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The most fruitful prevention and treatment tools for the COVID-19 pandemic have proven to be vaccines and therapeutic antibodies, which have reduced the spread of the disease to manageable proportions. The search for the most effective antibodies against the widest set of COV-2 variants has required a long time and substantial resources. It would be desirable to have a tool that will enable us to understand the structural basis on which mutants escape at least some of the epitope-bound antibodies, a tool that may substantially reduce the time and resources invested in this effort. In this work, we applied a computational-based tool (employed previously by us to understand COV-2 spike binding to its cognate cell receptor) to the study of the effect of Delta and Omicron mutations on the escape tendencies. Our binding energy predictions agree extremely well with the experimentally observed escape tendencies. They have also allowed us to set forth a structural explanation for the results that could be used for the screening of antibodies. Lastly, our results explain the differences in molecular interactions that govern interaction of the spike variants with the receptor as opposed to those with antibodies.

Inhalation allergies caused by cats and dogs can lead to a range of discomforting symptoms, such as rhinitis and asthma, in humans. With the increasing popularity of and care provided to these companion animals, the allergens they produce pose a growing threat to susceptible patients’ health. Allergens from cats and dogs have emerged as significant risk factors for triggering asthma and allergic rhinitis worldwide; however, there remains a lack of systematic measures aimed at assisting individuals in recognizing and preventing allergies caused by these animals. This review provides comprehensive insights into the classification of cat and dog allergens, along with their pathogenic mechanisms. This study also discusses implementation strategies for prevention and control measures, including physical methods, gene-editing technology, and immunological approaches, as well as potential strategies for enhancing allergen immunotherapy combined with immunoinformatics. Finally, it presents future prospects for the prevention and treatment of human allergies caused by cats and dogs. This review will improve knowledge regarding allergies to cats and dogs while providing insights into potential targets for the development of next-generation treatments.

Despite its medical relevance, there is no commercial vaccine that protects the population at risk from multidrug-resistant (MDR) Klebsiella pneumoniae infections. The availability of massive omic data and novel algorithms may improve antigen selection to develop effective prophylactic strategies. Up to 133 exposed proteins in the core proteomes, between 516 and 8666 genome samples, of the six most relevant MDR clonal groups (CGs) carried conserved B-cell epitopes, suggesting minimized future evasion if utilized for vaccination. Antigens showed a range of epitopicity, functional constraints, and potential side effects. Eleven antigens, including three sugar porins, were represented in all MDR-CGs, constitutively expressed, and showed limited reactivity with gut microbiota. Some of these antigens had important interactomic interactions and may elicit adhesion-neutralizing antibodies. Synergistic bivalent to pentavalent combinations that address expression conditions, interactome location, virulence activities, and clone-specific proteins may overcome the limiting protection of univalent vaccines. The combination of five central antigens accounted for 41% of all non-redundant interacting partners of the antigen dataset. Specific antigen mixtures represented in a few or just one MDR-CG further reduced the chance of microbiota interference. Rational antigen selection schemes facilitate the design of high-coverage and “magic bullet” multivalent vaccines against recalcitrant K. pneumoniae lineages.

Transmissible gastroenteritis virus (TGEV) is an etiological agent of enteric disease that results in high mortality rates in piglets. The economic impact of the virus is considerable, causing significant losses to the pig industry. The development of an efficacious subunit vaccine to provide promising protection against TGEV is of the utmost importance. The viral antigen, spike glycoprotein (S), is widely regarded as one of the most effective antigenic components for vaccine research. In this study, we employed immunoinformatics and molecular dynamics approaches to develop an ‘ideal’ multi-epitope vaccine. Firstly, the dominant, non-toxic, highly antigenic T (Th, CTL) and B cell epitopes predicted from the TGEV S protein were artificially engineered in tandem to design candidate subunit vaccines. Molecular docking and dynamic simulation results demonstrate that it exhibits robust interactions with toll-like receptor 4 (TLR4). Of particular significance was the finding that the vaccine was capable of triggering an immune response in mammals, as evidenced by the immune simulation results. The humoral aspect is typified by elevated levels of IgG and IgM, whereas the cellular immune aspect is capable of eliciting the robust production of interleukins and cytokines (IFN-γ and IL-2). Furthermore, the adoption of E. coli expression systems for the preparation of vaccines will also result in cost savings. This study offers logical guidelines for the development of a secure and efficacious subunit vaccine against TGEV, in addition to providing a novel theoretical foundation and strategy to prevent associated CoV infections.

The African swine fever virus (ASFV) is a large enveloped DNA virus that causes a highly pathogenic hemorrhagic disease in both domestic pigs and wild boars. The ASFV genome contains a double-stranded DNA encoding more than 150 proteins. The ASFV possesses only one protease, pS273R, which is important for virion assembly and host immune evasion. Therefore, the specific monoclonal antibody (mAb) against pS273R is useful for ASFV research. Here, we generated two specific anti-pS273R mAbs named 2F3 and 3C2, both of which were successfully applied for ELISA, Western blotting, and immunofluorescence assays. Further, we showed that both 2F3 and 3C2 mAbs recognize a new epitope of N terminal 1–25 amino acids of pS273R protein, which is highly conserved across different ASFV strains including all genotype I and II strains. Based on the recognized epitope, an indirect ELISA was established and was effective in detecting antibodies during ASFV infection. To conclude, the specific pS273R mAbs and corresponding epitope identified will strongly promote ASFV serological diagnosis and vaccine research.

African swine fever caused by African swine fever virus (ASFV) is an acute, highly contagious swine disease with high mortality. To facilitate effective vaccine development and find more serodiagnostic targets, fully exploring the ASFV antigenic proteins is urgently needed. In this study, the MGF_110-13L was identified as an immunodominant antigen among the seven transmembrane proteins. The main outer-membrane domain of MGF_110-13L was expressed and purified. Two monoclonal antibodies (mAbs; 8C3, and 10E4) against MGF_110-13L were generated. The epitopes of two mAbs were preliminary mapped with the peptide fusion proteins after probing with mAbs by enzyme-linked immunosorbent assay (ELISA) and Western blot. And the two target epitopes were fine-mapped using further truncated peptide fusion protein strategy. Finally, the core sequences of mAbs 8C3 and 10E4 were identified as 48WDCQDGICKNKITESRFIDS67, and 122GDHQQLSIKQ131, respectively. The peptides of epitopes were synthesized and probed with ASFV antibody positive pig sera by a dot blot assay, and the results showed that epitope 10E4 was an antigenic epitope. The epitope 10E4 peptide was further evaluated as a potential antigen for detecting ASFV antibodies. To our knowledge, this is the first report of antigenic epitope information on the antigenic MGF_110-13L protein of ASFV.

Spike protein sequences in SARS-CoV-2 have been employed for vaccine epitopes, but many short constituent sequences (SCSs) in the spike protein are present in the human proteome, suggesting that some anti-spike antibodies induced by infection or vaccination may be autoantibodies against human proteins. To evaluate this possibility of “molecular mimicry” in silico and in vitro, we exhaustively identified common SCSs (cSCSs) found both in spike and human proteins bioinformatically. The commonality of SCSs between the two systems seemed to be coincidental, and only some cSCSs were likely to be relevant to potential self-epitopes based on three-dimensional information. Among three antibodies raised against cSCS-containing spike peptides, only the antibody against EPLDVL showed high affinity for the spike protein and reacted with an EPLDVL-containing peptide from the human unc-80 homolog protein. Western blot analysis revealed that this antibody also reacted with several human proteins expressed mainly in the small intestine, ovary, and stomach. Taken together, these results showed that most cSCSs are likely incapable of inducing autoantibodies but that at least EPLDVL functions as a self-epitope, suggesting a serious possibility of infection-induced or vaccine-induced autoantibodies in humans. High-risk cSCSs, including EPLDVL, should be excluded from vaccine epitopes to prevent potential autoimmune disorders.

