Hypersensitivity in the small intestinal mucosa. V. Induction of cell-mediated immunity to a dietary antigen.

Feeding of a protein antigen to adult mice results in reduced humoral and cell-mediated immune (CMI) responses when that antigen is subsequently presented, and also causes activation of suppressor cells in the gut-associated lymphoid tissues (GALT). We have attempted to abrogate this tolerance to fed antigen by pretreating mice with 100 mg/kg cyclophosphamide before oral immunization and challenge with ovalbumin. Cyclophosphamide-pretreated mice did not develop serum haemagglutinating antibodies, nor systemic CMI (as assessed by skin testing) after ovalbumin feeding. However, evidence that CMI had been induced in the GALT was provided by the significant inhibition of migration and mesenteric lymph node cells from cyclophosphamide-pretreated animals, but not from other control groups. in the presence of ovalbumin. Our previous work on CMI reactions in the small intestine has shown that the cell production rate in the crypts of Lieberkuhn and the intraepithelial lymphocyte count are reliable although indirect measures of mucosal CMI. Cyclophosphamide-pretreated, ovalbumin-immunized animals, which had been fed 0 . 1 mg ovalbumin daily for 10 days before killing, had increased crypt cell mitoses, and increased intraepithelial lymphocyte counts, indicating the presence of mucosal CMI response to ovalbumin. Mechanisms whereby cyclophosphamide pretreatment leads to abrogation of tolerance and induction of mucosal CMI are discussed.     

The kinetics of oral hyposensitization to a protein antigen are determined by immune status and the timing, dose and frequency of antigen administration.

We have investigated the immunological consequences of feeding a protein antigen to previously immunized animals. BALB/c mice were systemically primed with ovalbumin (OVA) in complete Freund's adjuvant (CFA) and fed with high (10 mg/g body weight), medium (1 mg/g body weight) or low (1 microgram/g body weight) doses of OVA once (Day 1, 7 or 14) or sequentially for 5 days (Days 1-5, 7-11, 14-18). The specific IgG antibody response was suppressed only by early feeds of high-dose OVA (Days 1-5). Medium-dose OVA fed on Day 14 or low-dose OVA fed at any stage after immunization enhanced the IgG antibody response. In contradistinction, systemic delayed-type hypersensitivity responses (DTH) were usually suppressed by early feeds of high or medium doses of OVA but never after feeding low-dose OVA. The results suggest that systemic DTH and IgG antibody responses to oral antigen are subject to different control mechanisms in previously primed animals. Such responses depend on the immune status of the animal and are controlled by antigen dose, time and frequency of feeding. The immunological effects observed are also demonstrable following adoptive transfer of spleen cells collected 14 days after multiple feeds of high-dose OVA to immunized mice. Our findings suggest that oral hyposensitization after systemic immunization is regulated by (suppressor) spleen cells which are activated by gut-processed antigen.     

Oral tolerance in protein-deprived mice. I. Profound antibody tolerance but impaired DTH tolerance after antigen feeding.

We have examined the effects of protein deprivation on the induction of oral tolerance for systemic antibody and DTH responses to the protein antigen ovalbumin (OVA). Mice were fed 4% or 24% protein diets from weaning and given a single feed of OVA 2 weeks later (short-term deprivation) or after 10 weeks (long-term deprivation). Tolerance for serum antibody responses was more profound in protein-deprived animals than in 24% protein-fed control groups. Conversely, tolerance for DTH responses was impaired in protein-deprived mice. This was demonstrated both for short-term deprivation, where nutritional rehabilitation after OVA feeding was necessary to demonstrate this effect on DTH, and for long-term deprivation. Furthermore, the effect of short-term deprivation on tolerance for DTH responses was similar to that observed after cyclophosphamide pretreatment of OVA-fed mice. Protein deprivation has disparate effects on the humoral and cell-mediated limbs of oral tolerance, and our results support the hypothesis that this regime selectively depletes a population of suppressor T cells responsible for the fine control of DTH tolerance.     

Gliadin presented via the gut induces oral tolerance in mice.

