Novel "GaEl Antigenic Patches" Identified by a "Reverse Epitomics" Approach to Design Multipatch Vaccines against NIPAH Infection, a Silent Threat to Global Human Health.

Nipah virus (NiV) is a zoonotic virus that causes lethal encephalitis and respiratory disease with the symptom of endothelial cell-cell fusion. Several NiV outbreaks have been reported since 1999 with nearly annual occurrences in Bangladesh. The outbreaks had high mortality rates ranging from 40 to 90%. No specific vaccine has yet been reported against NiV. Recently, several vaccine candidates and different designs of vaccines composed of epitopes against NiV were proposed. Most of the vaccines target single protein or protein complex subunits of the pathogen. The multiepitope vaccines proposed also cover a largely limited number of epitopes, and hence, their efficiency is still uncertain. To address the urgent need for a specific and effective vaccine against NiV infection, in the present study, we have utilized the "reverse epitomics" approach ("overlapping-epitope-clusters-to-patches" method) to identify "antigenic patches" (Ag-Patches) and utilize them as immunogenic composition for multipatch vaccine (MPV) design. The designed MPVs were analyzed for immunologically crucial parameters, physiochemical properties, and interaction with Toll-like receptor 3 ectodomain. In total, 30 CTL (cytotoxic T lymphocyte) and 27 HTL (helper T lymphocyte) antigenic patches were identified from the entire NiV proteome based on the clusters of overlapping epitopes. These identified Ag-Patches cover a total of discrete 362 CTL and 414 HTL epitopes from the entire proteome of NiV. The antigenic patches were utilized as immunogenic composition for the design of two CTL and two HTL multipatch vaccines. The 57 antigenic patches utilized here cover 776 overlapping epitopes targeting 52 different HLA class I and II alleles, providing a global ethnically distributed human population coverage of 99.71%. Such large number of epitope coverage resulting in large human population coverage cannot be reached with single-protein/subunit or multiepitope based vaccines. The reported antigenic patches also provide potential immunogenic composition for early detection diagnostic kits for NiV infection. Further, all the MPVs and Toll-like receptor ectodomain complexes show a stable nature of molecular interaction with numerous hydrogen bonds, salt bridges, and nonbounded contact formation and acceptable root mean square deviation and fluctuation. The cDNA analysis shows a favorable large-scale expression of the MPV constructs in a human cell line. By utilizing the novel "reverse epitomics" approach, highly immunogenic novel "GaEl antigenic patches" (GaEl Ag-Patches), a synonym term for "antigenic patches", were identified and utilized as immunogenic composition to design four MPVs against NiV. We conclude that the novel multipatch vaccines are potential candidates to combat NiV, with greater effectiveness, high specificity, and large human population coverage worldwide.

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 that caused several serious outbreaks in the south asian region with high mortality rates ranging from 40 to 90% since 2001. NiV infection causes lethal encephalitis and respiratory disease with the symptom of endothelial cell-cell fusion. No specific and effective vaccine has yet been reported against NiV. To address the urgent need for a specific and effective vaccine against NiV infection, in the present study, we have designed two Multi-Epitope Vaccines (MEVs) composed of 33 Cytotoxic T lymphocyte (CTL) epitopes and 38 Helper T lymphocyte (HTL) epitopes. Out of those CTL and HTL combined 71 epitopes, 61 novel epitopes targeting nine different NiV proteins were not used before for vaccine design. Codon optimization for the cDNA of both the designed MEVs might ensure high expression potential in the human cell line as stable proteins. Both MEVs carry potential B cell linear epitope overlapping regions, B cell discontinuous epitopes as well as IFN-γ inducing epitopes. Additional criteria such as sequence consensus amongst CTL, HTL and B Cell epitopes was implemented for the design of final constructs constituting MEVs. Hence, the designed MEVs carry the potential to elicit cell-mediated as well as humoral immune response. Selected overlapping CTL and HTL epitopes were validated for their stable molecular interactions with HLA class I and II alleles and in case of CTL epitopes with human Transporter Associated with antigen Processing (TAP) cavity. The structure based epitope cross validation for interaction with TAP cavity was used as another criteria choosing final epitopes for NiV MEVs. Finally, human Beta-defensin 2 and Beta-defensin 3 were used as adjuvants to enhance the immune response of both the MEVs. Molecular dynamics simulation studies of MEVs-TLR3 ectodomain (Human Toll-Like Receptor 3) complex indicated the stable molecular interaction. We conclude that the MEVs designed and in silico validated here could be highly potential vaccine candidates to combat NiV infections, with great effectiveness, high specificity and large human population coverage worldwide.

