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Pneumococcal infections range from simple respiratory tract mucosal infections as otitis media and sinusitis to more severe diseases including pneumonia, septicemia and meningitis. There is a need for enhanced vaccines as a result of its epidemic outbreak and the lack of potential medications. In this study, epitope-based peptide vaccine components based on the Choline-binding protein A and Pneumococcal surface protein A of Streptococcus pneumoniae were developed using the reverse vaccinology method. These proteins were examined for cytotoxic T lymphocyte (CTL) epitopes using immunoinformatics tools. Common epitopes predicted by various methods were chosen and evaluated for immunogenicity score, toxicity, antigenicity, and allergenicity assessment. Two potent CTL epitopes in Choline binding protein A were identified as TENEGATQV and TKKRHTITV and one epitope in Pneumococcal surface protein A of Streptococcus pneumonia as KLQYEISTL. These predicted epitopes were conserved across different stains sequences. These discovered epitopes were expected to have a high potential for eliciting an immune response in the development of a Streptococcus pneumoniae vaccine components. Although, more experimental validation is required for final confirmation. [ABSTRACT FROM AUTHOR]

There has been progressive improvement in immunoinformatics approaches for epitope-based peptide design. Computational-based immune-informatics approaches were applied to identify the epitopes of SARS-CoV-2 to develop vaccines. The accessibility of the SARS-CoV-2 protein surface was analyzed, and hexa-peptide sequences (KTPKYK) were observed having a maximum score of 8.254, located between amino acids 97 and 102, whereas the FSVLAC at amino acids 112 to 117 showed the lowest score of 0.114. The surface flexibility of the target protein ranged from 0.864 to 1.099 having amino acid ranges of 159 to 165 and 118 to 124, respectively, harboring the FCYMHHM and YNGSPSG heptapeptide sequences. The surface flexibility was predicted, and a 0.864 score was observed from amino acids 159 to 165 with the hepta-peptide (FCYMHHM) sequence. Moreover, the highest score of 1.099 was observed between amino acids 118 and 124 against YNGSPSG. B-cell epitopes and cytotoxic T-lymphocyte (CTL) epitopes were also identified against SARS-CoV-2. In molecular docking analyses, -0.54 to -26.21 kcal/mol global energy was observed against the selected CTL epitopes, exhibiting binding solid energies of -3.33 to -26.36 kcal/mol. Based on optimization, eight epitopes (SEDMLNPNY, GSVGFNIDY, LLEDEFTPF, DYDCVSFCY, GTDLEGNFY, QTFSVLACY, TVNVLAWLY, and TANPKTPKY) showed reliable findings. The study calculated the associated HLA alleles with MHC-I and MHC-II and found that MHC-I epitopes had higher population coverage (0.9019% and 0.5639%) than MHC-II epitopes, which ranged from 58.49% to 34.71% in Italy and China, respectively. The CTL epitopes were docked with antigenic sites and analyzed with MHC-I HLA protein. In addition, virtual screening was conducted using the ZINC database library, which contained 3,447 compounds. The 10 top-ranked scrutinized molecules (ZINC222731806, ZINC077293241, ZINC014880001, ZINC003830427, ZINC030731133, ZINC003932831, ZINC003816514, ZINC004245650, ZINC000057255, and ZINC011592639) exhibited the least binding energy (-8.8 to -7.5 kcal/mol). The molecular dynamics (MD) and immune simulation data suggest that these epitopes could be used to design an effective SARS-CoV-2 vaccine in the form of a peptide-based vaccine. Our identified CTL epitopes have the potential to inhibit SARS-CoV-2 replication. [ABSTRACT FROM AUTHOR]

The rapid spread of the SARS-CoV-2 virus and its variants has created a catastrophic impact worldwide. Several variants have emerged, including B.1.351 (Beta), B.1.1.28/triple mutant (P.1), B.1.1.7 (Alpha), and B.1.429 (Epsilon). We performed comparative and comprehensive antigenicity mapping of the total S-glycoprotein using the Wuhan strain and the other variants and identified 9-mer, 15-mer, and 20-mer CTL epitopes through in silico analysis. The study found that 9-mer CTL epitope regions in the B.1.1.7 variant had the highest antigenicity and an average of the three epitope types. Cluster analysis of the 9-mer CTL epitopes depicted one significant cluster at the 70% level with two nodes (KGFNCYFPL and EGFNCYFPL). The phage-displayed peptides showed mimic 9-mer CTL epitopes with three clusters. CD spectra analysis showed the same band pattern of S-glycoprotein of Wuhan strain and all variants other than B.1.429. The developed 3D model of the superantigen (SAg)-like regions found an interaction pattern with the human TCR, indicating that the SAg-like component might interact with the TCR beta chain. The present study identified another partial SAg-like region (ANQFNSAIGKI) from the S-glycoprotein. Future research should examine the molecular mechanism of antigen processing for CD8+ T cells, especially all the variants' antigens of S-glycoprotein. [ABSTRACT FROM AUTHOR]

