Detection of naturally acquired, strain-transcending antibodies against rosetting Plasmodium falciparum strains in humans.

Strain-transcending antibodies against virulence-associated subsets of P. falciparum-infected erythrocyte surface antigens could protect children from severe malaria. However, the evidence supporting the existence of such antibodies is incomplete and inconsistent. One subset of surface antigens associated with severe malaria, rosette-mediating Plasmodium falciparum Erythrocyte Membrane Protein one (PfEMP1) variants, cause infected erythrocytes to bind to uninfected erythrocytes to form clusters of cells (rosettes) that contribute to microvascular obstruction and pathology. Here, we tested plasma from 80 individuals living in malaria-endemic regions for IgG recognition of the surface of four P. falciparum rosetting strains using flow cytometry. Broadly reactive plasma samples were then used in antibody elution experiments in which intact IgG was eluted from the surface of infected erythrocytes and transferred to heterologous rosetting strains to look for strain-transcending antibodies. We found that seroprevalence (percentage of positive plasma samples) against allopatric rosetting strains was high in adults (63%-93%) but lower in children (13%-48%). Strain-transcending antibodies were present in nine out of eleven eluted antibody experiments, with six of these recognizing multiple heterologous rosetting parasite strains. One eluate had rosette-disrupting activity against heterologous strains, suggesting PfEMP1 as the likely target of the strain-transcending antibodies. Naturally acquired strain-transcending antibodies to rosetting P. falciparum strains in humans have not been directly demonstrated previously. Their existence suggests that such antibodies could play a role in clinical protection and raises the possibility that conserved epitopes recognized by strain-transcending antibodies could be targeted therapeutically by monoclonal antibodies or vaccines.

Predictions of Immunodominant Epitope Peptides From the AsaA Type VI Secretion System in Acinetobacter baumannii: A Computational Approach.

Introduction Acinetobacter baumannii, designated as a priority pathogen by the World Health Organization (WHO), is responsible for recalcitrant infections in immunocompromised patients. The type VI secretion system (T6SS) is a class of macromolecular secretion machines, contributing to its virulence. The aim of this study is thus to predict the immune-dominant epitope peptides from the Acinetobacter T6SS-associated protein of A. baumannii (AsaA). Methods AsaA protein retrieval from the bacteria was carried out using computational platforms and the evaluation of antigenicity and allergenicity was performed. The T-cell epitopes of major histocompatibility complex class II binders were identified followed by molecular docking of the immune-dominant epitopes with human leukocyte antigen alleles using the ClusterPro server (https://cluspro.org/help.php). Additionally, the B-cell epitopes were predicted. Results Immune-informatic analysis showed immune-dominant peptides in the most favored regions with promising interactions with HLA alleles DP, DQ, DR, and toll-like receptor showing high binding capacity. Conclusion In the present investigation, epitope 1 (LILFLIGNY) was found to be a promising candidate for the synthesis of vaccines. However, it requires further experimentation for its immunological memory and response.

Deciphering a reliable synergistic bispecific strategy of rescuing antibodies for SARS-CoV-2 escape variants, including BA.2.86, EG.5.1, and JN.1.

The game between therapeutic monoclonal antibodies (mAbs) and continuously emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has favored the virus, as most therapeutic mAbs have been evaded. Addressing this challenge, we systematically explored a reproducible bispecific antibody (bsAb)-dependent synergistic effect in this study. It could effectively restore the neutralizing activity of the bsAb when any of its single mAbs is escaped by variants. This synergy is primarily attributed to the binding angle of receptor-binding domain (RBD)-5, facilitating inter-spike cross-linking and promoting cryptic epitope exposure that classical antibody cocktails cannot achieve. Furthermore, RBD-5 with RBD-2, RBD-6, and RBD-7, alongside RBD-8, also exhibit significantly enhanced effects. This study not only shifts the paradigm in understanding antibody interactions but paves the way for developing more effective therapeutic antibodies against rapidly mutating SARS-CoV-2, with Dia-19 already showing promise against emerging variants like BA.2.86, EG.5.1, and JN.1.

