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.     

Simultaneous presentation and cross-presentation of immune-stimulating complex-associated cognate antigen by antigen-specific B cells

We demonstrate that uptake of oligomeric cognate antigen (OVA-hen egg lysozyme, OVA-HEL) alone or incorporated in immune-stimulating complexes (ISCOMS) facilitates presentation and simultaneous cross-presentation of OVA by HEL-specific B cells in vitro. HEL-specific B cells stimulated CD8(+) T cell responses in vitro to the same extent as bone marrow-derived dendritic cells. Cross-presentation by specific B cells required endosomal acidification, proteasomal processing and classical MHC class I/peptide transport. Specific B cells also acquired both antigens rapidly in vivo and presented them to CD4(+) T cells. However, only HEL-specific B cells from OVA-HEL ISCOMS-immunised mice could cross-present OVA to naive OVA-specific CD8(+) T cells. Antigen-specific B cells were also activated selectively by OVA-HEL ISCOMS in vitro and importantly, the presence of HEL-specific B cells promoted the persistence of clonal expansion of OVA-specific CD8(+) T cells after in vivo immunisation with OVA-HEL ISCOMS. These results demonstrate preferential MHC class I and class II processing of cognate antigen incorporated in ISCOMS by specific B cells in vitro and in vivo, highlighting the ability of ISCOMS to target B cells and offering novel insights into the role of B cells in cross-presentation to CD8(+) T cells.     

Preservation of mucosal and systemic adjuvant properties of ISCOMS in the absence of functional interleukin-4 or interferon-gamma.

Adjuvants are a critical component of non-viable vaccine vectors, particularly for those to be used via mucosal routes. Although most adjuvants act by inducing local inflammatory responses, the molecular basis of many of these effects is unclear. Here we have investigated whether interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) are required for the induction of local and systemic immune responses by oral and parenteral administration of ovalbumin (OVA) in immune stimulating complexes (ISCOMS), a potent mucosal adjuvant vector. Our results show that after oral or systemic immunization with OVA ISCOMS, IL-4 knockout (IL4KO) and IFN-gamma receptor knockout (IFN-gamma RKO) mice develop an entirely normal range of immune responses including delayed-type hypersensitivity (DTH), serum immunoglobulin G (IgG) antibodies, T-cell proliferation and cytokine production, class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) activity and intestinal IgA antibodies. These responses were of a similar magnitude to those found in the wild-type mice, indicating that the immunogenicity of ISCOMS is not influenced by the presence of IL-4 or IFN-gamma and emphasizing the potential of ISCOMS as widely applicable mucosal adjuvants.     

Oral vaccination with immune stimulating complexes

There is a need for non-living adjuvant vectors which will induce a full range of local and systemic immune responses to orally administered purified antigens. Here we describe our experience with lipophilic immune stimulating complexes (ISCOMS) containing the saponin adjuvant Quil A. When given orally, ISCOMS containing the model protein antigen ovalbumin (OVA) induce a wide range of systemic immune responses, including Th1 and Th2 CD4 dependent activity, class I MHC restricted cytotoxic T-cell responses and local production of secretory IgA antibodies. More recent results indicate that ISCOMS may act partly by enhancing the uptake of protein from the gut. In addition, intraperitoneal injection of ISCOMS recruits and activates many components of the innate immune system. including neutrophils, macrophages, and dendritic cells. In parallel, there is increased production of nitric oxide (NO), reactive oxygen intermediates (ROI), interleukins (IL) 1, 6, 12, and gamma interferon (gammaIFN). Of these factors, only IL12 is essential for the immunogenicity of ISCOMS in vivo, as mucosal and systemic responses to ISCOMS are reduced in IL12KO mice, but not in IL4KO, IL6KO, inducible NO synthase (iNOS) KO, or gammaIFN receptor KO mice. We propose that ISCOMS act by targetting antigen and adjuvant to macrophages and/or dendritic cells. This pathway may be amenable to exploitation for vaccine development, especially if combined with another vector with a different mucosal adjuvant profile, such as cholera toxin.     

