Exploring the ability of the MD+FoldX method to predict SARS-CoV-2 antibody escape mutations using large-scale data.

Antibody escape mutations pose a significant challenge to the effectiveness of vaccines and antibody-based therapies. The ability to predict these escape mutations with computer simulations would allow us to detect threats early and develop effective countermeasures, but a lack of large-scale experimental data has hampered the validation of these calculations. In this study, we evaluate the ability of the MD+FoldX molecular modeling method to predict escape mutations by leveraging a large deep mutational scanning dataset, focusing on the SARS-CoV-2 receptor binding domain. Our results show a positive correlation between predicted and experimental data, indicating that mutations with reduced predicted binding affinity correlate moderately with higher experimental escape fractions. We also demonstrate that higher precision can be achieved using affinity cutoffs tailored to distinct SARS-CoV-2 antibodies from four different classes rather than a one-size-fits-all approach. Further, we suggest that the quartile values of optimized cutoffs reported for each class in this study can serve as a valuable guide for future work on escape mutation predictions. We find that 70% of the systems surpass the 50% precision mark, and demonstrate success in identifying mutations present in significant variants of concern and variants of interest. Despite promising results for some systems, our study highlights the challenges in comparing predicted and experimental values. It also emphasizes the need for new binding affinity methods with improved accuracy that are fast enough to estimate hundreds to thousands of antibody-antigen binding affinities.

Adenoviral fiber-knob based vaccination elicits efficient neutralizing antibodies and T cell responses against adenovirus infection.

BACKGROUND: Human adenoviruses (HAdVs) frequently cause common respiratory or gastrointestinal infections among children, adults, individuals with immune deficiencies, and other vulnerable populations with varying degree of symptoms, ranging from mild to server, and in some cases, even fatalities. Despite the significant clinical impact of HAdVs, there is currently no approved vaccine available. METHODS: This study explores the potential of the adenovirus type 5 fiber knob (Ad5-FK) to stimulate the production of Ad-specific neutralizing antibodies and T-cell responses in mice. Based on structure predictions, we first expressed Ad5-FK in E. coli and confirmed the assembly of FK into its trimeric form. After testing the binding capability of the trimeric FK to susceptible cells, the immunogenicity of the protein in combination with the c-di-AMP adjuvant was assessed in BALB/c mice. RESULTS: The purified Ad5-FK exhibited self-trimerization and maintained correct conformation akin to the authentic FK structure. This facilitated effective binding to susceptible HEK293 cells. Notably, the protein demonstrated significant inhibition of HEK293 cells infection by rAd5-GFP. Immunization of BALB/c mice with Ad5-FK, or Ad5-FK mixed with c-di-AMP yielded FK-specific antibodies with potent neutralization capacity. Significantly, Ad5-FK was found to elicit a vigorous CD4+ T-cell response in the immunized mice. CONCLUSION: Our findings underscore the efficacy of FK-based vaccine in eliciting anti-Ad humoral immune response and CD4 T-cell immune reactions essential for protection against viral infections.

Dengue in Pune city, India (2017-2019): a comprehensive analysis.

Objectives: To understand the dynamics of dengue disease with special reference to (1) age (2) primary/secondary infections (3) serostatus and (4) serotypes examined during three consecutive years. Methods: During 3 dengue seasons (2017-19), NS1/IgM ELISAs were used for dengue diagnosis in one of the 15 administrative wards of Pune City, India. Predefined symptoms were recorded at the time of diagnosis/hospitalization. IgG-capture ELISA (Panbio) was used to differentiate primary/secondary infections. DENV serotypes were determined for 260 viral RNA-positive patients. Results: During the 3 years, 3,014/6,786 (44.4%, 41.4-49.9%) suspected cases were diagnosed as dengue. Use of either NS1 or IgM would have missed 25.5% or 43% of the confirmed dengue cases, respectively. Notably, a higher proportion of secondary dengue cases remained mild while a substantial proportion of primary infections developed warning signs. The symptoms among Dengue/non-dengue patients and primary/secondary infections varied and influenced by age and serostatus. The number and proportion of dengue serotypes varied yearly. A remarkable decline in dengue cases was observed during the COVID-19 pandemic years. Conclusion: A substantial proportion of primary and secondary dengue patients progress to warning signs/severity or mild infection respectively, underscoring the possible role of non-ADE mechanisms in causing severe dengue that requires hospitalization. Both NS1 and IgM should be used for efficient diagnosis.