Elevated serum levels of soluble B-cell maturation antigen as a prognostic biomarker for multiple myeloma.

Serum B-cell maturation antigen (sBCMA) levels can serve as a sensitive biomarker in multiple myeloma (MM). In the research setting, sBCMA levels can be accurately detected by enzyme-linked immunosorbent assay (ELISA), but the approach has not been approved for clinical use. Here, we used a novel chemiluminescence method to assess sBCMA levels in 759 serum samples from 17 healthy donors and 443 patients with plasma cell (PC) diseases including AL amyloidosis, POEMS syndrome and MM. Serum BCMA levels were elevated 16.1-fold in patients with newly diagnosed MM compared to healthy donors and rare PC diseases patients. Specifically, the sBCMA levels in patients with progressive disease were 64.6-fold higher than those who showed partial response or above to treatment. The sBCMA level also correlated negatively with the response depth of MM patients. In newly diagnosed and relapsed MM patients, survival was significantly longer among those subjects whose sBCMA levels are below the median levels compared with those above the median value. We optimized the accuracy of the survival prediction further by integrating sBCMA level into the Second Revised International Staging System (R2-ISS). Our findings provide evidence that the novel chemiluminescence method is sensitive and practical for measuring sBCMA levels in clinical samples and confirm that sBCMA might serve as an independent prognostic biomarker for MM.

Multi-epitope protein production and its application in the diagnosis of opisthorchiasis.

BACKGROUND: Opisthorchiasis and cholangiocarcinoma (CCA) continue to be public health concerns in many Southeast Asian countries. Although the prevalence of opisthorchiasis is declining, reported cases tend to have a light-intensity infection. Therefore, early detection by using sensitive methods is necessary. Several sensitive methods have been developed to detect opisthorchiasis. The immunological detection of antigenic proteins has been proposed as a sensitive method for examining opisthorchiasis. METHODS: The Opisthorchis viverrini antigenic proteins, including cathepsin B (OvCB), asparaginyl endopeptidase (OvAEP), and cathepsin F (OvCF), were used to construct multi-antigenic proteins. The protein sequences of OvCB, OvAEP, and OvCF, with a high probability of B cell epitopes, were selected using BepiPred 1.0 and the IEDB Analysis Resource. These protein fragments were combined to form OvCB_OvAEP_OvCF recombinant DNA, which was then used to produce a recombinant protein in Escherichia coli strain BL21(DE3). The potency of the recombinant protein as a diagnostic target for opisthorchiasis was assessed using immunoblotting and compared with that of the gold standard method, the modified formalin-ether concentration technique. RESULTS: The recombinant OvCB_OvAEP_OvCF protein showed strong reactivity with total immunoglobulin G (IgG) antibodies against light-intensity O. viverrini infections in the endemic areas. Consequently, a high sensitivity (100%) for diagnosing opisthorchiasis was reported. However, cross-reactivity with sera from other helminth and protozoan infections (including taeniasis, strongyloidiasis, giardiasis, E. coli infection, enterobiasis, and mixed infection of Echinostome spp. and Taenia spp.) and no reactivity with sera from patients with non-parasitic infections led to a reduced specificity of 78.4%. In addition, the false negative rate (FNR), false positive rate (FPR), positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy were 0%, 21.6%, 81.4%, 100%, and 88.9%, respectively. CONCLUSIONS: The high sensitivity of the recombinant OvCB_OvAEP_OvCF protein in detecting opisthorchiasis demonstrates its potential as an opisthorchiasis screening target. Nonetheless, research on reducing cross-reactivity should be undertaken by detecting other antibodies in other sample types, such as saliva, urine, and feces.

Identification of a linear B-cell epitope on the "puff" loop of the Senecavirus A VP2 protein involved in receptor binding.

