Activation of mitogen-activated protein kinase cascades during priming of human neutrophils by TNF-alpha and GM-CSF

Oral administration of large doses of protein antigen generally induces a state of systemic unresponsiveness currently termed mucosally induced tolerance. In this study, we used human milk protein (HMP) without casein as a multi-protein antigen for the study of mucosally induced tolerance. The HMP utilized in this study mainly contained secretory (S) IgA, lactoferrin (Lf) and alpha-lactalbumin (Lact). When mice were given 1 or 25 mg of HMP orally 3 times or 25 mg orally four consecutive weeks prior to systemic immunization, antigen-specific serum IgG responses to HMP were induced by subsequent parenteral immunization with 100 microg of HMP. Analysis of IgG subclasses revealed that IgG1 followed by IgG2b accounted for the IgG responses noted. When both HMP and ovalbumin (OVA) were fed to mice, tolerance developed to OVA but not to HMP. To further investigate the nature of immune responses seen following oral gavage of HMP, we examined responses to individual protein of HMP. Brisk serum IgG1 and IgG2b responses to both S-IgA and Lf were induced by oral followed by systemic immunization with HMP. Analysis of splenic CD4+ T cells from mice given oral HMP revealed production of Th2- but not Th1-type cytokines. These results show that oral administration of HMP preferentially induces exclusive Th2-type immune responses, which may prevent the development of HMP (S-IgA and Lf)-specific mucosally induced tolerance.     

Effect of a monoclonal antibody against interleukin-4 on the induction of oral tolerance in mice

The present study was undertaken to investigate the effect of a monoclonal antibody against interleukin-4 on the induction of oral tolerance. Oral tolerance was induced by feeding mice with low and high doses (0.1, 1 and 10 mg) of hen egg lysozyme once a day for 5 days before immunization with the antigen. An anti-interleukin-4 monoclonal antibody was i.p. injected 30 min before each oral administration of hen egg lysozyme. The results showed that the oral administration of hen egg lysozyme suppressed immune responses to the antigen including delayed type hypersensitivity, production of both isotypes of immunoglobulin (Ig) G1 and IgG2a antibodies and proliferation of lymph node cells in a dose-dependent manner. The suppression of these responses by the oral antigen was associated with a marked reduction of interferon-gamma secretion and a moderate decrease in interleukin-4 production by lymphoid cells. The treatment with the anti-interleukin-4 monoclonal antibody blocked dose-dependently the suppression of the delayed type hypersensitivity response to hen egg lysozyme, anti-hen egg lysozyme IgG2a antibody production and interferon-gamma secretion. In contrast, the anti-interleukin-4 antibody facilitated the suppression of anti-hen egg lysozyme IgG1 antibody production and interleukin-4 secretion. Thus, the neutralization of interleukin-4 by anti-interleukin-4 antibodies appears to be effective in modulating the induction of oral tolerance.     

The induction and expression of IgA anti-DNP antibodies in rat bile and secretions

Pregnant rats were immunized with dinitrophenylated type III-pneumococcal vaccine by the intravenous, gastric, or intramammary routes. Anti-DNP antibody responses in the IgA, IgG and IgM isotypes were measured in serum, secretions and bile. Gastric intubation was most effective at eliciting IgA antibody responses in bile and secretions, whereas the other routes were more effective at inducing IgG responses in serum and bile. IgM antibody responses were infrequent and were found in fluids most closely associated with the immunization route. Isoelectric focusing studies of IgA antibodies appearing in secretions and bile revealed that the gastric route consistently elicited antibody spectrotypes with shared components. Intravenous and intramammary immunization resulted in IgA spectrotypes possessing less homology, suggesting that these protocols lead to independent antibody responses triggered in spleen, draining lymph nodes, or secretory sites. After gastric stimulation, the appearance of IgA antibodies with identical spectral components in secretions and bile favours the concept that IgA precursor cells with identical clonotype potential migrate from the gastrointestinal area to secretory sites. Antibody expression in bile appears to result from the selective transfer of IgA populations gaining access to serum after synthesis at a secretory site.     

