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Bovine Viral Diarrhea (BVD) is a major economic threat to the livestock industry due to its widespread infection among cattle around the world. Due to resistant strains and regulatory system inadequacies, traditional therapeutic vaccines failed to eradicate it. In this study, a Multi-Epitope Vaccine (MEV) with noticeable immunological properties is proposed that can mitigate these problems by decreasing cross-contamination, autoimmune responses, and inflammatory responses. Using Immunoinformatics approaches, putative CTL- and B-cell epitopes from four BVDV proteins (E1, E2, Erns, and NS3) were predicted and scrutinized after immunogenic profiling. The robust binding of chimeric vaccine to bovine TLR3 was validated through protein-protein docking investigations. The molecular dynamic simulation proved it to be stable in a biological system. The proposed MEV proved vital in strengthening the host immunity upon infection as the immunological simulation was projected to induce immunoglobulins, interleukins, interferons, helper T cells, and cytotoxic T cells. Our results therefore imply that the proposed vaccine will aid in inducing a strong and durable immunity in animals against BVDV infection. [ABSTRACT FROM AUTHOR]

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]

PPR is a small ruminant infectious illness that is wreaking havoc on economies in South Asia and Africa. Due to several hurdles, such as resistant strains and a limited regulatory framework, existing therapeutic vaccinations have failed to eradicate the disease. To overcome these challenges, a peptide-based vaccination with distinct immunological characteristics and less vulnerability to contamination, autoimmunity, and inflammatory diseases would be useful in accomplishing the suggested eradication goal. The possible CTL and B-cell epitopes of PPRV's four immunogenic proteins (H, F, M, and N) were predicted using a variety of immunoinformatic methods. Protein-protein docking confirmed that the proposed chimeric vaccine has a considerable affinity for the ovine toll-like receptor. In a biological system, the molecular dynamic simulation confirmed the in vivo stability of the protein. The immune simulation predicted the induction of immunoglobulins, interleukins, interferons, helper T cells, and cytotoxic T cells, which is critical for boosting host immunity during infection. Moreover, the translation efficiency was verified through in silico cloning of the vaccine in a bacterial expression system following codon optimization. Thus, our findings suggest that the candidate vaccine will help in provoking a robust and long-lasting immunity in animals against PPRV infection. [Display omitted] • Genotype IV of the PPR virus is currently circulating in Pakistan. • Designing of a multi-epitope vaccine against Peste des Petits Ruminants Virus (PPRV). • Chimeric vaccine has a strong binding affinity with ovine Toll-Like Receptor 6. • Both, molecular dynamic and immune simulations validated the vaccine's effectiveness. [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]

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 hepta-peptide 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.

Differences in outcome to COVID-19 infection in different individuals is largely attributed to genetic heterogeneity leading to differential immune responses across individuals and populations. HLA is one such genetic factor that varies across individuals leading to differences in how T-cell responses are triggered against SARS-CoV-2, directly influencing disease susceptibility. HLA alleles that influence COVID-19 outcome, by virtue of epitope binding and presentation, have been identified in cohorts worldwide. However, the heterogeneity in HLA distribution across ethnic groups limits the generality of such association. In this study, we address this limitation by comparing the recognition of CTL epitopes across HLA genotypes and ethnic groups. Using HLA allele frequency data for ethnic groups from Allele Frequency Net Database (AFND), we construct synthetic populations for each ethnic group and show that CTL epitope strength varies across HLA genotypes and populations. We also observe that HLA genotypes, in certain cases, can have high CTL epitope strengths in the absence of top-responsive HLA alleles. Finally, we show that the theoretical estimate of responsiveness and hence protection offered by a HLA allele is bound to vary across ethnic groups, due to the influence of other HLA alleles within the HLA genotype on CTL epitope recognition. This emphasizes the need for studying HLA-disease associations at the genotype level rather than at a single allele level. [ABSTRACT FROM AUTHOR]

