Deep Sequencing of the HIV-1 Polymerase Gene for Characterisation of Cytotoxic T-Lymphocyte Epitopes During Early and Chronic Disease Stages

Background Despite multiple attempts, there is still no effective HIV-1 vaccine available. The HIV-1 polymerase (pol) gene is highly conserved and encodes cytotoxic T-lymphocyte (CTL) epitopes. The aim of the study was to characterise HIV-1 Pol CTL epitopes in mostly sample pairs obtained during early and chronic stages of infection. Methods Illumina deep sequencing was conducted for all samples while Sanger sequencing was only performed on baseline samples. Codons under immune selection pressure were assessed by computing nonsynonymous to synonymous mutation ratios using MEGA. Minority CTL epitope variants occurring at \(\ge\)5% were detected using low-frequency variant tool in CLC Genomics. Los Alamos HIV database was used for mapping mutations to known HIV-1 CTL epitopes. Results Fifty-two participants were enrolled in the study. Their median age was 28 years (interquartile range: 24–32 years) and majority of participants (92.3%) were female. Illumina minority variant analysis identified a significantly higher number of CTL epitopes (n = 65) compared to epitopes (n = 8) identified through Sanger sequencing. Most of the identified epitopes mapped to reverse transcriptase (RT) and integrase (IN) regardless of sequencing method. There was a significantly higher proportion of minority variant epitopes in RT (n = 39, 60.0%) compared to IN (n = 17, 26.2%) and PR (n = 9, 13.8%), p = 0.002 and < 0.0001, respectively. However, no significant difference was observed between the proportion of minority variant epitopes in IN versus PR, p = 0.06. Some epitopes were detected in either early or chronic HIV-1 infection whereas others were detected in both stages. Different distribution patterns of minority variant epitopes were observed in sample pairs; with some increasing or decreasing over time, while others remained constant. Some of the identified epitopes have not been previously reported for HIV-1 subtype C. There were also variants that could not be mapped to reported CTL epitopes in the Los Alamos HIV database. Conclusion Deep sequencing revealed many Pol CTL epitopes, including some not previously reported for HIV-1 subtype C. The findings of this study support the inclusion of RT and IN epitopes in HIV-1 vaccine candidates as these proteins harbour many CTL epitopes.

Exploring the structural basis to develop efficient multi-epitope vaccines displaying interaction with HLA and TAP and TLR3 molecules to prevent NIPAH infection, a global threat to human health

Nipah virus (NiV) is an emerging zoonotic virus responsible to cause several serious outbreaks in South Asian region with high mortality rate of 40 to 90% since 2001. NiV infection causes lethal encephalitis and respiratory disease with the symptom of endothelial cell-cell fusion. No specific vaccine has yet been reported against NiV infection. Recently, some Multi-Epitope Vaccines (MEV) has been proposed but they involve limited number of epitopes which further limits the potential of vaccine. To address the urgent need for a specific and effective vaccine against NiV infection, in the present study, we have design two multi-epitope vaccines (MEVs) composed of 33 Cytotoxic T lymphocyte (CTL) epitopes and 38 Helper T lymphocyte (HTL) epitopes. Both the MEVs carry potential B cell linear epitope overlapping regions, B cell discontinuous epitopes as well as IFN-γ inducing epitopes. Hence the designed MEVs carry potential to elicit cell-mediated as well as humoral immune response. Selected CTL and HTL epitopes were validated for their stable molecular interactions with HLA class I and II alleles as well as in case of CTL epitopes, with human transporter associated with antigen processing (TAP). Human β-defensin 2 and β-defensin 3 were used as adjuvants to enhance the immune response of both the MEVs. The molecular dynamics simulation study of MEVs-TLR3(ECD) (Toll-Like Receptor 3 Ectodomain) complex indicated stable molecular interaction. Further, the codon optimized cDNA of both the MEVs has shown high expression potential in the mammalian host cell line (Human). Hence for further studies, both the design of CTL and HTL MEVs could be cloned, expressed and tried for in-vivo validations (animal trails) as potential vaccine candidates against NiV infection.