Rubella seroprevalence among unvaccinated school-aged children in Jos, North Central, Nigeria.

Introduction: rubella poses a significant public health threat, particularly in developing countries, where congenital rubella remains a preventable concern. This cross-sectional study examined rubella seroprevalence among children aged 10 and under from May to September 2016 in Jos, Nigeria. Methods: using a multistage sampling method, eligible participants who had not been vaccinated against the rubella virus and consented to participate in the study were recruited across schools in the city. Rubella-specific IgG and IgM antibodies were detected from eluted serum collected from the participants using the enzyme-linked immunosorbent assay (ELISA). Data analysis and visualization was done using the R software version 4.3.1. Results: of the 405 participants investigated in this study, 336 (82.96%) tested positive for rubella IgG, while 9 (2.22%) tested positive for rubella IgM. Factors such as age ≥ 5 years and lack of Western education showed significant associations with rubella seropositivity. Conclusion: this study highlights the seroprevalence of rubella IgG and IgM antibodies among children aged 10 and under in Jos, Nigeria. The significant associations between rubella seropositivity and factors such as age ≥ 5 years and lack of Western education underscore the necessity for an effective rubella vaccination program to prevent congenital rubella syndrome (CRS).

Detection of Neutralizing Antibodies in Serum Samples Using a SARS-CoV-2 Pseudotyped Virus Assay.

Conventional live virus research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease-19 (COVID-19), requires Biosafety Level 3 (BSL-3) facilities. SARS-CoV-2 pseudotyped viruses have emerged as valuable tools in virology, mimicking the entry process of the SARS-CoV-2 virus into human cells by expressing its spike glycoprotein in a surrogate system using recombinant plasmids. One significant application of this tool is in functional assays for the evaluation of neutralizing antibodies. Pseudotyped viruses have the advantage of being competent for only a single cycle of infection, providing better safety and versatility and allowing them to be studied in BSL-2 laboratories. Here, we describe three protocols for the detection of SARS-CoV-2 neutralizing antibodies through a pseudotyped virus assay. First, SARS-CoV-2 S pseudotyped viruses (PV SARS-CoV-2 S) are produced using a Moloney murine leukemia virus (MuLV) three-plasmid system. The plasmids are designed to express the GagPol packing proteins, enhanced green fluorescent protein (eGFP) as a readout system, and the SARS-CoV-2 S protein modified to remove the endoplasmic reticulum retention domain and to improve infection. Next, the internalization of PV SARS-CoV-2 S protein in human embryonic kidney 293T (HEK-293T) cells overexpressing angiotensin-converting enzyme 2 (HEK-293T-ACE2) is confirmed by fluorescence microscopy and quantified using flow cytometry. Finally, PV SARS-CoV-2 S is used to screen neutralizing antibodies in serum samples from convalescent COVID-19 patients; it can also be used for studying the cell entry mechanisms of different SARS-CoV-2 variants, evaluating antiviral agents, and designing vaccines. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Generation of PV SARS-CoV-2 S pseudotyped virus Basic Protocol 2: Assay of PV SARS-CoV-2 S internalization in target cells. Basic Protocol 3: Detection of neutralizing antibodies in serum samples.

COVID-19 mRNA booster vaccination induces robust antibody responses but few adverse events among SARS-CoV-2 naïve nursing home residents.

Residents in nursing homes face heightened COVID-19 risks. We aimed to assess the adverse events (AEs) rates and antibody responses after the first to the fifth dose of COVID-19 mRNA vaccination in a nursing home cohort. Ninety-five SARS-CoV-2 naïve participants consisted of 26 staff (median age, 51 years) and 69 residents (median age, 88 years). Life-threatening AEs were reported in neither residents nor staff. The severity of non-life-threatening AEs was graded, and severe AEs were reported only in staff. The AEs rates were considerably lower in residents, compared to those in staff. Anti-RBD IgG and the neutralizing titers (NTs) against Wuhan and Omicron BA.4/BA.5 did not differ significantly between those with 'any AE' and 'no AE' among both staff and residents two months after the second, third and fifth doses, while the anti-RBD IgG significantly differed between two groups after third dose in residents. These findings suggest that the anti-RBD IgG and the NTs increase regardless of the occurrence of AEs. Our study underscores a robust antibody response in both in staff and residents, and fewer AEs following COVID-19 vaccination in SARS-CoV-2 naïve residents than staff, supporting the recommendation for mRNA booster doses in older adults at high-risk care facilities.

