Using an Aluminum Hydroxide–Chitosan Matrix Increased the Vaccine Potential and Immune Response of Mice against Multi-Drug-Resistant Acinetobacter baumannii

Acinetobacter baumannii is a Gram-negative, immobile, aerobic nosocomial opportunistic coccobacillus that causes pneumonia, septicemia, and urinary tract infections in immunosuppressed patients. There are no commercially available alternative antimicrobials, and multi-drug resistance is an urgent concern that requires emergency measures and new therapeutic strategies. This study evaluated a multi-drug-resistant A. baumannii whole-cell vaccine, inactivated and adsorbed on an aluminum hydroxide–chitosan (mAhC) matrix, in an A. baumannii sepsis model in immunosuppressed mice by cyclophosphamide (CY). CY-treated mice were divided into immunized, non-immunized, and adjuvant-inoculated groups. Three vaccine doses were given at 0D, 14D, and 28D, followed by a lethal dose of 4.0 × 108 CFU/mL of A. baumannii. Immunized CY-treated mice underwent a significant humoral response, with the highest IgG levels and a higher survival rate (85%); this differed from the non-immunized CY-treated mice, none of whom survived (p < 0.001), and from the adjuvant group, with 45% survival (p < 0.05). Histological data revealed the evident expansion of white spleen pulp from immunized CY-treated mice, whereas, in non-immunized and adjuvanted CY-treated mice, there was more significant organ tissue damage. Our results confirmed the proof-of-concept of the immune response and vaccine protection in a sepsis model in CY-treated mice, contributing to the advancement of new alternatives for protection against A. baumannii infections.


mAhC and Vaccine Formulation
The mAhC was prepared with prior chitosan solubilization using chitosan powder (Polymar, 81% deacetylation degree) in 0.8% (v/v) acetic acid, 0.9% (w/v) saline solution, with a ratio of chitosan and aluminum hydroxide of 2:1, respectively. Groups of four animals were employed to assess the number of mAhC doses. Female Swiss mice were inoculated intramuscularly with 100 µL of the mAhC (without A. baumannii antigen) in a single shot, at time zero (0 D), with double doses at times (0 D, 14 D) and triple doses at 0 D, 14 D, and 28 D. After the last inoculation, the animals were challenged with a lethal dose of intraperitoneally administered A. baumannii (500 µL), and were then monitored for clinical manifestations; then, the Kaplan-Meier survival curve was determined. The vaccine formulation consisted of a cell concentration of A. baumannii of 1 × 10 9 cells.mL −1 at 1:1 (v/v) with chitosan matrix (1.25%, w/w) associated with aluminum hydroxide (1.6 mg) (Dinâmica ® , Belo Horizonte, Brazil) [29].

mAhC and Molecular Docking Calculations
The ligand selected for the molecular docking study was chitosan, and its 3D structures were built in neutral form using Marvin Sketch 18.10, ChemAxon (http://www. chemaxon.com, accessed on 31 January 2023). Acinetobacter baumannii lipopolysaccharide (LPS) amino acid sequences were obtained from the UniProt server database (http: //uniprot.org, accessed on 31 January 2023). The 3D structures of both proteins were constructed using the homology modeling approach with The Swiss Model Workspace (https://swissmodel.expasy.org/, accessed on 31 January 2023) after selecting their respective models using the BLASTp tool. The models were downloaded from The Protein Databank (https://www.rcsb.org/, accessed on 31 January 2023), considering quality parameters such as the experimental method, resolution, and R-value, and their complexation with a ligand. We used the Swiss model to verify protein structure breaks and amino acid positioning in the active site [35]. The generated models were validated by inspecting the Ramachandran plots [36,37], in which it was possible to analyze the distribution of the skeletal torsion angles ϕ and ψ responsible for the stereochemical quality of the protein studied, as well as the QMEAN factor [38]. Targets and ligands were prepared for the molecular docking process using Autodock Tools 1.5.7 [39], according to the methodology proposed by Moura et al. (2020) [40]. Using the AutoDock Vina [41] in the docking calculations, nine docking positions were generated for ligands interacting with the targets, returning affinity energy values (kcal/mol). The docking position results were analyzed using PyMOL 2.0 [42] and Discovery Studio 4.5 [43] to select the best position for each ligand inside the protein target using the parameters proposed by Moura et al. (2020) [40].

