Search Results (83)

Adeno-associated virus (AAV) vectors face a critical translational challenge in ocular gene therapy due to pre-existing neutralizing antibodies (NAbs) whose seroprevalence limits patient eligibility. Standard NAb detection using non-ocular cell models (Human Embryonic Kidney 293T) may inadequately predict retinal transduction inhibition due to cell type-related variations in receptor usage and immunogenicity. This study established parallel NAb detection platforms utilizing human retinal pigment epithelial (ARPE-19) cells and standard 293T cells to systematically evaluate clinical serum samples against ophthalmologically relevant AAV serotypes (2, 5, 8, 9) via luciferase reporter-based transduction inhibition assays. Comparative analysis demonstrated ARPE-19 exhibited 42–48% higher NAb titers against AAV5/9 compared to 293T cells, with distinct serotype-biased neutralization hierarchies observed between cellular models. Furthermore, female-derived sera exhibited significantly elevated NAbs against particular serotypes in the ARPE-19 system. Critically, inter-serotype cross-neutralization correlation patterns differed substantially between cellular platforms. These findings demonstrate that physiologically relevant retinal cellular models provide essential immunological profiling data, revealing NAb characteristics obscured in standard assays. Consequently, employing retinal cell-based platforms is crucial for optimizing AAV serotype selection, patient stratification, and predicting clinical outcomes in ocular gene therapy. 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

Background: Rabbit hemorrhagic disease (RHD) is an acute, hemorrhagic and highly lethal infectious disease caused by rabbit hemorrhagic disease virus (RHDV), which causes huge economic losses to the rabbit breeding industry. Moreover, there is limited cross-protection between the two different serotypes of classic RHDV (GI.1) and RHDV2 (GI.2). The shortcomings of traditional inactivated vaccines have led to the development of novel subunit vaccines that can protect against both strains, and the VP60 capsid protein is the ideal antigenic protein. This study focused on developing a bivalent RHDV vaccine that can prevent infection with both GI.1 and GI.2 strains. Methodology: Baculovirus vectors containing classic RHDV and RHDV2 VP60 were co-transfected with linearized baculovirus into sf9 cells and transferred to baculovirus via homologous recombination of the VP60 gene. Infected sf9 cells were lysed, and after purification via Ni-NTA chromatography, VLPs were observed using transmission electron microscopy (TEM). In order to evaluate the immunogenicity of the chimeric RHDV VLP vaccine in rabbits, the RHDV VP60-specific antibody, IL-4, IFN-γ and neutralizing antibody titers were analyzed in serum using ELISA and HI. Results: The recombinant baculovirus system successfully expressed chimeric RHDV VLPs with a diameter of 32–40 nm. After immunization, it could produce specific antibodies, IL-4 and IFN-γ. Following the second immunization, neutralizing antibodies, determined using hemagglutination inhibition (HI) assays, were elicited. Conclusions: These data show that the chimeric RHDV VLP bivalent vaccine for immunized New Zealand rabbits can induce humoral immunity and cellular immunity in vivo, and the immunization effect of the high-dose group is similar to that of the current commercial vaccine. Full article

Background: Clinical trials and serological studies have demonstrated that vaccine-induced antibodies can cross-react with some non-vaccine serotypes. However, there are limited longitudinal data on the impact of pneumococcal conjugate vaccines (PCV) on cross-reactive serotypes after implementation in immunization programs. This study examines the impact of PCVs on pneumococcal disease cases due to potential cross-reactive serotypes. Methods: Eleven countries with serotyped invasive pneumococcal disease (IPD) surveillance data that had introduced PCV10 or PCV13 were identified. The analysis focused on IPD cases due to serotypes included in PCV10 and PCV13 (PCV10/13 VTs: 6B, 9V, 19F, 23F; PCV13 only VTs: 6A, 19A) and to vaccine-related serotypes (VRTs: 6C, 9N, 23A, 23B) that may be immunologically related to VTs in children under 5 years old. For each country, the number of IPD cases were charted over time according to serogroup. Results: Following PCV introduction, reductions in VT IPD cases were observed in all countries, while some VRT IPD cases remained unchanged or increased. Serotype 19A cases declined in PCV13 countries but increased in countries that introduced PCV10. VRT 6C cases rose in PCV10 countries but showed minimal change in PCV13 countries. In PCV13 countries, 9N cases remained unchanged while 23A and 23B experienced modest increases. Conclusions: The inclusion of VT 19A in PCV13, but not in PCV10, may account for the significant increase in VRT 19A cases in PCV10 countries. The slight change in VRT 6C cases in PCV13 countries compared to the significant rise in PCV10 countries suggests that PCV13 provides cross-protection for serotype 6C through serotype 6A. Cross-protection could not be determined for other VRTs, as their cases increased or remained unchanged or had insufficient data for evaluation. Full article