Introduction: In an effort to combat SARS-CoV-2 through multi-subunit vaccine design, during studies using whole genome and immunome, ORF10, located at the 3′ end of the genome, displayed unique features. It showed no homology to any known protein in other organisms, including SARS-CoV. It was observed that its nucleotide sequence is 100% identical in the SARS-CoV-2 genomes sourced worldwide, even in the recent-most VoCs and VoIs of B.1.1.529 (Omicron), B.1.617 (Delta), B.1.1.7 (Alpha), B.1.351 (Beta), and P.1 (Gamma) lineages, implicating its constant nature throughout the evolution of deadly variants. Aim: The structure and function of SARS-CoV-2 ORF10 and the role it may play in the viral evolution is yet to be understood clearly. The aim of this study is to predict its structure, function, and understand evolutionary dynamics on the basis of mutations and likely heightened immune responses in the immunopathogenesis of this deadly virus. Methods: Sequence analysis, ab-initio structure modeling and an understanding of the impact of likely substitutions in key regions of protein was carried out. Analyses of viral T cell epitopes and primary anchor residue mutations was done to understand the role it may play in the evolution as a molecule with likely enhanced immune response and consequent immunopathogenesis. Results: Few amino acid substitution mutations are observed, most probably due to the ribosomal frameshifting, and these mutations may not be detrimental to its functioning. As ORF10 is observed to be an expressed protein, ab-initio structure modeling shows that it comprises mainly an α-helical region and maybe an ER-targeted membrane mini-protein. Analyzing the whole proteome, it is observed that ORF10 presents amongst the highest number of likely promiscuous and immunogenic CTL epitopes, specifically 11 out of 30 promiscuous ones and 9 out of these 11, immunogenic CTL epitopes. Reactive T cells to these epitopes have been uncovered in independent studies. Majority of these epitopes are located on the α-helix region of its structure, and the substitution mutations of primary anchor residues in these epitopes do not affect immunogenicity. Its conserved nucleotide sequence throughout the evolution and diversification of virus into several variants is a puzzle yet to be solved. Conclusions: On the basis of its sequence, structure, and epitope mapping, it is concluded that it may function like those mini-proteins used to boost immune responses in medical applications. Due to the complete nucleotide sequence conservation even a few years after SARS-CoV-2 genome was first sequenced, it poses a unique puzzle to be solved, in view of the evolutionary dynamics of variants emerging in the populations worldwide. [ABSTRACT FROM AUTHOR]

Foot-and-Mouth Disease (FMD) is causing 100% morbidity in adult animals with a high mortality rate in young animals due to cardiomyopathy. The disease-causing virus (FMDV) has been classified into seven serotypes and several topotypes. Amongst serotypes, the serotype O is predominantly present in Pakistan. A vaccine that should be safer and appears as an alternative to conventional vaccines that have failed to control the disease is required. Keeping in view, a multiepitope-based subunit vaccine (MESV) is designed using computational biology-based approaches. The sequence and structural characterization of capsid proteins and 3Cpro of O PanAsia-II sub-lineage are carried out for screening the antigenic proteins. Further, the B- and T-cell epitopes are selected based on toxicity, antigenicity, allergenicity, conservancy, and topology. The Cytotoxic T lymphocyte (CTL) epitopes are confirmed through molecular interactions with selected bovine alleles. The designed MESV is found stable, hydrophilic, antigenic, non-allergen, exposed extracellularly, non-toxic, and soluble. It will stimulate the protective immune response against the viral challenge as it has shown a strong binding affinity with the TLR4. These results suggest that the developed MESV will prove to be a potential candidate vaccine in controlling the disease against serotype O. For expression and accumulation of stable protein the sequence that encodes the multiepitope-based subunit vaccine is codonoptimized following the codon preference of E. coli and is cloned in pET-28a (+). [ABSTRACT FROM AUTHOR]