Ethical/Legal Requirements and Use of Animals in Experiments
This study used female Swiss mice aged 6 to 8 weeks weighing 17-23 g. They were acquired from the Central Animal Facility of the Federal University of Goiás and placed in the Laboratory of Biomolecules and Vaccines (LaBVac) pre-clinical testing room. The mice were housed in a pathogen-free primary enclosure with free access to food and water; the enclosure was maintained at 24 • C, with odor and light control with a light and dark cycle every 12 h. All animals in this experiment were previously dewormed with access to a 1:20 Ivermectin ® solution for seven days. The experiments were conducted following ethical recommendations established by the law of procedures for the scientific use of animals,  Every effort was made to avoid suffering or undue  pain; the mice were monitored for weight loss and gain and clinical manifestations such as lethargy, hypothermia, and difficulty breathing. The animals were euthanized using ketamine (300 mg/kg) (Vetbrands ® , Rio de Janeiro, Brazil) and xylazine hydrochloride (22.5 mg/kg) (Syntec ® , Piracicaba, Brazil) [17][18][19].

Experimental Design
Mice were given 3 intraperitoneal doses of CY (Genuxal ® Baxter, São Paulo, Brazil) (150 mg/Kg) for 7 days at 36 h intervals [29]. Blood samples were collected for hematologic analyses at the Pet Shop Dog Veterinary Clinic Center (Gurupi/TO-Brazil). Once the immunosuppression picture was established, 10 immunosuppressed Swiss mice were used to administer 100 µL of the vaccine composition in 3 doses at 0D, 14D, and 28D. Other groups of 10 immunosuppressed mice were inoculated with 100 µL of adjuvant solution (mAhC group) and phosphate-buffered saline (PBS) (non-immunized group) at the corresponding times of 0D, 14D, 28D. Then, 7 days after the final immunization (35D), mice were challenged with 500 µL of a previously defined and intraperitoneally administered lethal dose of A. baumannii [31]. The animals were monitored for clinical aspects, and biological samples were collected.

Enzyme-Linked Immunosorbent Assay Using A. baumannii Cultures
Blood samples from immunosuppressed mice after the last immunization (28D) and after the challenge test (35D) were evaluated to stimulate total IgG production. The ELISA was applied using A. baumannii culture, which used 10 mL of TSB for A. baumannii, under stirring at 120 rpm at 37 • C, for 8 h (pre-inoculum). Subsequently, the bacterial suspension was washed in saline solution, centrifuged to remove all the culture broth, and used for standardization and antigen fixation [33]. Cell concentration of 10 6 CFU/mL was used for overnight sensitivity of 96-well microplates (Nunc MaxiSorp during) at 4 • C. After blocking for 1 h with 2% casein-PBS buffer (blocking buffer) at room temperature, the plates were washed 4 times with 0.05% Tween-20-PBS buffer. Serum was diluted at 1:50 with blocking buffer and incubated for 16 h at 4 • C. After washing, anti-mouse IgG peroxidase conjugate (Sigma, St. Louis, MO, USA) was used at a ratio of 1:2000 in the blocking buffer. After washing, 3,3 ,5,5 -tetramethylbenzidine (BD Biosciences, San Diego, CA, USA) was added for 15 min and blocked with 2.5 M H 2 SO 4 (Dinâmica ® , Belo Horizonte, Brazil). Optical density was measured at 450 nm, and values above the cutoff were considered positive. The mean optical density of serum determined the cutoff point before immunization [17].

