Antitoxic immunity in experimental cholera: protection, and serum and local antibody responses in rabbits after enteral and parenteral immunization.

The protective effect of enternal and parenteral immunization with cholera toxin antigen against experimental cholera in rabbits was studied by using the small-bowel loop technique. Subcutaneous injection of crude toxin as well as purified toxin or toxoids gave rise to significant protection against toxin challenge. The enhanced resistance to toxin was found to correspond to a many-fold higher magnitude of protection against challenge with live vibrios. In the primary response the protection increased during the first month. Booster immunization gave rise to a further increased immunity which, however, declined rapidly. Multiple oral or repeated intraintestinal antigen administrations also induced protective antitoxic immunity although of less magnitude than that obtained by parenteral immunization. Enteral and, to a lesser extent, parenteral immunization gave rise to increased antitoxic antibody titers and immunoglobulin levels in intestinal washings and mucosa scraping. Immunoglobulin G (IgG) and IgG antitoxins predominated, but after enteral immunization total IgA and specific IgA antibodies occasionally reached levels similar to those for IgG. In serum, significantly increased antibody levels (IgG) were only recorded after parenteral immunization. Both the primary binding and the neutralizing antitoxin titers showed a stayistically significant correlation with the degree of protection against toxin challenge; however, for the neutralizing antibodies this correlation was not without exceptions. No relation to protection was found for intestinal antibodies. The results of the present study indicate that enternal as well as parenteral immunization with toxin antigen can give rise to effective cholera immunity. After enternal immunization, the protection appears to be medicated by locally synthesized antibodies. After parenteral vaccination both serum-derived and locally produced antibodies seem to be effective.     

Suppression of the intestinal immune response to cholera toxin by specific serum antibody.

The possibility that preexisting specific serum antibody could suppress a defined mucosal immune response to a topically applied antigen was studied in rats. Hyperimmune serum antibody induced by parenteral immunization of rats with cholera toxoid markedly suppressed the mucosal immune response to enterically applied cholera toxin. Such antibody was far more suppressive than antibody induced by primary parenteral immunization, apparently due to its greater avidity. Transfusion of small amounts (25 to 100 microliter) of hyperimmune serum suppressed the primary mucosal antitoxin response, the development of specific memory in the mucosal immune system, and, somewhat less effectively, the secondary mucosal antitoxin response. Suppression was due largely to a direct effect of serum antibody upon the interaction of absorbed enteric antigen with lymphoid tissue in Peyer's patches and, possible, mesenteric lymph nodes; interference with antigen absorption played little or no role in the observed suppression. These results do not explain the previously reported suppressive effect of primary parenteral immunization on the mucosal immune response to cholera toxin. However, they support the notions that repeated parenteral immunization can evoke avid serum antibody without necessarily stimulating mucosa-associated lymphoid tissue and that such antibody can markedly suppress primary and secondary phases of the local immune response to mucosally applied antigen. Thus, a mechanism is demonstrated by which repeated parenteral immunization may adversely affect efforts to initiate or sustain protective mucosal immune responses.     

Rapid analytical detection of microcystins using gold colloidal immunochromatographic strip

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is low and the detection method is usually complicated. We developed a rapid analytical detection method of microcystins gold colloidal immunochromatogeraphic strip. The sensitivity of the strip is about 1 ng/mL for microcystin LR; it is able to distinguish visually among different concentrations of microcystin solutions. The developed gold colloidal strip can detect microcystins within 15 min and does not require either a complicated extraction system, or trained or qualified experts.     