Southern Africa Territories 2 (SAT2) foot-and-mouth disease (FMD) has crossed long-standing regional boundaries in recent years and entered the Middle East. However, the existing vaccines offer poor cross-protection against the circulating strains in the field. Therefore, there is an urgent need for an alternative design approach for vaccines in anticipation of a pandemic of SAT2 Foot-and-mouth disease virus (FMDV). The porcine parvovirus (PPV) VP2 protein can embed exogenous epitopes into the four loops on its surface, assemble into virus-like particles (VLPs), and induce antibodies and cytokines to PPV and the exogenous epitope. In this study, chimeric porcine parvovirus VP2 VLPs (chimeric PPV-SAT2-VLPs) expressing the T-and/or B-cell epitopes of the structural protein VP1 of FMDV SAT2 were produced using the recombinant pFastBac™ Dual vector of baculoviruses in Sf9 and HF cells We used the Bac-to-Bac system to construct the recombinant baculoviruses. The VP2-VLP--SAT2 chimeras displayed chimeric T-cell epitope (amino acids 21–40 of VP1) and/or the B-cell epitope (amino acids 135–174) of SAT FMDV VP1 by substitution of the corresponding regions at the N terminus (amino acids 2–23) and/or loop 2 and/or loop 4 of the PPV VP2 protein, respectively. In mice, the chimeric PPV-SAT2-VLPs induced specific antibodies against PPV and the VP1 protein of SAT2 FMDV. The VP2-VLP-SAT2 chimeras induced specific antibodies to PPV and the VP1 protein specific epitopes of FMDV SAT2. In this study, as a proof-of-concept, successfully generated chimeric PPV-VP2 VLPs expressing epitopes of the structural protein VP1 of FMDV SAT2 that has a potential to prevent FMDV SAT2 and PPV infection in pigs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect human cells by first attaching to the ACE-2 receptor via its receptor-binding domain (RBD) in the spike protein. Here, we report the influence of N-glycosylation sites of the RBD and the membrane (M) protein on IgG antibody binding in serum samples from patients infected with the original SARS-CoV-2 strain in Germany. The RBDs of the wildtype, alpha, beta, gamma, and kappa variants expressed in HEK293S GnTI− cells were all N-glycosylated at Asn331, Asn334, Asn343, and Asn360 or Asn370, whereas the M-protein was glycosylated at Asn5. An ELISA using a coated RBD and probed with anti-RBD IgG antibodies gave a sensitivity of 96.3% and a specificity of 100% for the wildtype RBD, while the sensitivity decreased by 5% to 10% for the variants of concern, essentially in the order of appearance. Deglycosylation of the wildtype RBD strongly reduced antibody recognition by ~20%, considering the mean of the absorbances recorded for the ELISA. This effect was even stronger for the unglycosylated RBD expressed in Escherichia coli, suggesting structural changes affecting epitope recognition. Interestingly, the N-glycosylated M-protein expressed in HEK293S GnTI− cells gave good sensitivity (95%), which also decreased to 65% after deglycosylation, and selectivity (100%). In conclusion, N-glycosylation of the M-protein, the RBD, and most likely the spike protein are important for proper antibody binding and immunological assays, whereas the type of N-glycosylation is less relevant.

Acquired Immunodeficiency Syndrome is caused by the Human Immunodeficiency Virus (HIV), and a significant number of fatalities occur annually. There is a dire need to develop an effective vaccine against HIV-1. Understanding the structural proteins of viruses helps in designing a vaccine based on immunogenic peptides. In the current experiment, we identified gp120 epitopes using bioinformatic epitope prediction tools, molecular docking, and MD simulations. The Gb-1 peptide was considered an adjuvant. Consecutive sequences of GTG, GSG, GGTGG, and GGGGS linkers were used to bind the B cell, Cytotoxic T Lymphocytes (CTL), and Helper T Lymphocytes (HTL) epitopes. The final vaccine construct consisted of 315 amino acids and is expected to be a recombinant protein of approximately 35.49 kDa. Based on docking experiments, molecular dynamics simulations, and tertiary structure validation, the analysis of the modeled protein indicates that it possesses a stable structure and can interact with Toll-like receptors. The analysis demonstrates that the proposed vaccine can provoke an immunological response by activating T and B cells, as well as stimulating the release of IgA and IgG antibodies. This vaccine shows potential for HIV-1 prophylaxis. The in-silico design suggests that multiple-epitope constructs can be used as potentially effective immunogens for HIV-1 vaccine development.

Monoclonal antibody (mAb)-based and/or cell-based immunotherapies provide innovative approaches to cancer treatments. However, safety concerns over targeting normal cells expressing reactive antigens still exist. Therefore, the development of cancer-specific mAbs (CasMabs) that recognize cancer-specific antigens with in vivo antitumor efficacy is required to minimize the adverse effects. We previously screened anti-human epidermal growth factor receptor 2 (HER2) mAbs and successfully established a cancer-specific anti-HER2 mAb, H2Mab-250/H2CasMab-2 (IgG1, kappa). In this study, we showed that H2Mab-250 reacted with HER2-positive breast cancer cells but did not show reactivity to normal epithelial cells in flow cytometry. In contrast, a clinically approved anti-HER2 mAb, trastuzumab, recognized both breast cancer and normal epithelial cells. We further compared the affinity, effector activation, and antitumor effect of H2Mab-250 with trastuzumab. The results showed that H2Mab-250 exerted a comparable antitumor effect with trastuzumab in the mouse xenograft models of BT-474 and SK-BR-3, although H2Mab-250 possessed a lower affinity and effector activation than trastuzumab in vitro. H2Mab-250 could contribute to the development of chimeric antigen receptor-T or antibody–drug conjugates without adverse effects for breast cancer therapy.

We describe an investigation using structural mass spectrometry (MS) of the impact of two antibodies, 15497 and 15498, binding the highly flexible SARS-CoV-2 Nsp1 protein. We determined the epitopes and paratopes involved in the antibody–protein interactions by using hydrogen–deuterium exchange MS (HDX-MS). Notably, the Fab (Fragment antigen binding) for antibody 15498 captured a high energy form of the antigen exhibiting significant conformational changes that added flexibility over most of the Nsp1 protein. The Fab for antibody 15497, however, showed usual antigen binding behavior, revealing local changes presumably including the binding site. These findings illustrate an unusual antibody effect on an antigen and are consistent with the dynamic nature of the Nsp1 protein. Our studies suggest that this interaction capitalizes on the high flexibility of Nsp1 to undergo conformational change and be trapped in a higher energy state by binding with a specific antibody.

Parkinson’s disease (PD) is a chronic neurodegenerative disease that affects the central nervous system, specifically the motor system. It is mainly caused by the loss of dopamine due to the accumulation of α-synuclein (α-syn) protein in the striatum and substantia nigra pars compacta (SNpc). Previous studies have reported that immunization may be a potential preventive strategy for neurodegenerative diseases such as Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). Therefore, the aim of the study was to design an α-syn specific epitope vaccine and investigate its effect in PD-related pathophysiology using an α-syn-induced mouse model. We used an in silico model to identify and design a non-toxic α-syn-based peptide epitope vaccine and, to overcome poor immunogenicity, the vaccine was coupled with immunogenic carrier proteins, i.e., ovalbumin (OVA) and keyhole limpet haemocyanin (KLH). Our results showed that vaccinated PD mouse models, especially with vaccines with carrier proteins, improved in motor functions compared with the non-vaccinated PD model. Additionally, the vaccinated groups showed increased immunoglobulin G (IgG) levels in the spleen and plasma as well as decreased interleukin-10 (IL-10) levels in the plasma. Furthermore, vaccinated groups, especially OVA and KLH groups, showed decrease in α-syn levels and increased dopamine-related markers, i.e., tyrosine hydroxylase (TH), vesicle monoamine transporter 2 (VMAT2), and dopamine transporter (DAT), and autophagy activities in the striatum and SNpc. Lastly, our data showed decreased neuroinflammation by reducing the activation of microglia and astrocytes and pro-inflammatory cytokines in the immunized groups, especially with OVA and KLH carrier proteins. Overall, these results suggest that vaccination, especially with immunogenic carrier proteins, is effective in reducing the accumulation of α-syn aggregates in the brain and ameliorate PD-related pathophysiology. Hence, further development of this approach might have a potential role in preventing the development of PD.

