Search Results (1,277)

Influenza virus is a leading cause of global morbidity and mortality due to acute lower respiratory infection, even with the widespread use of multiple licensed influenza vaccines. However, antigenic drift during influenza replication can cause vaccine-induced antibodies to poorly neutralize influenza virus, thereby reducing vaccine effectiveness. To help overcome this problem, we leveraged a hydrogel platform with influenza hemagglutinin (HA) protein to induce prolonged antigen exposure. The hydrogel platform, Vaccine Self-Assembling Immune Matrix (VacSIM^®), in combination with recombinant influenza H1 or H3 HA protein antigens, increased antigen-specific antibody titers in vaccinated mice, which led to decreased disease severity after H1N1 infection for H1 HA-vaccinated mice and decreased lung viral titers after H3N2 challenge for H3 HA-vaccinated mice. Sera collected from mice immunized with VacSIM and HA also showed broader HAI activity, increasing by 1–3 log against a panel of influenza viruses. These results were consistent with the use of cocktail immunization, containing both an H1 and H3 HA, where mice immunized with VacSIM had an increase in antigen-specific antibody titers and decreased disease severity and lung viral titers against H1N1 and H3N2 influenza challenges, respectively. Finally, it was determined that a single immunization with VacSIM and H1 HA could provide protection against lethal H1N1 challenge compared to a group without VacSIM. In summary, we demonstrate that use of the slow-release platform VacSIM can improve the host immune response to vaccination and increase protection against influenza infection. Full article

Background: Type 1 diabetes (T1D) is an autoimmune disorder characterized by destruction of insulin-producing β-cells. While conventional insulin therapy manages hyperglycemia, it fails to halt autoimmunity. Oral immunotherapy targeting autoantigens like GAD65 offers potential for antigen-specific tolerance; however, its efficacy is limited by gastrointestinal degradation and poor mucosal uptake. Lactococcus lactis (L. lactis), a food-grade delivery vector, enables sustained antigen release and intestinal tract immune modulation, yet the differential transcriptomic mechanisms underlying mucosal versus systemic immune responses remain uncharacterized. Methods: Non-obese diabetic (NOD) mice were randomized into control and GAD65 groups, receiving oral PBS or the GAD65 recombinant L. lactis vaccine, respectively. Fasting blood glucose was monitored weekly. GAD65-specific IgA and IgG, along with immune tolerance-related factors, were quantified using ELISA. Lymphocyte subsets were analyzed by flow cytometry, alongside RNA sequencing and transcriptional profiling. Results: The study demonstrated that the orally administered GAD65-L. lactis vaccine could significantly induce GAD65-specific IgA antibody and TGF-β cytokine and alleviate hyperglycemia and diabetes symptoms in NOD mice. Our study facilitated the induction of GAD65-specific regulatory T cells within both intestinal lamina propria lymphocytes (LPLs) and splenic lymphocytes. Notably, antigen-specific tolerance was mainly observed in intestinal LPLs. Crucially, the immune responses elicited by the vaccine demonstrated significant disparities between intestinal LPLs and splenic lymphocytes, with intestinal LPLs exhibiting unique local immune tolerance transcriptomic profiles. Conclusions: Our findings have enhanced the comprehension of the mechanisms by which oral vaccines influence the interplay between mucosal and systemic immune responses, thereby establishing a foundational framework for the design of oral vaccines. This understanding is instrumental in advancing antigen-specific immune tolerance strategies for autoimmune diseases such as Type 1 Diabetes (T1D). Full article

