Search Results (896)

Influenza B viruses, divided into B/Victoria and B/Yamagata lineages, have not had B/Yamagata isolates after 2020. A study evaluated immunity to influenza B surface antigens hemagglutinin (HA) and neuraminidase (NA) in 138 patient sera from 2023 and 23 pairs of sera from 2018 to 2019 vaccine recipients. The phylogenetic tree of the influenza B virus, based on HA and NA genes, shows that the Yamagata lineage evolves gradually, while the Victoria lineage exhibits rapid mutations with short branches. In 2023, mean levels of antibodies to HA and NA of B/Yamagata virus were higher than to B/Victoria, despite no cases of B/Yamagata lineage isolation after 2020. The titers of antibodies to NA of B/Yamagata statistically significantly differed among individuals born before and after 1988. Among patients examined in 2018–2019, neuraminidase-inhibiting (NI) antibody titers before vaccination were higher to B/Yamagata than to B/Victoria, and NI antibodies to B/Victoria and B/Yamagata positively correlated with neutralizing antibodies to B/Victoria virus before and after vaccination. Immunity to B/Yamagata virus was stronger in 2023, despite no isolation since 2020, probably due to the presence of cross-reactive antibodies from B/Victoria infections or vaccinations. Antibodies to NA of B/Victoria and B/Yamagata in 2023 correlated significantly in patients born before 1988, potentially supporting the concept of ‘antigenic sin’ phenomenon for influenza B viruses. The fact that NI antibody titers to B/Victoria and B/Yamagata correlated with neutralizing antibody titers to B/Victoria may suggest broad cross-protection. Studying influenza B virus NA antigenic properties helps understand the evolution and antigenic competition of HA and NA. Full article

Breast milk can provide passive immunity to infants, serving as a valuable source of maternal antibodies while remaining a non-invasive sample for investigating maternal immune responses. To date, no studies have evaluated SARS-CoV-2 and potentially cross-reactive HCoV antibodies in breast milk following bamlanivimab administration. A 36-year-old postpartum female was PCR-positive for SARS-CoV-2 four days post-delivery. Bamlanivimab was administered intravenously two days later. Breast milk was collected before bamlanivimab infusion, daily for two weeks post-infusion, then weekly until 102 days post-infusion. Mother and infant sera were collected only at 102 days post-infusion. All milk and serum samples were tested for IgG antibodies against SARS-CoV-2 and HCoV. We observed two distinct SARS-CoV-2 antibody peaks at days 3 and 29 post-infusion, likely representing bamlanivimab transfer and the post-infection antibody response. Beta-HCoV antibodies showed two peaks at days 6 and 29, potentially representing backboosted beta-HCoV responses and/or antibody cross-reactivity with SARS-CoV-2. Infant seropositivity for SARS-CoV-2 102 days post-infusion may represent antibodies from passive transfer via breastfeeding or a subclinical infection. This case highlights the value of breast milk as a non-invasive and repeatable sample to help understand maternal immune responses post-infection, exogenous antibody infusion, and passive antibody transfer during breastfeeding, which can provide insights into maternal–infant health research. Full article

The C-kinase-activated protein phosphatase-1 (PP1) inhibitor of 17 kDa (CPI-17) is a specific inhibitor of the PP1 catalytic subunit (PP1c) and the myosin phosphatase (MP) holoenzyme. CPI-17 requires the phosphorylation of Thr38 in the peptide segment ³⁵ARV(P)TVKYDRREL⁴⁶ for inhibitory activity. CPI-17 regulates myosin phosphorylation in smooth muscle contraction and the tumorigenic transformation of several cell lines via the inhibition of MP. A phosphospecific antibody (anti-CPI-17^pThr38) against the phosphorylation peptide was used to determine the phosphorylation levels in cells. We found that phospho-CPI-17 and its closely related proteins are not present in HeLa and MCF7 cells after inducing phosphorylation by inhibiting phosphatases with calyculin A. In contrast, cross-reactions of proteins in the 40–220 kDa range with anti-CPI-17^pThr38 were apparent. Searching the protein database for similarities to the CPI-17 phosphorylation sequence revealed several proteins with 42–75% sequence identities. The LIMK2-1 isoform emerged as a possible PP1 inhibitor. Experiments with Flag-LIMK2-1 overexpressed in tsA201 cells proved that LIMK2-1 interacts with PP1c isoforms and is phosphorylated predominantly by protein kinase C. Phosphorylated LIMK2-1 inhibits PP1c and the MP holoenzyme with similar potencies (IC50 ~28–47 nM). In conclusion, our results suggest that LIMK2-1 is a novel phosphorylation-dependent inhibitor of PP1c and MP and may function as a CPI-17-like phosphatase inhibitor in cells where CPI-17 is present but not phosphorylated upon phosphatase inhibition. Full article

