Development of respiratory syncytial virus (RSV) live-attenuated vaccines for human use.

Abstract

Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and children. Efforts over the past 30 years to develop a satisfactory live or inactivated vaccine were not successful. In fact, the killed vaccine increased pulmonary pathology in children infected with RSV. Our RSV vaccine candidates are now ready for animal and clinical trials. Our research focused on the development of live attenuated RSV human vaccine lines. To date, 7 lines of RSV type A isolates from clinical cases at the University of Michigan have been successfully attenuated using three different approaches: (a) adaptation to suboptimal temperature by direct and stepwise passage; (b) high passage at 35$\sp\circ$C; and (c) adaptation to heterologous host. Lines 19, 48 and 49 type A RSV were cold adapted by direct passage in MRC-5 cells 20-40 times at 25$\sp\circ$C, or by stepwise lowering of the temperature to 25$\sp\circ$ or by passing in MRC-5 and Vero cells down to 20$\sp\circ$C. This process yielded 6 mutants with the cold adapted (ca) phenotype. In addition to the lines adapted to suboptimal temperature, line 19 was passed 100 times in MRC-5 cells until it grew to a very high titer. Seven candidate lines were characterized as temperature sensitive (ts), based on a titer of 2 logs lower replication at 39$\sp\circ$C than at 33$\sp\circ$C. These lines were plaque purified by a newly developed plaque system for RSV. All ts mutants are immunogenic in BALB/c mice using ELISA and neutralization antibody assays. In addition, we have begun investigating the genetic basis for the attenuation phenotype by comparing the genes coding for the F-protein of RSV lines 19 cold adapted in Vero and high passages with their wild type (wt) parent using f-mol sequencing techniques. The F genes of the two attenuated line 19 viruses have 66 nucleotides and 11 amino acids in common, but are different from that of the (wt) RSV at amino acid positions 66, 76, 79, 97, 119, 129, 191, 357, 384, 522, and 530. Any one amino acid or a combination might be necessary for attenuation.