Immune response effects of diverse vaccine antigen attachment ways based on the self-made nanoemulsion adjuvant in systemic MRSA infection

Abstract

Nanoemulsion adjuvants-based vaccines have potent induced immune responses against methicillin-resistant Staphylococcus aureus (MRSA) infection. However, the efficacies and immune responses of different antigen-attaching ways on self-made nanoemulsion adjuvants remain unknown. In this study, we designed three formulations of nanoemulsion adjuvants (encapsulation, mixture, and combination) to explore their immune response-enhancing effects and their underlying mechanism in a systemic infection model of MRSA. Our results showed that the three nanoemulsion-attachment ways formulated with a fusion antigen of MRSA (Hla_H35LIsdB_348-465) all improved humoral and cellular immune responses. When compared with the mixture and combination formulations, the nanoemulsion-encapsulation group effectively promoted the antigen uptake of dendritic cells (DCs) in vitro, the activation of DC in draining lymph nodes and the delayed release of antigen at injection sites in vivo. Moreover, the encapsulation group induced a more ideal protective efficacy in a MRSA sepsis model by inducing more potent antibody responses and a Th1/Th17 biased CD4⁺ T cell response when compared with the other two attachment ways. Our findings suggested that the encapsulated formulation of vaccine with nanoemulsion adjuvant is an effective attachment way to provide protective immunity against MRSA infection.

Fig. 1. Characterization of the encapsulation attachment way. Transmission electron micrographs (A), scanning electron micrographs (B) and atomic force micrographs (C) of the encapsulation attachment way. The size distribution (D) and zeta potential (E) were detected using nano ZS.

Fig. 2. Antibody responses by the three vaccine antigen attachment ways. (A) Serum (n = 6) were taken at days 7, 14 and 24. IgG responses were detected through ELISA. (B and C) Serum (n = 6) were taken at days 24. The responses of IgG1 and IgG2a were detected through ELISA. Antibody response is expressed as the mean absorbance at 450 nm ± SD. (D) IgG2a/IgG1 ratio was calculated. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3. Cellular immune responses by the three vaccine antigen attachment ways. Splenocytes of immunised mice (n = 6) were stimulated with the antigen for 3 days. (A) Splenocyte proliferation assay was performed using a CCK-8 kit. (B–D) IFN-γ, IL-4 and IL-17 levels were detected through ELISA. The results are reported as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4. Frequency of memory T cells induced by the three vaccine antigen attachment ways. Balb/c mice (n = 6) were immunised as previously described. On day 24, the splenocytes (1 × 10⁷ cells per mL) were stimulated with antigen (10 μg mL⁻¹) for 3 days. FACS plots in (A and B) are representative of the mean percentages of 6 mice in each group. The frequency of CD44^hiCD62^hi CD4⁺ T cells (C), CD44^hiCD62^low CD4⁺ T cells (D), CD44^hiCD62^hi CD8⁺ T cells (E) and CD44^hiCD62^low CD8⁺ T cells (F) were measured using flow cytometry. Data in (C), (D), (E), and (F) are expressed as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5. Antigen uptake by DCs and DC activation in the draining lymph nodes. (A) CLSM images of BMDCs after 30 minutes of incubation with the naïve GFP protein, GFP-combination nanoemulsion, GFP-mixture nanoemulsion and GFP-encapsulation nanoemulsion. For each panel, the images from left to right show Dil stained cell members by Lyso-Tracker (red), GFP fluorescence (green) and the overlays of the two images (red and green). (B) Results of the fluorescence intensity percentage (n = 3) are expressed as the mean ± SD. Balb/c mice (n = 6) were intramuscularly vaccinated with the different vaccine formulations. At 8 days after immunisation, the mice were euthanized and the popliteal lymph nodes were isolated. The percentage of CD86 (C), MHC I (D) and MHC II (E) expression on the CD11c⁺ DCs were determined using flow cytometry. Data are expressed as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6. Antigen persistence and depot at the injection sites. Nude mice (n = 3) were injected subcutaneously (left hind leg) or intramuscularly (right hind leg) with 100 μL of the different formulations of the nanoemulsion adjuvant vaccines containing 1 mg mL⁻¹ GFP protein. Antigen persistence at the injection sites was measured using a Carestream FX PRO in vivo imaging system. (A) Representative fluorescence images and (B and C) quantitative fluorescence intensity of the antigen persisting at the injection sites. Data are expressed as the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 7. Protective effects of three vaccine antigen attachment ways in a MRSA sepsis model. Balb/c mice (n = 6) were immunised as previously described. At days 21, mice were infected with 2.5 × 10⁸ CFUs MRSA 252. At 1 and 3 days post-infection, the bacterial burdens in the blood (A, D), spleen (B, E) and kidneys (C, F) were measured. (G) At 3 days post-infection, the kidneys were collected and the representative histopathological sections are shown (magnification = ×400). Arrowheads indicate Staphylococcal abscesses. (H) Severity scores of the kidneys (n = 6) from the six immunised groups at 3 days post-infection are shown. Data are presented as scatter plots and the means ± SD are shown. (I) The mice (n = 10) were intravenously infected with MRSA 252 (1 × 10⁹ CFUs) and the survival rates were monitored for 14 days. *P < 0.05; **P < 0.01; ***P < 0.001.