A novel skin test for an in vivo assessment of SARS-CoV-2-specific T-cell immunity was developed using CoronaDermPS, a multiepitope recombinant polypeptide encompassing MHC II–binding CD4+ T-cell epitopes of the SARS-CoV-2 structural proteins (S, E, M) and full length nucleocapsid (N). In silico epitope prediction and modeling guided antigen design, which was expressed in Escherichia coli, was purified (>95% purity) and formulated for intradermal administration. Preclinical evaluation in guinea pigs, mice, and rhesus macaques demonstrated a robust delayed type hypersensitivity (DTH) response at optimal doses (10–75 µg), with no acute or chronic toxicity, mutagenicity, or adverse effects on reproductive organs. An integrated clinical analysis included 374 volunteers stratified by vaccination status (EpiVacCorona, Gam-COVID-Vac, CoviVac) prior to COVID-19 infection (Wuhan/Alpha, Delta, Omicron variants), and SARS-CoV-2–naïve controls. Safety assessments across phase I–II trials recorded 477 adverse events, of which >88% were mild and self-limiting; no severe or anaphylactic reactions occurred. DTH responses were measured at 24 h, 72 h, and 144 h post-injection by papule and hyperemia measurements. Overall, 282/374 participants (75.4%) exhibited a positive skin test. Receiver operating characteristic analysis yielded an overall AUC of 0.825 (95% CI: 0.726–0.924), sensitivity 79.5% (95% CI: 75.1–83.3%), and specificity 85.5% (95% CI: 81.8–88.7%), with comparable diagnostic accuracy across vaccine, and variant subgroups (AUC range 0.782–0.870). CoronaDerm-PS–based skin testing offers a simple, reproducible, and low-cost method for qualitative evaluation of T-cell–mediated immunity to SARS-CoV-2, independent of specialized laboratory equipment (Eurasian Patent No. 047119). Its high safety profile and consistent performance across diverse cohorts support its utility for mass screening and monitoring of cellular immune protection following infection or vaccination. Full article

Background/Objectives: Single-domain immunoglobulins are small protein modules with specific affinities. Among them, the variable domains of heavy chains of heavy-chain-only antibodies (VHH) as the antigen-binding fragment of heavy-chain-only antibodies (also termed nanobodies) have been widely investigated for their applicability, e.g., therapeutics and immunodiagnostics. However, despite their advantageous biochemical and biophysical characteristics, protein aggregation throughout recombinant synthesis is a serious drawback in the development of nanobodies with application perspectives. Therefore, we aimed to develop a computational method to predict the aggregation propensity of VHH antibodies for the selection of promising candidates in early discovery. Methods: We employed a deep learning-based structure prediction for VHHs and derived from it likely biophysical and biochemical properties of the framework region 2 with relevance for aggregation. A total of 106 nanobody variants were produced by recombinant expression and characterized for their aggregation behavior using size exclusion chromatography (SEC). Results: Quantitative characteristics of framework region 2 patches were combined into a function that defines an aggregation score (AS) predicting the aggregation propensities of VHH variants. AS was evaluated for its capability to forecast recombinant VHH aggregation by experimentally studying VHH Fc-fusion proteins for their aggregation. We observed a clear correlation between the calculated aggregation score and the actual aggregation propensities of biochemically characterized VHHs Fc-fusion proteins. Moreover, we implemented an easily accessible pipeline of software modules to design nanobodies with desired solubility properties. Conclusions: AI-based prediction of VHH structures, followed by analysis of framework region 2 properties, can be used to predict the aggregation propensities of VHHs, providing a convenient and efficient tool for selecting stable recombinant nanobodies. Full article

