Latent Antigens Research Articles

Impact of Epstein-Barr virus co-infection on natural acquired Plasmodium vivax antibody response.

BackgroundThe simultaneous infection of Plasmodium falciparum and Epstein-Barr virus (EBV) could promote the development of the aggressive endemic Burkitt’s Lymphoma (eBL) in children living in P. falciparum holoendemic areas. While it is well-established that eBL is not related to other human malaria parasites, the impact of EBV infection on the generation of human malaria immunity remains largely unexplored. Considering that this highly prevalent herpesvirus establishes a lifelong persistent infection on B-cells with possible influence on malaria immunity, we hypothesized that EBV co-infection could have impact on the naturally acquired antibody responses to P. vivax, the most widespread human malaria parasite.Methodology/Principal findingsThe study design involved three cross-sectional surveys at six-month intervals (baseline, 6 and 12 months) among long-term P. vivax exposed individuals living in the Amazon rainforest. The approach focused on a group of malaria-exposed individuals whose EBV-DNA (amplification of balf-5 gene) was persistently detected in the peripheral blood (PersVDNA, n = 27), and an age-matched malaria-exposed group whose EBV-DNA could never be detected during the follow-up (NegVDNA, n = 29). During the follow-up period, the serological detection of EBV antibodies to lytic/ latent viral antigens showed that IgG antibodies to viral capsid antigen (VCA-p18) were significantly different between groups (PersVDNA > NegVDNA). A panel of blood-stage P. vivax antigens covering a wide range of immunogenicity confirmed that in general PersVDNA group showed low levels of antibodies as compared with NegVDNA. Interestingly, more significant differences were observed to a novel DBPII immunogen, named DEKnull-2, which has been associated with long-term neutralizing antibody response. Differences between groups were less pronounced with blood-stage antigens (such as MSP1-19) whose levels can fluctuate according to malaria transmission.Conclusions/SignificanceIn a proof-of-concept study we provide evidence that a persistent detection of EBV-DNA in peripheral blood of adults in a P. vivax semi-immune population may impact the long-term immune response to major malaria vaccine candidates.

EBNA2-EBF1 complexes promote MYC expression and metabolic processes driving S-phase progression of Epstein-Barr virus–infected B cells

Epstein-Barr virus (EBV) is a human tumor virus which preferentially infects resting human B cells. Upon infection invitro, EBV activates and immortalizes these cells. The viral latent protein EBV nuclear antigen 2 (EBNA2) is essential for B cell activation and immortalization; it targets and binds the cellular and ubiquitously expressed DNA-binding protein CBF1, thereby transactivating a plethora of viral and cellular genes. In addition, EBNA2 uses its N-terminal dimerization (END) domain to bind early B cell factor 1 (EBF1), a pioneer transcription factor specifying the B cell lineage. We found that EBNA2 exploits EBF1 to support key metabolic processes and to foster cell cycle progression of infected B cells in their first cell cycles upon activation. The α1-helix within the END domain was found to promote EBF1 binding. EBV mutants lacking the α1-helix in EBNA2 can infect and activate B cells efficiently, but activated cells fail to complete the early S phase of their initial cell cycle. Expression of MYC, target genes of MYC and E2F, as well as multiple metabolic processes linked to cell cycle progression are impaired in EBVΔα1-infected B cells. Our findings indicate that EBF1 controls B cell activation via EBNA2 and, thus, has a critical role in regulating the cell cycle of EBV-infected B cells. This is a function of EBF1 going beyond its well-known contribution to B cell lineage specification.

Abstract 3573: DNA immunotherapy targeting BARF1 demonstrates therapeutic impact in novel murine carcinoma models expressing EBV latent antigens