Transcutaneous immunization with cross-reacting material CRM(197) of diphtheria toxin boosts functional antibody levels in mice primed parenterally with adsorbed diphtheria toxoid vaccine

Transcutaneous immunization (TCI) capitalizes on the accessibility and immunocompetence of the skin, elicits protective immunity, simplifies vaccine delivery, and may be particularly advantageous when frequent boosting is required. In this study we examined the potential of TCI to boost preexisting immune responses to diphtheria in mice. The cross-reacting material (CRM(197)) of diphtheria toxin was used as the boosting antigen and was administered alone or together with either one of two commonly used mucosal adjuvants, cholera toxin (CT) and a partially detoxified mutant of heat-labile enterotoxin of Escherichia coli (LTR72). We report that TCI with CRM(197) significantly boosted preexisting immune responses elicited after parenteral priming with aluminum hydroxide-adsorbed diphtheria toxoid (DTxd) vaccine. In the presence of LTR72 as an adjuvant, toxin-neutralizing antibody titers were significantly higher than those elicited by CRM(197) alone and were comparable to the functional antibody levels induced after parenteral booster immunization with the adsorbed DTxd vaccine. Time course study showed that high levels of toxin-neutralizing antibodies persisted for at least 14 weeks after the transcutaneous boost. In addition, TCI resulted in a vigorous antigen-specific proliferative response in all groups of mice boosted with the CRM(197) protein. These findings highlight the promising prospect of using booster administrations of CRM(197) via the transcutaneous route to establish good herd immunity against diphtheria.     

Immunodulation of rat serum and mucosal antibody responses to Entamoeba histolytica trophozoites by beta-1,3-glucan and cholera toxin

Systemic and mucosal and immune responses can be manipulated with immunomodulators. Here we show the modulatory effects of cholera toxin (CT) and beta-1,3-glucan (GLU) on the rat antiamebic serum and fecal antibody responses to one or four intraperitoneal (IP) or intragastric (IG) doses of glutaraldehyde-fixed Entamoeba histolytica trophozoites (GFT). One IP dose of GFT maximized serum IgM and IgG antiamebic antibodies on days 4 and 9, respectively; CT coadministration increased IgM antibodies, whereas IgG titers increased with CT or GLU; coproantibodies were undetectable after GFT alone or coadministered with GLU, whereas CT coadministration maximized fecal IgA antibodies on day 6. One IG dose of GFT alone increased serum IgM and IgG antibodies 2.5 times and no further increases were detected using GLU, whereas CT doubled serum IgG antibodies; GFT did not affect the coproantibody responses, whereas GLU coadministration maximized IgG coproantibody levels on day 6 and CT increased IgG and IgA coproantibody levels on the same day. On the other hand, four IG doses of GFT alone or with GLU induced tolerance, whereas GFT alone via the IP route increased serum antibodies slightly and GLU coadministration increased serum IgG antibody titers 300-fold. CT coadministration by both routes increased IgA coproantibodies, and simultaneous CT+GLU coadministration induced lower responses than either CT or GLU. Different antiamebic immune responses might therefore be attained through the use of different immunization routes and immunomodulators to induce protective immunity against intestinal or extraintestinal amebiasis.     

Protection against experimental cholera by oral or parenteral immunization.

Comparisons were made between the antigenic potency and protective capacity of several cholera toxin derivatives. Rabbits were immunized parenterally with 50 microgram of cholera toxin, A subunit, B subunit, procholeragenoid, or Wyeth glutaraldehyde toxoid 20101. Examination of the antibody response curves revealed that cholera toxin elicited serum antitoxin responses that rose more quickly than in the subunit-immunized animals; however, antitoxin levels were of the same magnitude after 10 weeks. Parenteral immunization with procholeragenoid evoked antibody titers that were similar to the toxin, whereas Wyeth toxoid yielded only one-tenth the level of antitoxin. Oral immunization with procholeragenoid as well as Wyeth toxoid resulted in lower serum antitoxin titers than that achieved with parenteral immunization, despite the oral administration of 10 times the parenteral dose. Analysis of protection against live-cell challenge revealed that parenteral administration of procholeragenoid provided the best protection against fluid accumulation. Oral immunization with procholeragenoid also was very effective, whereas oral immunization with B subunit or Wyeth toxoid resulted in minimal protection. Also, the A subunit provided surprisingly more protection than did cholera toxin.     

