Antibodies and Antibody-Secreting Cells in the Female Genital Tract after Vaginal or Intranasal Immunization with Cholera Toxin B Subunit or Conjugates

We studied the antibody response including antibody-secreting cells (ASC) in the female genital tract of mice after mucosal immunizations with the recombinant B subunit of cholera toxin (rCTB) perorally, intraperitoneally, vaginally, and intranasally (i.n.). The strongest genital antibody responses as measured with a novel perfusion-extraction method were induced after vaginal and i.n. immunizations, and these routes also gave rise to specific immunoglobulin A (IgA) and IgG ASC in the genital mucosa. Specific ASC in the iliac lymph nodes, which drain the female genital tract, were seen only after vaginal immunization. Progesterone treatment increased the ASC response in the genital tissue after all mucosal immunizations but most markedly after vaginal immunization. We also tested rCTB as a carrier for human gamma globulin (HGG) and the effect of adding cholera toxin (CT) as an adjuvant for the induction of systemic and genital antibody responses to HGG after vaginal and i.n. immunizations. Vaginal immunizations with HGG conjugated to rCTB resulted in high levels of genital anti-HGG antibodies whether or not CT was added, while after i.n. immunization the strongest antibody response was seen with the conjugate together with CT. In summary, vaginal and i.n. immunization give rise to a specific mucosal immune response including ASC in the genital tissue, and vaginal immunization also elicits ASC in the iliac lymph nodes. We have also shown that rCTB can act as an efficient carrier for a conjugated antigen for induction of a specific antibody response in the genital tract of mice after vaginal or i.n. immunization.     

Antibody Responses in the Lower Respiratory Tract and Male Urogenital Tract in Humans after Nasal and Oral Vaccination with Cholera Toxin B Subunit

Nasal vaccine delivery is superior to oral delivery in inducing specific immunoglobulin A (IgA) and IgG antibody responses in the upper respiratory tract. Although an antibody response in the nasal passages is important in protecting against primary colonization with lung pathogens, antibodies in the lungs are usually required as well. We immunized 15 male volunteers twice nasally or orally with cholera toxin B subunit (CTB) and determined the specific antibody levels in serum, bronchoalveolar lavage (BAL) fluid, and urine before and 2 weeks after immunization. Nasal immunization induced fivefold increases in the levels of specific IgA antibodies in BAL fluid of most volunteers, whereas there were no significant specific IgA responses after oral immunization. The specific IgG antibody level increased eightfold in BAL fluid in the nasally vaccinated subjects, and the major part of IgG had most probably been transferred from serum. Since the specific IgG response in serum was lower in the individuals vaccinated orally, the IgG response in BAL fluid in this group was also lower and not significant. In conclusion, nasal immunization is also preferable to the oral route when vaccinating against lower respiratory tract infections, and a systemic immune response is considerably more important in the lower than in the upper respiratory tract. Moreover, both nasal and oral immunizations were able to stimulate 6- to 10-fold specific IgA and IgG responses in urine in about half of the individuals, which indicates that distant mucosal vaccination might be used to prevent adhesion of pathogens to the urogenital tract.     

Kinetics of Local and Systemic Immune Responses to an Oral Cholera Vaccine Given Alone or Together with Acetylcysteine

The possibility that a mucolytic drug, i.e., acetylcysteine, given orally may enhance the gut mucosal or systemic immune response to an oral B-subunit–whole-cell (B-WC) cholera vaccine was evaluated for 40 adult Swedish volunteers, and the kinetics of the immune responses were monitored for responding volunteers. Two doses of vaccine induced similar frequencies of immunoglobulin A (IgA) and IgG antitoxin responses (80 to 90%) and vibriocidal titer increases (60 to 65%) in serum irrespective of whether the vaccine was given alone or together with 2 g of acetylcysteine. In feces the frequencies of IgA antitoxin (67%) and antibacterial (33 to 40%) antibody responses were also comparable in the two immunization groups. Six months after vaccination, IgA and IgG antitoxin as well as vibriocidal antibody titer increases in serum could still be detected in approximately 80% of initially responding vaccinees. Significantly elevated fecal antitoxin and antibacterial IgA antibody levels were found in, respectively, 50 and 43% of those volunteers who initially had responded to the vaccine. Determination of IgA antibodies in feces does not seem to offer any advantages compared to determination in serum for assessment of immune responses after immunization with inactivated cholera vaccine.     