We evaluated cellular immune responses induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines in an immunized population based on HLA-E-restricted CD8+ T cell epitope identification. HLA-E-restricted SARS-CoV-2 CD8+ T cell nonamer peptides were predicted with software. An HLA-E-transfected K562 cell binding assay was used to screen for high-affinity peptides. IFN-γ enzyme-linked immunospot assays were used to identify HLA-E-restricted epitopes. An HLA-E/epitope tetramer was employed to detect the frequencies of epitope-specific CD8+ T cells. Four CD8+ T cell epitopes on the spike protein of SARS-CoV-2 restricted by both HLA-E*0101 and E*0103 were identified. HLA-E-restricted epitope-specific IFN-γ-secreting CD8+ T cell responses could be detected in individuals vaccinated with SARS-CoV-2 vaccines. Importantly, the frequencies of epitope-specific CD8+ T cells in Ad5-nCoV vaccinated individuals were higher than in individuals vaccinated with recombinant protein or inactivated vaccines. Moreover, the frequencies of epitope-specific CD8+ T cells could be maintained for at least 120 days after only one dose of Ad5-nCoV vaccine, while the frequencies of epitope-specific CD8+ T cells decreased in individuals after two doses of Ad5-nCoV vaccine. These findings may contribute to a more comprehensive evaluation of the protective effects of vaccines for SARS-CoV-2; meanwhile, they may provide information to characterize HLA-E-restricted CD8+ T cell immunity against SARS-CoV-2 infection.

African swine fever, which is induced by the African swine fever virus (ASFV), poses a significant threat to the global pig industry due to its high lethality in domestic pigs and wild boars. Despite the severity of the disease, there is a lack of effective vaccines and drugs against the ASFV. The p72 protein, constituting 31 to 33% of the total virus particle mass, serves as the primary capsid protein of ASFV. It is a crucial antigen for the development of ASF subunit vaccines and serological diagnostic methods. In this investigation, 27 monoclonal antibodies (mAbs) were generated through mouse immunization with the truncated C-terminal p72 protein expressed by Escherichia coli. Among these, six mAbs exhibited binding to the p72 trimer, with their respective recognized epitopes identified as 542VTAHGINLIDKF553, 568GNAIKTP574, and 584FALKPREEY592. All three epitopes were situated within the interval sequences of functional units of the C-terminal jelly-roll barrel of p72. Notably, two epitopes, 568GNAIKTP574 and 584FALKPREEY592, were internal to the p72 trimer, while the epitope 542VTAHGINLIDKF553 was exposed on the surface of the trimer and consistently conserved across all ASFV genotypes. These findings enhance our comprehension of the antigenic function and structure of the p72 protein, facilitating the utilization of p72 in the development of diagnostic techniques for ASFV.

Hemolysin II (HlyII)—one of the pathogenic factors of Bacillus cereus, a pore-forming β-barrel toxin—possesses a C-terminal extension of 94 amino acid residues, designated as the C-terminal domain of HlyII (HlyIICTD), which plays an important role in the functioning of the toxin. Our previous work described a monoclonal antibody (HlyIIC-20), capable of strain-specific inhibition of hemolysis caused by HlyII, and demonstrated the dependence of the efficiency of hemolysis on the presence of proline at position 324 in HlyII outside the conformational antigenic determinant. In this work, we studied 16 mutant forms of HlyIICTD. Each of the mutations, obtained via multiple site-directed mutagenesis leading to the replacement of amino acid residues lying on the surface of the 3D structure of HlyIICTD, led to a decrease in the interaction of HlyIIC-20 with the mutant form of the protein. Changes in epitope structure confirm the high conformational mobility of HlyIICTD required for the functioning of HlyII. Comparison of the effect of the introduced mutations on the effectiveness of interactions between HlyIICTD and HlyIIC-20 and a control antibody recognizing a non-overlapping epitope enabled the identification of the amino acid residues N339 and K340, included in the conformational antigenic determinant recognized by HlyIIC-20.

The avian coronavirus, infectious bronchitis virus (IBV), is an economically important infectious disease affecting chickens, with a diverse range of serotypes found globally. The major surface protein, spike (S), has high diversity between serotypes, and amino acid differences in the S1 sub-unit are thought to be responsible for poor cross-protection afforded by vaccination. Here, we attempt to address this, by using epitope mapping technology to identify shared and serotype-specific immunogenic epitopes of the S glycoprotein of three major circulating strains of IBV, M41, QX, and 4/91, via CLIPS peptide arrays based on peptides from the S1 sub-units. The arrays were screened with sera from chickens immunised with recombinant IBV, based on Beau-R backbone expressing heterologous S, generated in two independent vaccination/challenge trials. The screening of sera from rIBV vaccination experiments led to the identification of 52 immunogenic epitopes on the S1 of M41, QX, and 4/91. The epitopes were assigned into six overlapping epitope binding regions. Based on accessibility and location in the hypervariable regions of S, three sequences, 25YVYYYQSAFRPPNGWHLQGGAYAVVNSTN54, 67TVGVIKDVYNQSVASI82, and 83AMTVPPAGMSWSVS96, were selected for further investigation, and synthetic peptide mimics were recognised by polyclonal sera. These epitopes may have the potential to contribute towards a broader cross-protective IBV vaccine.

(1) Background: Renal transplantation (KT) is the most efficient treatment for chronic kidney disease among pediatric patients. Antigenic matching and epitopic load should be the main criteria for choosing a renal graft in pediatric transplantation. Our study aims to compare the integration of new histocompatibility predictive algorithms with classical human leukocyte antigen (HLA) matching regarding different types of pediatric renal transplants. (2) Methods: We categorized our cohort of pediatric patients depending on their risk level, type of donor and type of transplantation, delving into discussions surrounding their mismatching values in relation to both the human leukocyte antigen Matchmaker software (versions 4.0. and 3.1.) and the most recent version of the predicted indirectly identifiable HLA epitopes (PIRCHE) II score. (3) Results: We determined that the higher the antigen mismatch, the higher the epitopic load for both algorithms. The HLAMatchmaker algorithm reveals a noticeable difference in eplet load between living and deceased donors, whereas PIRCHE II does not show the same distinction. Dialysis recipients have a higher count of eplet mismatches, which demonstrates a significant difference according to the transplantation type. Our results are similar to those of four similar studies available in the current literature. (4) Conclusions: We suggest that an integrated data approach employing PIRCHE II and HLAMatchmaker algorithms better predicts histocompatibility in KT than classical HLA matching.

Recent progress in epitope prediction has shown promising results in the development of vaccines and therapeutics against various diseases. However, the overall accuracy and success rate need to be improved greatly to gain practical application significance, especially conformational epitope prediction. In this review, we examined the general features of antibody–antigen recognition, highlighting the conformation selection mechanism in flexible antibody–antigen binding. We recently highlighted the success and warning signs of antibody epitope predictions, including linear and conformation epitope predictions. While deep learning-based models gradually outperform traditional feature-based machine learning, sequence and structure features still provide insight into antibody–antigen recognition problems.