The current global outbreak of monkeypox (MPX) disease, caused by Monkeypox virus (MPXV), has resulted in 16 thousand infection cases, five deaths, and has been declared a global health emergency of international concern by the World Health Organization. Given current challenges in the safety of existing vaccines, a vaccine to prevent MPX infection and/or onset of symptoms would significantly advance disease management. In this context, a multi-epitope-based vaccine could be a well-suited approach. Herein, we searched a publicly accessible database (Virus Pathogen Database and Analysis Resource) for MPXV immune epitopes from various antigens. We prioritized a group of epitopes (10 CD8+ T cells and four B-cell epitopes) using a computeraided technique based on desirable immunological and physicochemical properties, sequence conservation criteria, and non-human homology. Three multi-epitope vaccines were constructed (MPXV-1-3) by fusing finalized epitopes with the aid of appropriate linkers and adjuvant (beta-defensin 3, 50S ribosomal protein L7/L12, and Heparin-binding hemagglutinin). Codon optimization and in silico cloning in the pET28a (+) expression vector ensure the optimal expression of each construct in the Escherichia Coli system. Two and three-dimensional structures of the constructed vaccines were predicted and refined. The optimal binding mode of the construct with immune receptors [Toll-like receptors (TLR2, TLR3, and TLR4)] was explored by molecular docking, which revealed high docking energies of MPXV-1-TLR3 (-99.09 kcal/mol), MPXV-2-TLR3 (-98.68 kcal/mol), and MPXV-3-TLR2 (-85.22 kcal/mol). Conformational stability and energetically favourable binding of the vaccine-TLR2/3 complexes were assessed by performing molecular dynamics simulations and free energy calculations (Molecular Mechanics/Generalized Born Surface Area method). In silico immune simulation suggested that innate, adaptive, and humoral responses will be elicited upon administration of such potent multi-epitope vaccine constructs. The vaccine constructs are antigenic, non-allergen, non-toxic, soluble, topographically exposed, and possess favourable physicochemical characteristics. These results may help experimental vaccinologists design a potent MPX vaccine. [ABSTRACT FROM AUTHOR]

Vaccination is the most effective means of protecting people from influenza virus infection. The effectiveness of existing vaccines is very limited due to antigenic drift of the influenza virus. Therefore, there is a requirement to develop a universal vaccine that provides broad and long-lasting protection against influenza. CD8+ T-cell response played a vital role in controlling influenza virus infection, reducing viral load, and less clinical syndrome. In this study, we optimized the HA sequences of human seasonal influenza viruses (H1N1, H3N2, Victoria, and Yamagata) by designing multivalent vaccine antigen sets using a mosaic vaccine design strategy and genetic algorithms, and designed an HA mosaic cocktail containing the most potential CTL epitopes of seasonal influenza viruses. We then tested the recombinant mosaic antigen, which has a significant number of potential T-cell epitopes. Results from genetic evolutionary analyses and 3D structural simulations demonstrated its potential to be an effective immunogen. In addition, we have modified an existing neutralizing antibody-based seasonal influenza virus vaccine to include a component that activates cross-protective T cells, which would provide an attractive strategy for improving human protection against seasonal influenza virus drift and mutation and provide an idea for the development of a rationally designed influenza vaccine targeting T lymphocyte immunity. [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]

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.

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 • Optimised FATT-CTL assay for the first time for sheep PBMC. • FATT-CTL assay was used to identify peptides that induced CTL. • Peptides from different antigens induced varied CTL responses in immune PBMC. • Peptides may be included in heartwater recombinant vaccine development. Abstract Since CD8+ T cells play an important role in resistance to infection with heartwater, effective vaccines against this disease will likely require identification of antigens that contain CD8+ T cell epitopes responsible for cytotoxic T lymphocyte (CTL) responses. With the use of the fluorescent antigen-transfected target cell (FATT)-CTL assay, IFN-γ ELISPOT and flow cytometry, peptides that induce CTL, proliferation of CD8 + T cells and IFN-γ production were identified as possible target antigens for vaccine development. Of particular relevance was the finding that different peptides from different antigens were able to elicit varied cytotoxic activities by immune peripheral blood mononuclear cells (PBMC) from heartwater immune tick-infected sheep. Several peptides derived from Erum0660, Erum2330, Erum2540, Erum2580 and Erum5000 induced CTL in immune sheep PBMC. Peptide Erum2540-6 was the only peptide that induced significant CTL, CD8+CD45RO+ and CD8+IFN-γ+ by PBMC from all three sheep, and Erum2540 and p2540-20 induced the highest % CTL response in all three outbred sheep. These results suggest that these epitopes may be of major importance in heartwater recombinant vaccine development. [ABSTRACT FROM AUTHOR]