Abstract Epstein Barr Virus (EBV) latent infection is associated with multiple types of cancer. Several clinical studies have targeted EBV antigens as immune therapeutic targets with limited efficacy of EBV+ malignancies, suggesting additional targets might be important. BamHI-A rightward frame 1 (BARF1) is an EBV antigen that is highly expressed in EBV-associated gastric carcinoma (EBVaGC) and EBV+ nasopharyngeal carcinoma (NPC) and has not been evaluated as an antigen in the context of therapeutic immunization. Here, we developed a synthetic DNA-based expression cassette as immunotherapy targeting BARF1 (pBARF1). Immunization with pBARF1 induced potent antigen-specific humoral and polyfunctional effector T cell responses in vivo. We observed significant antigen-specific increases of IFN-γ+, IFN-γ+ TNF-α+, and IFN-γ+ TNF-α+ IL-2+ populations in CD8+ T cells from the pBARF1 immunized animals in both C57BL/6 and BALB/c mice. Novel BARF1 expressing carcinoma lines that grow in immune-competent animals were developed as surrogates for human EBV+ tumors. We confirmed that BARF1 expression in these new cancer cell lines, MC38-BARF1 and CT26-BARF1, was within the range of human EBV+ cancer cell lines, SNU719 and C666-1. Immunization with pBARF1 plasmid demonstrated impact of tumor progression through induction of CD8+ T cell in these BARF1+ carcinoma models. We also observed long-term antitumor immunity, at least up to 446 days, against BARF1 in a CT26-BARF1 rechallenge study. Using IVIS in vivo imaging, we observed that pBARF1 immunized animals rapidly controlled and cleared cancer cells. These studies demonstrate the potential for pBARF1 immunity to induce antigen-specific immune responses impacting tumor progression. Further study of this immune target and these EBV+ tumor models is likely important as part of therapeutic approaches for EBV+ malignancies. Citation Format: Xizhou Zhu, Alfredo Perales-Puchalt, Krzysztof Wojtak, Ziyang Xu, Kun Yun, Pratik S. Bhojnagarwala, Devivasha Bordoloi, Daniel H. Park, Kevin Liaw, Mamadou A. Bah, Paul M. Lieberman, Ebony N. Gary, Ami Patel, David B. Weiner. DNA immunotherapy targeting BARF1 demonstrates therapeutic impact in novel murine carcinoma models expressing EBV latent antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3573.

Human Herpesvirus 8 seroprevalence among blood donors in Mali.

In sub-Saharan Africa, the Human Herpesvirus 8 (HHV-8) is endemic but with disparities between regions and population studied. Although the virus remains mostly latent, there is some evidence that blood transfusion may represents one of the transmission way for this virus. Here, we evaluated HHV-8 seroprevalence among blood donors in Mali. This cross-sectional study recruited blood donors from the Blood Transfusion Center at Gabriel Touré Hospital, Bamako. Serum was used for the detection of latent HHV-8 immunoglobulin G directed against latent associated nuclear antigen 1 by an indirect immunofluorescence assay.Human immunodeficiency virus 1(HIV-1), Hepatitis B Virus (HBV), HCV, and Treponema pallidum were also screened.HHV-8 seroprevalence was 10.4% in Malian blood donors. None of the sociodemographic characteristics were associated with HHV-8 infection, although there is a tendency of a higher HHV-8 seroprevalence among participants living in Bamako than those not living there. One individual had coinfection HHV-8/HBV, another HHV-8/HCV while another had HCV and T.pallidum. None has been tested positive for HIV infection. This intermediate seroprevalence in Malian blood donors suggests that the risk of HHV-8 transmission by transfusion should be considered. Further investigations are needed to assess impact of HHV-8 in polytransfused patients residing in an endemic area for this virus.

Epstein-Barr Virus, But Not Human Papillomavirus, Is Associated With Preinvasive and Invasive Ocular Surface Squamous Neoplasias in Zambian Patients.

