The virus production and neutralization core for this program will prepare Venezuelan equine encephalitis virus (VEE) preparations carrying replicons for expression of HIV and SIV envelope and gag genes and will conduct HIV-1 pseudovirus neutralization assays. All of the VEE preparations used in this program will be prepared in this core. We will maintain plasmid preparations of the helper plasmids and of pVR2 for production of replicon articles preparations carrying the recombinant pVR2 genomes of interest, and characterize the replicons for infectivity, expression of the antigens, and safety recombinant pVR2 genomes of interest, and characterize the replicons for infectivity, expression of the antigens, and safety. Tittered stocks of the replicon particles will be provided to the projects for use in immunogenicity experiments. Viruses for use in the neutralization assays will be prepared for pseudotyping envelopes of interest onto particles based on the pNL4-3.luc.R- in the neutralization assays will be prepared by pseudotyping envelopes of interest onto particles based on the NL4-3.luc.R.-E-reporter genome. Neutralization assays will be conducted in microtiter in HOS-CD4-CCR5 or HOS-CD4-CXCR4 cells, using luminescence as the endpoint readout. Stocks of pseudotyped viruses will be maintained using envelope expression constructs provided by Project 1, or as available in the laboratory.