Seroprevalence and risk factors for Lassa virus infection in South-West and North-Central Nigeria: a community-based cross-sectional study.

BACKGROUND: Understanding the level of exposure to Lassa virus (LASV) in at-risk communities allows for the administration of effective preventive interventions to mitigate epidemics of Lassa fever. We assessed the seroprevalence of LASV antibodies in rural and semiurban communities of two cosmopolitan cities in Nigeria with poorly understood Lassa epidemiology. METHODS: A cross-sectional study was conducted in ten communities located in the Abuja Municipal Area Council (AMAC), Abuja, and Ikorodu Local Government Area (LGA), Lagos, from February 2nd to July 5th, 2022. Serum samples collected from participants were analyzed for IgG and IgM antibodies using a ReLASV® Pan-Lassa NP IgG/IgM enzyme-linked immunosorbent assay (ELISA) kit. A questionnaire administered to participants collected self-reported sociodemographic and LASV exposure information. Seroprevalence of LASV IgG/IgM was estimated overall, and by study site. Univariate and multivariate log-binomial models estimated unadjusted and adjusted prevalence ratios (aPRs) and 95% confidence intervals (CI) for site-specific risk factors for LASV seropositivity. Grouped Least Absolute Shrinkage and Selection Operator (LASSO) was used for variable selection for multivariate analysis. RESULTS: A total of 628 participants with serum samples were included in the study. Most participants were female (434, 69%), married (459, 73%), and had a median age of 38 years (interquartile range 28-50). The overall seroprevalence was 27% (171/628), with a prevalence of 33% (126/376) in Abuja and 18% (45/252) in Lagos. Based on site-specific grouped LASSO selection, enrollment in the dry season (vs. wet; aPR, 95% CI: 1.73, 1.33-2.24), reported inconsistent washing of fruits and vegetables (aPR, 95% CI: 1.45, 1.10-1.92), and a positive malaria rapid test (aPR, 95% CI: 1.48, 1.09-2.00) were independently associated with LASV seropositivity in Abuja, whereas, only a self-reported history of rhinorrhea (PR, 95% CI: 2.21, 1.31-3.72) was independently associated with Lassa seropositivity in Lagos. CONCLUSIONS: The LASV seroprevalence was comparable to that in other areas in Nigeria. Our findings corroborate those from other studies on the importance of limiting human exposure to rodents and focusing on behavioral factors such as poor hygiene practices to reduce exposure to LASV.

Associations between clinical data, vaccination status, antibody responses, and post-COVID-19 symptoms in Thais infected with SARS-CoV-2 Delta and Omicron variants: a 1-year follow-up study.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), led to a global pandemic from 2020. In Thailand, five waves of outbreaks were recorded, with the fourth and fifth waves driven by the Delta and Omicron variants, resulting in over 20,000 new confirmed cases daily at their peaks. METHODS: This cross-sectional study investigated the associations between clinical symptoms, vaccination status, antibody responses, and post-COVID-19 sequelae in COVID-19 patients. Plasma samples and clinical data were collected from participants admitted to hospitals in Thailand between July 2021 and August 2022, with follow-ups conducted for one year. The study included 110 participants infected with either the Delta (n = 46) or Omicron (n = 64) variants. Virus genotypes were confirmed by RT-PCR of nasal swab RNA and partial nucleotide sequencing of the S gene. IgG and IgA antibody levels against the receptor-binding domain (RBD) of SARS-CoV-2 Delta and Omicron variants were measured in plasma samples using ELISA. RESULTS: Pneumonia was found to be associated with Delta variant infections, while sore throat, congestion or runny nose, and headache were linked to Omicron infections. Vaccination with fewer than two doses and diabetes mellitus were significantly associated with higher disease severity. Specific IgG and IgA antibodies against the RBD of the Delta variant generally rose by day 14 and were maintained for up to two months, whereas the pattern of antibody response to the Omicron variant was less clear. Antibody risings were found to be positively associated with pneumonia, certain underlying conditions (obesity, hypertension, dyslipidemia, and diabetes mellitus), and age ≥ 60 years. Delta variant infections were associated with forgetfulness, hair loss, and headache during the 1-year post-infection period. Females were more likely to experience hair loss, forgetfulness, and joint pain, while older age was associated with joint pain. CONCLUSIONS: This study enhances our understanding of SARS-CoV-2 infections in Thais, particularly concerning the Delta and Omicron variants. The findings can inform public health planning and response strategies for future outbreaks of SARS-CoV-2 or other emerging viral diseases.