We proposed a new HIV-1 therapeutic vaccine based on conserved cytotoxic T lymphocyte (CTL) epitopes of archived HIV-1 DNA according to their affinity to the dominant HLA-A and -B alleles of the population investigated. Our proposal (Hla Fitted VAC, HFVAC) was composed of 15 peptides originating from the RT, gag and nef parts of proviral DNA. Our aim was to investigate baseline immune reactivity to the vaccine in HIV-1 chronically infected patients at success of antiretroviral therapy (ART) who would be eligible for a therapeutic vaccine. Forty-one patients were tested. Most of them had been infected with HIV-1 subtype B and all had been receiving successful ART for 2 to 20 years. The predominant HLA-A and -B alleles were those of a Caucasian population. ELISPOT was carried out using the HFVAC peptides. In 22 patients, the PD-1 marker was investigated on CD4+ and CD8+ T cells by flow cytometry in order to evaluate global T cell exhaustion. ELISPOT positivity was 65% overall and 69% in patients exhibiting at least one HLA allele fitting with HFVAC. The percentages of CD4+ and CD8+ T cells expressing PD-1 were high (median values 23.70 and 32.60, respectively), but did not seem to be associated with an impairment of the immune response investigated in vitro. In conclusion, reactivity to HFVAC was high in this ART-treated population with dominant HLA alleles, despite potential cellular exhaustion associated with the PD-1 marker.

Tumor vaccine design requires prediction and validation of immunogenic MHC class I epitopes expressed by target cells as well as MHC class II epitopes expressed by antigen-presenting cells essential for the induction of optimal immune responses. Epitope prediction methods are based on different algorithms and are instrumental for a first screening of possible epitopes. However, their results do not reflect a one-to-one correlation with experimental data. We combined several in silico prediction methods to unravel the most promising C57BL/6 mouse-restricted Hepatitis C virus (HCV) MHC class I epitopes and validated these epitopes in vitro and in vivo. Cytotoxic T lymphocyte (CTL) epitopes within the HCV non-structural proteins were identified, and proteasomal cleavage sites and helper T cell (Th) epitopes at close proximity to these CTL epitopes were analyzed using multiple prediction algorithms. This combined in silico analysis enhances the precision of identification of functional HCV-specific CTL epitopes. This approach will be applicable to the design of human vaccines not only for HCV, but also for other antigens in which T-cell responses play a crucial role.

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]

The aim of this study is to evaluate the amino acid variability of HIV-1 Gp41, C2-V3, and Nef in a group of patients characterized by different disease progression rates. HIV-1 sequences were collected from 19 Long term non progressor patients (LTNPs), 9 slow progressors (SPs), and 11 rapid progressors (RPs). Phylogenetic trees were estimated by MEGA 6. Differences in amino acid variability among sequences belonging to the 3 groups have been evaluated by amino acid divergence, Shannon entropy analysis, and the number of amino acid mutations (defined as amino acid variations compared with HxB2). The involvement of amino acid mutations on epitope rich regions was also investigated. The population was mainly composed of males (74.3%) and HIV-1 subtype B strains (B: 92.32%, CRF_12BF, A1, C: 2.56% each). Viral load (log10 copies/mL) and CD4+T cell count (cells/mm3) were 3.9 (3.5-4.2) and 618 (504-857) in LTNPs, 3.3 (2.8-4.7) and 463 (333-627) in SPs, and 4.6 (4.3-5.3) and 201 (110-254) in RPs. Gp41 and C2-V3 amino acid divergence was lower in LTNP and SP strains compared to RPs (median value: 0.085 and 0.091 vs. 0.114, p = 0.005 and 0.042) and a trend of lower variability was observed for Nef (p = 0.198). A lower entropy value was observed at 10, 3, and 7 positions of Gp41, C2-V3, and Nef belonging to LTNPs and at 7, 3, and 1 positions of Gp41, C2-V3, and Nef belonging to SPs compared with RPs (p < 0.05). Focusing on epitope rich regions, again a higher degree of conservation was observed in Gp41 and C2-V3 sequences belonging to LTNPs and SPs compared to those belonging to RPs. This study shows that the extent of amino acid variability correlates with a different HIV-1 progression rate. This variability also involves CTL epitope rich regions, thus suggesting its involvement in the immune escape process modulation. [ABSTRACT FROM AUTHOR]

