A Promising Needle-Free Pyro-Drive Jet Injector for Augmentation of Immunity by Intradermal Injection as a Physical Adjuvant.

Current worldwide mRNA vaccination against SARS-CoV-2 by intramuscular injection using a needled syringe has greatly protected numerous people from COVID-19. An intramuscular injection is generally well tolerated, safer and easier to perform on a large scale, whereas the skin has the benefit of the presence of numerous immune cells, such as professional antigen-presenting dendritic cells. Therefore, intradermal injection is considered superior to intramuscular injection for the induction of protective immunity, but more proficiency is required for the injection. To improve these issues, several different types of more versatile jet injectors have been developed to deliver DNAs, proteins or drugs by high jet velocity through the skin without a needle. Among them, a new needle-free pyro-drive jet injector has a unique characteristic that utilizes gunpower as a mechanical driving force, in particular, bi-phasic pyrotechnics to provoke high jet velocity and consequently the wide dispersion of the injected DNA solution in the skin. A significant amount of evidence has revealed that it is highly effective as a vaccinating tool to induce potent protective cellular and humoral immunity against cancers and infectious diseases. This is presumably explained by the fact that shear stress generated by the high jet velocity facilitates the uptake of DNA in the cells and, consequently, its protein expression. The shear stress also possibly elicits danger signals which, together with the plasmid DNA, subsequently induces the activation of innate immunity including dendritic cell maturation, leading to the establishment of adaptive immunity. This review summarizes the recent advances in needle-free jet injectors to augment the cellular and humoral immunity by intradermal injection and the possible mechanism of action.

Elucidation of novel compounds and epitope-based peptide vaccine design against C30 endopeptidase regions of SARS-CoV-2 using immunoinformatics approaches.

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.

Pressured cytotoxic T cell epitope strength among SARS-CoV-2 variants correlates with COVID-19 severity.

Heterogeneity in susceptibility among individuals to COVID-19 has been evident through the pandemic worldwide. Cytotoxic T lymphocyte (CTL) responses generated against pathogens in certain individuals are known to impose selection pressure on the pathogen, thus driving emergence of new variants. In this study, we probe the role played by host genetic heterogeneity in terms of HLA-genotypes in determining differential COVID-19 severity in patients. We use bioinformatic tools for CTL epitope prediction to identify epitopes under immune pressure. Using HLA-genotype data of COVID-19 patients from a local cohort, we observe that the recognition of pressured epitopes from the parent strain Wuhan-Hu-1 correlates with COVID-19 severity. We also identify and rank list HLA-alleles and epitopes that offer protectivity against severe disease in infected individuals. Finally, we shortlist a set of 6 pressured and protective epitopes that represent regions in the viral proteome that are under high immune pressure across SARS-CoV-2 variants. Identification of such epitopes, defined by the distribution of HLA-genotypes among members of a population, could potentially aid in prediction of indigenous variants of SARS-CoV-2 and other pathogens.

Immunoinformatic prediction of potential immunodominant epitopes from cagW in order to investigate protection against Helicobacter pylori infection based on experimental consequences.

Helicobacter pylori is a leading cause of stomach cancer and peptic ulcers. Thus, identifying epitopes in H. pylori antigens is important for disease etiology, immunological surveillance, enhancing early detection tests, and developing optimal epitope-based vaccines. We used immunoinformatic and computational methods to create a potential CagW epitope candidate for H. pylori protection. The cagW gene of H. pylori was amplified and cloned into pcDNA3.1 (+) for injection into the muscles of healthy BALB/c mice to assess the impact of the DNA vaccine on interleukin levels. The results will be compared to a control group of mice that received PBS or cagW-pcDNA3.1 (+) vaccinations. An analysis of CagW protein antigens revealed 8 CTL and 7 HTL epitopes linked with AYY and GPGPG, which were enhanced by adding B-defensins to the N-terminus. The vaccine's immunogenicity, allergenicity, and physiochemistry were validated, and its strong activation of TLRs (1, 2, 3, 4, and 10) suggests it is antigenic. An in-silico cloning and immune response model confirmed the vaccine's expression efficiency and predicted its impact on the immune system. An immunofluorescence experiment showed stable and bioactive cagW gene expression in HDF cells after cloning the whole genome into pcDNA3.1 (+). In vivo vaccination showed that pcDNA3.1 (+)-cagW-immunized mice had stronger immune responses and a longer plasmid DNA release window than control-plasmid-immunized mice. After that, bioinformatics methods predicted, developed, and validated the three-dimensional structure. Many online services docked it with Toll-like receptors. The vaccine was refined using allergenicity, antigenicity, solubility, physicochemical properties, and molecular docking scores. Virtual-reality immune system simulations showed an impressive reaction. Codon optimization and in-silico cloning produced E. coli-expressed vaccines. This study suggests a CagW epitopes-protected H. pylori infection. These studies show that the proposed immunization may elicit particular immune responses against H. pylori, but laboratory confirmation is needed to verify its safety and immunogenicity.