Senecavirus A (SVA) is an important emerging swine pathogen that causes vesicular lesions in swine and acute death in newborn piglets. VP2 plays a significant role in the production of antibodies, which can be used in development of diagnostic tools and vaccines. Herein, the aim of the current study was to identify B-cell epitopes (BCEs) of SVA for generation of epitope-based SVA marker vaccine. Three monoclonal antibodies (mAbs), named 2E4, 1B8, and 2C7, against the SVA VP2 protein were obtained, and two novel linear BCEs, 177SLGTYYR183 and 266SPYFNGL272, were identified by peptide scanning. The epitope 177SLGTYYR183 was recognized by the mAb 1B8 and was fully exposed on the VP2 surface, and alanine scanning analysis revealed that it contained a high continuity of key amino acids. Importantly, we confirmed that 177SLGTYYR183 locates on "the puff" region within the VP2 EF loop, and contains three key amino acid residues involved in receptor binding. Moreover, a single mutation, Y182A, blocked the interaction of the mutant virus with the mAb 1B8, indicating that this mutation is the pivotal point for antibody recognition. In summary, the BCEs that identified in this study could be used to develop diagnostic tools and an epitope-based SVA marker vaccine.

Dominant B cell-T cell epitopes instigated robust immune response in-silico against Scrub Typhus.

Scrub typhus, a potentially life-threatening infectious disease, is attributed to bacteria Orientia tsutsugamushi (O. tsutsugamushi). The transmission of this illness to humans occurs through the bite of infected chiggers, which are the larval forms of mites belonging to the genus Leptotrombidium. In this research, we developed a subunit vaccine specifically designed to target outer membrane proteins. Immunodominant cytotoxic T-lymphocytes (CTLs), B- lymphocytes (BCLs), and major histocompatibility complex (MHC)- II epitopes were identified using machine learning and bioinformatics approaches. These epitopes were arranged in different combinations with the help of suitable linkers like AAY, KK, GPGPG and adjuvant (cholera toxin B) that resulted in a vaccine construct. Physiochemical properties were assessed, where the predicted solubility (0.571) was higher than threshold value. Tertiary structure was predicted using I-TASSER web server and evaluated using Ramachandran plot (94 % residues in most favourable region) and z-score (-6.04), which had shown the structure to have good stability and residue arrangement. Molecular docking with immune receptors, Toll-like receptor (TLR)-2 and -4 showed good residue interaction with 13 and 5 hydrogen bonds respectively. Molecular dynamics simulations of receptor-ligand complex provided the idea about the strong interaction having 1.524751 × 10-5 eigenvalue. Amino acid sequence of vaccine was converted to nucleotide sequence and underwent codon optimization. The optimized codon sequence was used for in-silico cloning, which provided idea about the possibility of synthesis of vaccine using E. coli as host. Overall, this study provided a promising blueprint for a scrub typhus vaccine, although experimental validation is needed for confirmation. Furthermore, it is crucial to acknowledge that while bioinformatics provides valuable insights, in-vitro and in-vivo studies are imperative for a comprehensive evaluation of vaccine candidate. Thus, the integration of computational predictions with empirical research is essential to validate the efficacy, safety, and real-world applicability of the designed vaccine against Scrub Typhus. Nevertheless, the findings are good to carry forward for in-vitro and in-vivo investigations.

A novel chimeric vaccine containing multiple epitopes for simulating robust immune activation against Klebsiella pneumoniae.

BACKGROUND: Due to antibiotic resistance, the Klebsiella genus is linked to morbidity and death, necessitating the development of a universally protective vaccine against Klebsiella pathogens. METHODS: Core sequence analysis prioritized non-redundant host molecules and expected lipid bilayer peptides from fully sequenced Klebsiella genomes. These proteins were refined to identify epitopes, examining their immunogenicity, toxicity, solubility, and interaction with MHC alleles. Epitopes were linked to CPG ODN C274 via EAAAK, HEYGAEALERAG, and GGGS linkers to enhance immunological responses. The vaccine's tertiary structure was modelled and docked with MHC-I and MHC-II. RESULTS: Fifty-five proteins were recognized in the Vaxign collection as having remarkable features. Twenty-three proteins with potential pathogenicity were then identified. Eight options for vaccines emerged after the immunogenicity of proteins was examined. The best antigens were three proteins: MrkD, Iron-regulated lipid membrane polypeptides, and RmpA. These compounds were selected for their sensitivity. The structural protein sequences of K. pneumoniae were utilized to identify seven CTL epitopes, seven HTL epitopes, and seven LBL epitopes, respectively. The produced immunization displayed a stable contact with the receptors, based on molecular dynamic simulations lasting 250 nanoseconds. Intermolecular binding free energies also indicated the dominance of the van der Waals and electrostatic energies. CONCLUSION: In summary, the results of this study might help scientists develop a novel vaccine to prevent K. pneumoniae infections.

