Efficient control of chronic LCMV infection by a CD4 T cell epitope-based heterologous prime-boost vaccination in a murine model

CD4^(+)T cells are essential for sustaining CD8^(+)T cell responses during a chronic infection.The adoptive transfer of virus-specific CD4^(+)T cells has been shown to efficiently rescue exhausted CD8^(+)T cells.However,the question of whether endogenous virus-specific CD4^(+)T cell responses can be enhanced by certain vaccination strategies and subsequently reinvigorate exhausted CD8^(+)T cells remains unexplored.In this study,we developed a CD4^(+)T cell epitope-based heterologous prime-boost immunization strategy and examined the efficacy of this strategy using a mouse model of chronic lymphocytic choriomeningitis virus(LCMV)infection.We primed chronically LCMV-infected mice with a Listeria monocytogenes vector that expressed the LCMV glycoprotein-specific I-Ab-restricted CD4^(+)T cell epitope GP61–80(LM-GP61)and subsequently boosted the primed mice with an influenza virus A(PR8 strain)vector that expressed the same CD4^(+)T cell epitope(IAV-GP61).This heterologous prime-boost vaccination strategy elicited strong anti-viral CD4^(+)T cell responses,which further improved both the quantity and quality of the virusspecific CD8^(+)T cells and led to better control of the viral loads.The combination of this strategy and the blockade of the programmed cell death-1(PD-1)inhibitory pathway further enhanced the anti-viral CD8^(+)T cell responses and viral clearance.Thus,a heterologous prime-boost immunization that selectively induces virus-specific CD4^(+)T cell responses in conjunction with blockade of the inhibitory pathway may represent a promising therapeutic approach to treating patients with chronic viral infections.

Screening HLA-A-restricted T cell epitopes of SARS-CoV-2 and the induction of CD8^(+)T cell responses in HLA-A transgenic mice

Since severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)-specific T cells have been found to play essential roles in host immune protection and pathology in patients with coronavirus disease 2019(COVID-19),this study focused on the functional validation of T cell epitopes and the development of vaccines that induce specific T cell responses.A total of 120 CD8^(+)T cell epitopes from the E,M,N,S,and RdRp proteins were functionally validated.Among these,110,15,6,14,and 12 epitopes were highly homologous with SARS-CoV,OC43,NL63,HKU1,and 229E,respectively;in addition,four epitopes from the S protein displayed one amino acid that was distinct from the current SARS-CoV-2 variants.Then,31 epitopes restricted by the HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C),R848 or poly(lactic-co-glycolic acid)nanoparticles,and these vaccines elicited robust and specific CD8^(+)T cell responses in HLA-A2/DR1 transgenic mice as well as wild-type mice.In contrast to previous research,this study established a modified DC-peptide-PBL cell coculture system using healthy donor PBMCs to validate the in silico predicted epitopes,provided an epitope library restricted by nine of the most prevalent HLA-A allotypes covering broad Asian populations,and identified the HLA-A restrictions of these validated epitopes using competitive peptide binding experiments with HMy2.CIR cell lines expressing the indicated HLA-A allotype,which initially confirmed the in vivo feasibility of 9-or 10-mer peptide cocktail vaccines against SARS-CoV-2.These data will facilitate the design and development of vaccines that induce antiviral CD8^(+)T cell responses in COVID-19 patients.

A novel recombinant DNA vaccine encodingMycobacterium tuberculosis ESAT-6 and FL protects againstMycobacterium tuberculosis challenge in mice

Mycobacterium tuberculosis 6-kDa early secretory antigenic target （ESAT-6） is a dominant target antigen for cell-mediated immunity in the early phase of tuberculosis. The fms-like tyrosine kinase 3 ligand （FL） that induces potent immune response has been used as an adjuvant in vaccine development. In this study, a new recombinant plasmid （plRES-epitope-peptides-FL） encoding three T cell epitopes of ESAT-6 and FL was constructed, and the immunogenicity of the DNA vaccine was assessed in C57BL/6 mice immunized with the plasmid DNA vaccine. Additionally, a strategy of intramuscular injection with the DNA vaccine （prime） and intranasal administration of the epitope peptides （boost） was employed to induce higher immune reaction of the mice. The results showed that mice vaccinated with the recombinant plasmid DNA vaccine and boosted with the peptides not only increased the levels of Thl cytokines （IFN-γ and IL-12）, the number of IFN-γ＋ T cells and activities of cytotoxic T lymphocytes as well as IgG, but also enhanced protection against Mycobacterium tuberculosis challenge. In conclusion, these data indicate that the novel recombinant plRES-epitope-peptides-FL plasmid is a useful DNA vaccine for pre- venting Mycobacterium tuberculosis infection.