Epidemiology of peste des petits ruminants in selected districts of Borena zone, Ethiopia.

BACKGROUND: Peste des petits ruminant (PPR) is a contagious disease caused by the peste des petits ruminants virus (PPRV). The disease poses a significant economic threat to small ruminant production in Ethiopia, particularly to the striving pastoral production system. A cross-sectional study was conducted to estimate the seroprevalence and associated risk factors of PPR in the small ruminants of the Borena Zone. A total of 384 serum samples were collected randomly from sheep and goats and examined for the presence of PPRV antibodies using competition enzyme-linked immune sorbent assay (c-ELISA). Additionally, a retrospective analysis of five years of outbreak data was performed to provide insight into the spatial and temporal distribution of the disease. RESULTS: The seroprevalence of PPRV antibodies in nonvaccinated, vaccinated, and unknown vaccination status of small ruminants in this study was found to be 32.1%, 68.8%, and 45.5%, respectively. Multivariable logistic analysis revealed a statistically significant association between PPRV seropositivity and several factors, including age, animal origin, flock size, and veterinary services status. A retrospective analysis revealed 53 PPR outbreaks in the Borena Zone from 2018 to 2022, exacerbated by low vaccination coverage relative to the at-risk animal population. CONCLUSION: The study revealed significant gaps in current vaccination efforts, with herd immunity levels falling below the FAO-WOAH recommended threshold of 80%. Despite Ethiopia's ambitious goal to eradicate PPR by 2027, the frequent outbreaks and insufficient herd immunity highlight the inadequacy of the existing strategies. To effectively move toward eradication, Ethiopia must align its approach with the global PPR eradication framework, which emphasizes a comprehensive strategy that includes diagnostics, surveillance, prevention, and the establishment of a robust veterinary regulatory system, rather than relying solely on vaccination. Overcoming logistical challenges, improving vaccination coverage, and optimizing the timing of vaccination campaigns, especially in hard-to-reach areas, will be crucial for reducing outbreaks and making progress toward eradication.

Analysis of the immunological response elicited by a polyvalent foot and mouth disease vaccine and its compatibility with a diva test in Jimma Town, Ethiopia.

The research was conducted in Jimma town, Oromiya Regional State, from October 2022 to June 2023, with the aim of assessing the immune response of polyvalent FMD (Foot and Mouth Disease) vaccine. The study involved 34 cattle in a longitudinal study, divided into two groups: 29 vaccinated and 5 unvaccinated. The vaccinated cattle received an inactivated polyvalent FMD virus vaccine produced by the National Veterinary Institute. Blood samples were collected on days 0, 14, 21, 35, 80, and 125 after vaccination and tested using Virus Neutralization Test and 3ABC ELISA. The results showed a significant increase in neutralizing antibodies against structural proteins in all vaccinated cattle on day 14 after vaccination for all three serotypes. (A/ETH/21/2000, p = 0.015; O/ETH/38/2005, p = 0.017; SAT2/ETH/64/2009, p = 0.007). On day, fourteen of post-vaccination vaccinated group showed immune response equal or above 1.5 log10 in a proportion of 69%, 73% and 94% for serotype A/ETH/21/2000, O/ETH/38/2005 and SAT2/ETH/64/2009 respectively. The status of raised antibody titer on day 125 post-vaccination showed decreasing by 14%, 18% and 4% for serotype A/ETH/21/2000, O/ETH/38/2005 and SAT2/ETH/64/2009 respectively. The DIVA test, or 3ABC ELISA, used to differentiate infected from vaccinated animals, revealed the absence of immune response to the Non-structural protein in the vaccinated cattle group. Conversely, the unvaccinated group showed no recorded antibody titer to both structural and non-structural proteins. In summary, the commercially available FMD vaccine, comprising serotype A, O, and SAT2, triggers an immune response to the structural protein rather than the non-structural protein after the initial administration. This outcome implies that FMD vaccines from the National Veterinary Institute align with the DIVA test. Nevertheless, additional efforts may be necessary to bolster the strength and duration of the vaccine-induced immune response.