Preparation and characterization of polyclonal and monoclonal antibodies against fusarenon‐X

Polyclonal and monoclonal antibodies against fusarenon‐X were prepared by using a fusarenon‐X oxime derivative coupled to human serum albumin as the antigen for the immunization of rabbits and BALB/c mice, respectively. The specificity and sensitivity of these antibodies were tested by using fusarenon‐X oxime coupled to horseradish peroxidase as an enzyme‐linked toxin in a competitive assay with a double antibody solid phase. The relative cross‐reactivities of the polyclonal antiserum with T‐2 toxin, diacetoxyscirpenol, fusarenon‐X and neosolaniol were 3.67, 2.75, 1.0 and 0.58, respectively. The monoclonal antibody, however, showed considerable cross‐reactivity only with neosolaniol (0.28). The detection limit for fusarenon‐X was 150 pg/ml (5 pg per assay) and 300 pg/ml (10 pg per assay), respectively, using the polyclonal and monoclonal antibodies.     

Parenteral adjuvant activities of Escherichia coli heat-labile toxin and its B subunit for immunization of mice against gastric Helicobacter pylori infection

The heat-labile toxin (LT) of Escherichia coli is a potent mucosal adjuvant that has been used to induce protective immunity against Helicobacter felis and Helicobacter pylori infection in mice. We studied whether recombinant LT or its B subunit (LTB) has adjuvant activity in mice when delivered with H. pylori urease antigen via the parenteral route. Mice were immunized subcutaneously or intradermally with urease plus LT, recombinant LTB, or a combination of LT and LTB prior to intragastric challenge with H. pylori. Control mice were immunized orally with urease plus LT, a regimen shown previously to protect against H. pylori gastric infection. Parenteral immunization using either LT or LTB as adjuvant protected mice against H. pylori challenge as effectively as oral immunization and enhanced urease-specific immunoglobulin G (IgG) responses in serum as effectively as aluminum hydroxide adjuvant. LT and LTB had adjuvant activity at subtoxic doses and induced more consistent antibody responses than those observed with oral immunization. A mixture of a low dose of LT and a high dose of LTB stimulated the highest levels of protection and specific IgG in serum. Urease-specific IgG1 and IgG2a antibody subclass responses were stimulated by all immunization regimens tested, but relative levels were dependent on the adjuvant used. Compared to parenteral immunization with urease alone, LT preferentially enhanced IgG1, while LTB or the LT-LTB mixture preferentially enhanced IgG2a. Parenteral immunization using LT or LTB as adjuvant also induced IgA to urease in the saliva of some mice. These results show that LT and LTB stimulate qualitatively different humoral immune responses to urease but are both effective parenteral adjuvants for immunization of mice against H. pylori infection.     

Lipopeptides as immunoadjuvants and immunostimulants in mucosal immunization

In previous studies we have shown that lipopeptides constitute potent immunoadjuvants in mice, rabbits and other species: in parenteral immunization, lipopeptide adjuvants were comparable, or in some cases superior to Freund's adjuvant, and were devoid of the side effects of this additive. Here we demonstrate that lipopeptides also constitute adjuvants for mucosal immunizations. The serum antibody responses against the wheat storage protein gliadin, the bee venom constituent melittin, or the hen egg protein ovalbumin could in most cases be enhanced more than 100-fold by the lipopeptide P3CSK4, applied via the nasal route. An enhanced specific antibody level could also be detected in supernatants of cell cultures prepared from spleens, Peyer's patches, lungs and mesenteric lymph nodes of immunized mice. Moreover, the lipopeptide P3CSK4 enhanced chemiluminescence in mouse spleen cells and peritoneal macrophages in vitro, indicating a macrophage-activating effect. Finally, nasal application of lipopeptide increased protection against a lethal infection of influenza. Our findings are of importance for the improvement of immunizations and might lead to more effective vaccines.     

