P22-Based Nanovaccines against Enterohemorrhagic Escherichia coli

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is an important causative agent of diarrhea in humans that causes outbreaks worldwide. Efforts have been made to mitigate the morbidity and mortality caused by these microorganisms; however, the global incidence is still high, causing hundreds of deaths per year. Several vaccine candidates have been evaluated that demonstrate some stability and therapeutic potential but have limited overarching effect. Virus-like particles have been used successfully as nanocontainers for the targeted delivery of drugs, proteins, or nucleic acids. In this study, phage P22 nanocontainers were used as a carrier for the highly antigenic T3SS structural protein EscC that is conserved between EHEC and other enteropathogenic bacteria. We were able to stably incorporate the EscC protein into P22 nanocontainers. The EscC-P22 particles were used to intranasally inoculate mice, which generated specific antibodies against EscC. These antibodies increased the phagocytic activity of murine macrophages infected with EHEC in vitro and reduced bacterial adherence to Caco-2 epithelial cells in vitro, illustrating their functionality. The EscC-P22-based particles are a potential nanovaccine candidate for immunization against EHEC O157:H7 infections. IMPORTANCE This study describes the initial attempt to use P22 viral-like particles as nanocontainers expressing enterohemorrhagic Escherichia coli (EHEC) proteins that are immunogenic and could be used as effective vaccines against EHEC infections.

Keywords: EHEC; P22-based nanovaccines; nanocontainer; pathogenic Escherichia coli.

FIG 1

Schematic representation of P22-based nanovaccine construction (modified from reference 39).

FIG 2

(A) SDS-PAGE showing the recombinant EscC protein (black arrows), EscC-P22 nanocontainers, and CP purified proteins (red arrow). (B) TEM micrographs of EscC-P22 nanocontainers (left) and P22 empty capsids (right). The high density inside EscC-P22 capsids (black arrow) indicates the presence of encapsulated protein, in comparison to low-density-empty capsids (red arrow).

FIG 3

Timeline of immunization protocol. After prime and boost immunizations, sera were collected to quantify antibody production, and lungs were removed and stored for further investigation (figure created in BioRender [BioRender.com]).

FIG 4

Antibody production after immunizations. ELISA was performed to measure total serum IgG titers using EscC protein as a target. Nano-EscC+adjuvant vaccine was successful in producing higher IgG titers (red line) than any other formulation, compared with preimmune sera (baseline). Significance was obtained by multiple unpaired t tests and shown in graph P values of < 0.001 (***). Statistical significance from all the dilutions can be found in Table S1.

FIG 5

Opsonophagocytic assay (A) and bacterial adherence inhibition assay (B). (A) RAW macrophage cells were infected with EHEC O157:H7 86-24 strain previously incubated with sera obtained from vaccinated or nonvaccinated (naive) mice. Sera from nano-EscC+adj immunized mice significantly increase bacterial killing, thus reducing the recovery of internalized bacteria. The intracellular survival was established as the recovered bacteria compared with the input (×100). (B) Caco-2 epithelial cells were infected with EHEC O157:H7 86-24 strain previously incubated with sera from mice immunized with nanovaccine formulations or nonimmunized mice (naive). The antibodies reduced dramatically the bacterial adherence to epithelial cells in all the tested formulations. The percentage of adhered bacteria was established as the output bacteria compared with the inoculum (×100). (A and B) Bars represent the mean of two independent experiments performed in triplicate ± SEM (standard error). Only statistically significant data relationships are shown, from a one-way ANOVA analysis. *, P < 0.05; **, P < 0.01.

FIG 6

Bactericidal activity of serum from mice vaccinated with different formulations or unvaccinated mice (naive). In each group, colored bars represent the active sera, white bars the inactive, and the striped bars show the inactive sera supplemented with mouse sera as an exogenous complement source. The survival percentage was established as follows: bacteria recovered/input × 100. Results are expressed as means of two independent experiments measured in triplicate + SEM.