Inserting Omp22 into the flagellin protein, replacing its hypervariable region, results in stronger protection against sepsis caused by Acinetobacter baumannii in a mouse model

doi:10.21203/rs.3.rs-4666148/v1

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Inserting Omp22 into the flagellin protein, replacing its hypervariable region, results in stronger protection against sepsis caused by Acinetobacter baumannii in a mouse model

https://doi.org/10.21203/rs.3.rs-4666148/v1

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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 sepsis model to assess the immunogenicity and protective efficacy of a vaccine 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, 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 the spleen, lungs, and liver of infected mice, thereby improving their survival against sepsis induced 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.

Biological sciences/Immunology

Biological sciences/Microbiology

Acinetobacter baumannii.

Omp22.

Flagellin.

FliC.

Immunization.

Sepsis

Acinetobacter baumannii can cause severe and fatal infections, including pneumonia, wound infections, and bloodstream infections, in immunocompromised patients, with mortality rates as high as 75 percent in untreated cases^1,2. The persistence and rapid dissemination of A. baumannii in hospital environments are primarily attributed to its resistance to disinfectants, broad resistance to antimicrobial agents, and production of diverse virulence factors, which facilitate prolonged colonization and transmissions^1,2. Therefore, both the US Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) consider it crucial to research new approaches against A. baumannii^3,4. This alarm highlights a significant threat to human health and emphasizes the urgent need for effective new antibiotics and vaccines. Treatment of multidrug-resistant (MDR) A. baumannii infection in patients admitted to ICUs presents substantial challenges because the bacterium can quickly spread to critical organs, resulting in sepsis and systemic infections associated with high mortality rates⁵. The increasing mortality rate associated with MDR A. baumannii sepsis, combined with the inadequate efficacy of existing antimicrobial agents, highlights the urgent need to develop effective vaccines to combat A. baumannii infections. Despite promising preclinical results from subunit vaccine candidates, including outer membrane, fimbrial, and biofilm-associated proteins, no authorized vaccine against A. baumannii infections has been approved^6–9. This may be attributed to the need to incorporate adjuvants or delivery systems into their formulations to enhance stability and immunogenicity^10,11.

Many A. baumannii strains isolated from patients express the highly conserved outer membrane protein 22 (Omp22), which possesses the outer membrane protein A (OmpA)-like domain, facilitates interactions with peptidoglycan and regulates the formation of outer membrane vesicles (OMVs), serving as carriers for the secretion of cytotoxic, virulence-enhancing, or resistance-inducing molecules^6,12. Furthermore, owing to its limited similarity to human proteins, antibodies against Omp22 do not cross-react with human proteins⁶. Previous studies of Omp22 vaccine candidates revealed that its immunogenicity is closely linked to its delivery method and the adjuvants usage^13–15. For instance, immunization of mice with 50 µg of Trx-Omp22 fusion protein with alum adjuvant protected against A. baumannii sepsis, whereas lower doses were less effective and showed cytotoxic effects on mammalian cell growth⁶. Another study demonstrated that immunization with 40 µg of Omp22 encapsulated in chitosan-poly (lactic-co-glycolic acid) increased survival rates against A. baumannii challenge compared to Omp22 alone¹³. Engineered OMVs displaying Omp22 also conferred dose-dependent protection against A. baumannii¹⁵. These findings highlight the importance of combining Omp22 with adjuvants or protein carriers to enhance its effectiveness and influence the immune response against A. baumannii.

Evidence indicates that flagellin, functioning as a pathogen-associated molecular pattern (PAMP), enhances the immunogenicity and protective efficacy of antigens through the activation of Toll-like receptor 5 (TLR5), which is expressed across diverse immune cell types^16–18. Flagellin's potent adjuvant activity is strictly linked to its high-affinity binding to TLR5 on CD11c⁺ dendritic cells (DCs), enhancing antigen uptake and presentation, thus leading to antigen-specific CD4⁺/CD8⁺ T cells and humoral immunity against pathogens^16–18. Utilization of the flagellin D3 hypervariable region for integrating foreign antigens has been an effective strategy for stimulating both innate and adaptive immune responses through improved antigen presentation and TLR5-dependent mechanisms^19,20. Integrating an envelope protein from the West Nile virus into the D3 domain of flagellin led to the developing of a vaccine capable of eliciting neutralizing and protective antibodies against this virus²⁰. Similarly, incorporating the hemagglutinin sequence of the avian influenza virus or Yersinia pestis V antigens into the hypervariable region of flagellin proved effective in developing vaccines against respective infections^19,21. Accordingly, modification of flagellin constructs, such as replacing the variable region of flagellin D3 with the antigen, enhances immunogenicity, and triggers specific immune responses, underscoring the versatility and effectiveness of flagellin-based vaccines.

Previous studies have indicated that the immunogenicity of Omp22 can be considerably enhanced by employing various adjuvants and display systems^13–15. In light of these findings, the current study aimed to investigate the potential of integrating Omp22 into the hypervariable D3 domain of flagellin (FliC-Omp22) as a vaccine candidate to enhance protective humoral and cellular immune responses against A. baumannii in a murine sepsis model.

Design and characterization of FliC-Omp22 protein

The construction and design of FliC-Omp22 involved deleting the D3 domain (residues 185 to 285) of the native FliC protein and inserting Omp22 in its place (Fig. 1A). The FliC-Omp22 fusion protein exhibited a half-life of > 20 h, as determined by Expasy's ProtParam analysis. Predictions indicated the stability of the chimeric FliC-Omp22 protein, with an instability index (II) of 28.04, classifying it as a stable protein. Computational modeling and prediction identified the surface-exposed loops of Omp22 spanning positions 103–113, 114–142, and 143–149 within the FliC-Omp22 fusion protein as linear and discontinuous B-cell epitopes (Fig. 1B). A 3D structural model of the FliC-Omp22 fusion protein was generated using the Robetta server (Fig. 1C). The FliC-Omp22 chimeric vaccine sequence had a VaxiJen score of 0.8431, indicating its potential as an antigen. SDS-PAGE revealed bands corresponding to approximately 25 kDa for Omp22, 51 kDa for FliC, and 64 kDa for FliC-Omp22 recombinant proteins (Fig. 1D-F). Additionally, recombinant proteins were identified in their purified form using a monoclonal anti-His-tag antibody (Fig. 1G). Full-length gels and blots are included in Supplementary Fig. S1.