Canine parvovirus (CPV-2) is one of the most important pathogens of dogs of all ages, causing pandemic infections that are characterized by fatal hemorrhagic enteritis. The CPV-2 vaccine is recommended as a core vaccine for pet animals. Despite the intensive practice of active immunization, CPV-2 remains a global threat. In this study, a multi-epitope vaccine against CPV-2 was designed, targeting the highly conserved capsid protein (VP2) via in silico approaches. Several immunoinformatics methods, such as epitope screening, molecular docking, and simulation were used to design a potential vaccine construct. The partial protein sequences of the VP2 gene of CPV-2 and protein sequences retrieved from the NCBI were screened to predict highly antigenic proteins through antigenicity, trans-membrane-topology screening, an allergenicity assessment, and a toxicity analysis. Homologous VP2 protein sequences typically linked to the disease were identified using NCBI BLAST, in which four conserved regions were preferred. Overall, 10 epitopes, DPIGGKTGI, KEFDTDLKP, GTDPDDVQ, GGTNFGYIG, GTFYFDCKP, NRALGLPP, SGTPTN, LGLPPFLNSL, IGGKTG, and VPPVYPN, were selected from the conserved regions to design the vaccine construct. The molecular docking demonstrated the higher binding affinity of these epitopes with dog leukocyte antigen (DLA) molecules. The selected epitopes were linked with Salmonella enterica flagellin FliC adjuvants, along with the PADRE sequence, by GGS linkers to construct a vaccine candidate with 272 nucleotides. The codon adaptation and in silico cloning showed that the generated vaccine can be expressed by the E. coli strain, K12, and the sequence of the vaccine construct showed no similarities with dog protein. Our results suggest that the vaccine construct might be useful in preventing canine parvoviral enteritis (CPE) in dogs. Further in vitro and in vivo experiments are needed for the validation of the vaccine candidate.

Anti-islet autoantibodies serve as key markers in immune-mediated type 1 diabetes (T1D) and slowly progressive T1D (SPIDDM), also known as latent autoimmune diabetes in adults (LADA). Autoantibodies to insulin (IAA), glutamic acid decarboxylase (GADA), tyrosine phosphatase-like protein IA-2 (IA-2A), and zinc transporter 8 (ZnT8A) are currently employed in the diagnosis, pathological analysis, and prediction of T1D. GADA can also be detected in non-diabetic patients with autoimmune diseases other than T1D and may not necessarily reflect insulitis. Conversely, IA-2A and ZnT8A serve as surrogate markers of pancreatic β-cell destruction. A combinatorial analysis of these four anti-islet autoantibodies demonstrated that 93–96% of acute-onset T1D and SPIDDM cases were diagnosed as immune-mediated T1D, while the majority of fulminant T1D cases were autoantibody-negative. Evaluating the epitopes and immunoglobulin subclasses of anti-islet autoantibodies help distinguish between diabetes-associated and non-diabetes-associated autoantibodies and is valuable for predicting future insulin deficiency in SPIDDM (LADA) patients. Additionally, GADA in T1D patients with autoimmune thyroid disease reveals the polyclonal expansion of autoantibody epitopes and immunoglobulin subclasses. Recent advancements in anti-islet autoantibody assays include nonradioactive fluid-phase assays and the simultaneous determination of multiple biochemically defined autoantibodies. Developing a high-throughput assay for detecting epitope-specific or immunoglobulin isotype-specific autoantibodies will facilitate a more accurate diagnosis and prediction of autoimmune disorders. The aim of this review is to summarize what is known about the clinical significance of anti-islet autoantibodies in the pathogenesis and diagnosis of T1D.

The African swine fever virus (ASFV) causes high mortality in domestic pigs. ASFV encodes an important protein target for subunit vaccine development, CD2v, but its most effective immunological regions are not known. Herein, we generated a monoclonal antibody (mAb) named IF3 by immunizing mice against the intracellular region of the CD2v protein (CD2v-IR). 1F3 specifically recognized CD2v, which is expressed transiently in transfected Sf9 cells and also in inactivated ASFV-infected porcine alveolar macrophage (PAM) cells. The epitope recognized by 1F3 is 264EPSPREP270, which is highly conserved in ASFV genotypes. Immunization of mice with this epitope elicited an increased IgG response, including IgG1 and IgG2a subtypes, and also increased CD8+ T cells and cytokine expression. Overall, these results indicate that this epitope induces both humoral and cellular immune responses that may be used for ASFV-related subunit vaccine design and development.

CD59 is an abundant immuno-regulatory human protein that protects cells from damage by inhibiting the complement system. CD59 inhibits the assembly of the Membrane Attack Complex (MAC), the bactericidal pore-forming toxin of the innate immune system. In addition, several pathogenic viruses, including HIV-1, escape complement-mediated virolysis by incorporating this complement inhibitor in their own viral envelope. This makes human pathogenic viruses, such as HIV-1, not neutralised by the complement in human fluids. CD59 is also overexpressed in several cancer cells to resist the complement attack. Consistent with its importance as a therapeutical target, CD59-targeting antibodies have been proven to be successful in hindering HIV-1 growth and counteracting the effect of complement inhibition by specific cancer cells. In this work, we make use of bioinformatics and computational tools to identify CD59 interactions with blocking antibodies and to describe molecular details of the paratope–epitope interface. Based on this information, we design and produce paratope-mimicking bicyclic peptides able to target CD59. Our results set the basis for the development of antibody-mimicking small molecules targeting CD59 with potential therapeutic interest as complement activators.

CC chemokine receptor 6 (CCR6) is one of the members of the G-protein-coupled receptor (GPCR) family that is upregulated in many immune-related cells, such as B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. The coordination between CCR6 and its ligand CC motif chemokine ligand 20 (CCL20) is deeply involved in the pathogenesis of various diseases, such as cancer, psoriasis, and autoimmune diseases. Thus, CCR6 is an attractive target for therapy and is being investigated as a diagnostic marker for various diseases. In a previous study, we developed an anti-mouse CCR6 (mCCR6) monoclonal antibody (mAb), C6Mab-13 (rat IgG1, kappa), that was applicable for flow cytometry by immunizing a rat with the N-terminal peptide of mCCR6. In this study, we investigated the binding epitope of C6Mab-13 using an enzyme-linked immunosorbent assay (ELISA) and the surface plasmon resonance (SPR) method, which were conducted with respect to the synthesized point-mutated-peptides within the 1–20 amino acid region of mCCR6. In the ELISA results, C6Mab-13 lost its ability to react to the alanine-substituted peptide of mCCR6 at Asp11, thereby identifying Asp11 as the epitope of C6Mab-13. In our SPR analysis, the dissociation constants (KD) could not be calculated for the G9A and D11A mutants due to the lack of binding. The SPR analysis demonstrated that the C6Mab-13 epitope comprises Gly9 and Asp11. Taken together, the key binding epitope of C6Mab-13 was determined to be located around Asp11 on mCCR6. Based on the epitope information, C6Mab-13 could be useful for further functional analysis of mCCR6 in future studies.

Porcine circovirus disease (PCVD) caused by porcine circovirus type 2 (PCV2) is widely distributed in pig farms. Up until now, nine genotypes of PCV2, PCV2a to 2i, have been identified in diseased pigs worldwide. This study analyzed 302 samples collected in the Jilin Province of China from 2016 to 2021, followed by genetic analysis of the PCV2 isolates. Meanwhile, the antigen epitopes, amino acid mutations, 3D structure of the PCV2 isolates and commercially available vaccine strains were evaluated and compared. The results showed that the predominant genotypes of PCV2 were PCV2b, followed by PCV2e and PCV2d in Jilin Province during 2016–2021. Although mutations were detected in the isolates, no recombination occurred in the PCV2 isolates, indicating a stable genotype of PCV2 in Jilin Province during these years. Moreover, the B cell epitopes in the Cap and Rep proteins of eighteen PCV2 isolates and T cell epitopes in the Cap of the isolates were changed compared to three currently used vaccine strains. The mutations in the Cap and Rep proteins did not affect their spatial conformation. Therefore, bivalent or multivalent vaccines with different genotypes of PCV2 might improve the protective effect of vaccines.