Aim: In silico approach was used to predict cytotoxic T lymphocyte (CTL) epitopes from the proteome of all four dengue serotypes.Methods: The immune epitope database analysis resource was used to analyze the CTL epitopes of dengue serotypes. The prediction of epitopes was done against nine high frequency HLA class I alleles occurring worldwide. Results: A total of 2784 epitopes were predicted from all four dengue virus proteomes. Immune epitope database analysis resource tool predicted 202 epitopes as positive for immunogenecity. A total of 39 of 257 consensus epitopes predicted were present in all four serotypes. This study identified nine new class I-restricted epitopes. Conclusion: Mapping of these potentially immunogenic dengue epitopes paves the way for future investigation of their utility as vaccine candidates to prevent or treat dengue virus infections. [ABSTRACT FROM AUTHOR]

The scientific and medical community is seeking to cure HIV. Several pathways have been or are being explored including therapeutic vaccination. Viroimmunological studies on primary infection as well as on elite controllers have demonstrated the importance of the cytotoxic CD8 response and have mainly oriented research on vaccine constructs toward this type of response. The results of these trials are clearly not commensurate with the hope placed in them. Might there be one or more uncontrolled variables? The genetics of patients need to be taken into consideration, especially their human lymphocyte antigen (HLA) alleles. There is a need to find a balance between the conservation of cytotoxic T lymphocyte (CTL) epitopes and presentation by HLA alleles. The pathway is a narrow one between adaptation of the virus to HLA I restriction and the definition of conserved proviral CTL epitopes presentable by HLA I alleles. It is likely that the genetics of patients will need to be considered for HIV-1 vaccine studies and that multidisciplinary collaboration will be essential in this field of infectious diseases. [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

Both the recognition of HIV-infected cells and the immunogenicity of candidate CTL vaccines depend on the presentation of a peptide epitope at the cell surface, which in turn depends on intracellular antigen processing. Differential antigen processing maybe responsible for the differences in both the quality and the quantity of epitopes produced, influencing the immunodominance hierarchy of viral epitopes. Previously, we showed that the magnitude of the HIV-2 gag-specific T-cell response is inversely correlated with plasma viral load, particularly when responses are directed against an epitope, 165DRFYKSLRA173, within the highly conserved Major Homology Region of gag-p26. We also showed that the presence of three proline residues, at positions 119, 159 and 178 of gag-p26, was significantly correlated with low viral load. Since this proline motif was also associated with stronger gag-specific CTL responses, we investigated the impact of these prolines on proteasomal processing of the protective 165DRFYKSLRA173 epitope. Our data demonstrate that the 165DRFYKSLRA173 epitope is most efficiently processed from precursors that contain two flanking proline residues, found naturally in low viral-load patients. Superior antigen processing and enhanced presentation may account for the link between infection with HIV-2 encoding the 'PPP-gag' sequence and both strong gag-specific CTL responses as well as lower viral load. [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]