Extensive Schmallenberg virus circulation in Germany, 2023.

Schmallenberg virus (SBV) and bluetongue virus (BTV) are both transmitted by Culicoides biting midges and infect predominantly ruminants. To investigate the extent of virus spread in the 2022 and 2023 vector seasons, we serologically tested wild ruminants from western Germany. While antibodies against BTV were not detected in any animal, regardless of age or sampling time, numerous wild ruminants tested positive for antibodies to SBV. In 2022, a low seroprevalence of 4.92% was measured. In sharp contrast, 40.15% of the animals tested positive in 2023. Of the young animals, about 31.82% were seropositive, clearly indicating large-scale SBV circulation in summer and autumn 2023.

Enhancing Omicron Sublineage Neutralization: Insights From Bivalent and Monovalent COVID-19 Booster Vaccines and Recent SARS-CoV-2 Omicron Variant Infections.

BACKGROUND: Omicron variants have rapidly diversified into sublineages with mutations that enhance immune evasion, posing challenges for vaccination and antibody responses. This study aimed to compare serum cross-neutralizing antibody responses against various SARS-CoV-2 Omicron sublineages (BA.1, BA.5, XBB.1.17.1, FK.1.1, and JN.1) in recipients of monovalent COVID-19 boosters, bivalent booster recipients, and individuals who had recovered from Omicron BA.5 infections. METHODS: We conducted a micro-neutralization assay on serum samples from monovalent BNT162b2 booster recipients (N = 54), bivalent BNT162b2 booster recipients (N = 24), and SARS-CoV-2 Omicron BA.5-recovered individuals (N = 13). The history of SARS-CoV-2 Omicron infection was assessed using ELISA against the SARS-CoV-2 NP protein. RESULTS: Bivalent booster recipients exhibited significantly enhanced neutralization efficacy against Omicron sublineages compared to those who had received monovalent booster vaccinations. Omicron BA.5-recovered individuals displayed similar neutralizing antibodies (NAbs) to the bivalent booster recipients. Despite the improved neutralization in bivalent recipients and BA.5-recovered individuals, there were limitations in neutralization against the recently emerged Omicron subvariants: XBB.1.17.1 FK.1.1, and JN.1. In both monovalent and bivalent booster recipients, a history of Omicron breakthrough infection was associated with relatively higher geometric mean titers of NAbs against Omicron BA.1, BA.5, and XBB.1.17.1 variants. CONCLUSION: This study underscores the intricate interplay between vaccination strategies, immune imprinting, and the dynamic landscape of SARS-CoV-2 variants. Although bivalent boosters enhance neutralization, addressing the challenge of emerging sublineages like XBB.1.17.1, FK.1.1, and JN.1 may necessitate the development of tailored vaccines, underscoring the need for ongoing adaptation to effectively combat this highly mutable virus.

A novel double antibody sandwich quantitative ELISA for detecting porcine epidemic diarrhea virus infection.

