Classical Flt3L-dependent dendritic cells control immunity to protein vaccine

DCs are critical for initiating immunity. The current paradigm in vaccine biology is that DCs migrating from peripheral tissue and classical lymphoid-resident DCs (cDCs) cooperate in the draining LNs to initiate priming and proliferation of T cells. Here, we observe subcutaneous immunity is Fms-like tyrosine kinase 3 ligand (Flt3L) dependent. Flt3L is rapidly secreted after immunization; Flt3 deletion reduces T cell responses by 50%. Flt3L enhances global T cell and humoral immunity as well as both the numbers and antigen capture capacity of migratory DCs (migDCs) and LN-resident cDCs. Surprisingly, however, we find immunity is controlled by cDCs and actively tempered in vivo by migDCs. Deletion of Langerin+ DC or blockade of DC migration improves immunity. Consistent with an immune-regulatory role, transcriptomic analyses reveals different skin migDC subsets in both mouse and human cluster together, and share immune-suppressing gene expression and regulatory pathways. These data reveal that protective immunity to protein vaccines is controlled by Flt3L-dependent, LN-resident cDCs.</jats:p

Cell cycle regulation by the Wee1 Inhibitor PD0166285, Pyrido [2,3-d] pyimidine, in the B16 mouse melanoma cell line

BACKGROUND: Wee1 kinase plays a critical role in maintaining G2 arrest through its inhibitory phosphorylation of cdc2. In previous reports, a pyridopyrimidine molecule PD0166285 was identified to inhibit Wee1 activity at nanomolar concentrations. This G2 checkpoint abrogation by PD0166285 was demonstrated to kill cancer cells, there at a toxic highest dose of 0.5 μM in some cell lines for exposure periods of no longer than 6 hours. The deregulated cell cycle progression may have ultimately damaged the cancer cells. We herein report one of the mechanism by which PD0166285 leads to cell death in the B16 mouse melanoma cell line. METHODS: Tumor cell proliferation was determined by counting cell numbers. Cell cycle distribution was determined by flow cytometry. Morphogenesis analysis such as microtubule stabilization, Wee1 distribution, and cyclin B location were observed by immunofluorescence confocal microscopy. An immunoblot analysis of cdc2-Tyr15, cyclin D, E, p16, 21, 27, and Rb. A real-time PCR of the mRNA of cyclin D were completed. RESULTS: In our experiment, B16 cells also dramatically abrogated the G2 checkpoint and were found to arrest in the early G1 phase by treatment with 0.5 μM for 4 hours observed by flow cytometry. Cyclin D mRNA decreased within 4 hours observed by Real-time PCR. Rb was dephosphrylated for 24 hours. However, B16 cells did not undergo cell death after 0.5 μM treatment for 24 hours. Immnofluoscence microscopy showed that the cells become round and small in the morphogenesis. More interesting phenomena were that microtubule stabilization was blocked, and Wee1 distribution was restricted after treatment for 4 hours. CONCLUSION: We analyzed the effect of Wee1 inhibitor PD0166285 described first by Wang in the G2 transition in the B16 melanoma cell line. The inhibitor PD0166285 abrogated G2/M checkpoint inducing early cell division. Moreover, we found that the treatment of cells with the inhibitor is related to microtubule stabilization and decrease in cyclin D transcription. These effects together suggest that Wee1 inhibitor may thus be a potentially useful anti-cancer therapy

Antiviral activity and mode of action of Griffithsin against HSV-2 and HPV: Preliminary studies of a potential non-ARV combination microbicide

Background: Griffithsin (GRFT) is a promising HIV microbicide candidate. Nixon et al. have shown that GRFT blocks herpes simplex 2 (HSV-2) infection in a mouse model, proposing inhibition of cell-to-cell spread as the mode of action (MOA). Using in vitro studies we further investigated the MOA of GRFT against HSV-2 and studied its antiviral activity against human papillomavirus (HPV). We also combined GRFT with zinc acetate (ZA) and/or carrageenan (CG) to render a more potent microbicide. Methods: We used XTT assay to define non-cytotoxic concentrations of GRFT, ZA, CG or their combinations. Assays for anti-HIV, anti-HPV and anti-HSV-2 activities were performed in TZM-bl cells or PBMCs using MAGI and p24 ELISA; in HeLa cells using a luciferase assay; and in Vero cells using plaque forming units (pfu) assay. We performed time-of-addition and temperature dependence experiments to differentiate inhibition of viral adsorption from entry. Surface plasmon resonance (SPR) was used to assess GRFT binding to viral glycoproteins and immunohistochemistry was used to determine the specific glycoprotein involved. Antiviral activities of prototype GRFT/CG (GC) and GRFT/ZA/CG (GZC) gels in a vaginal HSV-2 mouse model were evaluated. Results: GRFT shows modest in vitro antiviral activity against HSV-2 G (IC_50 = 5.8μg/ml) and HPV 6, 16, 18, 45 PsVs (IC_50 = 10.8-26.3μg/ml), compared to potent anti-HIV activity (IC_50 = 0.7-1.4ng/ml). As with HIV, GRFT blocks the entry but not the adsorption of HSV-2 and HPV to target cells. The combined analyses of SPR and immunohistochemistry for HSV-2 gD, suggest that GRFT binds to HSV-2 gD. GC and GZ had synergistic in vitro antiviral activity against HIV and HPV (CI \u3c 1). GC and GZC gels significantly reduced (p \u3c 0.05) HSV-2 vaginal infection in vivo when administered up to 2h before challenge with 10^6pfu/mouse. Conclusions: GRFT blocks HSV-2 and HPV entry to target cells and combination with CG and/or ZA may result in a potent/broad-spectrum non-ARV microbicide