The binding of peptides to classical major histocompatibility complex (MHC) class I proteins is the single most selective step in antigen presentation. However, the peptide-binding specificity of cattle MHC (bovine leucocyte antigen, BoLA) class I (BoLA-I) molecules remains poorly characterized. Here, we demonstrate how a combination of high-throughput assays using positional scanning combinatorial peptide libraries, peptide dissociation, and peptide-binding affinity binding measurements can be combined with bioinformatics to effectively characterize the functionality of BoLA-I molecules. Using this strategy, we characterized eight BoLA-I molecules, and found the peptide specificity to resemble that of human MHC-I molecules with primary anchors most often at P2 and P9, and occasional auxiliary P1/P3/P5/P6 anchors. We analyzed nine reported CTL epitopes from Theileria parva, and in eight cases, stable and high affinity binding was confirmed. A set of peptides were tested for binding affinity to the eight BoLA proteins and used to refine the predictors of peptide-MHC binding NetMHC and NetMHCpan. The inclusion of BoLA-specific peptide-binding data led to a significant improvement in prediction accuracy for reported T. parva CTL epitopes. For reported CTL epitopes with weak or no predicted binding, these refined prediction methods suggested presence of nested minimal epitopes with high-predicted binding affinity. The enhanced affinity of the alternative peptides was in all cases confirmed experimentally. This study demonstrates how biochemical high-throughput assays combined with immunoinformatics can be used to characterize the peptide-binding motifs of BoLA-I molecules, boosting performance of MHC peptide-binding prediction methods, and empowering rational epitope discovery in cattle. [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]

Vaccination is the most effective means of protecting people from influenza virus infection. The effectiveness of existing vaccines is very limited due to antigenic drift of the influenza virus. Therefore, there is a requirement to develop a universal vaccine that provides broad and long-lasting protection against influenza. CD8+ T-cell response played a vital role in controlling influenza virus infection, reducing viral load, and less clinical syndrome. In this study, we optimized the HA sequences of human seasonal influenza viruses (H1N1, H3N2, Victoria, and Yamagata) by designing multivalent vaccine antigen sets using a mosaic vaccine design strategy and genetic algorithms, and designed an HA mosaic cocktail containing the most potential CTL epitopes of seasonal influenza viruses. We then tested the recombinant mosaic antigen, which has a significant number of potential T-cell epitopes. Results from genetic evolutionary analyses and 3D structural simulations demonstrated its potential to be an effective immunogen. In addition, we have modified an existing neutralizing antibody-based seasonal influenza virus vaccine to include a component that activates cross-protective T cells, which would provide an attractive strategy for improving human protection against seasonal influenza virus drift and mutation and provide an idea for the development of a rationally designed influenza vaccine targeting T lymphocyte immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Zhao, Wang, Li, Sun and Shu.)

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]

Abstract: The classical antigen presentation pathway consists of two monomorphic (proteasome and TAP) and one polymorphic components (MHC Class I). Viruses can escape CTL responses by mutating an epitope so that it is no longer correctly processed by the pathway. Whereas escape mutations that affect MHC binding are typically no longer under selection pressure in the next host of the virus (as hosts differ in their MHC alleles), escape mutations that affect the antigen processing of epitope precursors prevent the use of those epitope precursors by any of the MHC alleles in a host population. Viruses might therefore be under selection pressure to adapt to the monomorphic proteasome and TAP. We designed an agent-based model of a host population, in which an HIV-1 like virus adapts to the antigen presentation pathway of individual hosts, as the virus spreads through the population. We studied how the polymorphism of the MHC and the monomorphism of the proteasome and TAP affected the level of adaptation to the host population that the virus could reach. We found that due to the polymorphism and high specificity of the MHC class I molecules, the CTL epitopes that are targeted by the CTL responses of different hosts do not share many epitope precursors. Therefore, escape mutations in epitope precursors are frequently released from immune selection pressure, and can revert back to the virus wildtype sequence. As a result, the selection pressure on the virus to adapt to the proteasome and TAP is relatively small, which explains the low level of adaptation of the virus to the monomorphic steps in the antigen presentation pathway. [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]