Antigenicity, structural integrity, and bioactivity of FliC-Omp22 fusion protein

To validate the appropriate display of the Omp22 protein and retention of FliC protein antigenicity in the FliC-Omp22 fusion protein, an ELISA assay was conducted using anti-Omp22 or anti-FliC specific antibodies. As illustrated in Fig. 2A, the FliC-Omp22 fusion protein effectively bound to both anti-Omp22 and anti-FliC-specific antibodies, indicating the accurate presentation of the Omp22 domain in the fusion protein and preservation of antigenicity for both Omp22 and FliC. Immunoblot analysis revealed that both anti-Omp22 and anti-FliC-specific antibodies specifically interacted with a ∼64 kDa protein, corresponding to the FliC-Omp22 protein (Fig. 2B and C). Additionally, the anti-FliC antibody displayed specific reactivity with ∼51 kDa and ∼64 kDa proteins, corresponding to FliC and FliC-Omp22 proteins, respectively (Fig. 2B). Similarly, the anti-Omp22 antibody showed specific reactivity with ∼25 kDa and ∼64 kDa proteins, corresponding to Omp22 and FliC-Omp22 proteins, respectively (Fig. 2B). Moreover, the anti-FliC-Omp22 antibody exhibited reactivity with ∼25 kDa, ∼51 kDa, and ∼64 kDa proteins, corresponding to Omp22, FliC, and FliC-Omp22 proteins, respectively (Fig. 2D). Additionally, anti-FliC and anti-Omp22 specific antibody did not show reactivity with the Omp22 and FliC proteins, respectively (Fig. 2B and C). To evaluate the TLR5-specific signaling potency of purified FliC-Omp22, TLR5-negative, and TLR5-positive RAW 264.7 cells were incubated with increasing concentrations of FliC-Omp22 or FliC (as a positive control). As illustrated in Fig. 2E, FliC-Omp22, similar to FliC, induced TNF-α production in a TLR5-specific manner and exhibited dose-dependent behavior. No significant difference was detected in the capacity of varying concentrations of FliC-Omp22 and FliC proteins to stimulate TNF-α production in TLR5-positive RAW 264.7 cells (P > 0.05; Fig. 2E). The effectiveness of both proteins was comparable, implying that inclusion of the Omp22 sequence into FliC did not substantially influence the TLR5 binding domain of flagellin. In contrast, Omp22 and PBS did not stimulate TNF-α production in TLR5-positive RAW 264.7 cells. No statistically significant differences were detected among FliC-Omp22, FliC, Omp22, and PBS in inducing TNF-α production in TLR5-negative RAW 264.7 cells. Full-length immunoblots are given in Supplementary Fig. S2.

FliC-Omp22 immunization induced robust serum IgG, IgG1, and IgG2a levels

To evaluate the humoral responses elicited by the FliC-Omp22 vaccine, we conducted a comparative analysis of serum IgG levels in immunized mice against whole-cell A. baumannii, Omp22, FliC, or FliC-Omp22 recombinant proteins (Fig. 3A-D). FliC-Omp22-immunized mice exhibited notably higher IgG levels against the Omp22 recombinant protein on days 14, 28, and 42 post-immunization than the other groups (P < 0.05; Fig. 3A). IgG levels in the sera of mice injected with FliC or PBS against the Omp22 recombinant protein were similar; however, they were significantly lower in the sera from Omp22-immunized mice (P < 0.05). Furthermore, serum IgG levels in FliC-Omp22-immunized mice increased significantly by 1–2 fold at four and six weeks post-booster doses against the Omp22 recombinant protein compared to those in sera from Omp22-immunized mice (P < 0.05; Fig. 3A). The IgG levels in FliC-Omp22-immunized mice against the FliC-Omp22 recombinant protein on days 14, 28, and 42 post-immunization were significantly higher than those in other groups (P < 0.05; Fig. 3B). At the same time, the IgG levels of FliC- or Omp22-immunized mice sera against FliC-Omp22 protein were similar; however, these levels were significantly higher in PBS-injected mouse sera (P < 0.05; Fig. 3B). The serum IgG levels in FliC-Omp22-immunized mice significantly increased (P < 0.05) four and six weeks following the booster dose against FliC-Omp22 recombinant protein compared with that in FliC or Omp22 -immunized mice sera (Fig. 3B). The IgG levels against the FliC protein in mice immunized with FliC were significantly higher on days 14, 28, and 42 post-immunization than those in the other groups (P < 0.05; Fig. 3C). Conversely, IgG levels in the sera of mice immunized with Omp22 or PBS against FliC remained similar (P > 0.05) but were significantly lower in FliC-Omp22-immunized mice sera (P < 0.05; Fig. 3C). Furthermore, the serum IgG levels in FliC-immunized mice increased significantly by 2 fold six weeks post-booster dose against FliC recombinant protein compared to those in FliC-Omp22-immunized mice sera (P < 0.05). Moreover, anti-A. baumannii IgG levels in the sera of FliC-Omp22-immunized mice were significantly higher than those in other groups (P < 0.05; Fig. 3D). The anti-A. baumannii IgG levels detected in the sera of Omp22-immunized mice were notably higher than those in mice receiving FliC or PBS (P < 0.05; Fig. 3D).