Histopathological Analysis
After finishing the lethal dose analysis test, the histopathological analysis of organs was performed by collecting the liver, lung, and spleen from three non-immunized, mAhCtreated, and immunized animals. The organs were weighed and fixed in a 10% formalinbuffered solution. Subsequently, they were dehydrated in alcohol (Qhemis ® , São Paulo, Brazil), diaphanized in xylene (Synth ® , Porto Alegre, Brazil), and embedded in histological paraffin (Synth ® , Porto Alegre, Brazil). Then, 5-micron sections were made with a microtome, stained in hematoxylin/eosin, and examined under a Leica DM1000 LED optical microscope (Software Application Suite version 4.9.0) with 10, 40, and 100× objectives.
Histological slides of the liver, lung, and spleen were analyzed for tissue damage caused by A. baumannii infection, evaluating the presence of edema, degeneration, necrosis, acute inflammation, and hyperemia. Histopathological damage was classified using the lesion score considering the degree of alteration (if absent) when there was no evidence of damage, discrete if it was evident in up to 25% of the field of observation, moderate if greater than 25% but less than 50%, or severe if greater than 50% [17]. As previously described, expansion analyses of white pulp from the spleens of immunosuppressed mice were performed [44]. Histological sections of the spleen were analyzed by capturing images using a Leica Application Suite 4.9.0 photomicroscope. Histological images of white pulp were measured in 10 fields using a 10× objective and ImageJ ® 1.47v-2012 software (Bethesda, MD, USA) [44].

Statistical Analysis
Statistical analysis was performed using the GraphPad Prism software GraphPad Software Inc. San Diego, CA, USA. The Kruskal-Wallis analysis of variance test was used to compare non-immunized, mAhC-treated, and immunized animals (regarding immunization efficiency). The following parameters were compared: the number of surviving animals according to the Kaplan-Meier survival curves was used with the logrank test (Mantel-Cox test) and those with symptoms, white blood cell count, and IgG production. Histopathological damage and white pulp expansion were used to compare non-immunized versus adjuvant-treated and non-immunized versus immunized animals. The significance level was p < 0.05, and we included the 95% confidence interval. The Student's t-test was used for comparisons of means of experimental parameters.

Sepsis Model of Infection Using A. baumannii in CY-Untreated Mice
The survival curves of mice after inoculation with an active culture of A. baumannii revealed percentages corresponding to 100%, 50%, and 0% when active suspensions were inoculated intraperitoneally at 10 −3 (OD 600 0.02), 10 −2 (OD 600 0.15), and 10 −1 (OD 600 1.24), respectively, p < 0.01 ( Figure 1A). After that, mice infections were performed using A. baumannii culture at 10 9 -10 7 CFU/mL. After the first 24 h, A. baumannii-infected (4.0 × 10 8 CFU/mL) mice showed 75% lethality, and this dose was used for the evaluations of vaccine efficacy ( Figure 1B), p < 0.001. sis, acute inflammation, and hyperemia. Histopathological damage was classified using the lesion score considering the degree of alteration (if absent) when there was no evidence of damage, discrete if it was evident in up to 25% of the field of observation, moderate if greater than 25% but less than 50%, or severe if greater than 50% [17].
As previously described, expansion analyses of white pulp from the spleens of immunosuppressed mice were performed [44]. Histological sections of the spleen were analyzed by capturing images using a Leica Application Suite 4.9.0 photomicroscope. Histological images of white pulp were measured in 10 fields using a 10× objective and ImageJ ® 1.47v-2012 software [44].

Statistical Analysis
Statistical analysis was performed using the GraphPad Prism software GraphPad Software Inc. San Diego, CA, USA. The Kruskal-Wallis analysis of variance test was used to compare non-immunized, mAhC-treated, and immunized animals (regarding immunization efficiency). The following parameters were compared: the number of surviving animals according to the Kaplan-Meier survival curves was used with the log-rank test (Mantel-Cox test) and those with symptoms, white blood cell count, and IgG production. Histopathological damage and white pulp expansion were used to compare non-immunized versus adjuvant-treated and non-immunized versus immunized animals. The significance level was p < 0.05, and we included the 95% confidence interval. The Student's ttest was used for comparisons of means of experimental parameters.

Determination of mAhC Dose in CY-Untreated Mice
Mice (CY-untreated mice) inoculated with one or more doses of mAhC showed bodyweight gain ( Figure 2A). However, after inoculation with the lethal dose of A. baumannii Vaccines 2023, 11, 669 6 of 15 (4.0 × 10 8 CFU/mL), differences in survival percentages were observed, with the highest survival rate obtained when a higher number of doses was applied ( Figure 2B). No survivors were observed after an A. baumannii challenge test in CY-untreated mice that received a single dose of mAhC or were administered only 1.6 mg/mL aluminum hydroxide solution. CY-untreated mice inoculated with 2 or 3 doses showed a survival rate of 45% (p < 0.01), suggesting the potential stimulation and protection of mAhC ( Figure 2B).