Binding of peptides to major histocompatibility complex (MHC) molecules is the single most selective step in the recognition of pathogens by the cellular immune system. The human MHC genomic region (called HLA) is extremely polymorphic comprising several thousand alleles, each encoding a distinct MHC molecule. The potentially unique specificity of the majority of HLA alleles that have been identified to date remains uncharacterized. Likewise, only a limited number of chimpanzee and rhesus macaque MHC class I molecules have been characterized experimentally. Here, we present NetMHCpan-2.0, a method that generates quantitative predictions of the affinity of any peptide–MHC class I interaction. NetMHCpan-2.0 has been trained on the hitherto largest set of quantitative MHC binding data available, covering HLA-A and HLA-B, as well as chimpanzee, rhesus macaque, gorilla, and mouse MHC class I molecules. We show that the NetMHCpan-2.0 method can accurately predict binding to uncharacterized HLA molecules, including HLA-C and HLA-G. Moreover, NetMHCpan-2.0 is demonstrated to accurately predict peptide binding to chimpanzee and macaque MHC class I molecules. The power of NetMHCpan-2.0 to guide immunologists in interpreting cellular immune responses in large out-bred populations is demonstrated. Further, we used NetMHCpan-2.0 to predict potential binding peptides for the pig MHC class I molecule SLA-1*0401. Ninety-three percent of the predicted peptides were demonstrated to bind stronger than 500 nM. The high performance of NetMHCpan-2.0 for non-human primates documents the method’s ability to provide broad allelic coverage also beyond human MHC molecules. The method is available at . [ABSTRACT FROM AUTHOR]

Abstract: Gag genetic heterogeneity of equine infectious anemia virus (EIAV) variants in naturally infected horses in Canada was studied since very limited information is available on the variability of EIAV Gag sequences in public database. A phylogenetic analysis based on 414nts of Gag gene sequences amplified by a nested polymerase chain reaction (PCR) revealed the distinct divergence of these variants compared to other published strains in a corresponding region. Significant predicted amino acid sequence variations were also identified in an immunorelevant region within this fragment which corresponded to a previously characterized cytotoxic T lymphocytes (CTL) epitope cluster (EC2, aa 77–119). Furthermore, alignment of the predicted full-length Gag protein gene sequences of some of these variants associated with clinical cases of EIA in Canada with the published sequences of EIAV originating from other countries revealed conserved and variant sequences in regions corresponding to other characterized CTL epitope clusters, EC1, EC3 and EC4. Conserved sequences identified among different variant strains might have an important implication for their screening and selection of putative peptide epitopes to mediate relevant immune response and cross protection against divergent field strains of EIAV. [Copyright &y& Elsevier]

Abstract: Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease caused by a novel coronavirus (SARS-CoV), which has overwhelmed more than 30 countries claiming nearly 8400 cases with over 800 fatalities. Thanks to the unprecedented international collaboration, the whole-genomes of SARS-CoVs were successfully deciphered shortly after the identification of the causative pathogen for outbreak of SARS in southern China, in 2003. Hitherto, the SARS-CoV, as a viral paradigm of emerging infectious entities, has been extensively studied that has ranged from epidemiology, molecular virology/immunology to structural genomics. Also, several lines of breakthroughs have been record-brokenly obtained, that included the finding of ACE2, a functional receptor for the SARS-CoV, solution of the 3CLpro structure, a first crystal structure of SARS-related macromolecules, revealing of bats as natural reservoirs for SARS-like viruses and the possible involvement of civet cats in the SARS emergence. This review intends to outline the major progress in the journey of SARS-related exploration, by emphasizing those inaugurated studies with milestone-like significance contributed by Chinese research groups. [Copyright &y& Elsevier]

To simplify the identification method of cytotoxicity T lymphocytek (CTL) epitopes, the binding affinity relationship between peptide and MHC (major histocompatibility complex) molecular was quantitatively studied with the modified artificial neural networks method. The model of predicting the epitopes of CTL was established, and the optimum parameters for the prediction model were obtained. The binding affinity prediction to the 805 peptides samples of HLA-A ∗ 0201 molecular was carried on with this model, and the accuracy of prediction was 73. 8%. The affinity between nonamer of melanin MAGE-2 and MHC is predicted, and the results are reliable. [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]