Cow’s milk protein allergy is one of the most common pediatric food allergies. It poses a significant socioeconomic burden in industrialized countries and has a profound effect on the quality of life of affected individuals and their families. Diverse immunologic pathways can lead to the clinical symptoms of cow’s milk protein allergy; some of the pathomechanisms are known in detail, but others need further elucidation. A comprehensive understanding of the development of food allergies and the features of oral tolerance could have the potential to unlock more precise diagnostic tools and novel therapeutic approaches for patients with cow’s milk protein allergy.

Millions of people have been vaccinated with Gam-COVID-Vac but fine specificities of induced antibodies have not been fully studied. Plasma from 12 naïve and 10 coronavirus disease 2019 (COVID-19) convalescent subjects was obtained before and after two immunizations with Gam-COVID-Vac. Antibody reactivity in the plasma samples (n = 44) was studied on a panel of micro-arrayed recombinant folded and unfolded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and 46 peptides spanning the spike protein (S) and by immunoglobulin G (IgG) subclass enzyme-linked immunosorbent assay (ELISA). The ability of Gam-COVID-Vac-induced antibodies to inhibit binding of the receptor-binding domain (RBD) to its receptor angiotensin converting enzyme 2 (ACE2) was investigated in a molecular interaction assay (MIA). The virus-neutralizing capacity of antibodies was studied by the pseudo-typed virus neutralization test (pVNT) for Wuhan-Hu-1 and Omicron. We found that Gam-COVID-Vac vaccination induced significant increases of IgG1 but not of other IgG subclasses against folded S, spike protein subunit 1 (S1), spike protein subunit 2 (S2), and RBD in a comparable manner in naïve and convalescent subjects. Virus neutralization was highly correlated with vaccination-induced antibodies specific for folded RBD and a novel peptide (i.e., peptide 12). Peptide 12 was located close to RBD in the N-terminal part of S1 and may potentially be involved in the transition of the pre- to post-fusion conformation of the spike protein. In summary, Gam-COVID-Vac vaccination induced S-specific IgG1 antibodies in naive and convalescent subjects in a comparable manner. Besides the antibodies specific for RBD, the antibodies induced against a peptide close to the N-terminus of RBD were also associated with virus-neutralization.

T-cell immunity plays an important role in the control of SARS-CoV-2 and has a great cross-protective effect on the variants. The Omicron BA.1 variant contains more than 30 mutations in the spike and severely evades humoral immunity. To understand how Omicron BA.1 spike mutations affect cellular immunity, the T-cell epitopes of SARS-CoV-2 wild-type and Omicron BA.1 spike in BALB/c (H-2d) and C57BL/6 mice (H-2b) were mapped through IFNγ ELISpot and intracellular cytokine staining assays. The epitopes were identified and verified in splenocytes from mice vaccinated with the adenovirus type 5 vector encoding the homologous spike, and the positive peptides involved in spike mutations were tested against wide-type and Omicron BA.1 vaccines. A total of eleven T-cell epitopes of wild-type and Omicron BA.1 spike were identified in BALB/c mice, and nine were identified in C57BL/6 mice, only two of which were CD4+ T-cell epitopes and most of which were CD8+ T-cell epitopes. The A67V and Del 69-70 mutations in Omicron BA.1 spike abolished one epitope in wild-type spike, and the T478K, E484A, Q493R, G496S and H655Y mutations resulted in three new epitopes in Omicron BA.1 spike, while the Y505H mutation did not affect the epitope. These data describe the difference of T-cell epitopes in SARS-CoV-2 wild-type and Omicron BA.1 spike in H-2b and H-2d mice, providing a better understanding of the effects of Omicron BA.1 spike mutations on cellular immunity.

Molecularly imprinted polymers (MIPs) are really interesting for nanomedicine. To be suitable for such application, they need to be small, stable in aqueous media and sometimes fluorescent for bioimaging. We report herein, the facile synthesis of fluorescent, small (below 200 nm), water-soluble and water-stable MIP capable of specific and selective recognition of their target epitope (small part of a protein). To synthesize these materials, we used dithiocarbamate-based photoiniferter polymerization in water. The use of a rhodamine-based monomer makes the resulting polymers fluorescent. Isothermal titration calorimetry (ITC) is used to determine the affinity as well as the selectivity of the MIP for its imprinted epitope, according to the significant differences observed when comparing the binding enthalpy of the original epitope with that of other peptides. The toxicity of the nanoparticles is also tested in two breast cancer cell lines to show the possible use of these particle for future in vivo applications. The materials demonstrated a high specificity and selectivity for the imprinted epitope, with a Kd value comparable with the affinity values of antibodies. The synthesized MIP are not toxic, which makes them suitable for nanomedicine.

Clostridioides difficile (CD) is a Gram-positive, anaerobic bacterium that infects mainly hospitalized and elderly people who have been treated with long-term antibiotic therapy leading to dysbiosis. The deteriorating demographic structure and the increase in the number of antibiotics used indicate that the problem of CD infections (CDI) will continue to increase. Thus far, there is no vaccine against CD on the market. Unfortunately, clinical trials conducted using the CD toxin-based antigens did not show sufficiently high efficacy, because they did not prevent colonization and transmission between patients. It seems that the vaccine should also include antigens found in the bacterium itself or its spores in order not only to fight the effects of toxins but also to prevent the colonization of the patient. This literature review summarizes the latest advances in research into vaccine antigens that do not contain CD toxins.

The analysis of T-cell responses in HIV-1-infected controllers may contribute to a better understanding of the protective components of the immune system. Here, we analyzed the HIV-1-specific T-cell response in a 59-year-old HIV-1-infected controller, infected for at least seven years, who presented with low viral loads ranging from 800 cells/µL. In γ-IFN-ELISpot assays using freshly isolated PBMCs, he displayed a very strong polyclonal T-cell response to eight epitopes in Gag, Nef and Rev; with the dominant responses directed against the HLA-B*57-epitope AISPRTLNAW and against a so-far-unknown epitope within Rev. Further analyses using peptide-stimulated T-cell lines in γ-IFN-ELISpot assays delineated the peptide RQRQIRSI (Rev-RI8) as a newly defined HLA-B*52-restricted epitope located within a functionally important region of Rev. Peptide-stimulation assays in 15 HLA-B*52-positive HIV-1-infected subjects, including the controller, demonstrated recognition of the Rev-RI8 epitope in 6/15 subjects. CD4 counts before the start of antiviral therapy were significantly higher in subjects with recognition of the Rev-RI8 epitope. Targeting of the Rev-RI8 epitope in Rev by CTL could contribute to the positive association of HLA-B*52 with a more favorable course of HIV-1-infection.

African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs of all breeds and ages, caused by African swine fever virus (ASFV). Due to the absence of a safe and efficacious vaccine, accurate laboratory diagnosis is critical for the control of ASF prevention. The p30 protein is immunogenic and stimulates a high level of antibody response to ASFV infection. We developed a panel of 4 monoclonal antibodies (mAbs) against p30 protein, and mAb-2B4 showed the highest percent of inhibition (PI) of 70% in the solid phase blocking ELISA (bELISA). Epitope mapping revealed the mAb-2B4 recognized the epitope of aa 12–18 of p30, which is conserved among various ASFV genotypes. Subsequently, a competitive enzyme-linked immunosorbent assay (cELISA) was established using HRP-labeled mAb-2B4. The cutoff for discrimination between 98 negative sera and 40 positive sera against ASFV was determined by plotting a receiver operating characteristic (ROC) curve. It yielded the area under the curve (AUC) of 0.998, and a diagnostic specificity of 97.96% and a sensitivity of 97.5% were achieved when the cutoff value was determined at 37.1%. Furthermore, the results showed an excellent repeatability of the established cELISA and no cross-reaction to antisera against six other pig pathogens. Additionally, the cELISA detected a titer of 1:256 in the positive standard serum. Overall, mAb-2B4 showed a conserved epitope and high ability to be inhibited by positive sera in ASFV antibody detection. The cELISA based on HRP-labeled mAb-2B4 offers an alternative to other assays for a broader diagnostic coverage of ASFV infection.