Adjuvant activity of a nontoxic mutant of Escherichia coli heat-labile enterotoxin on systemic and mucosal immune responses elicited against a heterologous antigen carried by a live Salmonella enterica serovar Typhimurium vaccine strain

Systemic and mucosal antibody responses against both the major subunit of colonization factor antigen I (CFA/I) of enterotoxigenic Escherichia coli (ETEC) and the somatic lipopolysaccharide expressed by recombinant bivalent Salmonella vaccine strains were significantly enhanced by coadministration of a detoxified derivative with preserved adjuvant effects of the ETEC heat-labile toxin, LT((R192G)). The results further support the adjuvant effects of LT((R192G)) and represent a simple alternative to improve responses against passenger antigens expressed by orally delivered Salmonella vaccine strains.     

Biliary Immune Response to Orally Presented Food Antigen, Ovomucoid, and its Potentiation by Cholera Toxin B Subunit

To clarify the biliary immune response against food antigen, we studied biliary antibody response to intravenous and oral primary immunization with ovomucoid (OM) and the effects of cholera toxin B subunit (CTB) on the oral response in mice. Specific antibodies against OM were induced in biliary and serum immunoglobulin (Ig) A, IgG and IgM by intravenous (i.v.) administration of the antigen. However, the antibodies were induced only in biliary Igs, but not in serum Igs, by oral intubation of the antigen. The higher levels of anti-OM in bile than in serum observed in the oral group may favour the assumption that antigen-stimulated lymphoid cells migrate to the liver, gall bladder or bile duct where they produce antibody. Both serum and biliary anti-OM responses to oral immunization were potentiated remarkably by oral administration of CTB with the antigen, the IgM anti-OM response being potentiated to the largest extent. In the CTB-treated mice, the IgA anti-OM level was higher in bile than in serum. Serum level of IgG anti-OM was much lower in the CTB-treated mice than in the i.v.-immunized mice, but the biliary level of IgG anti-OM in the CTB-treated mice was comparable to that in the i.v.-immunized mice. The relationship between serum and biliary IgA and IgG antibodies suggests that CTB potentiates biliary anti-OM responses not solely through increasing systemic levels of the antibodies but through modulating the processes specific to mucosal presentation of antigen.     

Cholera antibody production in vitro by peripheral blood lymphocytes following oral immunization of humans and mice.

We have studied specific antibody production from peripheral blood lymphocytes (PBL) after oral cholera immunization of humans and mice. Two oral immunizations with cholera toxin (CT) in mice or a single dose of the combined cholera B-subunit/whole cell vaccine in humans gave rise to PBL which spontaneously secreted cholera-specific antibodies when cultured in vitro. A high proportion of IgA antibodies was seen in contrast to antibodies produced by PBL after parenteral immunization which were predominantly IgG. Cultured PBL produced antitoxin as well as anti-lipopolysaccharide antibodies after oral immunization, whereas serum only revealed titre rises for anti-CT. Antibody-secreting PBL appeared in the blood 2-4 days after immunization and persisted for about two weeks with a peak after 6-8 days. Mitogen stimulation in vitro of PBL from multiply-orally vaccinated humans activated a population of specific IgM antibody-secreting cells which persisted for several months following immunization, suggesting the presence of long-lived memory cells. The analysis of IgA antibody production from in-vitro cultured PBL seems to be a promising technique to assess the local immunogenicity of oral vaccines.     

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.     

Absence of amplification role of the protein KLH on antibody response generated by a MAP Staphyloccocus aureus enterotoxin A (SEA) peptide comparing with the corresponding monomeric peptide

Staphuloccocus aureus enterotoxin A (SEA) is a toxin involved in numerous food poisoning cases. To develop a detection test specific for this toxin, different approaches have been envisaged to obtain the highest antibody titer sera against a peptide sequence from this protein. The present work compares the properties of antibodies raised against the peptide epitope by classical and multiple antigen peptide (MAP) system approaches. Different immunization protocols were used: BALB/c mice were immunized either with the peptide or the MAP alone in Freund's adjuvant, co-immunized with the keyhole limpet hemocyanin (KLH) protein or, with the peptide, conjugated to this carrier KLH protein. The extent of reaction of the antibodies to the MAP construct with the parent protein was found to be significantly less than the antibodies raised against the monomeric peptide co-immunized with or conjugated to a carrier protein but more that the antibodies raised against the peptide alone. Inversely, co-immunization of the MAP with the KLH was not able to raise the immune response as it was observed with the monomeric peptide. The results suggest that, for the epitope chosen here, MAP constructs were not the most effective option to induce sera with high levels of antibodies that react with the native protein.     

