Laboratory Studies of Human Respiratory Syncytial Virus and Other Pneumoviruses

National Institute Of Allergy And Infectious Diseases

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Abstract

We previously performed codon-pair deoptimization (CPD) of various open reading frames (ORFs) of RSV. This is done by rearranging codons of RSV open reading frames using computer algorithms and de novo gene synthesis to increase the content of normally underrepresented codon-pairs without changing amino acid coding or overall codon usage. CPD typically is attenuating, because under-represented codon pairs are thought to function suboptimally. CDP leads to poor ORF expression and attenuation, providing a new path to vaccine candidates. Due to the large number of genetic changes, it is generally thought that attenuation by CPD is refractory to de-attenuation. In previous years, we produced RSV mutants, attenuated by CPD of various combinations of ORFs, and showed that these viruses unexpectedly were temperature-sensitive. We used this characteristic to evaluate the genetic stability of these vaccine candidates when cultured at restrictive temperatures. These studies revealed a surprising level of adaptability of these attenuated viruses, identifying different routes to de-attenuation. Improved CPD RSV strains have been licensed for vaccine development by Codagenix, Inc., and are being evaluated in clinical studies. During this fiscal year, we further improved RSV vaccine candidates and their genetic stability. In addition, we developed several candidate live-attenuated RSV vaccine viruses using reverse genetics. Attenuation is based on deletion of accessory protein genes and point mutations that were previously developed and had been designed to be refractory to de-attenuation. Lead candidates are attenuated by deletion of the M2-2 ORF, or the NS1 or NS2 genes. In experiments that are currently in progress, we are further characterizing the basis of attenuation of these live-attenuated RSV vaccine candidates. A deeper understanding of the basis of attenuation will guide the development and prioritization of backup candidates for clinical evaluation. While lead candidates are being evaluated in Phase 1 and 2 clinical studies (see our report "Clinical Trials of Vaccines for Respiratory Syncytial Virus and Related Viruses"), it is important that additional candidates are being optimized. If clinical study results suggest that a different level of attenuation is needed, backup candidates are needed that can be advanced rapidly to clinical development. In other experiments that are currently in progress, we are improving RSV serology assays. These assays will support preclinical and clinical development of RSV vaccine candidates.