Human respiratory syncytial virus N, P and M protein interactions in HEK-293T cells

Characterization of Human Respiratory Syncytial Virus (HRSV) protein interactions with host cell components is crucial to devise antiviral strategies. Viral nucleoprotein, phosphoprotein and matrix protein genes were optimized for human codon usage and cloned into expression vectors. HEK-293T cells were transfected with these vectors, viral proteins were immunoprecipitated, and co-immunoprecipitated cellular proteins were identified through mass spectrometry. Cell proteins identified with higher confidence scores were probed in the immunoprecipitation using specific antibodies. The results indicate that nucleoprotein interacts with arginine methyl-transferase, methylosome protein and Hsp70. Phosphoprotein interacts with Hsp70 and tropomysin, and matrix with tropomysin and nucleophosmin. Additionally, we performed immunoprecipitation of these cellular proteins in cells infected with HRSV, followed by detection of co-immunoprecipitated viral proteins. The results indicate that these interactions also occur in the context of viral infection, and their potential contribution for a HRSV replication model is discussed.

Activation of PAF-receptor induces regulatory dendritic cells through PGE2 and IL-10

Activation of the platelet-activating factor receptor (PAFR) in macrophages is associated with suppressor phenotype. Here, we investigated the PAFR in murine dendritic cells (DC). Bone marrow-derived dendritic cells (BALB/c) were cultured with GM-CSF and maturation was induced by LPS. The PAFR antagonists (WEB2086, WEB2170, PCA4248) and the prostaglandin (PG) synthesis inhibitors (indomethacin, nimesulide and NS-398) were added before LPS. Mature and immature DCs expressed PAFR. LPS increased MHCII, CD40, CD80, CD86, CCR7 and induced IL-10, IL-12, COX-2 and PGE2 expression. IL-10, COX-2 and PGE2 levels were reduced by PAFR antagonists and increased by cPAF. The IL-10 production was independent of PGs. Mature DCs induced antigen-specific lymphocyte proliferation. PAFR antagonists or PG-synthesis inhibitors significantly increased lymphocyte proliferation. It is proposed that PAF has a central role in regulatory DC differentiation through potentiation of IL-10 and PGE2 production.

Bicistronic DNA vaccines simultaneously encoding HIV, HSV and HPV antigens promote CD8⁺ T cell responses and protective immunity

Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⁺ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⁺ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.