BackgroundThe etiopathogenesis of ocular surface squamous neoplasia (OSSN) is not fully understood. We assessed the frequency of oncogenic viruses in OSSN by immunohistochemistry (IHC) and polymerase chain reaction (PCR) for human papillomavirus (HPV), Epstein–Barr virus (EBV), Merkel cell polyomavirus (MCPyV), Kaposi sarcoma virus, and adenovirus. Cases from Zambia were prospectively enrolled using a cross-sectional study design between November 2017 and March 2020.MethodsDemographic and clinical data [age, sex, HIV status, antiretroviral therapy (ART) history, CD4 count, plasma viral load] and tumor biopsies were collected from 243 consenting patients. Tumor samples were bisected, and half was used for DNA isolation, while the other half was formalin fixed and paraffin embedded (FFPE) for histopathology analysis. The expressions of latent EBV nuclear antigen 1 (EBNA1), CDKN2A/p16INK4A (p16), and MCPyV large T-antigen (LT) were tested by IHC. Multiplex PCR was used to detect 16 HPV genotypes and four other DNA tumor viruses [Kaposi’s sarcoma-associated herpesvirus (KSHV), EBV, MCPyV, and adenovirus]. Relationships between HIV status, viral DNA and protein expression, and tumor grades were determined by statistical analysis.ResultsOSSN tumors from patients were 29.6% preinvasive and 70.4% invasive. Patients presented with unilateral tumors that were 70.4% late stage (T3/T4). OSSN patients were HIV positive (72.8%). IHC on 243 FFPE biopsies resulted in the detection of EBNA1 (EBV), p16 high-risk HPV (HR-HPV), and MCPyV LT expression in 89.0%, 4.9%, and 0.0%, respectively. EBNA1 was expressed in all grades of preinvasive [cornea–conjunctiva intraepithelial neoplasia (CIN)1, 100%; CIN2, 85.7%; CIN3, 95.8%; and carcinoma in situ (CIS), 83.8%] and in invasive (89.2%) OSSN. PCR on 178 samples detected EBV, HR-HPV, and MCPyV in 80.3%, 9.0%, and 13.5% of tumors, respectively. EBV was detected in all grades of preinvasive and invasive OSSN. EBV detection was associated with high HIV viral loads (p = 0.022). HR-HPV was detected in 0.0% CIN1, 0.0% CIN2, 5.6% CIN3, 13.0% CIS, and 7.0% invasive OSSN.ConclusionsOur findings of EBV DNA and EBNA1 protein in all the grades of preinvasive and especially invasive OSSN are consistent with a potential causal role for EBV in OSSN. A role of HPV in OSSN was not clearly established in this study.

The Biological and Clinical Aspects of a Latent Tuberculosis Infection

Tuberculosis (TB), caused by bacilli from the Mycobacterium tuberculosis complex, remains a serious global public health problem, representing one of the main causes of death from infectious diseases. About one quarter of the world’s population is infected with Mtb and has a latent TB infection (LTBI). According to the World Health Organization (WHO), an LTBI is characterized by a lasting immune response to Mtb antigens without any TB symptoms. Current LTBI diagnoses and treatments are based on this simplified definition, although an LTBI involves a broad range of conditions, including when Mtb remains in the body in a persistent form and the immune response cannot be detected. The study of LTBIs has progressed in recent years; however, many biological and medical aspects of an LTBI are still under discussion. This review focuses on an LTBI as a broad spectrum of states, both of the human body, and of Mtb cells. The problems of phenotypic insusceptibility, diagnoses, chemoprophylaxis, and the necessity of treatment are discussed. We emphasize the complexity of an LTBI diagnosis and its treatment due to its ambiguous nature. We consider alternative ways of differentiating an LTBI from active TB, as well as predicting TB reactivation based on using mycobacterial “latency antigens” for interferon gamma release assay (IGRA) tests and the transcriptomic analysis of human blood cells.

Enhanced immunogenicity of the tuberculosis subunit Rv0572c vaccine delivered in DMT liposome adjuvant as a BCG-booster

COVID-19 has affected the progress made in the prevention and treatment of tuberculosis (TB); hence, the mortality of tuberculosis has risen. Different strategies-based novel TB vaccine candidates have been developed. This study identifies strategies to overcome the limitations of Bacille Calmette-Guérin (BCG) in preventing latent infection and reactivation of TB. The latency antigen Rv0572c was selected based on the mechanism of interaction between Mycobacterium tuberculosis and its host. The rRv0572c protein was used to stimulate whole blood samples derived from patients with clinically diagnosed active TB (ATBs) or latent TB infections (LTBIs) and healthy control (HCs) donors, confirming that this protein can be recognized by T cells in patients with TB, especially LTBIs. C57BL/6 mice were used to investigate the immunogenicity of the rRv0572c protein emulsified in the liposome adjuvant dimethyldioctadecylammonium [DDA], monophosphoryl lipid A [MPLA], trehalose-6, 6′-dibehenate [TDB] (DMT). The results demonstrated that rRv0572c/DMT could boost BCG-primed mice to induce antigen-specific CD4+ T cell production and generate functional T cells dominated by antigen-specific CD8+ T cells. The rRv0572c/DMT vaccine could also trigger limited Th2 humoral immune responses. These findings suggest that rRv0572c/DMT is a potential subunit vaccine candidate that can be used as a booster vaccine for BCG.

The Role of Epstein-Barr Virus in Modulating Key Tumor Suppressor Genes in Associated Malignancies: Epigenetics, Transcriptional, and Post-Translational Modifications.

