The Immunogenicity and Properties of a Whole-Cell ETEC Vaccine Inactivated with Psoralen and UVA Light in Comparison to Formalin

Abstract

Inactivated whole-cell vaccines present a full repertoire of antigens to the immune system. Formalin treatment, a standard method for microbial inactivation, can modify or destroy protein antigenic epitopes. We tested the hypothesis that photochemical inactivation with psoralen and UVA light (PUVA), which targets nucleic acid, would improve the immunogenicity of an Enterotoxigenic E. coli (ETEC) vaccine relative to a formalin-inactivated counterpart. Exposure of ETEC H10407 to PUVA using the psoralen drug 4′-Aminomethyltrioxsalen hydrochloride (AMT) yielded replication-incompetent bacteria that retained their metabolic activity. CFA/I-mediated mannose-resistant hemagglutination (MRHA) was equivalent for PUVA-inactivated and live ETEC, but was severely reduced for formalin–ETEC, indicating that PUVA preserved fimbrial protein functional integrity. The immunogenicity of PUVA–ETEC and formalin–ETEC was compared in mice ± double mutant heat-labile enterotoxin (dmLT) adjuvant. Two weeks after an intramuscular prime/boost, serum anti-ETEC IgG titers were similar for the two vaccines and were increased by dmLT. However, the IgG responses raised against several conserved ETEC proteins were greater after vaccination with PUVA–ETEC. In addition, PUVA–ETEC generated IgG specific for heat-labile toxin (LT) in the absence of dmLT, which was not a property of formalin–ETEC. These data are consistent with PUVA preserving ETEC protein antigens in their native-like form and justify the further testing of PUVA as a vaccine platform for ETEC using murine challenge models.

Photochemical inactivation as an alternative method to produce a whole-cell vaccine for uropathogenic Escherichia coli (UPEC)

“…However, sera were screened against the full repertoire of protein and polysaccharide antigens presented by whole UPEC, which would not have revealed differences in IgG levels produced against individual proteins. These results are consistent with our recently reported results in which ETEC-specific polyclonal anti-ETEC IgG levels in the serum were not different after vaccination of mice with PUVA-ETEC and formalin-ETEC, but IgG specific for several conserved, outer membrane proteins was significantly greater after vaccination with PUVA-ETEC ( 44 ). A similar screen against UPEC proteins that give rise to protective immune responses such as fimbrial and iron-binding proteins and proteins that are conserved across strains ( 64 ) would provide key information on the potential of psoralen inactivation to improve whole-cell UPEC vaccines.…”

“…As demonstrated previously for other bacterial species ( 37 , 42 , 43 , 52 , 65 ) and our recent study ( 44 ), UPEC was KBMA after PUVA treatment as measured by sustained activity of cellular dehydrogenase enzymes. Previous reports of the KBMA property of psoralen-killed bacteria [reviewed in reference ( 41 )] focused on strains with mutations in the uvrABC locus, which renders bacteria highly sensitive to UV damage by disabling the nucleotide excision repair (NER) system ( 66 ).…”

“…We chose the psoralen drug AMT to photochemically inactivate UPEC according to a protocol that was used to inactivate ETEC ( 44 ). An AMT dose-response experiment was performed with doses of 0–100 µg/mL AMT (0–340 µM), followed by irradiation with 2 J/cm 2 UVA light.…”

“…The use of uvr mutants reduced the frequency of psoralen-induced crosslinks required to disable replication, which allowed for maximal production of endogenous or engineered protein antigens. We based PUVA inactivation of UPEC on our work with ETEC ( 44 ) in which wild-type bacteria were rendered replication-defective using the psoralen drug AMT. AMT is one of a group of psoralen drugs ( 39 ) that had previously been used to inactivate viruses ( 32 , 38 ) but not bacteria.…”

“…The KBMA property was observed in a study in which E. coli was photochemically inactivated with the psoralen drug 8-MOP for the purpose of UTI vaccine development, but no immunogenicity data were reported ( 43 ). We recently reported that enterotoxigenic E. coli (ETEC) inactivated with the psoralen drug AMT and UVA light were KBMA, retained surface adhesins in native-like form, and demonstrated superior immunogenicity in mice relative to formalin-killed ETEC ( 44 ). Here, we have extended the approach to UPEC.…”