Phosphatidylinositol mannosides are efficient mucosal adjuvants

The development of defined sub-unit vaccines requires the inclusion in the vaccine of an immunological adjuvant. The most important property of adjuvants for vaccines aimed at inducing optimal protection against intracellular bacteria such as Mycobacterium tuberculosis or M. bovis is the ability to enhance cell-mediated immunity, specifically Th1 responses. In this paper, we describe a system where transgenic mice expressing a high proportion of T cells specific for an ovalbumin (OVA) peptide are used to assess the ability of a novel class of adjuvants to positively modulate cell-mediated immune responses. Defined fractions containing purified native or synthetic phosphatidylinositol mannosides (PIMs) from mycobacteria were assessed for their adjuvant activities in response to the model antigen (OVA). Purified PIM preparations given to mice with OVA by the subcutaneous route were shown to elicit an enhanced release of interferon-gamma (IFN-gamma) in cellular responses to OVA peptide in vitro. Very little interleukin-4 (IL-4) was released by cells from mice immunized with PIMs and OVA, whereas cells from animals immunized with complete Freund's adjuvant (CFA) and OVA released IL-4 as well as IFN-gamma. Synthetic preparations of PIM2 and PIM4 also acted as adjuvants in the mouse model studied. In addition, PIM preparations were shown to generate an efficient cell-mediated immune response to OVA, when the antigen/adjuvant preparations were administered via the oral route or intranasal route. PIM preparations elicited substantial release of interleukin-12 (IL-12) from dendritic cells (DCs). These data suggest that purified or synthetic PIMs act as adjuvants when administered at mucosal surfaces and represent a new class of adjuvants for mucosal immunization against intracellular pathogens.     

Phosphatidylinositol Mannosides are Efficient Mucosal Adjuvants

The development of defined sub-unit vaccines requires the inclusion in the vaccine of an immunological adjuvant. The most important property of adjuvants for vaccines aimed at inducing optimal protection against intracellular bacteria such as Mycobacterium tuberculosis or M. bovis is the ability to enhance cell-mediated immunity, specifically Th1 responses. In this paper, we describe a system where transgenic mice expressing a high proportion of T cells specific for an ovalbumin (OVA) peptide are used to assess the ability of a novel class of adjuvants to positively modulate cell-mediated immune responses. Defined fractions containing purified native or synthetic phosphatidylinositol mannosides (PIMs) from mycobacteria were assessed for their adjuvant activities in response to the model antigen (OVA). Purified PIM preparations given to mice with OVA by the subcutaneous route were shown to elicit an enhanced release of interferon-gamma (IFN-γ) in cellular responses to OVA peptide in vitro. Very little interleukin-4 (IL-4) was released by cells from mice immunized with PIMs and OVA, whereas cells from animals immunized with complete Freund's adjuvant (CFA) and OVA released IL-4 as well as IFN-γ. Synthetic preparations of PIM2 and PIM4 also acted as adjuvants in the mouse model studied. In addition, PIM preparations were shown to generate an efficient cell-mediated immune response to OVA, when the antigen/adjuvant preparations were administered via the oral route or intranasal route. PIM preparations elicited substantial release of interleukin-12 (IL-12) from dendritic cells (DCs). These data suggest that purified or synthetic PIMs act as adjuvants when administered at mucosal surfaces and represent a new class of adjuvants for mucosal immunization against intracellular pathogens.     

Yersinia pestis caf1 variants and the limits of plague vaccine protection

Yersinia pestis, the highly virulent agent of plague, is a biological weapon. Strategies that prevent plague have been sought for centuries, and immunization with live, attenuated (nonpigmented) strains or subunit vaccines with F1 (Caf1) antigen is considered effective. We show here that immunization with live, attenuated strains generates plague-protective immunity and humoral immune responses against F1 pilus antigen and LcrV. Y. pestis variants lacking caf1 (F1 pili) are not only fully virulent in animal models of bubonic and pneumonic plague but also break through immune responses generated with live, attenuated strains or F1 subunit vaccines. In contrast, immunization with purified LcrV, a protein at the tip of type III needles, generates protective immunity against the wild-type and the fully virulent caf1 mutant strain, in agreement with the notion that LcrV can elicit vaccine protection against both types of virulent plague strains.     