Somatic antigens of adult Dicrocoelium dendriticum recognised by bile antibodies of naturally infected cattle

Bile samples, from slaughtered cattle harbouring between 120 and 280 adult lancet flukes, were used to investigate the range of somatic proteins inducing local antibody responses in naturally infected animals. Lancet fluke infections induced local (bile) antibody responses against Tris-buffered saline (TBS) soluble, sodium dodecyl sulphate (SDS) soluble and 2-mercaptoethanol (2-Me) soluble somatic proteins of adult Dicrocoelium dendriticum. IgA antibody isotypes predominated in the response against buffer-soluble somatic antigens, whereas SDS-soluble and 2-Me-soluble proteins induced similar level of both IgA and IgG1 antibodies. Analysis of the antigens recognised by particular isotype-specific bile antibodies suggests that different antigens preferentially induce isotype restricted antibody responses. The bile antibody response was highly species specific, only one antigen from somatic protein extracts of Fasciola hepatica being precipitated by bile samples showing the highest reactivity against D. dendriticum.     

Differential kinetics and distribution of antibodies in serum and nasal and vaginal secretions after nasal and oral vaccination of humans

Although nasal vaccination has emerged as an interesting alternative to systemic or oral vaccination, knowledge is scarce about the immune responses after such immunization in humans. In the present study, we have compared the kinetics and organ distribution of the antibody responses after nasal and oral vaccination. We immunized female volunteers nasally or orally with cholera toxin B subunit (CTB) and determined the specific antibody levels in serum and nasal and vaginal secretions, as well as the number of circulating antibody-secreting cells, before immunization and 1, 2, 3, 6, and 26 weeks thereafter. Nasal vaccination induced 9-fold CTB-specific immunoglobulin A (IgA) and 56-fold specific IgG antibody increases in nasal secretions, whereas no significant IgA increase was seen after oral vaccination. Both oral and nasal vaccination resulted in 5- to 6-fold CTB-specific IgA and 20- to 30-fold specific IgG increases in vaginal secretions. Strong serum responses to CTB were also induced by both routes of vaccination. A notable difference between nasal and oral vaccination was that the nasal route elicited a specific antibody response with a later onset but of much longer duration than did the oral route. We conclude from this study that the nasal route is superior to the oral route for administering at least nonliving vaccines against infections in the upper respiratory tract, whereas either oral or nasal vaccination might be used for eliciting antibody responses in the female genital tract.     

Evidence for serum immunoglobulin G (IgG) antibody responses in Macaca fascicularis identified by monoclonal antibodies to human IgG subclasses

This investigation determined the capacity of murine monoclonal antibodies directed to human immunoglobulin G (IgG) subclasses to identify molecules with conserved epitopes in the serum of the nonhuman primate, Macaca fascicularis. We subsequently utilized this cross-reactivity to document the characteristics of IgG subclass antibody responses in M. fascicularis to parenteral immunization with intact oral microorganisms, antigens from oral microorganisms, and finally a defined protein toxin, tetanus toxoid. The IgG response in nonhuman primates immunized with tetanus toxoid showed a 40-fold and 110-fold increase after primary and secondary immunizations, respectively. The major IgG subclass responses were IgG1 and IgG3, with little, though significant, responses in the IgG4 and IgG2 subclasses. Seventy-five to 94% of the natural IgG antibody in nonhuman primate sera to Porphyromonas gingivalis, Prevotella intermedia and Campylobacter rectus was IgG1. IgG2 and IgG3 predominated to Bacteroides fragilis, IgG4 to Actinomyces viscosus and an equal distribution among the subclasses was noted in response to Fusobacterium nucleatum. Parenteral immunization of nonhuman primates with intact P. gingivalis elicited primarily IgG3 and IgG4, while the post-immunization IgG response to P. intermedia was largely IgG1. Nonhuman primates were also parenterally immunized with cell envelope antigens of P. gingivalis, P. intermedia, or a combination of cell envelope antigen from C. rectus and F. nucleatum and cell wall antigens of A. viscosus. The greatest IgG antibody response seen post-immunization was reactive with anti-human IgG1 for all of these antigens except to C. rectus which bound nonhuman primate antibody reactive with anti-human IgG2. It appears that the bacteria and their products exhibit unique differences in their induction of serum IgG subclass antibody responses. The characteristics of their immunogenicity as detected by the nonhuman primate may contribute to the ability of the immune responses to effectively interact with these pathogens.     

