Anti-HIV-1 Activity of a New Scorpion Venom Peptide Derivative Kn2-7

For over 30 years, HIV/AIDS has wreaked havoc in the world. In the absence of an effective vaccine for HIV, development of new anti-HIV agents is urgently needed. We previously identified the antiviral activities of the scorpion-venom-peptide-derived mucroporin-M1 for three RNA viruses (measles viruses, SARS-CoV, and H5N1). In this investigation, a panel of scorpion venom peptides and their derivatives were designed and chosen for assessment of their anti-HIV activities. A new scorpion venom peptide derivative Kn2-7 was identified as the most potent anti-HIV-1 peptide by screening assays with an EC50 value of 2.76 µg/ml (1.65 µM) and showed low cytotoxicity to host cells with a selective index (SI) of 13.93. Kn2-7 could inhibit all members of a standard reference panel of HIV-1 subtype B pseudotyped virus (PV) with CCR5-tropic and CXCR4-tropic NL4-3 PV strain. Furthermore, it also inhibited a CXCR4-tropic replication-competent strain of HIV-1 subtype B virus. Binding assay of Kn2-7 to HIV-1 PV by Octet Red system suggested the anti-HIV-1 activity was correlated with a direct interaction between Kn2-7 and HIV-1 envelope. These results demonstrated that peptide Kn2-7 could inhibit HIV-1 by direct interaction with viral particle and may become a promising candidate compound for further development of microbicide against HIV-1

Targeting sphingosine 1-phosphate receptor 3 inhibits T-cell exhaustion and regulates recruitment of proinflammatory macrophages to improve antitumor efficacy of CAR-T cells against solid tumor

Backgrounds Chimeric antigen receptor (CAR)-modified T cells (CAR-T) are limited in solid tumors due to the hostile tumor microenvironment (TME). Combination therapy could be a promising approach to overcome this obstacle. Recent studies have shown that sphingosine 1-phosphate receptor (S1PR)3 has tremendous potential in regulating the immune environment. However, the functional significance of S1PR3 in T-cell-based immunotherapies and the molecular mechanisms have not been fully understood.Methods Here, we studied the combination of EpCAM-specific CAR T-cell therapy with pharmacological blockade of S1PR3 against solid tumor. We have applied RNA sequencing, flow cytometry, ELISA, cellular/molecular immunological technology, and mouse models of solid cancers.Results Our study provided evidence that S1PR3 high expression is positively associated with resistance to programmed cell death protein-1 (PD-1)-based immunotherapy and increased T-cell exhaustion. In addition, pharmacological inhibition of S1PR3 improves the efficacy of anti-PD-1 therapy. Next, we explored the possible combination of S1PR3 antagonist with murine EpCAM-targeted CAR-T cells in immunocompetent mouse models of breast cancer and colon cancer. The results indicated that the S1PR3 antagonist could significantly enhance the efficacy of murine EpCAM CAR-T cells in vitro and in vivo. Mechanistically, the S1PR3 antagonist improved CAR-T cell activation, regulated the central memory phenotype, and reduced CAR-T cell exhaustion in vitro. Targeting S1PR3 was shown to remodel the TME through the recruitment of proinflammatory macrophages by promoting macrophage activation and proinflammatory phenotype polarization, resulting in improved CAR-T cell infiltration and amplified recruitment of CD8+T cells.Conclusions This work demonstrated targeting S1PR3 could increase the antitumor activities of CAR-T cell therapy at least partially by inhibiting T-cell exhaustion and remodeling the TME through the recruitment of proinflammatory macrophages. These findings provided additional rationale for combining S1PR3 inhibitor with CAR-T cells for the treatment of solid tumor

Unpolarized release of vaccinia virus and HIV antigen by colchicine treatment enhances intranasal HIV antigen expression and mucosal humoral responses.

The induction of a strong mucosal immune response is essential to building successful HIV vaccines. Highly attenuated recombinant HIV vaccinia virus can be administered mucosally, but even high doses of immunization have been found unable to induce strong mucosal antibody responses. In order to solve this problem, we studied the interactions of recombinant HIV vaccinia virus Tiantan strain (rVTT-gagpol) in mucosal epithelial cells (specifically Caco-2 cell layers) and in BALB/c mice. We evaluated the impact of this virus on HIV antigen delivery and specific immune responses. The results demonstrated that rVTT-gagpol was able to infect Caco-2 cell layers and both the nasal and lung epithelia in BALB/c mice. The progeny viruses and expressed p24 were released mainly from apical surfaces. In BALB/c mice, the infection was limited to the respiratory system and was not observed in the blood. This showed that polarized distribution limited antigen delivery into the whole body and thus limited immune response. To see if this could be improved upon, we stimulated unpolarized budding of the virus and HIV antigens by treating both Caco-2 cells and BALB/c mice with colchicine. We found that, in BALB/c mice, the degree of infection and antigen expression in the epithelia went up. As a result, specific immune responses increased correspondingly. Together, these data suggest that polarized budding limits antigen delivery and immune responses, but unpolarized distribution can increase antigen expression and delivery and thus enhance specific immune responses. This conclusion can be used to optimize mucosal HIV vaccine strategies

