CTL Epitopes Research Articles

In Silico Characterization of an Important Metacyclogenesis Marker in Leishmania donovani, HASPB1, as a Potential Vaccine Candidate.

Visceral leishmaniasis is a life-threatening infectious disease worldwide. Extensive experiments have been done to introduce potential vaccine candidates to combat leishmaniasis. The present study was done to evaluate Leishmania donovani hydrophilic acylated surface protein B1 as a potential vaccine candidate using in silico methods. For this aim, server-based predictions were performed regarding physicochemical characteristics, solubility, antigenicity, allergenicity, signal peptide, transmembrane domain, and posttranslational modifications (PTMs). Also, secondary and tertiary structures were predicted using NetSurfP-3.0 and I-TASSER, respectively. The 3D model was further subjected to refinement and validation, and promising B-cell, cytotoxic T-lymphocyte (CTL; human, dog), and helper T-lymphocyte (HTL; human) epitopes were predicted. The protein had a molecular weight of 42.19 kDa, with high solubility (0.749), stability (instability index: 21.34), and hydrophilicity (GRAVY: -2.322). No signal peptide or transmembrane domain was predicted, and the most abundant PTMs were phosphorylation, O-glycosylation, and acetylation. Many coils and disordered regions existed in the secondary structure analysis, and the tertiary model had a good confidence score (-0.79). Next, the ProSA-web and PROCHECK tools showed adequate improvements in the refined model compared to the crude model. Only 4 shared B-cell epitopes among three web servers (ABCpred, BepiPred 2.0, and SVMTriP) were shown to be antigenic, nonallergenic, and with good water solubility. Also, five potent CTL epitopes in dogs and five in humans were predicted. Notably, two HTL epitopes were found to be potential IFN-γ inducers. In conclusion, our results demonstrated several immunogenic epitopes in this protein, which could be directed towards multiepitope vaccine design.

Sporozoite glycoprotein antigen (SGA) of Cryptosporidium parvum is a promising vaccine target for prevention of cryptosporidiosis In silico analysis of C. parvum SGA protein

Cryptosporidiosis is a zoonotic parasitic infection causing diarrhea in humans and farm animals. The present study was done to computationally analyze the Cryptosporidium parvum sporozoite glycoprotein antigen (SGA) regarding immunogenic epitopes, as a potential vaccine candidate. Physico-chemical properties, solubility, antigenicity, allergenicity, signal peptide, transmembrane domain and post-translational modifications (PTMs) along with secondary and tertiary structure analyses were predicted. Also, linear B-cell epitopes, cytotoxic T-lymphocyte (CTL; human, cattle) and helper T-lymphocyte (HTL; human) epitopes were forecasted. The molecular weight of this 265-residue protein was 27.14 kDa, with hydrophilicity (GRAVY: 0.700), high solubility (0.892), and aliphatic index (46.83). No signal peptide and transmembrane domain was predicted, and the most abundant PTMs were phosphorylation (n = 68) and O-glycosylation (n = 53). Coils were dominant in secondary structure analysis, and the tertiary model refinement showed enhancement in the refined than crude model. Only 4 shared B-cell epitopes among three web servers (ABCpred, BepiPred 2.0, and SVMTriP) were shown to be antigenic, non-allergenic, with good water solubility. Also, five potent CTL epitopes in humans and two in cattle were predicted. Notably, four HTL epitopes were found as inducers of IFN-γ, IL-4 or both. In conclusion, our results demonstrated several immunogenic epitopes in this protein, which could be directed towards multi-epitope vaccine design.

An In-Silico Reverse Vaccinology Approach in Predicting Vaccine Candidate Against Mycobacterium tuberculosis CCDC5180

A quarter of the world’s population is thought to be latently infected with Mycobacterium tuberculosis, creating a vast pool of possible future illnesses. Combined with the rising prevalence of multi-drug resistant Mycobacterium tuberculosis (Mtb) strains, TB poses one of the biggest challenges for world health, and the TB epidemic can only be eradicated with a new and more effective vaccine strategy. Since the Bacille Calmette-Guérin (BCG) vaccine is the only one used to treat tuberculosis (TB), Reverse vaccine technology aims to accelerate the development of subunit vaccines by identifying specific proteins in a pathogenic bacterial proteome that may be protective antigens. This approach was employed on four extracellular proteins namely Secreted fibronectin-binding protein C antigen 85-C, PE-PGRS family protein, Invasion protein RipA, and Invasion protein RipB. The shortlisted proteins were subjected to B-cell, CTL, and HTL epitope prediction. To further identify the most promising epitopes, specialized filtering techniques were employed to narrow down CTL epitopes that were non-allergenic, non-toxic, and antigenic as well as B-cell epitopes that produced antibodies. Similarly, HTL epitopes that generate IFN- γ but not IL-10 were selected. The analysis revealed invasion protein RipA as a potential candidate for Mycobacterium tuberculosis CCDC5180 immunization.

