Cross-Neutralisation of Novel Bombali Virus by Ebola Virus Antibodies and Convalescent Plasma Using an Optimised Pseudotype-Based Neutralisation Assay

Ebolaviruses continue to pose a significant outbreak threat, and while Ebola virus (EBOV)-specific vaccines and antivirals have been licensed, efforts to develop candidates offering broad species cross-protection are continuing. The use of pseudotyped virus in place of live virus is recognised as an alternative, safer, high-throughput platform to evaluate anti-ebolavirus antibodies towards their development, yet it requires optimisation. Here, we have shown that the target cell line impacts neutralisation assay results and cannot be selected purely based on permissiveness. In expanding the platform to incorporate each of the ebolavirus species envelope glycoprotein, allowing a comprehensive assessment of cross-neutralisation, we found that the recently discovered Bombali virus has a point mutation in the receptor-binding domain which prevents entry into a hamster cell line and, importantly, shows that this virus can be cross-neutralised by EBOV antibodies and convalescent plasma.

A functional mammalian display screen identifies rare antibodies that stimulate NK cell–mediated cytotoxicity

Therapies that boost the antitumor immune response have shown a great deal of success. Although most of these therapies have focused on enhancing T cell functions, there is a growing interest in developing therapies that can target other immune cell subsets. Like T cells, natural killer (NK) cells are cytotoxic effector cells that play a key role in the antitumor response. To advance the development of NK-based therapies, we developed a functional screen to rapidly identify antibodies that can activate NK cells. We displayed antibodies on a mammalian target cell line and probed their ability to stimulate NK cell–mediated cytotoxicity. From this screen, we identified five antibodies that bound with high affinity to NK cells and stimulated NK cell–mediated cytotoxicity and interferon-γ (IFN-γ) secretion. We demonstrate that these antibodies can be further developed into bispecific antibodies to redirect NK cell–mediated cytotoxicity toward CD20+ B cell lymphoma cells and HER2+ breast cancer cells. While antibodies to two of the receptors, CD16 and NCR1, have previously been targeted as bispecific antibodies to redirect NK cell–mediated cytotoxicity, we demonstrate that bispecific antibodies targeting NCR3 can also potently activate NK cells. These results show that this screen can be used to directly identify antibodies that can enhance antitumor immune responses.

Modulation of Macrophage Polarization by Carbon Nanodots and Elucidation of Carbon Nanodot Uptake Routes in Macrophages

Atherosclerosis represents an ever-present global concern, as it is a leading cause of cardiovascular disease and an immense public welfare issue. Macrophages play a key role in the onset of the disease state and are popular targets in vascular research and therapeutic treatment. Carbon nanodots (CNDs) represent a type of carbon-based nanomaterial and have garnered attention in recent years for potential in biomedical applications. This investigation serves as a foremost attempt at characterizing the interplay between macrophages and CNDs. We have employed THP-1 monocyte-derived macrophages as our target cell line representing primary macrophages in the human body. Our results showcase that CNDs are non-toxic at a variety of doses. THP-1 monocytes were differentiated into macrophages by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) and co-treatment with 0.1 mg/mL CNDs. This co-treatment significantly increased the expression of CD 206 and CD 68 (key receptors involved in phagocytosis) and increased the expression of CCL2 (a monocyte chemoattractant and pro-inflammatory cytokine). The phagocytic activity of THP-1 monocyte-derived macrophages co-treated with 0.1 mg/mL CNDs also showed a significant increase. Furthermore, this study also examined potential entrance routes of CNDs into macrophages. We have demonstrated an inhibition in the uptake of CNDs in macrophages treated with nocodazole (microtubule disruptor), N-phenylanthranilic acid (chloride channel blocker), and mercury chloride (aquaporin channel inhibitor). Collectively, this research provides evidence that CNDs cause functional changes in macrophages and indicates a variety of potential entrance routes.