Background and objective. Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) affects small- to medium-sized vessels and is characterized by the production of ANCAs. The ANCA-negative term is used if the patient otherwise fulfills the definition for AAV but has negative results on serologic testing for ANCAs. The objective of this study was to compare ANCA-positive and -negative vasculitis patients and to evaluate the main differences possibly related to the presence of ANCAs. Material and methods. A cross-sectional study of 73 patients treated at the tertiary Rheumatology Centre of University Hospital from the 1 January, 2001, to the 31August, 2023, with diagnoses of AAV was carried out. Clinical characteristics and laboratory data were collected at the onset or at the first year of the disease. Results. Forty-eight (65.8%) patients were ANCA-positive, while twenty-five (34.3%) were ANCA-negative. Distribution by gender was similar in both groups, with a female–male ratio of 2:1. C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were elevated for all AAV patients, but values were higher in the ANCA-positive patients’ group. The median hemoglobin was 106 g/L in the seropositive group and 127 g/L in the seronegative group. A higher prevalence of kidney involvement (60.4%) with elevated serum creatinine level (93.5 µmol/L) was observed in the ANCA-positive group compared with 24% and 70 µmol/l in the ANCA-negative group (p < 0.05). Neurological involvement was more frequently found in the ANCA-positive patient group, too: 29.2% compared to 20%. Among patients with ANCA-negative vasculitis, 88% had pulmonary; 92% ear, nose, throat (ENT); 48% joint; and 28% skin presentation. In comparison, involvement of these organs was less common in the ANCA-positive patients’ group, at 79.2%, 60.4%, 31.3%, and 25 %, respectively. Conclusions. ANCA-positive patients appear to be in a more difficult clinical situation in terms of organ involvement and laboratory changes. Full article

Porcine Rotavirus (PoRV), a predominant causative agent of neonatal diarrhea in piglets, shares substantial genetic homology with human rotavirus and represents a considerable threat to both public health and the global swine industry in the absence of specific antiviral interventions. The VP6 protein, an internal capsid component, is characterized by exceptional sequence conservation and robust immunogenicity, rendering it an ideal candidate for viral genotyping and vaccine development. In the present study, the recombinant plasmid pET28a(+)-VP6 was engineered to facilitate the high-yield expression and purification of the VP6 antigen. BALB/c mice were immunized to generate monoclonal antibodies (mAbs) through hybridoma technology, and the antigenic specificity of the resulting mAbs was stringently validated. Subsequently, a panel of truncated protein constructs was designed to precisely map linear B-cell epitopes, followed by comparative conservation analysis across diverse PoRV strains. Functional validation demonstrated that all three mAbs exhibited high-affinity binding to VP6, with a peak detection titer of 1:3,000,000 and exclusive specificity toward PoRVA. These antibodies effectively recognized representative genotypes such as G3 and X1, while exhibiting no cross-reactivity with unrelated viral pathogens; however, their reactivity against other PoRV serogroups (e.g., types B and C) remains to be further elucidated. Epitope mapping identified two novel linear B-cell epitopes, ¹²⁸YIKNWNLQNR¹³⁷ and ¹³⁸RQRTGFVFHK¹⁴⁷, both displaying strong sequence conservation among circulating PoRV strains. Collectively, these findings provide a rigorous experimental framework for the functional dissection of VP6 and reinforce its potential as a valuable diagnostic and immunoprophylactic target in PoRV control strategies. Full article

Background: This study aimed to assess the prevalence of SARS-CoV-2 infection, vaccination, and immune status among a population, both Dentists and University Professors, within a clinical setting at one and at 12 months after COVID-19 vaccination. Methods: A cross-sectional study involving 47 professionals (aged 27–52) was conducted in the University Fernando Pessoa. Participants completed an online survey on SARS-CoV-2 infection status and vaccination, received and provided plasma samples for serological analysis. The protocol was approved by the UFP-Ethics Committee. Anti-S1-RBD SARS-CoV-2 IgM and IgG antibody titration values (AU/mL) were measured, by enzyme-linked-immunosorbent assay (ELISA), with reactive immunoglobulins (Ig) seropositivity for values ≥1 AU/mL. Results: SARS-CoV-2 infection rate increased from 8.5% in July 2021 to 48.9% in June 2022, with 8.5% experiencing reinfection. Vaccination rate was 91.5% by July 2021 and increased slightly to 93.6% by June 2022; 72.3% of the sample received a third dose. IgG seropositivity increased from 91.5% to 95.7% in June 2022. After one-year, significant associations were found between IgG seropositivity and both participant’s age (p = 0.009; <50 years) and vaccine doses (p = 0.003; 1–3 doses) received. Conclusions: SARS-CoV-2 infection rate, vaccination, and IgG seropositivity rates were high and increased over one year. The age and vaccination status were associated with the immunity status at 12th month follow-up. Findings highlight variability in IgG seroprevalence due to multiple influencing factors, which justifies future studies. Full article