Background: African swine fever (ASF) is a highly contagious and often deadly disease that poses a major threat to swine production worldwide. The lack of a commercially available vaccine underscores the critical need for innovative immunization strategies to combat ASF. Methods: Six ASFV antigenic proteins (K78R, A104R, E120R, E183L, D117L, and H171R) were fused with the Lactiplantibacillus plantarum WCFS1 surface anchor LP3065 (LPxTG motif) to generate recombinant Lactiplantibacillus plantarum NC8 (rNC8) strains. The surface expression was confirmed using immunofluorescence and Western blotting assays. Additionally, the dendritic cell-targeting peptides (DCpep) were co-expressed with each antigen protein. Mice were immunized at a dosage of 10⁹ colony-forming units (CFU) per strain per mouse via intragastric (I.G.), intranasal (I.N.), and intravenous (I.V.) routes. The bacterial mixture was heat-inactivated by boiling for 15 min to destroy viable cells while preserving antigenic structures. I.V. administration caused no hypersensitivity, confirming the method’s safety and effectiveness. Results: Following I.G. administration, rNC8-E120R, rNC8-E183L, rNC8-K78R, and rNC8-A104R induced significant levels of secretory immunoglobulin A (sIgA) in fecal samples, whereas rNC8-H171R and rNC8-D117L failed to induce a comparable response. Meanwhile, rNC8-D117L, rNC8-K78R, and rNC8-A104R also elicited significant levels of sIgA in bronchoalveolar lavage fluid (BALF). Following I.N. immunization, rNC8-E120R, rNC8-K78R, and rNC8-A104R significantly increased sIgA levels in both fecal and BALF immunization. In contrast, I.V. immunization with heat-inactivated rNC8-K78R and rNC8-A104R induced robust serum IgG titers, whereas the remaining antigens elicited minimal or insignificant responses. Flow cytometry analysis revealed expanded CD3⁺CD4⁺ T cells in mice immunized via the I.N. and I.G. and CD3⁺CD4⁺ T cells only in those immunized via the I.N. route. Th1 responses were also significant in the sera of mice immunized via the I.G. and I.N. routes. Conclusions: The rNC8 multiple-antigen cocktail elicited strong systemic and mucosal immune responses, providing a solid foundation for the development of a probiotic-based vaccine against ASF. Full article

SSB proteins play essential roles in DNA replication, recombination, and repair in bacteria, archaea, and eukarya. This study investigates the transcript levels, identification, expression and purification, subcellular localization, and immune protective potential of the SSB-like proteins of Eimeria necatrix (EnSSB), exploring its role in the development of E. necatrix and its potential as a candidate antigen for a subunit vaccine against avian coccidiosis. The level of EnSSB gene transcription was highest in unsporulated oocysts (UO), followed by gametocytes (GAM) (p < 0.05). The gene consisted of an open reading frame of 1488 nucleotides encoding a protein of 495 amino acid residues with a predicted molecular weight of 53.31 kDa. EnSSB contained a SSB domain with a conserved OB (oligonucleotide/oligosaccharide binding) fold. The molecular mass of the native protein, as determined by Western blot analysis, was ~58 kDa in second-generation merozoites (MZ-2) and UO. In addition to the 58 kDa band, four other bands (~98 kDa, ~82 kDa, ~36 kDa and ~28 kDa) were detected in GAM. No bands were detected in MZ-3. Indirect immunofluorescence and immuno-electron microscopy localized EnSSB in the cytoplasm of macrogametocytes but not in wall-forming bodies and oocyst wall. Animal challenge experiments demonstrated that rEnSSB elicited robust IgY responses, increased splenic T lymphocytes and body weight gain, reduced intestinal lesion scores and oocyst shedding, and presented anticoccidial index (ACI) more than 160. These findings not only offer a foundation for understanding the role of EnSSB protein in regulating the development of E. necatrix, but also present a potential protective antigen of E. necatrix for the development of a subunit vaccine against avian coccidiosis. Full article

Influenza is a serious and global health issue, and it is a major cause of morbidity, fatality, and economic loss every year. Seasonal vaccines exist but are not very effective due to strain mismatches, delays in production, and antigenic drift. This comprehensive overview discusses the current situation of influenza vaccination, including the numerous types of vaccines—inactivated, live attenuated, and recombinant vaccines—and their effectiveness, efficacy, and associated challenges. It highlights the effects of the COVID-19 pandemic on the trends of influenza vaccination and the level to which innovation should be practiced. In the future universal influenza vaccines will be developed that target conserved viral antigens to provide long-term protection to people. In the meantime, novel vaccine delivery platforms, such as mRNA technology, virus-like particle (VLP), and nanoparticle-based systems, and less cumbersome and invasive administration routes, as well as immune responses are also under development to increase access and production capacity. Collectively, these innovations have the potential to not only reduce the global influenza epidemic but also to change the way influenza is prevented and prepare the world for a pandemic. Full article