Epstein-Barr virus (EBV) is ubiquitous and carried by approximately 90% of the world’s adult population. Several mechanisms and pathways have been proposed as to how EBV facilitates the pathogenesis and progression of malignancies, such as Hodgkin’s lymphoma, Burkitt’s lymphoma, nasopharyngeal carcinoma, and gastric cancers, the majority of which have been linked to viral proteins that are expressed upon infection including latent membrane proteins (LMPs) and Epstein-Barr virus nuclear antigens (EBNAs). EBV expresses microRNAs that facilitate the progression of some cancers. Mostly, EBV induces epigenetic silencing of tumor suppressor genes, degradation of tumor suppressor mRNA transcripts, post-translational modification, and inactivation of tumor suppressor proteins. This review summarizes the mechanisms by which EBV modulates different tumor suppressors at the molecular and cellular levels in associated cancers. Briefly, EBV gene products upregulate DNA methylases to induce epigenetic silencing of tumor suppressor genes via hypermethylation. MicroRNAs expressed by EBV are also involved in the direct targeting of tumor suppressor genes for degradation, and other EBV gene products directly bind to tumor suppressor proteins to inactivate them. All these processes result in downregulation and impaired function of tumor suppressors, ultimately promoting malignances.

The Influence of Underlying Disease on Rituximab Pharmacokinetics May be Explained by Target-Mediated Drug Disposition.

Rituximab is an anti-CD20 monoclonal antibody approved in several diseases, including chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), rheumatoid arthritis (RA), and anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV). The influence of underlying disease on rituximab pharmacokinetics has never been investigated for several cancer and non-cancer diseases simultaneously. This study aimed at assessing this influence using an integrated semi-mechanistic model accounting for target-mediated elimination of rituximab. Rituximab concentration-time data from five studies previously published in patients with CLL, DLBCL, FL, RA, and AAV were described using a two-compartment model with irreversible binding of rituximab to its target antigen. Both underlying disease and target antigen measurements were assessed as covariates. Central volume of distribution was [95% confidence interval] 1.7-fold [1.6-1.9] higher in DLBCL than in RA, FL, and CLL, and it was 1.8-fold [1.6-2.1] higher in RA, FL, and CLL than in AAV. First-order elimination rate constants were 1.8-fold [1.7-2.0] and 1.3-fold [1.2-1.5] higher in RA, DLBCL, and FL than in CLL and AAV, respectively. Baseline latent antigen level (L0) was 54-fold [30-94], 20-fold [11-36], and 29-fold [14-64] higher in CLL, DLBCL, and FL, respectively, than in RA and AAV. In lymphoma, L0 increased with baseline total metabolic tumor volume (p = 6.10-7). In CLL, the second-order target-mediated elimination rate constant (kdeg) increased with baseline CD20 count on circulating B cells (CD20cir, p = 0.0081). Our results show for the first time that rituximab pharmacokinetics is strongly influenced by underlying disease and disease activity. Notably, neoplasms are associated with higher antigen amounts that result in decreased exposure to rituximab compared to inflammatory diseases. Our model might be used to estimate unbound target amounts in upcoming studies.

CAR-T Cells Targeting TSHR Demonstrate Safety and Potent Preclinical Activity Against Differentiated Thyroid Cancer.

Chimeric antigen receptor T cells (CAR-Ts) have demonstrated remarkable efficacy in hematological cancers but have not yet translated in treating solid tumors. The significant hurdles limiting CAR-T therapy were from a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present TSH receptor (TSHR) as a putative target for CAR-T therapy of differentiated thyroid cancer (DTC). We undertook a large-scale screen on thyroid cancer tissues and multiple internal organs through bioinformatical analysis and immunohistochemistry to date TSHR expression. Using 3 previously described monoclonal antibodies, we generated 3 third-generation CAR-Ts. We tested anti-TSHR CAR-T in vitro activity by T-cell function and killing assay. Then we tested preclinical therapeutical efficacy in a xenograft mouse model of DTC and analyzed mice's physical conditions and histological abnormalities to evaluate anti-TSHR CAR-T's safety. TSHR is highly and homogeneously expressed on 90.8% (138/152) of papillary thyroid cancer, 89.2% (33/37) of follicular thyroid cancer, 78.2% (18/23) of cervical lymph node metastases, and 86.7% of radioactive iodine resistance diseases. We developed 3 novel anti-TSHR CAR-Ts from monoclonal antibodies M22, K1-18, and K1-70; all 3 CAR-Ts mediate significant antitumor activity in vitro. Among these, we demonstrate that K1-70 CAR-T can have therapeutical efficacy in vivo, and no apparent toxicity has been observed. TSHR is a latent target antigen of CAR-T therapy for DTC. Anti-TSHR CAR-T could represent a therapeutic option for patients with locoregional relapsed or distant metastases of thyroid cancer and should be tested in carefully designed clinical trials.