When an antigen is first presented via the gut, either priming or suppression of the systemic immune response may result. Many factors influence the outcome, including physico-chemical properties of the antigen. The aim of this study is to establish if wheat gliadin behaves as an oral immunogen or tolerogen. Mice reared on a gluten-free diet were fed gliadin, either as wheat flour in a standard rodent diet or as the purified molecule. Immune status (tolerant or sensitized) was then defined by measuring specific systemic immune responses after parenteral immunization of gliadin-fed and control mice. A single feed of 25 or 125 mg of purified gliadin resulted in a dose-dependent suppression of both cell-mediated and humoral immune responses. Similar oral tolerance was achieved by feeding mice with a gluten-containing diet for a week. Finally, mice reared on a normal, gluten-containing diet showed evidence of established oral tolerance, with significantly lower systemic immune response to gliadin than mice reared on gluten-free diet. These results indicate that gliadin is an effective oral tolerogen. In vivo studies on the immunogenicity of gliadins should be conducted in animals from gluten-free colonies.     

Suppression of an established DTH response to ovalbumin in mice by feeding antigen after immunization

Experiments were designed to examine whether systemic delayed-type hypersensitivity responses (DTH) to ovalbumin (OVA) can be suppressed when antigen is fed after immunization, and to investigate the immunological mechanisms involved. A single 25 mg feed of OVA given 7 or 14 days after immunization with OVA in complete Freund's adjuvant (CFA) suppressed the DTH response of BDF1 mice, but had no significant effect on the serum IgG antibody response. DTH suppression was greatest when antigen was fed soon after immunization, and became less pronounced as the time interval between feeding and immunization increased. The phenomenon was also demonstrated in mice of the BALB/c strain. Cell transfer experiments suggested that the post-immunization suppression was not due to a population of suppressor cells that have been described previously in association with classical oral tolerance for DTH. We conclude that there are separate and distinct mechanisms for the prevention of induction of DTH by antigen feeding in naive mice and the suppression of expression of DTH in sensitized animals.     

Priming of systemic and local delayed-type hypersensitivity responses by feeding low doses of ovalbumin to mice.

We have examined the effects on both systemic and intestinal immunity of feeding different doses of ovalbumin (OVA) to mice. A single feed of doses of more than 1 mg OVA produced significant suppression of subsequent delayed-type hypersensitivity (DTH) and IgG antibody responses. Feeding 100 micrograms-1 mg OVA had no net effect on systemic immunity, but mice fed 10-50 micrograms OVA had consistently enhanced systemic DTH responses when immunized subsequently with OVA in adjuvant. Oral challenge of these mice with OVA produced alterations in mucosal architecture and in intra-epithelial lymphocyte counts, consistent with the presence of an intestinal DTH response. Similar changes were not found in mice fed tolerogenic doses of OVA. Although feeding low doses of OVA primed both systemic and intestinal DTH responses, this had no effect on serum IgG responses and very little systemic DTH could be revealed in OVA-fed mice without systemic challenge with OVA in adjuvant. We conclude that feeding certain low doses of protein antigens can induce priming of local and systemic DTH responses rather than the immune tolerance which is normally found. The development of clinical food hypersensitivities may be highly dependent on the dose of dietary antigen at the time of first encounter.     

HUMORAL IMMUNE-MEDIATED ACUTE, ANTIGEN-INDUCED ARTHRITIS IN RATS IS SUPPRESSED BY THE INDUCING ANTIGEN ADMINISTERED ORALLY BEFORE, BUT NOT AFTER, IMMUNIZATION

Acute ovalbumin-induced arthritis (OIA), which is mediated by Arthus reaction to ovalbumin (OVA) in the joint space, can be induced by immunization of rats with OVA followed by the intraarticular injection of OVA. Because oral administration of antigen induces immunological unresponsiveness, we studied for the first time the effects of oral administration of OVA on acute OIA. The oral administration of OVA before immunization significantly suppressed the development of acute OIA, in accordance with decreases in both the anti-OVA IgG antibody production and in vitro lymphocyte proliferative responses to OVA. However, the oral administration of OVA after immunization did not show any decrease in antibody production or in vitro lymphocyte proliferation to OVA, or in the severity of acute OIA. These results indicate that the induction of oral tolerance to OVA in OIA is possible by oral administration of OVA before, but not after, immunization with the antigen. This supports the concept of using antigen feeding as a treatment for certain humoral immune-mediated diseases.     

Irradiated mice lose the capacity to ''process'' fed antigen for systemic tolerance of delayed-type hypersensitivity.