Human plasma protein binding of the angiotensin II receptor antagonist losartan potassium (DuP 753/MK 954) and its pharmacologically active metabolite EXP3174

Immunological tolerance can be induced by the oral administration of antigen. We induced oral tolerance rats to experimental allergic conjunctivitis by ovalbumin and investigated the suppression of inflammation. In groups which had started eating antigen both before and after the immunization, the serum anti-ovalbumin IgE level measured by passive cutaneous anaphylaxis reaction was significantly lower than in a group that had not been fed antigen. The intensity of experimental allergic conjunctivitis in the group which had started eating antigen before immunization, was significantly suppressed in regard to the leakage of Evans Blue from conjunctival vessels 30 minutes after the challenge and the neutrophil infiltration 6 hours after. The dye leakage of the group which had started feeding after immunization was also significantly suppressed. There was a positive correlation between the serum IgE level and the leakage of Evans Blue (r = 0.90, p < 0.001). These results suggest that the suppression of antigen-specific IgE antibody production caused by oral tolerance affected the decrease of local inflammation on the conjunctiva.     

Detection of specific and 'constitutive' antibody secreting cells in the gills, head kidney and peripheral blood leucocytes of dab (Limanda limanda)

The relative immunological importance of the gills of fish was investigated in terms of antibody production by enumerating antibody secreting cells (ASC) in the gills, head kidney and blood of dab (Limanda limanda) using the ELISPOT assay. The contribution of 'constitutive' ASC in the gill appeared more substantial than that of elicited specific ASC. The gills were found to contain a mean (+/- SD) of 4227 +/- 1029 'constitutive' ASC/10(6) cells which was fewer than the head kidney which contained a mean (+/- SD) of 15617 +/- 3723 'constitutive' ASC/10(6) cells but more than peripheral blood leucocytes which contained a mean (+/- SD) of 2650 +/- 212 'constitutive' ASC/10(6) cells. The number of specific anti-human gamma globulin (HGG) ASC following parenteral or oral administration of HGG was also determined. Anti-HGG ASC were detected in all three tissues following parenteral immunization, peaking simultaneously, 4 weeks post-immunization. The strongest response was found in the head kidney. After oral immunization, responses were much weaker: again the head kidney was the most active but the gill response was barely detectable. These data were complemented by measurement of specific antibody in the serum by ELISA. Serum antibody titres following immunization were found to correlate closely with the number of specific ASC in the head kidney following parenteral immunization whereas serum antibody titres after oral administration of antigen most closely followed the number of specific ASC in the blood. In the light of these data it is suggested that the primary immunological role of the leucocytes in the gill may be in the earliest stages of defence against infection.     

Mapping of B epitopes in GRA4, a dense granule antigen of Toxoplasma gondii and protection studies using recombinant proteins administered by the oral route

GRA4, a dense granule protein of Toxoplasma gondii elicits both mucosal and systemic immune responses following oral infection of mice with cysts. We studied the antigenicity and immunogenicity of truncated and soluble forms of GRA4 expressed as glutathione S-transferase fusion proteins in Escherichia coli. Protein C (amino-acids 297-345) was particularly well recognized by serum IgG antibodies, milk IgA antibodies and intestinal IgA antibodies from T. gondii infected mice and by serum IgG antibodies from T. gondii infected humans and T. gondii infected sheep. One major B epitope was localized within the last 11 C-terminal residues of GRA4. A second epitope, recognized with lower frequency, was mapped within the region 318-334. In contrast, the N domain of GRA4 (amino acids 25-276) was poorly recognized. Oral immunization of C57BL/6 mice with N, C or NC (amino acids 25-276 fused to 297-345) in association with cholera toxin induced a significant production of serum anti-GRA4 IgG antibodies but a weak and inconsistent intestinal anti-GRA4 IgG antibody response and afforded partial resistance to oral infection with T. gondii. These results provide new molecular and immunological understanding of GRA4 and indicate that it is a potential candidate for oral vaccination against T. gondii.     

