Design a laboratory experiment to confirm that deletion of gene X is responsible for attenuation in a candidate live vaccine strain. Which combination of approaches will most convincingly demonstrate causality?

• A. Only measure antibody titers generated by the ∆geneX strain in vitro without any animal testing, because immunogenicity data alone proves gene X deletion causes attenuation.
• B. Sequence the ∆geneX strain to confirm deletion and then immediately deploy it in human volunteers without further functional testing because sequencing alone proves attenuation.
• C. Co-infect cells with wild-type and ∆geneX strains and conclude gene X is responsible if the mixed culture shows an intermediate colorimetric change in any assay, without any complementation or animal studies.
• D. Compare phenotypes of wild-type, ∆geneX strain, and a complemented strain (∆geneX + geneX restored) in vitro for replication and in an appropriate animal model for virulence; rescue of the virulent phenotype by complementation would support causality.

Answer: (D)