Global Stability of a MERS-CoV Infection Model with CTL Immune Response and Intracellular Delay

In this paper, we propose and study a Middle East respiratory syndrome coronavirus (MERS-CoV) infection model with cytotoxic T lymphocyte (CTL) immune response and intracellular delay. This model includes five compartments: uninfected cells, infected cells, viruses, dipeptidyl peptidase 4 (DPP4), and CTL immune cells. We obtained an immunity-inactivated reproduction number (Formula presented.) and an immunity-activated reproduction number (Formula presented.). By analyzing the distributions of roots of the corresponding characteristic equations, the local stability results of the infection-free equilibrium, the immunity-inactivated equilibrium, and the immunity-activated equilibrium were obtained. Moreover, by constructing suitable Lyapunov functionals and combining LaSalle's invariance principle and Barbalat's lemma, some sufficient conditions for the global stability of the three types of equilibria were obtained. It was found that the infection-free equilibrium is globally asymptotically stable if (Formula presented.) and unstable if (Formula presented.) ;the immunity-inactivated equilibrium is locally asymptotically stable if (Formula presented.) and globally asymptotically stable if (Formula presented.) and condition (H1) holds, but unstable if (Formula presented.) ;and the immunity-activated equilibrium is locally asymptotically stable if (Formula presented.) and is globally asymptotically stable if (Formula presented.) and condition (H1) holds. © 2023 by the authors.

Synthesis of Killer T Cell Epitopes for Peptide-Based Vaccine of SARS-CoV-2

COVID-19 is a current global pandemic, which has prompted many countries to develop ways to deal with it. Peptides have many medicinal and diagnostic benefits, so recently, many researchers have been developing peptide-based vaccines against COVID-19. In peptide-based vaccines, peptides act as specific antigens that will provide a faster immune response because they do not go through the process of cutting proteins in the Major Histocompatibility complex (MHC) antigen-presenting cells (APC) and can be directly presented outside the cells so that they can be recognized by the host killer T cells (CTL). Vaccine development can be accelerated with the help of immunoinformatics to predict specific epitopes to induce CTL. We have predicted the CTL epitope through the immunoinformatic method. This study aims to synthesize candidate CTL epitopes as a candidate for the SARS-CoV-2 vaccine using the SPPS method with the Fmoc/t-Bu strategy. In this study, two CTL epitopes were synthesized through a conventional solid-phase peptide synthesis (SPPS) method, and another CTL epitope was synthesized using a semi-automated peptide synthesizer. The SPPS method is faster because the purification is only carried out at the final stage, while the Fmoc/t-Bu strategy was applied because it provides a mild reaction condition. Both synthetic approaches were compared. The semi-automated peptide synthesizer made the synthesis faster and more efficient due to using an inert gas (N2) during the synthesis. The synthetic peptides were characterized by TOF-ESI-MS. The three peptides showed ion peaks at m/z 1137.5509 (M+H)+, 1064.3468 (M+H)+, and 916.5859 (M+H)+, indicating correct molecular ion peaks for EILDITPCSF, IPIGAGICASY, and FIAGLIAIV, respectively. © 2022 National Information and Documentation Center.

Designing of cytotoxic T lymphocyte-based multi-epitope vaccine against SARS-CoV2: a reverse vaccinology approach.

SARS-CoV2 is a single-stranded RNA virus, gaining much attention after it out broke in China in December 2019. The virus rapidly spread to several countries around the world and caused severe respiratory illness to humans. Since the outbreak, researchers around the world have devoted maximum resources and effort to develop a potent vaccine that would offer protection to uninfected individuals against SARS-CoV2. Reverse vaccinology is a relatively new approach that thrives faster in vaccine research. In this study, we constructed Cytotoxic T Lymphocytes (CTL)-based multi-epitope vaccine using hybrid epitope prediction methods. A total of 121 immunogenic CTL epitopes were screened by various sequence-based prediction methods and docked with their respective HLA alleles using the AutoDock Vina v1.1.2. In all, 17 epitopes were selected based on their binding affinity, followed by the construction of multi-epitope vaccine by placing the appropriate linkers between the epitopes and tuberculosis heparin-binding hemagglutinin (HBHA) adjuvant. The final vaccine construct was modeled by the I-TASSER server and the best model was further validated by ERRAT, ProSA, and PROCHECK servers. Furthermore, the molecular interaction of the constructed vaccine with TLR4 was assessed by ClusPro 2.0 and PROtein binDIng enerGY prediction (PRODIGY) server. The immune simulation analysis confirms that the constructed vaccine was capable of inducing long-lasting memory T helper (Th) and CTL responses. Finally, the nucleotide sequence was codon-optimized by the JCAT tool and cloned into the pET21a (+) vector. The current results reveal that the candidate vaccine is capable of provoking robust CTL response against the SARS-CoV2.Communicated by Ramaswamy H. Sarma.