The Parallel Presentation of Two Functional CTL Epitopes Derived from the O and Asia 1 Serotypes of Foot-and-Mouth Disease Virus and Swine SLA-2*HB01: Implications for Universal Vaccine Development.

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

PP 1.13 – 00078 CTL epitopes from structurally important hiv proteins are identified in rebound HIV

PP 1.13 – 00078 CTL epitopes from structurally important hiv proteins are identified in rebound HIV

Design of a multi-epitope protein as a subunit vaccine against lumpy skin disease using an immunoinformatics approach

Lumpy skin disease (LSD) is a transboundary viral disease of cattle that causes substantial economic loss globally. There is no specific treatment and subunit vaccine for this disease to date. Reports of the global spread of this disease are worrisome. We designed a multi-epitope protein using an immunoinformatics approach in this study. We analyzed the proteome of LSDV and found 32 structural/surface proteins. Four of these 32 proteins were predicted as antigenic and non-homologous to bovine and highly conserved in 26 LSDV isolates. The predicted B-cell epitopes and CTL epitopes were stitched together with the help of an AAY linker leading to the formation of a multi-epitope protein. The in silico study revealed that the modeled subunit vaccine candidate and TLR4 receptor interact with high affinity. This interaction was also found to be stable using a molecular dynamics simulation study. Our study demonstrates a leap towards developing a subunit vaccine against LSD.

Candidate Multi-Epitope Vaccine against Corona B.1.617 Lineage: In Silico Approach.

Various mutations have accumulated since the first genome sequence of SARS-CoV2 in 2020. Mutants of the virus carrying the D614G and P681R mutations in the spike protein are increasingly becoming dominant all over the world. The two mutations increase the viral infectivity and severity of the disease. This report describes an in silico design of SARS-CoV-2 multi-epitope carrying the spike D614G and P681R mutations. The designed vaccine harbors the D614G mutation that increases viral infectivity, fitness, and the P681R mutation that enhances the cleavage of S to S1 and S2 subunits. The designed multi-epitope vaccine showed an antigenic property with a value of 0.67 and the immunogenicity of the predicted vaccine was calculated and yielded 3.4. The vaccine construct is predicted to be non-allergenic, thermostable and has hydrophilic nature. The combination of the selected CTL and HTL epitopes in the vaccine resulted in 96.85% population coverage globally. Stable interactions of the vaccine with Toll-Like Receptor 4 were tested by docking studies. The multi-epitope vaccine can be a good candidate against highly infecting SARS-CoV-2 variants.

Altered MUC1 epitope-specific CTLs: A potential target for immunotherapy of pancreatic cancer.

The efficacy of conventional treatments for pancreatic cancer remains unsatisfactory, and immunotherapy is an emerging option for adjuvant treatment of this highly deadly disorder. The tumor-associated antigen (TAA) MUC1 is expressed in a variety of human cancers and is overexpressed in more than 90% of pancreatic cancer, which makes it an attractive target for cancer immunotherapy. As a self-protein, MUC1 shows a low immunogenicity because of immune tolerance, and the most effective approach to breaking immune tolerance is alteration of the antigen structure. In this study, the altered MUC11068-1076Y1 epitope (YLQRDISEM) by modification of amino acid residues in sequences presented a higher immunogenicity and elicited more CTLs relative to the wild-type (WT) MUC11068-1076 epitope (ELQRDISEM). In addition, the altered MUC11068-1076Y1 epitope was found to cross-recognize pancreatic cancer cells expressing WT MUC1 peptides in an HLA-A0201-restricted manner and trigger stronger immune responses against pancreatic cancer via the perforin/granzyme apoptosis pathway. As a potential HLA-A0201-restricted CTL epitope, the altered MUC11068-1076Y1 epitope is considered as a promising target for immunotherapy of pancreatic cancer. Alteration of epitope residues may be feasible to solve the problem of the low immunogenicity of TAA and break immune tolerance to induce immune responses against human cancers.

