Evaluation of serum Epstein-Barr virus envelope glycoproteins antibodies and their association with systemic autoimmune diseases.

Systemic autoimmune diseases (SADs) are a growing spectrum of autoimmune disorders that commonly affect multiple organs. The role of Epstein-Barr virus (EBV) infection or reactivation as a trigger for the initiation and progression of SADs has been established, while the relationship between EBV envelope glycoproteins and SADs remains unclear. Here, we assessed the levels of IgG, IgA, and IgM against EBV glycoproteins (including gp350, gp42, gHgL, and gB) in serum samples obtained from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), and found that RA and SLE patients exhibited a statistically significant increase in the levels of 8 and 11 glycoprotein antibodies, respectively, compared to healthy controls (p < 0.05). The LASSO model identified four factors as significant diagnostic markers for RA: gp350 IgG, gp350 IgA, gHgL IgM, and gp42 IgA; whereas for SLE it included gp350 IgG, gp350 IgA, gHgL IgA, and gp42 IgM. Combining these selected biomarkers yielded an area under the curve (AUC) of 0.749 for RA and 0.843 for SLE. We subsequently quantified the levels of autoantibodies associated with SADs in mouse sera following immunization with gp350. Remarkably, none of the tested autoantibody levels exhibited statistically significant alterations. Elevation of glycoprotein antibody concentration suggests that Epstein-Barr virus reactivation and replication occurred in SADs patients, potentially serving as a promising biomarker for diagnosing SADs. Moreover, the absence of cross-reactivity between gp350 antibodies and SADs-associated autoantigens indicates the safety profile of a vaccine based on gp350 antigen.

Induction of Broadly Cross-Reactive Antibody Responses to SARS-CoV-2 Variants by S1 Nanoparticle Vaccines.

Despite the rapid deployment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, the emergence of SARS-CoV-2 variants and reports of their immune evasion characteristics have led to an urgent need for novel vaccines that confer potent cross-protective immunity. In this study, we constructed three different SARS-CoV-2 spike S1-conjugated nanoparticle vaccine candidates that exhibited high structural homogeneity and stability. Notably, these vaccines elicited up to 50-times-higher neutralizing antibody titers than the S1 monomer in mice. Crucially, it was found that the S1-conjugated nanoparticle vaccine could elicit comparable levels of neutralizing antibodies against wild-type or emerging variant SARS-CoV-2, with cross-reactivity to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), the effect of which could be further enhanced using our designed nanoparticles. Our results indicate that the S1-conjugated nanoparticles are promising vaccine candidates with the potential to elicit potent and cross-reactive immunity against not only wild-type SARS-CoV-2, but also its variants of concern, variants of interest, and even other pathogenic betacoronaviruses. IMPORTANCE The emergence of SARS-CoV-2 variants led to an urgent demand for a broadly effective vaccine against the threat of variant infection. The spike protein S1-based nanoparticle designed in our study could elicit a comprehensive humoral response toward different SARS-CoV-2 variants of concern and variants of interest and will be helpful to combat COVID-19 globally.

Vesicular Stomatitis Virus-Based Epstein-Barr Virus Vaccines Elicit Strong Protective Immune Responses.