The use of a cationic liposome formulation (DOTAP) mixed with a recombinant tumor-associated antigen to induce immune responses and protective immunity in mice

The cationic liposome DOTAP is a well-known transfection reagent. It has been manufactured and approved for clinical use, is readily available, and can be easily used as an adjuvant. These characteristics prompted us to investigate the effectiveness of DOTAP as an adjuvant to induce immune responses and protective immunity in mice using baculovirus-derived carcinoembryonic antigen (bV-CEA) as a model antigen. Two routes of administration and a dose-response study of bV-CEA were used in BALB/c mice to define the magnitude of the immune response as well as the most effective route of immunization. The results demonstrate differences in antibody titers, immunoglobulin (Ig)G isotype, and T-cell responses between the intravenous (i.v.) or subcutaneous (s.c.) route of immunization. The titer of the anti-CEA antibodies induced by the s.c. immunization was greater than the response by i.v. immunization. The s.c. route enhanced the IgG2a/2b isotype, whereas i.v. immunization elicited primarily IgG1. T-cell proliferation responses and cytokine production paralleled the humoral response (i.e., production was higher in the s.c. immunized animals). No differences in immunological responses were seen using either 25 or 10 microg of bV-CEA three times. An amount of 25 microg of bV-CEA/DOTAP given by s.c. immunization was sufficient in protecting mice from the transplant of syngeneic tumor cells transduced with the human CEA gene. We conclude that the cationic liposome DOTAP may be a useful immunoadjuvant for active anti-tumor immunotherapy in future clinical trials. This study will help to define the most effective way to use such an adjuvant.     

Comparison of the new matrix system with traditional fixed prosthodontic impression procedures

Conjugation of proteins with polyethylene glycol (PEG) has been reported to make the proteins tolerogenic. Native proteins are also potentially tolerogenic when given without adjuvants. We compared immunotolerogenic activities between PEG-conjugated and native hen egg-white lysozyme (HEL). BALB/c mice were given consecutive weekly intraperitoneal administrations of PEG-conjugated HEL, unmodified HEL or phosphate-buffered saline (PBS), for 3 weeks, then challenged with HEL in Freund's complete adjuvant. The pretreatment with PEG-HEL tolerized both T-cell and humoral responses, while native HEL tolerized only the T-cell response. Immunoglobulin G1 (IgG1) antibody was already elevated in HEL-pretreated mice prior to challenge immunization, followed by suppressed IgG2a and IgG2b, but spared IgG1 production after the antigen challenge, whereas PEG-HEL-pretreated mice produced no antibody in all IgG subclasses prior and subsequent to the antigen-challenge. Production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by lymphoid cells in response to HEL in vitro was markedly suppressed in both the antigen-pretreated groups, while suppression of IL-4 production was evident only in PEG-HEL-, not in HEL-pretreated animals. Involvement of suppressor cells in these tolerance states was found to be unlikely, and the immunological property of PEG-HEL differed from deaggregated HEL that was similar to the original HEL. These results suggest a unique immunotolerogenic activity of PEG-conjugated proteins to suppress both T-helper type-1 (Th1)- and Th2-type responses, the result being extensive inhibition of all IgG subclass responses, while tolerance induction by unconjugated soluble proteins tends to be targeted on Th1-, but spares Th2-type responses.     

Evaluation of subcutaneous chambers as an alternative to conventional methods of antibody production in chickens

