A Systematic Review of T Cell Epitopes Defined from the Proteome of Hepatitis B Virus

Hepatitis B virus (HBV) infection remains a worldwide health problem and no eradicative therapy is currently available. Host T cell immune responses have crucial influences on the outcome of HBV infection, however the development of therapeutic vaccines, T cell therapies and the clinical evaluation of HBV-specific T cell responses are hampered markedly by the lack of validated T cell epitopes. This review presented a map of T cell epitopes functionally validated from HBV antigens during the past 33 years; the human leukocyte antigen (HLA) supertypes to present these epitopes, and the methods to screen and identify T cell epitopes. To the best of our knowledge, a total of 205 CD8+ T cell epitopes and 79 CD4+ T cell epitopes have been defined from HBV antigens by cellular functional experiments thus far, but most are restricted to several common HLA supertypes, such as HLA-A0201, A2402, B0702, DR04, and DR12 molecules. Therefore, the currently defined T cell epitope repertoire cannot cover the major populations with HLA diversity in an indicated geographic region. More researches are needed to dissect a more comprehensive map of T cell epitopes, which covers overall HBV proteome and global patients.


Introduction
Hepatitis B virus (HBV) infection still disseminates across the world and causes the most common and fatal liver diseases including acute liver failure, chronic hepatitis, liver cirrhosis (LC), and hepatocellular carcinoma (HCC) [1,2]. Nucleoside analogs and/or interferon are widely utilized antiviral drugs, which can effectively suppress virus replication, decrease serum HBV DNA to undetectable levels, mitigate liver fibrosis, and reduce HCC risk [3][4][5], however cannot eliminate the virus in patients. Recurrence after therapy discontinuation is emerging to be a common etiology of morbidity and mortality in patients with chronic HBV infection [6].
Numerous researches have demonstrated the important influence of HBV-specific T cell responses on virus clearance [7], disease progression [8][9][10], antiviral efficacy [11,12], and recurrence [13][14][15], particularly the CD8 + T cells, which act as vital effector cells to kill virus-infected hepatocytes and secret cytokines. Patients with acute-resolving HBV infection show robust HBV-specific CD8 + T cell responses, while the patients with chronic HBV infection present a phenomenon termed CD8 + T cell functional exhaustion with multifactorial heterogeneity [9], and differs depending on the targeted antigen for HLA-A02 restricted epitopes located in the core antigen versus polymerase [16]. Furthermore, the heterogeneity of HBV-specific T cells also responds differently to therapeutic stimuli [17]. Therefore, T cells specific for HBV not only are the potential markers for monitoring the effects of antiviral therapy and predicting the recurrence [18], but also are the promising modulators in specific immunotherapy. Identifying the T cell epitopes as many as possible from HBV antigens will greatly contribute to the design and development of epitope-based and T cell-based therapies and the detection of host HBV-specific T cell immunity. Although negatively related to persistence to chronic HBV infection in the populations of Africans [26], Europeans [27], Koreans [28] and Northwestern Chinese [29]. In addition, HLA-A*33 is closely associated with susceptibility to persisting HBV infection, and HLA-DRB1*13 is closely related to protection against persisting HBV infection in an Iranian population [30]. A*0301 and DRB1*1302 are relevant to viral clearance and B*08 is associated with viral persistence in Caucasians [31]. However, although the correlation between HBV infection and HLA alleles has been studied for several decades, in accordance with what we described in the above review, it often has conflicting results. These variations partly result from host HLA polymorphism in different races and regions [32,33]. Further studies should be explored in different regions to reduce the heterogeneity of results.