Designing of a multi-epitopes based vaccine against Haemophilius parainfluenzae and its validation through integrated computational approaches.

Haemophilus parainfluenzae is a Gram-negative opportunist pathogen within the mucus of the nose and mouth without significant symptoms and has an ability to cause various infections ranging from ear, eye, and sinus to pneumonia. A concerning development is the increasing resistance of H. parainfluenzae to beta-lactam antibiotics, with the potential to cause dental infections or abscesses. The principal objective of this investigation is to utilize bioinformatics and immuno-informatic methodologies in the development of a candidate multi-epitope Vaccine. The investigation focuses on identifying potential epitopes for both B cells (B lymphocytes) and T cells (helper T lymphocytes and cytotoxic T lymphocytes) based on high non-toxic and non-allergenic characteristics. The selection process involves identifying human leukocyte antigen alleles demonstrating strong associations with recognized antigenic and overlapping epitopes. Notably, the chosen alleles aim to provide coverage for 90% of the global population. Multi-epitope constructs were designed by using suitable linker sequences. To enhance the immunological potential, an adjuvant sequence was incorporated using the EAAAK linker. The final vaccine construct, comprising 344 amino acids, was achieved after the addition of adjuvants and linkers. This multi-epitope Vaccine demonstrates notable antigenicity and possesses favorable physiochemical characteristics. The three-dimensional conformation underwent modeling and refinement, validated through in-silico methods. Additionally, a protein-protein molecular docking analysis was conducted to predict effective binding poses between the multi-epitope Vaccine and the Toll-like receptor 4 protein. The Molecular Dynamics (MD) investigation of the docked TLR4-vaccine complex demonstrated consistent stability over the simulation period, primarily attributed to electrostatic energy. The docked complex displayed minimal deformation and enhanced rigidity in the motion of residues during the dynamic simulation. Furthermore, codon translational optimization and computational cloning was performed to ensure the reliability and proper expression of the multi-Epitope Vaccine. It is crucial to emphasize that despite these computational validations, experimental research in the laboratory is imperative to demonstrate the immunogenicity and protective efficacy of the developed vaccine. This would involve practical assessments to ascertain the real-world effectiveness of the multi-epitope Vaccine.

Recombinant linear multiple epitopes of σB protein protect Muscovy ducks against novel duck reovirus infection.

Infection by the novel duck reovirus (NDRV) in ducklings causes high mortality, which leads to substantial economic losses in the duck industry in China. To date, no commercial vaccine is available for this disease. In this study, linear B cell epitopes of the σB protein of the NDRV were predicted and recombinant multiple linear B cell epitopes (MLBEs) were constructed through linkers. The recombinant MLBEs were then expressed and purified. One-day-old Muscovy ducklings were immunized with different doses of MLBEs and challenged with 5 × 104 ELD50 of the virulent CHY strain of NDRV 14 days after immunization. The ducklings vaccinated with 20 and 40 µg of MLBE performed no clinical signs or gross or histopathological lesions, indicating 100% protection against infection. The viral load in the liver and spleens of these birds was significantly lower than that in the control group. Additionally, these ducklings exhibited positive seroconversion at 7 days after vaccination on enzyme-linked immunosorbent assay. These results indicate that MLBE of σB could be used as a candidate for developing vaccines against NDRV infection.

