viruses Article A Novel MVA-Based HIV Vaccine Candidate (MVA-gp145-GPN) Co-Expressing Clade C Membrane-Bound Trimeric gp145 Env and Gag-Induced Virus-Like Particles (VLPs) Triggered Broad and Multifunctional HIV-1-Specific T Cell and Antibody Responses Beatriz Perdiguero 1 , Cristina Sánchez-Corzo 1 , Carlos Oscar S. Sorzano 2 , Lidia Saiz 1 , Pilar Mediavilla 1 , Mariano Esteban 1, * and Carmen Elena Gómez 1, * 1 Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CNB-CSIC), Campus de Cantoblanco, 28049 Madrid, Spain; perdigue@cnb.csic.es (B.P.); cscorzo@cnb.csic.es (C.S.-C.); lidiasm1995@gmail.com (L.S.); pilarmediavilla15@hotmail.com (P.M.) 2 Biocomputing Unit, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CNB-CSIC), Campus de Cantoblanco, 28049 Madrid, Spain; coss@cnb.csic.es * Correspondence: mesteban@cnb.csic.es (M.E.); cegomez@cnb.csic.es (C.E.G.); Tel.: +34-915854553 (M.E.); +34-915854560 (C.E.G.) Received: 22 January 2019; Accepted: 13 February 2019; Published: 16 February 2019   Abstract: The development of an effective Human Immunodeficiency Virus (HIV) vaccine that is able to stimulate both the humoral and cellular HIV-1-specific immune responses remains a major priority challenge. In this study, we described the generation and preclinical evaluation of single and double modified vaccinia virus Ankara (MVA)-based candidates expressing the HIV-1 clade C membrane-bound gp145(ZM96) trimeric protein and/or the Gag(ZM96)-Pol-Nef(CN54) (GPN) polyprotein that was processed to form Gag-induced virus-like particles (VLPs). In vitro characterization of MVA recombinants revealed the stable integration of HIV-1 genes without affecting its replication capacity. In cells that were infected with Env-expressing viruses, the gp145 protein was inserted into the plasma membrane exposing critical epitopes that were recognized by broadly neutralizing antibodies (bNAbs), whereas Gag-induced VLPs were released from cells that were infected with GPN-expressing viruses. VLP particles as well as purified MVA virions contain Env and Gag visualized by immunoelectron microscopy and western-blot of fractions that were obtained after detergent treatments of purified virus particles. In BALB/c mice, homologous MVA-gp145-GPN prime/boost regimen induced broad and polyfunctional Env- and Gag-specific CD4 T cells and antigen-specific T follicular helper (Tfh) and Germinal Center (GC) B cells, which correlated with robust HIV-1-specific humoral responses. Overall, these results support the consideration of MVA-gp145-GPN vector as a potential vaccine candidate against HIV-1. Keywords: HIV-1; MVA vaccine; Env-gp145; Gag-Pol-Nef; VLPs; immunogenicity; CD4 T cells; Tfh; GC B cells; humoral responses 1. Introduction The AIDS (Acquired Immunodeficiency Syndrome) pandemic that is caused by Human Immunodeficiency Virus (HIV) represents a global health problem with enormous disease control Viruses 2019, 11, 160; doi:10.3390/v11020160 www.mdpi.com/journal/viruses