Generation of female genital tract antibody responses by local or central (common) mucosal immunization

Genital antibody responses were compared in female mice immunized intravaginally (i.vag.) or intranasally (i.n.) with a bacterial protein antigen (AgI/II of Streptococcus mutans) coupled to the B subunit of cholera toxin. Serum and salivary antibodies were also evaluated as measures of disseminated mucosal and systemic responses. Although i.vag. immunization induced local vaginal immunoglobulin A (IgA) and IgG antibody responses, these were not disseminated to a remote secretion, the saliva, and only modest levels of serum antibodies were generated. In contrast, i.n. immunization was substantially more effective at inducing IgA and IgG antibody responses in the genital tract and in the circulation, as well as at inducing IgA antibodies in the saliva. Moreover, mucosal and systemic antibodies induced by i.n. immunization persisted for at least 12 months. Analysis of the molecular form of genital IgA indicated that the majority of both total IgA and specific IgA antibody was polymeric, and likely derived from the common mucosal immune system.     

Induction of neutralizing antibodies by a tobacco chloroplast-derived vaccine based on a B cell epitope from canine parvovirus

The 2L21 epitope of the VP2 protein from the canine parvovirus (CPV), fused to the cholera toxin B subunit (CTB-2L21), was expressed in transgenic tobacco chloroplasts. Mice and rabbits that received protein-enriched leaf extracts by parenteral route produced high titers of anti-2L21 antibodies able to recognize the VP2 protein. Rabbit sera were able to neutralize CPV in an in vitro infection assay with an efficacy similar to the anti-2L21 neutralizing monoclonal antibody 3C9. Anti-2L21 IgG and seric IgA antibodies were elicited when mice were gavaged with a suspension of pulverized tissues from CTB-2L21 transformed plants. Combined immunization (a single parenteral injection followed by oral boosters) shows that oral boosters help to maintain the anti-2L21 IgG response induced after a single injection, whereas parenteral administration of the antigen primes the subsequent oral boosters by promoting the induction of anti-2L21 seric IgA antibodies. Despite the induced humoral response, antibodies elicited by oral delivery did not show neutralizing capacity in the in vitro assay. The high yield of the fusion protein permits the preparation of a high number of vaccine doses from a single plant and makes feasible the oral vaccination using a small amount of crude plant material. However, a big effort has still to be done to enhance the protective efficacy of subunit vaccines by the oral route.     

Natural and immune antibodies for Vibrio fetus in serum and secretions of cattle

Naturally occurring antibodies for Vibrio fetus with whole-cell and O antigen specificities were detected in sera of mature cattle by means of the agglutination and indirect immunofluorescence reactions. O antibodies occurred in each of 21 sera examined, and whole-cell antibodies, in lower concentrations, occurred in 15 of the 21 sera. Natural antibodies were detected in the immunoglobulin G, M, and A (IgG, IgM, IgA) classes. Parenteral immunization with whole cells in Freund's complete adjuvant caused an increase in antibodies, predominantly IgG, with both whole-cell and O specificities; genital infection produced minimal alterations in levels of serum antibody. O antibodies, predominantly of the IgA class, occurred in nasal secretions of most of these animals but only infrequently in saliva or tears. Nasal secretion titers of O antibodies fluctuated widely from week to week but appeared to be unaffected by parenteral immunization or experimental infection. It is hypothesized that the nasal passages serve as a site of antigenic stimulation and synthesis of naturally occurring V. fetus antibodies.     

Comparative immunogenicities of Vi polysaccharide-protein conjugates composed of cholera toxin or its B subunit as a carrier bound to high- or lower-molecular-weight Vi.