In-silico study of influence of HLA heterogeneity on CTL responses across ethnicities to SARS-CoV-2

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.

High frequency of CD8 escape mutations in elite controllers as new obstacle for HIV cure

ABSTRACT Accumulation of mutations in epitopes of cytolytic-T-lymphocytes immune response (CTL) in HIV-reservoir seems to be one of the reasons for shock-and-kill strategy failure. Ten non-controller patients on successful cART (TX) and seven elite controllers (EC) were included. HIV-Gag gene from purified resting memory CD4+ T-cells was sequenced by Next-Generation-Sequencing. HLA class-I alleles were typed to predict optimal HIV-Gag CTL epitopes. For each subject, the frequency of mutated epitopes in the HIV-Gag gene, the proportion of them considered as CTL-escape variants as well as their effect on antigen recognition by HLA were assessed. The proportion (%) of mutated HIV-Gag CTL epitopes in the reservoir was high and similar in EC and TX (86%[50–100] and 57%[48–82] respectively, p=0.315). Many of them were predicted to negatively impact antigen recognition. Moreover, the proportion of mutated epitopes considered to be CTL-escape variants was also similar in TX and EC (77%[49–92] vs. 50%[33–75] respectively, p=0.117). Thus, the most relevant finding of our study was the high and similar proportions of HIV-Gag CTL-escape mutations in the reservoir of both HIV-noncontroller patients with cART (TX) and patients with spontaneous HIV-control (EC). Our findings suggest that escape mutations of CTL-response may be another obstacle to eliminate the HIV reservoir and constitute a proof of concept that challenges HIV cure strategies focused on the reactivation of reservoirs. Due to the small sample size that could impact the robustness of the study, further studies with larger cohorts of elite controller patients are needed to confirm these results.

Strategies targeting hemagglutinin cocktail as a potential universal influenza vaccine.

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.

Immunoinformatic analysis of the whole proteome for vaccine design: An application to Clostridium perfringens.

Clostridium perfringens is a dangerous bacterium and known biological warfare weapon associated with several diseases, whose lethal toxins can produce necrosis in humans. However, there is no safe and fully effective vaccine against C. perfringens for humans yet. To address this problem, we computationally screened its whole proteome, identifying highly immunogenic proteins, domains, and epitopes. First, we identified that the proteins with the highest epitope density are Collagenase A, Exo-alpha-sialidase, alpha n-acetylglucosaminidase and hyaluronoglucosaminidase, representing potential recombinant vaccine candidates. Second, we further explored the toxins, finding that the non-toxic domain of Perfringolysin O is enriched in CTL and HTL epitopes. This domain could be used as a potential sub-unit vaccine to combat gas gangrene. And third, we designed a multi-epitope protein containing 24 HTL-epitopes and 34 CTL-epitopes from extracellular regions of transmembrane proteins. Also, we analyzed the structural properties of this novel protein using molecular dynamics. Altogether, we are presenting a thorough immunoinformatic exploration of the whole proteome of C. perfringens, as well as promising whole-protein, domain-based and multi-epitope vaccine candidates. These can be evaluated in preclinical trials to assess their immunogenicity and protection against C. perfringens infection.

Designing a Multi-Epitope Vaccine against Toxoplasma gondii: An Immunoinformatics Approach.

Infection with the intracellular apicomplexan parasite Toxoplasma gondii causes serious clinical outcomes in both human and veterinary settings worldwide. Although approximately one-third of the world’s population is infected with T. gondii, an effective human vaccine for this disease remains unavailable. We aimed to design a potential T. gondii vaccine candidate that consisted of the B- and T-lymphocyte epitopes of three parasite immunogenic antigens. Firstly, the immunodominant epitopes expressed within the ROP2, MIC3, and GRA7 proteins of T. gondii were identified. Subsequently, six B-cell epitopes, five CTL epitopes, and five HTL epitopes were combined to generate a multi-epitope vaccine, and the 50S ribosomal protein L7/L12 was added as an adjuvant to boost the vaccine’s immunogenicity. All these epitopes were found to be antigenic, nonallergenic, nontoxic, and nonhuman homologs. The designed vaccine construct has a molecular weight of 51 kDa, an antigenicity score of 0.6182, and a solubility of 0.903461. Likewise, the candidate vaccine was immunogenic, nonallergenic, and stable. Molecular docking analysis revealed stable interactions between the vaccine construct and the TLR-4 immune receptor. Meanwhile, the stability of the developed vaccine was validated using molecular dynamics simulation. In silico, the vaccine construct was able to trigger primary immune responses. However, further laboratory-based assessments are needed to confirm its efficacy and safety.