Moreover, IgG1 and IgG2a titers were evaluated to investigate the potential of the FliC-Omp22 vaccine to induce class switching of IgG subtypes. Serum levels of IgG1 and IgG2a were significantly higher in all immunized mice than in those administered PBS (P < 0.05; Fig. 4). In FliC-Omp22-immunized mice, IgG1 and IgG2a levels against Omp22 protein were remarkably higher than those in other groups (P < 0.05; Fig. 4). IgG1 levels predominated over IgG2a levels in FliC-Omp22-immunized mice against the Omp22 protein (P < 0.05). While no significant differences were observed in IgG1 and IgG2a levels in mice immunized with Omp22 (P > 0.05), they were significantly higher than those in FliC- or PBS-injected mice (P < 0.05). Conversely, there were no significant differences in IgG1 and IgG2a levels between mice injected with FliC or PBS and those injected with Omp22 (P > 0.05). IgG1 and IgG2a levels in the sera of mice immunized with FliC-Omp22 against the FliC-Omp22 protein were significantly higher than those in the other groups (P < 0.05; Fig. 4). In FliC-Omp22-vaccinated mice, IgG1 levels against FliC-Omp22 were markedly higher than IgG2a levels (P < 0.05). Although no significant differences were noted in IgG1 and IgG2a levels between mice injected with FliC or Omp22 against the FliC-Omp22 protein, these levels were significantly higher than those in PBS-injected mice (P < 0.05; Fig. 4). In addition, in mice receiving FliC or Omp22 antigens, IgG1 levels against FliC-Omp22 surpassed IgG2a levels (P < 0.05). Moreover, the serum levels of IgG1 and IgG2a in mice immunized with FliC were notably higher than those in the other groups (P < 0.05). Furthermore, IgG1 and IgG2a levels in the sera of FliC-Omp22-immunized mice against FliC were significantly higher than those in mice immunized with Omp22 or PBS alone (P < 0.05). In mice receiving the FliC or FliC-Omp22 vaccine, IgG1 levels against FliC were higher than IgG2a levels (P < 0.05; Fig. 4). In addition, no significant differences were observed in IgG1 and IgG2a levels between mice injected with Omp22 or PBS against FliC protein (P > 0.05).

FliC-Omp22 immunization enhanced splenocyte proliferation and cytokine production

The proliferation stimulation index (SI) and cytokine production of splenocytes from experimental mice were examined after stimulation with Omp22, FliC, or FliC-Omp22 proteins. As shown in Fig. 5, the SI of FliC-Omp22 immune splenocytes upon stimulation with Omp22 was markedly higher than that in the other experimental groups (P > 0.05). Moreover, the SI of Omp22 immune splenocytes stimulated with Omp22 was significantly greater (up to 2.7 times) than that of FliC or PBS splenocytes (P < 0.01). In addition, no significant differences were observed in the SI of FliC or PBS splenocyte cultures following stimulation with Omp22 (P > 0.05; Fig. 5). FliC-Omp22 immune splenocyte cultures showed a notably higher proliferation rate when stimulated with FliC-Omp22 compared to the other groups. The SI of FliC-Omp22 or FliC immune splenocytes stimulated with FliC-Omp22 demonstrated a significant increase, up to 2.4 times greater than that of PBS splenocyte cultures (P < 0.01). In addition, no significant differences were observed in the SI of FliC or Omp22 splenocyte cultures stimulated with FliC-Omp22 (P > 0.05). As shown in Fig. 5, the stimulation index (SI) of FliC immune splenocytes exposed to FliC was notably higher, peaking at three times greater than that of FliC-Omp22 splenocyte cultures (P < 0.05). For FliC-Omp22 immune splenocytes stimulated with FliC, there was a marked increase, up to 2 times greater than that of Omp22 or PBS splenocyte cultures (P < 0.01). No significant differences were observed in the SI of Omp22 or PBS splenocyte cultures stimulated with FliC (P > 0.05; Fig. 5).

The cytokine response of splenocytes was assessed by quantifying the production of IL-4 and IFN-γ. As shown in Fig. 6, IL-4 and IFN-γ concentrations in the culture supernatant of splenocytes from mice immunized with FliC-Omp22 and subsequently stimulated with Omp22 proteins were significantly elevated compared to those in the other experimental groups (P > 0.05). Specifically, the IL-4 and IFN-γ concentrations of splenocytes immunized with Omp22 and then stimulated with Omp22 were notably higher (up to 2 times) than those of splenocytes from mice treated with FliC or PBS (P < 0.01). Moreover, there were no significant differences in the concentrations of IL-4 and IFN-γ in splenocytes from mice treated with FliC or PBS, and subsequently stimulated with Omp22 (P > 0.05; Fig. 6). In splenocytes from mice immunized with FliC-Omp22 or Omp22 and stimulated with Omp22, IL-4 levels surpassed IFN-γ levels (P < 0.05).

As shown in Fig. 6, IL-4 and IFN-γ concentrations in the culture supernatant of FliC-Omp22 immune mouse splenocytes, when stimulated with FliC-Omp22 proteins, were significantly higher than those in the other groups (P > 0.05). The IL-4 and IFN-γ concentrations of FliC immune splenocytes following stimulation with FliC-Omp22 were notably elevated (up to twice as high) compared to those of Omp22 or PBS immune splenocytes (P < 0.01; Fig. 6). IL-4 and IFN-γ levels in Omp22 immune splenocytes following FliC-Omp22 stimulation were notably elevated (up to twice as high) compared to those in PBS splenocyte cultures (P < 0.01). In FliC-Omp22 and FliC immune splenocytes stimulated with FliC-Omp22, IL-4 levels surpassed those of IFN-γ (P < 0.05). No significant differences were observed in IL-4 and IFN-γ concentrations of Omp22 or PBS splenocyte cultures upon stimulation with FliC-Omp22 (P > 0.05).

The concentrations of IL-4 and IFN-γ in the culture supernatant of splenocytes from mice vaccinated with FliC, upon stimulation with FliC protein, were notably elevated compared to those in the other experimental groups (P > 0.05; Fig. 6). The concentrations of IL-4 and IFN-γ in FliC-Omp22-immune splenocytes following stimulation with FliC were notably elevated (up to twice as high) compared to those in Omp22- or PBS-immune splenocytes (P < 0.01). In FliC-Omp22 and FliC immune splenocytes stimulated with FliC, IL-4 levels exceeded IFN-γ levels (P < 0.05). No significant differences were observed in IL-4 and IFN-γ concentrations of Omp22 or PBS splenocyte cultures upon stimulation with FliC (P > 0.05; Fig. 6).

