| Abstrakt: |
Respiratory syncytial virus (RSV) is the most important viral agent of severe pediatric respiratory illness worldwide, but there is no approved pediatric vaccine. Here, we describe the development of the live-attenuated RSV vaccine candidate Min AL as well as engineered derivatives. Min AL was attenuated by codon-pair deoptimization (CPD) of seven of the 11 RSV open reading frames (ORFs) (NS1, NS2, N, P, M, SH and L; 2,073 silent nucleotide substitutions in total). Min AL replicated efficiently in vitro at the permissive temperature of 32°C but was highly temperature sensitive (shut-off temperature of 36°C). When serially passaged at increasing temperatures, Min AL retained greater temperature sensitivity compared to previous candidates with fewer CPD ORFs. However, whole-genome deep-sequencing of passaged Min AL revealed mutations throughout its genome, most commonly missense mutations in the polymerase cofactor P and anti-termination transcription factor M2-1 (the latter was not CPD). Reintroduction of selected mutations into Min AL partially rescued its replication in vitro at temperatures up to 40°C, confirming their compensatory effect. These mutations restored the accumulation of positive-sense RNAs to wild-type (wt) RSV levels, suggesting increased activity by the viral transcriptase, whereas viral protein expression, RNA replication, and virus production were only partly rescued. In hamsters, Min AL and derivatives remained highly restricted in replication in the upper and lower airways, but induced serum IgG and IgA responses to the prefusion form of F (pre F) that were comparable to those induced by wt RSV, as well as robust mucosal and systemic IgG and IgA responses against RSV G. Min AL and derivatives were fully protective against challenge virus replication. The derivatives had increased genetic stability compared to Min AL. Thus, Min AL and derivatives with selected mutations are stable, attenuated, yet highly-immunogenic RSV vaccine candidates that are available for further evaluation. Author summary: Synonymous recoding of codon-pair usage is an emerging strategy for expedited development of live-attenuated vaccines. Respiratory syncytial virus (RSV) is the most important viral agent of severe respiratory illness in children worldwide, but a pediatric vaccine for active immunization is not yet available. Here, we developed a new genome-scale codon-pair deoptimized (CPD) RSV vaccine candidate, namely Min AL, containing 2,073 silent nucleotide mutations spread over seven different ORFs. Min AL replicated efficiently at the permissive temperature of 32°C but was highly temperature sensitive. When serially passaged at increasing temperatures, Min AL remained highly temperature sensitive at 39–40°C. However, sequence analysis showed that it had acquired numerous missense mutations in several CPD and non-CPD genes. Reintroduction of several of these mutations into Min AL decreased its temperature sensitivity, rescued viral transcription to wt RSV levels, partially increased RNA replication, protein expression, and virus replication, albeit not to wt levels, and increased genetic stability. In hamsters, intranasal immunization with Min AL and derivatives induced robust mucosal and systemic anti-RSV antibody responses and full protection against challenge virus replication. This study identified Min AL and derivatives as highly attenuated, yet highly immunogenic and genetically stabilized CPD RSV vaccine candidates appropriate for further evaluation. [ABSTRACT FROM AUTHOR] |