Epstein-Barr virus (EBV), the first identified human tumor virus, is etiologically associated with various kinds of malignant and benign diseases, accounting for 265,000 cancer incident cases and 164,000 cancer deaths in 2017. EBV prophylactic vaccine development has been gp350 centered for several decades. However, clinical studies show that gp350-centered vaccines fail to prevent EBV infection. Advances in the EBV infection mechanisms shed light on gB and gHgL, the two key components of the infection apparatus. In this study, for the first time, we utilized recombinant vesicular stomatitis virus (VSV) to display EBV gB (VSV-ΔG-gB/gB-G) or gHgL (VSV-ΔG-gHgL). In vitro studies confirmed successful virion production and glycoprotein presentation on the virion surface. In mouse models, VSV-ΔG-gB/gB-G or VSV-ΔG-gHgL elicited potent humoral responses. Neutralizing antibodies elicited by VSV-ΔG-gB/gB-G were prone to prevent B cell infection, while those elicited by VSV-ΔG-gHgL were prone to prevent epithelial cell infection. Combinatorial vaccination yields an additive effect. The ratio of endpoint neutralizing antibody titers to the endpoint total IgG titers immunized with VSV-ΔG-gHgL was approximately 1. The ratio of IgG1/IgG2a after VSV-ΔG-gB/gB-G immunization was approximately 1 in a dose-dependent, adjuvant-independent manner. Taken together, VSV-based EBV vaccines can elicit a high ratio of epithelial and B lymphocyte neutralizing antibodies, implying their unique potential as EBV prophylactic vaccine candidates. IMPORTANCE Epstein-Barr virus (EBV), one of the most common human viruses and the first identified human oncogenic virus, accounted for 265,000 cancer incident cases and 164,000 cancer deaths in 2017 as well as millions of nonmalignant disease cases. So far, no prophylactic vaccine is available to prevent EBV infection. In this study, for the first time, we reported the VSV-based EBV vaccines presenting two key components of the EBV infection apparatus, gB and gHgL. We confirmed potent antigen-specific antibody generation; these antibodies prevented EBV from infecting epithelial cells and B cells, and the IgG1/IgG2a ratio indicated balanced humoral-cellular responses. Taken together, we suggest VSV-based EBV vaccines are potent prophylactic candidates for clinical studies and help eradicate numerous EBV-associated malignant and benign diseases.

Immunization with a Self-Assembled Nanoparticle Vaccine Elicits Potent Neutralizing Antibody Responses against EBV Infection.

Epstein-Barr virus (EBV) infection is a global health concern infecting over 90% of the population. However, there is no currently available vaccine. EBV primarily infects B cells, where the major glycoprotein 350 (gp350) is the main target of neutralizing antibodies. Given the advancement of nanoparticle vaccines, we describe rationally designed vaccine modalities presenting 60 copies of gp350 on self-assembled nanoparticles in a repetitive array. In a mouse model, gp350s on lumazine synthase (LS) and I3-01 adjuvanted with MF59 or aluminum hydroxide (Alhydrogel) elicited over 65- to 133-fold higher neutralizing antibody titers than the corresponding gp350 monomer to EBV. Furthermore, immunization with gp350D123-LS and gp350D123-I3-01 vaccine induced a Th2-biased response. For the nonhuman primate model, gp350D123-LS in MF59 elicited higher titers of total IgG and neutralizing antibodies than the monomeric gp350D123. Overall, these results support gp350D123-based nanoparticle vaccine design as a promising vaccine candidate for potent protection against EBV infection.

mRNA-based Vaccines Targeting the T-cell Epitope-rich Domain of Epstein Barr Virus Latent Proteins Elicit Robust Anti-Tumor Immunity in Mice.

Epstein-Barr virus (EBV) is associated with various malignancies and infects >90% of the global population. EBV latent proteins are expressed in numerous EBV-associated cancers and contribute to carcinogenesis, making them critical therapeutic targets for these cancers. Thus, this study aims to develop mRNA-based therapeutic vaccines that express the T-cell-epitope-rich domain of truncated latent proteins of EBV, including truncatedlatent membrane protein 2A (Trunc-LMP2A), truncated EBV nuclear antigen 1 (Trunc-EBNA1), and Trunc-EBNA3A. The vaccines effectively activate both cellular and humoral immunity in mice and show promising results in suppressing tumor progression and improving survival time in tumor-bearing mice. Furthermore, it is observed that the truncated forms of the antigens, Trunc-LMP2A, Trunc-EBNA1, and Trunc-EBNA3A, are more effective than full-length antigens in activating antigen-specific immune responses. In summary, the findings demonstrate the effectiveness of mRNA-based therapeutic vaccines targeting the T-cell-epitope-rich domain of EBV latent proteins and providing new treatment options for EBV-associated cancers.

Non-overlapping epitopes on the gHgL-gp42 complex for the rational design of a triple-antibody cocktail against EBV infection.