Fruit bats in the genus Pteropus are the natural reservoirs for zoonotic paramyxoviruses, notably henipaviruses and pararubulaviruses, which are found across Southeast Asia and Oceania. The genetic and antigenic diversity of viruses in both genera, and region specificity, are ill-defined, limiting health security measures aimed at minimizing spillover. For example, Nipah virus has been isolated from bats in the Battambang province of western Cambodia, and surveys suggest bat foraging behaviors occur in close proximity to human settlements. However, there have been no historical cases of Nipah virus in Cambodia. Here, we use a multiplex microsphere immunoassay to identify cryptic human exposure to selected henipaviruses and pararubulaviruses in Cambodia. Convalescent human sera from persons presenting with acute respiratory illness were screened to detect the presence or absence of antibodies reactive with attachment glycoprotein antigens from Nipah virus, Hendra virus, Cedar virus, and Ghana virus, and a hemagglutinin-neuraminidase antigen from Menangle virus. In this sero-survey, we detected antibodies that were specifically reactive with Cedar virus and Menangle virus, including one serum sample that neutralized a recombinant Cedar virus. Additionally, we detected a pattern of cross-reactivity with Hendra virus, Cedar virus, and Ghana virus, suggesting previous infection by an antigenically-related henipavirus. We did not detect high antibody reactivity with the NiV glycoprotein. Future studies should expand serological surveillance for these transboundary pathogens, including genetic surveillance to aid in henipavirus discovery, and focused biosurveillance where interfaces with livestock and humans occur. Full article

Objectives: The construction of subunit vaccines based on antigens that can induce strong cellular immunity is a widely accepted strategy to develop new tuberculosis vaccines. This study screens immunogens with potential for subunit vaccine development from seven candidate antigens and then verifies their vaccine efficacy. Design: C57BL/6 mice were immunized subcutaneously with purified PPE19, PPE50, FadD21, Rv1505c, Rv1506c, Rv2035, and Rv0976c proteins formulated with Freund’s adjuvant to evaluate both the antigen-specific Th1 cellular immune responses and IgG level. After the vaccination of mice with recombined pcDNA3.1 expressing Rv0976c, intravenous or aerosol infection with M. tb were further challenged to assess protective efficacy. Results: Purified PPE19, PPE50, FadD21, and Rv0976c proteins generated strong antigen-specific Th1 cellular immune responses in mice. Compared to Ag85A, Rv0976c also stimulated higher IgG antibody level in mice. In particular, Rv0976c stimulated high and specific IgG antibody levels in serum from TB patients. The vaccination of mice with DNA vaccines expressing Rv0976c, followed by intravenous challenge with Bacillus Calmette–Guerin (BCG) Pasteur or M. tb, resulted in significant levels of protection that are comparable to or better than that afforded by the two leading antigens, Ag85A and PPE18. Conclusions: These results indicated that Rv0976c was a better protective antigen. Future studies to combine Rv0976c with other antigens and evaluate its effectiveness as a booster of BCG or as a therapeutic vaccine are warranted. Full article

Background: Toxoplasma gondii (T. gondii) infection causes serious diseases in immunocompromised patients and causes congenital toxoplasmosis in infants. T. gondii microneme protein 8 (MIC8) and apical membrane antigen 1 (AMA1) are essential proteins involved in parasitic invasion. Methods: In this study, we generated virus-like particles (VLPs) and recombinant vaccinia virus (rVV) containing MIC8 or AMA1 proteins. Vaccine efficacy was evaluated in mice (BALB/c) upon challenge infection with T. gondii ME49. Results: Intramuscular immunization with heterologous vaccines (rVV + VLPs; rVV for prime and VLPs for boost) elicited T. gondii-specific IgG antibody responses in mice. Four weeks after the boost, all mice were orally challenged with T. gondii ME49, and protective immunity was assessed. The responses of antibody-secreting cells for IgG2a and IgG2b and those of memory B cells and CD4+ and CD8+ T cells were higher in the rVV + VLP group than in the VLP + VLP group. The rVV + VLP group exhibited a significant reduction in cyst count in the brain. Conclusions: These findings indicate that heterologous vaccination with vaccinia viruses and VLPs improves vaccine efficacy. Full article

