Research scientists at CDC have developed compositions and methods that can be used to develop attenuated vaccines having well-defined levels of replicative fitness and enhanced genetic stabilities. Infections by intracellular pathogens, such as viruses, bacteria, and parasites, are cleared in most cases after activation of specific T-cell immune responses that recognize foreign antigens and eliminate infected cells. Vaccines against those infectious organisms traditionally have been developed by administration of whole live attenuated or inactivated microorganisms. Although research has been performed using subunit vaccines, the levels of cellular immunity induced are usually low and not capable of eliciting complete protection against diseases caused by intracellular microbes. CDC inventors discovered that replacement of one or more natural (or native) codons in a pathogen with synonymous unpreferred codons can decrease the replicative fitness of the pathogen, thereby attenuating the pathogen. The unpreferred synonymous codon(s) encode the same amino acid as the native codon(s), but have nonetheless been found to reduce a pathogen's replicative fitness.