Deep sequencing of the HIV-1 polymerase gene for characterisation of cytotoxic T-lymphocyte epitopes during early and chronic disease stages

Background Despite multiple attempts, there is still no effective HIV-1 vaccine available. The HIV-1 polymerase (pol) gene is highly conserved and encodes cytotoxic T-lymphocyte (CTL) epitopes. In this study, deep sequencing was employed for characterisation of HIV-1 Pol CTL epitopes in mostly paired samples obtained during early and chronic stages of infection. Deep sequencing data was then compared to Sanger sequencing data only in samples obtained at baseline. Results Fifty-two participants were enrolled in the study. Their median age was 28 years (interquartile range: 24–32 years) and the majority of participants (92.3%) were female. Illumina minority variant analysis identified a significantly higher number of CTL epitopes (n = 65) compared to epitopes (n = 8) identified through Sanger sequencing. Most of the identified epitopes mapped to reverse transcriptase (RT) and integrase (IN) regardless of the method of sequencing. There was a significantly higher proportion of minority variant epitopes in RT (n = 39, 60.0%) compared to IN (n = 17, 26.2%) and PR (n = 9, 13.8%), p = 0.002 and < 0.0001, respectively. However, no significant difference was observed between the proportion of minority variant epitopes in IN versus PR, p = 0.06. Some epitopes were detected in either early or chronic HIV-1 infection whereas others were detected in both stages. Different distribution patterns of minority variant epitopes were observed in sample pairs; with some increasing or decreasing over time, while others remained constant. Some of the identified epitopes have not been previously reported for HIV-1 subtype C. There were also variants that could not be mapped to reported CTL epitopes in the Los Alamos HIV database. Conclusion Deep sequencing revealed many Pol CTL epitopes, including some not previously reported for HIV-1 subtype C. The findings of this study support the inclusion of RT and IN epitopes in HIV-1 vaccine candidates as these proteins harbour many CTL epitopes. Variants that were not mapped within CTL epitopes could represent new epitopes.

Predicting Multi-Epitope Peptide Cancer Vaccine from Novel TAA Topo48

Cancer is one of the most lethal diseases. Recently, cancer immunotherapy has a tremendous achievement in cancer treatment. A certain number of cancer based epitope vaccines with different moiety have been discovered. In japan, several clinical tests of cancer based epitope vaccine derived from tumor associated antigens (TAAs) are now ongoing or have recently been completed. a novel of TAAs potentially as cancer vaccine have been retrieved from a fragment weighed 48kDa derived from human DNA-topoisomerase 1 (TOP1) called Topo48. Therefore, it is still critical to discover a derived Topo48 epitope based cancer vaccine. Immuno-informatics considered as a methods noted to have better accuracy to design promising vaccine candidates. Here, continuous and discontinuous B-cell epitopes following with CTL epitopes and their docking interaction to major histocompatibility complex (MHC) class I Human Leukocyte Antigens (HLA)- A0201 were predicted. Kolaskar-Tongaonkar’s, Emini’s, Karpus-Schulz’s, and Parker’s methods were used to predict continuous B-cell epitopes while ElliPro was used for prediction of discontinued B-cell epitopes. Those considered methods marked to have better accuracy to design promising vaccine candidates. Similarly, CTL epitopes was also predicted by using NetCTL server and the best candidates were further investigated their binding affinity by mean of PEP-FOLD3, PatchDock rigid-body docking server, and FireDock server. Total 27 continuous epitopes and 7 discontinuous B-cell epitopes were predicted. In the other hand, 9 peptides were predicted as CTL epitopes. Whereas, three predicted CTL epitope in range 263MLDHEYTTK27, 755AIDMADEDY763, 715ALGTSKLNY724) exhibited good interactions to HLA-A0201. Moreover, we also found residues His266, Thr270, Ala755, Tyr723, Thr718, Ser719, Lys720 from Topo48 and residues Thr163, Asp757, His70, Glu63 from HLA- A0201 were indicated to be antigenic. Ultimately, our proposed continuous/discontinuous B-cell epitopes, and also CTL epitopes can be potential vaccines for cancer immunotherapy.