Lichen planus after COVID-19 infection and vaccination.

Lichen planus (LP) is an inflammatory disorder believed to result from CD8 + cytotoxic T-cell (CTL)-mediated autoimmune reactions against basal keratinocytes. We present a review of LP following COVID-19 infection and vaccination. Literature searches were conducted on PubMed and Google Scholar from 2019 to 7/2022. 36 articles were selected based on subject relevance, and references within articles were also screened. 39 cases of post-vaccination LP and 6 cases of post-infection LP were found among case reports and case series. 152 cases of post-vaccination LP and 12 cases of post-infection LP were found in retrospective and prospective studies. LP is a rare complication of COVID-19 infection and vaccination that may be mediated by overstimulation of T-cell responses and proinflammatory cytokine production. However, it does not represent a limitation against COVID-19 vaccination, and the benefits of vaccination considerably outweigh the risks.

Global stability of a general HTLV-I infection model with Cytotoxic T-Lymphocyte immune response and mitotic transmission

Mathematical models have been considered as a robust tool to support biological and medical studies of human viral infections. The global stability of viral infection models remains an important and largely open research problem. Such results are necessary to evaluate treatment strategies for infections and to establish thresholds for treatment rates. Human T-lymphotropic virus class I (HTLV-I) is a retrovirus which infects the CD4+T cells and causes chronic and deadly diseases. In this article, we developed a general nonlinear system of ODEs which describes the within-host dynamics of HTLV-I under the effect Cytotoxic T-Lymphocytes (CTLs) immunity. The mitotic division of actively infected cells are modeled. We consider general nonlinear functions for the generation, proliferation and clearance rates for all types of cells. The incidence rate of infec-tion is also modeled by a general nonlinear function. These general functions are assumed to satisfy a set of suitable conditions and include several forms presented in the literature. We determine a bounded domain for the system's solutions. We prove the existence of the system's equilibrium points and determine two threshold numbers, the basic reproductive number R0 and the CTL immunity stimulation number R1. We establish the global stability of all equilibrium points by con-structing Lyapunov function and applying Lyapunov-LaSalle asymptotic stability theorem. Under certain conditions it is shown that if R0 <= 1, then the infection-free equilibrium point is globally asymptotically stable (GAS) and the HTLV-I infection is cleared. If R1 < 1 < R0, then the infected equilibrium point without CTL immunity is GAS and the HTLV-I infection becomes chronic with no sustained CTL immune response. If R1 > 1, then the infected equilibrium point with CTL immu-nity is GAS and the infection becomes chronic with persistent CTL immune response. We present numerical simulations for the system by choosing special shapes of the general functions. The effect of Crowley-Martin functional response and mitotic division of actively infected cells on the HTLV-I progression are studied. Our results cover and improve several ones presented in the literature.(c) 2022 THE AUTHORS. Published by Elsevier BV on behalf of Faculty of Engineering, Alexandria University. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/ 4.0/).

Immunotherapeutic strategies to induce inflection in the immune response: therapy for cancer and COVID-19.

Cancer has agonized the human race for millions of years. The present decade witnesses biological therapeutics to combat cancer effectively. Cancer Immunotherapy involves the use of therapeutics for manipulation of the immune system by immune agents like cytokines, vaccines, and transfection agents. Recently, this therapeutic approach has got vast attention due to the current pandemic COVID-19 and has been very effective. Concerning cancer, immunotherapy is based on the activation of the host's antitumor response by enhancing effector cell number and the production of soluble mediators, thereby reducing the host's suppressor mechanisms by induction of a tumour killing environment and by modulating immune checkpoints. In the present era, immunotherapies have gained traction and momentum as a pedestal of cancer treatment, improving the prognosis of many patients with a wide variety of haematological and solid malignancies. Food supplements, natural immunomodulatory drugs, and phytochemicals, with recent developments, have shown positive trends in cancer treatment by improving the immune system. The current review presents the systematic studies on major immunotherapeutics and their development for the effective treatment of cancers as well as in COVID-19. The focus of the review is to highlight comparative analytics of existing and novel immunotherapies in cancers, concerning immunomodulatory drugs and natural immunosuppressants, including immunotherapy in COVID-19 patients.