'Intestinal antigen processing' is a function of the gastro-intestinal tract whereby shortly after an animal has been fed an immunogenic protein antigen, such as ovalbumin (OVA), a tolerogenic form of the protein is generated and can be detected in the circulation. The effect of damage to the intestinal epithelium on the processing of OVA has been examined in lethally irradiated mice. Irradiated animals were fed 25 mg OVA and their serum collected 1 h later. When this serum was transferred intraperitoneally into naive recipient mice, this did not induce the typical suppression of systemic delayed-type hypersensitivity. Results were similar when the serum donors were at 2 days after irradiation, with crypt hypoplasia, and at 5 days after irradiation when there was reactive crypt hyperplasia. However reconstitution of donors with normal spleen cells immediately after irradiation restored their capacity to generate a tolerogenic form of the antigen. Immunoreactive OVA was detected by ELISA in both tolerizing and non-tolerizing sera, and the immunological properties of these sera were not related to serum levels of OVA after feeding. Thus subtle immunochemical alterations in the nature of a protein antigen are likely to be more important than the quantity of absorbed antigen, in influencing systemic cell-mediated immune responses after feeding. The lack of generation of a tolerogenic form of the protein in irradiated mice, unrelated to the pattern of epithelial cell kinetics, and the restoration of this function by normal spleen cells, suggests that lymphoid cells may be involved in the phenomenon of antigen processing.     

Mucosal and Systemic Immune Responses in Humans after Primary and Booster Immunizations with Orally Administered Invasive and Noninvasive Live Attenuated Bacteria

The mucosal and systemic immune responses after primary and booster immunizations with two attenuated live oral vaccine strains derived from a noninvasive (Vibrio cholerae) and an invasive (Salmonella typhi) enteric pathogen were comparatively evaluated. Vaccination with S. typhi Ty21a elicited antibody-secreting cell (ASC) responses specific for S. typhi O9, 12 lipopolysaccharide (LPS), as well as significant increases in levels of immunoglobulin G (IgG) and IgA antibodies to the same antigen in serum. A strong systemic CD4(+) T-helper type 1 cell-mediated immune (CMI) response was also induced. In contrast to results with Ty21a, no evidence of a CMI response was obtained after primary immunization with V. cholerae CVD 103-HgR in spite of the good immunogenicity of the vaccine. Volunteers who received a single dose of CVD 103-HgR primarily developed an IgM ASC response against whole vaccine cells and purified V. cholerae Inaba LPS, and seroconversion of serum vibriocidal antibodies occurred in four of five subjects. Serum IgG anti-cholera toxin antibody titers were of lower magnitude. For both live vaccines, the volunteers still presented significant local immunity 14 months after primary immunization, as revealed by the elevated baseline antibody titers at the time of the booster immunization and the lower ASC, serum IgG, and vibriocidal antibody responses after the booster immunization. These results suggest that local immunity may interfere with colonization of the gut by both vaccine strains at least up to 14 months after basis immunization. Interestingly, despite a low secondary ASC response, Ty21a was able to boost both humoral (anti-LPS systemic IgG and IgA) and CMI responses. Evidence of a CMI response was also observed for one of three volunteers given a cholera vaccine booster dose. The direct comparison of results with two attenuated live oral vaccine strains in human volunteers clearly showed that the capacity of the vaccine strain to colonize specific body compartments conditions the pattern of vaccine-induced immune responses.     

Divergent effects of bacterial lipopolysaccharide on immunity to orally administered protein and particulate antigens in mice.

We have investigated whether bacterial lipopolysaccharide (LPS) influences immune responses to dietary protein antigens in experimental animals. Simultaneous intravenous administration of LPS to normal mice fed ovalbumin (OVA) prevented the induction of tolerance for serum IgG antibody responses but did not alter the tolerance of systemic delayed-type hypersensitivity (DTH). In addition, exogenous LPS did not enhance the ability of spleen accessory cells to present OVA to primed T cells. LPS-unresponsive C3H/HeJ mice developed full tolerance of both humoral and cell-mediated immunity after feeding a range of doses of OVA that was equal in degree and persistence to that seen in normal, congenic C3H/HeOla mice and also had normal antigen-presenting cell (APC) activity for OVA. In contrast, C3H/HeJ mice were primed by feeding SRBC instead of developing the systemic tolerance found in normal C3H mice. Our results indicate the complexity of mechanisms that may regulate systemic immunity to orally administered antigens of different forms. Nevertheless, LPS does not modulate DTH responses to fed OVA and does not enhance APC activity, and we conclude that bacterial LPS may be unable to influence hypersensitivity to dietary proteins in man.     