We compared antibody levels among serum, egg yolk extract, and granuloma fluid in chickens immunized with bovine serum albumin (BSA). One group of hens was immunized by intramuscular and subcutaneous injection of bovine serum albumin in complete Freund's adjuvant, followed by two subsequent booster injections in incomplete Freund's adjuvant. Two other groups were surgically implanted with plastic, perforated wiffle balls (subcutaneous chambers). After a 30-day recovery period, one of the groups with subcutaneous chambers was immunized with BSA in sterile water with two subsequent boosts. The other group was injected with only sterile water. Serum samples, eggs, and granuloma fluid were collected biweekly and analyzed to determine specific IgG, total IgG, and total protein. The subcutaneous chambers were well tolerated. Quantitative ELISAs of serum, egg yolk extract, and granuloma fluid specimens disclosed that specific antibody levels were present in all specimens by 2 weeks after primary immunization. During the course of the experiment, specific antibody levels of serum and egg yolk specimens were significantly higher than those of granuloma fluid (P less than 0.05). However, an additional injection of antigen into the subcutaneous chambers resulted in specific antibody levels in granuloma fluid specimens that were comparable to those of serum and egg yolk extract. Use of subcutaneous chambers in chickens may be a viable alternative to routine antibody production methods.     

Immune response in the hamster. VIII. Ig class differences in susceptibility to tolerance induction

A hemolytic plaque assay was used to quantitate the antibody response of the Syrian hamster after immunization with hen egg albumin (HEA). Whereas HEA in complete Freund's adjuvant (HEA-CF) induced a prolonged heterogeneous (IgM, IgG1 and IgG2) antibody response, the response to soluble HEA in saline (HEA-S) differed in that: 1)both the primary and secondary responses were restricted to the IgG1 class; 2)the IgG1 primary response was cyclical with PFC peaks on days 9 and 16; 3)although an anamnestic secondary response was demonstrated, no further augmentation was noted after tertiary and quaternary boosters; 4)the booster response was transient reaching a peak after 48 hr and declining to low levels within 7 days. Adoptive transfer of lymph node cells to irradiated recipients followed by challenge with HEA-CF revealed: 1)that HEA-S-treated donor cells were primed for an IgG1 response because anamnesis was seen 7 days after challenge, yet on day 21, IgG1 PFC were 20-fold less than that of controls; 2)recipients of HEA-S treated cells showed profound suppression of both IgM and IgG2 PFC on days 14 and 21. These studies indicate that soluble antigen induced in hamsters a state of complete tolerance of IgM and IgG2 classes whereas the anamnestic response of the IgG1 class remained intact.     

Differences in immune responses induced by oral and rectal immunizations with Salmonella typhi Ty21a: evidence for compartmentalization within the common mucosal immune system in humans

Based on the concept of the common mucosal immune system, immunization at various inductive sites can induce an immune response at other, remote mucosal surfaces. The immune responses elicited through rectal and oral routes of antigen delivery were compared with respect to (i) measurement of antibody responses in serum and various external secretions of the vaccinees and (ii) characterization of the nature and homing potentials of circulating antibody-secreting cells (ASC). Specific ASC appeared in the circulation in 4 of 5 volunteers after oral and 9 of 11 volunteers after rectal immunization with Salmonella typhi Ty21a. The kinetics, magnitude, and immunoglobulin isotype distribution of the ASC responses were similar in the two groups. In both groups, almost all ASC (99 or 95% after oral or rectal immunization, respectively) expressed alpha4 beta7, the gut homing receptor (HR), whereas L-selectin, the peripheral lymph node HR, was expressed only on 22 or 38% of ASC, respectively. Oral immunization elicited a more pronounced immune response in saliva and vaginal secretion, while rectal immunization was more potent in inducing a response in nasal secretion, rectum, and tears. No major differences were found in the abilities of the two immunization routes to induce a response in serum or intestinal secretion. Thus, the rectal antigen delivery should be considered as an alternative to the oral immunization route. The different immune response profiles found in various secretions after oral versus rectal antigen administration provide evidence for a compartmentalization within the common mucosal immune system in humans.     

