A killed, genetically engineered derivative of a wild-type extraintestinal pathogenic E. coli strain is a vaccine candidate

Abstract

Infections due to extraintestinal pathogenic E. coli (ExPEC) result in significant morbidity, mortality and increased healthcare costs. An efficacious vaccine against ExPEC would be desirable. In this report, we explore the use of killed-whole E. coli as a vaccine immunogen. Given the diversity of capsule and O-antigens in ExPEC, we have hypothesized that alternative targets are viable vaccine candidates. We have also hypothesized that immunization with a genetically engineered strain that is deficient in the capsule and O-antigen will generate a greater immune response against antigens other than the capsular and O-antigen epitopes than a wild-type strain. Lastly, we hypothesize that mucosal immunization with killed E. coli has the potential to generate a significant immune response. In this study, we demonstrated that nasal immunization with a formalin-killed ExPEC derivative deficient in capsule and O-antigen results in a significantly greater overall humoral response compared to its wild-type derivative (which demonstrates that capsule and/or the O-antigen impede the development of an optimal humoral immune response) and a significantly greater immune response against non-capsular and O-antigen epitopes. These antibodies also bound to a subset of heterologous ExPEC strains and enhanced neutrophil-mediated bactericidal activity against the homologous and a heterologous strain. Taken together, these studies support the concept that formalin-killed genetically engineered ExPEC derivatives are whole cell vaccine candidates to prevent infections due to ExPEC.

Figure Legend 1

The serum IgG and IgA antibody response after nasal immunization with CP9 (w.t.). After pre-immunization serum was obtained, C57 black6 mice were nasally immunized (2 week intervals × 3) with 1 × 10⁹ cfu live CP9, 1 × 10⁹ formalin-killed CP9, or received 1X PBS (non-immunized controls). IgG and IgA were measured in pre and post-immunization sera by ELISA using the homologous strain CP9, as described in Methods. There was a significant increase in IgG and IgA concentrations between the pre and post CP9 (live) and pre and post CP9 (formalin-killed) groups (* P < 0.0001; # P = 0.01, paired t-test). There was no significant difference in the post-immunization concentrations of IgG and IgA between the CP9 (live) and CP9 (formalin-killed) groups (P > 0.1, unpaired t-test).

Figure Legend 2

The serum IgG antibody response after nasal immunization of mice with formalin-killed CP9 (w.t.) and CP923 (capsule and O-antigen minus). C57 black6 mice were nasally immunized (2 week intervals × 3) with 1 × 10⁹ cfu formalin-killed CP9 or 1 × 10⁹ formalin-killed CP923. A quantitative assessment of serum IgG (by ELISA, as described in Methods) was performed using pooled post-immunization sera from 5 female and 10 male mice immunized with formalin-killed CP9 and 9 female and 10 male C57 mice immunized with formalin-killed CP923. In panels A and B the ELISA plates were coated with 1 × 10⁷ cfu of CP923 and CP9 respectively. In all instances, a significantly greater amount of CP923-specific IgG was present after CP923 was used as the immunogen compared to CP9-specific IgG when CP9 was used. (* P < 0.0001, # P = 0.0003, & P = 0.02; unpaired t-test).

Figure Legends 3

The serum IgG antibody response after nasal immunization of mice with formalin-killed CP9 (w.t.) and CP923 (capsule and O-antigen minus). After pre-immunization serum was obtained, C57 black6 mice were nasally immunized (2 week intervals × 3) with 1 × 10⁹ cfu formalin-killed CP9, 1 × 10⁹ formalin-killed CP923, or received 1X PBS. IgG concentrations were measured against varying concentrations (10²-10⁸) of both CP9 and CP923 in pre and post-immunization sera by ELISA, as described in Methods. 1:1000 dilutions of CP9 and CP923-specific anti-sera were used. Panels A and B depict the response in male mice and panels C and D depict the response in female mice. The serum IgG response was greater in mice immunized with formalin-killed CP923 compared to formalin-killed CP9.

Figure Legend 4

Binding of antibodies generated by immunization with either live or formalin-killed CP923 to CP923 (capsule and O-antigen minus). C57 black6 mice were nasally immunized (2 week intervals × 3) with either 1 × 10⁹ cfu of live CP923 or 1 × 10⁹ of formalin-killed CP923. Binding of these antisera to live CP923 (homologous strain) was measured by flow cytometry as described in Methods and was expressed as geometric mean fluorescence. The binding of antibodies generated by immunization with either live or formalin-killed CP923 was similar (P > 0.1).

Figure Legend 5

Random amplified polymorphic DNA (RAPD)-based phylogenetic analysis of 29 ExPEC strains and the binding of CP923-specific antiserum to these strains. Genomic profiles, as generated for each isolate using RAPD primer 1254, were used for cluster analysis. Antibody binding was performed on live bacteria via flow cytometry and was reported as the geometric mean fluorescence (GMF). A GMF ≥10 (in bold) was above non-specific background levels and therefore represents specific binding. Antibody binding is listed to the right of each strain. CP9 is the homologous strain. The heterologous strains are: 104, 159, 161, 163, 165, 167, 169, 170, 171, 173, 175, 177, 184, 187, 189, 195, 197 (bacteremic isolates from Buffalo, NY); 470(O25/K5), 743(O6/K2) (bacteremic isolates kindly provided by Alan Cross); J96(O4/K-), R28(O4/K3), 518(O4/K10, K54/96) (3 strains previously established to be highly related to CP9 (O4 J96-like strains)(17); R45(O4/K12, V31(O4/K12), PM8(O4/K12)(3 strains previously established to be less related to CP9 (other O4 strains)(17); and K1/Y(O7/K1)(55), IA2 (O4/K12)(15), CFT073(O6/K2)(56)(3 strains that have served as model pathogens for investigations on ExPEC pathogenesis).

Figure Legend 6

Bactericidal assay assessing the effects of CP923-specific antiserum on neutrophil-mediated bactericidal activity against the homologous strain CP9 (w.t.). Approximately 1 × 10⁵ cfu of CP9 that were: 1) not pre-opsonized (n=8), 2) preopsonized with a 1:100 dilution of pre-immune serum (Δ56°C) (n =5) or, 3) opsonized with a 1:100 dilution of CP923-specific antiserum (Δ56°C) (n = 12) were added to wells did or did not contain 5 × 10⁵ purified human neutrophils. Bacterial titers were determined at 60 minutes and bactericidal activity was calculated as the difference between bacterial log titers in the presence and absence of neutrophils. * P = 0.002 compared to CP9 pre-opsonized with pre-immune sera; * P = 0.0006 compared to CP9 that was not pre-opsonized.

Figure Legend 7

Bactericidal assay comparing the effects of CP9-specific antiserum to CP923-specific antiserum on neutrophil-mediated bactericidal activity against the heterologous strain K1/Y. Approximately 1 × 10⁵ cfu of K1/Y that were: 1) not pre-opsonized (N=9), 2) pre-opsonized with a 1:100 dilution of CP9-specific mouse or rabbit antiserum(Δ56°C)(N=9) or, 3) opsonized with a 1:100 dilution of CP923-specific mouse or rabbit antiserum (Δ56°C)(N=9) were added to wells with and without neutrophils. Bacterial titers were determined at 60 minutes and bactericidal activity was calculated as the difference between bacterial log titers in the presence and absence of neutrophils. * P < 0.0003 compared to K1/Y opsonized with CP9-specific antiserum sera; * P < 0.0001 compared to K1/Y that was not pre-opsonized.