Porcine epidemic diarrhea (PED), a contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), has caused significant economic losses to the global pig farming industry due to its rapid course and spread and its high mortality among piglets. In this study, we prepared rabbit polyclonal antibody and monoclonal antibody 6C12 against the PEDV nucleocapsid (N) protein using the conserved and antigenic PEDV N protein as an immunogen. A double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) was established to detect PEDV using rabbit polyclonal antibodies as capture antibodies and horseradish peroxidase (HRP)-labeled 6C12 as the detection antibody. Using DAS-qELISA, recombinant PEDV N protein, and virus titer detection limits were approximately 0.05 ng/mL and 103.02 50% tissue culture infective dose per mL (TCID50/mL), respectively. There was no cross-reactivity with porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (PoRV), porcine pseudorabies virus (PRV), porcine deltacoronavirus (PDCoV), or porcine circovirus (PCV). The reproducibility of DAS-qELISA was verified, and the coefficient of variation (CV) for intra- and inter-batch replicates was less than 10%, indicating good reproducibility. When testing anal swab samples from PEDV-infected piglets using DAS-qELISA, the coincidence rate was 92.55% with a kappa value of 0.85 when using reverse transcription-polymerase chain reaction (RT-PCR) and 94.29% with a kappa value of 0.88 when using PEDV antigen detection test strips, demonstrating the reliability of the method. These findings provide fundamental material support for both fundamental and practical studies on PEDV and offer a crucial diagnostic tool for clinical applications. KEY POINTS: • A new anti-PEDV N protein monoclonal antibody strain was prepared • Establishment of a more sensitive double antibody sandwich quantitative ELISA • DAS-qELISA was found to be useful for controlling the PEDV spread.

Development of an indirect ELISA for the serologic detection of bovine viral diarrhea virus based on E2 antigen sub-genotypes 1b, 1e, and 1d.

Bovine viral diarrhea virus (BVDV) causes ongoing economic losses to cattle industries, directly through reduced herd performance or indirectly through control program costs. ELISA assays, one of the most widely used techniques due to their ease of implementation, have been a valuable tool for mass surveillance and detection of BVDV. In this study, we developed a new indirect ELISA (rE2-ELISA) for serologic detection of BVDV. The assay considers three recombinant E2 protein subtypes as antigens, allowing serologic diagnosis of BVDV-1b (high prevalence worldwide), BVDV-1d and 1e (high prevalence in southern Chile) sub-genotypes. Recombinant E2 (rE2) proteins were successfully expressed in stably transfected CHO cells. Conditions for rE2 ELISAs were established after determining appropriate concentrations of antigen, blocking agent, secondary antibody, and serum dilutions to achieve maximum discrimination between positive and negative serum samples. The developed rE2-ELISA showed a sensitivity of 92.86% and a specificity of 98.33%. Clinical testing of 180 serum samples from herds in southern Chile showed high accuracy (kappa > 0.8) compared to the commercial BVDV Total Ab kit (IDEXX), with 95.37% positive and 87.5% negative predictive value. In addition, the rE2 ELISA has shown the capability to detect anti-BVDV antibodies from naturally infected animals with sub-genotypes 1b, 1e, or undetermined. These results indicate that the developed indirect ELISA could serve as a valid, and efficient alternative for identifying BVDV-infected animals, thus contributing to the success of disease control and eradication programs.

Studying the Humoral Response against SARS-CoV-2 in Cuban COVID-19 Recovered Patients.

Understanding the immune response generated by SARS-CoV-2 is critical for assessing efficient therapeutic protocols and gaining insights into the durability of protective immunity. The current work was aimed at studying the specific humoral responses against SARS-CoV-2 in Cuban COVID-19 convalescents. We developed suitable tools and methods based on ELISA methodology, for supporting this evaluation. Here, we describe the development of an ELISA for the quantification of anti-RBD IgG titers in a large number of samples and a similar test in the presence of NH4SCN as chaotropic agent for estimating the RBD specific antibody avidity. Additionally, a simple and rapid ELISA based on antibody-mediated blockage of the binding RBD-ACE2 was implemented for detecting, as a surrogate of conventional test, the levels of anti-RBD inhibitory antibodies in convalescent sera. In a cohort of 273 unvaccinated convalescents, we identified higher anti-RBD IgG titer (1 : 1,330, p < 0.0001) and higher levels of inhibitory antibodies blocking RBD-ACE2 binding (1 : 216, p < 0.05) among those who had recovered from severe illness. Our results suggest that disease severity, and not demographic features such as age, sex, and skin color, is the main determinant of the magnitude and neutralizing ability of the anti-RBD antibody response. An additional paired longitudinal assessment in 14 symptomatic convalescents revealed a decline in the antiviral antibody response and the persistence of neutralizing antibodies for at least 4 months after the onset of symptoms. Overall, SARS-CoV-2 infection elicits different levels of antibody response according to disease severity that declines over time and can be monitored using our homemade serological assays.