Foot-and-mouth disease (FMD) is a devastating infectious viral disease of cloven-hoofed animals. Differentiating FMD from other vesicular diseases is difficult based on only clinical symptoms, requiring an appropriate laboratory diagnostic test. The double-antibody sandwich (DAS)-ELISA is a reliable diagnostic technique for antigen detection and serotyping of FMDV. However, classical DAS-ELISAs use polyclonal antibodies (pAbs), which are inconsistent in yields and limited in large-scale applications compared to hybridoma cell-secreted laboratory-made monoclonal antibodies (mAbs). Therefore, this study aimed to develop simplified and sensitive FMD serotype-specific DAS-ELISAs using HRP-conjugated mAbs and a TMB substrate. Six FMDV serotype-specific mAb-DAS-ELISAs were developed. All assays were optimized using BEI-inactivated FMD antigens. Real-time reverse-transcriptase PCR (RRT-PCR) was also used to verify the detection efficiency of all assays. Known negative and positive 10% tissue suspensions of different animal origins were examined to calculate the diagnostic specificity (DSp) and sensitivity (DSe). Serotype-specific mAb-DAS-ELISAs demonstrated 100%, 97%, 97%, 99%, 99%, and 94% DSp and 100%, 95%, 90%, 95%, 100%, and 100% DSe for serotypes O, A, Asia-1, SAT-1, SAT-2, and SAT-3, respectively. The detection efficiency of mAb-DAS-ELISAs was better than that of classical DAS-ELISAs. Also, all assays demonstrated minimal cross-reactivity and optimal reproducibility. Therefore, the mAb-DAS-ELISAs developed in this study could be useful for detecting and serotyping FMDV and ultimately replacing the classical DAS-ELISA. Full article

Background/Objectives: Intact (146S) foot-and-mouth disease virus (FMDV) particles easily dissociate into 12S particles with a concomitant decreased immunogenicity. Vaccine quality control with 146S-specific single-domain antibodies (VHHs) is hampered by the high strain specificity of most 146S-specific VHHs. This study aimed to isolate 146S-specific VHHs that recognize all serotype O strains. Methods: Biopanning was performed with the FMDV strain O/SKR/7/2010 146S, using a secondary library of mutagenized M170F VHH that did not recognize O/SKR/7/2010 or using phage-display libraries from llamas immunized with other serotype O strains. Novel VHHs were yeast-produced and their strain-, particle-, and antigenic-site specificities were determined by ELISA. Results: M170F mutagenesis did not improve the cross-reaction with O/SKR/7/2010. However, selection from immune libraries resulted in four VHHs that exhibited high 146S specificity for all five serotype O strains analyzed. These VHHs presumably recognize all serotype O strains since the five strains analyzed represent different phylogenetic clades. They bind the same antigenic site as M170F, which was previously shown to be a conserved site in serotypes A and O, and which has an altered 3D structure when 146S dissociates into 12S particles. M916F had the lowest limit of detection, which varied from 0.7 to 5.9 ng/mL 146S particles for three serotype O strains. Conclusions: We identified four VHHs (M907F, M910F, M912F, and M916F) that specifically bind 146S particles of probably all serotype O strains. They enable further improved FMDV vaccine quality control. Full article