Orally administered OVA/CpG-ODN induces specific mucosal and systemic immune response in young and aged mice

We have previously demonstrated that subcutaneously administered ovalbumin (OVA) plus synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG-ODN) as adjuvant stimulate cellular and humoral immunity and promote T helper cell type 1 differentiation in aged mice. The present study assessed the ability of CpG-ODN to induce an OVA-specific immune response after oral immunization in young (3-month-old) and aged (18-month-old) BALB/c mice. Oral OVA/CpG-ODN immunization induces a similar OVA-specific T cell-proliferative response (in mucosal and systemic tissues), immunoglobulin G (IgG) in plasma, and IgA in intestinal washes in both groups of ages. The OVA-specific humoral immune response observed in aged mice was similar to the one observed in young mice, peaking at day 7 after the last oral immunization and was present over 40 days after the last oral immunization. The pattern of cytokines released in culture supernatants in both groups of mice was similar, with specific interferon-gamma secretion in the absence of interleukin-5 responses. These results provide evidence that orally administered OVA/CpG-ODN induces a young-like, specific, immune response against OVA in aged mice, showing that CpG-ODN might be used as a mucosal adjuvant during aging.     

The pH-Sensitive Fusogenic 3-Methyl-Glutarylated Hyperbranched Poly(Glycidol)-Conjugated Liposome Induces Antigen-Specific Cellular and Humoral Immunity

We examined the ability of a novel liposome, surface modified by 3-methyl-glutarylated hyperbranched poly(glycidol) (MGlu-HPG), to enhance antigen-specific immunity in vitro and in vivo and to function as a vaccine carrier. Murine bone marrow-derived dendritic cells took up ovalbumin (OVA) encapsulated in MGlu-HPG-modified liposomes more effectively than free OVA or OVA encapsulated in unmodified liposomes. Immunization of mice with OVA-containing MGlu-HPG-modified liposomes induced antigen-specific splenocyte proliferation and production of gamma interferon (IFN-γ) more strongly than did immunization with free OVA or OVA encapsulated in unmodified liposomes. The immune responses induced by OVA encapsulated in MGlu-HPG-modified liposomes were significantly suppressed by addition of anti-major histocompatibility complex (MHC) class I and class II monoclonal antibodies, indicating the involvement of antigen presentation via MHC class I and II. Furthermore, delayed-type hypersensitivity responses and OVA-specific antibodies were induced more effectively in mice immunized with OVA encapsulated by MGlu-HPG-modified liposomes than with unencapsulated OVA or OVA encapsulated in unmodified liposomes. These results suggested that MGlu-HPG-modified liposomes effectively induced both cell-mediated and humoral immune responses. Collectively, this study is the first to demonstrate the induction of both cell-mediated and humoral immune responses in vivo by MGlu-HPG-modified liposomes.     

Maternal determinants of neonatal immune response to ovalbumin: effect of breast feeding on development of anti-ovalbumin antibody in the neonate

Inbred Brown-Norway female rats were immunized intraperitoneally with ovalbumin (OVA) or sham-immunized 14 days before and 10 days after mating. In subsequent studies with OVA, babies fed by immunized mothers, regardless of whether they were born from immunized or sham-immunized mothers, showed suppression of IgG, IgM and IgE anti-OVA responses. In additional studies, these babies developed OX-8-positive but W3/25-negative phenotypic suppressor T-cells specific for anti-OVA antibody production. However, these regulatory cells did not react with OVA itself when tested for in vitro proliferative response to OVA. Subsequent immunization of the neonates with OVA appeared to abrogate suppression of IgG and IgM antibody responses. However, maternally induced suppression of IgE persisted and was not influenced by subsequent immunization.     