FliC-Omp22-immunized sera inhibited the adhesion and invasiveness of A. baumannii

Antibodies directed against FliC-Omp22 exhibited a dose-dependent inhibitory effect on the adhesion and invasion of A549 cells by A. baumannii. Sera from mice immunized with FliC-Omp22 at a dilution of 1:10, demonstrated a significant decrease in both adhesion and invasion of A. baumannii compared to the control groups (P < 0.05; Fig. 7). Notably, at a 1:10 dilution, FliC-Omp22 immunized sera showed a more marked reduction in the adhesion (55% vs. 72%) and invasion (42 vs. 62%) of A. baumannii than at a 1:100 dilution (P < 0.05). Similarly, Omp22-immunized sera at dilutions of 1:10 and 1:100 significantly reduced adhesion and invasion compared with the controls (P < 0.05). Omp22 immunized sera at a 1:10 dilution exhibited a more substantial decrease in A. baumannii adhesion and invasion than sera at a 1:100 dilution. No significant differences were observed between the sera of FliC-immunized and PBS-immunized mice in reduction of A. baumannii adhesion and invasion (P > 0.05; Fig. 7).

FliC-Omp22-immune sera have high opsonophagocytic activity against A. baumannii

Given the importance of opsonophagocytic activity in eliminating systemic A. baumannii infection, we compared the effectiveness of sera from mice vaccinated with the FliC-Omp22 or Omp22 vaccines against those injected with FliC or PBS. FliC-Omp22-immunized sera significantly improved opsonophagocytic killing of A. baumannii compared to Omp22-immunized sera (P < 0.05; Fig. 8). Sera from FliC-Omp22-immunized mice at dilutions of 1:10 and 1:100 exhibited 59% and 45% opsonophagocytic killing activity against A. baumannii, respectively (P < 0.05). Omp22-immunized sera at dilutions of 1:10 and 1:100 also significantly enhanced opsonophagocytic killing of A. baumannii compared to controls (P < 0.05). There were no notable differences between the FliC and PBS controls in enhancing the opsonophagocytic killing of A. baumannii (P > 0.05; Fig. 8).

FliC-Omp22 immunization increased survival and reduced bacterial load of infected mice

To assess the effectiveness of the FliC-Omp22 vaccine against lethal challenge by A. baumannii, we examined the survival rates of both immunized and non-immunized mice. FliC-Omp22-immunized mice exhibited significantly higher survival rates of 75% (6/8) than the other experimental groups and were protected from fatal A. baumannii sepsis (P < 0.05; Fig. 9A). In contrast, all mice in the control group succumbed to A. baumannii infection within two days, resulting in 100% mortality. Furthermore, the Omp22-immunized mice displayed a survival rate of 33% (3/8) post-infection (P < 0.05; Fig. 9A). Furthermore, we evaluated the effectiveness of the FliC-Omp22 vaccine in reducing bacterial colonization in the liver, spleen, and lungs of mice challenged with A. baumannii, and compared it with that of mice immunized with Omp22, FliC, or PBS (Fig. 9B). Notably, mice immunized with FliC-Omp22 exhibited the lowest bacterial load among those challenged with A. baumannii. Immunization with FliC-Omp22 resulted in the most significant reduction (up to 10²-fold) in the liver, spleen, and lung bacterial counts following A. baumannii infection (P < 0.05; Fig. 9B), surpassing the Omp22 group. Moreover, immunization with the Omp22 antigen led to nearly 10³- to 10⁴-fold reductions in the liver, spleen, and lung bacterial loads compared to mice injected with FliC and PBS (P < 0.05). No significant differences were observed in the liver, spleen, and lung bacterial loads between the FliC and PBS controls (P > 0.05; Fig. 9B).

Furthermore, we assessed the efficacy of passive immunization using FliC-Omp22-immunized sera against lethal A. baumannii sepsis. As illustrated in Fig. 9C, mice injected with FliC-Omp22-immunized sera displayed the highest survival rates following challenge with A. baumannii (50% survival rates, P < 0.05), whereas all mice injected with FliC or PBS immunized sera succumbed to A. baumannii sepsis within one day (100% mortality). Half of the mice injected with Omp22-immunized mouse sera survived after A. baumannii infection (Fig. 9C). Moreover, mice administered FliC-Omp22-immunized sera demonstrated the greatest reduction in bacterial burden compared to the other groups (P < 0.01; Fig. 9D). The bacterial load in the spleen of Omp22-immunized sera-treated mice was significantly lower than that in FliC- and PBS-immunized sera-treated mice (P < 0.05). No significant differences were observed among the FliC and PBS controls in spleen bacterial loads (P > 0.05).