Objective: Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) is one of the deadliest diseases causing millions of deaths worldwide. Bacillus Calmette-Guérin (BCG) is the only vaccine that has been used in many countries where TB is prevalent. Despite vaccination, this disease prevails in many of the developing countries, necessitating the development of an effective vaccine against TB. Since M. tb acts as an intracellular pathogen, cell-mediated immune response plays an important role in disease control. Therefore, screening of CD8+ T cell epitopes of M. tb antigens could aid in the development of an effective vaccine against TB. In the current study, a reverse vaccinology approach was utilized to predict and map cytotoxic T lymphocyte (CTL) epitopes in the virulent proteins that are also essential for M. tb. Materials and Methods: Database of Essential Genes and Virulence Factor Database were used for identifying the virulent proteins of M. tb and their antigenicity was assessed using VaxiJen server. Various immunoinformatics tools were used to predict MHC class I binding, MHC processing, immunogenicity, toxicity and allergenicity. Results: Twelve M. tb antigens were selected for the prediction analyses using various tools. The results indicated the presence of 20 novel CTL epitopes predicted against human HLA-A alleles. This study has also screened for multiple allele binding epitopes that could be used as a vaccine component. Conclusion: This study has yielded a few hitherto unreported CTL epitopes binding to class I HLA-A alleles. Further experimental validation is necessary for confirming their potential as vaccine candidates. [ABSTRACT FROM AUTHOR]

Highlights • Four novel CTL epitope peptides of Rv2629 showed high-affinity and stability of binding with T2 cells. • Their ability of inducing CTLs was higher in TB patients than that in healthy controls and LTBI individuals. • These peptides could induce the CTLs to generate specific cytotoxic activity. • They showed higher immunogenicity in TB patients and had the potential to become candidate targets for TB vaccine. Abstract There is an urgent need for a more effective vaccine against tuberculosis (TB). Cytotoxic T lymphocytes (CTLs) play a critical role in combating Mycobacterium tuberculosis (M.tb). The identification of novel CTL epitopes is essential for the design of peptide-based vaccines. In this study, we predicted CTL epitope peptides of M.tb antigen Rv2629 restricted by HLA-A2, using bioinformatics methods. The affinity and stability of binding of these peptides with HLA-A2 molecules were detected by flow cytometry. Their ability to induce CTLs generation was determined in peripheral blood mononuclear cells (PBMCs) from healthy uninfected subjects, Latent tuberculosis infection (LTBI) subjects, and TB patients ex vivo. The cytotoxic activity induced by the epitope peptides was tested by lactate dehydrogenase (LDH) release assay. Finally, we found four novel CTL epitope peptides, Rv2629-p190-2L, Rv2629-p190-1Y2L, Rv2629-p274, and Rv2629-p315, which had high-affinity and stability of binding with T2 cells. Their ability of inducing CTLs was highest in PBMCs from TB patients (P < 0.05). In addition, these peptides could induce the CTLs to generate specific cytotoxic activity. They showed higher immunogenicity in TB patients and had the potential to become candidate vaccines for TB therapy. [ABSTRACT FROM AUTHOR]

Viral epitopes have a distinct codon usage that reflects their dual role in infection and immunity. On the one hand, epitopes are part of proteins important to viral function; on the other hand, they are targets of the immune response. Studies of selection are most commonly based on changes of amino acid and seen through the accumulation of non-synonymous mutations. An independent measure of selection is the codon usage and underlying changeability of the nucleotide sequences. We here use multiple tools and a large-scale analysis of viral genomes to demonstrate that viral epitopes have a distinct codon usage and that this codon usage reflects distinct short- and long-term types of selection during viral evolution. We show that CD8 T cell epitopes are encoded by codons more distant from stop codons and more changeable than codons outside epitopes. This biased codon usage reflects the viral population toggling back and forth from a wild-type sequence to an escape mode, which enable them to avoid immune detection when needed, and go back to the functionally favorable form when the threat is removed (i.e., in a new host). [ABSTRACT FROM AUTHOR]

