Nalini Srinivasan

Polysaccharide-protein complex from Lycium barbarum L. is a novel stimulus of dendritic cell immunogenicity.

Dendritic cell (DC) immunogenicity correlates with its maturation, which can be induced by toxic microbial products such as LPS. In this study, we report that a nontoxic polysaccharide-protein complex isolated from a Chinese medicinal herb, Lycium barbarum (LBP), induces phenotypic and functional maturation of DCs with strong immunogenicity. LBP up-regulated DC expression of CD40, CD80, CD86, and MHC class II molecules; down-regulated DC uptake of Ag; enhanced DC allostimulatory activity; and induced IL-12p40 and p70 production. All of its five fractions were active. LBP developed enhanced Th1 response, and LBP-treated DCs enhanced Th1 and Th2 responses in vitro and in vivo. Our study provides evidence and rationale on using LBP in various clinical conditions to enhance host immunity and suggests LBP as a potent adjuvant for the design of DC-based vaccines.

Activation of macrophages by polysaccharide–protein complex from Lycium barbarum L.

Macrophages play crucial roles in innate immunity. This paper reports that a polysaccharide–protein complex isolated from Lycium barbarum (LBP) is able to activate macrophages. LBP was isolated from Lycium barbarum fruit and separated to five homogenous fractions, designated LBPF1, LBPF2, LBPF3, LBPF4 and LBPF5. It was found that LBP (50 mg/kg, i.p.) markedly upregulated the expressions of CD40, CD80, CD86 and MHC class II molecules on peritoneal macrophages. In vitro studies showed that LBP and LBPF1‐5 activated transcription factors NF‐κB and AP‐1 by RAW264.7 macrophage cells, induced TNF‐α, IL‐1β, IL‐12p40 mRNA expression, and enhanced TNF‐α production in a dose‐dependent manner. Furthermore, LBP (50 mg/kg, i.p.) significantly enhanced macrophage endocytic and phagocytic capacities in vivo. These results indicate that LBP enhances innate immunity by activating macrophages. The mechanism may be through activation of transcription factors NF‐κB and AP‐1 to induce TNF‐α production and upregulation of MHC class II costimulatory molecules. Copyright

Extended Loop Region of Hcp1 is Critical for the Assembly and Function of Type VI Secretion System in Burkholderia pseudomallei

The Type VI Secretion System cluster 1 (T6SS1) is essential for the pathogenesis of Burkholderia pseudomallei, the causative agent of melioidosis, a disease endemic in the tropics. Inside host cells, B. pseudomallei escapes into the cytosol and through T6SS1, induces multinucleated giant cell (MNGC) formation that is thought to be important for bacterial cell to cell spread. The hemolysin-coregulated protein (Hcp) is both a T6SS substrate, as well as postulated to form part of the T6SS secretion tube. Our structural study reveals that Hcp1 forms hexameric rings similar to the other Hcp homologs but has an extended loop (Asp40-Arg56) that deviates significantly in position compared to other Hcp structures and may act as a key contact point between adjacent hexameric rings. When two residues within the loop were mutated, the mutant proteins were unable to stack as dodecamers, suggesting defective tube assembly. Moreover, infection with a bacterial mutant containing in situ substitution of these hcp1 residues abolishes Hcp1 secretion inside infected cells and MNGC formation. We further show that Hcp has the ability to preferentially bind to the surface of antigen-presenting cells, which may contribute to its immunogenicity in inducing high titers of antibodies seen in melioidosis patients.

SYNTHESIS AND ANTI-CANCER ACTIVITIES OF A PAIR OF ENANTIOMERIC GOLD(I) COMPLEXES CONTAINING SULFANYL-SUBSTITUTED P-STEREOGENIC PHOSPHINES

Abstract A pair of enantiomeric gold(I) complexes containing enantiomerically pure 2,3-dimethyl-7-phenyl-5-(phenylsulfanyl)-7-( R/S )-phosphabicyclo[2.2.1]hept-2-ene has been prepared and in vitro cytotoxicity tests showed that both the enantiomers are relatively nontoxic against healthy lymphocytes, but are highly potent against suspension and monolayer human tumor cell lines.

