Attachment of class B CpG ODN onto DOTAP/DC-chol liposome in nasal vaccine formulations augments antigen-specific immune responses in mice - PubMed (original) (raw)

Attachment of class B CpG ODN onto DOTAP/DC-chol liposome in nasal vaccine formulations augments antigen-specific immune responses in mice

Rui Tada et al. BMC Res Notes. 2017.

Abstract

Background: To overcome infectious diseases, the development of mucosal vaccines would be an effective strategy, since mucosal surfaces are the entry site for most pathogens. In general, protein antigens show inherently poor immunogenicity when administered by the mucosal route. Therefore, co-administration of an appropriate mucosal adjuvant is required to exert immune responses toward pathogen-derived antigens effectively. However, the development of a safe and effective mucosal adjuvant system is still challenging. Although, recent studies reported that oligodeoxynucleotides (ODNs) containing immunostimulatory CpG motifs (CpG ODNs) act as potent mucosal adjuvants and are useful in the formulation of nasal vaccines, there are some disadvantages. For instance, the administration of phosphorothioate (PS)-modified CpG ODNs can induce adverse systemic effects, such as splenomegaly, in a dose-dependent manner. Therefore, a reduced dose of CpG ODN might be crucial when used as vaccine adjuvant for clinical purposes. Therefore, we prepared a CpG ODN-loaded cationic liposome, and evaluated its mucosal adjuvant activity.

Results: We prepared a CpG ODN-loaded DOTAP/DC-chol liposome that was stable during our experiments, by mixing CpG ODNs and liposomes at an N/P ratio of 4. Further, we demonstrated that the attachment of class B CpG ODN to the DOTAP/DC-chol liposomes synergistically enhanced antigen-specific IgA production in the nasal area than that induced by CpG ODN and DOTAP/DC-chol liposomes alone. The endpoint titers were more than tenfolds higher than that induced by either single CpG ODN or single DOTAP/DC-chol liposomes. Additionally, although serum IgG1 responses (indicated as a Th2 response) remained unchanged for DOTAP/DC-chol liposomes and CpG ODN-loaded DOTAP/DC-chol liposomes, the CpG ODN-loaded DOTAP/DC-chol liposomes synergistically induced the production of serum IgG2a (indicated as a Th1 response) than that by the individual liposomes.

Conclusions: We conclude that the advantage of using DOTAP/DC-chol liposome harboring CpG ODN is it induces both antigen-specific mucosal IgA responses and balanced Th1/Th2 responses. Therefore, such a combination enables us to resolve the adverse effects of using CpG ODNs (as a mucosal adjuvant) by reducing the overall dose of CpG ODNs. Further, the biodegradable and essentially non-antigenic nature of the liposomes makes it superior than the other existing mucosal adjuvants.

Keywords: Cationic liposome; CpG ODN; Intranasal immunization; Mucosal adjuvant; Vaccine.

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Figures

Fig. 1

Fig. 1

Agarose gel retardation assay of the prepared CpG ODN-loaded DOTAP/DC-chol liposomes with various N/P ratios. The prepared CpG ODN-loaded DOTAP/DC-chol liposomes were loaded onto a 2% agarose gel and then ODN bands were visualized by staining with GelRed Nucleic Acid Gel Stain

Fig. 2

Fig. 2

Distribution of particle size (a) and ζ-potential (b) of the prepared CpG ODN-loaded DOTAP/DC-chol liposomes with various N/P ratios. The particle size and ζ-potential of the prepared CpG ODN-loaded DOTAP/DC-chol liposomes were measured by NICOMP 380 ZLS. The particle size and ζ-potential are expressed as the mean ± standard error for samples assayed in triplicate

Fig. 3

Fig. 3

Mucosal adjuvant activity of CpG ODN assessed by the induction of OVA-specific nasal IgA and serum IgG in BALB/c mice. BALB/c female mice were immunized intranasally with OVA (5 µg/mouse) alone, or OVA (5 µg/mouse) plus CpG ODN (1 or 10 µg/mouse) on days 0 and 7. Serum and nasal washes were collected on day 14. The anti-OVA IgA level in nasal washes and anti-OVA IgG, IgG1, and IgG2a levels in serum were detected by ELISA assay as described in the “Methods” section. The data were obtained from three independent experiments. The box-plot shows the median value with the 25th–75th percentiles and the error bars indicate the 5th–95th percentiles. Symbols in the box plots represent individual mice (PBS, n = 8; OVA, n = 8; OVA plus CpG ODN (1 µg/mouse), n = 8; OVA plus CpG ODN (10 µg/mouse), n = 4). Significance was assessed using the Kruskal–Wallis with Dunn’s post-hoc test: *p < 0.05, NS not significant

Fig. 4

Fig. 4

Mucosal adjuvant activity of CpG ODN-loaded DOTAP/DC-chol liposomes assessed by the induction of OVA-specific nasal IgA and serum IgG in BALB/c mice. BALB/c female mice were immunized intranasally with PBS, OVA (5 µg/mouse) alone, OVA (5 µg/mouse) plus DOTAP/DC-chol liposomes, OVA (5 µg/mouse) plus CpG ODN (1 µg/mouse), or OVA (5 µg/mouse) plus CpG ODN-loaded DOTAP/DC-chol liposomes on days 0 and 7. Serum and nasal washes were collected on day 14. The anti-OVA IgA level in nasal washes and anti-OVA IgG, IgG1, and IgG2a levels in serum were detected by ELISA assay as described in the “Methods” section. The data were obtained from three independent experiments. The box-plot shows the median value with the 25th–75th percentiles and the error bars indicate the 5th–95th percentiles. The symbols in box plots represent individual mice (PBS, n = 10; OVA, n = 10; OVA plus liposome, n = 10; OVA plus CpG-loaded liposome, n = 12). Significance was assessed using the Kruskal–Wallis with Dunn’s post–hoc test: *p < 0.05, NS not significant

Fig. 5

Fig. 5

Serum IgG subclasses induced by intranasal immunization of OVA plus CpG ODN-loaded DOTAP/DC-chol liposomes in BALB/c mice. BALB/c female mice were immunized intranasally with PBS, OVA (5 µg/mouse) alone, OVA (5 µg/mouse) plus DOTAP/DC-chol liposomes, or OVA (5 µg/mouse) plus CpG ODN-loaded DOTAP/DC-chol liposomes on days 0 and 7. Serum samples were collected on day 14. The anti-OVA IgG1 and IgG2a levels in serum were detected by ELISA assay as described in the “Methods” section. The data were obtained from three independent experiments. The box-plot shows the median value with the 25th–75th percentiles and the error bars indicate the 5th–95th percentiles. The symbols in box plots represent individual mice (PBS, n = 10; OVA, n = 10; OVA plus liposome, n = 10; OVA plus CpG-loaded liposome, n = 12; OVA plus CpG ODN, n = 8). Significance was assessed using the Kruskal–Wallis with Dunn’s post hoc test: *p < 0.05, NS not significant

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