PCEP enhances IgA mucosal immune responses in mice following different immunization routes with influenza virus antigens

Abstract

Background: We previously demonstrated that polyphosphazenes, particularly PCEP, enhance immune responses in mice immunized subcutaneously and intranasally. The objective of the present study was to investigate the efficacy of polyphosphazenes as adjuvants when delivered through different routes of vaccine administration.

Methods: BALB/c mice were immunized through intranasal, subcutaneous, oral and intrarectal delivery with vaccine formulations containing either influenza X:31 antigen alone or formulated in PCEP. Serum and mucosal washes were collected and assayed for antigen-specific antibody responses by ELISA, while splenocytes were assayed for antigen-specific cytokine production by ELISPOT.

Results: Intranasal immunization with PCEP+X:31 induced significantly higher IgA titers in all mucosal secretions (lung, nasal, and vaginal) compared to the other routes. Serum analysis showed that all mice given the PCEP+X:31 combination showed evidence of enhanced IgG2a titers in all administered routes, indicating that PCEP can be effective as an adjuvant in enhancing systemic immune responses when delivered via different routes of administration.

Conclusions: We conclude that PCEP is a potent and versatile mucosal adjuvant that can be administered in a variety of routes and effectively enhances systemic and local immune responses. Furthermore, intranasal immunization was found to be the best administration route for enhancing IgA titers, providing further evidence for the potential of PCEP as a mucosal adjuvant.

Figure 1

PCEP enhances IgG1 and IgG2a X:31-specific serum titers when PCEP+X:31 is administered through intranasal, subcutaneous, and oral routes, but not after intrarectal immunization. BALB/c mice were given PCEP+X:31 (closed symbols) or X:31 (open symbols) through IN (A, E, squares), SC (B, F, circles), oral (C, G, triangles) and IR (D, H, diamonds) immunization. X:31-specific IgG1 (A-D) and IgG2a (E-H) were assayed in mouse serum. Groups given PCEP+X:31 with different letters are significantly different from each other within each IgG subtype (p < 0.05). A single asterisk indicates an adjuvant effect of PCEP during the course of the experiment, while a double asterisk indicates an adjuvant effect only at the specified time point. Arrows indicate the time of delivery of a secondary immunization using the same delivery route as the primary immunization.

Figure 2

PCEP enhances IgA and IgG antigen-specific titers in lung washes of mice following intranasal and subcutaneous immunization. At the end of the 8 week experiment, lung washes from all the mice were collected and analyzed for X:31-specific IgA (A) and total IgG (B) titers by ELISA after receiving either PCEP+X:31 (closed bars) or X:31 alone (open bars) following IN, SC, oral, or IR immunization. Different letters indicate significant differences between groups that received PCEP+X:31, while asterisks indicate significant differences (i.e. adjuvant effect) between PCEP+X:31 compared to X:31 antigen alone (p < 0.05).

Figure 3

Intranasal and subcutaneous immunization of PCEP + X:31 is effective in enhancing IgA and IgG titers in nasal secretions. Nasal secretions were collected from mice receiving either PCEP+X:31 (closed bars) or X:31 (open bars) following IN, SC, oral, or IR immunization. Antigen-specific IgA (A) and IgG (B) was assayed from the nasal washes. Different letters indicate significant differences between groups that received PCEP+X:31, while asterisks indicate significant differences between PCEP+X:31 compared to X:31 antigen alone (p < 0.05).

Figure 4

Enhanced IgA and IgG titers from immunized mice are also found in vaginal secretions. Vaginal mucosal washes were also collected at the end of the 8 week experiment where X:31-specific IgA (A) and IgG (B) was assayed from mice that were immunized by IN, SC, oral, or IR immunization with either PCEP+X:31 (closed bars) or X:31 alone (open bars). Different letters indicate significant differences between groups that received PCEP+X:31, while asterisks indicate significant differences between PCEP+X:31 compared to X:31 antigen alone (p < 0.05).

Figure 5

PCEP significantly enhances IFN-g and IL-4 cytokine production in mice immunized via intranasal route. The spleens from mice immunized with PCEP+X:31 (closed bars) or X:31 alone (open bars) were collected and splenocytes were subsequently isolated. The splenocytes were stimulated with X:31 antigen and antigen-specific IFN-γ (A) and IL-4 (B) were assayed to determine Th1/Th2 responses. Differences in cytokine production as a result of IN, SC, oral, or IR immunization were also examined. Different letters indicate significant differences between groups that received PCEP+X:31, while asterisks indicate significant differences between PCEP+X:31 compared to X:31 antigen alone (p < 0.05).