During the last decade, a large number of tumor-associated antigens (TAA) have been identified, which can be recognized by T cells. This has led to renewed interest in the use of active immunization as a modality for the treatment of cancer. HER-2/neu is a 185-KDa receptor-like glycoprotein that is overexpressed by a variety of tumors including breast, ovarian, lung, prostate and colorectal carcinomata. Several immunogenic HER-2/neu peptides recognized by cytotoxic T lymphocytes (CTL) or helper T lymphocytes (TH) have been identified thus far. Patients with HER-2/neu over-expressing cancers exhibit increased frequencies of peripheral blood T cells recognizing immunogenic HER-2/neu peptides. Various protocols for generating T cell-mediated immune responses specific for HER-2/neu peptides have been examined in pre-clinical models or in clinical trials. Vaccination studies in animals utilizing HER-2/neu peptides have been successful in eliminating tumor growth. In humans, however, although immunological responses have been detected against the peptides used for vaccination, no clinical responses have been described. Because HER-2/neu is a self-antigen, functional immune responses against it may be limited through tolerance mechanisms. Therefore, it would be interesting to determine whether abrogation of tolerance to HER-2/neu using appropriate adjuvants and/or peptide analogs may lead to the development of immune responses to HER-2/neu epitopes that can be of relevance to cancer immunotherapy. Vaccine preparations containing mixtures of HER-2/neu peptides and peptide from other tumor-related antigens might also enhance efficacy of therapeutic vaccination. [ABSTRACT FROM AUTHOR]

Abstract: We previously showed that the known HLA-B27-restricted influenza A epitope identified from human studies, NP.383–391, was recognized by CTLs following influenza A infection of transgenic (Tg) HLA-B27/H2 class I-deficient (H2 DKO) mice. Here, we examined the kinetics of the primary NP.383–391-specific response in Tg HLA-B27/H2 DKO mice at the site of respiratory infection, along with the profile of additional influenza A epitopes recognized. While the temporal kinetics of the Tg HLA-B27/NP.383–391-specific CD8+ T cell response paralleled the H2-Db/NP.366–374-specific response of non-Tg H2b mice, the magnitude was less. Using epitope prediction programs, we identified three novel B27-restricted influenza A epitopes, PB2.702–710, PB1.571–579, and PB2.368–376, recognized during both the primary and secondary response to infection. Although the secondary NP.383–391-specific response was dominant, PB1.571–579 and PB2.368–376 stimulated stronger proliferative expansion in memory T cells. Our results indicate a broader B27/influenza A CTL repertoire than previously known. Together with results for other HLA class I alleles, this information will become important in improving vaccine strategies for influenza A and other human pathogens. [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]

The generation of cytotoxic T lymphocyte (CTL) epitopes from an antigenic sequence involves number of intracellular processes, including production of peptide fragments by proteasome and transport of peptides to endoplasmic reticulum through transporter associated with antigen processing (TAP). In this study, 409 peptides that bind to human TAP transporter with varying affinity were analyzed to explore the selectivity and specificity of TAP transporter. The abundance of each amino acid from P1 to P9 positions in high-, intermediate-, and low-affinity TAP binders were examined. The rules for predicting TAP binding regions in an antigenic sequence were derived from the above analysis. The quantitative matrix was generated on the basis of contribution of each position and residue in binding affinity. The correlation of r = 0.65 was obtained between experimentally determined and predicted binding affinity by using a quantitative matrix. Further a support vector machine (SVM)-based method has been developed to model the TAP binding affinity of peptides. The correlation ( r = 0.80) was obtained between the predicted and experimental measured values by using sequence-based SVM. The reliability of prediction was further improved by cascade SVM that uses features of amino acids along with sequence. An extremely good correlation ( r = 0.88) was obtained between measured and predicted values, when the cascade SVM-based method was evaluated through jackknife testing. A Web service, TAPPred ( or ), has been developed based on this approach. [ABSTRACT FROM AUTHOR]