Major histocompatibility complex ( MHC) molecules play a key role in cell-mediated immune responses presenting bounded peptides for recognition by the immune system cells. Several in silico methods have been developed to predict the binding affinity of a given peptide to a specific MHC molecule. One of the current state-of-the-art methods for MHC class I is NetMHCpan, which has a core ingredient for the representation of the MHC class I molecule using a pseudo-sequence representation of the binding cleft amino acid environment. New and large MHC-peptide-binding data sets are constantly being made available, and also new structures of MHC class I molecules with a bound peptide have been published. In order to test if the NetMHCpan method can be improved by integrating this novel information, we created new pseudo-sequence definitions for the MHC-binding cleft environment from sequence and structural analyses of different MHC data sets including human leukocyte antigen ( HLA), non-human primates (chimpanzee, macaque and gorilla) and other animal alleles (cattle, mouse and swine). From these constructs, we showed that by focusing on MHC sequence positions found to be polymorphic across the MHC molecules used to train the method, the NetMHCpan method achieved a significant increase in the predictive performance, in particular, of non-human MHCs. This study hence showed that an improved performance of MHC-binding methods can be achieved not only by the accumulation of more MHC-peptide-binding data but also by a refined definition of the MHC-binding environment including information from non-human species. [ABSTRACT FROM AUTHOR]

Abstract: In this study, 12 nonamers derived from the nonstructural protein NS4A and NS4B of a Chinese attenuated Yellow fever 17D (YF-17D) vaccine strain were screened for their ability to elicit a recall interferon-γ (IFN-γ) response from the splenocytes of BALB/c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus expressing NS4A or NS4B protein. Three peptides (amino acid residues S106YIMLIFFV from NS4A protein and V43YVGIVTML and L174YLLLALSL from NS4B protein) were identified as CD8+ cytotoxic T lymphocyte (CTL) epitopes. These peptides elicited a response from a significant numbers of IFN-γ secreting cells compared with the other nonamers of NS4A and NS4B and compared to one irrelevant nonamer derived from infectious bronchitis virus (IBV) H52 strain. Bioinformatics analysis demonstrated that these three nonamers are H-2K d -restricted CTL epitopes. The multiple amino acid sequence alignments among different YFV NS4A, and NS4B sequences submitted to GenBank indicated that these three CTL epitopes are strongly conserved; this novel finding will potentially aid further studies on cellular immunity against YF virus and YFV-based expression vectors. [Copyright &y& Elsevier]

Twenty-seven nanopeptides derived from the matrix (M) protein of porcine reproductive and respiratory syndrome virus (PRRSV) were screened for their ability to elicit a recall interferon-γ (IFN-γ) response from the splenocytes of BALB/c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus rWR-PRRSV-M. We identified two peptides (amino acid residues K93FITSRCRL and F57GYMTFVHF) as CD8+ cytotoxic T lymphocyte (CTL) epitopes. These peptides elicited significant numbers of IFN-γ secreting cells, compared with other M nonapeptides and one irrelevant nonapeptide. Bioinformatics analysis showed that the former is an H-2Kd-restricted CTL epitope, and the latter is an H-2Dd-restricted CTL epitope. Multiple amino acid sequence alignment among different PRRSV M sequences submitted to GenBank indicated that these two CTL epitopes are strongly conserved, and they should therefore be considered for further research on the mechanisms of cellular immune responses to PRRSV. [ABSTRACT FROM AUTHOR]

Heparanase is expressed in almost all advanced tumors, and therefore it may serve as a potential target for tumor therapy. Our previous study has shown that heparanase can serve as a potential universal tumor-associated antigen (TAA) for the immunotherapy of advanced tumors. Further study demonstrated that the HLA-A*0201-restricted Cytotoxic T lymphocytes (CTL) epitopes Hpa525 (PAFSYSFFV), Hpa277 (KMLKSFLKA) and Hpa405 (WLSLLFKKL) from human heparanase could induce a potent anti-tumor immune response in vitro. The present study was designed to investigate whether the above peptides could induce immune responses in mice. Our results demonstrated that the effectors from heparanase peptide-immunized mice could effectively lyse various tumor cells that were heparanase positive and HLA-A*0201 matched. We also found that these peptide-specific CTLs did not lyse autologous lymphocytes that had low heparanase activity. Further study revealed that Hpa525, Hpa277, and Hpa405 peptides increased the frequency of IFN-γ-producing T cells as compared to a negative peptide. These results suggest that Hpa525, Hpa277, and Hpa405 peptides are novel HLA-A*0201-restricted CTL epitopes capable of inducing heparanase-specific CTLs in mice. Because heparanase is expressed in most advanced malignant tumors, Hpa525, Hpa277, and Hpa405 peptide-based vaccines may be useful for the immunotherapy of patients with advanced tumors. [ABSTRACT FROM AUTHOR]