Establishment of Tree Shrew Animal Model for Kaposi's Sarcoma-Associated Herpesvirus (HHV-8) Infection.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the most common cause of Kaposi’s sarcoma (KS) and other malignant growths in humans. However, the lack of a KSHV-infected small animal model has hampered understanding of the mechanisms of KSHV infection, virus replication, pathogenesis, and persistence. This study was designed to explore the susceptibility of tree shrews as a possible KSHV-infected small animal model. A recombinant GFP (latent)/RFP (lytic)-positive rKSHV.219 strain was used to infect primary cells cultured from different tissues of tree shrews as an in vitro model and adult tree shrews as an in vivo model. KSHV latent nuclear antigen (LANA) and DNA were successfully detected in primary cells of tree shrews. Among them, tree shrew kidney epithelial cells (TSKEC) were the most susceptible cells to KSHV infection compared to other cells. KSHV genomic DNA, mRNA, and KSHV-specific proteins were readily detected in the TSKEC cultured up to 32 dpi. Moreover, KSHV DNA and mRNA transcription were also readily detected in the peripheral blood mononuclear cells (PBMCs) and various tissues of tree shrews infected with KSHV. Haematoxylin and eosin (HE) staining showed lymphocyte infiltration, lymphoid tissue focal aggregation, alveolar wall thickening, hepatocyte edema, hepatic necrosis in the spleen, lung, and liver of KSHV-infected animals. Additionally, immune-histochemical (IHC) staining showed that LANA or ORF62-positive cells were present in the spleen, lung, liver, and kidney of KSHV-infected tree shrews. Here, we have successfully established in vitro and in vivo KSHV latent infection in tree shrews. This small animal model is not only useful for studying the pathogenesis of KSHV in vivo but can also be a useful model to study transmission routes of viral infection and a useful platform to characterize the novel therapeutics against KSHV.

NDRG1 facilitates lytic replication of Kaposi's sarcoma-associated herpesvirus by maintaining the stability of the KSHV helicase.

The presumed DNA helicase encoded by ORF44 of Kaposi’s sarcoma-associated herpesvirus (KSHV) plays a crucial role in unwinding viral double-stranded DNA and initiating DNA replication during lytic reactivation. However, the regulatory mechanism of KSHV ORF44 has not been fully elucidated. In a previous study, we identified that N-Myc downstream regulated gene 1 (NDRG1), a host scaffold protein, facilitates viral genome replication by interacting with proliferating cell nuclear antigen (PCNA) and the latent viral protein latency-associated nuclear antigen (LANA) during viral latency. In the present study, we further demonstrated that NDRG1 can interact with KSHV ORF44 during viral lytic replication. We also found that the mRNA and protein levels of NDRG1 were significantly increased by KSHV ORF50-encoded replication and transcription activator (RTA). Remarkably, knockdown of NDRG1 greatly decreased the protein level of ORF44 and impaired viral lytic replication. Interestingly, NDRG1 enhanced the stability of ORF44 and inhibited its ubiquitin-proteasome-mediated degradation by reducing the polyubiquitination of the lysine residues at positions 79 and 368 in ORF44. In summary, NDRG1 is a novel binding partner of ORF44 and facilitates viral lytic replication by maintaining the stability of ORF44. This study provides new insight into the mechanisms underlying KSHV lytic replication.