A role for dendritic cells in the priming of antigen-specific CD4+ and CD8+ T lymphocytes by immune-stimulating complexes in vivo

Immune-stimulating complexes (ISCOMS) are adjuvant vectors which are unusual in being able to prime both CD4(+) and CD8(+) T cells by parenteral and mucosal routes. However, their mode of action is unclear and to define better the cellular interactions involved we have studied the ability of ISCOMS containing ovalbumin (OVA) to prime TCR transgenic CD4(+) or CD8(+) T cells in vivo. Immunization with OVA ISCOMS caused activation and clonal expansion of CD4(+) and CD8(+) T cells in the T cell areas of the draining lymph nodes, followed by the migration of both CD4(+) and CD8(+) T cells into the B cell follicle. The T cells were primed to proliferate and secrete IFN-gamma after re-stimulation in vitro with the appropriate OVA peptide and CD8(+) T cell priming occurred in the absence of CD4(+) T cells. Increasing the number of dendritic cells (DC) in vivo with flt3 ligand augmented the expansion and activation of the OVA-specific T cells, particularly CD8(+) T cells. These studies indicate DC play a central role in the priming of both CD4(+) and CD8(+) T cells in vivo, and suggest that an ability to target DC may allow ISCOMS to be powerful vaccine vectors for stimulating protective immunity.     

Prolonged oral treatment with low doses of allergen conjugated to cholera toxin B subunit suppresses immunoglobulin E antibody responses in sensitized mice

BACKGROUND: Oral tolerance is a long recognized method for inducing systemic immunological tolerance. However, large doses of antigen and frequent administrations are often required. By linking the antigen to the nontoxic mucosa-binding B subunit of cholera toxin (CTB), the required amount can be dramatically reduced. We have previously shown that mucosal administration of small amounts of antigens coupled to CTB can suppress peripheral Th1 cell-reactivity and associated inflammatory immunopathology in both naive and systemically-immunized animals. Induction of oral tolerance by repeated feeding of relatively small doses of antigen has, in some cases been shown to involve the generation of regulatory Th2-like CD4+ T cells, and hence could promote rather than suppress type I immunoglobulin (Ig) E-mediated allergic responses. OBJECTIVES: We examined whether oral prophylactic or therapeutic administration of a model allergen coupled to CTB would modulate allergen-specific IgE responses in high IgE responder Balb/c mice. METHODS: Ovalbumin (OVA) was used as a model allergen. Mice were treated perorally with free or CTB-coupled OVA before or after systemic priming with alum-adsorbed OVA. Allergen-specific IgE levels in serum were measured with the passive cutaneous anaphylaxis test at various time-points. RESULTS: Oral administration of a single low dose of CTB-linked OVA, prior to systemic sensitization and challenge with OVA, suppressed allergen-specific serum IgE antibody responses. Treatment with comparable doses of free OVA was much less effective. Most importantly, oral treatment with CTB-OVA conjugate could also suppress an already initiated IgE antibody response, but to achieve such a 'therapeutic effect', administration of multiple low doses of conjugate over a long time was required. Oral treatment with CTB-OVA conjugate could also effectively suppress antigen-specific Th1-mediated delayed-type hypersensitivity. Thus treatment with a CTB-conjugated model allergen can affect a broad range of T-cell-driven immune responses, even in antigen-experienced animals. CONCLUSION: These results may impact on the development of therapeutic vaccines against type I allergies.     

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.     

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.     