Griffithsin and carrageenan combination to target herpes simplex virus 2 and human papillomavirus

Extensive preclinical evaluation of griffithsin (GRFT) has identified this lectin to be a promising broad-spectrum microbicide. We set out to explore the antiviral properties of a GRFT and carrageenan (CG) combination product against herpes simplex virus 2 (HSV-2) and human papillomavirus (HPV) as well as determine the mechanism of action (MOA) of GRFT against both viruses. We performed the experiments in different cell lines, using time-of-addition and temperature dependence experiments to differentiate inhibition of viral attachment from entry and viral receptor internalization. Surface plasmon resonance (SPR) was used to assess GRFT binding to viral glycoproteins, and immunoprecipitation and immunohistochemistry were used to identify the specific glycoprotein involved. We determined the antiviral activity of GRFT against HSV-2 to be a 50% effective concentration (EC_50) of 230 nM and provide the first evidence that GRFT has moderate anti-HPV activity (EC_50 = 0.429 to 1.39 μM). GRFT blocks the entry of HSV-2 and HPV into target cells but not the adsorption of HSV-2 and HPV onto target cells. The results of the SPR, immunoprecipitation, and immunohistochemistry analyses of HSV-2 combined suggest that GRFT may block viral entry by binding to HSV-2 glycoprotein D. Cell-based assays suggest anti-HPV activity through α6 integrin internalization. The GRFT-CG combination product but not GRFT or CG alone reduced HSV-2 vaginal infection in mice when given an hour before challenge (P = 0.0352). While GRFT significantly protected mice against vaginal HPV infection when dosed during and after HPV16 pseudovirus challenge (P \u3c 0.026), greater CG-mediated protection was afforded by the GRFT-CG combination for up to 8 h (P \u3c 0.0022). These findings support the development of the GRFT-CG combination as a broad-spectrum microbicide

Dendritic cell targeted HIV gag protein vaccine provides help to a DNA vaccine including mobilization of protective CD8+ T cells

To improve the efficacy of T cell–based vaccination, we pursued the principle that CD4+ T cells provide help for functional CD8+ T cell immunity. To do so, we administered HIV gag to mice successively as protein and DNA vaccines. To achieve strong CD4+ T cell immunity, the protein vaccine was targeted selectively to DEC-205, a receptor for antigen presentation on dendritic cells. This targeting helped CD8+ T cell immunity develop to a subsequent DNA vaccine and improved protection to intranasal challenge with recombinant vaccinia gag virus, including more rapid accumulation of CD8+ T cells in the lung. The helper effect of dendritic cell-targeted protein vaccine was mimicked by immunization with specific MHC II binding HIV gag peptides but not peptides from a disparate Yersinia pestis microbe. CD4+ helper cells upon adoptive transfer allowed wild-type, but not CD40−/−, recipient mice to respond better to the DNA vaccine. The transfer also enabled recipients to more rapidly accumulate gag-specific CD8+ T cells in the lung following challenge with vaccinia gag virus. Thus, complementary prime boost vaccination, in which prime and boost favor distinct types of T cell immunity, improves plasmid DNA immunization, including mobilization of CD8+ T cells to sites of infection

Griffithsin carrageenan fast dissolving inserts prevent SHIV HSV-2 and HPV infections in vivo

Human immunodeficiency virus (HIV) pre-exposure prophylaxis (PrEP) strategies with proven in vivo efficacy rely on antiretroviral drugs, creating the potential for drug resistance and complicated treatment options in individuals who become infected. Moreover, on-demand products are currently missing from the PrEP development portfolio. Griffithsin (GRFT) is a non-antiretroviral HIV entry inhibitor derived from red algae with an excellent safety profile and potent activity in vitro. When combined with carrageenan (CG), GRFT has strong activity against herpes simplex virus-2 (HSV-2) and human papillomavirus (HPV) in vitro and in vivo. Here, we report that GRFT/CG in a freeze-dried fast dissolving insert (FDI) formulation for on-demand use protects rhesus macaques from a high dose vaginal SHIV SF162P3 challenge 4 h after FDI insertion. Furthermore, the GRFT/CG FDI also protects mice vaginally against HSV-2 and HPV pseudovirus. As a safe, potent, broad-spectrum, on-demand non-antiretroviral product, the GRFT/CG FDI warrants clinical development