The increasing prevalence of multi- or pan-resistant strains of A. baumannii, combined with their diverse virulence factors, poses significant challenges in healthcare, especially for sepsis caused by A. baumannii²². Consequently, there is an urgent need for alternative strategies such as vaccination. The combination of high immunogenicity, protective efficacy against clinical isolates, and involvement in pathogenesis makes Omp22 a promising antigen candidate for the development of effective subunit vaccines against A. baumannii infections^6,13. However, achieving effective immune protection against A. baumannii with the Omp22 antigen often necessitates its combination with adjuvants, conjugation to vaccine carriers, or utilization of advanced vaccine delivery platform ^6,13,14. The D3 hypervariable region of flagellin provides an effective platform for integrating foreign antigens, facilitating improved antigen presentation and TLR5-dependent activation of innate and adaptive immune responses^23–26. In the present study, we successfully showed that incorporation of Omp22 into the hypervariable D3 domain of flagellin improves the immunogenic response and protects against A. baumannii in a murine sepsis model. The observed enhancement can be ascribed to the multifunctional role of flagellin, acting both as a carrier and an adjuvant within the fusion FliC-Omp22 construct, which facilitates the efficient display and presentation of the Omp22 antigen to antigen presenting cells (APCs)^27,28. Furthermore, flagellin exerts its immunostimulatory properties by stimulating APCs and promoting a robust adaptive immune response against the incorporated Omp22 antigen, leading to increased protection against A. baumannii-induced sepsis in a murine model. The FliC-Omp22 vaccine at a concentration as low as 10 µg demonstrated remarkable survival rates among immunized mice upon infection with MDR A. baumannii strain 58ST. Despite the efficacy of the previous Omp22 vaccines, a potential limitation arises from the cytotoxicity observed in mammalian cells with recombinant Omp22 at a protective dose of 50 µg/mL against A. baumannii infections⁶. Flagellin fusion proteins appear to require a lower injection dose to elicit immune responses due to flagellin signaling via TLR5, which is expressed by several cell types, including epithelial cells, monocytes, and DCs, thereby enhancing the immune response to co-delivered antigens in vaccine formulations^29,30. In the FliC-Omp22 vaccine, not only is the cytotoxicity of Omp22 reduced by decreasing the injection dose but its immunogenicity is also enhanced owing to the adjuvant activity of flagellin, which interacts with immune cells, offering a promising strategy against A. baumannii infections²⁹. The immunoassay results provided evidence that the FliC-Omp22 fusion protein effectively presented the Omp22 antigen while preserving the adjuvant capabilities of flagellin. Notably, the fusion construct exhibited the anticipated molecular weight and antigenic characteristics of both constituents, thereby confirming its structural integrity as a potential vaccine candidate against A. baumannii^6,31. Flagellin-Omp22 vaccine demonstrated effectiveness in eliciting the generation of heightened levels of IL-4 in comparison to IFN-γ cytokines from splenocytes of immunized mice upon stimulation with Omp22, indicating a Th2-biased response^18,32,33. In addition, elevated IL-4 levels promote IgG isotype class switching to IgG1 antibodies, which is important for opsonization, neutralization, and protection against A. baumannii^34,35. Similarly, the substitution of the hypervariable domain of flagellin with the V and F1 antigens of Yersinia pestis led to potent antigen-specific humoral immune responses, conferring protection against lethal challenges in animal models. This was facilitated by the potent adjuvant activity of the flagellin molecule within the chimeric protein, which augmented the immunogenicity of F1 and V plague antigens²⁷. Substituting the hypervariable domain of flagellin with the HIV-1 p24 antigen elicits an IgA-biased antibody response and attenuates systemic inflammatory responses²⁸. Incorporating the HIV gp41 membrane-proximal external region (MPER) into FliC, functioning as both an adjuvant and scaffold, significantly boosted MPER-specific antibody responses in a TLR5-dependent manner³¹. Immunization with four tandem copies of conserved influenza HA2 integrated into the hypervariable region of flagellin elicited robust antibody responses, resulting in complete protection against various lethal challenges posed by influenza A viruses in a murine model³⁶. Substitution of the hypervariable region of flagellin with a viral envelope protein from the dengue virus induces both T cell-dependent and T cell-independent antibody responses²³. The integration of the influenza M2 protein into the flagellin molecule, observed to be safe and well-tolerated in a human clinical trial while enhancing the production of anti-M2e-specific antibodies, holds promise for the development of a universal influenza vaccine²⁵. Consequently, the FliC-Omp22 vaccine demonstrated effectiveness in eliciting the generation of elevated levels of IgG1/IgG2a and IL-4/IFN-γ ratios, which align with the recognized adjuvant properties of flagellin in promoting Th2-type immune responses^25,38,50,57. This is crucial for effective vaccine-induced protection of A. baumannii^10,37. Furthermore, the increased IFN-γ levels detected in the splenocytes of mice vaccinated with FliC-Omp22, compared to those vaccinated with Omp22 alone, which promotes IgG isotype class switching to IgG2a antibodies, implies the initiation of a Th1-type response. This response is correlated with macrophage activation, improved antigen presentation, and facilitation of bacterial clearance, thereby aiding in the establishment of enduring protective immunity against A. baumannii infection³⁸. In addition, the higher proliferation rate of FliC-Omp22 immune splenocyte cultures when stimulated with the Omp22 antigen indicates a robust antigen-specific amnestic proliferative response of FliC-Omp22 immune splenocytes, which can rapidly respond to subsequent exposure to A. baumannii, providing long-term protective immunity³⁹. Consistently, immunization of mice with chimeric malaria antigen fused with the hypervariable region of truncated flagellin, serving as a carrier-adjuvant, stimulated lymphocyte proliferation and facilitated the generation of memory T-cells²⁶. The cellular immune response elicited by the FliC-Omp22 vaccine, as evidenced by the upregulation of IFN-γ cytokine production and increased proliferation rate of immune splenocytes, synergistically complements the antibody response, facilitating enhanced opsonization and clearance of A. baumannii by phagocytes^35,40,41.

By assessing the in vitro protective efficacy of antibodies elicited by the vaccine, we observed that FliC-Omp22-immunized sera demonstrated potent inhibition of both adhesion and invasion, along with increased opsonic killing activity against A. baumannii. This highlights the potential of the FliC-Omp22 vaccine to induce antibodies that can bind to the Omp22 protein of A. baumannii, thereby inhibiting bacterial adhesion and invasion to host cells, facilitating opsonization, and ultimately preventing colonization and infection^37,42,43. This finding corresponds with the recognized adjuvant properties of flagellin, which promote Th2-type immune responses^{25,32,38,50,51}, leading to the production of antibodies capable of binding to the Omp22 and inhibits A. baumannii adhesion and invasion to host cells, facilitates opsonization, and ultimately prevents colonization and infection^37,42,43. The significant decrease in bacterial load observed in critical organs underscores the efficacy of FliC-Omp22 vaccination in inhibiting the ability of A. baumannii to establish systemic dissemination and infection^13,44. Passive immunization with sera from FliC-Omp22-immunized subjects provided significant protection against lethal A. baumannii sepsis, as demonstrated by elevated survival rates and reduced bacterial dissemination^6,45. This finding further emphasizes the adjuvant effect of flagellin in enhancing effective antibody responses targeting Omp22 when integrated into the fusion construct^18,32,46. The mechanism of protection provided by Flagellin-Omp22-immunized sera against lethal A. baumannii sepsis involves antibody-mediated opsonization and inhibition of bacterial adhesion and invasion to host cells^42,43,47. Active immunization with the FliC-Omp22 vaccine provides superior and enduring protection against A. baumannii compared with passive immunization. It stimulates prolonged antibody production and memory B cell formation, ensuring sustained protection^24,48. Moreover, active immunization triggers cell- and humoral-mediated immune responses, providing a comprehensive defense mechanism^47,49. In contrast, passive immunization offers only a temporary antibody boost without inducing cellular immunity, resulting in less durable protection^40,50. These findings underscore the potential of active immunization with FliC-Omp22 as a promising strategy to reduce the morbidity and mortality associated with A. baumannii infection.