Cyclic constrained immunoreactive peptides from crucial P. falciparum proteins: potential implications in malaria diagnostics.

Malaria is still a global challenge with significant morbidity and mortality, especially in the African, South-East Asian, and Latin American regions. Malaria diagnosis is a crucial pillar in the control and elimination efforts, often accomplished by the administration of mass-scale Rapid diagnostic tests (RDTs). The inherent limitations of RDTs- insensitivity in scenarios of low transmission settings and deletion of one of the target proteins- Histidine rich protein 2/3 (HRP-2/3) are evident from multiple reports, thus necessitating the need to explore novel diagnostic tools/targets. The present study used peptide microarray to screen potential epitopes from 13 antigenic proteins (CSP, EXP1, LSA1, TRAP, AARP, AMA1, GLURP, MSP1, MSP2, MSP3, MSP4, P48/45, HAP2) of P. falciparum. Three cyclic constrained immunoreactive peptides- C6 (EXP1), A8 (MSP2), B7 (GLURP) were identified from 5458 cyclic constrained peptides (in duplicate) against P. falciparum-infected sera. Peptides (C6, A8, B7- cyclic constrained) and (G11, DSQ, NQN- corresponding linear peptides) were fairly immunoreactive towards P. falciparum-infected sera in dot-blot assay. Using direct ELISA, cyclic constrained peptides (C6 and B7) were found to be specific to P. falciparum-infected sera. A substantial number of samples were tested and the peptides successfully differentiated the P. falciparum positive and negative samples with high confidence. In conclusion, the study identified 3 cyclic constrained immunoreactive peptides (C6, B7, and A8) from P. falciparum secretory/surface proteins and further validated for diagnostic potential of 2 peptides (C6 and B7) with field-collected P. falciparum-infected sera samples.

Computationally validated SARS-CoV-2 CTL and HTL Multi-Patch vaccines, designed by reverse epitomics approach, show potential to cover large ethnically distributed human population worldwide.

The SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is responsible for the COVID-19 outbreak. The highly contagious COVID-19 disease has spread to 216 countries in less than six months. Though several vaccine candidates are being claimed, an effective vaccine is yet to come. A novel reverse epitomics approach, 'overlapping-epitope-clusters-to-patches' method is utilized to identify the antigenic regions from the SARS-CoV-2 proteome. These antigenic regions are named as 'Ag-Patch or Ag-Patches', for Antigenic Patch or Patches. The identification of Ag-Patches is based on the clusters of overlapping epitopes rising from SARS-CoV-2 proteins. Further, we have utilized the identified Ag-Patches to design Multi-Patch Vaccines (MPVs), proposing a novel method for the vaccine design. The designed MPVs were analyzed for immunologically crucial parameters, physiochemical properties and cDNA constructs. We identified 73 CTL (Cytotoxic T-Lymphocyte) and 49 HTL (Helper T-Lymphocyte) novel Ag-Patches from the proteome of SARS-CoV-2. The identified Ag-Patches utilized to design MPVs cover 768 overlapping epitopes targeting 55 different HLA alleles leading to 99.98% of world human population coverage. The MPVs and Toll-Like Receptor ectodomain complex shows stable complex formation tendency. Further, the cDNA analysis favors high expression of the MPVs constructs in a human cell line. We identified highly immunogenic novel Ag-Patches from the entire proteome of SARS CoV-2 by a novel reverse epitomics approach and utilized them to design MPVs. We conclude that the novel MPVs could be a highly potential novel approach to combat SARS-CoV-2, with greater effectiveness, high specificity and large human population coverage worldwide. Communicated by Ramaswamy H. Sarma.

Computational study of novel inhibitory molecule, 1-(4-((2S,3S)-3-amino-2-hydroxy-4-phenylbutyl)piperazin-1-yl)-3-phenylurea, with high potential to competitively block ATP binding to the RNA dependent RNA polymerase of SARS-CoV-2 virus.