HBV Proteome and the Approaches Identifying T Cell Epitopes
HBV is one of the smallest viruses with a genome length of 3.2 Kb [34]. Its genome contains four open reading frames (ORFs) coding four partially overlapping proteins as displayed in Figure 1: (1) preS/S ORF encodes large (L), middle (M), and small (S) surface antigens (HBsAg). HBsAg is being widely investigated in clinical fields and quantified as a diagnostic marker of HBV infection as it can reflect the level of covalently closed circular DNA (cccDNA) and intrahepatic HBV DNA in chronic infection [35,36]. (2) Pre-core/core ORF encodes hepatitis B e antigen (HBeAg), core antigen (HBcAg) or in combination corerelated antigen (HBcrAg). HBeAg has long been advocated as a serum marker for guiding the clinical practice of chronic hepatitis B virus [37,38]. HBcrAg has been demonstrated more recently as a potential surrogate marker of cccDNA [39]. (3) X ORF encodes HBx antigen (HBxAg), which plays an important role in virus genome transcription and is correlated with liver cancer. The expression of HBxAg in HBV-associated HCC patients is significantly higher than other viral proteins [40]. (4) P ORF encodes the viral DNA polymerase (HBpol), which is responsible for the replication of the viral genome and is an effective target for the therapeutic intervention of chronic HBV infection [41]. Human HBV strains occur in nine genotypes A-I, and its major HBV surface antigen (HBsAg) has several immune protective conformational B cell epitopes a, d or y, w1-4 or r [42]. The entire amino acid sequences of each protein from different genotypes were obtained from the UniProt database and aligned in Figure 2.   The process of T cell epitope identification begins with the selection of candidate epitope peptides. The first strategy is using overlapping peptides (OLPs) spanning the entire proteome or selected antigens of interest (peptide scanning). Chen et al. expanded HBVspecific T cells in vitro by co-culturing the overlapping peptide pools spanning the entire sequence of HBV genotypes B and C and the peripheral blood mononuclear cells (PBMCs) from patients with chronic HBV infection, followed by the detection of T cell response in each co-culture using IFN-γ enzyme-linked immunospot (IFN-γ ELISpot) assay, IFN-γ intracellular staining and flow cytometry [43]. However, peptide scanning is a high-cost and laborious method due to a large amount of OLPs spanning overall HBV proteins. For CD8 + T cell epitopes, HBsAg, HBeAg, HBx and HBpol contain 131, 68, 49, and 279 OLPs, respectively, when overlapping 6 amino acids in each 9-mer peptide. An alternative strategy is to focus on the in silico predicted T cell epitopes binding to the indicated HLA supertypes as calculated by multiple epitope prediction tools and algorithms. Brinck-Jensen et al. predicted 20 HBV-specific epitopes using combined in silico methods and evaluated for the immunogenicity of these epitopes through exposure to patients' PBMCs by IFN-γ ELISpot [44]. More recently, a similar in silico approach was also employed to assess all previously verified HBx-and HBpol-derived epitopes and to predict novel HLA-binding peptides for 6 HLA supertypes. Then, a part of reported epitopes were chosen for experimental validation. A total of 13 HLA binders derived from HBx and 33 binders from HBpol were described across HLA subtypes by this strategy [45]. Predicted epitopes are based on the indicated HLA restrictions and limit the number of research objects with diverse HLA subtypes to a reasonable range, yet the inaccuracy of theoretical prediction may omit some real-world epitopes.
The methodologies to validate the immunogenicity of candidate epitope peptides have been improved remarkably over the last two decades. Different assays are utilized for the detection of peptide-induced T cell response or peptide-specific T cells with individual advantages and disadvantages in terms of practicability, cost, sensitivity, function evaluation. The following approaches are currently widely used, such as cytotoxicity assay, proliferation assay, intracellular cytokine staining (ICS), ELISpot/FluoroSpot, and peptide-MHC multimers staining (tetramers, pentamers, or dextramers). The cytotoxicity assay was initially performed to validate CD8 + T cell epitopes by co-culturing patients' PBMCs with target cells labeled with Chromium-51, after the PBMCs were stimulated by the indicated candidate epitope peptides [46]. Additionally, lymphocyte proliferation assay is mostly applied to CD4 + T cell epitopes validation. The PBMCs from HBV-infected or HBV-vaccinated individuals were co-cultured with HBV-derived peptides for several days and 3 H-thymidine pulses were administered eventually followed by quantifying the incorporated radioactivity [47]. One more common approach currently utilized is ICS or ELISpot/FluoroSpot. Patient's PBMCs are in vitro or ex vivo stimulated with the candidate epitope peptides and simultaneously cytokine release is blocked followed by ICS and flow cytometry to define whether CD4 + T cells or CD8 + T cells activation [48]. The ELISpot or FluoroSpot technology enables the detection of single activated cells among one million PBMCs. The accuracy, sensitivity, reproducibility and durability have led to its widespread applications in researches and the broad prospects in the clinical detection of antigen-specific T cells [49,50]. An issue encountered with ELISpot, FluoroSpot, ICS, and related assays is that they may ignore T cells that produce different cytokines or trace cytokines during the window of time of the assay (e.g., Follicular helper CD4 + T cells generally produce very low amounts of cytokines). Peptide-MHC tetramer staining has been the gold standard to quantify antigen-specific T cells with high sensitivity and precision, thus is often used to identify T cell epitopes in many researches. However, the preparation of peptide-MHC tetramers or multimers is high-cost, complicated, and timeconsumption [51,52]. A pioneering study focused on all possible peptides of the entire HBV genome and 484 unique HLA-A1101-restricted epitopes predicted by NetMHC algorithms were validated using mass cytometry and multiplex peptide-tetramers staining [53]. Many researchers also have established a transgenic mouse model to map HLA-restricted epi-topes. Ru et al. developed and immunized HLA-A2/DP4 mice with epitopes derived from HBsAg to identify four new HLA-DP4-restricted epitopes [54]. Besides cellular functional experiments, peptide-HLA molecule binding and stabilization assays were commonly used to identify epitopes. Pan et al. defined 16 HBV epitopes by analyzing the different binding affinities of candidate epitope peptides with HLA-A3303 using RMA-S cells binding and stability assay. More recently, Ferretti et al. used a high-throughput genome-wide screening technology to identify the target cells expressing candidate epitopes productively recognized by T cells (T-Scan) and determined 29 epitopes in SARS-CoV-2 for the six most prevalent HLA types [55]. Chikata et al. employed immunocapture and liquid chromatography mass spectrometry (LC-MS) subsequent to pre-treatment of the target protein to disrupt its three-dimensional structure to characterize HIV-1 epitope peptides on a large scale presented by HLA-C1202 [56]. A variety of epitope assay strategies have been utilized with their own features and potential. Table 1 collected the CD8 + T cell epitopes and CD4 + T cell epitopes defined from HBV proteome during the past 33 years and displayed their HLA restrictions and the methods used to validate their immunogenicity. Notably, we performed manual management in this review, only the epitopes of 8-14 or 12-25 amino acids in length presented by HLA class I molecules or class II molecules are displayed since they reflect the standard size of the peptide-binding groove of HLA molecules. According to the previous report, if the epitope peptides are too short or long, the experiment tends to represent false positives instead of the result caused by the binding of peptide and HLA molecule [57].