Precise location of three novel linear epitopes using the generated monoclonal antibodies against the Knob domain of FAdV-4 surface structural protein, fiber1.

Background: Fowl adenovirus serotype 4 (FAdV-4) is the main pathogen of hepatitis-hydropericardium syndrome (HHS), which brings huge economic losses to the poultry industry worldwide. Fiber-1 protein plays an important role in viral infection and pathogenesis by binding directly to cellular receptors of FAdV-4. In particular, the knob domain of fiber-1 protein has been reported to induce the production of neutralizing antibodies and arouse protection against the lethal challenge of chickens with FAdV-4. Methods: The fiber-1 knob (F1K) protein was expressed in a prokaryotic expression system and purified using Ni-NTA affinity chromatography. Monoclonal antibodies (mAbs) against FAdV-4 were generated by immunizing BALB/c mice with the purified F1K protein and screened using a series of immunoassays. Potential B cell epitopes on the knob domain of fiber-1 protein were mapped using enzyme-linked immunosorbent assay (ELISA) and dot-blot. Precious location and crucial amino acids of the identified epitopes were determined using peptide array scanning, truncations and alanine-scanning mutagenesis. The epitopes were analyzed and visualized on the knob trimer of FAdV-4 fiber-1 protein using the PyMOL software. Results: Water-soluble recombinant fiber-1 knob (F1K) protein was obtained with the assistance of chaperone. Four monoclonal antibodies (5C10, 6F8, 8D8, and 8E8) against FAdV-4 were generated and characterized using indirect ELISA, Western blot, dot-blot, and immunological fluorescence assay (IFA). The mAbs were demonstrated to be from different hybridoma cell lines based on the sequences of the variable regions. Meanwhile, three distinct novel linear B-cell epitopes (319SDVGYLGLPPH329, 328PHTRDNWYV336, and 407VTTGPIPFSYQ417) on the knob domain of fiber-1 protein were identified and the key amino acid residues in the epitopes were determined. Structural analysis showed that the two adjacent epitopes 319SDVGYLGLPPH329 and 328PHTRDNWYV336 were exposed on the surface of the fiber-1 knob trimer, whereas the epitope 407VTTGPIPFSYQ417 was located inside of the spatial structure. Conclusion: This was the first identification of B-cell epitopes on the knob domain of fiber-1 protein and these findings provided a sound basis for the development of subunit vaccines, therapeutics, and diagnostic methods to control FAdV infections.

Improved deep learning prediction of antigen-antibody interactions.

Identifying antibodies that neutralize specific antigens is crucial for developing effective immunotherapies, but this task remains challenging for many target antigens. The rise of deep learning-based computational approaches presents a promising avenue to address this challenge. Here, we assess the performance of a deep learning approach through two benchmark tests aimed at predicting antibodies for the receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Three different strategies for constructing input sequence alignments are employed for predicting structural models of antigen-antibody complexes. In our initial testing set, which comprises known experimental structures, these strategies collectively yield a significant top-ranked prediction for 61% of cases and a success rate of 47%. Notably, one strategy that utilizes the sequences of known antigen binders outperforms the other two, achieving a precision of 90% in a subsequent test set of ~1,000 antibodies, balanced between true and control antibodies for the antigen, albeit with a lower recall of 25%. Our results underscore the potential of integrating deep learning methods with single B cell sequencing techniques to enhance the prediction accuracy of antigen-antibody interactions.

Seropositivity of West Nile virus among acute febrile patients in Ilorin, Nigeria.