Local (Immunoglobulin A) Immune Response by the Intestine to Cholera Toxin and Its Partial Suppression with Combined Systemic and Intra-Intestinal Immunization

Chronically isolated Thiry-Vella (T-V) ileal loops in rabbits were used to study the local and systemic immune response to purified cholera toxin (CT). Immunization consisted of intraloop (i.l.), subcutaneous (s.c.), or combined i.l. and s.c. inoculation of CT. Fluid from the loops and sera were tested for neutralization of CT by the blueing test and for relative content of isotype-specific (immunoglobulins A [IgA] and G [IgG]) anti-CT. To demonstrate protection against CT, fluid production by the chronic T-V loops was measured after challenge with CT; an "acute" loop prepared from adjacent intestine at the time of challenge was also tested in some animals. The highest neutralizing titers in loop fluids were found in animals receiving i.l. or i.l. and s.c. inoculations, whereas titers in sera were highest in rabbits receiving s.c. or i.l. and s.c. inoculations. IgA anti-CT in fluids became greatest after i.l. inoculation alone and was lowest in s.c. animals. Combined s.c. and i.l. immunization was accompanied by reduced content of IgA anti-CT in fluids as compared with that obtained with i.l. inoculation alone. This finding strongly suggested a suppressive effect on local immunization by s.c. inoculation. While this suppression may have been due to a direct (toxigenic) effect of CT on lymphocytes, an immunogenic mechanism, probably mediated through suppressor T cells, is favored. Little IgG anti-CT was detected in any loop fluids, but high levels were found in sera after two s.c. inoculations or four i.l. inoculations. Neutralization titers for the fluid specimens showed much better correlation with IgA anti-CT values than with IgG anti-CT values. The chronic and acute T-V loops showed protection against fluid production after exposure to CT in systemically and locally immunized animals. However, IgG anti-CT usually appeared in both loops; leakage of serum antibodies because of surgical manipulation was felt, therefore, to invalidate these protection results as a demonstration of local immunity. In challenge studies in undisturbed chronic loops, only local immunization alone was found to result in definite protection.     

Enteric Immunization with Live Adenovirus Type 21 Vaccine II. Systemic and Local Immune Responses Following Immunization

Studies of the immunologic responses following administration of a live, enteric-coated adenovirus (ADV) type 21 vaccine showed that nine of ten vaccinees and none of five controls developed neutralizing antibody. Antibody activity of serum and secretory immunoglobulins was assayed by using a (14)C-labeled ADV-21 antigen in a radioimmunodiffusion system. Increases in immunoglobulin M, A and G (IgM, IgA, IgG) activity were detected in sera from vaccinees but not in those from controls. IgA copro antibody activity was also shown in vaccinees but not in controls. Nasal secretions showed no detectable IgA antibody responses by this method. These studies show marked differences in serum and local IgA antibody activity in induced enteric ADV infection compared to previously reported responses after natural infection. The protective role of secretory IgA in adenovirus infections is obscure. However, absence of nasal IgA responses may indicate that protection against disease with enteric ADV vaccines depends primarily upon humoral antibody.     

Oral Immunization of Dogs with Purified Cholera Toxin, Crude Cholera Toxin, or B Subunit: Evidence for Synergistic Protection by Antitoxic and Antibacterial Mechanisms

