Recent trends of human papillomavirus (HPV) infections in head and neck carcinomas, especially oropharyngeal carcinomas, have been incorporated into NPC research. HPV was detected in two of the 58 NPC tissues at our institute. The HPV-positive cases were classified as WHO II, and EBERs were detected using in situ hybridization. One case had HPV18, and the others had HPV16 and 18 ^[9][14]. Reports have been inconsistent, likely because of the limited number of patients and the ethnic and geographic differences among the study populations. Several studies have detected HPV in NPC, with some demonstrating a dichotomy between EBV and HPV infections predominantly in non-endemic regions ^[10][11][12][15,16,17]. Others have reported cases of EBV and HPV co-infection, predominantly in patients from endemic regions ^[13][14][15][18,19,20]. Similar to EBV, a serological test has been developed for HPV and applied to screening for HPV-associated diseases ^[16][21]. EBV-associated NPC is characterized by the prominent infiltration of lymphocytes in the tumor-surrounding area and the so-called lymphoepithelioma. Based on the unique pathological features of NPC, the relationship between the immune system and NPC tumor cells has been intensively investigated. The nasopharynx contains a unique lymphoid tissue called the nasopharynx-associated lymphoid tissue (NALT), which differs from other lymphoid tissues involved in organogenesis. NALT plays an important role in the mucosal immune system ^[17][94]. The secretion of anti-EBV IgA from the NALT into the airway lumen and serum is a marker for NPC screening and diagnosis. New insights into the tumor microenvironment of NPC have been growing, including the tumor–immune microenvironment (TIME). The tumor microenvironment is mainly composed of heterogeneous cellular components such as tumor cells, fibroblasts, endothelial cells, and leukocytes. It also contains various mediators, such as cytokines and exosomes, which influence the behavior of NPC tumors and, eventually, the prognosis of NPC patients. The TIME consists of immunostimulant and immunosuppressive components. Thus, the TIME component also serves as a prognostic biomarker and a potential target for novel therapies ^[18][95]. Tumor-infiltrating lymphocytes (TIL) in NPC tissue contain various immune cells, and the proportion of the immune population dynamically changes with tumor progression and treatment. However, TIL levels usually reflect the treatment outcomes of patients. Abundant intratumoral and stromal TILs are predictive markers of favorable outcomes in patients with NPC ^[19][96].

2. Immune Evasion Mechanism in NPC

Malignant tumor cells begin the construction of the TIME once they are recognized as targets of immune cells. Subsequently, the tumor cells escape the immune attack. Generally, viral proteins are presented as antigens combined with MHC class I molecules to recruit CD 8-positive cytotoxic T cells (CTL) to the virus-infected tumor cells. Three EBV genes, EBNA1, LMP1, and LMP2, are expressed in NPC. However, EBV-encoded gene products expressed in NPC cells must escape from immune cells, mainly from CTL, for the development of NPC. In the EBV latent infection program, the most efficient strategy for immune escape is to keep the level of expression of the EBV antigen per cell as low as possible. Maintaining low antigen expression leads to low CTL epitope presentation in EBV-infected cells. Crotzer et al. reported that the presentation of PRIYDLIEL-like epitopes in EBNA3C is less than one epitope per cell ^[20][97], resulting in very low or no detection of EBV-transformed B-cells by CTL clones with high affinity to the peptide ^[21][98]. In addition to the low levels of latent gene expression, EBNA1 contains a Gly-Ara repeat domain that suppresses the translation of EBNA1 and prevents the processing of EBNA1, resulting in the inhibition of antigen presentation by MHC class I ^[22][99]. Upon activation, LMP1 localizes and aggregates within lipid rafts on the cell membrane ^[23][100]. LMP1 mutants that lose their aggregating properties at lipid rafts are recognized by HLA epitope-sensitized CTL, whereas wild-type and LMP mutants that retain their aggregating properties at lipid rafts are not. These results suggest that the aggregation of LMP1 in lipid rafts protects LMP1 from being processed and presented with MHC class I, which allows LMP1 expression in NPC cells to escape immune surveillance ^[24][101]. In contrast, lytic infection involves the expression of more than 60 viral gene products with high copy numbers per cell ^[25][102]. For the transmission of EBV from carriers who have established T-cell immunity to the EBV gene to other individuals, cells with EBV lytic infection must escape T-cell recognition long enough to produce mature EBV particles. Even in cells under abortive lytic infection, the expression of immunogenic ZEBRA should affect the tumor–immune cell interactions. BZLF1 suppresses the transcription of inflammatory factors TNF-α and IFN-γ and prevents their response during the EBV lytic infection. This implies that the EBV lytic cycle employs a distinct strategy to evade the antiviral inflammatory response ^[26][103].