The effect of molecular weight or size of the components on the immunogenicity of polysaccharide-protein conjugates prepared with the native Vi capsular polysaccharide (Vi) (approximately 3 x 10(3) kilodaltons) or lower-molecular-weight Vi (Vis; approximately 46 kilodaltons) abound to cholera toxin (CT) or to its B subunit (CTB) was studied. In mice, Vi-CT, Vi-CTB, and Vis-CTB elicited higher Vi antibody levels than the Vi alone (P less than 0.0001). Vi-CT and Vi-CTB were more immunogenic than Vis-CTB (P less than 0.01). CT or Vi-CT elicited higher levels of CT antibodies, as measured by enzyme-linked immunosorbent assay, than did CTB or Vi-CTB. In rhesus monkeys, the Vi conjugates elicited higher Vi antibody levels than the Vi alone (P less than 0.01). Vi-CTB elicited higher levels of Vi antibody after each injection than did Vis-CTB. Similar levels of CT antibodies, as measured by enzyme-linked immunosorbent assay, were elicited by all three conjugates. In contrast, Vi-CT elicited higher levels of neutralizing antibodies than Vi-CTB or Vis-CTB when either CT or the related heat-labile toxin of Escherichia coli was used as the antigen. These results indicate that the holotoxin and the native Vi provide the most immunogenic components for conjugates designed to induce both Vi and CT antibodies.     

Intranasal Immunization with Heat-Inactivated Streptococcus pneumoniae Protects Mice against Systemic Pneumococcal Infection

In order to study the mucosal and serum antibody response to polysaccharide-encapsulated bacteria in mice, a preparation of heat-inactivated Streptococcus pneumoniae type 4 was administered, with and without cholera toxin, at various mucosal sites. It appeared that intranasal immunization of nonanesthesized animals was superior to either oral, gastric, or colonic-rectal antigen delivery with regard to the induction of serum immunoglobulin G (IgG) and IgA, as well as saliva IgA antibodies specific for pneumococci. The marked IgA antibody response in feces after intranasal, but not after oral or gastric, immunization is suggestive of a cellular link between the nasal induction site and the distant mucosal effector sites. Intranasal immunization also induced antibodies in serum and in mucosal secretions against type-specific capsular polysaccharide. IgA and IgG antibody levels in pulmonary lavage fluids correlated well with saliva IgA and serum IgG antibodies, respectively. Antibody determinations in pulmonary secretions may therefore be redundant in some cases, and the number of experimental animals may be reduced accordingly. After intraperitoneal challenge with type 4 pneumococci, mice immunized intranasally were protected against both systemic infection and death, even without the use of cholera toxin as a mucosal adjuvant. Thus, an efficient intranasal vaccine against invasive pneumococcal disease may be based on a very simple formulation with whole killed pneumococci.     

Local and Systemic Neutralizing Antibody Responses Induced by Intranasal Immunization with the Nontoxic Binding Domain of Toxin A from Clostridium difficile

Fourteen of the 38 C-terminal repeats from Clostridium difficile toxin A (14CDTA) were cloned and expressed either with an N-terminal polyhistidine tag (14CDTA-HIS) or fused to the nontoxic binding domain from tetanus toxin (14CDTA-TETC). The recombinant proteins were successfully purified by bovine thyroglobulin affinity chromatography. Both C. difficile toxin A fusion proteins bound to known toxin A ligands present on the surface of rabbit erythrocytes. Intranasal immunization of BALB/c mice with three separate 10-microg doses of 14CDTA-HIS or -TETC generated significant levels of anti-toxin A serum antibodies compared to control animals. The coadministration of the mucosal adjuvant heat labile toxin (LT) from Escherichia coli (1 microg) significantly increased the anti-toxin A response in the serum and at the mucosal surface. Importantly, the local and systemic antibodies generated neutralized toxin A cytotoxicity. Impressive systemic and mucosal anti-toxin A responses were also seen following coadministration of 14CDTA-TETC with LTR72, an LT derivative with reduced toxicity which shows potential as a mucosal adjuvant for humans.