Immunological characterization of chimeras of high specificity antigens from Mycobacterium tuberculosis H37Rv

Tuberculosis remains a serious global health problem. BCG is the only prophylactic TB vaccine and it shows variable protective efficacy. Chimeric protein subunit vaccines hold great potential as stand-alone vaccines or heterologous BCG prime boosters. We have designed a protein chimera, PP31, by combining Mtb ESAT-6 family antigen Rv1198 and MoCo biosynthesis family antigen Rv3111. Further, PP31 was extended by addition of latency antigen Rv1813c to yield PP43. Immunization of BALB/c mice with PP31 or PP43 with FIA adjuvant elicited strong humoral immune response. Restimulation of splenocytes of the immunized mice lead to significant proliferation of lymphocytes, secretion of cytokines IFN-γ, TNF, IL-2 of the Th1 class, IL-17A of the Th17 class, and IL-6. PP31 and PP43 also induced intracellular cytokine expression (IFN-γ, TNF, and IL-2) from both CD4+-CD44high and CD8+-CD44high T-cells. Antigen-specific IFN-γ+/IL-2+ double positive CD4+ T-cells were significantly higher in case of PP43 than PP31-immunized mice and control group. PP43 showed protection equivalent to heat-inactivated BCG in response to challenge of the immunized mice with Mtb H37Ra. Based on its immunogenicity and protective efficacy, PP43 appears to be a potential candidate for further development as a subunit vaccine against TB.

Risk of Kaposi's Sarcoma-Associated Herpes Virus Transmission From Donor Allografts Among Italian Posttransplant Kaposi's Sarcoma Patients

AbstractKaposi's sarcoma–associated herpesvirus (KSHV) is a newly discovered herpes virus found in all forms of Kaposi's sarcoma (KS) including KS among immunosuppressed transplant patients. It is unknown whether this virus is transmitted by organ transplantation or is reactivated during immunosuppression among those patients infected before transplantation. To investigate the risk of KSHV transmission during organ transplantation, we conducted a case-control study of transplant recipients with and without KS matched to their respective donors. Sera were collected at time of transplantation and tested in a randomized and blinded fashion using four KSHV serologic assays testing for antibodies to both latent and lytic phase antigens. Ten (91%) of 11 organ recipients who developed KS were seropositive prior to transplantation by one or more of the assays compared with two (12%) of 17 control organ recipients (OR = 75, 95% CI = 4.7, 3500). KS cases were more likely to have been born in southern Italy where KS is endemic than the recipient controls or either donor group. Only four (36%) of 11 donors to case patients and three (18%) of 17 donors to control patients were seropositive (P = .38, two-tailed Fisher's exact test). KSHV transmission could not be ruled out for the single KS patient seronegative at transplantation and clear evidence for organ-related transmission was found for another KS patient outside of the case-control study. Antibodies to KSHV are detectable in the sera from most transplant recipients before initiation of immunosuppressive treatment suggesting that KS among immunosuppressed transplant patients is primarily due to virus reactivation. KSHV transmission, however, from an infected allograft can occur, and our study reports the first documented case of person-to-person transmission of KSHV.

Lack of serologic association of human herpesvirus-8 (KSHV) in patients with monoclonal gammopathy of undetermined significance with and without progression to multiple myeloma

AbstractBecause human herpesvirus-8 (HHV-8) DNA has been found in multiple myeloma (MM) patients by polymerase chain reaction, it was suggested that HHV-8 may play a role in the transformation of monoclonal gammopathy of undetermined significance (MGUS) to MM. Therefore, 362 MGUS sera with and without progression to MM were tested for IgG antibody to HHV-8. Only 7.8% of the MGUS sera contained HHV-8 antibody to lytic proteins, and IgG antibody to HHV-8 latent antigen was even lower than lytic antibody (2.9%). No differences were observed in the distribution of antibody to HHV-8 in sera from MGUS patients who progressed to MM. The seroprevalences of HHV-8 in MGUS (7.8%), MM (5.4%), and healthy donors (5.9%) were similar, thus arguing for the lack of epidemiologic evidence of HHV-8 participation in the pathogenesis of MM. MGUS patients were immune competent in response to Epstein-Barr virus (EBV) infection because 97% contained antibody to EBV virus capsid antigen.

Identification and in silico functional prediction of lineage-specific SNPs distributed in DosR-related proteins and resuscitation-promoting factor proteins of Mycobacterium tuberculosis