3. Superantigen Induction by EBV

One theory states that despite these mechanisms of immune evasion, some EBV gene expression leads to lymphocyte infiltration. This is one theory. The other theory is the induction of superantigens by EBV genes. Superantigens are microbial pathogen-derived proteins that induce strong T-cell responses. HERV-K18 was the first human endogenous provirus to originate from a retrovirus with superantigen activity ^[27][104]. HERV-K18 preferentially activates human TCRBV13 T cells ^[28][105]. Essentially, it exists in a dormant state. However, the expression of its env gene was observed in latently infected EBV cells. Sutkowski showed that both LMP1 and LMP2A are able to transactivate the HERV-K18 superantigen. Both LMP1 and LMP2A are expressed in NPC cells ^[29][106]. HERV-K18 may induce heavy T-cell infiltration, and the secretion of cytokines by the interaction of superantigen-expressing tumor cells with activated T-cells can contribute to the highly metastatic features of NPC ^[30][31][107,108].

4. Modulation of Immune Checkpoint in NPC

Generally, the number of intratumoral and stromal TILs is associated with a favorable prognosis in patients with NPC ^[19][96]. The programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) axis plays an important role in T-cell tolerance and immune escape of tumor cells. PD-L1 expression is higher in EBV-positive than in EBV-negative NPC cell lines. It can be due to the abundant IFN-γ induction by EBV infection. In addition, LMP1 and IFN-γ pathways cooperate to regulate PD-L1 ^[32][44]. LMP1 knockdown suppresses PD-L1 expression in EBV-positive cell lines. IFN-γ upregulates PD-L1 expression independently and synergistically with LMP1 in NPC tissue ^[33][109]. PD-1 is expressed on TIL, while PD-L1 is expressed in tumor and stromal cells. The interaction between PD-1 and PD-L1 suppresses CTL attacks on tumor cells ^[33][109]. Thus, the co-expression of PD-1 and PD-L1 in the tumor microenvironment usually predicts the recurrence and metastasis of NPC after initial therapy. Antibodies that block this pathway have been clinically used against various cancers, including NPC ^[18][34][95,110]. Adoptive T-cell therapy targeting the EBV gene products is a potential therapeutic strategy for NPC. PD-L1 expression in cancer cells can inhibit the effector functions of adoptively transferred EBV-specific T-cells. The combination of EBV-specific adoptive T-cell and PD-L1 blockade therapies has been reported to be more effective ^[35][111]. Several immunotherapies have been explored for NPC; however, these trials have yielded inconsistent conclusions, probably due to different immune microenvironments ^{[33][34][36][37][38][39]}[109,110,112,113,114,115]. For example, LMP1 promotes myeloid-derived suppressor cell (MDSC) expansion in the tumor microenvironment by promoting extra-mitochondrial glycolysis in malignant cells. In addition to RAGE, LMP1 promotes the expression of multiple glycolytic genes, such as GLUT1. This metabolic reprogramming induces the NOD-like receptor family protein 3 inflammasome and activates the arachidonic cascade, which in turn activates various cytokines such as IL-1β, IL-6, and GM-CSF. These changes in the tumor environment result in NPC-derived MDSC induction ^[40][116].

5. Challenge for EBV Vaccine Development

The close association of EBV infection with NPC suggests that controlling EBV infection and targeting EBV-infected cells are effective strategies for preventing NPC development and managing developed NPC. However, despite the urgent need for prophylactic or therapeutic solutions, there has been no licensed EBV vaccine thus far ^[41][117]. The major obstacles to developing a prophylactic vaccine include the global prevalence of EBV and the fact that infection typically occurs during early childhood to adolescence, resulting in a limited number of control cohorts without EBV infection. In addition, the complexity of the EBV replication system and its infectivity to T lymphocytes and NK cells present challenges in eliminating all EBV target cells ^[41][117]. With recent advancements in messenger RNA vaccines, exemplified by their successful application in the COVID-19 pandemic, various viruses, including EBV, have been considered candidates for this technique ^[42][118]. Moderna has announced the initiation of a phase 1 study for its mRNA Epstein-Barr virus (EBV) vaccine, code-named mRNA-1189. mRNA-1189 comprises five mRNAs encoding envelope glycoproteins (gp320, gH, gL, gp42, and gp220) that bind to target cell surface receptors, playing key roles in initiating EBV infection of target cells. The administration of mRNA-1189 aims to induce a broad immune response that could prevent EBV infection in various types of cells, ultimately reducing the symptoms of infectious mononucleosis. According to the manufacturer, preclinical testing of mRNA-1189 in mice and nonhuman primates demonstrated high and durable levels of antigen-specific antibodies against B cell and epithelial cell infection ^[43][119]. The announced phase 1 clinical trial (NCT05164094) will assess the safety and tolerability of three different doses of mRNA-1189 in healthy adults aged 18 to 30. The humoral immune response will be evaluated up to day 197 ^[44][120]. (https://clinicaltrials.gov/study/NCT05164094 (accessed on 11 October 2023)). mRNA-1189, which encodes additional latent EBV genes, may serve as a candidate for therapeutic vaccines. Certainly, mRNA technology is poised to bring about significant progress in the future of vaccinology.