For coronaviruses, RNA-dependent RNA polymerase (RdRp) is an essential enzyme that catalyses the replication from RNA template and therefore remains an attractive therapeutic target for anti-COVID drug discovery. In the present study, we performed a comprehensive in silico screening for 16,776 potential molecules from recently established drug libraries based on two important pharmacophores (3-amino-4-phenylbutan-2-ol and piperazine). Based on initial assessment, 4042 molecules were obtained suitable as drug candidates, which were following Lipinski's rule. Molecular docking implemented for the analysis of molecular interactions narrowed this number of compounds down to 19. Subsequent to screening filtering criteria and considering the critical parameters viz. docking score and MM-GBSA binding free energy, 1-(4-((2S,3S)-3-amino-2-hydroxy-4-phenylbutyl)piperazin-1-yl)-3-phenylurea (compound 1) was accomplished to score highest in comparison to the remaining 18 shortlisted drug candidates. Notably, compound 1 displayed higher docking score (-8.069 kcal/mol) and MM-GBSA binding free energy (-49.56 kcal/mol) than the control drug, remdesivir triphosphate, the active form of remdesivir as well as adenosine triphosphate. Furthermore, a molecular dynamics simulation was carried out (100 ns), which substantiated the candidacy of compound 1 as better inhibitor. Overall, our systematic in silico study predicts the potential of compound 1 to exhibit a more favourable specific activity than remdesivir triphosphate. Hence, we suggest compound 1 as a novel potential drug candidate, which should be considered for further exploration and validation of its potential against SARS-CoV-2 in wet lab experimental studies.Communicated by Ramasawamy H. Sarma.

Vaccines Targeting Numerous Coronavirus Antigens, Ensuring Broader Global Population Coverage: Multi-epitope and Multi-patch Vaccines.

Coronaviruses are causative agents of different zoonosis including SARS, MERS, or COVID-19 in humans. The high transmission rate of coronaviruses, the time-consuming development of efficient anti-infectives and vaccines, the possible evolutionary adaptation of the virus to conventional vaccines, and the challenge to cover broad human population worldwide are the major reasons that made it challenging to avoid coronaviruses outbreaks. Although, a plethora of different approaches are being followed to design and develop vaccines against coronaviruses, most of them target subunits, full-length single, or only a very limited number of proteins. Vaccine targeting multiple proteins or even the entire proteome of the coronavirus is yet to come. In the present chapter, we will be discussing multi-epitope vaccine (MEV) and multi-patch vaccine (MPV) approaches to design and develop efficient and sustainably successful strategies against coronaviruses. MEV and MPV utilize highly conserved, potentially immunogenic epitopes and antigenic patches, respectively, and hence they have the potential to target large number of coronavirus proteins or even its entire proteome, allowing us to combat the challenge of its evolutionary adaptation. In addition, the large number of human leukocyte antigen (HLA) alleles targeted by the chosen specific epitopes enables MEV and MPV to cover broader global population.

Structural Basis for Designing Multiepitope Vaccines Against COVID-19 Infection: In Silico Vaccine Design and Validation.

BACKGROUND: The novel coronavirus disease (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to the ongoing 2019-2020 pandemic. SARS-CoV-2 is a positive-sense single-stranded RNA coronavirus. Effective countermeasures against SARS-CoV-2 infection require the design and development of specific and effective vaccine candidates. OBJECTIVE: To address the urgent need for a SARS-CoV-2 vaccine, in the present study, we designed and validated one cytotoxic T lymphocyte (CTL) and one helper T lymphocyte (HTL) multi-epitope vaccine (MEV) against SARS-CoV-2 using various in silico methods. METHODS: Both designed MEVs are composed of CTL and HTL epitopes screened from 11 Open Reading Frame (ORF), structural and nonstructural proteins of the SARS-CoV-2 proteome. Both MEVs also carry potential B-cell linear and discontinuous epitopes as well as interferon gamma-inducing epitopes. To enhance the immune response of our vaccine design, truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1 was used as an adjuvant at the N termini of both MEVs. The tertiary models for both the designed MEVs were generated, refined, and further analyzed for stable molecular interaction with toll-like receptor 3. Codon-biased complementary DNA (cDNA) was generated for both MEVs and analyzed in silico for high level expression in a mammalian (human) host cell line. RESULTS: In the present study, we screened and shortlisted 38 CTL, 33 HTL, and 12 B cell epitopes from the 11 ORF protein sequences of the SARS-CoV-2 proteome. Moreover, the molecular interactions of the screened epitopes with their respective human leukocyte antigen allele binders and the transporter associated with antigen processing (TAP) complex were positively validated. The shortlisted screened epitopes were utilized to design two novel MEVs against SARS-CoV-2. Further molecular models of both MEVs were prepared, and their stable molecular interactions with toll-like receptor 3 were positively validated. The codon-optimized cDNAs of both MEVs were also positively analyzed for high levels of overexpression in a human cell line. CONCLUSIONS: The present study is highly significant in terms of the molecular design of prospective CTL and HTL vaccines against SARS-CoV-2 infection with potential to elicit cellular and humoral immune responses. The epitopes of the designed MEVs are predicted to cover the large human population worldwide (96.10%). Hence, both designed MEVs could be tried in vivo as potential vaccine candidates against SARS-CoV-2.