The immunogenicity and safety of purified cholera toxin (CT), its B subunit, and a crude culture filtrate of toxigenic Vibrio cholerae (CrT) were compared in dogs immunized orally and challenged with virulent V. cholerae. CT and CrT caused marked protection in two- or three-dose regimens. Protection due to CT occurred only with doses that caused transient, sometimes severe, diarrhea in most dogs; this protection was proportional to the peak antitoxin response in jejunal mucosa and lasted at least 15 weeks. In contrast, minimum protective doses of CrT contained much less cholera toxin, caused very mild diarrhea in only 21% of the dogs, and evoked protection that was greater than predicted from the modest jejunal antitoxin response. B subunit caused smaller jejunal antitoxin responses than did similar doses of CT and was poorly protective, the 50% protective dose being >40-fold greater than that of CT. Two observations indicated that protection due to CrT involved synergy between antibacterial and antitoxic immune responses. First, the 50% protective dose of CrT was 24-fold and >36-fold smaller than the 50% protective doses of its CT and non-CT antigenic components, respectively, when tested separately. Second, protection was greater in CrT-immunized dogs than in CT-immunized dogs for a given mucosal antitoxin response. Low doses of CrT evoked serotype-specific protection, indicating that the serotype-specific O somatic antigen contributed significatly to antibacterial protection. These results suggest that a simple, effective, nonliving oral vaccine for cholera based on combined antibacterial and antitoxic immunity can probably be achieved. However, further studies are needed to determine how a protective antitoxic response can be evoked without causing diarrhea during immunization.     

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 G_M1 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.     

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.     

Oral Immunization with Recombinant Lactobacillus acidophilus Expressing the Adhesin Hp0410 of Helicobacter pylori Induces Mucosal and Systemic Immune Responses

Helicobacter pylori infection is relatively common worldwide and is closely related to gastric mucosa-associated lymphoid tissue (MALT) lymphoma, chronic gastritis, and stomach ulcers. Therefore, a safe and effective method for preventing H. pylori infection is urgently needed. Given that developing an effective vaccine against H. pylori is one of the best alternatives, H. pylori adhesin Hp0410 was expressed in the food-grade bacterium Lactobacillus acidophilus. The recombinant live bacterial vaccine was then used to orally vaccinate mice, and the immunoprotective effects of Hp0410-producing strains were investigated. H. pylori colonization in the stomach of mice immunized with the recombinant L. acidophilus was significantly reduced, in comparison with that in control groups. Furthermore, mucosal secretory IgA antibodies were elicited in the mucosal tissue of mice immunized with the recombinant bacteria, and specific anti-Hp0410 IgG responses were also detected in mouse serum. There was a significant increase in the level of protection against gastric Helicobacter infection following a challenge with H. pylori Sydney strain 1 (SS1). Our results collectively indicate that adhesin Hp0410 is a promising candidate vaccine antigen, and recombinant L. acidophilus expressing Hp0410 is likely to constitute an effective, low-cost, live bacterial vaccine against H. pylori.     

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.     

Whole Cholera Toxin and B Subunit Act Synergistically as an Adjuvant for the Mucosal Immune Response of Mice to Keyhole Limpet Haemocyanin

Cholera toxin (CT) is a potent stimulator of IgA responses when administered orally and has been shown to promote IgA responses to a second protein such as keyhole limpet haemocyanin (KLH) if this is fed simultaneously. In this paper we show that whilst feeding 5 mg KLH with either 0.5 micrograms CT or 10 micrograms B subunit fails to stimulate a mucosal IgA response to KLH, feeding 0.5 microgram CT and 10 micrograms B subunit together with 5 mg KLH produces a local IgA anti-KLH response as great as that produced by 10 micrograms of whole CT. In addition to stimulating IgA responses in the lamina propria, preliminary results indicate that cellular responses are also stimulated, as we have demonstrated KLH antigen-driven proliferation of cells isolated from groups of mice fed either 10 micrograms CT + 5 mg KLH or 0.5 micrograms CT + 10 micrograms CTB + 5 mg KLH but not mice fed KLH alone or with either 10 micrograms CTB or 0.5 micrograms CT. These results indicate that the mucosal adjuvant action of CT is due to a synergistic effect involving both the GM1 binding of the B subunit and adenylate cyclase activation by the A subunit.     

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.     

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.     