One-third of the world population is infected by Mycobacterium tuberculosis, which may persist in the latent or dormant state. Bacteria can shift to dormancy when encountering harsh conditions such as low oxygen, nutrient starvation, high acidity and host immune defenses. Genes related to the dormancy survival regulator (DosR) regulon are responsible for the inhibition of aerobic respiration and replication, which is required to enter dormancy. Conversely, resuscitation-promoting factor (rpf) proteins participate in reactivation from dormancy and the development of active tuberculosis (TB). Many DosR regulon and rpf proteins are immunodominant T cell antigens that are highly expressed in latent TB infection. They could serve as TB vaccine candidates and be used for diagnostic development. We explored the genetic polymorphisms of 50 DosR-related genes and 5 rpf genes among 1,170 previously sequenced clinical M. tuberculosis genomes. Forty-three lineage- or sublineage-specific nonsynonymous single nucleotide polymorphisms (nsSNPs) were identified. Ten nsSNPs were specific to all Mtb isolates belonging to lineage 1 (L1). Two common sublineages, the Beijing family (L2.2) and EAI2 (L1.2.1), differed at as many as 26 lineage- or sublineage-specific SNPs. DosR regulon genes related to membrane proteins and the rpf family possessed mean dN/dS ratios greater than one, suggesting that they are under positive selection. Although the T cell epitope regions of DosR-related and rpf antigens were quite conserved, we found that the epitopes in L1 had higher rates of genetic polymorphisms than the other lineages. Some mutations in immunogenic epitopes of the antigens were specific to particular M. tuberculosis lineages. Therefore, the genetic diversity of the DosR regulon and rpf proteins might impact the adaptation of M. tuberculosis to the dormant state and the immunogenicity of latency antigens, which warrants further investigation.

A Bank of CD30.CAR-Modified, Epstein-Barr Virus-Specific T Cells That Lacks Host Reactivity and Resists Graft Rejection for Patients with CD30-Positive Lymphoma

While autologous T cell therapies can effectively treat B-cell leukemia and lymphoma, the personalized manufacturing process is difficult to scale, expensive and may fail. Even when autologous products are successfully manufactured, they are not immediately available to acutely ill patients. "Off-the-shelf" T cell products derived from healthy donors that can rapidly be administered, would improve accessibility and reduce the cost of T cell therapy. However, major obstacles to successful allogeneic T cell products include their potential for graft-versus-host disease (GVHD) and graft rejection, mediated by host and recipient alloreactive T cells respectively. To address GVHD, we are using Epstein-Barr Virus-specific T cells (EBVSTs) as our platform since they are virus specific rather than allospecific and have not produced GVHD in more than 300 allogeneic recipients. To prevent graft rejection we have introduced into these EBVSTs, a chimeric antigen receptor for CD30 (CD30.CAR). CD30 is upregulated during the activation of alloreactive T cells, which leads to them becoming targets. The CD30.CAR provides the additional advantage of targeting CD30-positive lymphoma and has proved safe and effective in prior clinical trials (NCT02917083) using autologous CAR-T cells. Hence, we expect off-the-shelf CD30.CAR EBVSTs to eliminate the alloreactive T cells they elicit in allogeneic hosts, and therefore persist for sufficient time to eliminate CD30-positive lymphoma, without causing GVHD. Here we show that CD30.CAR-EBVSTs resist fratricide by masking their own CD30 molecules expressed in cis, but are nonetheless protected from rejection when co-cultured with alloreactive T cells expressing CD30 in trans. Notably, CD30.CAR EBVSTs preserve the function of both their TCR and the CD30.CAR, with retention of EBV specificity and the ability to eliminate CD30-positive tumor cells. We have manufactured a bank of clinical grade CD30.CAR EBVSTs from donors with HLA types designed to provide a partial HLA match for our diverse recipients. Clinical grade CD30.CAR EBVST cultures readily expanded to sufficient numbers for a planned clinical trial and expressed the CD30.CAR on 77% to 99% of cells. All of the lines passed functional release criteria of having greater than 100 IFNɣ spot-forming units (SFU) per 105 cells in response to both latent and lytic EBV antigens, and greater than 20% specific cytolysis against a CD30-positive Hodgkin lymphoma cell line, HDLM2, at an effector to target ratio of 20:1. Although CD30.CAR killing is not HLA restricted, we will select the CD30.CAR EBVST product for each recipient, based on the best HLA class I and class II match. This will allow endogenous EBV to boost the in vivo activity of CD30.CAR EBVSTs, and will provide additional reactivity for patients with CD30-positive and EBV-positive tumors. The IND for the clinical trial (NCT04288726) has been approved and we will recruit patients with CD30-positive lymphomas including Hodgkin lymphoma, diffuse large B cell lymphoma and NK/T cell lymphoma. In summary, we present an approach to making an off-the-shelf T cell therapy that can rapidly translate to the clinic, requires no gene editing, and can serve as a platform for other CAR/TCRs to target a multiplicity of malignancies. Disclosures Quach: Tessa Therapeutics: Research Funding. Brenner:Memmgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Maker Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Tumstone: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees. Heslop:Tessa Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; Gilead Biosciences: Consultancy; PACT Pharma: Consultancy; Kiadis: Consultancy; AlloVir: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Ramos:Novartis: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding; Kuur Therapeutics: Research Funding. Rouce:Tessa Therapeutics: Other, Research Funding; Novartis: Honoraria. Rooney:Marker Therapeutics: Current equity holder in publicly-traded company, Other: co-founder; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Allovir: Current equity holder in publicly-traded company, Other: co-founder.