Design of novel multi-epitope vaccines against severe acute respiratory syndrome validated through multistage molecular interaction and dynamics.

Severe acute respiratory syndrome (SARS) is endemic in South China and is continuing to spread worldwide since the 2003 outbreak, affecting human population of 37 countries till present. SARS is caused by the severe acute respiratory syndrome Coronavirus (SARS-CoV). In the present study, we have designed two multi-epitope vaccines (MEVs) composed of cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL) and B cell epitopes overlap, bearing the potential to elicit cellular as well as humoral immune response. We have used truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1 as molecular adjuvants at N-terminal of both the MEVs. Selected overlapping epitopes of both the MEVs were further validated for stable molecular interactions with their respective human leukocyte antigen class I and II allele binders. Moreover, CTL epitopes were further studied for their molecular interaction with transporter associated with antigen processing. Furthermore, after tertiary structure modelling, both the MEVs were validated for their stable molecular interaction with Toll-like receptors 2 and 4. Codon-optimized cDNA of both the MEVs was analysed for their potential high level of expression in the mammalian cell line (Human) needed for their further in vivo testing. Overall, the present study proposes in silico validated design of two MEVs against SARS composed of specific epitopes with the potential to cause a high level of SARS-CoV specific cellular as well as humoral immune response. Communicated by Ramaswamy H. Sarma.

Structural basis of development of multi-epitope vaccine against Middle East respiratory syndrome using in silico approach.

BACKGROUND: Middle East respiratory syndrome (MERS) is caused by MERS coronavirus (MERS-CoV). Thus far, MERS outbreaks have been reported from Saudi Arabia (2013 and 2014) and South Korea (2015). No specific vaccine has yet been reported against MERS. PURPOSE: To address the urgent need for an MERS vaccine, in the present study, we have designed two multi-epitope vaccines (MEVs) against MERS utilizing several in silico methods and tools. METHODS: The design of both the multi-epitope vaccines (MEVs) are composed of cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) epitopes, screened form thirteen different proteins of MERS-CoV. Both the MEVs also carry potential B-cell linear epitope regions, B-cell discontinuous epitopes as well as interferon-γ-inducing epitopes. Human ß-defensin-2 and ß-defensin-3 were used as adjuvants to enhance the immune response of MEVs. To design the MEVs, short peptide molecular linkers were utilized to link screened most potential CTL epitopes, HTL epitopes and the adjuvants. Tertiary models for both the MEVs were generated, refined, and further studied for their molecular interaction with toll-like receptor 3. The cDNAs of both MEVs were generated and analyzed in silico for their expression in a mammalian host cell line (human). RESULTS: Screened CTL and HTL epitopes were found to have high propensity for stable molecular interaction with HLA alleles molecules. CTL epitopes were also found to have favorable molecular interaction within the cavity of transporter associated with antigen processing. The selected CTL and HTL epitopes jointly cover upto 94.0% of worldwide human population. Both the CTL and HTL MEVs molecular models have shown to have stable binding and complex formation propensity with toll-like receptor 3. The cDNA analysis of both the MEVs have shown high expression tendency in mammalian host cell line (human). CONCLUSION: After multistage in silico analysis, both the MEVs are predicted to elicit humoral as well as cell mediated immune response. Epitopes of the designed MEVs are predicted to cover large human population worldwide. Hence both the designed MEVs could be tried in vivo as potential vaccine candidates against MERS.