RESUMO
The structure of the HIV-1 envelope membrane-proximal external region (MPER) is influenced by its association with the lipid bilayer on the surface of virus particles and infected cells. To develop a replicating vaccine vector displaying MPER sequences in association with membrane, Env epitopes recognized by the broadly neutralizing antibodies 2F5, 4E10, or both were grafted into the membrane-proximal stem region of the vesicular stomatitis virus (VSV) glycoprotein (G). VSV encoding functional G-MPER chimeras based on G from the Indiana or New Jersey serotype propagated efficiently, although grafting of both epitopes (G-2F5-4E10) modestly reduced replication and resulted in the acquisition of one to two adaptive mutations in the grafted MPER sequence. Monoclonal antibodies 2F5 and 4E10 efficiently neutralized VSV G-MPER vectors and bound to virus particles in solution, indicating that the epitopes were accessible in the preattachment form of the G-MPER chimeras. Overall, our results showed that the HIV Env MPER could functionally substitute for the VSV G-stem region implying that both perform similar functions even though they are from unrelated viruses. Furthermore, we found that the MPER sequence grafts induced low but detectable MPER-specific antibody responses in rabbits vaccinated with live VSV, although additional vector and immunogen modifications or use of a heterologous prime-boost vaccination regimen will be required to increase the magnitude of the immune response.
Assuntos
Anticorpos Anti-HIV/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Vesiculovirus/fisiologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Feminino , Glicoproteínas de Membrana/imunologia , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Vesiculovirus/genética , Vesiculovirus/crescimento & desenvolvimento , Vesiculovirus/imunologia , Proteínas do Envelope Viral/imunologia , Replicação Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4⺠cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.
Assuntos
Quimera/fisiologia , Vírus da Encefalite Equina Venezuelana/fisiologia , Vetores Genéticos/fisiologia , Replicação Viral , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Animais , Linhagem Celular , Quimera/genética , Vírus da Encefalite Equina Venezuelana/genética , Expressão Gênica , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Vetores Genéticos/genética , Infecções por HIV/prevenção & controle , Humanos , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Montagem de VírusRESUMO
Poliovirus 2A(pro) is required for the inhibition of host cell protein synthesis and efficient viral replication. We investigated the role of 2A(pro) in regulating viral RNA stability, translation and replication in HeLa S10 reactions. The protease activity of 2A(pro) or its polyprotein precursors, 2AB or P2, was required to increase the stability of viral RNA and prolong translation. Since other viral proteins were not required for the observed effects of 2A(pro), it is likely that a cellular protein(s) modified by 2A(pro) mediated these effects on stability and translation. In addition, the protease activity of 2A(pro) stimulated negative-strand initiation by approximately five-fold but had no effect on positive-strand initiation. The 2A(pro) stimulation of negative-strand synthesis was independent of its effect on stability and translation. These findings further extend the previously known functions of protein 2A(pro) to include its role in increasing RNA stability, prolonging translation and stimulating negative-strand synthesis.