Epstein-Barr virus (EBV) is closely associated with cancer, multiple sclerosis, and post-acute coronavirus disease 2019 (COVID-19) sequelae. There are currently no approved therapeutics or vaccines against EBV. It is noteworthy that combining multiple EBV glycoproteins can elicit potent neutralizing antibodies (nAbs) against viral infection, suggesting possible synergistic effects. Here, we characterize three nAbs (anti-gp42 5E3, anti-gHgL 6H2, and anti-gHgL 10E4) targeting different glycoproteins of the gHgL-gp42 complex. Two antibody cocktails synergistically neutralize infection in B cells (5E3+6H2+10E4) and epithelial cells (6H2+10E4) in vitro. Moreover, 5E3 alone and the 5E3+6H2+10E4 cocktail confer potent in vivo protection against lethal EBV challenge in humanized mice. The cryo-EM structure of a heptatomic gHgL-gp42 immune complex reveals non-overlapping epitopes of 5E3, 6H2, and 10E4 on the gHgL-gp42 complex. Structural and functional analyses highlight different neutralization mechanisms for each of the three nAbs. In summary, our results provide insight for the rational design of therapeutics or vaccines against EBV infection.

A gB nanoparticle vaccine elicits a protective neutralizing antibody response against EBV.

Epstein-Barr virus (EBV) is a global public health concern, as it is known to cause multiple diseases while also being etiologically associated with a wide range of epithelial and lymphoid malignancies. Currently, there is no available prophylactic vaccine against EBV. gB is the EBV fusion protein that mediates viral membrane fusion and participates in host recognition, making it critical for EBV infection in both B cells and epithelial cells. Here, we present a gB nanoparticle, gB-I53-50 NP, that displays multiple copies of gB. Compared with the gB trimer, gB-I53-50 NP shows improved structural integrity and stability, as well as enhanced immunogenicity in mice and non-human primate (NHP) preclinical models. Immunization and passive transfer demonstrate a robust and durable protective antibody response that protects humanized mice against lethal EBV challenge. This vaccine candidate demonstrates significant potential in preventing EBV infection, providing a possible platform for developing prophylactic vaccines for EBV.

How EBV Infects: The Tropism and Underlying Molecular Mechanism for Viral Infection.

The Epstein-Barr virus (EBV) is associated with a variety of human malignancies, including Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma and gastric cancers. EBV infection is crucial for the oncogenesis of its host cells. The prerequisite for the establishment of infection is the virus entry. Interactions of viral membrane glycoproteins and host membrane receptors play important roles in the process of virus entry into host cells. Current studies have shown that the main tropism for EBV are B cells and epithelial cells and that EBV is also found in the tumor cells derived from NK/T cells and leiomyosarcoma. However, the process of EBV infecting B cells and epithelial cells significantly differs, relying on heterogenous glycoprotein-receptor interactions. This review focuses on the tropism and molecular mechanism of EBV infection. We systematically summarize the key molecular events that mediate EBV cell tropism and its entry into target cells and provide a comprehensive overview.

Quadrivalent mosaic HexaPro-bearing nanoparticle vaccine protects against infection of SARS-CoV-2 variants.

Emerging SARS-CoV-2 variants of concern (VOCs) harboring multiple mutations in the spike protein raise concerns on effectiveness of current vaccines that rely on the ancestral spike protein. Here, we design a quadrivalent mosaic nanoparticle vaccine displaying spike proteins from the SARS-CoV-2 prototype and 3 different VOCs. The mosaic nanoparticle elicits equivalent or superior neutralizing antibodies against variant strains in mice and non-human primates with only small reduction in neutralization titers against the ancestral strain. Notably, it provides protection against infection with prototype and B.1.351 strains in mice. These results provide a proof of principle for the development of multivalent vaccines against pandemic and potential pre-emergent SARS-CoV-2 variants.

Parallel profiling of antigenicity alteration and immune escape of SARS-CoV-2 Omicron and other variants.

SARS-CoV-2 variants have evolved a variety of critical mutations, leading to antigenicity changes and immune escape. The recent emerging SARS-CoV-2 Omicron variant attracted global attention due to its significant resistance to current antibody therapies and vaccines. Here, we profiled the mutations of Omicron and other various circulating SARS-CoV-2 variants in parallel by computational interface analysis and in vitro experimental assays. We identified critical mutations that lead to antigenicity changes and diminished neutralization efficiency of a panel of 14 antibodies due to diverse molecular mechanisms influencing the antigen-antibody interaction. Our study identified that Omicron exhibited extraordinary potency in immune escape compared to the other variants of concern, and explores the application of computational interface analysis in SARS-CoV-2 mutation surveillance and demonstrates its potential for the early identification of concerning variants, providing preliminary guidance for neutralizing antibody therapy.

The Status and Prospects of Epstein-Barr Virus Prophylactic Vaccine Development.