The diversity of HIV-1 and its propensity to generate escape mutants present fundamental challenges to control efforts, including HIV vaccine design. Intra-host diversification of HIV is determined by immune responses elicited by an HIV-infected individual over the course of the infection. Complex and dynamic patterns of transmission of HIV lead to an even more complex population viral diversity over time, thus presenting enormous challenges to vaccine development. To address inter-patient viral evolution over time, a set of 653 unique HIV-1 subtype C gag sequences were retrieved from the LANL HIV Database, grouped by sampling year as <2000, 2000, 2001-2002, 2003, and 2004-2006, and analyzed for the site-specific frequency of translated amino acid residues. Phylogenetic analysis revealed that a total of 289 out of 653 (44.3%) analyzed sequences were found within 16 clusters defined by aLRT of more than 0.90. Median (IQR) intersample diversity of analyzed gag sequences was 8.7% (7.7%; 9.8%). Despite the heterogeneous origins of analyzed sequences, the gamut and frequency of amino acid residues in wild-type Gag were remarkably stable over the last decade of the HIV-1 subtype C epidemic. The vast majority of amino acid residues demonstrated minor frequency fluctuation over time, consistent with the conservative nature of the HIV-1 Gag protein. Only 4.0% (20 out of 500; HXB2 numbering) amino acid residues across Gag displayed both statistically significant (p<0.05 by both a trend test and heterogeneity test) changes in amino acid frequency over time as well as a range of at least 10% in the frequency of the major amino acid. A total of 59.2% of amino acid residues with changing frequency of 10%+ were found within previously identified CTL epitopes. The time of the most recent common ancestor of the HIV-1 subtype C was dated to around 1950 (95% HPD from 1928 to 1962). This study provides evidence for the overall stability of HIV-1 subtype C Gag among viruses circulating in the epidemic over the last decade. However selected sites across HIV-1C Gag with changing amino acid frequency are likely to be under selection pressure at the population level. [ABSTRACT FROM AUTHOR]

Recent human immunodeficiency virus type 1 (HIV-1) vaccination strategies aim at targeting a broad range of cytotoxic T lymphocyte (CTL) epitopes from different HIV-1 proteins by immunization with multiple CTL epitopes simultaneously. However, this may establish an immune hierarchical response, where the immune system responds to only a small number of the epitopes administered. To evaluate the feasibility of such vaccine strategies, we used the human leukocyte antigen (HLA)-A*0201 transgenic (tg) HHD murine in vivo model and immunized with dendritic cells pulsed with seven HIV-1-derived HLA-A*0201 binding CTL epitopes. The seven peptides were simultaneously presented on the same dendritic cell (DC) or on separate DCs before immunization to one or different lymphoid compartments. Data from this study showed that the T-cell response, as measured by cytolytic activity and γ-interferon (IFN-γ)-producing CD8+ T cells, mainly focused on two of seven administered epitopes. The magnitude of individual T-cell responses induced by immunization with multiple peptides correlated with their individual immunogenicity that depended on major histocompatibility class I binding and was not influenced by mode of loading or mode of immunization. These findings may have implications for the design of vaccines based on DCs when using multiple epitopes simultaneously. [ABSTRACT FROM AUTHOR]

Cytotoxic T-lymphocytes (CTL) are critical for immune control of infection with human immunodeficiency virus type-1 (HIV-1) and searches for relevant CTL epitopes for immune therapy are ongoing. Recently, we identified 28 HLA-A2-binding HIV-1 CTL epitopes (1). In this follow-up study we fully genome sequenced HIV-1 from 11 HLA-A2+ patients to examine the sequence variation of these natural epitopes and compared them with the patient's CD8+ T-cell recall response. Often the epitope was conserved but only a few patients showed a CD8+ T-cell recall response. This infrequent targeting may be explained by immune subdominance. CD8+ T-cell recall response to a natural epitope could be measured despite sequence differences in the patient's virus. T-cell cross-reaction between such variants could be demonstrated in HLA-A2 transgenic mice. Nine infrequently targeted but conserved or cross-reacting epitopes were identified in seven HIV-1 proteins. More immunogenic anchor amino acid optimized immunogens were designed that induced T-cell cross-reaction with these natural epitopes. It is concluded that most of the new CTL epitopes are conserved but subdominant during the infection. It is suggested that T-cell promiscuity may explain the observed CD8+ T-cell reaction to epitope variants and it may be possible to use the selected immune optimized epitope peptides for therapeutic vaccination. [ABSTRACT FROM AUTHOR]