Determination of mAhC Dose in CY-Untreated Mice
Mice (CY-untreated mice) inoculated with one or more doses of mAhC showed bodyweight gain (Figure 2A). However, after inoculation with the lethal dose of A. baumannii (4.0 × 10 8 CFU/mL), differences in survival percentages were observed, with the highest survival rate obtained when a higher number of doses was applied ( Figure 2B). No survivors were observed after an A. baumannii challenge test in CY-untreated mice that received a single dose of mAhC or were administered only 1.6 mg/mL aluminum hydroxide solution. CY-untreated mice inoculated with 2 or 3 doses showed a survival rate of 45% (p < 0.01), suggesting the potential stimulation and protection of mAhC ( Figure 2B).

Molecular Docking from MAhC and Bacterial Lipopolysaccharide
The selected templates for homology modeling are shown, highlighting the identities and the validation results with the corresponding Ramachandran favored values and QMEAN. (Table 1). The chitosan complexed with the receptor and formed various interactions with varying affinity energies, as indicated by the docking assays ( Table 2). The chitosan ligand showed better affinity energy with target Acinetobacter baumannii at −8.4 kcal/mol. The complex formed between the chitosan ligand and the LPS target receptor of the organism Acinetobacter baumannii presents a strong interaction with the receptor's amino acids ( Figure 3A). This complex showed interactions between the target and ligand of the type: van der Waals, conventional hydrogen bond, carbon-hydrogen bond and pidonor hydrogen bond ( Figure 3B).

Molecular Docking from MAhC and Bacterial Lipopolysaccharide
The selected templates for homology modeling are shown, highlighting the identities and the validation results with the corresponding Ramachandran favored values and QMEAN. ( Table 1). The chitosan complexed with the receptor and formed various interactions with varying affinity energies, as indicated by the docking assays ( Table 2). The chitosan ligand showed better affinity energy with target Acinetobacter baumannii at −8.4 kcal/mol. The complex formed between the chitosan ligand and the LPS target receptor of the organism Acinetobacter baumannii presents a strong interaction with the receptor's amino acids ( Figure 3A). This complex showed interactions between the target and ligand of the type: van der Waals, conventional hydrogen bond, carbon-hydrogen bond and pi-donor hydrogen bond ( Figure 3B).

Immunosuppressive Model
We established an immunosuppressive model in Swiss mice using doses of CY, an agent with immunosuppressive properties that causes a decrease in hematologic parameters in the blood and leukopenia. CY promoted a progressive change in the clinical picture after three doses, causing ruffled hair, hair loss, and weight loss (p < 0.05) ( Figure 4A). Furthermore, about 70% of the CY-treated mice survived and experienced a gradual recovery in body-weight gain ( Figure 4B). In addition, these animals showed an exacerbated reduction in the number of total leukocytes, 79% (p < 0.001) ( Figure 4C), neutrophils (p <

Immunosuppressive Model
We established an immunosuppressive model in Swiss mice using doses of CY, an agent with immunosuppressive properties that causes a decrease in hematologic parameters in the blood and leukopenia. CY promoted a progressive change in the clinical picture after three doses, causing ruffled hair, hair loss, and weight loss (p < 0.05) ( Figure 4A). Furthermore, about 70% of the CY-treated mice survived and experienced a gradual recovery in body-weight gain ( Figure 4B). In addition, these animals showed an exacerbated reduction in the number of total leukocytes, 79% (p < 0.001) ( Figure 4C), neutrophils (p < 0.01) ( Figure 4D), lymphocytes (p < 0.001) ( Figure 4E), and monocytes (p < 0.05) ( Figure 4F), compared to the group of non-treated mice. Erythrocytes reduction was also evidenced with a decline of 51% compared to the group of CY-treated mice (p < 0.01) ( Figure 4G) and a drop in hemoglobin concentration (p < 0.05) ( Figure 4H) and hematocrit (p < 0.05) ( Figure 4I). 0.01) ( Figure 4D), lymphocytes (p < 0.001) ( Figure 4E), and monocytes (p < 0.05) ( Figure  4F), compared to the group of non-treated mice. Erythrocytes reduction was also evidenced with a decline of 51% compared to the group of CY-treated mice (p < 0.01) ( Figure  4G) and a drop in hemoglobin concentration (p < 0.05) ( Figure 4H) and hematocrit (p < 0.05) ( Figure 4I).