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

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

双价志贺疫苗滴鼻免疫小鼠后粘膜免疫与系统免疫应答持续时间的观察

本文探讨双价志贺疫苗滴鼻免疫小鼠一段时间后,粘膜免疫和系统免疫应答的变化。将BALB/c小鼠随机分为三组,每组30只。PBS、FSM-2117和FS-5416(菌量为5×10~6、1×10~7、4×10~7和4×10~7CFU/只/次)经滴鼻途径免疫小鼠。间隔2周,4次免疫后7、30和90d活杀,收集鼻咽、肺、肠、生殖道冲洗液和血清。采用ELISA法检测其中特异性抗福氏、宋内LPSIgA和IgG。结果是两株疫苗经鼻内免疫后,诱发鼻咽、肺、胃肠道和生殖道等不同粘膜部位及血清中特异性抗福氏、宋内LPSIgA、IgG的显著增加(P<0.01)。特异性抗体水平虽然在免疫后30、90d明显下降,但仍明显高于PBS对照组水平。故认为两株双价志贺疫苗滴鼻免疫小鼠后能有效诱导粘膜免疫和系统免疫应答,并持续较长时间。     

Receptor-Dependent Immune Responses in Pigs after Oral Immunization with F4 Fimbriae

F4 receptor-positive (F4R⁺) and F4 receptor-negative (F4R⁻) pigs were orally vaccinated with purified F4 fimbriae of enterotoxigenic Escherichia coli (ETEC). Serum immunoglobulin G (IgG) and IgA responses were readily detected in F4R⁺ animals, whereas immune responses were not detected in F4R⁻ animals. Even after a subsequent oral infection with virulent F4⁺ ETEC and a booster immunization with F4, the F4R⁻ animals remained F4 seronegative whereas the unvaccinated F4R⁺ pigs exhibited clear IgA and IgG responses. These results clearly demonstrate that F4Rs are a prerequisite for an immune response following oral immunization. Furthermore, indications that oral F4 vaccination can induce mucosal protection were obtained, since the experimental ETEC infection did not induce a systemic booster response or fecal ETEC excretion in orally vaccinated F4R⁺ pigs, in contrast to the clear immune response and ETEC excretion of unvaccinated F4R⁺ animals. F4-specific IgA antibodies could be found in the feces of the vaccinated F4R⁺ pigs. They are secreted at the intestinal mucosal surface and appear to prevent ETEC infection. The F4R-dependent induction of a mucosal immune response can be used as a model to better understand mucosal immunization and mucosal immune responses and can contribute to the development of oral vaccines in veterinary as well as in human medicine.     

Local and Systemic Immune and Inflammatory Responses to Helicobacter pylori Strains

Colonization with Helicobacter pylori eventuates in varied clinical outcomes, which relate to both bacterial and host factors. Here we examine the relationships between cagA status, serum and gastric juice antibody responses, and gastric inflammation in dyspeptic patients. Serum, gastric juice, and gastric biopsy specimens were obtained from 89 patients undergoing endoscopy. H. pylori colonization and cagA status were determined by histology, culture, and PCR methods, and acute inflammation and chronic inflammation in the gastric mucosa were scored by a single pathologist. Serum and gastric juice antibodies to H. pylori whole-cell and CagA antigens were determined by enzyme-linked immunosorbent assay. Relationships between variables were sequentially analyzed using univariate and multivariate statistical methods. Of the 89 subjects, 62 were colonized by H. pylori. By univariate analyses, levels of serum immunoglobulin G (IgG) and IgA and gastric juice IgA antibodies against whole-cell and CagA antigens each were significantly higher in the H. pylori-positive group than in the H. pylori-negative group (P < 0.001). H. pylori and CagA seropositivities were both significantly associated with enhanced inflammation in gastric antrum and body (P < 0.02). The presence of gastric juice antibodies to H. pylori antigens was associated with more severe gastric inflammation. However, in multivariate analyses, only the presence of serum antibodies against CagA and, to a lesser extent, whole-cell antigens remained significantly associated with acute and chronic inflammation in antrum and body (P < 0.05). Thus, serum antibody response to CagA correlates with severity of gastric inflammation. Furthermore, given the relationships demonstrated by multivariate analysis, determination of gastric juice antibodies may provide a better representation of serum, rather than secretory, immune response.     