Background/Objectives: Vaccine development for the prevention of ASF has been very challenging due to the extensive genetic and largely unknown antigenic diversity. Inactivated vaccines, using different inactivation methods and a variety of adjuvants, have been consistently inefficacious. Historically, animals recovering from an infection with an attenuated virus became protected from the development of a clinical disease caused by an antigenically related strain. Therefore, immunization of susceptible animals with attenuathe ted virus strains has become a common method of vaccination with the first two commercially available vaccines based on recombinant live-attenuated viruses (LAVs). An important limitation is that the efficacy of the LAV is restricted to those strains that are antigenically related and, in most cases, only provide protection against homologous strains. Due to the unknown antigenic heterogeneity among all ASFV field isolates, the development of broad-spectrum vaccines is a challenge. Besides the anecdotal data, there is not a large amount of information describing patterns of cross-protection between different ASFV strains. Methods: We evaluated the cross-protection induced by the ASFV live-attenuated vaccine ASFV-G-ΔI177L against different biotypes of ASFV and compared their genomic sequences to determine potential genetic mutations that could cause the lack of cross-protection. Results: Results presented here demonstrate different patterns of protection when ASFV-G-ΔI177L vaccinated pigs were challenged with six different ASFV field isolates belonging to different biotypes. Conclusions: The presence of cross-protection cannot be predicted solely by the classical methodology for genotyping-based B646L ORF only. Biotyping, considering the entire virus proteome, appears to be a more promising prediction tool, although additional gathering of experimental data will be necessary to fully validate it; until then, the presence of cross-protection needs to be confirmed in efficacy trials challenging vaccinated animals. Full article

Bubaline alphaherpesvirus 1 (BuHV-1) is a virus that belongs to the Varicellovirus genus within the Alphaherpesvirinae subfamily. While BuHV-1 infections in water buffaloes (Bubalus bubalis) are often subclinical, clinical manifestations have been reported. This study provides complete genome sequences of five BuHV-1 strains isolated in Argentina, marking the first genomic characterization of BuHV-1 from the Americas. Phylogenetic reconstructions based on whole-genome and coding sequences, along with analyses of glycoproteins C, D, and E, identified a distinct clade and divergent strains. Comparative genomic analyses with publicly available BuHV-1 and Bovine alphaherpesvirus 5 (BoHV-5) sequences showed nucleotide divergence of up to 1.3% among BuHV-1 strains, indicating significant intraspecific genetic diversity. Cross-neutralization assays revealed variable relationships between BuHV-1 and BoHV-5 strains. Some Argentinian BuHV-1 strains exhibited significant antigenic subtype differences compared to Bovine alphaherpesvirus 1 (BoHV-1). Recombination analyses uncovered events between BuHV-1 and bovine herpesviruses, suggesting a complex evolutionary history within mixed farming systems. The findings indicate that the monophyletic BuHV-1 clade, including the reference BuHV-1 isolate, is representative of the BuHV-1 species. The remaining strains, provisionally classified as BuHV-1 indeterminate (BuHV-1i), can be categorized based on specific clinical and antigenic properties. The identified heterogeneity has significant implications for diagnostic accuracy, vaccine development, and disease management strategies in buffalo populations worldwide. Full article

Although Torque Teno Viruses (TTVs) were initially considered to be ubiquitous members of the mammalian virome, the finding that swine TTVs (TTSuV) can act as primary pathogens elevates the possible status of swine TTVs (TTSuVs) to an emerging swine pathogen. Since their discovery, the molecular mechanisms of TTV–host interactions remain largely unknown as robust in vitro culture systems and in vivo animal models have not been available. This study was undertaken to address some of these long-standing gaps. Recombinant TTSuV1 rescued from an infectious clone was used to infect C57BL/J6 mice. Infected mice seroconverted within 15 days post-infection and mounted virus neutralizing antibody responses. Viral DNA was detected in blood and lung tissue for the duration of the study. TTSuV1 isolated from the lung tissue of infected mice productively and serially infected PK-15 cells in vitro, indicating that the treatment produced viable, replicative viral particles in the host. TTSuV1 antigen was also detected by flow cytometry in lymphocytes, including the T and B lymphocyte subsets. Infected mice exhibited mild splenic hyperplasia and lymphopenia. The ability to respond to mitogenic stimuli was highly diminished in infected mice and a striking lack of virus-specific recall responses was observed for the 30-day duration of the study. Therefore, this study is the first to provide experimental evidence that recombinant TTSuV1 rescued from an infectious clone is infective and induces immune responses in laboratory mice. This model provides a critical tool for advancing research on TTV immunopathogenesis. Full article

