Oral administration of polymer-grafted starch microparticles activates gut-associated lymphocytes and primes mice for a subsequent systemic antigen challenge.

The mucosal and systemic humoral immune systems can function essentially independent of each other, responding to mucosal and parenteral antigens, respectively. Nevertheless, antigen administered by one route can modify responsiveness to subsequent immunization by an alternate route. Here we demonstrated, in mice, in addition to stimulating rapid and robust sera antibody responses, intragastric (i.g.) immunization with human serum albumin (HSA)-containing starch microparticles (MP) grafted with 3-(triethoxysilyl)-propyl-terminated polydimethylsiloxane (TS-PDMS) primed for enhanced specific sera IgG following a parenteral antigen boost. After as little as one i.g. immunization with microentrapped, but not with soluble, HSA antigen-specific proliferation and antibody secretion were detected in Peyer's patches (PP); this activity peaked after three i.g. MP immunizations. We observed a progressive dissemination of antigen-specific lymphocyte reactivity from PP to splenic tissue following oral MP immunization. Similarly, we observed a shift in HSA-specific antibody-secreting cells from PP and mesenteric lymph nodes to splenic tissue following i.g. MP immunization. We also demonstrated that oral immunization with microentrapped, but not with soluble HSA, resulted in enhanced numbers of spontaneous Th2-cytokine secreting lymphocytes which disseminated from mucosal to systemic lymphoid compartments. This observation coincided with our findings that HSA-specific sera IgG1 responses in animals given HSA in MP were significantly higher than those detected in the sera of mice given soluble HSA i.g., both before and after parenteral antigen challenge. These findings suggest that orally-administered TS-PDMS-grafted MP, by stimulating elements of the mucosal immune system, are a valuable addition to mucosal and systemic vaccine delivery systems.

Polymer-grafted starch microparticles for oral and nasal immunization.

Microparticle delivery systems for oral vaccine administration are receiving considerable attention. A novel silicone polymer-grafted starch microparticle system was developed that is efficacious both orally and intranasally. Unlike most other microparticle systems, this novel system does not appear to retard the release of antigen or to protect antigen from degradation. The results indicate that a unique physiochemical relationship occurs between protein antigen and silicone in a starch matrix that facilitates the mucosal immunogenicity of antigen. This leads to predominance of Th2 antibody response. Taken together, these findings indicate that this novel microparticle system may be advantageous for the delivery of small quantities of antigen, especially intranasally, and may be useful for the induction of oral tolerance.

Intranasal immunization with polymer-grafted microparticles activates the nasal-associated lymphoid tissue and draining lymph nodes.

Waldeyer's ring is located at the juncture of the respiratory and alimentary tracts, where it is bombarded by inhaled and ingested antigens. However, knowledge of its exact function or consequences of its removal is incomplete. Recently, the murine nasal-associated lymphoid tissue (NALT) has been reported to have functional similarities to Waldeyer's ring and, thus, might be a suitable model to examine the function of oronasopharyngeal lymphoid tissues. To explore the capability of NALT to incite local mucosal and systemic immunity, we immunized mice intranasally (i.n.) with 3-(triethoxysilyl)-propyl-terminated polydimethylsiloxane (TS-PDMS)-grafted microparticles (MP), an inoculant previously shown to induce robust systemic and mucosal humoral immunity following intragastric (i.g.) administration. We demonstrated that i.n. immunization with low doses of microentrapped, but not soluble, human serum albumin (HSA) evoked robust circulating IgG responses (P < 0.05). Additionally, NALT cells isolated from MP-treated mice proliferated in vitro when restimulated with HSA (P < 0.05), suggesting that i.n. immunization with HSA-containing MP incited specific immunity via NALT cell activation. Coinciding with these observations, after i.n. MP administration HSA-specific spot-forming cells (SFC) were observed in NALT, and later posterior cervical lymph nodes (pCLN) and spleen (SPL), suggesting that the observed MP-induced specific systemic antibody responses emanated from the NALT. We also showed that i.n. immunization with HSA-containing TS-PDMS-grafted MP stimulated interleukin-4 (IL-4)-secreting lymphocytes in the NALT. This cytokine microenvironment was probably responsible for driving the IgG1 sera response observed after i.n. MP administration, via the migration of NALT-derived IgG1-committed B cells. Interestingly, unlike i.g. MP administration, i.n. immunization with HSA-containing MP did not evoke detectable specific IgA in any lymphoid tissue examined, or in nasal secretions, probably reflecting differences between NALT and other mucosae-associated lymphoid tissues (MALT).

Comparison of murine nasal-associated lymphoid tissue and Peyer's patches.

The nasal mucosal is the first site of contact with inhaled antigens. However, the nature of local immune responses and the role of nasal-associated lymphoid tissue (NALT) in those responses have rarely been studied. To characterize the cells involved in mucosally derived immune responses, NALT and Peyer's patch (PP) cells from normal mice, and mice immunized intragastrically or intranasally with cholera toxin (CT), were isolated and analyzed. Compared with PP cells, unstimulated NALT cells contained a higher proportion of T-cells. The CD4:CD8 ratio in NALT cell preparations was less than that observed in PP and more closely resembled that seen in spleen. Additionally, the total B-cell frequency in NALT cell isolates was 20% lower than that observed in PP cell preparations. Although NALT and PP cell isolates contained both mature B-cells and cells undergoing activation to express surface IgA, unlike PP, NALT showed no significant frequency of IgA-switched cells. After intranasal immunization with CT, toxin-specific IgA antibody-forming cells (AFCs) were detected in NALT cell preparations. The numbers of these cells correlated with CT-specific IgA in nasal, but not in gut washes or sera, thus suggesting local nasal production of antigen-specific mucosal antibodies. There was no evidence of anti-CT AFCs in NALT or CT-specific antibody in nasal washes after intragastric CT administration. These results support the notion that nasal mucosal antibody production is best achieved via direct stimulation of IgA-committed, NALT-derived B-cells.

Novel polymer-grafted starch microparticles for mucosal delivery of vaccines.

Recent studies have demonstrated that systemic and mucosal administration of soluble antigens in biodegradable microparticles can potentiate antigen-specific humoral and cellular immune responses. However, current microparticle formulations are not adequate for all vaccine antigens, necessitating the further development of microparticle carrier systems. In this study, we developed a novel microparticle fabrication technique in which human serum albumin (HSA) was entrapped in starch microparticles grafted with 3-(triethoxysilyl)-propyl-terminated polydimethylsiloxane (TS-PDMS), a biocompatible silicone polymer. The immunogenicity of HSA was preserved during the microparticle fabrication process. Following intraperitoneal immunization of mice, TS-PDMS-grafted microparticles (MP) dramatically enhanced serum IgG responses compared with ungrafted MP and soluble HSA alone (P < 0.001). When delivered orally, both TS-PDMS-grafted and ungrafted microparticles elicited HSA-specific IgA responses in gut secretions, in contrast to orally administered soluble antigen. Indeed, TS-PDMS-grafted microparticles stimulated significantly stronger serum IgG (P < 0.005) and IgA (P < 0.001) responses compared with those elicited by ungrafted microparticles. These findings indicate that TS-PDMS-grafted starch microparticles have potential as systemic and mucosal vaccine delivery vehicles.