Abstract: Ebola virus (EBOV) is known to cause a severe hemorrhagic fever resulting in high mortality. Although the precise host defense mechanism(s) that afford protection against EBOV is not completely understood, T cell-mediated immune responses is believed to play a pivotal role in controlling virus replication and EBOV infection. There have been no reports on mapping of MHC Class I-binding CTL epitopes for EBOV till to date. In this study, we identified five HLA-A2-binding 9-mer peptides of EBOV nucleoprotein (NP) using computer-assisted algorithm. The peptides were synthesized and examined for their ability to bind to MHC class I molecules using a flow cytometry based MHC stabilization assay. Three of the EBOV-NP peptides tested (FLSFASLFL, RLMRTNFLI and KLTEAITAA) stabilized HLA-A2. The ability of the HLA-A2-binding EBOV-NP peptides to generate peptide-specific CTLs was evaluated in HLA-A2.1 transgenic mice. Epitope-specific CTL responses were confirmed by cytotoxic assays against peptide-pulsed target cells and interferon-γ ELISPOT assay. Each of the EBOV-NP peptides induced CTL responses in HLA-A2-transgenic mice. Interestingly, all the three peptides were conserved in three different strains of Ebola (Zaire and Reston and Sudan). Taken together, these findings provide direct evidence for the existence of EBOV-derived NP epitopes that may be useful in the development of protective immunogens for this hemorrhagic virus. [Copyright &y& Elsevier]

Cytotoxic T lymphocyte (CTL) epitopes are potential candidates for subunit vaccine design for various diseases. Most of the existing T cell epitope prediction methods are indirect methods that predict MHC class I binders instead of CTL epitopes. In this study, a systematic attempt has been made to develop a direct method for predicting CTL epitopes from an antigenic sequence. This method is based on quantitative matrix (QM) and machine learning techniques such as Support Vector Machine (SVM) and Artificial Neural Network (ANN). This method has been trained and tested on non-redundant dataset of T cell epitopes and non-epitopes that includes 1137 experimentally proven MHC class I restricted T cell epitopes. The accuracy of QM-, ANN- and SVM-based methods was 70.0, 72.2 and 75.2%, respectively. The performance of these methods has been evaluated through Leave One Out Cross-Validation (LOOCV) at a cutoff score where sensitivity and specificity was nearly equal. Finally, both machine-learning methods were used for consensus and combined prediction of CTL epitopes. The performances of these methods were evaluated on blind dataset where machine learning-based methods perform better than QM-based method. We also demonstrated through subgroup analysis that our methods can discriminate between T-cell epitopes and MHC binders (non-epitopes). In brief this method allows prediction of CTL epitopes using QM, SVM, ANN approaches. The method also facilitates prediction of MHC restriction in predicted T cell epitopes. The method is available at . [Copyright &y& Elsevier]

Tumor immunotherapy is currently receiving close scrutiny. However, with the identification of tumor antigens and their production by recombinant means, the use of cytokines and knowledge of major histocompatibility complex (MHC) class I and class II presentation has provided ample reagents for use and clear indications of how they should be used. At this time, much attention is focused on using peptides to be presented by MHC class I molecules to both induce and be targets for CD8+ cytolytic T cells. Many peptides generated endogenously or given exogenously can enter the class I pathway, but a number of other methods of entering this pathway are also known and are discussed in detail herein. While the review concentrates on inducing cytotoxic T cells (CTLs), it is becoming increasingly apparent that other modes of immunotherapy would be desirable, such as class II presentation to induce increased helper activity (for CTL), but also activating macrophages to be effective against tumor cells. [ABSTRACT FROM AUTHOR]