Epstein-Barr Virus Facilitates Expression of KLF14 by Regulating the Cooperative Binding of the E2F-Rb-HDAC Complex in Latent Infection.

Epstein-Barr virus (EBV) was discovered as the first human tumor virus more than 50 years ago. EBV infects more than 90% of the human population worldwide and is associated with numerous hematologic malignancies and epithelial malignancies. EBV establishes latent infection in B cells, which is the typical program seen in lymphomagenesis. Understanding EBV-mediated transcription regulatory networks is one of the current challenges that will uncover new insights into the mechanism of viral-mediated lymphomagenesis. Here, we describe the regulatory profiles of several cellular factors (E2F6, E2F1, Rb, HDAC1, and HDAC2) together with EBV latent nuclear antigens using next-generation sequencing (NGS) analysis. Our results show that the E2F-Rb-HDAC complex exhibits similar distributions in genomic regions of EBV-positive cells and is associated with oncogenic super-enhancers involving long-range regulatory regions. Furthermore, EBV latent antigens cooperatively hijack this complex to bind at KLFs gene loci and facilitate KLF14 gene expression in lymphoblastoid cell lines (LCLs). These results demonstrate that EBV latent antigens can function as master regulators of this multisubunit repressor complex (E2F-Rb-HDAC) to reverse its suppressive activities and facilitate downstream gene expression that can contribute to viral-induced lymphomagenesis. These results provide novel insights into targets for the development of new therapeutic interventions for treating EBV-associated lymphomas.IMPORTANCE Epstein-Barr virus (EBV), as the first human tumor virus, infects more than 90% of the human population worldwide and is associated with numerous human cancers. Exploring EBV-mediated transcription regulatory networks is critical to understand viral-associated lymphomagenesis. However, the detailed mechanism is not fully explored. Now we describe the regulatory profiles of the E2F-Rb-HDAC complex together with EBV latent antigens, and we found that EBV latent antigens cooperatively facilitate KLF14 expression by antagonizing this multisubunit repressor complex in EBV-positive cells. This provides potential therapeutic targets for the treatment of EBV-associated cancers.

Functional interplay of Epstein-Barr virus oncoproteins in a mouse model of B cell lymphomagenesis

Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation. We find aberrant activation of early B cell factor 1 (EBF1) to promote transformation of LMP1-expressing B cells by inhibiting their differentiation to plasma cells. EBV EBNA3A phenocopies EBF1 activities in LMP1-expressing B cells, promoting transformation while inhibiting differentiation. In cells expressing LMP1 together with LMP2A, EBNA3A only promotes lymphomagenesis when the EBNA2 target Myc is also overexpressed. Collectively, our data support a model where proproliferative activities of LMP1, LMP2A, and EBNA2 in combination with EBNA3A-mediated inhibition of terminal plasma cell differentiation critically control EBV-mediated B cell lymphomagenesis.

Redirecting T Cells against Epstein-Barr Virus Infection and Associated Oncogenesis.

The Epstein–Barr virus (EBV) is associated with lymphomas and carcinomas. For some of these, the adoptive transfer of EBV specific T cells has been therapeutically explored, with clinical success. In order to avoid naturally occurring EBV specific autologous T cell selection from every patient, the transgenic expression of latent and early lytic viral antigen specific T cell receptors (TCRs) to redirect T cells, to target the respective tumors, is being developed. Recent evidence suggests that not only TCRs against transforming latent EBV antigens, but also against early lytic viral gene products, might be protective for the control of EBV infection and associated oncogenesis. At the same time, these approaches might be more selective and cause less collateral damage than targeting general B cell markers with chimeric antigen receptors (CARs). Thus, EBV specific TCR transgenic T cells constitute a promising therapeutic strategy against EBV associated malignancies.