A genetically determined lack of oral tolerance to ovalbumin is due to failure of the immune system to respond to intestinally derived tolerogen

In this study we have examined whether differences between mouse strains in the induction of tolerance after feeding ovalbumin (OVA) are due to differences in intestinal processing of OVA or are determined by the systemic immune system. Compared with major histocompatibility complex (MHC)-congenic BALB/c mice, BALB/B mice develop much less tolerance of systemic delayed-type hypersensitivity (DTH) and humoral immunity after feeding OVA and this defect is also expressed partially in (BALB/B x BALB/c)F1 animals. Serum taken from either BALB/c or BALB/B mice fed OVA 1 h before produced significant suppression of systemic DTH responses in BALB/c, but not in BALB/B mice. Although OVA-fed BALB/B serum was slightly less tolerogenic than BALB/c serum, we conclude that the defective induction of oral tolerance in BALB/B mice is due primarily to a MHC-influenced defect with the immune system. These findings support the idea that clinical food-sensitive enteropathy reflects an immune response gene-controlled defect in tolerance to dietary proteins.     

Oral administration of bovine whey proteins to mice elicits opposing immunoregulatory responses and is adjuvant dependent

Most studies investigating the induction of oral tolerance (OT) use purified proteins such as ovalbumin (OVA), bovine serum albumin (BSA) and beta-lactoglobulin (beta-LG). Little information is available regarding the induction of OT to a protein mixture, e.g. cow's milk. In this study we compared the regulatory mechanisms induced after the oral administration of a whey protein concentrate (WP) derived from cow's milk following immunization with two different adjuvants, complete Freund's adjuvant (CFA) and alum. OVA was used as a control antigen. Animals were given a single feed of these proteins at an equivalent dose of 1 mg/g body weight before they were immunized seven days later with the antigen in Freund's adjuvant or alum. Delayed type hypersensitivity (DTH) responses were suppressed by both a feed of WP and OVA after immunization with CFA. However, only OVA feeding suppressed antigen specific IgG responses. In an attempt to investigate whether WP would tolerize the more susceptible IgE responses, alum immunization replaced CFA as the adjuvant used for systemic immunizations. WP, after a single feed, significantly primed for DTH and IgE responses indicating oral sensitization to WP. In contrast, OVA suppressed DTH, IgE and IgG responses. Antigen specific proliferation of mononuclear cells was suppressed in mice fed OVA, but primed in those fed with WP. In addition cells taken from sensitized mice fed WP up-regulated levels of specific interleukin (IL) -4, -10 and -12 in vitro whereas these cytokines were suppressed in cultures from tolerant WP fed mice. Global suppression was obtained in cultures from tolerant OVA fed mice. TGF-beta was not detected in draining PLN cell cultures of either tolerant or sensitized mice. These data suggest that a whey protein mixture induces divergent responses following immunization with either CFA or alum despite being fed at an identical dose. We suggest that that the choice of the adjuvant may determine the immunoregulatory outcome and this is also reflected by the systemic cytokine profile.     

Induction of mucosal and systemic immune responses by immunization with ovalbumin entrapped in poly(lactide-co-glycolide) microparticles.

We have examined the range of mucosal and systemic immune responses induced by oral or parenteral immunization with ovalbumin (OVA) entrapped in poly(D,L-lactide-co-glycolide) (PLG) microparticles. A single subcutaneous immunization with OVA-PLG primed significant OVA-specific IgG and delayed-type hypersensitivity (DTH) responses. The DTH responses were of similar magnitude to those obtained using immunostimulating complexes (ISCOMS) as a potent control adjuvant, although ISCOMS stimulated higher serum IgG responses. Both vectors also primed OVA-specific in vitro proliferative responses in draining lymph node cells following a single immunization and strong OVA-specific CTL responses were found after intraperitoneal (i.p.) immunization. ISCOMS were more efficient in inducing cytotoxic T lymphocytes (CTL), requiring much less antigen and only ISCOMS could stimulate primary OVA-specific CTL responses in the draining lymph nodes. Multiple oral immunizations with OVA in PLG microparticles or in ISCOMS resulted in OVA-specific CTL responses and again ISCOMS seemed more potent as fewer feeds were necessary. Lastly, multiple feeds of OVA in PLG microparticles generated significant OVA-specific intestinal IgA responses. This is the first demonstration that PLG microparticles can stimulate CTL responses in vivo and our results highlight their ability to prime a variety of systemic and mucosal immune responses which may be useful in future oral vaccine development.     