Enterotoxigenic Escherichia coli (ETEC) is one of the primary pathogens causing diarrhea in piglets, causing significant economic losses in the swine farming industry. Due to the numerous serotypes of ETEC, traditional vaccines fail to provide sufficient cross-protection, and subunit vaccines based on epitope design have emerged as a safer and more effective approach for prevention and control. Unlike vaccine development strategies that involve the tandem arrangement of multiple antigenic epitopes, this study used the K88-FaeG protein as a backbone and incorporated the antigenic epitopes of K99-FanC to achieve a better immunogenicity. By using bioinformatics software to predict B-cell linear epitopes (score of over 0.6), B-cell epitopes from three-dimensional structures (50% amino acid score of ≥0.2), and B-cell epitope IgG antibody subtypes, as well as docking analysis with Sus scrofa aminopeptidase N (APN) receptors, six antigenic epitopes of K99-FanC were selected. Through Western blotting and competitive ELISA, we confirmed that all six recombinant proteins exhibited binding capabilities to K88- and K99-positive serum. The ELISA results showed that the serum levels of specific IgG and IgA antibodies increased after immunization, with FaeG-Ep3 and FaeG-Ep5 inducing the highest antibody titers against FanC-IgG (Log2 = 14.96) and FaeG-IgG (Log2 = 17.96), respectively. Bacterial adhesion assays revealed that only FaeG-Ep3 effectively blocked the adhesion of both K99 and K88 to IPEC-J2 cells. Immunization challenge experiments showed that, in the unimmunized group, mice infected with K88 and K99 experienced weight loss (p < 0.05) with intestinal villus shedding and intestinal wall structural damage. However, in the FaeG-Ep3-immunized group, no significant weight loss occurred after infection, and the villus protection rate (83%) was the same as that in the FaeG and FanC immunized groups. Overall, the FaeG-Ep3 recombinant protein identified in this study shows potential vaccine application value and provides new insights for developing multivalent vaccines against ETEC. Full article

Dengue is a neglected disease mainly affecting tropical and subtropical countries. The diagnosis of dengue fever is still a problem since most of it is made from whole or recombinant DENV proteins, which present cross-reactions with other members of the Flavivirus family. Therefore, there is still a huge demand for new diagnostic methods that provide rapid, low-cost, easy-to-use confirmation. Thus, in this study, we developed an affordable electrochemical biosensor for rapidly detecting immunoglobulin G (IgG) serological antibodies in the sera of DENV-infected patients. An identified linear B-cell epitope (DENV/18) specific for DENV 1–4 serotypes recognized by IgG in patient sera was selected as a target molecule after a microarray of peptides using the SPOT-synthesis methodology. After chemical synthesis, the DENV/18-peptide was immobilized on the surface of the working electrode of a commercially available screen-printed gold electrode (SPGE). The capture of DENV-specific IgG allowed for the formation of an immunocomplex that was measured by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) using a potassium ferrocyanide/ferricyanide ([Fe(CN)₆]^3−/4−) electrochemical probe. An evaluation of the biosensor’s performance showed a detection limit of 100 µg mL⁻¹ for the synthetic peptides (DENV/18) and 1.21 ng mL⁻¹ in CV and 0.43 ng mL⁻¹ in DPV for human serum, with a sensitivity of 7.21 µA in CV and 8.79 µA in DPV. The differentiation of infected and uninfected individuals was possible even at a high dilution factor that reduced the required sample volumes to a few microliters. The final device proved suitable for diagnosing DENV by analyzing real serum samples, and the results showed good agreement with molecular biology diagnostics. The flexibility to conjugate other antigenic peptides to SPEs suggests that this technology could be rapidly adapted to diagnose other pathogens. Full article

In this study, a novel rapid immunochromatographic (IC) test for African swine fever virus (ASFV) antibodies is presented. An immunochromatographic test (IC) is a detection technique that combines membrane chromatography with immunolabeling. This approach saves time for antibody preparation, resulting in a shorter production cycle. p54 is an important structural protein of African swine fever, and an ideal protein for serotype diagnosis. Gold nanoparticles are attached to the ASFV p54-Fc fusion protein, and the ASFV-specific antigen p54 and Staphylococcus aureus protein A (SPA) are labeled on a nitrocellulose membrane, at positions T and C, respectively. We developed a SPA double sandwich IC test strip, and assessed its feasibility using ASFV p54 and p54-Fc fusion proteins as antigens. ASFV p54 and p54-Fc fusion proteins were expressed and purified. A sandwich cross-flow detection method for p54, which is the primary structural protein of ASFV, was established, using colloidal gold conjugation. Our method can detect ASFV antibodies in field serum samples in about 15 min using a portable colloidal gold detector, demonstrating high specificity and sensitivity (1:320), and the coincidence rate was 98% using a commercial ELISA kit. The dilution of the serum sample can be determined by substituting the absorbance (T-line) interpreted by portable devices into the calibration curve function formula of an African swine fever virus standard serum. In summary, our method is rapid, cost-effective, precise, and highly selective. Additionally, it introduces a new approach for constructing IC test strips using SPA protein without antibody preparation, making it a reliable on-site antibody test for ASFV. Full article