Epstein-Barr virus (EBV) is a human herpesvirus that is common among the global population, causing an enormous disease burden. EBV can directly cause infectious mononucleosis and is also associated with various malignancies and autoimmune diseases. In order to prevent primary infection and subsequent chronic disease, efforts have been made to develop a prophylactic vaccine against EBV in recent years, but there is still no vaccine in clinical use. The outbreak of the COVID-19 pandemic and the global cooperation in vaccine development against SARS-CoV-2 provide insights for next-generation antiviral vaccine design and opportunities for developing an effective prophylactic EBV vaccine. With improvements in antigen selection, vaccine platforms, formulation and evaluation systems, novel vaccines against EBV are expected to elicit dual protection against infection of both B lymphocytes and epithelial cells. This would provide sustainable immunity against EBV-associated malignancies, finally enabling the control of worldwide EBV infection and management of EBV-associated diseases.

PD-1+CXCR5-CD4+ Th-CXCL13 cell subset drives B cells into tertiary lymphoid structures of nasopharyngeal carcinoma.

BACKGROUND: A major current challenge is to exploit tertiary lymphoid structures (TLSs) to promote the lymphocyte infiltration, activation and differentiation by tumor antigens to increase antitumor immune responses. The mechanisms that underlie the role of TLS formation in the adaptive immune responses against nasopharyngeal carcinoma (NPC) remain largely unknown. METHODS: Cell populations and the corresponding markers were identified by single-cell RNA sequencing and fluorescence-activated cell sorting analysis. In vitro differentiation experiments were used to simulate the generation, regulation and function of the Th-CXCL13 cell subset in the tumor microenvironment of NPC. These were followed by histological evaluation of the colocalization of tumor-associated B cells (TABs) and Th-CXCL13 cells within TLSs, and statistical analysis of the relationship between the cells in TLSs and overall survival. RESULTS: A PD-1+CXCR5-CD4+ Th-CXCL13 cell subset was identified in NPC. This subset was a major source of CXCL13, representing the majority of the CD4+ T cells at levels comparable with Th1 and Tfh cells present in the TLSs. Monocytes activated by toll-like receptor 4 agonists served as the antigen-presenting cells that most efficiently triggered the expansion of Th-CXCL13 cells. Transforming growth factor beta 1 (TGF-ß1) stimulation and activation of Sox4 were critical for the induction and polarization of Th-CXCL13 cells in this process. The potential functional contributions of TABs recruited by Th-CXCL13 cells which induced plasma cell differentiation and immunoglobulin production via interleukin-21 and CD84 interactions in the TLSs demonstrated improved survival. CONCLUSIONS: Induction of Th-CXCL13 cells links innate inflammation to immune privilege in tumor-associated TLSs and might predict better survival.

Rapid Development of SARS-CoV-2 Spike Protein Receptor-Binding Domain Self-Assembled Nanoparticle Vaccine Candidates.

The coronavirus disease pandemic of 2019 (COVID-19) caused by the novel SARS-CoV-2 coronavirus resulted in economic losses and threatened human health worldwide. The pandemic highlights an urgent need for a stable, easily produced, and effective vaccine. SARS-CoV-2 uses the spike protein receptor-binding domain (RBD) to bind its cognate receptor, angiotensin-converting enzyme 2 (ACE2), and initiate membrane fusion. Thus, the RBD is an ideal target for vaccine development. In this study, we designed three different RBD-conjugated nanoparticle vaccine candidates, namely, RBD-Ferritin (24-mer), RBD-mi3 (60-mer), and RBD-I53-50 (120-mer), via covalent conjugation using the SpyTag-SpyCatcher system. When mice were immunized with the RBD-conjugated nanoparticles (NPs) in conjunction with the AddaVax or Sigma Adjuvant System, the resulting antisera exhibited 8- to 120-fold greater neutralizing activity against both a pseudovirus and the authentic virus than those of mice immunized with monomeric RBD. Most importantly, sera from mice immunized with RBD-conjugated NPs more efficiently blocked the binding of RBD to ACE2 in vitro, further corroborating the promising immunization effect. Additionally, the vaccine has distinct advantages in terms of a relatively simple scale-up and flexible assembly. These results illustrate that the SARS-CoV-2 RBD-conjugated nanoparticles developed in this study are a competitive vaccine candidate and that the carrier nanoparticles could be adopted as a universal platform for a future vaccine development.