Currently, there is a lack of suitable pre-clinical mouse models for testing and optimization of experimental cancer vaccines. Here, in situ developed mammary tumors of MMTV-v-Ha-ras and MMTV-c-myc transgenic mice and normal mammary, liver, spleen, and testis were screened for expression of tumor-associated antigens (TAA) Mage-b1/2/3 by reverse-transcriptase polymerase chain reaction (RT-PCR) and Southern blot hybridization. Mage-b1/2/3 are homologues of the human TAA MAGE-B1/2/3. Expression of these human MAGE genes has been found in tumors of various histological types, including breast cancer [39]. Mage-specific RT-PCR products (using primers that amplify all three Mage-b1/2/3) were detected in mammary tumors of the MMTV-v-Ha-ras and MMTV-c-myc transgenic mice and in testis, but not in other normal tissues. RT-PCR products obtained from the mammary tumors (using primers that amplify the complete protein-encoding region of Mage-b1/2/3) were cloned and sequenced, and appeared to be most homologous with Mage-b3. Comparison of the Mage-b3 gene in mammary tumors and normal tissues suggest that somatic mutations did not occur in the Mage-b3 gene of the ras- and myc-induced mammary tumors. In addition, no differences were found between the Mage-b3 cDNA of testis, the only normal tissue that expresses Mage-b3, and Mage-b3 in genomic DNA of normal kidney, where Mage-b3 is silent. The MMTV-v-Ha-ras and MMTV-c-myc transgenic mice of this study are the first immune competent mouse models with in situ developed mammary tumors in which the expression of Mage-b3 TAA has been demonstrated. This makes them potentially suitable as a mouse model for pre-clinical testing of Mage-specific cancer vaccines in vivo. [ABSTRACT FROM AUTHOR]

Focuses on the importance of simian immunodeficiency virus (SIV) infection of rhesus macaques as a model for the study of HIV infection in humans. Importance in controlling viral replication in infected hosts; Expansion on the utility of the rhesus macaque as a model for vaccine and pathogenesis study; Understanding on the importance of cytotoxic T lymphocytes responses during SIV infection.

Abstract: Several cytotoxic T lymphocyte peptide-based vaccines against hepatitis B, human immunodeficiency virus and melanoma were recently studied in clinical trials. One interesting melanoma vaccine candidate alone or in combination with other tumor antigens, is the decapeptide ELA. This peptide is a Melan-A/MART-1 antigen immunodominant peptide analog, with an N-terminal glutamic acid. It has been reported that the amino group and γ-carboxylic group of glutamic acids, as well as the amino group and γ-carboxamide group of glutamines, condense easily to form pyroglutamic derivatives. To overcome this stability problem, several peptides of pharmaceutical interest have been developed with a pyroglutamic acid instead of N-terminal glutamine or glutamic acid, without loss of pharmacological properties. Unfortunately compared with ELA, the pyroglutamic acid derivative (PyrELA) and also the N-terminal acetyl-capped derivative (AcELA) failed to elicit cytotoxic T lymphocyte (CTL) activity. Despite the apparent minor modifications introduced in PyrELA and AcELA, these two derivatives probably have lower affinity than ELA for the specific class I major histocompatibility complex. Consequently, in order to conserve full activity of ELA, the formation of PyrELA must be avoided. Furthermore, this stability problem is worse in the case of clinical grade ELA, produced as an acetate salt, like most of the pharmaceutical grade peptides. We report here that the hydrochloride salt, shows higher stability than the acetate salt and may be suitable for use in man. Similar stability data were also obtained for MAGE-3, another N-terminal glutamic acid containing CTL peptide in clinical development, leading us to suggest that all N-terminal glutamic acid and probably glutamine-containing CTL peptide epitopes may be stabilized as hydrochloride salts. [ABSTRACT FROM AUTHOR]

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]