Immunoinformatics and Molecular Docking Studies Predicted Potential Multiepitope-Based Peptide Vaccine and Novel Compounds against Novel SARS-CoV-2 through Virtual Screening

Background. Coronaviruses (CoVs) are enveloped positive-strand RNA viruses which have club-like spikes at the surface with a unique replication process. Coronaviruses are categorized as major pathogenic viruses causing a variety of diseases in birds and mammals including humans (lethal respiratory dysfunctions). Nowadays, a new strain of coronaviruses is identified and named as SARS-CoV-2. Multiple cases of SARS-CoV-2 attacks are being reported all over the world. SARS-CoV-2 showed high death rate; however, no specific treatment is available against SARS-CoV-2. Methods. In the current study, immunoinformatics approaches were employed to predict the antigenic epitopes against SARS-CoV-2 for the development of the coronavirus vaccine. Cytotoxic T-lymphocyte and B-cell epitopes were predicted for SARS-CoV-2 coronavirus protein. Multiple sequence alignment of three genomes (SARS-CoV, MERS-CoV, and SARS-CoV-2) was used to conserved binding domain analysis. Results. The docking complexes of 4 CTL epitopes with antigenic sites were analyzed followed by binding affinity and binding interaction analyses of top-ranked predicted peptides with MHC-I HLA molecule. The molecular docking (Food and Drug Regulatory Authority library) was performed, and four compounds exhibiting least binding energy were identified. The designed epitopes lead to the molecular docking against MHC-I, and interactional analyses of the selected docked complexes were investigated. In conclusion, four CTL epitopes (GTDLEGNFY, TVNVLAWLY, GSVGFNIDY, and QTFSVLACY) and four FDA-scrutinized compounds exhibited potential targets as peptide vaccines and potential biomolecules against deadly SARS-CoV-2, respectively. A multiepitope vaccine was also designed from different epitopes of coronavirus proteins joined by linkers and led by an adjuvant. Conclusion. Our investigations predicted epitopes and the reported molecules that may have the potential to inhibit the SARS-CoV-2 virus. These findings can be a step towards the development of a peptide-based vaccine or natural compound drug target against SARS-CoV-2.

Immunoinformatic based identification of cytotoxic T lymphocyte epitopes from the Indian isolate of SARS-CoV-2

The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has turned into a pandemic with about thirty million confirmed cases worldwide as of September 2020. Being an airborne infection, it can be catastrophic to populous countries like India. This study sets to identify potential cytotoxic T lymphocyte (CTL) epitopes in the SARS-CoV-2 Indian isolate which can act as an effective vaccine epitope candidate for the majority of the Indian population. The immunogenicity and the foreignness of the epitopes towards the human body have to be studied to further confirm their candidacy. The top-scoring epitopes were subjected to molecular docking studies to study their interactions with the corresponding human leukocyte antigen (HLA) system. The CTL epitopes were observed to bind at the peptide-binding groove of the corresponding HLA system, indicating their potency as an epitope candidate. The candidacy was further analyzed using sequence conservation studies and molecular dynamics simulation. The identified epitopes can be subjected to further studies for the development of the SARS-CoV-2 vaccine.

Rational Design of a Pan-Coronavirus Vaccine Based on Conserved CTL Epitopes

With the rapid global spread of the Coronavirus Disease 2019 (COVID-19) pandemic, a safe and effective vaccine against human coronaviruses (HCoVs) is believed to be a top priority in the field of public health. Due to the frequent outbreaks of different HCoVs, the development of a pan-HCoVs vaccine is of great value to biomedical science. The antigen design is a key prerequisite for vaccine efficacy, and we therefore developed a novel antigen with broad coverage based on the genetic algorithm of mosaic strategy. The designed antigen has a potentially broad coverage of conserved cytotoxic T lymphocyte (CTL) epitopes to the greatest extent, including the existing epitopes from all reported HCoV sequences (HCoV-NL63, HCoV-229E, HCoV-OC43, HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2). This novel antigen is expected to induce strong CTL responses with broad coverage by targeting conserved epitopes against multiple coronaviruses.