Protective Effect of A. baumannii Whole-Cell Vaccine Inactivated and Adsorbed on mAhC in CY-Treated Mice
Analysis of the post-immunization humoral response revealed greater stimulation of IgG levels in CY-treated mice inoculated with mAhC alone (adjuvant group) compared to non-immunized CY-treated mice (p < 0.05) ( Figure 5). However, CY-treated mice immunized with inactivated A. baumannii whole-cell vaccine adsorbed with mAhC had IgG levels significantly higher than the levels obtained from CY-treated mice in the adjuvant group (p < 0.05), suggesting a greater capacity to stimulate the immune response against A. baumannii ( Figure 5).
The study of vaccine efficacy after applying the lethal dose of active A. baumannii (4 × 10 8 CFU/mL) revealed a lethality rate of 100%, corresponding to non-immunized and

Protective Effect of A. baumannii Whole-Cell Vaccine Inactivated and Adsorbed on mAhC in CY-Treated Mice
Analysis of the post-immunization humoral response revealed greater stimulation of IgG levels in CY-treated mice inoculated with mAhC alone (adjuvant group) compared to non-immunized CY-treated mice (p < 0.05) ( Figure 5). However, CY-treated mice immunized with inactivated A. baumannii whole-cell vaccine adsorbed with mAhC had IgG levels significantly higher than the levels obtained from CY-treated mice in the adjuvant group (p < 0.05), suggesting a greater capacity to stimulate the immune response against A. baumannii ( Figure 5).
The study of vaccine efficacy after applying the lethal dose of active A. baumannii (4 × 10 8 CFU/mL) revealed a lethality rate of 100%, corresponding to non-immunized and CY-treated mice (control group) (p < 0.001). In contrast, in immunized CY-treated mice, the survival rate was 85%, a value that differed from the survival percentages of CY-treated mice in the mAhC group (adjuvant) (45%) (p < 0.05) ( Figure 6A). In addition, immunized CY-treated mice showed higher levels of IgG compared to the control and adjuvant groups ( Figure 6B) (p < 0.05), as well as a more significant expansion of white pulp ( Figure 6C,D). CY-treated mice (control group) (p < 0.001). In contrast, in immunized CY-treated mice, the survival rate was 85%, a value that differed from the survival percentages of CYtreated mice in the mAhC group (adjuvant) (45%) (p < 0.05) ( Figure 6A). In addition, immunized CY-treated mice showed higher levels of IgG compared to the control and adjuvant groups ( Figure 6B) (p < 0.05), as well as a more significant expansion of white pulp ( Figure 6C,D).

Histopathological Analysis of the Sepsis Model of A. baumannii Infection in CY-Treated Mice
The sepsis model of A. baumannii infection showed tissue damage in the liver, spleens, and lungs in CY-treated mice, the non-immunized mice, and the mAhC ( Figure  7). By comparison, there were no significant differences between the liver and lung weights of these groups (Figure 7 A,B). In terms of the weight of the spleens of immunized CY-treated mice, there were significant differences from the non-immunized CY-treated or adjuvant groups (p < 0.05) ( Figure 7C). In addition, more substantial expansion of the white pulp was observed in this group (p < 0.01) and the adjuvant group (p < 0.05) ( Figure  7D). The expansion of the white pulp is related to the presence of periarteriolar and follicular hyperplasia caused by the antigenic stimulus and immune response (Figures 6D and  7D). The histopathological findings revealed greater severity in the liver with hydropic degeneration and decreased sinusoids, and interstitial pneumonia with a mixed Figure 6. Efficiency test of whole-cell vaccine inactivated and adsorbed on mAhC in CY-treated mice. (A) Survival curve of experimental groups using CY-treated mice after a lethal dose of A. baumannii (4.0 × 10 8 CFU/mL). The log-rank test (Mantel-Cox test, with six animals per group) determined the p-value. (*) p < 0.05 represents a comparative analysis between non-immunized CY-treated mice vs. CY-treated mice inoculated with mAhC using six animals per group. (***) p < 0.001 represents a comparative analysis between non-immunized CY-treated mice vs. immunized CY-treated mice and (#) p < 0.05 represents a comparative analysis between immunized CY-treated mice vs. CY-treated mice inoculated with mAhC. (B) Humoral response after a lethal dose of A. baumannii (4.0 × 10 8 CFU/mL). Data refer to the total IgG levels produced from immunized CY-treated mice, non-immunized CY-treated mice, and post-lethal CY-treated mice inoculated with mAhC. (*) p < 0.05 represents a comparative analysis between non-immunized CY-treated mice vs. CY-treated mice inoculated with mAhC and non-immunized CY-treated mice vs. immunized CY-treated mice. p < 0.05 (*#) represents a comparative analysis between immunized CY-treated mice vs. CY-treated mice inoculated with mAhC. (C,D) Measurement and imaging of white pulp expansion of immunosuppressed mice. (*) p < 0.05 represents a comparative analysis between non-immunized CY-treated mice vs. those inoculated with mAhC. (***) p < 0.01 represents a comparative analysis between immunized CYtreated mice vs. CY-treated mice inoculated with mAhC. ImageJ employee software is available (https://imagej.nih.gov/ij/ accessed on 31 January 2023).