Nipah virus (NiV) and Hendra virus (HeV), which both belong to the genus henipavirus, are zoonotic pathogens that cause severe systemic, neurological, and/or respiratory disease in humans and a variety of mammals. Therefore, monitoring viral prevalence in natural reservoirs and rapidly diagnosing cases of henipavirus infection are critical to limiting the spread of these viruses. Current laboratory methods for detecting NiV and HeV include virus isolation, reverse transcription quantitative real-time PCR (RT-qPCR), and antigen detection via an enzyme-linked immunosorbent assay (ELISA), all of which require highly trained personnel and specialized equipment. Here, we describe the development of a point-of-care customized immunochromatographic lateral flow (ILF) assay that uses recombinant human ephrin B2 as a capture ligand on the test line and a NiV-specific monoclonal antibody (mAb) on the conjugate pad to detect NiV and HeV. The ILF assay detects NiV and HeV with a diagnostic specificity of 94.4% and has no cross-reactivity with other viruses. This rapid test may be suitable for field testing and in countries with limited laboratory resources. Full article

Background/Objectives: Since the World Health Organization (WHO) recommended HIV self-testing as an alternative to traditional facility-based testing in 2016, it has been increasingly adopted worldwide. This study aimed to evaluate the performance of the ConfiSign HIV Self-Test (GenBody Inc., Republic of Korea), a newly developed blood-based immunochromatographic assay for the qualitative detection of total antibodies (IgG and IgM) against HIV-1/HIV-2. Methods: The evaluation included four components: (1) retrospective analysis of 1400 archived serum samples (400 HIV-positive and 1000 HIV-negative samples), (2) prospective self-testing by 335 participants (112 HIV-positive participants and 223 individuals with an unknown HIV status, including healthy volunteers), (3) assessment using seroconversion panels and diverse HIV subtypes, and (4) analytical specificity testing for cross-reactivity and interference. The Elecsys HIV combi PT and Alinity I HIV Ag/Ab Combo assays were used as reference assays. Results: In retrospective testing, the ConfiSign HIV Self-Test achieved a positive percent agreement (PPA) of 100%, a negative percent agreement (NPA) of 99.2%, and a Cohen’s kappa value of 0.986, showing excellent agreement with the reference assays. In the prospective study, the test showed 100% sensitivity and specificity, with a low invalid result rate of 1.8%. All HIV-positive samples, including those with low signal-to-cutoff (S/Co) values in the Alinity I assay, were correctly identified. The test also reliably detected early seroconversion samples and accurately identified a broad range of HIV-1 subtypes (A, B, C, D, F, G, CRF01_AE, CRF02_AG, and group O) as well as HIV-2. No cross-reactivity or interference was observed with samples that were positive for hepatitis viruses, cytomegalovirus, Epstein–Barr virus, varicella zoster virus, influenza, HTLV-1, HTLV-2, or malaria. Conclusions: The ConfiSign HIV Self-Test demonstrated excellent sensitivity, specificity, and robustness across diverse clinical samples, supporting its reliability and practicality as a self-testing option for HIV-1/2 antibody detection. Full article

Synthetic biology has fundamentally advanced cell engineering and helped to develop effective therapeutics such as chimeric antigen receptor (CAR)-T cells. For these applications, the detection, localization, and quantification of heterologous fusion proteins assembled from interchangeable building blocks is of high importance. The V5 tag, a 14-residue epitope tag, offers promising characteristics for these applications but has only rarely been used in this context. Thus, we have systematically evaluated the murine anti-V5 tag antibody mu_SV5-Pk1 as well as its humanized version, hu_SV5-Pk1, to analyze cells expressing V5-tagged receptors in samples from various in vitro and in vivo experiments. We found that the V5 tag signal on cells is affected by certain fixation and detachment reagents. Immunohistochemistry (IHC) on formalin-fixed paraffin-embedded (FFPE) mouse tissue samples was performed to sensitively detect cells in tissue. We improved IHC by applying the hu_SV5-Pk1 monoclonal antibody (mAb) to avoid cross-reactivity within and unspecific background signals arising on fixed mouse tissue. Conversely, the absence of unspecific binding by the mu_SV5-Pk1 mAb was evaluated on 46 human normal or cancer tissues. Our findings present a robust toolbox for utilizing the V5 tag and cognate antibodies in synthetic biology applications. Full article