Effectiveness of liposomes possessing surface-linked recombinant B subunit of cholera toxin as an oral antigen delivery system

Liposomes appear to be a promising oral antigen delivery system for the development of vaccines against infectious diseases, although their uptake efficiency by Peyer's patches in the gut and the subsequent induction of mucosal immunoglobulin A (IgA) responses remain a major concern. Aiming at targeted delivery of liposomal immunogens, we have previously reported the conjugation via a thioether bond of the GM1 ganglioside-binding subunit of cholera toxin (CTB) to the liposomal outer surface. In the present study, we have investigated the effectiveness of liposomes containing the saliva-binding region (SBR) of Streptococcus mutans AgI/II adhesin and possessing surface-linked recombinant CTB (rCTB) in generating mucosal (salivary, vaginal, and intestinal) IgA as well as serum IgG responses to the parent molecule, AgI/II. Responses in mice given a single oral dose of the rCTB-conjugated liposomes were compared to those in mice given one of the following unconjugated liposome preparations: (i) empty liposomes, (ii) liposomes containing SBR, (iii) liposomes containing SBR and coadministered with rCTB, and (iv) liposomes containing SBR plus rCTB. Three weeks after the primary immunization, significantly higher levels of mucosal IgA and serum IgG antibodies to AgI/II were observed in the rCTB-conjugated group than in mice given the unconjugated liposome preparations, although the latter mice received a booster dose at week 9. The antibody responses in mice immunized with rCTB-conjugated liposomes persisted at high levels for at least 6 months, at which time (week 26) a recall immunization significantly augmented the responses. In general, mice given unconjugated liposome preparations required one or two booster immunizations to develop a substantial anti-AgI/II antibody response, which was more prominent in the group given coencapsulated SBR and rCTB. These data indicate that conjugation of rCTB to liposomes greatly enhances their effectiveness as an antigen delivery system. This oral immunization strategy should be applicable for the development of vaccines against oral, intestinal, or sexually transmitted diseases.     

A two-stage screening test for pregnancy-induced hypertension and preeclampsia

PURPOSE: To define the inductive pathways leading to rat tear IgA antibody responses. METHODS: Fluoresceinated dinitrophenylated bovine serum albumin was encapsulated in poly(lactide-co-glycolide) microparticles and was administered by intranasal, ocular topical, or gastrointestinal routes. Histologic methods were used to determine the microparticles' ability to access tissues associated with mucosal inductive pathways. Rats were immunized with microencapsulated antigen by intranasal or ocular topical routes. Tear IgA and serum IgG antibody concentrations were assessed by radioimmunoassay. The frequency of antibody-secreting cells in tissues, postulated to function in tear IgA induction, was measured by enzyme-linked immunospot assay. RESULTS: Although uptake of microencapsulated antigen was greatest at the site of delivery, ocular topical administration resulted in antigen uptake in the conjunctiva and in nasal-associated lymphoid tissue. Intranasal immunization resulted in earlier and significantly higher tear IgA and serum IgG antibody responses and in higher frequencies of antibody-secreting cells in corresponding draining cervical lymph nodes and lacrimal glands than did ocular topical immunization. CONCLUSIONS: Nasal-associated lymphoid tissue functions as a primary inductive site for tear IgA antibody responses by contributing triggered IgA-committed B cells to the lacrimal gland.     

Induction of mucosal immunity by intranasal application of a streptococcal surface protein antigen with the cholera toxin B subunit

The level and distribution of isotype-specific antibodies in various secretions and of antibody-secreting cells in corresponding lymphoid organs and tissues were compared in mice immunized with Streptococcus mutans surface protein antigen I/II (AgI/II) conjugated to the cholera toxin B subunit (CTB), given intranasally (i.n.) or intragastrically (i.g.), with or without free cholera toxin (CT) as an adjuvant. Immunization i.n. induced stronger initial antibody responses to AgI/II in both serum and saliva than immunization i.g., but salivary immunoglobulin A (IgA)-specific antibody responses to immunization about 3 months later were not increased relative to total salivary IgA concentrations. Specific antibodies induced by i.n. immunization were as widely distributed in serum, saliva, tracheal wash, gut wash, and vaginal wash as those induced by i.g. immunization. Likewise, specific antibody-secreting cells were generated in the spleen, salivary glands, intestinal lamina propria, and mesenteric and cervical lymph nodes by either route of immunization. The strongest salivary IgA antibody response was induced by AgI/II-CTB conjugate given i.n., but the addition of CT did not further enhance it. However, free CTB could effectively replace CT as an adjuvant in i.n. immunization with unconjugated AgI/II. Booster i.n. immunization with AgI/II plus either free CT or CTB induced stronger recall serum antibody responses than conjugated AgI/II-CTB with or without CT as an adjuvant. Therefore, i.n. immunization with a protein antigen and free or coupled CTB is an effective means of generating IgA antibody responses expressed at several mucosal sites where protective immunity may be beneficial.     