This study investigated the IgG and IgA antibody response against recombinant S1 and receptor binding domains (RBD) of the spike (S-) protein and the membrane (M-) protein using a set of 115 serum samples collected from patients infected with SARS-CoV-2 in Germany before April 2021 using protein and peptide ELISA. As S1- and RBD-proteins expressed in Escherichia coli provided poor sensitivities in ELISA, they were replaced by proteins expressed in HEK cells. The RBD-ELISA provided a sensitivity of 90.6% (N = 85) for samples collected from patients with confirmed SARS-CoV-2 infections more than 14 days after symptom onset or a positive PCR test. In population-based controls, the specificity was 97.9% (N = 94). In contrast, the sensitivities were only 41.2% and 72.6% for M- and N-proteins, respectively, while the specificities were 88.5% and 100%, respectively. Considering also 20 samples collected during the first two weeks of symptom onset or PCR confirmation, the sensitivity of RBD- and N-protein ELISA decreased to 82.6% and 72.6%, respectively. The combination of two data sets, i.e., N- and RBD-, N- and M-, or RBD- and M-proteins increased the sensitivity to 85.8%, 77.9%, and 87.8%, respectively. Peptide mapping mostly confirmed epitopes previously reported for S1- and M-proteins, but they were only recognized by a few samples already tested positive in the corresponding protein ELISA indicating that peptide-based assays will not improve the diagnostic sensitivity.

Foot-and-mouth disease virus (FMDV) poses a significant threat to the livestock industry. Through their recognition of the conserved epitopes presented by the swine leukocyte antigen (SLA), T cells play a pivotal role in the antiviral immunity of pigs. Herein, based on the peptide binding motif of SLA-2*HB01, from an original SLA-2 allele, a series of functional T-cell epitopes derived from the dominant antigen VP1 of FMDV with high binding capacity to SLA-2 were identified. Two parallel peptides, Hu64 and As64, from the O and Asia I serotypes, respectively, were both crystallized with SLA-2*HB01. Compared to SLA-1 and SLA-3, the SLA-2 structures showed the flexibility of residues in the P4, P6, and P8 positions and in their potential interface with TCR. Notably, the peptides Hu64 and As64 adopted quite similar overall conformation when bound to SLA-2*HB01. Hu64 has two different conformations, a more stable ‘chair’ conformation and an unstable ‘boat’ conformation observed in the two molecules of one asymmetric unit, whereas only a single ‘chair’ conformation was observed for As64. Both Hu64 and As64 could induce similar dominant T-cell activities. Our interdisciplinary study establishes a basis for the in-depth interpretation of the peptide presentation of SLA-I, which can be used toward the development of universal vaccines.

Several human monoclonal Abs for treating Influenza have been evaluated in clinical trials with limited success despite demonstrating superiority in preclinical animal models including mice. To conduct efficacy studies in mice, human monoclonal Abs are genetically engineered to contain mouse heavy chain constant domain to facilitate the engagement of Fc-receptors on mouse immune effector cells. Although studies have consistently reported discrepancies in Ab effectiveness following genetic engineering, the structural and mechanistic basis for these inconsistencies remain uncharacterized. Here, we use homology modeling to predict variable region (VR) analogous monoclonal Abs possessing human IgG1, mouse IgG1, and mouse IgG2a heavy chain constant domains. We then examine predicted 3D structures for variations in the spatial location and orientation of corresponding paratope amino acid residues. By structurally aligning crystal structures of Fabs in complex with hemagglutinin (HA), we show that corresponding paratope amino acid residues for VR-analogous human IgG1, mouse IgG1, and mouse IgG2a monoclonal Abs interact differentially with HA suggesting that their epitopes might not be identical. To demonstrate that variations in the paratope 3D fine architecture have implications for Ab specificity and effectiveness, we genetically engineered VR-analogous human IgG1, human IgG4, mouse IgG1, and mouse IgG2a monoclonal Abs and explored their specificity and effectiveness in protecting MDCK cells from infection by pandemic H1N1 and H3N2 Influenza viruses. We found that VR-analogous monoclonal Abs placed on mouse heavy chain constant domains were more efficacious at protecting MDCK cells from Influenza virus infection relative to those on human heavy chain constant domains. Interestingly, mouse but not human heavy chain constant domains increased target breadth in some monoclonal Abs. These data suggest that heavy chain constant domain sequences play a role in shaping Ab repertoires that go beyond class or sub-class differences in immune effector recruitment. This represents a facet of Ab biology that can potentially be exploited to improve the scope and utilization of current therapeutic or prophylactic candidates for influenza.

Since 2018, the outbreak and prevalence of the African swine fever virus (ASFV) in China have caused huge economic losses. Less virulent ASFVs emerged in 2020, which led to difficulties and challenges for early diagnosis and control of African swine fever (ASF) in China. An effective method of monitoring ASFV antibodies and specific antibodies against ASFV to promote the development of prevention techniques are urgently needed. In the present study, ASFV p17 was successfully expressed in CHO cells using a suspension culture system. An indirect enzyme-linked immunosorbent assay (ELISA) based on purified p17 was established and optimized. The monoclonal antibody (mAb) against p17 recognized a conservative linear epitope (3TETSPLLSH11) and exhibited specific reactivity, which was conducive to the identification of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) expressing p17. The ELISA method efficiently detected clinical ASFV infection and effectively monitored the antibody levels in vivo after recombinant PRRSV live vector virus expressing p17 vaccination. Overall, the determination of the conserved linear epitope of p17 would contribute to the in-depth exploration of the biological function of the ASFV antigen protein. The indirect ELISA method and mAb against ASFV p17 revealed efficient detection and promising application prospects, making them ideal for epidemiological surveillance and vaccine research on ASF.

Vaccines are an upcoming medical intervention for breast cancer. By targeting the tumor antigen, cancer vaccines can be designed to train the immune system to recognize tumor cells. Therefore, along with technological advances, the vaccine design process is now starting to be carried out with more rational methods such as designing epitope-based peptide vaccines using immunoinformatics methods. Immunoinformatics methods can assist vaccine design in terms of antigenicity and safety. Common protocols used to design epitope-based peptide vaccines include tumor antigen identification, protein structure analysis, T cell epitope prediction, epitope characterization, and evaluation of protein–epitope interactions. Tumor antigen can be divided into two types: tumor associated antigen and tumor specific antigen. We will discuss the identification of tumor antigens using high-throughput technologies. Protein structure analysis comprises the physiochemical, hydrochemical, and antigenicity of the protein. T cell epitope prediction models are widely available with various prediction parameters as well as filtering tools for the prediction results. Epitope characterization such as allergenicity and toxicity can be done in silico as well using allergenicity and toxicity predictors. Evaluation of protein–epitope interactions can also be carried out in silico with molecular simulation. We will also discuss current and future developments of breast cancer vaccines using an immunoinformatics approach. Finally, although prediction models have high accuracy, the opposite can happen after being tested in vitro and in vivo. Therefore, further studies are needed to ensure the effectiveness of the vaccine to be developed. Although epitope-based peptide vaccines have the disadvantage of low immunogenicity, the addition of adjuvants can be a solution.