Defining the expression hierarchy of latent T-cell epitopes in Epstein-Barr virus infection with TCR-like antibodies

Epstein-Barr virus (EBV) is a gamma herpesvirus that causes a life-long latent infection in human hosts. The latent gene products LMP1, LMP2A and EBNA1 are expressed by EBV-associated tumors and peptide epitopes derived from these can be targeted by CD8 Cytotoxic T-Lymphocyte (CTL) lines. Whilst CTL-based methodologies can be utilized to infer the presence of specific latent epitopes, they do not allow a direct visualization or quantitation of these epitopes. Here, we describe the characterization of three TCR-like monoclonal antibodies (mAbs) targeting the latent epitopes LMP1125–133, LMP2A426–434 or EBNA1562–570 in association with HLA-A0201. These are employed to map the expression hierarchy of endogenously generated EBV epitopes. The dominance of EBNA1562–570 in association with HLA-A0201 was consistently observed in cell lines and EBV-associated tumor biopsies. These data highlight the discordance between MHC-epitope density and frequencies of associated CTL with implications for cell-based immunotherapies and/or vaccines for EBV-associated disease.

Targeting Epstein-Barr virus transformed B lymphoblastoid cells using antibodies with T cell receptor-like specificities

Epstein-Barr virus (EBV) is an oncovirus associated with several human malignancies including posttransplant lymphoproliferative disease in immunosuppressed patients. We show here that anti-EBV T-cell receptor-like monoclonal antibodies (TCR-like mAbs) E1, L1, and L2 bound to their respective HLA-A*0201-restricted EBV peptides EBNA1562-570, LMP1125-133, and LMP2A426-434 with high affinities and specificities. These mAbs recognized endogenously presented targets on EBV B lymphoblastoid cell lines (BLCLs), but not peripheral blood mononuclear cells, from which they were derived. Furthermore, these mAbs displayed similar binding activities on several BLCLs, despite inherent heterogeneity between different donor samples. A single weekly administration of the naked mAbs reduced splenomegaly, liver tumor spots, and tumor burden in BLCL-engrafted immunodeficient NOD-SCID/Il2rg(-/-) mice. In particular, mice that were treated with the E1 mAb displayed a delayed weight loss and significantly prolonged survival. In vitro, these TCR-like mAbs induced early apoptosis of BLCLs, thereby enhancing their Fc-dependent phagocytic uptake by macrophages. These data provide evidence for TCR-like mAbs as potential therapeutic modalities to target EBV-associated diseases.

TCR–like antibodies mediate complement and antibody-dependent cellular cytotoxicity against Epstein-Barr virus–transformed B lymphoblastoid cells expressing different HLA-A*02 microvariants

Epstein-Barr virus (EBV) is a common gammaherpesvirus associated with various human malignancies. Antibodies with T cell receptor-like specificities (TCR-like mAbs) provide a means to target intracellular tumor- or virus-associated antigens by recognising their processed peptides presented on major histocompatibility complex (MHC) class I (pMHC) complexes. These antibodies are however thought to be relevant only for a single HLA allele. Here, we show that HLA-A*02:01-restricted EBV antigenic peptides EBNA1562-570, LMP1125-133 and LMP2A426-434 display binding degeneracy towards HLA-A*02 allelic microvariants, and that these pMHC complexes are recognised by anti-EBV TCR-like mAbs E1, L1 and L2 raised in the context of HLA-A*02:01. These antibodies bound endogenously derived pMHC targets on EBV–transformed human B lymphoblastoid cell lines expressing A*02:01, A*02:03, A*02:06 and A*02:07 alleles. More importantly, these TCR-like mAbs mediated both complement-dependent and antibody-dependent cellular cytotoxicity of these cell lines in vitro. This finding suggests the utility of TCR-like mAbs against target cells of closely related HLA subtypes, and the potential applicability of similar reagents within populations of diverse HLA-A*02 alleles.