Comparison of distribution of differently treated cell layers after rVTT-gagpol infection <i>in vitro</i>.

<p>Caco-2 cell layers were infected with rVTT-gagpol 14–16 days after polarization to form the polarized group. A similar group was treated with colchicine (0.08 mg/ml) apically 72 hours before rVTT-gagpol infection to form the polarized + colchicine group. A 3-day Caco-2 transwell culture was infected with rVTT-gagpol to form the unpolarized group. The data shown are the sums of the 48-hour and 72-hour values. (A) p24 distribution. (B) Virus distribution. (C) TER. Apical, cellular, and basolateral samples of the three groups were compared. T-testing was conducted to compare the polarized + colchicine group and unpolarized group to the polarized group. Statistical significance: (<i>P</i><0.05) in an unpaired test is indicated by an asterisk. There were two wells for every group. The experiments were performed twice.</p

Comparison of p24 and virus distribution between untreated and colchicine-treated rVTT-gagpol immunization groups in mice.

<p>The colchicine-treated group included 6-week-old female BALB/c mice that were i.n. inoculated with colchicine (20 µg) 72 hours before rVTT-gagpol immunization (1×10⁷ PFU). The rVTT-gagpol immunization group was given identical treatment except that PBS was used as a substitute for colchicine. Infection and expression kinetics in nose samples, nasal washes, lung samples, and lung bronchial washes were evaluated (A, B, C, and D). Immunoperoxidase staining of p24 antigen in the nose (E, F, G, and H). In detail, p24 distribution in (A) nose samples and nasal washes and (C) lung samples and lung bronchial washes. Virus distribution in (B) nose samples and nasal washes and (D) lung samples and lung bronchial washes. Nose sections of the rVTT-gagpol immunization group after (E) 24 hours and (G) 72 hours. Nose sections of the colchicine-treated rVTT-gagpol immunization group after (F) 24 hours and (H) 72 hours. (▹) designates epithelium, () designates lamina propria, and () designates lumen. There were five mice in each group and the images shown are typical of those observed. Magnification, 100×. Two independent experiments were conducted with similar results. One set of results is shown.</p

Immunization schemes.

<p>(A) I.n. immunization. (B) Colchicine i.n. immunization for distribution of virus and antigen. (C) Colchicine i.n. immunization for antibody tests.</p

Humoral immune responses to high doses of rVTT-gagpol after i.n. inoculation.

<p>Mice were i.n. inoculated with rVTT-gagpol at weeks 0 and 4 and euthanized during week 6. Sera, lung washes, and vaginal washes taken during week 6 were collected to ascertain anti-p24 IgG and IgA by ELISA. There were seven mice in each group. Anti-p24 (A) serum IgG, (B) serum IgA, lung wash IgA, and (C) vaginal wash IgA are displayed above. Three independent experiments were conducted with similar results. The results of one representative experiment are shown.</p

Comparison of anti-p24 humoral immune responses between untreated and colchicine-treated rVTT-gagpol immunization groups in mice.

<p>The colchicine-treated group consisted of five 6-week-old female BALB/c mice that were given i.n. rVTT-gagpol (10⁷PFU) and colchicine (40 µg) at the same time. During week 4, mice were given colchicine boosters (4 µg) 24 hours before rVTT-gagpol. The mice were euthanized during week 6. The rVTT-gagpol immunization group was given identical treatment except that PBS was used as a substitute for colchicine. Serum and mucosal samples were collected to test for the presence of anti-p24 antibody by ELISA. Anti-p24 levels of (A) serum IgG, (B) serum IgA, bronchial wash IgA, and (C) vaginal wash IgA are displayed. T-testing was conducted between the colchicine-treated group and the rVTT-gagpol immunization group. Statistical significance (P<0.05 in an unpaired test) is indicated by an asterisk. Two independent experiments were conducted with similar results. One set of results is shown.</p