B.1.1.7 (Alpha) variant is the most antigenic compared to Wuhan strain, B.1.351, B.1.1.28/triple mutant and B.1.429 variants.

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.

Engineering a novel immunogenic chimera protein utilizing bacterial infections associated with atherosclerosis to induce a deviation in adaptive immune responses via Immunoinformatics approaches.

Recent studies have established the role of bacteria including Streptococcus pneumoniae, Helicobacter pylori, Chlamydia pneumonia, Mycobacterium tuberculosis, and Porphyromonas gingivalis in the development of atherosclerosis. These bacteria contribute to plaque formation via promoting Th1 immune responses and speeding up ox-LDL formation. Hence, we employed computational reverse vaccinology (RV) approaches to deviate immune response toward Th2 via engineering a novel immunogenic chimera protein. Prominent atherogenic antigens from related bacteria were identified. Then, machine learning-based servers were employed for predicting CTL and HTL epitopes. We selected epitopes from a wide variety of HLAs. Then, a chimeric protein sequence containing TAT peptide, adjuvant, IL-10 inducer, and linker-separated epitopes was designed. The conformational structure of the vaccine was built via multiple-template homology modelling using MODELLER. The initial structure was refined and validated by Ramachandran plot. The vaccine was also docked with TLR4. After that, molecular dynamics (MD) simulation of the docked vaccine-TLR4 was conducted. Finally, the immune simulation of the vaccine was conducted via the C-ImmSim server. A chimera protein with 629 amino acids was built and, classified as a non-allergenic probable antigen. An improved ERRAT score of 80.95 for the refined structure verified its stability. Additionally, validation via the Ramachandran plot showed 98.09% of the residues were located in the most favorable and permitted regions. MD simulations showed the vaccine-TLR4 complex reached a stable conformation. Also, RMS fluctuations analysis revealed no sign of protein denaturation or unfolding. Finally, immune response simulations indicated a promising response by innate and adaptive immunity. In summary, we built an immunogenic vaccine against atherosclerosis and demonstrated its favorable properties via advanced Immunoinformatics analyses. This study may pave the path for combat against atherosclerosis.

Exploratory Algorithm of a Multi-epitope-based Subunit Vaccine Candidate Against Cryptosporidium hominis: Reverse Vaccinology-Based Immunoinformatic Approach.

Cryptosporidiosis is the leading protozoan-induced cause of diarrheal illness in children, and it has been linked to childhood mortality, malnutrition, cognitive development, with retardation of growth. Cryptosporidium hominis, the anthroponotically transmitted species within the Cryptosporidium genus, contributes significantly to the global burden of infection, accounting for the majority of clinical cases in numerous nations, as well as its emergence in the last decade is largely due to detections obtained through noteworthy epidemiologic research. Nevertheless, there is no vaccine available, and the only licensed medication, nitazoxanide, has been demonstrated to have efficacy limitations in a number of patient groups recognized to be at high risk of complications. Therefore, current study delineates the computational vaccine design for Cryptosporidium hominis, the notable pathogen for enteric diarrhea. Firstly, a comprehensive literature search was conducted to identify six proteins based on their toxigenicity, allergenicity, antigenicity, and prediction of transmembrane helices to make up a multi-epitope-based subunit vaccine. Following that, antigenic non-toxic HTL epitope, CTL epitope with B cell epitope were predicted from the selected proteins and construct a vaccine candidate with adding an adjuvant and some linkers with immunologically superior epitopes. Afterwards, the constructed vaccine candidates and TLR2 receptor were put into the ClusPro server for molecular dynamic simulation to know the binding stability of the vaccine-TLR2 complex. Following that, Escherichia coli strain K12 was used as a cloning host for the chosen vaccine construct via the JCat server. As a result of the findings, it was resolute that the proposed chimeric peptide vaccine could improve the immune response to Cryptosporidium hominis.

Mining of Marburg Virus Proteome for Designing an Epitope-Based Vaccine.