Recombinant cholera toxin B subunit acts as an adjuvant for the mucosal and systemic responses of mice to mucosally co-administered bovine serum albumin

We examined the mucosal adjuvant activity of recombinant cholera toxin B subunit (rCTB) produced by Bacillus brevis carrying pNU212-CTB by intranasal or oral co-administration of bovine serum albumin (BSA). Intranasal administration stimulated a high level of BSA-specific serum IgG antibody response and BSA-specific IgA antibody responses in the nasal and pulmonary lavages. Oral administration induced a moderate level of BSA-specific serum IgG antibody and a low level of BSA-specific IgA antibody in the large intestinal washes. These results show that CTB alone can act as an intranasal or oral delivery carrier; it also has strong adjuvant properties for stimulating serum IgG and mucosal IgA immune responses to unrelated, non-coupled antigens after intranasal or oral co-immunization.     

Attempts to immunize chickens with Cryptosporidium baileyi oocyst extract

In order to study the possibility of immunization against Cryptosporidium baileyi with extracted crude antigen, Arbor Acres chickens were injected intramuscularly with 80 micrograms of C. baileyi oocyst-derived proteins (uninfected immunized, UI) or inoculated orally with 8 x 10(5) viable C. baileyi oocysts (infected control, IC) at 1 wk of age. The immunization was repeated in the UI group at 2 wk of age. Uninfected (UC) birds served as controls. All animals in UI, IC, and UC groups were challenged orally with 8 x 10(5) C. baileyi oocysts at the age of 4 wk. Blood samples were collected when birds were 4 and 6 wk of age, and sera were examined by enzyme-linked immunosorbent assay for the presence of antibodies against C. baileyi. Total oocyst output of UI chickens was about 60% of that of UC birds after challenge, and the prepatent and patent periods were nearly identical in the latter 2 groups. In contrast, IC birds developed complete resistance to challenge infections. These results suggest that immunization with the oocyst extract of C. baileyi may confer some degree of protection against oral challenge; however, the protection is less effective than that induced by primary oral infection. The lack of significant difference between the antibody responses of IC and UI animals to C. baileyi at 2 wk of age suggests that serum antibodies play little role in acquired resistance to challenge infection.     

Influence of cellular location of expressed antigen on the efficacy of DNA vaccination: cytotoxic T lymphocyte and antibody responses are suboptimal when antigen is cytoplasmic after intramuscular DNA immunization

We examined the role of the cellular localization of antigen on the immune response after DNA immunization of mice with three forms of ovalbumin (OVA). DNA encoding OVA which was secreted (sOVA) generated 10- to 100-fold higher IgG responses with 50-and 100-fold higher levels of IgG1 than the cytoplasmic (cOVA) or membrane bound (mOVA) forms. An IgG2a predominance was seen only in cOVA and mOVA immunized mice. Although the antibody response was CD4+ T cell dependent, the differences in the antibody response could not be compensated for by provision of excess CD4+ T cell help in TCR transgenic mice. Together with our hapten-carrier studies, this would indicate that membrane or intracellular localization limits the availability of antigen for B cell priming which affects the magnitude and form of the antibody response. Surprisingly, stronger cytotoxic T lymphocyte (CTL) responses were generated for sOVA or mOVA than for cOVA via intramuscular (i.m.) injection. Since a cytoplasmic antigen should have best access to the canonical class I pathway for antigen presentation, our results indicate that priming of CTL responses after i.m. DNA immunization is probably by cross-presentation of antigen by non-transfected professional antigen-presenting cells. In contrast, intradermal immunization with cOVA produced optimal CTL responses but, as with mOVA, suboptimal antibody responses. This, together with our ex vivo RT-PCR analysis showing similar mRNA levels from all three constructs 7 days post-immunization, argues against the differential CTL response for i.m. injection to be due to dose.