Oral immunization with a recombinant malaria protein induces conformational antibodies and protects mice against lethal malaria

The increasing death toll from malaria, due to the decreasing effectiveness of current prophylactic and therapeutic regimens, has sparked a search for alternative methods of control, such as vaccines. Although several single proteins have shown some promise as subunit vaccines against sexual blood stages in experimental systems, it is clear that multicomponent vaccines are required. Many logistic difficulties make such an approach prohibitively expensive. In an effort to try to overcome some of these issues, we examined the possibility of oral immunization as a route for inducing host protective immunity. We report here that oral feeding of a malaria protein induced serum antibody levels similar to those induced by intraperitoneal immunization with Freund's adjuvant. Further, responses to conformational epitopes were induced. In the rodent challenge system, significant levels of protection to lethal challenge with malaria were induced in mice. The protective efficacy was highly correlated with antibody levels, which depended on the antigen dosage and required cholera toxin subunit B as an oral adjuvant. These findings offer new approaches to the development of a malaria vaccine and provide justification for the investigation of transgenic plants as a means of vaccine delivery.     

Induction of Systemic and Mucosal Immune Responses Following Immunization with Somatostatin-Avidin Complexes Incorporated Into Iscoms

Synthetic, biotinylated somatostatin-14 (Somatotropin Release-Inhibiting Factor; SRIF) was conjugated to avidin, and the resulting complex incorporated into immune-stimulating complexes (ISCOMS). The ISCOMS were used to study the systemic and mucosal immune responses induced by parenteral and gastrointestinal vaccination. Mice were immunized by intraperitoneal (IP) and intragastric (IG) routes and subsequently by either IP or IG secondary immunizations (groups-IP/IP; IP/IG; IG/IG). Antigen specific IgG and IgA antibody secreting cells (ASC) from the spleen, mesenteric lymph nodes (MLN) and Peyer's patches (PP's) were studied by an enzyme-linked immunospot assay (ELISPOT). Specific proliferative responses of spleen cells to avidin and to SRIF were measured.

Immunization IP/IP evoked the highest serum IgG levels to avidin and to SRIF as well as the highest numbers of splenic IgG isotype ASC. The greatest IgA response in MLN and PP's was induced by IP/IG immunization. Only marginal mucosal immunity and no splenic cell specific proliferative responses were found by IG/IG immunization.

These results indicate that ISCOMS are an effective delivery system for protein-peptide antigens. The ISCOMS system described elicited systemic and mucosal antibody immune responses, and primed specific proliferative response when administered IP/IG. This offers another approach for the design and delivery of mucosally administered peptide vaccines.     

Targeting the allergen to oral dendritic cells with mucoadhesive chitosan particles enhances tolerance induction

Background: Sublingual immunotherapy (SLIT) efficacy could be improved by formulations facilitating allergen contact with the oral mucosa and uptake by antigen‐presenting cells (APCs). Methods: Two types of chitosan microparticles, differing in size and surface charge, were tested in vitro for their capacity to improve antigen uptake and presentation by murine bone marrow‐derived dendritic cells (BMDCs) or purified oral APCs. T‐cell priming in cervical lymph nodes (LNs) was assessed by intravenous transfer of carboxyfluorescein diacetate succinimidyl ester‐labelled ovalbumin (OVA)‐specific CD4+ T cells and flow cytometry analysis. Ovalbumin‐sensitized BALB/c mice were treated sublingually with soluble or chitosan‐formulated OVA twice a week for 2 months. Airway hyperresponsiveness (AHR), lung inflammation and T‐cell responses in cervical and mediastinal LNs were assessed by whole‐body plethysmography, lung histology and Cytometric Bead Array technology, respectively. Results: Only a mucoadhesive (i.e. highly positively charged) and microparticulate form of chitosan enhances OVA uptake, processing and presentation by murine BMDCs and oral APCs. Targeting OVA to dendritic cells with this formulation increases specific T‐cell proliferation and IFN‐γ/IL‐10 secretion in vitro, as well as T‐cell priming in cervical LNs in vivo. Sublingual administration of such chitosan‐formulated OVA particles enhances tolerance induction in mice with established asthma, with a dramatic reduction of both AHR, lung inflammation, eosinophil numbers in bronchoalveolar lavages, as well as antigen‐specific Th2 responses in mediastinal LNs. Conclusions: Mucoadhesive chitosan microparticles represent a valid formulation for sublingual allergy vaccines.     