Establishment of an ELISA Based on a Recombinant Antigenic Protein Containing Multiple Prominent Epitopes for Detection of African Swine Fever Virus Antibodies.

African swine fever virus (ASFV) poses a significant threat to the global pig industry, necessitating accurate and efficient diagnostic methods for its infection. Previous studies have often focused on a limited number of epitopes from a few proteins for detecting antibodies against ASFV. Therefore, the current study aimed to use multiple B-cell epitopes in developing an indirect Enzyme-Linked Immunosorbent Assay (ELISA) for enhanced detection of ASFV antibodies. For the expression of recombinant protein, k3 derived from 27 multiple peptides of 11 ASFV proteins, such as p72, pA104R, pB602L, p12, p14.5, p49, pE248R, p30, p54, pp62, and pp220, was used. To confirm the expression of the recombinant protein, we used the Western blotting analysis. The purified recombinant K3 protein served as the antigen in our study, and we employed the indirect ELISA technique to detect anti-ASFV antibodies. The present finding showed that there was no cross-reactivity with antibodies targeting Foot-and-mouth disease virus (FMDV), Porcine circovirus type 2 (PCV2), Pseudorabies virus (PRV), Porcine reproductive and respiratory syndrome virus (PRRSV), and Classical swine fever virus (CSFV). Moreover, the current finding was sensitive enough to find anti-ASFV in serum samples that had been diluted up to 32 times. The test (k3-iELISA) showed diagnostic specificity and sensitivity of 98.41% and 97.40%, respectively. Moreover, during the present investigation, we compared the Ingenasa kit and the k3-iELISA to test clinical pig serum, and the results revealed that there was 99.00% agreement between the two tests, showing good detection capability of the k3-iELISA method. Hence, the current finding showed that the ELISA kit we developed can be used for the rapid detection of ASFV antibodies and used as an alternative during serological investigation of ASF in endemic areas.

Immunogenicity and Protective Efficacy of Psoralen-Inactivated SARS-CoV-2 Vaccine in Nonhuman Primates.

COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly impacted public health and the economy worldwide. Most of the currently licensed COVID-19 vaccines act by inhibiting the receptor-binding function of the SARS-CoV-2 spike protein. The constant emergence of SARS-CoV-2 variants resulting from mutations in the receptor-binding domain (RBD) leads to vaccine immune evasion and underscores the importance of broadly acting COVID-19 vaccines. Inactivated whole virus vaccines can elicit broader immune responses to multiple epitopes of several antigens and help overcome such immune evasions. We prepared a psoralen-inactivated SARS-CoV-2 vaccine (SARS-CoV-2 PsIV) and evaluated its immunogenicity and efficacy in nonhuman primates (NHPs) when administered with the Advax-CpG adjuvant. We also evaluated the SARS-CoV-2 PsIV as a booster shot in animals vaccinated with a DNA vaccine that can express the full-length spike protein. The Advax-CpG-adjuvanted SARS-CoV-2 PsIV elicited a dose-dependent neutralizing antibody response in the NHPs, as measured using a serum microneutralization assay against the SARS-CoV-2 Washington strain and the Delta variant. The animals vaccinated with the DNA vaccine followed by a boosting dose of the SARS-CoV-2 PsIV exhibited the highest neutralizing antibody responses and were able to quickly clear infection after an intranasal challenge with the SARS-CoV-2 Delta variant. Overall, the data show that the Advax-CpG-adjuvanted SARS-CoV-2 PsIV, either by itself or as a booster shot following nucleic acid (NA) vaccines, has the potential to protect against emerging variants.

Exploring the S Protein of SARS-CoV-2 to Design a Novel Multi-Epitope Vaccine against COVID-19 Based on Immunoinformatics Approaches.