CD8+ T-cell immune escape by SARS-CoV-2 variants of concern.

Despite the efficacy of antiviral drug repositioning, convalescent plasma (CP), and the currently available vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the worldwide coronavirus disease 2019 (COVID-19) pandemic is still challenging because of the ongoing emergence of certain new SARS-CoV-2 strains known as variants of concern (VOCs). Mutations occurring within the viral genome, characterized by these new emerging VOCs, confer on them the ability to efficiently resist and escape natural and vaccine-induced humoral and cellular immune responses. Consequently, these VOCs have enhanced infectivity, increasing their stable spread in a given population with an important fatality rate. While the humoral immune escape process is well documented, the evasion mechanisms of VOCs from cellular immunity are not well elaborated. In this review, we discussed how SARS-CoV-2 VOCs adapt inside host cells and escape anti-COVID-19 cellular immunity, focusing on the effect of specific SARS-CoV-2 mutations in hampering the activation of CD8⁺ T-cell immunity.

Periodic oscillation for a class of in-host MERS-CoV infection model with CTL immune response.

The purpose of this paper is to give some sufficient conditions for the existence of periodic oscillation of a class of in-host MERS-Cov infection model with cytotoxic T lymphocyte (CTL) immune response. A new technique is developed to obtain a lower bound of the state variable characterizing CTL immune response in the model. Our results expand on some previous works.

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.

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.

Faster cytotoxicity with age: Increased perforin and granzyme levels in cytotoxic CD8+ T cells boost cancer cell elimination.

A variety of intrinsic and extrinsic factors contribute to the altered efficiency of CTLs in elderly organisms. In particular, the efficacy of antiviral CD8+ T cells responses in the elderly has come back into focus since the COVID-19 pandemic outbreak. However, the exact molecular mechanisms leading to alterations in T cell function and the origin of the observed impairments have not been fully explored. Therefore, we investigated whether intrinsic changes affect the cytotoxic ability of CD8+ T cells in aging. We focused on the different subpopulations and time-resolved quantification of cytotoxicity during tumor cell elimination. We report a surprising result: Killing kinetics of CD8+ T cells from elderly mice are much faster than those of CD8+ T cells from adult mice. This is true not only in the total CD8+ T cell population but also for their effector (TEM ) and central memory (TCM ) T cell subpopulations. TIRF experiments reveal that CD8+ T cells from elderly mice possess comparable numbers of fusion events per cell, but significantly increased numbers of cells with granule fusion. Analysis of the cytotoxic granule (CG) content shows significantly increased perforin and granzyme levels and turns CD8+ T cells of elderly mice into very efficient killers. This highlights the importance of distinguishing between cell-intrinsic alterations and microenvironmental changes in elderly individuals. Our results also stress the importance of analyzing the dynamics of CTL cytotoxicity against cancer cells because, with a simple endpoint lysis analysis, cytotoxic differences could have easily been overlooked.

Computational Structural and Functional Analyses of ORF10 in Novel Coronavirus SARS-CoV-2 Variants to Understand Evolutionary Dynamics.

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.

Activation of Anti-SARS-CoV-2 Human CTLs by Extracellular Vesicles Engineered with the N Viral Protein.

We propose an innovative anti-SARS-CoV-2 immune strategy based on extracellular vesicles (EVs) inducing an anti-SARS-CoV-2 N CD8+ T cytotoxic lymphocyte (CTL) immune response. We previously reported that the SARS-CoV-2 N protein can be uploaded at high levels in EVs upon fusion with Nefmut, i.e., a biologically inactive HIV-1 Nef mutant incorporating into EVs at quite high levels. Here, we analyze the immunogenic properties in human cells of EVs engineered with SARS-CoV-2 N fused at the C-terminus of either Nefmut or a deletion mutant of Nefmut referred to as NefmutPL. The analysis of in vitro-produced EVs has supported the uploading of N protein when fused with truncated Nefmut. Mice injected with DNA vectors expressed each fusion protein developed robust SARS-CoV-2 N-specific CD8+ T cell immune responses. When ex vivo human dendritic cells were challenged with EVs engineered with either fusion products, the induction of a robust N-specific CTL activity, as evaluated by both CD107a and trogocytosis assays, was observed. Through these data we achieved the proof-of-principle that engineered EVs can be instrumental to elicit anti-SARS-CoV-2 CTL immune response in human cells. This achievement represents a mandatory step towards the upcoming experimentations in pre-clinical models focused on intranasal administration of N-engineered EVs.