Marburg virus (MARV) is one of the most harmful zoonotic viruses with deadly effects on both humans and nonhuman primates. Because of its severe outbreaks with a high rate of fatality, the world health organization put it as a risk group 4 pathogen and focused on the urgent need for the development of effective solutions against that virus. However, up to date, there is no effective vaccine against MARV in the market. In the current study, the complete proteome of MARV (seven proteins) was analyzed for the antigenicity score and the virulence or physiological role of each protein where we nominated envelope glycoprotein (Gp), Transcriptional activator (VP30), and membrane-associated protein (VP24) as the candidates for epitope prediction. Following that, a vaccine construct was designed based on CTL, HTL, and BCL epitopes of the selected protein candidates and to finalize the vaccine construct, several amino acid linkers, β-defensin adjuvant, and PADRE peptides were incorporated. The generated potential vaccine was assessed computationally for several properties such as antigenicity, allergenicity, stability, and other structural features where the outcomes of these assessments nominated this potential vaccine to be validated for its binding affinity with two molecular targets TLR-8 and TLR-4. The binding score and the stability of the vaccine-receptor complex, which was deeply studied through molecular docking-coupled dynamics simulation, supported the selection of our designed vaccine as a putative solution for MARV that should be validated through future wet-lab experiments. Here, we describe the computational approach for designing and analysis of this potential vaccine.

Efficient Identification of Tembusu Virus CTL Epitopes in Inbred HBW/B4 Ducks Using a Novel MHC Class I-Restricted Epitope Screening Scheme.

The identification of MHC class I-restricted CTL epitopes in certain species, particularly nonmammals, remains a challenge. In this study, we developed a four-step identification scheme and confirmed its efficiency by identifying the Anpl-UAA*76-restricted CTL epitopes of Tembusu virus (TMUV) in inbred haplotype ducks HBW/B4. First, the peptide binding motif of Anpl-UAA*76 was determined by random peptide library in de novo liquid chromatography-tandem mass spectrometry, a novel nonbiased, data-independent acquisition method that we previously established. Second, a total of 38 TMUV peptides matching the motif were screened from the viral proteome, among which 11 peptides were conserved across the different TMUV strains. Third, the conserved TMUV peptides were refolded in vitro with Anpl-UAA*76 and Anpl-β2-microglobulin to verify the results from the previous two steps. To clarify the structural basis of the obtained motif, we resolved the crystal structure of Anpl-UAA*76 with the TMUV NS3 peptide LRKRQLTVL and found that Asp34 is critical for the preferential binding of the B pocket to bind the second residue to arginine as an anchor residue. Fourth, the immunogenicity of the conserved TMUV peptides was tested in vivo using specific pathogen-free HBW/B4 ducks immunized with the attenuated TMUV vaccine. All 11 conserved TMUV epitopes could bind stably to Anpl-UAA*76 in vitro and stimulate the secretion of IFN-γ and lymphocyte proliferation, and three conserved and one nonconserved peptides were selected to evaluate the CTL responses in vivo by flow cytometry and their tetramers. We believe that this new scheme could improve the identification of MHC class I-restricted CTL epitopes, and our data provide a foundation for further study on duck anti-TMUV CTL immunity.

P345: AN IMMUNE SCREEN IDENTIFIES 5-NONLOXYTRYPTAMINE AS A NOVEL ANTI-CANCER AGENT CAPABLE OF IMPROVING ANTI-TUMOR IMMUNITY IN COMBINATION WITH ANTI-PD1 THERAPY