Anaplasma phagocytophilum Major surface protein 4 (MSP4) plays a role during infection and multiplication in host neutrophils and tick vector cells. Recently, vaccination trials with the A. phagocytophilum antigen MSP4 in sheep showed only partial protection against pathogen infection. However, in rabbits immunized with MSP4, this recombinant antigen was protective. Differences between rabbit and sheep antibody responses are probably associated with the recognition of non-protective epitopes by IgG of immunized lambs. To address this question, we applied quantum vaccinomics to identify and characterize MSP4 protective epitopes by a microarray epitope mapping using sera from vaccinated rabbits and sheep. The identified candidate protective epitopes or immunological quantum were used for the design and production of a chimeric protective antigen. Inhibition assays of A. phagocytophilum infection in human HL60 and Ixodes scapularis tick ISE6 cells evidenced protection by IgG from sheep and rabbits immunized with the chimeric antigen. These results supported that the design of new chimeric candidate protective antigens using quantum vaccinomics to improve the protective capacity of antigens in multiple hosts.

Outbreaks of monkeypox virus infections have imposed major health concerns worldwide, with high morbidity threats to children and immunocompromised adults. Although repurposed drugs and vaccines are being used to curb the disease, the evolving traits of the virus, exhibiting considerable genetic dynamicity, challenge the limits of a targeted treatment. A pan-genome-based reverse vaccinology approach can provide fast and efficient solutions to resolve persistent inconveniences in experimental vaccine design during an outbreak-exigency. The approach encompassed screening of available monkeypox whole genomes (n = 910) to identify viral targets. From 102 screened viral targets, viral proteins L5L, A28, and L5 were finalized based on their location, solubility, and antigenicity. The potential T-cell and B-cell epitopes were extracted from the proteins using immunoinformatics tools and algorithms. Multiple vaccine constructs were designed by combining the epitopes. Based on immunological properties, chemical stability, and structural quality, a novel multi-epitopic vaccine construct, V4, was finalized. Flexible-docking and coarse-dynamics simulation portrayed that the V4 had high binding affinity towards human HLA-proteins (binding energy < −15.0 kcal/mol) with low conformational fluctuations (

Milk protein hydrolysates are common in infant formula, but some of them retain allergenicity due to incomplete hydrolysis of the epitopes for milk allergens. This study explored a method for screening proteases that could specifically hydrolyze the epitope of αs1-casein allergen. Firstly, the αs1-casein epitope AA83-105 was synthesized by the solid-phase synthesis method. Then, after purification and identification, the complete antigen was prepared through coupling with bovine serum albumin (BSA) and was used to raise monoclonal antibodies (mAb) in BALB/c mice. Additionally, an indirect competitive-enzyme-linked immunosorbent assay (icELISA) was established. The mAb raised against αs1-casein protein was used as a control. The results showed that the purity of the synthetic epitope was >90%, and the coupling rate with BSA was 6.31. The mAb subtype is IgG1, with a titer of 1:320,000. The mAb reacted specifically with αs1-casein but did not cross-react with soybean protein. The linear regression equation of the competitive inhibition curve was y = −9.22x + 100.78 (R2 = 0.9891). The detection limit of icELISA method was more sensitive, and the method showed good accuracy and repeatability. The amounts of antigen residues in papain protease hydrolysates were relatively small, and the epitope fragment was detected in papain hydrolysate via mass spectrometry. This study provides ideas and methods for screening proteases that specifically hydrolyze the epitopes of milk allergens and also provides a superior foundation for the development of an advanced hypoallergenic formula.

Zika virus (ZIKV)-specific T cells are activated by different peptides derived from virus structural and nonstructural proteins, and contributed to the viral clearance or protective immunity. Herein, we have depicted the profile of CD8+ and CD4+ T cell immunogenicity of ZIKV proteins in C57BL/6 (H-2b) and BALB/c (H-2d) mice, and found that featured cellular immunity antigens were variant among different murine alleles. In H-2b mice, the proteins E, NS2, NS3 and NS5 are recognized as immunodominant antigens by CD8+ T cells, while NS4 is dominantly recognized by CD4+ T cells. In contrast, in H-2d mice, NS1 and NS4 are the dominant CD8+ T cell antigen and NS4 as the dominant CD4+ T cell antigen, respectively. Among the synthesized 364 overlapping polypeptides spanning the whole proteome of ZIKV, we mapped 91 and 39 polypeptides which can induce ZIKV-specific T cell responses in H-2b and H-2d mice, respectively. Through the identification of CD8+ T cell epitopes, we found that immunodominant regions E294-302 and NS42351-2360 are hotspots epitopes with a distinct immunodominance hierarchy present in H-2b and H-2d mice, respectively. Our data characterized an overall landscape of the immunogenic spectrum of the ZIKV polyprotein, and provide useful insight into the vaccine development.

After the outbreak of SARS-CoV-2 by the end of 2019, the vaccine development strategies became a worldwide priority. Furthermore, the appearances of novel SARS-CoV-2 variants challenge researchers to develop new pharmacological or preventive strategies. However, vaccines still represent an efficient way to control the SARS-CoV-2 pandemic worldwide. This review describes the importance of bioinformatic and immunoinformatic tools (in silico) for guide vaccine design. In silico strategies permit the identification of epitopes (immunogenic peptides) which could be used as potential vaccines, as well as nonacarriers such as: vector viral based vaccines, RNA-based vaccines and dendrimers through immunoinformatics. Currently, nucleic acid and protein sequential as well structural analyses through bioinformatic tools allow us to get immunogenic epitopes which can induce immune response alone or in complex with nanocarriers. One of the advantages of in silico techniques is that they facilitate the identification of epitopes, while accelerating the process and helping to economize some stages of the development of safe vaccines.

Gallbladder cancer (GBC) is an aggressive and difficult to treat biliary tract carcinoma with a poor survival rate. The aim of this study was to design a peptide-based multi-epitope vaccine construct against GBC using immunoinformatics approaches. Three proteins implicated in the progression of GBC were selected for B and T cell epitope prediction and the designing of the potential vaccine construct. Seven CTL, four HTL and six Bcell epitopes along with a suitable adjuvant were selected and connected using linkers for designing the vaccine construct. The secondary and tertiary models of the designed vaccine were generated and satisfactorily validated. A Ramachandran plot of the final 3D model showed more than 90% of the residues in allowed regions and only 0.4% in disallowed regions. The binding affinity of a vaccine construct with TLR 2, 3 and 4 receptors was assessed through molecular docking and simulation. The average numbers of hydrogen bonds for vaccine-TLR 2, 3 and 4 complexes in the simulation were 15.36, 16.45, and 11.98, respectively, and remained consistent over a 100 ns simulation period, which is critical for their function. The results of this study provide a strong basis for further evaluation through in vitro/in vivo experimental validation of the safety and efficacy of the designed vaccine construct.

Introduction: The ongoing coronavirus disease 2019 (COVID-19), which emerged in December 2019, is a serious health concern throughout the world. Despite massive COVID-19 vaccination on a global scale, there is a rising need to develop more effective vaccines and drugs to curb the spread of coronavirus. Methodology: In this study, we screened the amino acid sequence of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 (the causative agent of COVID-19) for the identification of B and T cell epitopes using various immunoinformatic tools. These identified potent B and T cell epitopes with high antigenicity scores were linked together to design the multi-epitope vaccine construct. The physicochemical properties, overall quality, and stability of the designed vaccine construct were confirmed by suitable bioinformatic tools. Results: After proper in silico prediction and screening, we identified 3 B cell, 18 CTL, and 10 HTL epitopes from the RdRp protein sequence. The screened epitopes were non-toxic, non-allergenic, and highly antigenic in nature as revealed by appropriate servers. Molecular docking revealed stable interactions of the designed multi-epitope vaccine with human TLR3. Moreover, in silico immune simulations showed a substantial immunogenic response of the designed vaccine. Conclusions: These findings suggest that our designed multi-epitope vaccine possessing intrinsic T cell and B cell epitopes with high antigenicity scores could be considered for the ongoing development of peptide-based novel vaccines against COVID-19. However, further in vitro and in vivo studies need to be performed to confirm our in silico observations.