Path to Success and Future Impact of Nucleic Acid Vaccines: DNA and mRNA

The rapid development of mRNA vaccines for COVID-19 has both astonished the world and raised concerns about their safety, perhaps because many people do not realize the decades’ long efforts for nucleic acid vaccines, both mRNA and DNA vaccines, including the licensure of several veterinary DNA vaccines. This manuscript traces the milestones for nucleic acid vaccine research and development (R&D), with a focus on the immune and safety issues they both raised and answered. The characteristics of the two entities are compared, demonstrating the similarities and differences between them, the advantages and disadvantages, which might lead toward using one or the other technology for different indications. In addition, as the SARS-CoV-2 pandemic has once again highlighted the importance of One Health, that is, the interactions between animal and human pathogens, focus will also be given to how DNA vaccine utilization and studies both in large domestic animals and in wildlife pave the way for more integrated approaches for vaccines to respond quickly to, and prevent, the global impacts of emerging diseases.

Identification of HLA-A*24:02-Restricted CTL Candidate Epitopes Derived from the Nonstructural Polyprotein 1a of SARS-CoV-2 and Analysis of Their Conservation Using the Mutation Database of SARS-CoV-2 Variants.

COVID-19 vaccines are currently being administered worldwide and playing a critical role in controlling the pandemic. They have been designed to elicit neutralizing antibodies against Spike protein of the original SARS-CoV-2, and hence they are less effective against SARS-CoV-2 variants with mutated Spike than the original virus. It is possible that novel variants with abilities of enhanced transmissibility and/or immunoevasion will appear in the near future and perfectly escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Several lines of evidence suggest the contribution of CTLs on the viral control in COVID-19, and CTLs target a wide range of proteins involving comparatively conserved nonstructural proteins. Here, we identified 22 HLA-A*24:02-restricted CTL candidate epitopes derived from the nonstructural polyprotein 1a (pp1a) of SARS-CoV-2 using computational algorithms, HLA-A*24:02 transgenic mice and the peptide-encapsulated liposomes. We focused on pp1a and HLA-A*24:02 because pp1a is relatively conserved and HLA-A*24:02 is predominant in East Asians such as Japanese. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by a number of mutations in the Sequence Read Archive database of SARS-CoV-2 variants. The information of such conserved epitopes might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any SARS-CoV-2 variants by the induction of both anti-Spike neutralizing antibodies and CTLs specific for conserved epitopes. IMPORTANCE COVID-19 vaccines have been designed to elicit neutralizing antibodies against the Spike protein of the original SARS-CoV-2, and hence they are less effective against variants. It is possible that novel variants will appear and escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Here, we identified 22 HLA-A*24:02-restricted CTL candidate epitopes derived from the nonstructural polyprotein 1a (pp1a) of SARS-CoV-2. We focused on pp1a and HLA-A*24:02 because pp1a is conserved and HLA-A*24:02 is predominant in East Asians. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by mutations in the database of SARS-CoV-2 variants. The information might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any variants.

Peptides Derived From S and N Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 Induce T Cell Responses: A Proof of Concept for T Cell Vaccines.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that escape vaccine-induced neutralizing antibodies has indicated the importance of T cell responses against this virus. In this study, we highlight the SARS-CoV-2 epitopes that induce potent T cell responses and discuss whether T cell responses alone are adequate to confer protection against SARS-CoV-2 and describe the administration of 20 peptides with an RNA adjuvant in mice. The peptides have been synthesized based on SARS-CoV-2 spike and nucleocapsid protein sequences. Our study demonstrates that immunization with these peptides significantly increases the proportion of effector memory T cell population and interferon-γ (IFN-γ)-, interleukin-4 (IL-4)-, tumor necrosis factor-α (TNF-α)-, and granzyme B-producing T cells. Of these 20 peptides, four induce the generation of IFN-γ-producing T cells, elicit CD8+ T cell (CTL) responses in a dose-dependent manner, and induce cytotoxic T lymphocytes that eliminate peptide-pulsed target cells in vivo. Although it is not statistically significant, these peptide vaccines reduce viral titers in infected hamsters and alleviate pulmonary pathology in SARS-CoV-2-infected human ACE2 transgenic mice. These findings may aid the design of effective SARS-CoV-2 peptide vaccines, while providing insights into the role of T cells in SARS-CoV-2 infection.