In conclusion, integrating the Omp22 protein into the hypervariable D3 domain of flagellin holds great promise for enhancing the efficacy of an A. baumannii vaccine, which presents multiple advantages. First, the D3 domain of flagellin serves as an optimal site for antigen integration, facilitating proper presentation of Omp22 to APCs. Consequently, the FliC-Omp22 fusion protein can elicit robust Th2 immune responses against Omp22, characterized by increased IgG1/IgG2a and IL-4/IFN-γ levels. Moreover, vaccination with FliC-Omp22 immunization demonstrated a notable capacity to hinder the adhesion and invasion of A. baumannii, concurrently augmenting opsonic killing activity against the pathogen. This synergistic effect has the potential to hinder the establishment and spread of infection, resulting in diminished bacterial loads in vital organs and heightened survival rates of MDR A. baumannii-infected mice. These observations highlight the potential of the vaccine in mitigating A. baumannii infections, particularly in the face of increasing antibiotic resistance. These findings necessitate a comprehensive assessment of the efficacy of the FliC-Omp22 vaccine against a wide range of A. baumannii isolates. In addition, our data suggest that the FliC-Omp22 vaccine can be applied to other models of A. baumannii infection. These results underscore the potential of the FliC-Omp22 vaccine to provide long-term protection against A. baumannii infection, thus reducing the substantial morbidity and mortality associated with sepsis globally.

Bacterial strains

Acinetobacter baumannii ATCC19606 and Salmonella enterica serovar Typhimurium PTCC1735 (S. Typhimurium) were used to produce recombinant Omp22 and flagellin (FliC) recombinant proteins, respectively. Molecular cloning and expression of the recombinant proteins were performed using Escherichia coli strains DH5α and BL21 (DE3), respectively. Escherichia coli strains, A. baumannii ATCC19606 and a previously characterized multidrug-resistant (MDR) hospital strain of A. baumannii strain 58ST³⁷, were obtained from the Department of Biology, Shahed University (Tehran, Iran). Salmonella enterica serovar Typhimurium was kindly provided by the Bacteriology Department of the Pasteur Institute of Iran.

Ethics statement

We confirm that this study is conducted following ARRIVE guidelines. The principles stated in the Guide for the Care and Use of Laboratory Animals were followed. All protocols involving animal and human cell experiments were approved by the Research Ethics Committees of Shahed University (approval number: R.SHAHED.REC.1400.197). In summary, female 6–8 weeks BALB/C mice (Pasteur Institute of Iran) were allocated to experimental and control groups, each consisting of three to five mice for serum antibody titration, in vitro lymphocyte proliferation and cytokine assays, and bacterial load assays. Survival studies of infected mice included 8 mice per group. The number of mice assigned to each group was determined using power analysis, set at 80% power, with a significance level of P < 0.05. Polymorphonuclear leukocytes (PMNs) were isolated from heparinized venous blood with the consent of healthy laboratory personnel and purified using the Ficoll-Hypaque density gradient method⁵¹. A549 human non-small cell lung cancer and RAW 264.7 murine macrophage cell lines were obtained from the Iranian Biological Resource Center (Tehran, Iran).

Bioinformatics analysis of FliC-Omp22 chimeric protein

The amino acid sequences of FliC (GenBank Accession No: AAL20871.1) and Omp22 proteins (GenBank Accession No: ABO11316.1) were retrieved from the National Center for Biotechnology Information (NCBI). Following the removal of the predicted signal peptide region using SignalP 5.1 (https://services.healthtech.dtu.dk/services/SignalP-5.0/), computational tools were employed to analyze the FliC-Omp22 chimeric protein. ProtParam (http://web.expasy.org/protparam/) and IEDB tools (http://tools.iedb.org/bcell/) were used to determine the physicochemical properties of the chimeric protein, as summarized in (Supplementary Table S1). SYMPRED (https://www.ibi.vu.nl/programs/sympredwww/), PSIPRED 4.0 (http://bioinf.cs.ucl.ac.uk/psipred), and SSPro (https://download.igb.uci.edu/sspro4.html) servers were used to predict the α-helix, β-sheet, and coil structures within the FliC-Omp22 chimeric protein (Supplementary Fig. S3). BepiPred-1.0 (http://tools.iedb.org/bcell/), BepiPred-2.0 (https://services.healthtech.dtu.dk/services/BepiPred-2.0/) ABCpred (http://webs.iiitd.edu.in/raghava/abcpred/), LBtope (http://crdd.osdd.net/raghava/lbtope/), BcePred (http://crdd.osdd.net/raghava/bcepred/), and BCPred (http://ailab.cs.iastate.edu/bcpreds/) were used to predict potential linear B-cell epitopes within the chimeric protein sequence. The Robetta online server (http://robetta.bakerlab.org/) was used to generate the 3D structure of FliC-Omp22 chimeric protein. The predicted structure was refined and validated using the QMEAN tool (https://swissmodel.expasy.org/qmean/). ElliPro (http://tools.iedb.org/ellipro/) was used to identify both linear and discontinuous epitopes on the surface of the FliC-Omp22 chimeric protein, based on its predicted 3D structure (Supplementary Fig. S4). The VaxiJen server (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html/) was used to assess antigenicity of the FliC-Omp22 chimeric protein.