Background: Exploiting the immune system, particularly CD8+ T cells to eliminate tumors has revolutionized the treatment landscape. However, despite the evident successes of CD8+ T cell-targeting immunotherapies such as anti-PD1 checkpoint inhibitors, many patients fail to respond especially those with poorly immunogenic tumors. Additional obstacles to successful immunotherapy responses include therapy-induced toxicity, lack of bio-markers of response, an immunosuppressive environment leading to T cell dysfunction and exhaustion and poor immune infiltration highlighting a need for novel combinatorial approaches to augment immunotherapeutic activity. Aims: We aimed to uncover novel therapeutic agents capable of augmenting the anti-tumoral responses of CD8+ T cells in ex vivo systems and in vivo syngenetic poorly immunogenic tumor models. Methods: To uncover novel potentiators of T cell anti-tumor immunity, we carried out an ex vivo pharmacological screen, using Lymphocytic choriomeningitis virus (LCMV)-primed splenic T cells that were combined tumor cells expressing the LCMV gp33 (B16) peptide CTL epitope and incubated with an NIH 770 compound library. Hits that increased tumor-cell killing when combined with LCMV-primed splenic T cells were further validated ex vivo. The key candidate was assessed in vivo in C57BL/6J syngeneic tumor models. Immunocompromised NSG mice as well as antibody depletion were used validate CD8+ T cell dependence. In vitro and in vivo immunomodulatory effects were deciphered using flow cytometry, immunoblot, CRISPR/Cas9 and histological analyses. Results: We identified 5-Nonyloxytryptamine (5-NL), a serotonin receptor (HTR) agonist, as increasing the ability of T cells to target tumor cells. In vitro, 5-NL induced apoptosis in melanoma and childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) at low micromolar levels. 5-NL delayed tumor growth in vivo and the phenotype was dependent on the hosts’ immune system, specifically CD8+ T cells as their depletion abrogated the phenotype (Figure 1A-B). 5-NL’s pro-immune effects were attributed to the upregulation of antigen presenting machinery in melanoma, BCP-ALL and other poorly immunogenic tumors with low MHC-I/HLAA-C expression (Figure 1C). Importantly, the upregulation of MHC-I/HLAA-C occurred without concomitant increases in PD-L1 expression and was linked to upregulation of cAMP Response Element-Binding Protein (CREB) in vitro and in vivo. As demonstrated through CRISPR/Cas9 HTR1D knockout, the immunomodulatory and pro-apoptotic effects of 5-NL occurred independently of receptor target signaling. 5-NL was successfully combined with an anti-PD1 antibody in vivo for maximal tumor growth inhibition. Figure 1: The serotonin agonist 5-Nonyloxytryptamine (5-NL) was identified as potentiating T cell mediated anti-tumor immunity. (A) C57BL/6J or (B) CD8 depleted C57BL/6J mice were subcutaneously injected with 5 x 105 gp33 expressing tumor cells. 7 days post-tumor injection, mice were randomized and treated daily 6.25 mg/kg of 5-NL or vehicle for five consecutive days and tumor volume was measured (n = 9-12). H2-Db (MHC-I, murine cell lines) and HLAA-C (human cells lines) were assessed by FACS analysis following treatment with 5-NL for 24 hours (n = 3-9). Error bars in all experiments indicate SEM; *P < 0.05 as determined by a Student´s t-test (unpaired, 2 tailed). Image:Summary/Conclusion: This study demonstrates novel therapeutic opportunities for augmenting immune responses in poorly immunogenic tumors and increasing their responsiveness to immunotherapies.

In silico Investigation of Immunodominant Antigenic Regions, Helper T Lymphocyte, and Cytotoxic T Lymphocyte Epitopes Credentials for SARS-CoV-2 Vaccination

Background: In recent days, COVID-19 cases are increasing globally at an alarming rate due to the COVID-19 second wave despite the mass vaccination programs. Search for the potential vaccine for SARS-CoV-2 is still under progress. The epitope-based vaccine is effective and is a cornerstone in vaccine development. The quick prediction of epitopes could be a proficient way to monitor vaccine development during a global health crisis. Objective: This study is designed to predict the potential epitopes with computational tools for vaccine development. Methods: NetCTLpan v. 1.1 and NetMHCIIpan v. 3.2 servers were used for T-cell epitope analysis. IEDB servers were employed for HLA and DRB1 peptide calculations. The epitope Immunogenicity, toxicity, physiochemical character, and other features are measured by immunogen evaluation. Furthermore, the top-ranked immunogenic epitopes were computationally validated by molecular docking analysis. The epitopes are docked to Toll-like receptors (TLRs), which is helpful to generate an immune response against SARS-CoV-2. Results: Overall, six HTL and CTL epitopes were predicted (IDGYFKIYSKH, HPLSHFVNLDNL, RIGNNYKLNT, and WTAGAAAYYVG, MACLVGLMWLS, FRLKGGAPIKGVT), which had good immunogenicity scores, and stable interaction with Toll-like receptor (TLR). Therefore, these epitopes can bind with HLA and DRB1 molecules, respectively. Conclusion: The computationally predicted antigenic regions might be considered for epitope-based vaccine against SARS-CoV-2 after in vitro