• Eight T-cell immunodominant epitopes of PRRSV-2 M protein were validated. • 3 potential CTL peptides and their SLA-2 binding domain residues were identified. • PRRSV-2 M protein peptides M 2−5 & M 6−1 bound SLA-2*0502; M 9−1 bound SLA-2*1201. • PRRSV-2 M protein peptides were tested for SLA binding via rapid in vitro assays. • ELISPOT, in-silico prediction, ELISA and peptide complex refolding tests were used. CD8+ cytotoxic T lymphocytes (CTLs), are essential for clearance of porcine reproductive and respiratory syndrome virus (PRRSV) infection and regulation of host immune responses. Identification of SLA I-restricted CD8+ CTL epitopes would facilitate PRRSV vaccine development. Here, cells isolated from peripheral blood mononuclear cells (PBMCs) of PRRSV-immunized Large White pigs (JXA1-R strain) were screened for immunodominant PRRSV-2 M protein T cell epitopes via ELISPOT assay. Of nine immunodominant epitopes detected, eight elicited significant IFN-γ secretion responses that varied among individual pigs and according to epitope. To predict which epitopes harbored potential CTL epitopes, swine leukocyte antigen (SLA) class I genes of Large White pigs were cloned and sequenced, yielding fourteen distinct SLA class I gene sequences. Based on ELISPOT and SLA genotyping results, SLA-restricted binding of the fourteen predicted class I proteins to peptides derived from the eight immunodominant epitopes were predicted in-silico. After evaluation of 42 pET-peptide-SLA-I-β2m complexes containing predicted restricted peptides, extracellular SLA class I domains and β2m, ELISA testing of 33 peptide-SLA-I-β2m complexes detected four complexed peptides. These four peptides were evaluated using in vitro complex refolding assays that confirmed that M 2−5 and M 6−1 peptides each formed complexes with SLA-2*0502 and sβ2m, while M 9−1 formed a complex with SLA-2*1201 and sβ2m. ELISPOT results confirmed these three 9-mer potential CTL epitopes efficiently stimulated IFN-γ secretion when presented by SLA class I molecules specified here. This study describes effective CTL epitope identification methods for use in future investigations of swine cellular immunity toward T cell-based vaccine development. [ABSTRACT FROM AUTHOR]

An immunoinformatics-based approach was used to identify potential multivalent subunit CTL vaccine candidates for SARS-CoV-2. Criteria for computational screening included antigen processing, antigenicity, allergenicity, and toxicity. A total of 2604 epitopes were found to be strong binders to MHC class I molecules when analyzed using IEDB tools. Further testing for antigen processing yielded 826 peptides of which 451 were 9-mers that were analyzed for potential antigenicity. Antigenic properties were predicted for 102 of the 451 peptides. Further assessment for potential allergenicity and toxicity narrowed the number of candidate CTL epitopes to 50 peptide sequences, 45 of which were present in all strains of SARS-CoV-2 that were tested. The predicted CTL epitopes were then tested to eliminate those with MHC class II binding potential, a property that could induce hyperinflammatory responses mediated by T H 2 cells in immunized hosts. Eighteen of the 50 epitopes did not show class II binding potential. To our knowledge this is the first comprehensive analysis on the proteome of SARS-CoV-2 for prediction of CTL epitopes lacking binding properties that could stimulate unwanted T H 2 responses. Future studies will be needed to assess these epitopes as multivalent subunit vaccine candidates which stimulate protective CTL responses against SARS-COV-2. • Immunoinformatics-based approach to identify potential CTL vaccine candidates for SARS-CoV-2 • 50 peptides predicted as CTL epitopes and to our knowledge, first comprehensive analysis of SARS-CoV-2 for CTL prediction • 18 of the 50 peptides lack predicted binding to class II MHC, property that could otherwise stimulate unwanted Th2 response [ABSTRACT FROM AUTHOR]

Abstract: The use of cytotoxic T lymphocyte (CTL)-inducing vaccines could afford both homo- and heterosubtypic immunity. However, amino acid variation in CTL epitopes associated with escape from CTL-mediated immunity might undermine the use of these vaccines. To assess the impact of amino acid substitutions in highly conserved epitopes on viral fitness and recognition by specific CTL, we performed a mutational analysis of various CTL epitopes. Our findings indicated that fitness costs limited variation in functionally constrained epitopes, especially at anchor residues. [Copyright &y& Elsevier]