To identify Mycobacterium tuberculosis (Mtb) antigens as candidates for a subunit vaccine against tuberculosis (TB), we have employed a CD4+ T-cell expression screening method. Mtb-specific CD4+ T-cell lines from nine healthy PPD positive donors were stimulated with different antigenic substrates including autologous dendritic cells (DC) infected with Mtb, or cultured with culture filtrate proteins (CFP), and purified protein derivative of Mtb (PPD). These lines were used to screen a genomic Mtb library expressed in Escherichia coli and processed and presented by autologous DC. This screening led to the recovery of numerous T-cell antigens, including both novel and previously described antigens. One of these novel antigens, referred to as Mtb9.8 (Rv0287), was recognized by multiple T-cell lines, stimulated with either Mtb-infected DC or CFP. Using the mouse and guinea pig models of TB, high levels of IFN-gamma were produced, and solid protection from Mtb challenge was observed following immunization with Mtb9.8 formulated in either AS02A or AS01B Adjuvant Systems. These results demonstrate that T-cell screening of the Mtb genome can be used to identify CD4+ T-cell antigens that are candidates for vaccine development.