Effect of neonatal sublingual vaccination with native or denatured ovalbumin and adjuvant CpG or cholera toxin on systemic and mucosal immunity in mice

Sublingual immunotherapy has been applied for allergic diseases, but whether sublingual immunization in neonates can prevent sensitization has not been studied. In this study, we evaluate the effect of neonatal sublingual vaccination with native or denatured allergens alone or plus adjuvant on allergy prevention. Newborn BALB/ c mice were sublingually vaccinated daily for the first 3 days with native or denatured ovalbumin (OVA) only, or combined adjuvant CpG or cholera toxin (CT). They were sensitized with OVA adsorbed onto alum 7 weeks after the last vaccination. Specific secretory IgA antibody responses were readily induced by neonatal vaccination with antigen plus CpG or CT, but not with antigen alone. Whereas vaccination with denatured OVA plus CpG markedly enhanced T helper 1 (Th1) responses and inhibited IgE production, vaccination with denatured OVA plus CT increased cervical lymph node cell production of interleukin-4 (IL-4), IL-5, IL-6, and serum IgG1 responses. These data demonstrate that neonatal sublingual vaccination with denatured OVA and CpG not only preferentially induces systemic Th1 responses and mucosal immunity, but also simultaneously abrogates IgE production. Neonatal sublingual vaccines may play a role for the strategy of allergy prevention.     

Dendritic cell maturation enhances CD8+ T-cell responses to exogenous antigen via a proteasome-independent mechanism of major histocompatibility complex class I loading

Immune stimulating complexes (ISCOMS) containing the saponin adjuvant Quil A are vaccine adjuvants that induce a wide range of immune responses in vivo, including strong class I major histocompatibility complex (MHC)-restricted cytotoxic T-lymphocyte activity. However, the antigen-presenting cell responsible for the induction of these responses has not been characterized. Here we have investigated the role of dendritic cells (DC) in the priming of antigen-specific CD8+ T cells in vitro by ISCOMS containing ovalbumin. Resting bone marrow DC pulsed with ovalbumin ISCOMS efficiently prime resting CD8+ T cells through a mechanism that is transporter associated with antigen processing (TAP) dependent, but independent of CD40 ligation and CD4+ T-cell help. Lipopolysaccharide-induced maturation of DC markedly enhances their ability to prime CD8+ T cells through a mechanism which is also independent of CD4+ T-cell help, but is dependent on CD40 ligation. Furthermore, DC maturation revealed a TAP-independent mechanism of CD8+ T-cell priming. Our results also show that class I MHC-restricted presentation of ovalbumin in ISCOMS by DC is sensitive to chloroquine and brefeldin A but insensitive to lactacystin. We suggest that DC may be the principal antigen-presenting cells responsible for the priming of CD8+ T cells by ISCOMS in vivo and that targeting these vectors to activated DC may enhance their presentation via a novel pathway of class I antigen processing.     

Induction of Systemic and Mucosal Immune Responses Following Immunization with Somatostatin-Avidin Complexes Incorporated Into Iscoms

Synthetic, biotinylated somatostatin-14 (Somatotropin Release-Inhibiting Factor; SRIF) was conjugated to avidin, and the resulting complex incorporated into immune-stimulating complexes (ISCOMS). The ISCOMS were used to study the systemic and mucosal immune responses induced by parenteral and gastrointestinal vaccination. Mice were immunized by intraperitoneal (IP) and intragastric (IG) routes and subsequently by either IP or IG secondary immunizations (groups-IP/IP; IP/IG; IG/IG). Antigen specific IgG and IgA antibody secreting cells (ASC) from the spleen, mesenteric lymph nodes (MLN) and Peyer's patches (PP's) were studied by an enzyme-linked immunospot assay (ELISPOT). Specific proliferative responses of spleen cells to avidin and to SRIF were measured.