Background: Measuring frequencies of antigen-specific memory B cells (B_mem), their immunoglobulin (Ig) class and subclass usage, cross-reactivity, and affinity can provide insights into the efficacy of future antibody responses in case of antigen re-encounter. B cell ImmunoSpot^® assays can provide such information; however, like most cell-based tests, they require considerable amounts of blood to be drawn from the donor and this has hindered their inclusion in clinical trials and routine immune diagnostics. Methods: We introduce strategies for reducing the cell numbers required to 2–3 million peripheral blood mononuclear cells (PBMCs) per antigen, obtainable from 2–3 mL of blood from healthy adult donors. Results: Except when B_mem frequencies were very low, we found that testing PBMCs in singlet wells, but in serial dilution, enables as reliable B_mem frequency assessments as when testing replicate wells at a single fixed cell number. Additionally, B cell ImmunoSpot^® assays can be multiplexed for detecting four Ig classes, or IgG subclasses, simultaneously and without loss of sensitivity. The requirement for low cell numbers and the retention of B cell functionality by cryopreserved PBMCs equivalent to freshly isolated material implies that fewer than the standard 10 million PBMCs per vial can be frozen. This would reduce the number of individuals who could not be tested for B_mem due to insufficient availability of PBMCs, a common problem with such assays. Conclusions: The predictable need for and recovery of cryopreserved PBMCs facilitates planning of and optimal cell utilization in B cell ImmunoSpot^® assays and increases the practical feasibility of extensive B_mem characterization in larger cohorts. Full article

(1) Background: The ongoing global health threat posed by SARS-CoV-2 requires reliable and accessible diagnostic tools, especially in resource-limited settings where RT-qPCR may be impractical. This study describes the development and validation of two enzyme-linked immunosorbent assays (ELISA) designed to detect anti-SARS-CoV-2 IgG antibodies employing recombinant S1 and S2 spike protein subunits. (2) Methods: The assays were optimized and validated using serum samples from 354 RT-qPCR-confirmed hospitalized patients and 337 pre-pandemic blood donors. (3) Results: The S1-based ELISA achieved a 52.8% sensitivity and a specificity of 93.5%, with an area under the ROC curve (AUC) of 71.6%. In contrast, the S2-based ELISA demonstrated superior diagnostic performance, with a sensitivity of 63.7%, a specificity of 99.7%, and an AUC of 83.1%. Cross-reactivity analysis using sera from individuals with unrelated infectious diseases confirmed the high specificity of the S2-ELISA. Time-stratified analysis revealed that sensitivity increased with time, peaking between 15 and 21 days post-symptom onset. Compared to commercial serological assays, the S2-ELISA demonstrated comparable or improved performance, particularly in specificity and diagnostic odds ratio. (4) Conclusions: The S2-ELISA offers a robust, highly specific, and operationally simple tool for serological detection of SARS-CoV-2 infection. Its strong diagnostic performance and accessibility make it well-suited for implementation in diverse epidemiological settings, particularly where molecular testing is limited. The development of affordable, validated serological assays such as this is critical for strengthening surveillance, understanding transmission dynamics, and informing public health responses. Full article

Co-infections of dengue serotypes and dengue with other flaviviruses pose substantial hurdles in disease diagnosis, treatment options, and disease management. The overlapping geographic distributions and mosquito vectors significantly enhance the probability of co-infections. Co-infections may result in more severe disease outcomes due to elevated viral loads, modulation of the immune response, and antibody enhancement. Cross-reactivity in serological assays and the likeness of clinical presentations add to the ongoing challenges in disease diagnosis. Molecular diagnostics such as reverse transcription polymerase chain reaction (RT-PCR) and next-generation sequencing (NGS) are, therefore, employed for more specific disease diagnosis although requiring substantial resources. Despite the advancements, specific anti-flaviviral therapy is still limited, hence the urgency for further investigative research into various therapeutic approaches, including peptide inhibitors, host-targeted therapies, and RNA-based interventions. This review discusses the epidemiology, clinical ramifications, and diagnostic obstacles associated with flavivirus co-infections whilst assessing prospective strategies for better disease prevention, treatment, and management. Addressing these critical gaps is essential for disease mitigation whilst improving patient management especially in regions where co-circulation of flaviviruses is common and their diseases are highly endemic. Full article

