"Aim of the project is to search for antigens that induce neutralizing antibodies in mice, which prevent entry of Human cytomegalovirus (HCMV) in vitro, and to characterize the identified candidate antigen with respect to its function. We will employ the “reverse vaccinology approach” with two robust technological platforms. A published genomic DNA sequence of HCMV has been analyzed and identified putative genes encoding surface exposed or secreted proteins using computer algorithms. Based on this analysis, candidate DNA sequences have been synthesized and are ready for cloning into expression vectors. •The selected genes will be expressed in eukaryotic cells in vitro and characterized to compare with information obtained from the bioinformatic analyses. •To screen immunogenicity of the candidate proteins, we will insert candidate genes into alphavirus vector to prepare pseudo-virion (alphavirus replicon particles), which induce robust immune responses in mice against the expressed gene product. Sera from immunized mice will be collected and submitted to immunological assays, including western blotting, immunofluorescence studies and in vitro neutralization assays. •In vitro neutralization assays will be set up with bacterial artificial chromosome (BAC) system that produce recombinant HCMV expressing a reporter gene to detect infection to cultured cells. •Sera from HCMV seropositive individuals will be analyzed to detect existence of specific antibodies to selected antigens by western blotting or the protein microarray technology. Screening of vaccine targets against HCMV infection in MEGALOVAX includes the reverse vaccinology approach applied for the first time to viral pathogens with the two abovementioned technological platforms. This new approach is expected to offer a breakthrough of discovery of new HCMV antigens for protection. The results from this project will also contribute to further understanding of viral-host interactions during HCMV infection."