Immunization IP/IP evoked the highest serum IgG levels to avidin and to SRIF as well as the highest numbers of splenic IgG isotype ASC. The greatest IgA response in MLN and PP's was induced by IP/IG immunization. Only marginal mucosal immunity and no splenic cell specific proliferative responses were found by IG/IG immunization.

These results indicate that ISCOMS are an effective delivery system for protein-peptide antigens. The ISCOMS system described elicited systemic and mucosal antibody immune responses, and primed specific proliferative response when administered IP/IG. This offers another approach for the design and delivery of mucosally administered peptide vaccines.     

Antibody response to polyhistidine-tagged peptide and protein antigens attached to liposomes via lipid-linked nitrilotriacetic acid in mice

Particulate delivery systems enhance antibody responses to subunit antigens. However, covalent attachment of protein antigens can disrupt protein structure and mask critical epitopes, altering the antibody response to the antigen. In this report, we evaluate noncovalent metal chelation via nitrilotriacetic acid (NTA) as a nondestructive method to attach peptide and protein antigens to liposomes. Two model antigens, ovalbumin (OVA) and a peptide derived from the membrane-proximal region of HIV-1 gp41 (N-MPR), were polyhistidinylated and attached to liposomes via monovalent NTA (mono-NTA; K(D) [equilibrium dissociation constant], ～10 μM), trivalent NTA (tris-NTA; K(D), ～1 nM), or a covalent linkage. Attachment of N-MPR, but not OVA, to liposomes via an NTA lipid elicited stronger antibody responses in BALB/c mice than a formulation in which unassociated antigen was simply admixed with control liposomes lacking NTA. However, the tris-NTA linkage did not increase antibody responses to either N-MPR or OVA compared to the level for the mono-NTA linkage, despite the greater liposomal association of the antigen. For both antigens, covalently attaching them to a lipid elicited significantly stronger antibody responses than NTA-anchored antigens (OVA titer, 3.4 × 10(6) versus 1.4 × 10(6) to 1.6 × 10(6) [P < 0.001]; N-MPR titer, 4.4 × 10(4) versus 5.5 × 10(2) to 7.6 × 10(2) [P < 0.003]). The data indicate that NTA linkages may increase antibody titers to weak antigens such as N-MPR, but NTA-mediated attachment remains inferior to covalent conjugation. Moreover, enhancements in antigen-liposome affinity do not result in increased antibody titers. Thus, additional improvements of NTA-mediated conjugation technology are necessary to achieve an effective, nondestructive method for increasing the humoral response to antigens in particulate vaccines.     

Examination of oral sensitization with ovalbumin in Brown Norway rats and three strains of mice

We studied the conditions needed to sensitize animals to the oral feeding of food allergens, without induction of tolerance, in order to investigate the allergenicity of orally ingested food proteins. Brown Norway (BN) rats were sensitized by daily OVA (ovalbumin)-gavage or by drinking OVA containing water ad libitum and the ASA (active systemic anaphylaxis) response, as the immediate hypersensitivity response to antigen stimulation after oral sensitization, was examined. The oral administration of OVA by gavage produced a higher OVA-specific IgE response and an increase in serum histamine after antigen challenge, as compared to those produced by drinking water. Next, we examined the effect of murine age, the oral feeding technique and the oral feeding dose on sensitization using BALB/c, B10A and ASK mice. Twenty-week-old mice showed the strongest OVA-specific IgE and IgG1 responses and ASA-associated serum histamine contents increased with gavage in the three different age groups of BALB/c mice. Administering 0.1 mg of OVA by gavage daily for 9 weeks appeared to induce a higher response than administering 1 mg of OVA, in terms of OVA-specific IgE and IgG1 antibody responses and ASA responses. Among the three strains of mice, B10A mice exhibited the highest response in terms of OVA-specific IgE and IgG1 antibody and ASA responses. These findings suggested BN rats and B10A mice were suitable models for oral sensitization with antigen protein and that oral sensitization in mice requires low dose, intermittent antigen intakes.