Inserting Omp22 into the flagellin protein, replacing its hypervariable region, results in stronger protection against lethal Acinetobacter baumannii infection

Abstract

Acinetobacter baumannii, a common nosocomial pathogen, is known for its rapid acquisition of antimicrobial resistance, underscoring the urgent need to develop an effective vaccine against this pathogen. Outer membrane protein 22 (Omp22) regulates the biogenesis of outer membrane vesicles to transport virulence-promoting factors into the host cells and facilitates the progression of A. baumannii infection. In this study, we used a mouse model to assess a vaccine's immunogenicity and protective efficacy using recombinant Omp22 protein within the hypervariable region of flagellin (FliC-Omp22). FliC-Omp22 demonstrated superior protection following challenge with a lethal dose of multidrug-resistant (MDR) A. baumannii strain 58ST compared to Omp22 alone. In addition, it elicited increased IgG1/IgG2a and IL-4/IFN-γ ratios, indicating a predominant Th2 immune response. Furthermore, the FliC-Omp22 vaccination elicited strong specific antibodies that inhibited the adhesion and invasion of A. baumannii 58ST and enhanced the opsonic killing activity against the pathogen. FliC-Omp22 immunization significantly reduced bacterial loads in infected mice's spleen, lungs, and liver, thereby improving their survival against the lethal infection caused by MDR A. baumannii 58ST. This study suggests that integrating Omp22 into the hypervariable domain of flagellin holds promise for developing an effective vaccine against A. baumannii infections.

Keywords: Acinetobacter baumannii.; Flagellin.; FliC.; Immunization.; Omp22.; Sepsis.

Fig. 1

Design and evaluation of fusion FliC-Omp22 protein. (A) Diagram illustrating the design and construction of FliC-Omp22. The D3 domain of FliC protein (residues 185 to 285) was removed and substituted with the Omp22 protein. (B) Schematic representation of the linear B-cell epitopes identified by BepiPred-1.0, BepiPred-2.0, ABCpred, LBtope, BcePred and BCPred tools. (C) Robetta’s prediction for the 3D structure of FliC-Omp22. (D) Western blot analysis shows purified recombinant Omp22, FliC, and FliC-Omp22 proteins in lanes 1, 2, and 3, respectively, identified using an anti-His tag antibody.

Fig. 2

Antigenicity, integrity and bioactivity of FliC-Omp22 fusion protein. (A) In the protein ELISA assay, recombinant FliC-Omp22 showed binding to anti-Omp22 and anti-FliC specific antibodies, compared to normal mouse serum, which showed no binding. (B) In the immunoblot analysis, anti-FliC antibody specifically reacted with FliC (lane 2, ∼51 kDa) and FliC-Omp22 (lane 3, ∼64 kDa) proteins and did not show reactivity with Omp22 (lane 1). (C) Anti-Omp22 antibody specifically bound to Omp22 (lane 1, ∼25 kDa) and FliC-Omp22 (lane 2, ∼64 kDa) proteins and did not show reactivity with FliC proteins (lane 3). (D) Anti-FliC-Omp22 antibody exhibited reactivity with FliC (lane 1, ∼51 kDa), Omp22 (lane 2, ∼25 kDa), and FliC-Omp22 (lane 3, ∼64 kDa) proteins. Lane M denotes the positions of molecular weight markers. (E) FliC-Omp22, similar to FliC, induced TNF-α production in TLR5-positive RAW 264.7 cells in a concentration-dependent manner. The data are represented as the mean ± standard deviation (SD).

Fig. 3

Antibody responses in in the sera of both immunized and non-immunized. (A) Total IgG levels were measured against Omp22, FliC-Omp22, FliC, and the whole cell of A. baumannii. (B) IgG1 and IgG2a subclass titers were assessed. The data are represented as the mean ± SD. Datasets marked with distinct superscript letters exhibit statistical differences from each other.

Fig. 4

In vitro immune response of splenocytes from immunized and non-immunized mice. Two weeks post the final immunization, spleens from mice were processed and stimulated with Omp22, FliC-Omp22, and FliC antigens in vitro for 72 h. (A) The proliferative assay of splenocytes was measured with the MTT method. (B) The IL-4 and IFN-γ cytokine production in splenocytes stimulated by antigens were assessed. The data are represented as the mean ± SD. Datasets marked with distinct superscript letters exhibit statistical differences from each other.

Fig. 5

Comparative analysis of the opsonophagocytic killing activity and the inhibition of A. baumannii adhesion and invasion by sera from immunized and non-immunized mice. The data are represented as the mean ± SD. Datasets marked with distinct superscript letters exhibit statistical differences from each other.

Fig. 6

Active immunization with FliC-Omp22 protected mice against lethal challenges with A. baumannii. (A) On day 56, 21 days after the last immunization, the mice (n = 8 mice/group) were intraperitoneally challenged with A. baumannii and monitored twice daily for seven days. (B) Active immunization with FliC-Omp22 notably reduced bacterial burdens in the liver, spleen and lung, assessed 16 h post-infection. (C) Passive immunization with FliC-Omp22-immunized sera protected mice against lethal challenge by A. baumannii (n = 8 mice/group). (D) Passive immunization with FliC-Omp22-immunized sera resulted in a significant reduction in bacterial burdens in the spleen. Survival rates were monitored for seven days. *P < 0.05 and **P < 0.01. Datasets marked with distinct superscript letters exhibit statistical differences from each other.