Plasmodium vivax Ookinete Surface Protein Pvs25 Linked to Cholera Toxin B Subunit Induces Potent Transmission-Blocking Immunity by Intranasal as Well as Subcutaneous Immunization

The nontoxic cholera toxin B subunit (CTB) was evaluated as a potential delivery molecule for the Plasmodium vivax ookinete surface protein, Pvs25. Recombinant Pvs25 was expressed as a secreted protein in the yeast Pichia pastoris, as a mixture of isoforms including multimers and the A and B monomers. The A isoform with the presumed native protein fold was the most abundant, accounting for more than 40% of all expressed protein. The molecularly uniform A isoform was chemically conjugated to CTB via its primary amines, and the fusion protein, retaining GM1-ganglioside affinity, was administered to BALB/c mice by the subcutaneous (s.c.) or intranasal (i.n.) route. Immunization of mice with conjugated Pvs25 without supplemental adjuvant induced antisera that specifically recognized P. vivax ookinetes in vitro. Furthermore, the antisera, when mixed with parasitized blood isolated from P. vivax patients from Thailand, was found to reduce parasite transmission to mosquitoes, conferring a 93 to 98% (s.c.) or a 73 to 88% (i.n.) decrease in oocyst number. Unconjugated Pvs25 alone conferred only a 23 to 60% (s.c.) or a 0 to 6% (i.n.) decrease in oocyst number. Coadministration of extraneous adjuvants, however, further enhanced the vaccine efficacy up to complete blockade. Taken together, we conclude that a weakly immunogenic Pvs25 by itself, when linked to CTB, transforms into a potent transmission-blocking antigen in both i.n. and s.c. routes. In addition, the present study is, to the best of our knowledge, the first demonstration of the immune potentiating function of CTB for a vaccine antigen delivered by the s.c. route.Malaria is one of the most serious infectious diseases, with high mortality and morbidity, especially in tropical regions of the world. The disease causes 350 to 500 million clinical cases every year, and the estimated annual mortality exceeds 1.1 million (28). Implementation of many malaria control measures, including chemotherapy and insecticide-treated bed nets, have made a significant contribution to the reduction of malaria cases worldwide; however, these control measures are suboptimal, and hence new tools, particularly vaccines, should be used for local elimination and the ultimate eradication of malaria from the globe (9, 10, 24). The development of effective and affordable malaria vaccines is therefore likely to benefit global public health (Malaria Vaccine Technology Roadmap [MVTR], 2006 [http://www.malariavaccineroadmap.net/pdfs/Malaria_Vaccine_TRM_Final.pdf]).Although Plasmodium falciparum causes the highest mortality rates among the four Plasmodium species known to infect humans (18), P. vivax malaria has the highest morbidity and is an important cause of recurrent malaria. This species is therefore an important target of malaria control efforts (4-6; MVTR). Furthermore, because global malaria eradiation is the ultimate goal, the value of developing vaccines against P. vivax cannot be underestimated (4-6; MVTR). Several promising vaccine candidates have been intensively investigated, such as those targeting the asexual stages, i.e., the sporozoite, hepatic and erythrocytic stages, which are designed to prevent infection and to reduce disease severity. On the other hand, transmission-blocking vaccines (TBVs) that target the sexual stage, in which the parasite undergoes sporogonic development in anopheline mosquitoes, prevent parasite transmission from mosquitoes to humans (7, 14, 25). TBVs induce antibodies that react with the ookinete surface proteins (OSPs) of malaria parasites within the mosquito midgut, and as such they do not directly protect vaccinated individuals from infection. They could, however, contribute to elimination of the disease by lowering the parasite transmission frequency below the threshold at which the parasite can maintain its life cycle (4, 6). In addition, TBVs, when combined with vaccines targeting other stages of the infection, could prevent transmission of parasites that have escaped the immune response. Furthermore, TBVs could also prevent transmission of drug-resistant parasites, which will likely to emerge when mass administration of primaquine is initiated. Therefore, TBVs might function as a “safety net” for pre-erythrocytic and erythrocytic vaccines, as well as other nonvaccine interventions.We have recently tested whether a mucosal vaccination regime can be applied to TBVs, on the premise that noninvasive and easy-to-administer mucosal vaccines are advantageous in a mass vaccination campaign in a region where malaria is endemic (1-3). In these animal studies, we have demonstrated the potential of mucosal vaccines to block parasite transmission, but enhancement of the mucosal immunogenicity of recombinant antigens was found to be critically dependent upon the use of cholera toxin (CT) adjuvant. CT is well known for its high immune potentiating function for admixed antigens administered through the mucosal, particularly the intranasal (i.n.), route (13). However, its clinical application is hampered by its severe toxicity (26). Thus, alternative vaccine formulations not using the CT holotoxin are highly desirable.Here, we extended our previous studies to test our hypothesis that the immunogenicity of a Plasmodium vivax malaria OSP, Pvs25, becomes substantially augmented when physically linked to the nontoxic B subunit of CT (CTB), even without supplementation with extraneous adjuvants. This should, in theory, effectively reduce parasite transmission to mosquitoes. Furthermore, we tested the TBV efficacy of the engineered fusion complex in a subcutaneous (s.c.) immunization regimen to test the immune potentiating function of CTB with this particular immunization route.     