BACKGROUND: Developing a novel COVID-19 multi-epitope vaccine (CoVMEV) is essential to containing the SARS-CoV-2 pandemic. METHODS: The virus's immunodominant B and T cell epitopes from the S protein were found and joined to create the CoVMEV. Bioinformatics techniques were used to investigate the secondary and tertiary structures, as well as the physical and chemical properties of CoVMEV. RESULTS: CoVMEV exhibited high antigenicity and immunogenicity scores, together with good water solubility and stability. Toll-like receptor 2 (TLR2) and toll-like receptor4 (TLR4), which are critical in triggering immunological responses, were also strongly favoured by CoVMEV. Molecular dynamics simulation and immune stimulation studies revealed that CoVMEV effectively activated T and B lymphocytes, and increased the number of active CD8+ T cells than similar vaccines. CONCLUSION: CoVMEV holds promise as a potential vaccine candidate for COVID-19, given its robust immunogenicity, stability, antigenicity, and capacity to stimulate a strong immune response. This study presents a significant design concept for the development of peptidyl vaccines targeting SARS-CoV-2. Further investigation and clinical trials will be crucial in assessing the efficacy and safety of CoVMEV as a potential vaccine for COVID-19.

A novel Candida albicans Als3, Hwp1 and Met6 derived complex peptide protects mice against hematogenously induced candidiasis.

Candida albicans can cause superficial or systemic infections in humans, particularly in immunocompromised individuals. Vaccination strategies targeting specific antigens of C. albicans have shown promise in providing protection against invasive candidiasis. This study aimed to evaluate the immuno-protective capacity of a KLH conjugated complex peptide, 3P-KLH, containing epitopes from C. albicans antigens Als3, Hwp1, and Met6 in a murine model of hematogenously induced candidiasis. Mice immunized with 3P-KLH raised a specific antibody response, and protection against C. albicans infection was assessed. Immunized mice exhibited significantly lower fungal load in their kidneys compared to the control group. Moreover, 37.5 % of immunized mice survived 21 days after the infection, while all control animals died within the first nine days. These findings suggest that the 3P-KLH complex peptide, targeting C. albicans key antigens, elicits a protective immune response and reduces the severity of systemic Candida infection. In addition, the high binding affinity of the selected epitopes with MHC II alleles further supports the potential immunogenicity of this peptide in humans. This research provides insights into the development of novel immunotherapeutic approaches against invasive candidiasis.

Technical pitfalls when collecting, cryopreserving, thawing, and stimulating human T-cells.

The collection, cryopreservation, thawing, and culture of peripheral blood mononuclear cells (PBMCs) can profoundly influence T cell viability and immunogenicity. Gold-standard PBMC processing protocols have been developed by the Office of HIV/AIDS Network Coordination (HANC); however, these protocols are not universally observed. Herein, we have explored the current literature assessing how technical variation during PBMC processing can influence cellular viability and T cell immunogenicity, noting inconsistent findings between many of these studies. Amid the mounting concerns over scientific replicability, there is growing acknowledgement that improved methodological rigour and transparent reporting is required to facilitate independent reproducibility. This review highlights that in human T cell studies, this entails adopting stringent standardised operating procedures (SOPs) for PBMC processing. We specifically propose the use of HANC's Cross-Network PBMC Processing SOP, when collecting and cryopreserving PBMCs, and the HANC member network International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) PBMC Thawing SOP when thawing PBMCs. These stringent and detailed protocols include comprehensive reporting procedures to document unavoidable technical variations, such as delayed processing times. Additionally, we make further standardisation and reporting recommendations to minimise and document variability during this critical experimental period. This review provides a detailed overview of the challenges inherent to a procedure often considered routine, highlighting the importance of carefully considering each aspect of SOPs for PBMC collection, cryopreservation, thawing, and culture to ensure accurate interpretation and comparison between studies.

The impact of SARS-CoV-2 spike mutation on peptide presentation is HLA allomorph-specific.