Construction of Omp22, FliC, FliC-Omp22 recombinant plasmids

Specific primers (Supplementary Table S2) were designed for the fliC and omp22 genes using the genomic DNA of S. Typhimurium LT2 and A. baumannii ATCC17978 as templates, respectively. Pfu DNA polymerase-amplified omp22 and fliC amplicons were purified, digested, and ligated into the NdeI-BamHI and NcoI-HindIII (Thermo Scientific, UK) sites of the pET-28a vector (Novagen, USA), respectively. Overlap extension PCR was used to construct the FliC-Omp22 fusion fragment. Briefly, full-length fliC was amplified from the verified pET-28a/fliC vector in two fragments to remove the D3 hypervariable region for insertion of the omp22 gene. The first fragment (F1) was amplified from the first 184 amino acid residues of FliC using a forward primer with a flanking 5' NcoI site and a reverse primer with a 5' sequence overlapping the 5' sequence of the omp22 gene (Supplementary Table S2). The second fragment (F2) was amplified from residues 285–494 of FliC using a forward primer with a 3' sequence overlapping the 3' end of the omp22 gene and a restriction site for HindIII at the 5' end (Supplementary Table S2). Then, F1, omp22, and F2 fragments were overlapped using the conventional splicing by overlap extension (SOEing) PCR method⁵². Briefly, fragments were combined in equimolar amounts and subjected to PCR with 10 cycles of the program (1 min at 95°C, 45 sec at 50°C, and 2 min at 72°C) for overlap extension without primers, which produced the full-length FliC-Omp22 DNA, and then external primers were added to the mixture, and PCR was performed (50°C for 1 min, 30 cycles of 95°C for 30 sec, 50°C for 45 sec, and 72°C for 5 min). The purified chimeric DNA fragment was subsequently ligated into the NcoI-HindIII sites of the pET-28a vector. Recombinant plasmids were verified by DNA sequencing.

Preparation of recombinant proteins

Recombinant plasmids were transformed into E. coli BL21 (DE3) cells, followed by induction with IPTG (Sigma-Aldrich, USA) at 37°C for 4 h. Subsequently, bacterial cultures were harvested, treated with 20 mM Tris-HCl (pH 8.0), sonicated on ice, and centrifuged. The resulting pellet, containing inclusion bodies, underwent resuspension in cold buffer solutions A (2 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton™ X-100; pH 8.0) and B (20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride, 1 mM 2-mercaptoethanol; pH 8.0), sonication, and centrifugation steps. The sample was then mixed with Ni-NTA agarose in buffer B, applied to a column, and washed with buffer solutions B and D (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol, 6 M urea; pH 8.0). Urea removal was achieved through a stepwise decrease in concentration using buffer D. Finally, the recombinant proteins were eluted using buffer E (20 mM Tris-HCl, 0.5 M NaCl, 250 mM imidazole, 1 mM 2-mercaptoethanol; pH 8.0) and subsequently dialyzed against phosphate-buffered saline (PBS) pH 7.4 overnight. SDS-PAGE and western blotting with an anti-His tag antibody assessed the identification and correctness of the recombinant proteins⁵¹. The PYROGENT™ Plus Gel Clot LAL Assay (LONZA, USA) was used to analyze the endotoxin levels of each protein, which were found to be below 10 EU/mL.

In vitro assessment of TLR5-specific signaling

The examination of TLR5-specific signaling triggered by FliC-Omp22 was carried out through an in vitro bioassay employing two distinct cell lines: RAW 264.7 cells lacking TLR5 transfection exhibited negligible tumor necrosis factor alpha (TNF-α) synthesis upon flagellin stimulation, whereas RAW 264.7 cells transfected with TLR5 exhibited notable responsiveness to flagellin-induced TNF-α synthesis. RAW 264.7 cells were cultured in 24-well plates with RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and then treated with different concentrations of either FliC-Omp22 or FliC for 4 h. Subsequently, the culture supernatant from each well was collected and subjected to TNF-α quantification using a specific ELISA assay (PeproTech, USA).

Mouse immunizations and bacterial challenges

BALB/c mice were subcutaneously administered 10 µg of Omp22, FliC, or FliC-Omp22 recombinant proteins at various sites on days 0, 14, and 28. Control mice were injected with PBS. Serum samples were obtained from orbital sinus blood to analyze antigen-specific antibodies. Infection challenge experiments were carried out using A. baumannii 58ST, as described previously⁶. On day 56, the mice were intraperitoneally (i.p.) challenged with a lethal dose (LD) of A. baumannii 58ST (1.9 × 10⁷ CFU) with 10% porcine mucin (w/v; Sigma-Aldrich). Sixty hours post-infection, the spleen, lung, and liver were collected, weighed, homogenized, and cultured for bacterial load quantification. Mice were observed and monitored for seven days to assess their survival rates.

For passive immunization, BALB/c mice were intravenously administered pooled sera from immunized and non-immunized mice (100 µL) and then subjected to A. baumannii strain 58ST challenge described above. Sixty hours post-infection, spleen samples were collected and cultured on LB plates to assess the bacterial loads. Daily monitoring of survival rates continued for one week.

For in vivo experiments, mice were anesthetized using a combination of Ketamine (80mg/kg) and Xylazine (10 mg/kg). Euthanasia was performed by exposing the mice to carbon dioxide (CO₂) inhalation from a pressurized tank.

ELISA and western blot assays of FliC-Omp22 fusion protein

ELISA and western blot assays were performed to confirm the identity, integrity, and binding capacity of the FliC-Omp22 fusion protein to anti-OmpA or anti-FliC-specific antibodies, as described previously^21,23. Different concentrations of FliC-Omp22 recombinant protein were coated onto 96-well ELISA plates (Nunc, USA) overnight at 4°C in carbonate-bicarbonate buffer. The plates were washed with PBS containing 0.5% Tween-20 (PBS-T) and blocked with PBS containing 3% bovine serum albumin (BSA, Sigma-Aldrich) for 2 h at room temperature (RT). Next, anti-OmpA- or anti-FliC-specific antibodies were added and incubated for 2 h at RT. Subsequently, the HRP-conjugated goat anti-mouse antibody (Sigma-Aldrich) was added and incubated for 1 h at RT. After the addition of TMB substrate (Invitrogen, USA) and subsequent termination of the enzymatic color reaction with 2 N H₂SO₄, the absorbance at 450 nm was measured.