A newly formatted enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bluetongue virus (BTV) was developed and validated for bovine and ovine sera and plasma. Validation of the new sandwich ELISA (sELISA) was achieved with 949 negative bovine and ovine sera from BTV endemic and non-endemic areas of Australia and 752 BTV positive (field and experimental) sera verified by VNT and/or PCR. The test diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were 99.70% and 99.20%, respectively, for bovine sera, and 97.80% and 99.50%, respectively, for ovine sera. Comparable diagnostic performances were noted for the sELISA compared to four competition ELISAs. While the sensitivity of the sELISA remained unaffected by BTV-15 positive sera, the cELISAs were not as sensitive. BTV-15 is endemic to Australia, and early warning depends on sensitive diagnoses of all serotypes: endemic or incurring. The sELISA failed to discriminate against epizootic hemorrhagic disease virus (EHDV) antibodies, the most serologically related orbivirus to BTV. The ACDP cELISA and the IDEXX kit showed cross-reactivity with some EHDV serotypes, with the least cross-reactive being the VMRD and the IDVet kits. Cross-reactivities, however, were also detected in sera raised experimentally from 10 isolates of the 21 known non-BTV orbiviruses. In this case, the sELISA was the least affected, followed equally by the VMRD and IDVet kits, and the IDEXX kit and the ACDP cELISA were the least discriminatory. In addition to exclusivity assessment of the ELISAs, an inclusivity assessment was made for all ELISAs using well characterized reference sera positive for antibodies to all serotypes BTV-1 to BTV-24. Full article

African horse sickness (AHS) is an acute, fatal, contagious disease of animals of the family Equidae and is caused by infection with the African horse sickness virus (AHSV). Based on the outer capsid protein VP2, AHSV is classified into nine serotypes (AHSV−1 to −9) with little or no serological cross-reactivity between them. In 2020, AHS outbreaks caused by AHSV−1 were reported in Thailand and Malaysia, marking the first occurrences of AHS in Southeast Asia. However, little is known about the antigenic profile of AHSV−1 VP2. In this study, a recombinant VP2 protein was expressed in Escherichia coli and used as an immunogen, and three monoclonal antibodies (mAbs), designated 7D11, 10A9, and 9E7, against AHSV−1 VP2, were generated. These three mAbs were then successfully used in IFA, WB, and ELISA for the detection of AHSV−1 VP2. Two overlapping linear epitopes, ⁶⁷⁰NEFDFE⁶⁷⁵ (E670–675) recognized by 9E7 and ⁶⁷⁰NEFDF⁶⁷⁴ (E670–674) recognized by 7D11 and 10A9, were identified through truncation of GST-fused VP2. Amino acid sequence alignment shows that the ⁶⁷⁰NEFDFE⁶⁷⁵ motif is completely conserved within AHSV−1 but is highly divergent in other AHSV serotypes. Our studies provide an important tool for basic research into AHSV−1 and for the diagnosis of AHSV−1. Full article

The high antigenic variability of the foot-and-mouth disease virus (FMDV) represents a challenge for developing prophylactic strategies, stressing the need for research into vaccines offering broad protection against a range of virus strains. Here, the heterotypic cross-reaction using different vaccine schemes against serotype O strains was studied, evaluating the impact of revaccination, antigen dose, and incorporation of additional FMDV serotypes. Naïve cattle were immunized with seven distinct FMDV vaccines, receiving three doses of the same formulation at 0, 28, and 56 days post-primary vaccination (dpv). Serum samples were collected up to 70 dpv and tested by a virus-neutralizing test against serotype O strains from a South American lineage and two strains representative of two Asian lineages. Our results showed that vaccines containing the ME-SA topotype O1/Campos strain developed cross-neutralizing responses against the two Asian viruses after the first vaccination. In contrast, significant heterotypic neutralizing antibody titers against the homologous topotype strain were only found after the second vaccination, indicating that the phylogenic relationship may differ from the antigenic profiles for these two viruses. The amount of the O1/Campos strain and the revaccination were essential factors for neutralization against the homologous- and heterologous-type O FMDV viruses. The strain composition of the vaccine was only relevant for cross-neutralization against one of the Asian strains, suggesting potential intra-serotypic divergences for this pattern. Full article