Identification of cross-reactive CD8^(+)T cell receptors with high functional avidity to a SARS-CoV-2 immunodominant epitope and its natural mutant variants

Despite the growing knowledge of T cell responses in COVID-19 patients,there is a lack of detailed characterizations for T cell-antigen interactions and T cell functions.Here,with a predicted peptide library from SARS-CoV-2 S and N proteins,we found that specific CD8+T cell responses were identified in over 75%of COVID-19 convalescent patients(15/20)and an epitope from the N protein,N361-369(KTFPPTEPK),was the most dominant epitope from our selected peptide library.Importantly,we discovered 2 N361-369-specific T cell receptors(TCRs)with high functional avidity that were independent of the CD8 co-receptor.These TCRs exhibited complementary cross-reactivity to several presently reported N361-369 mutant variants,as to the wild-type epitope.Further,the natural functions of these TCRs in the cytotoxic immunity against SARS-CoV-2 were determined with dendritic cells(DCs)and the lung organoid model.We found that the N361-369 epitope could be normally processed and endogenously presented by these different types of antigen presenting cells,to elicit successful activation and effective cytotoxicity of CD8+T cells ex vivo.Our study evidenced potential mechanisms of cellular immunity to SARS-CoV-2,and illuminated potential ways of viral clearance in COVID-19 patients.These results indicate that utilizing CD8-independent TCRs against SARS-CoV-2-associated antigens may provide functional superiority that is beneficial for the adoptive cell immunotherapies based on natural or genetically engineered T cells.Additionally,this information is highly relevant for the development of the next-generation vaccines with protections against continuously emerged SARS-CoV-2 mutant strains.

MHC Class Ⅰ Antigen Presentation-Recently Trimmed and Well Presented

Presentation of antigenic peptide to T cells by major histocompatibility complex (MHC) class I molecules is the key to the cellular immune response.Non-self intracellular proteins are processed into short peptides and transported into endoplasmic reticulum (ER) where they are assembled with class I molecules assisted by several chaperone proteins to form trimeric complex.MHC class I complex loaded with optimised peptides travels to the cell surface of antigen presentation cells to be recognised by T cells.The cells presenting non-self peptides are cleared by CD8 positive T cells.In order to ensure that T cells detect an infection or mutation within the target cells the process of peptide loading and class I expression must be carefully regulated.Many of the cellular components involved in antigen processing and class I presentation are known and their various functions are now becoming clearer.Cellular & Molecular Immunology.2004;1(1):22-30.

Yeast display of MHC-II enables rapid identification of peptide ligands from protein antigens(RIPPA)

CD4^(+)T cells orchestrate adaptive immune responses via binding of antigens to their receptors through specific peptide/MHC-II complexes.To study these responses,it is essential to identify protein-derived MHC-II peptide ligands that constitute epitopes for T cell recognition.However,generating cells expressing single MHC-II alleles and isolating these proteins for use in peptide elution or binding studies is time consuming.Here,we express human MHC alleles(HLA-DR4 and HLA-DQ6)as native,noncovalentαβdimers on yeast cells for direct flow cytometry-based screening of peptide ligands from selected antigens.We demonstrate rapid,accurate identification of DQ6 ligands from pre-pro-hypocretin,a narcolepsy-related immunogenic target.We also identify 20 DR4-binding SARS-CoV-2 spike peptides homologous to SARS-CoV-1 epitopes,and one spike peptide overlapping with the reported SARS-CoV-2 epitope recognized by CD4^(+)T cells from unexposed individuals carrying DR4 subtypes.Our method is optimized for immediate application upon the emergence of novel pathogens.

Computational Analysis of Cysteine Proteases(Clan CA,Family C1) of Leishmania major to Find Potential Epitopic Regions

Leishmania is associated with a broad spectrum of diseases, ranging from simple cutaneous to invasive visceral leishmaniasis. Here, the sequences of ten cysteine proteases of types A, B and C of Leishmania major were obtained from GeneDB database. Prediction of MHC class I epitopes of these cysteine proteases was performed by NetCTL program version 1.2. In addition, by using BcePred server, different structural properties of the proteins were predicted to find out their potential B cell epitopes. According to this computational analysis, nine regions were predicted as B cell epitopes. The results provide useful information for designing peptide-based vaccines.