Histopathological Analysis of the Sepsis Model of A. baumannii Infection in CY-Treated Mice
The sepsis model of A. baumannii infection showed tissue damage in the liver, spleens, and lungs in CY-treated mice, the non-immunized mice, and the mAhC (Figure 7). By comparison, there were no significant differences between the liver and lung weights of these groups (Figure 7A,B). In terms of the weight of the spleens of immunized CY-treated mice, there were significant differences from the non-immunized CY-treated or adjuvant groups (p < 0.05) ( Figure 7C). In addition, more substantial expansion of the white pulp was observed in this group (p < 0.01) and the adjuvant group (p < 0.05) ( Figure 7D). The expansion of the white pulp is related to the presence of periarteriolar and follicular hyperplasia caused by the antigenic stimulus and immune response (Figures 6D and 7D). The histopathological findings revealed greater severity in the liver with hydropic degeneration and decreased sinusoids, and interstitial pneumonia with a mixed inflammatory infiltrate and congestion (of minor severity in the mAhC and immunized groups) in the lung. Additionally, hemorrhage, congestion, and white pulp lymphoid hyperplasia were detected in the spleen ( Figure 7D). inflammatory infiltrate and congestion (of minor severity in the mAhC and immunized groups) in the lung. Additionally, hemorrhage, congestion, and white pulp lymphoid hyperplasia were detected in the spleen ( Figure 7D).

Discussion
A. baumannii is a critical emerging pathogen that is responsible for nosocomial pneumonia in healthcare settings; it has become a global emergency [45][46][47]. Murine models have contributed to elucidating the mechanisms associated with pathogenesis and