Protection against Cholera Toxin after Oral Immunization is Thymus-Dependent and Associated with Intestinal Production of Neutralizing IgA Antitoxin

We studied whether gut mucosal IgA antitoxin production as well as the acquired protection against cholera toxin (CT) after oral immunization with CT are both thymus-dependent immune manifestations. In contrast to normal BALB/c mice, nude, athymic mice did not respond to oral immunizations with CT with either IgA antitoxin-producing cells (SFC) in the lamina propria or protection against challenge with CT in ligated intestinal loops. However, when nude mice were first reconstituted by grafting of syngeneic thymus glands, both IgA antitoxin SFC in the lamina propria and protection were stimulated by oral immunizations with CT and the response were of similar magnitude to those of normal mice after immunizations. During in vitro culture, isolated lamina propria lymphocytes from immunized but not from control mice concomitantly and proportionally produced IgA antitoxin and CT-neutralizing activity. We conclude that intestinal antitoxin formation and protection against toxin challenge after oral immunization with CT are both critically thymus-dependent and therefore likely to be under T-cell control.     

Cell-Mediated Immune Responses in Four-Year-Old Children after Primary Immunization with Acellular Pertussis Vaccines

Cell-mediated immune (CMI) responses to Bordetella pertussis antigens (pertussis toxin [PT], pertactin [PRN], and filamentous hemagglutinin [FHA]) were assessed in 48-month-old recipients of acellular pertussis [aP] vaccines (either from Chiron-Biocine [aP-CB] or from SmithKline Beecham [aP-SB]) and compared to CMI responses to the same antigens at 7 months of age, i.e., 1 month after completion of the primary immunization cycle. None of the children enrolled in this study received any booster of pertussis vaccines or was affected by pertussis during the whole follow-up period. Overall, around 75% of 4-year-old children showed a CMI-positive response to at least one B. pertussis antigen, independently of the type of aP vaccine received, and the proportion of CMI responders were at least equal at 48 and 7 months of age. However, longitudinal examination of individual responses showed that from 20 (against PT) to 37% (against FHA) of CMI responders after primary immunization became negative at 48 months of age. This loss was more than compensated for by conversion to positive CMI responses, ranging from 36% against FHA to 69% against PRN, in other children who were CMI negative at 7 months of age. In 60 to 80% of these CMI converters, a lack of decline or even marked elevation of antibody (Ab) titers against B. pertussis antigens also occurred between 20 and 48 months of age. In particular, the frequency of seropositivity to PRN and FHA (but not to PT) was roughly three times higher in CMI converters than in nonconverters. The acquisition of CMI response to B. pertussis antigens in 48-month-old children was not associated with a greater frequency of coughing episodes lasting >/=7 days and was characterized by a prevalent type 1 cytokine profile, with high gamma interferon and low or no production of interleukin-5, reminiscent of cytokine patterns following immunization with whole-cell pertussis vaccine or natural infection. Our data imply that vaccination-induced systemic CMI may wane by 4 years of age but may be acquired or naturally boosted by symptomless or minor clinical infection by B. pertussis. This might explain, at least in part, the persistence of protection against typical pertussis in aP vaccine recipients despite a substantial waning of both Ab and CMI responses induced by the primary immunization.