Chlamydia trachomatis infection is a public health problem. Serological tests can determine the disease burden and serve as a biomarker for identifying patients with infertility due to tubal obstruction. However, cross-reactions between chlamydial species have been reported, which causes problems with diagnosis. A real-time PCR commercial test for the detection of endocervical infection and two ELISAs with the recombinant major outer membrane protein (rMOMP) from C. trachomatis and C. abortus as antigens were used to diagnose both infections. The prevalence of endocervical infection by C. trachomatis was 7.77%, and that of IgG antibodies against C. trachomatis and C. abortus was 31.1% and 10.7%, respectively. The ELISA with C. trachomatis rMOMP showed a sensitivity of 75% and a specificity of 72.5%. The lowest sensitivity (25%) and high specificity (76.8%) were obtained with anti-C. abortus rMOMP ELISAs. A low cross-reactivity of 7% between ELISA tests was observed. Conclusion. The recombinant MOMP ELISA could help identify women who had contact with C. trachomatis or C. abortus and could be a tool to lower the costs of performing molecular testing on all patients attending an infertility clinic. Full article

Background: The Human Papillomavirus (HPV) vaccine generates high antibody titers against targeted HPV types. This study investigated vaccine-induced anti-HPV18 immunoglobulin (IgG) antibody titers and subsequent HPV18/45 infections. Methods: We performed a nested matched case-control study leveraging a prospective longitudinal cohort of adolescent and young adult women (AYW) vaccinated with the quadrivalent HPV vaccine (4vHPV) attending the Mount Sinai Adolescent Health Center (MSAHC) in Manhattan, NY. The case individuals included AYW who had an incident detection of cervical HPV18 (n = 3) or HPV45 (n = 34) DNA after vaccination and were compared to two vaccinated control individuals (HPV18/45-negative); one random control (RC, n = 37) and one high-risk control (HRC, n = 37) selected from the upper quartile of a sexual risk behavior score. Serological titers against HPV18 were measured by end-point dilution and enzyme-linked immunosorbent assay (ELISA) in serum collected before the incident detection of HPV. Matching was performed based on age at first dose, follow-up time, and sexual risk behavior score. Conditional logistic regression was used to assess the association between case-control status and anti-HPV antibody titers, consistent with the matched-pair design. Results: Antibody titers for HPV18 were most different between AYW who developed an HPV18/45 infection compared to high-risk controls OR = 1.66, 95% CI: 0.96–2.85 (p = 0.1629). Analyses of pooled data from vaccinated recipients including who developed HPV16/31 or HPV18/45 infections demonstrated that the odds of a one-log unit increase in anti-HPV16 or 18 antibody titers, respectively, were 40% higher in the combined control groups (RC + HRC, n = 160) (OR = 1.40, 95% CI: 1.09–1.79, p = 0.0135) and 73% higher in the HRC (n = 80) (OR 1.73, 95% CI: 1.34, 2.52, p = 0.0117) compared to HPV16/18/31/45 cases (n = 80). Conclusions: Overall, these findings suggest that higher IgG antibodies to HPV16/18 after vaccination represent an increased likelihood of protection from homologous and cross-reactive HPV types (HPV16/18/31/45). These results show that differences in antibody titers are associated with breakthrough infection after vaccination, suggesting that further study of long-term antibody titers and infection should be pursued. Full article

Equine Infectious Anemia Virus (EIAV) poses significant diagnostic challenges due to its genetic variability and the limitations of conventional nucleic acid detection methods. This study developed an antigen-capture, enzyme-linked immunosorbent assay (AC-ELISA) for the detection and quantification of the EIAV capsid protein p26. The assay utilized a monoclonal antibody (1G11) specific to the p26 protein as the capture antibody and a polyclonal antibody as the detection antibody, forming a highly specific and sensitive detection system. Under optimized conditions, the detection limit of the AC-ELISA was 1.95 ng/mL, with a good linear relationship observed between 1.95 ng/mL and 60.5 ng/mL of p26 protein. Additionally, the AC-ELISA effectively distinguished EIAV from other equine viruses, including equine herpesvirus 1 (EHV-1), equine arteritis virus (EAV), and equine influenza virus (EIV), without cross-reactivity. Importantly, the AC-ELISA demonstrated the ability to detect multiple EIAV strains, including virulent strains, attenuated strains, and strains from other countries, highlighting its broad applicability across diverse EIAV isolates. Compared to western blot and reverse transcriptase assays, the AC-ELISA exhibited higher sensitivity and strong correlation in quantifying the EIAV p26 protein. The assay is simple, rapid, and cost-effective, making it suitable for both laboratory research and clinical applications. It provides a powerful tool for EIAV detection and quantification, supporting future vaccine development and clinical trials. Full article