The goal of this project is for Crucell to manufacture clinical-grade master virus seed stocks and trivalent, clinical-grade rAd26 and rAd5HVR48 vaccine products for the clinical trials described in Project 2. Crucell Holland BV is a medium-sized biotechnology company that is dedicated to the development of vaccines for infectious diseases. In particular, Crucell has pioneered the development of rAd vector-based vaccines and has developed vectors from Ad serotypes that have low seroprevalence in human populations. Crucell has also been a leader in the preclinical-to-clinical translational development of rAd vector-based vaccines for a variety of pathogens. In particular, Crucell has developed robust production and purification methods to manufacture clinical-grade rAd vaccines. Crucell has also standardized and validated release assays and has developed several packaging cell lines that support pharmaceutical-scale manufacturing in full compliance with Good Manufacturing Practice (GMP) guidelines. Vaccine candidates based on several different Ad serotypes have been manufactured and released by Crucell and have recently entered clinical trials for Ebola, malaria, and tuberculosis in both the United States and South Africa. Crucell will guarantee uniform, high quality manufacturing of the rAd vaccines as well as strict adherence to Standard Operating Procedures (SOPs) and rigorous quality control / quality assurance (QC/QA) standards in full compliance with all pertinent regulatory authorities. Research-grade vaccines will be provided for the GLP preclinical toxicology studies in rabbits, and clinical-grade vaccines will be manufactured for the phase 1 and phase 2a clinical trials described in Project 2. In our current IPCAVD AI066305 program, we have developed rAd26 and rAd5HVR48 GMP manufacturing processes and release assays that have already been approved by the FDA. In Project 3, we propose to utilize and to improve these existing production processes and analytical assays to manufacture and release the multivalent rAd26 and rAd5HVR48 clinical vaccine products. To accomplish these goals, we propose the following three Specific Aims: 1. To manufacture and release clinical-grade master virus seed stocks for rAd26 and rAd5HVR48 vectors expressing the optimal HIV-1 Gag-Nef, Pol, and Env antigens under GMP conditions; 2. To improve our current manufacturing processes for rAd26 and rAd5HVR48 vectors by optimizing vector yields at harvest and downstream purification steps; and 3. To manufacture, purify, release, and monitor the stability of trivalent, clinical-grade rAd26 and rAd5HVR48 vaccines expressing the optimal HIV Gag-Nef, Pol, and Env antigens under GMP conditions.