A Bowman-Birk type trypsin-chymotrypsin inhibitor was isolated from seeds of the legume green lentil (Lens culinaris) by means of affinity chromatography on Affi-gel blue gel, ion exchange chromatography on Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q and Mono S, and gel filtration by FPLC on Superdex 75. The trypsin-chymotrypsin inhibitor was bound on the first three types of chromatographic media. It appeared as a single 16-kDa peak in gel filtration and a single 16-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The trypsin inhibitory activity of the inhibitor was sensitive to the reducing agent dithiothreitol. It was completely abrogated after treatment with 10 mM dithiothreitol for 20 minutes. The protease inhibitor did not exert any inhibitory effect on hepatoma (Hep G2) and breast cancer (MCF 7) cell lines. There was no suppressive action on several fungal species including Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola. It slightly inhibited the activity of HIV-1 reverse transcriptase, with an IC50 of 30 mM.
Three trypsin-chymotrypsin inhibitors were isolated from seeds of the black gram (Vigna mungo) with a procedure that entailed cation exchange chromatography on SP-Sepharose, anion exchange chromatography on Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q and Mono S, and gel filtration by FPLC on Superdex 75. Two of the trypsin-chymotrypsin inhibitors were adsorbed on the first four types of chromatographic media. All three inhibitors have a molecular mass of 16 kDa as judged by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The trypsin inhibitory activity of the inhibitors was attenuated in the presence of the reducing agent dithiothreitol. The remaining inhibitor was unadsorbed on SP-Sepharose but adsorbed on Q-Sepharose, Mono Q and Mono S. The protease inhibitors did not exert any inhibitory effect on hepatoma (Hep G2) and breast cancer (MCF 7) cells or antifungal action toward Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola. Two of the inhibitors slightly inhibited the activity of HIV-1 reverse transcriptase, with an IC50 in the millimolar range.
Inducing an effective CD8+ T cell immunity is important in the protection and elimination of infectious pathogens. Programmed death-1 (PD1) up-regulation in in chronic infections (e.g. HIV-1 and TB) results in “exhausted” function of CD8+ T cells, but is restored by blockade of the PD1/PD-L pathway with antibodies or a soluble form of (s)PD1. Apart from sPD1, the other three spliced variants previously identified currently have no known function. In this study, we identified a new isoform of human PD1 that contains a 42-nucleotide in-frame deletion located at exon 2 near its IgV domain found expressed in PBMCs, named Δ42PD1. We found that Δ42PD1 is distinct from PD1 as it does not engage PD-L1 or PD-L2 and capable of inducing the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β). Next, we used Δ42PD1 as an intramolecular adjuvant to construct a DNA fusion vaccine with HIV-1 Gag p24 antigen. Following immunization in Balb/c mice, a significantly enhanced level of anti-p24 antibody titer and p24-specific CD8+ T cell responses were elicited that persisted for at least 7.5 months. Furthermore, vaccinated mice were protected against pathogenic vaccinia-Gag virus challenge, likely due to the improved proliferative and cytotoxic functions of the elicited CD8+ T cells. Therefore, our study demonstrates that a novel Δ42PD1 variant that amplifies the generation of antigen-specific CD8+ T cell immunity when used in a DNA vaccine.
Previously a well-defined dendritic cell (DC)-targeting strategy of anti-DEC205 based vaccine shows highly enhanced CD4+ T cell immunity. We have demonstrated that a DNA vaccine with soluble (s)PD1 fused with HIV-1 p24 antigen can greatly enhance p24-specific humoral and cellular immune responses in mice by targeting DCs. Consequently, we compared the efficacies of the two vaccines in our system and found that sPD1-based DNA vaccination induced higher functional CD8+ T cell responses. Although the binding and uptake of encoded fusion proteins are similar between sPD1-p24 and anti-DEC205-p24 in DCs in vitro, transfer of DCs pulsed with these proteins into mice resulted in the sPD1-p24 group eliciting higher IFN-γ releasing CD8+ T cells. These data suggests that different mechanistic pathways exist between the two DC-targeting strategies. We examined the route of antigen processing/presentation by confocal microscopy and found that sPD1-p24 protein co-localized to both Rab14 and Lamp1 endosomal compartments used for MHC class I and II presentation, respectively, while anti-DEC205-p24 was only found in the latter. In addition, sPD1 DNA vaccination resulted in draining lymph node DCs with elevated CD40 and MHC class II expression with higher production of IL-12 compared to anti-DEC205. The mechanism of sPD1-based vaccination in enhancing CD8+ T cells immunity is likely dependent on DC-targeting and activation, production of Th1 cytokines and antigen cross-presentation.
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