| Products/Services Used | Details | Operation |
|---|---|---|
| Codon Optimization | The discontinuous sequence encoding gB Domain II (AA 112-133 + 343-438) was joined with the flexible linker Ile-Ala-Gly-Ser-Gly. For Merlin strain gB Domain I (AA133-343), the 5’ HA and 3’ avidin/polyhistidine tags omitted due to hypothesized steric hinderance. Nucleotides were codon optimized for mammalian cells, synthesized de novo (Genscript), then cloned into pcDNA3.1(+) mammalian expression vector (Invitrogen) via BamHI at the 5′ end and EcoRI site at the 3′ end. Plasmids were transiently transfected into 293F suspension cells as previously described using polyethyleneimine transfection reagent (Sigma-Aldrich) [25]. Supernatant was harvested 5 days later, and purified using Nickel-NTA resin for gB Domain II (Thermo Fisher Scientific), and lectin resin (VWR) for gB Domain I. Purity and identity were confirmed by Western blot using polyclonal CMV IgG (Cytogam – CSL Behring) or monoclonal antibodies SM10 (Domain I) and SM5-1 (Domain II). | Get A Quote |
Human cytomegalovirus (HCMV) is the most common congenital infection, and the leading nongenetic cause of sensorineural hearing loss (SNHL) in newborns globally. A gB subunit vaccine administered with adjuvent MF59 (gB/MF59) is the most efficacious tested to-date, achieving 50% efficacy in preventing infection of HCMV-seronegative mothers. We recently discovered that gB/MF59 vaccination elicited primarily non-neutralizing antibody responses, that HCMV strains acquired by vaccinees more often included strains with gB genotypes that are distinct from the vaccine antigen, and that protection against HCMV acquisition correlated with ability of vaccine-elicited antibodies to bind to membrane associated gB. Thus, we ... More