Respiratory Tract Cell-Mediated Immunity: Comparison of Primary and Secondary Response

A secondary local and splenic cell-mediated immune response was observed and compared to the primary response. Previous studies have demonstrated cell-mediated immunity (CMI) by lymphocytes from bronchopulmonary washings and have shown that its appearance is to a large extent inedpendent of splenic CMI. This study evaluated the secondary as compared to the primary response, with respect to both cellular and humoral immune responses. Guinea pigs were immunized with influenza virus vaccine either nasally or parenterally, booster immunizations were given by the same route, and animals were killed at various times after immunization or booster. The inhibition of macrophage migration was used to assess CMI. As in previous studies, local application of antigen led to mainly local appearance of CMI, whereas parenteral immunization led to mainly systemic CMI. Both pulmonary and splenic lymphocytes showed an inhibition of macrophage migration that appeared 2 to 3 days sooner after the booster, as compared to the primary immunization. There was no evidence, however, for the earlier production or increased amount of antibody in the bronchial secretions in the boosted animals. The results suggest that pulmonary as well as splenic T lymphocytes exhibit memory, but that pulmonary B lymphocytes do not.     

Suppression of delayed-type hypersensitivity mediated by macrophage-like cells in mice with experimental liver injury.

Systemic hyporesponsiveness after ingestion of a protein antigen (oral tolerance) depends on antigen processing by the gut and the actions of immunoregulatory T cells. We have examined the effects of a graft-versus-host reaction (GvHR) on oral tolerance, since both immune status and intestinal function are altered in GvHR. The GvHR was induced in unirradiated (CBA X BALB/c)F1 mice by intraperitoneal injection of CBA spleen cells. The tolerance of systemic humoral immunity and of delayed-type hypersensitivity normally found in mice fed 25 mg ovalbumin (OVA) was partially abrogated from 1 to 3 weeks after induction of the GvHR. In addition, mice with GvHR had a persistent enhancement of systemic immunity to OVA, and this was associated with an augmented ability of spleen cells to present OVA to primed T cells. The phagocytic activity of the reticuloendothelial system, as established by carbon clearance tests, was not altered by the GvHR. These findings suggest that enhanced antigen-presenting cell activity interferes with the induction of oral tolerance, and may be another pathogenetic mechanism of intestinal hypersensitivity disease.     

Immune-stimulating complexes containing Quil A and protein antigen prime class I MHC-restricted T lymphocytes in vivo and are immunogenic by the oral route

Induction of all forms of protective immunity by oral immunization with subunit vaccines is an ideal goal for the development of novel vaccines, but creates several theoretical problems from the point of view of antigen processing mechanisms. We show here that incorporation of the protein antigen ovalbumin (OVA) in lipophilic immune-stimulating complexes (ISCOMS) induces very strong primary immune responses in mice and requires very small amounts of antigen. OVA ISCOMS were particularly efficient at stimulating T-cell-mediated immunity in vivo, including delayed-type hypersensitivity (DTH) and potent class I major histocompatibility complex (MHC)-restricted cytotoxic T-cell responses. Furthermore, unlike native protein, OVA in ISCOMS was immunogenic when given orally. Thus, ISCOMS seem to allow protein to enter both the endogenous and exogenous pathways of antigen processing and overcome the usual induction of tolerance after feeding antigen. ISCOMS could provide potentially useful adjuvants for the development of oral subunit vaccines.     

Humoral immune responses to model antigen co-delivered with biomaterials used in tissue engineering

A model shed antigen, ovalbumin (OVA), was co-delivered with polymeric biomaterial carrier vehicles in C57BL6 mice to test whether the presence of the biomaterial acted as an adjuvant in the immune response towards the associated antigen. The biomaterials tested were non-biodegradable polystyrene microparticles and biodegradable 50:50 or 75:25 poly(lactic-co-glycolic acid) (PLGA) microparticles or scaffolds. For each biomaterial carrier vehicle, to assess the resulting time-dependent systemic humoral immune response towards the co-delivered OVA, the OVA-specific IgG concentration and isotypes (IgG2a or IgG1, indicating a predominant Th1 or Th2 response, respectively) were determined using ELISA. OVA co-delivered with biomaterial carrier vehicles supported a moderate humoral immune response that was maintained for the 18-week duration of the experiment. This humoral immune response was primarily Th2 helper T cell-dependent as indicated by the predominant IgG1 isotype. Furthermore, this humoral immune response was not material chemistry-dependent within the material set tested here. With the presence of the biomaterial resulting in an enhancement of the humoral immune response to co-delivered antigen, it appears that the biomaterial acts as an adjuvant in the development of an adaptive immune response to co-delivered antigen.