Additionally, an immunoblotting assay was used to assess the specificity and reactivity of anti-Omp22 or anti-FliC-specific antibodies. Recombinant Omp22, FliC, and FliC-Omp22 proteins were electrophoresed and then transferred onto PVDF membranes (Amersham, USA). Subsequently, the PVDF membranes were subsequently incubated with anti-Omp22 or anti-FliC-specific antibodies, followed by HRP-conjugated goat anti-mouse antibodies. Following the addition of 3,3'-diaminobenzidine substrate (DAB, Sigma-Aldrich), the color-developed blots were recorded using a digital camera.

Evaluation of antigen-specific antibody responses

To evaluate humoral responses, we measured IgG titers against whole-cell A. baumannii, Omp22, FliC, and FliC-Omp22 antigens, as described previously⁵¹. The ELISA plates were coated with antigens, washed with PBS-T, and blocked with PBS containing 3% BSA. Serially diluted mouse sera were then added, washed with PBS-T, and incubated with HRP-conjugated goat anti-mouse IgG. The absorbance at 450 nm was measured after the addition of the TMB substrate and termination of the enzymatic color reaction. Serum IgG1 and IgG2a subtypes were determined as described above using rabbit primary anti-mouse IgG1 or IgG2a antibodies (BioLegend, USA) followed by HRP-conjugated goat secondary anti-rabbit IgG antibody (Sigma-Aldrich). The endpoint titer was determined as the highest dilution at which the optical density at OD₄₅₀ exceeded that of the background wells by at least 0.1. The background wells contained PBS instead of the serum samples⁵³.

Lymphocyte proliferation and cytokine assays

Two weeks after the last immunization, splenocytes from experimental mice were removed, homogenized, suspended in cold Hanks' balanced salt solution (HBSS, Sigma), and subsequently treated with RBC lysis buffer (BioLegend) to remove red blood cells. Splenocytes (1×10⁶ cells/mL) were resuspended in RPMI 1640 supplemented with 5% fetal bovine serum (FBS) (both from Gibco) in 96-well or 24-well plates (Nunc) and incubated with Omp22, FliC, or FliC-Omp22 antigens for 72 h. Phytohemagglutinin-A (PHA, Gibco) and unstimulated cells in RPMI 1640 were used as the positive and negative control groups, respectively. Subsequently, the culture supernatant from each well was collected and analyzed for interferon-γ (IFN-γ) and interleukin 4 (IL-4) levels using a specific ELISA assay (PeproTech). MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay was performed to assess the proliferation of mouse splenocytes stimulated with antigens, as described previously⁵⁴. MTT solution was added to each well and incubated at 37°C for 4 h. Dimethyl sulfoxide (DMSO, Sigma-Aldrich) was then added to each well to dissolve the purple formazan crystals. Absorbance was measured at 550 nm, and the stimulation index (SI) was calculated by dividing the average absorbance of stimulated cells by the average absorbance of unstimulated cells (negative control).

Adhesion and invasion assays

The effectiveness of FliC-Omp22-immunized sera in inhibiting the adhesion and invasion of A. baumannii 58ST on the A549 cell line was assessed, as described previously^37,55. Briefly, A. baumannii 58ST was exposed to different dilutions of heat-inactivated serum from experimental mice for 1 h at 37°C. A549 cells were infected at a multiplicity of infection (MOI) of 100 for 2 h at 37°C and subsequently lysed with PBS containing 0.1% Triton X-100 to evaluate bacterial adhesion. To determine bacterial invasion, infected A549 cells were treated with gentamicin (250 µg/mL) to eradicate extracellular bacteria and dissolved in PBS with 0.1% Triton X-100. Diluted samples of adhesive and invasive lysates were cultured on LB agar plates for bacterial quantification.

Opsonophagocytic assays

Acinetobacter baumannii 58ST (10⁶ CFU) was treated with different dilutions of heat-inactivated mouse serum and infant rabbit serum for 30 min. Afterward, the suspension was added to PMNs at a concentration of 10⁵ cells/mL and incubated for 90 min, followed by serial dilution and culture on LB agar plates for bacterial quantification^42,46. Opsonic activity was calculated using the following formula:

Opsonophagocytic activity (%) = [1 − (bacterial CFUs of immune serum at 90 min/bacterial CFUs of pre-immune serum at 90 min)] × 100.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software Inc., USA). Data were analyzed using a two-way analysis of variance with Tukey’s multiple comparison test. Survival data were analyzed using the Mantel-Cox log-rank test. Results are expressed as the mean ± Standard Deviation (SD), and a p-value of < 0.05 was considered statistically significant.

Competing interests

The authors declare no competing interests.

Author Contribution

I.R. and F.B. conceived, planned, and supervised the project. B.B. carried out the experiments and wrote the first draft of the manuscript. All authors were involved in the preparation of the manuscript.

Acknowledgments

The authors would like to thank Shahed University for supporting the conduct of this research.

Data Availability

No data were generated. All data supporting the findings of this study are available in the paper and its Supplementary Information section.

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Inserting Omp22 into the flagellin protein, replacing its hypervariable region, results in stronger protection against sepsis caused by Acinetobacter baumannii in a mouse model

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Version 1

Abstract

Figures

Introduction

Results

Design and characterization of FliC-Omp22 protein

Antigenicity, structural integrity, and bioactivity of FliC-Omp22 fusion protein

FliC-Omp22 immunization induced robust serum IgG, IgG1, and IgG2a levels

FliC-Omp22 immunization enhanced splenocyte proliferation and cytokine production

FliC-Omp22 immunization increased survival and reduced bacterial load of infected mice

Discussion

Materials and methods

Bacterial strains

Ethics statement

Bioinformatics analysis of FliC-Omp22 chimeric protein

Construction of Omp22, FliC, FliC-Omp22 recombinant plasmids

Preparation of recombinant proteins

Mouse immunizations and bacterial challenges

ELISA and western blot assays of FliC-Omp22 fusion protein

Evaluation of antigen-specific antibody responses

Lymphocyte proliferation and cytokine assays

Adhesion and invasion assays

Opsonophagocytic assays

Statistical analysis

Declarations

Competing interests

Author Contribution

Acknowledgments

Data Availability

References

Additional Declarations

Supplementary Files

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