PD-1+ Tim3+ tumor-infiltrating CD8 T cells sustain the potential for IFN-γ production, but lose cytotoxic activity in ovarian cancer

Persistent exposure to tumor antigens results in exhausted tumor-infiltrating T cells (TILs) that express the immune checkpoint molecules, PD-1 and Tim3, and lack anti-tumor immunity. To examine the exhausted status of TILs in ovarian cancer, the potential for cytokine production, proliferation and cytotoxicity by purified PD-1+ Tim3+ CD8 TILs was assessed. The production of IFN-γ and TNF-α by PD-1+ Tim3+ CD8 TILs remained the same in an intracellular cytokine staining assay and was higher in a cytokine catch assay than that by PD-1− Tim3− and PD-1+ Tim3− CD8 TILs. %Ki67+ was higher in PD-1+ Tim3+ CD8 TILs than in PD-1− Tim3− CD8 TILs. However, patients with high PD-1+ Tim3+ CD8 TILs had a poor prognosis. The potential for cytotoxicity was then examined. %Perforin+ and %granzyme B+ were lower in PD-1+ Tim3+ CD8 TILs than in PD-1− Tim3− and PD-1+ Tim3− CD8 TILs. To observe the potential for direct cytotoxicity by T cells, a target cell line expressing membrane-bound anti-CD3scFv was newly established and a cytotoxic assay targeting these cells was performed. The cytotoxicity of PD-1+ Tim3+ CD8 TILs was significantly lower than that of PD-1− Tim3− and PD-1+ Tim3− CD8 TILs. Even though PD-1+ Tim3+ CD8 TILs in ovarian cancer showed a sustained potential for cytokine production and proliferation, cytotoxicity was markedly impaired, which may contribute to the poor prognosis of patients with ovarian cancer. Among the impaired functions of exhausted TILs, cytotoxicity may be an essential target for cancer immunotherapy.

Biologic and Immunologic Effects of Knockout of Human Cytomegalovirus pp65 Nuclear Localization Signal

ABSTRACT The human cytomegalovirus (CMV) pp65 protein contains two bipartite nuclear localization signals (NLSs) at amino acids (aa) 415 to 438 and aa 537 to 561 near the carboxy terminus of CMV pp65 and a phosphate binding site related to kinase activity at lysine-436. A mutation of pp65 with K436N (CMV pp65mII) and further deletion of aa 537 to 561 resulted in a novel protein (pp65mIINLSKO, where NLSKO indicate NLS knockout) that is kinaseless and that has markedly reduced nuclear localization. The purpose of this study was to biologically characterize this protein and its immunogenicity compared to that of native pp65. Unlike the native CMV pp65, following either DNA- or recombinant adeno-associated virus-based transduction of CMV pp65mIINLSKO into cells in vitro, the first observation of pp65mIINLSKO expression was in the cytoplasm and pp65mIINLSKO was expressed at higher levels than the native protein. The CMV pp65mIINLSKO mRNA was more abundant earlier than CMV pp65 mRNA (at 4 h and 8 h, respectively), but the half-lives of the proteins were the same. This modification altered the antigenic processing of CMV pp65 in vitro, as measured by the improved efficiency of cytotoxic killing in a pp65mIINLSKO-transduced human HLA A*0201 target cell line. In HHDII mice expressing HLA A*0201, pp65mIINLSKO was as immunogenic as CMV pp65. By RNA microarray analysis, expression of the CMV pp65mIINLSKO had less of an effect on cell cycle pathways than the native CMV pp65 did and a greater effect on cell surface signaling pathways involving immune activity. It is concluded that the removal of the primary NLS motif from pp65 does not impair its immunogenicity and should be considered in the design of a vaccine.

Enhancement of Lymphokine-Activated Killer Cell Cytotoxicity by Mistletoe Plant Extracts.

Anthroposophical cancer therapies focus on the use of mistletoe (MT) preparations and extracts from European mistletoe (Viscum album l.) belong to the complementary therapeutic regimens. Although widely used in Europe as an additional therapy in the treatment of patients suffering from different malignancies, these cancer therapies have remained relatively unknown in North America. To date, mistletoe is the most commonly used complementary cancer drug in Germany, applied as monotherapy or in combination with conventional therapies. ABNOBAviscum fraxini is a standardized extract from all parts of fresh mistle, obtained from the ash tree. This study aims to investigate the effects of MT on the activity of lymphokine-activated killer (LAK) cell cytotoxicity in vivo. Here, we demonstrate that in vivo treatment of mice with a standardized MT plant extract results in activation and function of LAK cells. We show an enhanced cytotoxicity against the NK-sensitive target cell line YAC-1 by flow cytometry. Our data show that in vivo application of MT can double the cytotoxic activity against malignant disease and suggest that one mechasim of action of MT is the induction of LAK-cell mediated immune response.