Shigellosis represents a significant global health concern particularly affecting children under 5 years in low- and middle-income countries (LMICs) and is associated with stunting and antimicrobial resistance. There is a critical need for an effective vaccine offering broad protection against the different Shigella serotypes. A correlate of protection has not yet been established but there is a general consensus about the relevant role of anti-O-Antigen-specific IgG and its functionality evaluated by the Serum Bactericidal Assay (SBA). This study aims to characterize a high-throughput luminescence-based SBA (L-SBA) against seven widespread Shigella serotypes. The assay was previously developed and characterized for S. sonnei and S. flexneri 1b, 2a, and 3a and has now been refined and extended to an additional five serotypes (S. flexneri 4a, 5b, 6, X, and Y). The characterization of the assay with human sera confirmed the repeatability, intermediate precision, and linearity of the assays; both homologous and heterologous specificity were verified as well; finally, limit of detection and quantification were established for all assays. Moreover, different sources of baby rabbit complement showed to have no impact on L-SBA output. The results obtained confirm the possibility of extending the L-SBA to multiple Shigella serotypes, thus enabling analysis of the functional response induced by natural exposure to Shigella in epidemiological studies and the ability of candidate vaccines to elicit cross-functional antibodies able to kill a broad panel of prevalent Shigella serotypes in a complement-mediated fashion. Full article

Objectives: The aim of this study was to prepare monoclonal antibodies (mAbs) that broadly target Acinetobacter baumannii and protect against infection by multi-drug-resistant (MDR) A. baumannii from different sources. Methods: mAb 8E6 and mAb 1B5 were prepared by sequentially immunizing mice with a sublethal inoculation of three heterogeneous serotypes of pan-drug-resistant (PDR) A. baumannii, ST-208, ST-195, and ST-229. Results: The cross-recognition of heterogeneous bacteria (n = 13) by two mAbs and potential targets was verified, and the in vitro antibacterial efficacy of mAbs was assessed. The median killing rate of mAb 8E6 against A. baumannii in the presence of complement and dHL-60 cells was found to be 61.51%, while that of mAb 1B5 was 41.96%. When only dHL-60 cells were present, the killing rate of mAb 8E6 was 65.73%, while that of mAb 1B5 was 69.93%. We found that mAb 8E6 and mAb 1B5 broadly targeted MDR A. baumannii on the ATP synthase complex and were equipped with an antibacterial killing ability by enhancing the innate immune bacteriolytic effect of ST-208 and ST-195 strains. Both monoclonal antibodies were validated to protect against respiratory infection at 4 and 24 h via enhancing the release of innate immune substances and inflammatory cytokines, effectively shortening the disease period in mice. Conclusions: mAb 8E6 and mAb 1B5 significantly enhanced the opsonization process of phagocytosis against A. baumannii strains prevalent in southern China by targeting ATP synthase antigens thereof, resulting in protective effects in mice. Full article

Foot-and-mouth disease (FMD) is one of the most infectious viral transboundary diseases of livestock, which causes devastating global economic losses. Different enzyme-linked immunosorbent assays (ELISAs) are used for sero-surveillance of the foot-and-mouth disease virus (FMDV). However, more sensitive, accurate, and convenient ELISAs are still required to detect antibodies against FMDV serotypes. The primary goal of this study was to establish serotype-specific monoclonal antibody (mAb)-based blocking ELISAs (mAb-bELISAs) that would provide better performance characteristics or be equivalent in performance characteristics compared with a conventional polyclonal antibody (pAb)-based competitive ELISA (pAb-cELISA). Four mAb-bELISAs were developed using FMDV serotype-specific mAbs for the detection of anti-FMDV/O/A/Asia1/SAT2 antibodies. Using a 50% cut-off, all four mAb-bELISAs exhibited species-independent 99.74%, 98.01%, 96.59%, and 98.55% diagnostic specificity (DSp) and 98.93%, 98.25%, 100%, and 87.50% diagnostic sensitivity (DSe) for FMDV serotypes O, A, Asia1, and SAT2, respectively. In addition, a 100% DSe of serotypes O- and SAT2-specific mAb-bELISAs was observed for porcine sera when the cut-off was 30%. All mAb-bELISAs developed in this study displayed high repeatability/reproducibility without cross-reactivity. Finally, the diagnostic performance of mAb-bELISAs was found to be better than or equivalent to compared with pAb-cELISAs, suggesting that mAb-bELISAs can be used to replace existing pAb-ELISAs for the detection of antibodies against these four FMDV serotypes. Full article