CD8+ T cells are crucial for viral elimination and recovery from viral infection. Nonetheless, the current understanding of the T cell response to SARS-CoV-2 at the antigen level remains limited. The Spike protein is an external structural protein that is prone to mutations, threatening the efficacy of current vaccines. Therefore, we have characterised the immune response towards the immunogenic Spike-derived peptide (S976-984, VLNDILSRL), restricted to the HLA-A*02:01 molecule, which is mutated in both Alpha (S982A) and Omicron BA.1 (L981F) variants of concern. We determined that the mutation in the Alpha variant (S982A) impacted both the stability and conformation of the peptide, bound to HLA-A*02:01, in comparison to the original S976-984. We identified a longer and overlapping immunogenic peptide (S975-984, SVLNDILSRL) that could be presented by HLA-A*02:01, HLA-A*11:01 and HLA-B*13:01 allomorphs. We showed that S975-specific CD8+ T cells were weakly cross-reactive to the mutant peptides despite their similar conformations when presented by HLA-A*11:01. Altogether, our results show that the impact of SARS-CoV-2 mutations on peptide presentation is HLA allomorph-specific, and that post vaccination there are T cells able to react and cross-react towards the variant of concern peptides.

Repeated human leukocyte antigens eplets, importance of typing the partner.

INTRODUCTION: Antibody-mediated rejection (AMR) is the most common cause of immune-mediated allograft failure after kidney transplant and impacts allograft survival. Previous sensitization is a major risk factor for development of donor specific antibodies (DSA). AMR can have a wide range of clinical features such as impaired kidney function, proteinuria/hypertension or can be subclinical. HLA molecules have specific regions of antigens binding antibodies called epitopes and eplets are considered essential components responsible for immune recognition. We present a patient with subclinical AMR 1 week post transplantation. CASE REPORT: A 48-year-old, caucasian woman with end-stage kidney disease (ESKD) secondary to autosomal dominant polycystic kidney disease (ADPKD) on peritoneal dialysis was registered in deceased donor waitlist. She was a hypersensitized patient from 3 prior pregnancies with a calculated panel reactive antibody of 93,48%. She was transplanted through kidney paired exchange donation with no evidence of DSA pre transplantation. Surgery and post-op were unremarkable with excellent and immediate graft function. Per protocol DSA levels on the 5th day was DR1 of 3300 MFI, with an increase in MFI by day 13 with 7820 MFI and a new B41 1979MFI. Allograft kidney biopsy findings were diagnostic of AMR and she was treated with immunoglobulin and plasmapheresis. As early onset AMR post transplantation was observed an anamnestic response was hypothesized from a previous exposure to allo-HLA. We decided to type her husband, her son's father, which was presented with DSA. Mismatch eplet analysis revealed a shared 41 T and 67LQ eplets between the donor and husband, responsible for the reactivity and new HLA class I B41 and HLA class II DR1 DSA, respectively. DISCUSSION: Shared eplets between the patient husband and donor was responsible for the alloimmune response and early development of DSAs. This case highlights the importance of early monitoring DSA levels in highly sensitized patients after transplant in order to promptly address and lower inflammatory damage. Mismatch eplet analysis can provide a thorough and precise evaluation of immune compatibility providing a useful technique to immune risk stratification, donor selection and post-transplant immunosuppressive therapy and monitoring.

Bioinformatics approach for structure modeling, vaccine design, and molecular docking of Brucella candidate proteins BvrR, OMP25, and OMP31.

