase peptide synthesis (SPPS) to maximize the process to facilitate the synthesis of a desired peptide sequence, without extensive optimization or resynthesis. Over the last 25 years, a set of synthetic protocols developed by Kent and Alewood has proven to be robust and efficient for Boc/Bzl SPPS and has been widely adopted by the research community. In this chapter, we describe a variation of manual in situ neutralization protocols for Boc-SPPS that are highly effective for the rapid synthesis of peptides with different C-terminal functionalities.