The promoting role of aluminum oxide nanoparticles in enhancing Pseudomonas aeruginosa antigen immunogenicity

Abstract

Background: Pseudomonas aeruginosa is considered one of the major opportunistic bacteria that cause infections in both animals and humans. It is a significant problem in global health care due to its high inherent resistance to numerous antibiotics. Pathologic microorganisms are resistant to the number of available treatments, highlighting the pressing need for efficient immunization and immunotherapeutic approaches.

Aim: Data regarding the use of nanoparticles as delivery are limited; therefore, we sought to develop an adjuvant vaccine to lessen the chance of developing a P. aeruginosa infection.

Methods: Thirty mice were randomly assigned to three groups: T1 (Naïve control), T2 (non-nave control), and T3 (non-nave control) received two intraperitoneal (IP) doses (2-week interval) of 0.5 mg/ml of killed whole sonicated P. aeruginosa antigens (KWSPAgs) and two IP doses (2-week interval) of 1 mg/ml of KWSPAgs and aluminum oxide nanoparticles. At 14 days post-immunization, cell-mediated immunity was evaluated by delayed-type hypersensitivity, and at day 28, humoral immunity was assessed via Enzyme-Linked Immune Sorbent Assay to measure serum levels of Immunoglobulins (IgG), toll-like receptor 2 (TLR2), and IL-2. All groups were challenged IP with P. aeruginosa (0.2 × 10⁷ CFU/ml). After 14 days post-immunization, mice were sacrificed for bacterial isolation, and internal organs were collected for histopathological and immunohistochemical analysis.

Results: Pseudomonas aeruginosa antigens were sufficient to elicit immunogenicity. Moreover, the use of Nanoparticles (NPs) markedly enhanced immunogenicity through the delivery of KWSPAgs. Our results have shown that group with NP with KWSPAgs were able to induce inflammation and cellular immunity by increasing interleukin 2 and foot pad thickness, both of which are indicators of T lymphocyte proliferation. In addition, our data indicated that group with NP were able to induce humoral immunity by increasing the IgG and TLR2 levels in the immunized groups compared with Naïve control. The histopathological lesions and immune-positive cells in the infected and non-infected groups were different. Lesions were significantly higher in the infected group than in the noninfected control group at the observed time points.

Conclusion: Overall, these findings highlight that NPs are a good activator for inducing cellular and humoral immunity, enhancing the delivery of future vaccine designs and/or target therapies against P. aeruginosa.

Keywords: Aluminum oxide; Bacterial vaccine; Immune responses; Immunohistochemistry; Phonological changes.

Fig. 1.. Nanoparticles enhancing KWSPAgs- immunization would be more fit than using infection. Mice were exposed to a double dose of 0.5 ml KWSPAgs (IP) at 2-week intervals. On day 1, DDW as a VEH was administered to these mice (IP) in the control group and KWSPAgs alone and KWSPAgs +NPs on day 14. The mice were euthanized on day 28 for various studies. (A) IL2 cytokine level from serum obtained on day 28. (B and C) Compression between the clinical macroscopic changes between the naive and immunized groups. Five mice in each group were used, and the data were confirmed in three independent experiments. (D) Comparison of the foot pad thickness between the groups at 24 and 48 hours *p ≤ 0.05, p** ≤ 0.01, ***p ≤ 0.001.

Fig. 2.. (A and B) We further investigated humoral immunity after we noticed a cellular immune response after exposure of mice. Injections of KWSPAgs and KWSPAgs + NPs, both IgG and TLR2 were significantly increased after KWSPAgs compared with Naïve group suggesting the successful preparation of KWSPAgs for inducing the immune response. At the same time, there was a further upregulation of both IgG and TLR in KWSPAg +NPs compared with KWSPAgs alone, suggesting the successful role of NPs in promoting the immunogenicity of KWSPAgs compared with KWSPAgs alone. *p ≤ 0.05, p** ≤ 0.01.

Fig. 3.. Microscopic assessment of control, immunized, and non-immunized groups at 28 days post-infection with P. aeruginosa H and E stain (400, 100X). (A) The control group had no liver lesions. (B) The liver of the KWSPAgs immunized group showed severe inflammatory reaction with infiltration of inflammatory cells (IC), necrosis of hepatocytes (NH), narrowing of sinusoidal (NS), and hepatocyte swelling (HS). Cellular edema (CE) was severe and there was a slight vacuolar fatty change. (C) The liver of the immunized group had perivascular cuffing (PC) followed by inflammatory edema (IE), central vein distension, and few mononuclear cells. (D) Control group with no lesion in spleen tissue. (E) Infected group with a reactive lymphoid follicle, white pulp with a prominent necrotic area of lymphoid tissue suppuration (LS). The white and red pulp has mononuclear cells infiltrating it (MI). (F) Mononuclear cell infiltration with two foci of CFU (MC) in spleen with small area of necrotic lesion (N). (G) Kidney of control group with no lesion. (H) Kidney tissue of infected group with edematous fluid between tubules (IF) with mononuclear cells infiltration (M) and narrowing of distal and proximal tubules (T) and some of it with coagulative necrosis (N). (I) Kidney of immunized group, the cortex showed hypertrophy in glomeruli (G) with polymorphic cells infiltration (P).

Fig. 4.. Immunohistochemical staining of the liver of naive, KWSPAgs, and KWSPAgs+NPs at 14 days post-infection with P. aeruginosa. Naïve immunohistochemistry showed negative staining cells (Fig. 4A), KWSPAgs showed potent positive staining represented by pink-red-brown cells (black arrow) (Fig. 4B), and KWSPAgs+NPs showed scattered pink-red-brown cells (yellow head arrow) (Fig. 4C). Immunohistochemical staining, the lung of Naïve, KWSPAgs, and KWSPAgs+NPs at 14 days post-infection with P. aeruginosa (Fig. 4D–F).