Defined T Cell Epitopes in HBV Proteins during the Past 33 Years
Overall, 82 and 19 studies reported the epitopes presented by HLA class I molecules and class II molecules, respectively, and totally contained 284 unique epitopes including 205 CD8 + T cell epitopes and 79 CD4 + T cell epitopes (Table 1). Of these, 121 (59.0%) CD8 + T cell epitopes are restricted by HLA-A0201, A2402 or B0702 ( Figure 3A), which are common supertypes in Caucasians and less predominant in Asia and Africa [58,59]. The remainder are restricted mainly by 12 HLA-A, 5 HLA-B and 1 HLA-C supertypes. For the CD4 + T cell epitopes, the majority of currently described restrictions apply to 8 DRB1 supertypes ( Figure 3B). The cumulative frequency of the HLA-A supertypes described in Figure 3A was highest in Europe (66.6%), followed by Asia (53.1%), Africa (50.7%), and North America (52.3%) while the HLA-B supertypes showed an accumulative frequency of 32.7% in Europe, 20.1% in Asia, 19.2% in Africa and 18.8% in North America. The DRB1 supertypes in Figure 3B displayed little difference in the cumulative gene frequency in Europe (30.5%), Asia (32.2%), Africa (31.1%) and North America (34.1%). (Data from http://www.allelefrequencies.net/, assessed on 11 November 2021). Obviously, the 284 validated T cell epitopes of HBV cannot cover the major populations in an indicated geographic region. More T cell epitopes restricted by more HLA supertypes are urgently needed. Further efforts are required to identify more T cell epitopes restricted to the regional prevalent HLA supertypes, especially for the HLA alleles prevalent in Asian populations with a high HBV incidence [59,60].
In addition, although the validated T cell epitopes have derived from all HBV proteins, the CD8 + T cell epitopes mainly distribute in HBpol and HBsAg (72%) ( Figure 3C), while the majority of CD4 + T cell epitopes concentrate in HBeAg and HBsAg (78%) ( Figure 3D). The biased distribution of epitopes in proteome may be caused partially by the different lengths of proteins (HBpol 843aa, HBsAg 400 aa, HBeAg 212 aa, HBx 154 aa) and the pitfalls of screening methods.
As displayed in the sixth column of Table 1, most studies used the in silico prediction strategy to screen the candidate CD8 + T cell epitopes (92% of studies) and CD4 + T cells epitopes (63% of studies). Relatively, overlapping peptides were more often used in selecting candidate CD4 + T cell epitopes (7 of 19 studies; 37%) than CD8 + T cell epitopes (7 of 82 studies; 8%), partially due to the lower accuracy and efficacy of predicting HLA class II molecule-binding epitopes as compared with class I molecule-binding epitopes.

Conclusions
Here, we have taken an effort to present a reliable and updated T cell epitope repertoire of HBV. We summarized the statistics of 205 unique CD8 + T cell epitopes and 79 unique CD4 + T cell epitopes that have been experimentally validated and reported during the past 33 years, corresponding restricting HLA-molecule, and the methods to screen candidate epitopes and validate candidate epitopes. We hope that this review will be used as a tool for the design and development of therapeutic vaccines and T cell detection kits for HBV-infected patients.