Abstract B095: Targeting Epstein-Barr virus transformed B lymphoblastoid cells using antibodies with T cell receptor-like specificities

Introduction: Epstein-Barr virus (EBV) is a gamma herpesvirus that is found in the majority of the human population. Though commonly established as a life-long, asymptomatic infection, EBV has also been implicated in a number of human malignancies including Hodgkin9s and Burkitt9s lymphomas, nasopharyngeal carcinoma as well as post-transplant lymphoproliferative disease in immunosuppressed patients. While EBV displays restricted gene expression during different latency programs, the viral gene products that are frequently detected in EBV-associated tumors include Epstein-Barr virus Nuclear Antigen 1 (EBNA1), Latent Membrane Protein 1 (LMP1) and Latent Membrane Protein 2A (LMP2A). Our lab has previously described the generation and characterization of monoclonal antibodies with T cell receptor-like specificities (TCR-like mAbs) targeting EBV latent epitopes in association with HLA-A0201. These antibodies have been shown to bind to their targets with high specificities and affinities, and were capable of recognizing antigens displayed on human nasopharyngeal carcinoma biopsies, highlighting their immunotherapeutic potential for targeting EBV-associated tumors. Methods: EBV is particularly capable of transforming resting B cells into latently infected, actively proliferating B lymphoblastoid cell lines (BLCL) in vitro. As a proof-of-concept, we first generated EBV BLCLs from HLA-A0201+ human peripheral blood mononuclear cells (PBMCs) and compared the ability of our TCR-like mAbs to detect their endogenous targets before and after establishment of the cell lines. The xenograft model involving the inoculation of EBV BLCL into immunodeficient mice resembles B cell lymphoma that develops in post-transplant, immunosuppressed patients. Hence to test the ability of our TCR-like mAbs to target the BLCLs in vivo, we next inoculated PBMC-derived BLCLs into immunodeficient NSG mice and administered weekly dosage of TCR-like mAbs. Mice were assessed for changes in weight, survival as well as end-point organs analysis. Results: Here, we showed that endogenous EBV latent epitopes could be detected by our TCR-like mAbs on PBMC-derived BLCLs after EBV establishment of the cell line. Despite the inherent heterogeneities between different donors PBMC-derived BLCLs, several of the HLA-A0201+ BLCLs tested displayed similar binding activities with these TCR-like mAbs. In addition, BLCL-injected NSG mice that received weekly treatment of TCR-like mAbs displayed reduced tumor burden, splenomegaly and liver tumor spots in comparison to mice that received isotype antibody or PBS control. Importantly, BLCL-injected NSG mice that received the TCR-like mAb targeting the EBNA1 latent epitope exhibited delayed weight loss and survival advantage. Conclusion: We have utilized TCR-like mAbs with specificities against EBV latent epitopes expressed on HLA-A0201 and showed that these antibodies could recognize endogenous targets on EBV transformed, PBMC-derived BLCLs. Furthermore, weekly administration of TCR-like mAbs into BLCL xenograft NSG mice resulted in an overall reduction of tumor load and improved survival in one of the three described antibodies. Taken together, our data provide preliminary evidence for the therapeutic usage of antibodies with TCR-like specificities for the targeting EBV transformed BLCL in vivo. Citation Format: Junyun Lai, Wei Jian Tan, Chien Tei Too, Nalini Srinivasan, Lan Hiong Wong, Fatimah Mustafa, Soh Ha Chan, Jianzhu Chen, Paul Anthony MacAry. Targeting Epstein-Barr virus transformed B lymphoblastoid cells using antibodies with T cell receptor-like specificities. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B095.