Background: Toxoplasma gondii (T. gondii) is a significant opportunistic zoonotic protozoan, presenting a substantial risk to human health and livestock. Consequently, the development of an effective vaccine against toxoplasmosis is imperative. This study focuses on the GRA12 protein as a target for developing a recombinant protein vaccine, with its efficacy evaluated through immunization trials in cats. Methods: We expressed recombinant GRA12 protein in E. coli and immunized cats with the purified antigen. The cats were categorized into four groups: G1 (PBS control), G2 (ISA 201 adjuvant alone), G3 (rGRA12 vaccine), and G4 (rGRA12 combined with ISA 201 adjuvant). All cats underwent subcutaneous immunizations on days 0, 14, and 28. Subsequently, serum levels of IgG (including IgG1 and IgG2a subclasses) and cytokines (IFN-γ, IL-2, TNF-α, IL-4, IL-10) were measured by enzyme-linked immunosorbent assay (ELISA). Two weeks after the third immunization (42 DPI), each cat was intraperitoneally infected with 1 × 10⁶T. gondii RH tachyzoites. Oocyst shedding, survival duration, and T. gondii burden were monitored to assess vaccine-induced immunity. Results: The results indicate that immunization with recombinant rGRA12 protein significantly elevated IgG, IgG1, and IgG2a antibody levels in cats. G4 displayed elevated IgG levels post-immunization compared to G1 and G2, with an IgG1/IgG2a ratio > 1, indicating a mixed Th1/Th2 immune response. G4 also showed significantly increased IFN-γ, IL-2, TNF-α, and IL-4 levels compared to G1 (p < 0.05), while IL-10 remained unchanged. After T. gondii infection, total oocyst counts were 4.61 × 10⁶ (G1), 4.49 × 10⁶ (G2), 3.58 × 10⁶ (G3), and 2.59 × 10⁶ (G4), with G3/G4 showing 20.1–27.9% reduction relative to G1 (p < 0.05). Survival analysis revealed that groups G3 and G4 exhibited significantly longer median survival times (38 and 60 days, respectively; G4 with no mortality) compared to G1 and G2 (19 and 26 days, respectively). Additionally, parasite burdens in the brain, heart, lungs, liver, and spleen were significantly reduced in G3/G4 compared to G1/G2 (p < 0.01). Conclusions: In summary, the recombinant GRA12 vaccine significantly enhanced host survival and reduced parasite burden, demonstrating its potential as an effective toxoplasmosis vaccine candidate. These findings provide valuable data for future toxoplasmosis vaccine development. Full article

Canine visceral leishmaniasis (CVL) is a major zoonosis that poses a growing challenge to public health services, as successful disease management requires sensitive, specific, and rapid diagnostic methods capable of identifying infected animals even at a subclinical level. The objective of this study was to evaluate the performance of the recombinant chimeric protein rMELEISH3 as an antigen in ELISA assays for the robust diagnosis of CVL. The protein was expressed in a bacterial system, purified by affinity chromatography, and evaluated through a series of serological assays using serum samples from dogs infected with Leishmania infantum. ROC curve analysis revealed a diagnostic sensitivity of 96.4%, a specificity of 100%, and an area under the curve of 0.996, indicating excellent discriminatory power. Furthermore, rMELEISH3 was recognized by antibodies present in the serum of dogs with low parasite loads, reinforcing the diagnostic potential of the assay in asymptomatic cases. It is concluded that the use of the recombinant antigen rMELEISH3 could significantly contribute to the improvement of CVL surveillance and control programs in endemic areas of Brazil and other countries, by offering a safe, reproducible and effective alternative to the methods currently recommended for the serological diagnosis of the disease. Full article