Brucellosis is a zoonotic disease with significant economic and healthcare costs. Despite the eradication efforts, the disease persists. Vaccines prevent disease in animals while antibiotics cure humans with limitations. This study aims to design vaccines and drugs for brucellosis in animals and humans, using protein modeling, epitope prediction, and molecular docking of the target proteins (BvrR, OMP25, and OMP31). Tertiary structure models of three target proteins were constructed and assessed using RMSD, TM-score, C-score, Z-score, and ERRAT. The best models selected from AlphaFold and I-TASSER due to their superior performance according to CASP 12 - CASP 15 were chosen for further analysis. The motif analysis of best models using MotifFinder revealed two, five, and five protein binding motifs, however, the Motif Scan identified seven, six, and eight Post-Translational Modification sites (PTMs) in the BvrR, OMP25, and OMP31 proteins, respectively. Dominant B cell epitopes were predicted at (44-63, 85-93, 126-137, 193-205, and 208-237), (26-46, 52-71, 98-114, 142-155, and 183-200), and (29-45, 58-82, 119-142, 177-198, and 222-251) for the three target proteins. Additionally, cytotoxic T lymphocyte epitopes were detected at (173-181, 189-197, and 202-210), (61-69, 91-99, 159-167, and 181-189), and (3-11, 24-32, 167-175, and 216-224), while T helper lymphocyte epitopes were displayed at (39-53, 57-65, 150-158, 163-171), (79-87, 95-108, 115-123, 128-142, and 189-197), and (39-47, 109-123, 216-224, and 245-253), for the respective target protein. Furthermore, structure-based virtual screening of the ZINC and DrugBank databases using the docking MOE program was followed by ADMET analysis. The best five compounds of the ZINC database revealed docking scores ranged from (- 16.8744 to - 15.1922), (- 16.0424 to - 14.1645), and (- 14.7566 to - 13.3222) for the BvrR, OMP25, and OMP31, respectively. These compounds had good ADMET parameters and no cytotoxicity, while DrugBank compounds didn't meet Lipinski's rule criteria. Therefore, the five selected compounds from the ZINC20 databases may fulfill the pharmacokinetics and could be considered lead molecules for potentially inhibiting Brucella's proteins.

Identification of HLA-A*11:01 and A*02:01-Restricted EBV Peptides Using HLA Peptidomics.

Epstein-Barr Virus (EBV) is closely linked to nasopharyngeal carcinoma (NPC), notably prevalent in southern China. Although type II latency of EBV plays a crucial role in the development of NPC, some lytic genes and intermittent reactivation are also critical for viral propagation and tumor progression. Since T cell-mediated immunity is effective in targeted killing of EBV-positive cells, it is important to identify EBV-derived peptides presented by highly prevalent human leukocyte antigen class I (HLA-I) molecules throughout the EBV life cycle. Here, we constructed an EBV-positive NPC cell model to evaluate the presentation of EBV lytic phase peptides on streptavidin-tagged specific HLA-I molecules. Utilizing a mass spectrometry (LC-MS/MS)-based immunopeptidomic approach, we characterized eleven novel EBV peptides as well as two previously identified peptides. Furthermore, we determined these peptides were immunogenic and could stimulate PBMCs from EBV VCA/NA-IgA positive donors in an NPC endemic southern Chinese population. Overall, this work demonstrates that highly prevalent HLA-I-specific EBV peptides can be captured and functionally presented to elicit immune responses in an in vitro model, which provides insight into the epitopes presented during EBV lytic cycle and reactivation. It expands the range of viral targets for potential NPC early diagnosis and treatment.

Regional Variation of the CD4 and CD8 T Cell Epitopes Conserved in Circulating Dengue Viruses and Shared with Potential Vaccine Candidates.

As dengue expands globally and many vaccines are under trials, there is a growing recognition of the need for assessing T cell immunity in addition to assessing the functions of neutralizing antibodies during these endeavors. While several dengue-specific experimentally validated T cell epitopes are known, less is understood about which of these epitopes are conserved among circulating dengue viruses and also shared by potential vaccine candidates. As India emerges as the epicenter of the dengue disease burden and vaccine trials commence in this region, we have here aligned known dengue specific T cell epitopes, reported from other parts of the world with published polyprotein sequences of 107 dengue virus isolates available from India. Of the 1305 CD4 and 584 CD8 epitopes, we found that 24% and 41%, respectively, were conserved universally, whereas 27% and 13% were absent in any viral isolates. With these data, we catalogued epitopes conserved in circulating dengue viruses from India and matched them with each of the six vaccine candidates under consideration (TV003, TDEN, DPIV, CYD-TDV, DENVax and TVDV). Similar analyses with viruses from Thailand, Brazil and Mexico revealed regional overlaps and variations in these patterns. Thus, our study provides detailed and nuanced insights into regional variation that should be considered for itemization of T cell responses during dengue natural infection and vaccine design, testing and evaluation.