Discussion
A. baumannii is a critical emerging pathogen that is responsible for nosocomial pneumonia in healthcare settings; it has become a global emergency [45][46][47]. Murine models have contributed to elucidating the mechanisms associated with pathogenesis and understanding the pathogen-host relationship of infections caused by A. baumannii in sepsis, pneumonia, and immunosuppression models [17,19,24]. CY is a synthetic antineoplastic agent widely used as an immunosuppressive drug in mice, which causes a cytostatic effect and inhibits the rapid cycle of immune cells, consequently causing granulopenia [23,25,26]. A. baumannii-infected mice may show changes consistent with pneumonia, acute inflammation with mild-to-severe inflammatory infiltration of polymorphonuclear cells, segmental abscess formation, and mild-to-moderate infiltration of alveolar macrophages when treated with an immunosuppressant [48]. Here, we used an A. baumannii sepsis model in CYtreated mice to understand the behavior of multi-drug-resistant strains in infections and to evaluate new vaccine strategies based on inactivated bacterial strains adsorbed on mAhC.
Preliminarily, we revealed the immunosuppression of immunocompetent mice that showed signs of infection and presented with ruffled and shedding animal hair, loss in body weight, and lethargy, accompanied by a reduction in leukocytes (neutrophils, lymphocytes, and monocytes), erythrocytes, and hemoglobin and the percentage of hematocrit ( Figure 4). Our data corroborate those of other studies that show low concentrations of circulating polymorphonuclear neutrophils, followed by a marked and progressive increase leading to greater susceptibility of immunosuppressed mice [23,29,49] and distinct alterations of macrophages and lymphocytes with lower participation in the first hours of infection, followed by baseline levels.
The expansion of the follicular area is characterized by increased cellularity of rounded and regular nuclear membranes stimulated by immunization, leading to subsequent B cell proliferation [17,28,50,51]. The present study revealed the expansion of spleen white pulp in the immunized CY-treated mice groups with the production of IgG antibodies. In addition, the adequate protection of 85% after a lethal dose of multi-drug-resistant A. baumannii suggests that our murine model was effective in vaccine protection in immunized animals ( Figure 6B,C), as reported in previous studies [23,28,30].
Another relevant aspect observed here was the greater tissue protection of groups of immunized CY-treated mice because non-immunized CY-treated mice or adjuvantinoculated-CY-treated mice presented histopathological findings of edema, hemorrhage, hydropic degeneration, necrosis, and hyperemia in liver sections, as well as edema, hemorrhage, and hyperemia in lung sections. There were signs of acute infection in the nonimmunized CY-treated mice and adjuvant-inoculated-CY-treated mice groups after infection, revealing characteristic signs of the pathology with death within 24 h. In contrast, the immunized CY-treated mice groups showed adequate protection after receiving a lethal dose of multi-drug-resistant A. baumannii. Our data corroborate previous infection studies in murine sepsis models [49,52,53].
Splenic alterations caused by A. baumannii infections are accompanied by white pulp damage, leukocyte apoptosis, embolus formation, clusters of degenerated or necrotic leukocytes in the white pulp areas, and the appearance of moderate congestion in the medullary region [49,54]. Infiltration of mixed inflammatory cells in the perivascular and peribronchial space, with the presence of polymorphonuclear granulocytes and mononuclear cells in the airway lumen, especially in the first hours of infection, is followed by vasodilation, congestion, and hemorrhage, which leads to the destruction of alveolar structures of the lung after 48 h of infection [49,52,53,55,56]. Our histopathological findings highlight the importance and adequacy of the sepsis model, evidencing vaccine protection, the stimulation of the immune response, and the consequent tissue protection of the livers, spleens, and lungs of immunized animals.
The mAhC used here contributed to the stimulation of IgG production of immunized CY-treated mice groups and was superior to non-immunized CY-treated mice groups when used at three doses/animal. Previous studies demonstrated the stimulation of IgG produc-tion in immunized mice using protein antigens adsorbed exclusively on chitosan [34,57,58] or exclusively on aluminum hydroxide or Freund's adjuvant [15,30,33,59]. However, the present study is the first to associate aluminum hydroxide and chitosan for the adsorption of A. baumannii antigens on mAhC, revealing a vaccine adjuvant that stimulates the immune system response of immunosuppressed mice.
Despite the relevance of the efficacy of a new vaccine formulation with inactivated multi-drug-resistant A. baumannii that is whole-cell adsorbed on mAhC, issues remain that have not been fully elucidated. As described here, we recorded high stimulation of the immune response by measuring total IgG levels. However, we did not determine IgG1 and IgG2c levels, which participate in eliminating pathogens and reducing postoperative bacterial loads [17,32,33]. We did not measure cytokine profiles, which may be necessary for elucidating the mechanism of pathogen elimination and vaccine protection. Thus, future studies should address these issues.

Conclusions
We demonstrated the role of an A. baumannii sepsis model in mice and the reduction in neutrophil numbers before immunization (treated with CY). The inactivated and adsorbed multi-drug A. baumannii whole-cell vaccine (mAhC) stimulated the immune response and protected immunized animals, even after a challenge with a lethal dose. These findings provide the basis for studies on minimizing the impacts of A. baumannii infections.

Institutional Review Board Statement:
The experiments were conducted following ethical recommendations established by the law of procedures for the scientific use of animals, being approved for execution by the Animal Ethics and Research Committee of the Federal University of Tocantins under protocol number 23101.002359/2020-31. All efforts were made to avoid suffering or undue pain. The animals were monitored for clinical aspects, loss and gain in body weight, and the first clinical signs, including lethargy, hypothermia, and difficulty breathing. The animals were euthanized using the protocol described in the material and methods.