INTRODUCTION: West Nile Virus (WNV), a member of Flaviviridae family, is one of the most widely distributed arboviruses in the world. In developing countries like Nigeria, fever resulting from the WNV infection is often presumptively ascribed to malaria or typhoid due to misdiagnosis and low-level awareness of the viral infection. This study determined the prevalence of WNV IgM and IgG antibodies among febrile patients in the Ilorin metropolis. MATERIALS AND METHODS: A total of two hundred (200) blood samples were collected from consenting patients and each serum was screened for anti-WNV IgM and IgG antibodies using indirect enzyme-linked immunosorbent assay (ELISA). Statistical correlation and logistic regression analysis were conducted. RESULTS: Overall, 6% (12/200) anti-WNV IgM seropositivity rate was recorded amongst the acute febrile patients with higher prevalence (6.30%) in females than in males (5.45%). Anti-WNV IgG positivity rate of 52% (104/200) was recorded, with 50.67% positivity rate in males and 38.95% in female participants. The convalescence phase posited by the 5.4% (11/200) co-detection of anti-WNV IgG and IgM antibodies among the participants was recorded. A statistical correlation was noticed with the age and religion of respondents to WNV serological positivity while gender, occupation, use of mosquito nets and formal education had no positive correlation at p < 0.05. However, based on odd ratio at 95% CI and logistic regression coefficients, the evaluated risk factors such as blood transfusion, residency, malaria parasite, and proximity to stagnant water and bush were significant to anti-WNV IgG and IgM positivity. CONCLUSION: The findings of this study show the circulation of WNV in the study area. There is an urgent need for clinicians/physicians to include screening for the West Nile virus in cases of febrile patients before the commencement of treatment.

[The Molecular and Biological Patterns Underlying Sustained SARS-CoV-2 Circulation in the Human Population].

INTRODUCTION: For four years, SARS-CoV-2, the etiological agent of COVID-19, has been circulating among humans. By the end of the second year, an absence of immunologically naive individuals was observed, attributable to extensive immunization efforts and natural viral exposure. This study focuses on delineating the molecular and biological patterns that facilitate the persistence of SARS-CoV-2, thereby informing predictions on the epidemiological trajectory of COVID-19 toward refining pandemic countermeasures. The aim of this study was to describe the molecular biological patterns identified that contribute to the persistence of the virus in the human population. MATERIALS AND METHODS: For over three years since the beginning of the COVID-19 pandemic, molecular genetic monitoring of SARS-CoV-2 has been conducted, which included the collection of nasopharyngeal swabs from infected individuals, assessment of viral load, and subsequent whole-genome sequencing. RESULTS: We discerned dominant genetic lineages correlated with rising disease incidence. We scrutinized amino acid substitutions across SARS-CoV-2 proteins and quantified viral loads in swab samples from patients with emerging COVID-19 variants. Our findings suggest a model of viral persistence characterized by 1) periodic serotype shifts causing substantial diminutions in serum virus-neutralizing activity (> 10-fold), 2) serotype-specific accrual of point mutations in the receptor-binding domain (RBD) to modestly circumvent neutralizing antibodies and enhance receptor affinity, and 3) a gradually increasing amount of virus being shed in mucosal surfaces within a single serotype. CONCLUSION: This model aptly accounts for the dynamics of COVID-19 incidence in Moscow. For a comprehensive understanding of these dynamics, acquiring population-level data on immune tension and antibody neutralization relative to genetic lineage compositions is essential.

Development, study, and comparison of models of cross-immunity to the influenza virus using statistical methods and machine learning.

INTRODUCTION: The World Health Organization considers the values of antibody titers in the hemagglutination inhibition assay as one of the most important criteria for assessing successful vaccination. Mathematical modeling of cross-immunity allows for identification on a real-time basis of new antigenic variants, which is of paramount importance for human health. MATERIALS AND METHODS: This study uses statistical methods and machine learning techniques from simple to complex: logistic regression model, random forest method, and gradient boosting. The calculations used the AAindex matrices in parallel to the Hamming distance. The calculations were carried out with different types and values of antigenic escape thresholds, on four data sets. The results were compared using common binary classification metrics. RESULTS: Significant differentiation is shown depending on the data sets used. The best results were demonstrated by all three models for the forecast autumn season of 2022, which were preliminary trained on the February season of the same year (Auroc 0.934; 0.958; 0.956, respectively). The lowest results were obtained for the entire forecast year 2023, they were set up on data from two seasons of 2022 (Aucroc 0.614; 0.658; 0.775). The dependence of the results on the types of thresholds used and their values turned out to be insignificant. The additional use of AAindex matrices did not significantly improve the results of the models without introducing significant deterioration. CONCLUSION: More complex models show better results. When developing cross-immunity models, testing on a variety of data sets is important to make strong claims about their prognostic robustness.