Direct-acting antivirals (DAAs) have recently revolutionized the eradication of chronic hepatitis C virus (HCV) infection. However, the effects of DAAs on the development of hepatocellular carcinoma (HCC) remain unknown. Therefore, the present study aimed to investigate immune responses to HCC influenced by DAAs in HCV-infected patients and elucidate the underlying mechanisms. We compared immune responses to 19 different HCC-related tumor-associated antigen (TAA)-derived peptides and host immune cell profiles before and 24 weeks after a treatment with DAAs in 47 HLA-A24-positive patients. The relationships between the different immune responses and phenotypic changes in immune cells were also examined. The treatment with DAAs induced four types of immune responses to TAAs and markedly altered host immune cell profiles. Prominently, reductions in the frequencies of PD-1+CD4+ and PD-1+CD8+ T cells by DAAs were associated with enhanced immune responses to TAAs. The HCV F protein was identified as contributing to the increased frequency of PD-1+ T cells, which may be decreased after eradication by DAAs. DAAs altered the immune responses of patients to HCC by decreasing the frequency of PD-1-expressing CD4+ and CD8+ T cells.

Epitopes are short amino acid sequences that define the antigen signature to which an antibody or T cell receptor binds. In light of the current pandemic, epitope analysis and prediction are paramount to improving serological testing and developing vaccines. In this paper, known epitope sequences from SARS-CoV, SARS-CoV-2, and other Coronaviridae were leveraged to identify additional antigen regions in 62K SARS-CoV-2 genomes. Additionally, we present epitope distribution across SARS-CoV-2 genomes, locate the most commonly found epitopes, and discuss where epitopes are located on proteins and how epitopes can be grouped into classes. The mutation density of different protein regions is presented using a big data approach. It was observed that there are 112 B cell and 279 T cell conserved epitopes between SARS-CoV-2 and SARS-CoV, with more diverse sequences found in Nucleoprotein and Spike glycoprotein.

Vaccination is an environmentally-friendly alternative for tick control. The tick antigen Subolesin (SUB) has shown protection in vaccines for the control of multiple tick species in cattle. Additionally, recent approaches in quantum vaccinomics have predicted SUB-protective epitopes and the peptide sequences involved in protein–protein interactions in this tick antigen. Therefore, the identification of B-cell–reactive epitopes by epitope mapping using a SUB peptide array could be essential as a novel strategy for vaccine development. Subolesin can be used as a model to evaluate the effectiveness of these approaches for the identification of protective epitopes related to vaccine protection and efficacy. In this study, the mapping of B-cell linear epitopes of SUB from three different tick species common in Uganda (Rhipicephalus appendiculatus, R. decoloratus, and Amblyomma variegatum) was conducted using serum samples from two cattle breeds immunized with SUB-based vaccines. The results showed that in cattle immunized with SUB from R. appendiculatus (SUBra) all the reactive peptides (Z-score > 2) recognized by IgG were also significant (Z-ratio > 1.96) when compared to the control group. Additionally, some of the reactive peptides recognized by IgG from the control group were also recognized in SUB cocktail–immunized groups. As a significant result, cattle groups that showed the highest vaccine efficacy were Bos indicus immunized with a SUB cocktail (92%), and crossbred cattle were immunized with SUBra (90%) against R. appendiculatus ticks; the IgG from these groups recognized overlapping epitopes from the peptide SPTGLSPGLSPVRDQPLFTFRQVGLICERMMKERESQIRDEYDHVLSAKLAEQYDTFVKFTYDQKRFEGATPSYLS (Z-ratio > 1.96), which partially corresponded to a Q38 peptide and the SUB protein interaction domain. These identified epitopes could be related to the protection and efficacy of the SUB-based vaccines, and new chimeras containing these protective epitopes could be designed using this new approach.

Dengue is a viral mosquito-borne disease that rapidly spreads in tropical and subtropical countries, including the Philippines. One of its most distinguishing characteristics is the ability of the Dengue Virus (DENV) to easily surpass the innate responses of the body, thus activating B cells of the adaptive immunity to produce virus-specific antibodies. Moreover, Dengvaxia® is the only licensed vaccine for DENV, but recent studies showed that seronegative individuals become prone to increased disease severity and hospitalization. Owing to this limitation of the dengue vaccine, this study determined and compared consensus and unique B cell epitopes among each DENV (1–4) Philippine isolate to identify potential areas of interest for future vaccine studies and therapeutic developments. An in silico-based epitope prediction of forty (40) DENV 1–4 strains, each serotype represented by ten (10) sequences from The National Center for Biotechnology Information (NCBI), was conducted using Kolaskar and Tongaonkar antigenicity, Emini surface accessibility, and Parker hydrophilicity prediction in Immune Epitope Database (IEDB). Results showed that five (5) epitopes were consensus for DENV-1 with no detected unique epitope, one (1) consensus epitope for DENV-2 with two (2) unique epitopes, one (1) consensus epitope for DENV-3 plus two (2) unique epitopes, and two (2) consensus epitopes and one (1) unique epitope for DENV-4. The findings of this study would contribute to determining potential vaccine and diagnostic marker candidates for further research studies and immunological applications against DENV (1–4) Philippine isolates.

Humoral immunity is crucial for protection against invading pathogens. Broadly neutralizing antibodies (bnAbs) provide sterilizing immunity by targeting conserved regions of viral variants and represent the goal of most vaccination approaches. While antibodies can be selected to bind virtually any region of a given antigen, the consistent induction of bnAbs in the context of influenza and HIV has represented a major roadblock. Many possible explanations have been considered; however, none of the arguments proposed to date seem to fully recapitulate the observed counter-selection for broadly protective antibodies. Antibodies can influence antigen presentation by enhancing the processing of CD4 epitopes adjacent to the binding region while suppressing the overlapping ones. We analyze the relative positioning of dominant B and T cell epitopes in published antigens that elicit strong and poor humoral responses. In strong immunogenic antigens, regions bound by immunodominant antibodies are frequently adjacent to CD4 epitopes, potentially boosting their presentation. Conversely, poorly immunogenic regions targeted by bnAbs in HIV and influenza overlap with clusters of dominant CD4 epitopes, potentially conferring an intrinsic disadvantage for bnAb-bearing B cells in germinal centers. Here, we propose the theory of immunodominance relativity, according to which the relative positioning of immunodominant B and CD4 epitopes within a given antigen drives immunodominance. Thus, we suggest that the relative positioning of B-T epitopes may be one additional mechanism that cooperates with other previously described processes to influence immunodominance. If demonstrated, this theory can improve the current understanding of immunodominance, provide a novel explanation for HIV and influenza escape from humoral responses, and pave the way for a new rational design of universal vaccines.

Vaccinia virus (VACV) is a promising oncolytic agent because it exhibits many characteristic features of an oncolytic virus. However, its effectiveness is limited by the strong antiviral immune response induced by this virus. One possible approach to overcome this limitation is to develop deimmunized recombinant VACV. It is known that VACV p35 is a major protein for B- and T-cell immune response. Despite the relevance of p35, its epitope structure remains insufficiently studied. To determine neutralizing epitopes, a panel of recombinant p35 variants was designed, expressed, and used for mice immunization. Plaque-reduction neutralization tests demonstrated that VACV was only neutralized by sera from mice that were immunized with variants containing both N- and C- terminal regions of p35. This result was confirmed by the depletion of anti-p35 mice sera with recombinant p35 variants. At least nine amino acid residues affecting the immunogenic profile of p35 were identified. Substitutions of seven residues led to disruption of B-cell epitopes, whereas substitutions of two residues resulted in the recognition of the mutant p35 solely by non-neutralizing antibodies.