Enhanced mucosal and systemic immune responses to Helicobacter pylori antigens through mucosal priming followed by systemic boosting immunizations

It is estimated that Helicobacter pylori infects the stomachs of over 50% of the world's population and if not treated may cause chronic gastritis, peptic ulcer disease, gastric adenocarcinoma and gastric B-cell lymphoma. The aim of this study was to enhance the mucosal and systemic immune responses against the H. pylori antigens cytotoxin-associated gene A (CagA) and neutrophil-activating protein (NAP), through combinations of mucosal and systemic immunizations in female BALB/c mice. We found that oral or intranasal (i.n.) followed by i.m. immunizations induced significantly higher serum titres against NAP and CagA compared to i.n. alone, oral alone, i.m. alone, i.m. followed by i.n. or i.m. followed by oral immunizations. However, only oral followed by i.m. immunizations induced anti-NAP antibody-secreting cells in the stomach. Moreover, mucosal immunizations alone or in combination with i.m., but not i.m. immunizations alone, induced mucosal immunoglobulin A (IgA) responses in faeces. Any single route or combination of immunization routes with NAP and CagA preferentially induced antigen-specific splenic interleukin-4-secreting cells and far fewer interferon-gamma-secreting cells in the spleen. Moreover, i.n. immunizations alone or in combination with i.m. immunizations induced predominantly serum IgG1 and far less serum IgG2a. Importantly, we found that while both i.n. and i.m. recall immunizations induced similar levels of serum antibody responses, mucosal IgA responses in faeces were only achieved through i.n. recall immunization. Collectively, our data show that mucosal followed by systemic immunization significantly enhanced local and systemic immune responses and that i.n. recall immunization is required to induce both mucosal and systemic memory type responses.     

Soluble invasion plasmid antigen C (IpaC) from Shigella flexneri elicits epithelial cell responses related to pathogen invasion.

Shigella flexneri invades colonic epithelial cells by pathogen-induced phagocytosis. The three proposed effectors of S. flexneri internalization are invasion plasmid antigens B (IpaB), IpaC, and IpaD, which are encoded on the pathogen's 230-kb virulence plasmid and translocated to the extracellular milieu via the Mxi-Spa translocon. To date, there are no definitive functional data for any purified Ipa protein. Here, we describe the first characterization of highly purified recombinant IpaC, which elicits numerous epithelial cell responses related to events that take place during pathogen invasion. 125I-labeled IpaC binds cultured Henle 407 intestinal cells with an apparent dissociation constant in the low micromolar range. Moreover, incubation of epithelial cells with IpaC results in general changes in cellular phosphoprotein content, demonstrating this protein's ability to influence cellular protein kinase activities. These results contrast dramatically with those seen for recombinant IpaD, which does not bind to or induce detectable changes in the normal activities of cultured epithelial cells. In addition to influencing host cell activities, preincubation of epithelial cells with purified IpaC enhances uptake of S. flexneri by host cells. A similar result is seen when the cells are preincubated with a highly concentrated water extract of virulent S. flexneri 2a (strain 2457T). Interestingly, soluble IpaC also appears to promote uptake of the noninvasive S. flexneri 2a strain BS103. Purified IpaD failed to enhance the uptake of virulent S. flexneri and did not facilitate uptake of BS103. Taken together, the data suggest that IpaC is a potential effector of the host cell biological activities and may be responsible for entry of S. flexneri into target cells.     

Shigella flexneri invasion plasmid antigens B and C: epitope location and characterization with monoclonal antibodies.

Invasion plasmid antigens B (IpaB) and C (IpaC) are associated with the ability of shigellae to invade cultured mammalian cells. Monoclonal antibodies against IpaB and IpaC polypeptides were produced and used in a whole-cell enzyme-linked immunosorbent assay to show that both IpaB and IpaC polypeptides were exposed on the surface of virulent shigellae. Moreover, these surface epitopes were shown to be highly conserved among different serotypes of Shigella spp. and enteroinvasive Escherichia coli. Cross-reactive epitopes were not found on noninvasive Shigella strains or on other enteric bacteria including Salmonella, Yersinia, Campylobacter, Vibrio, and Aeromonas spp. and various pathogenic strains of E. coli. The monoclonal antibodies were used in competitive binding assays to define three unique epitopes of the IpaB polypeptide and four unique epitopes of the IpaC polypeptide. Epitope locations and their corresponding DNA-encoding regions were defined by examining the IpaB and IpaC products expressed by lambda gt11 recombinants and by constructing a genetic map of the insert DNAs of these recombinants. Three IpaB epitopes (2F1, 1H4, 4C8) were found to be encoded on three contiguous DNA regions comprising a 700-base-pair (bp) segment that corresponded to the amino-terminal end of the IpaB polypeptide. Similarly, a 640-bp DNA segment that corresponded to the amino-terminal end of the IpaC polypeptide was found to encode three clustered IpaC epitopes (5H1, 9B6, 5B1). Approximately 50 bp downstream from this region a fourth IpaC epitope-encoding region (2G2) was found. The effect of the monoclonal antibodies on plaque formation by virulent Shigella flexneri on a monolayer of cultured mammalian cells (a sensitive measure of invasiveness) was determined. Only the IpaB-specific monoclonal antibody 2F1 was able to reduce the plaque-forming capacity by greater than 50%, suggesting that this epitope of the IpaB polypeptide is involved in the invasion process.     

Gastric invasion by Trypanosoma cruzi and induction of protective mucosal immune responses.

Trypanosoma cruzi is an intracellular parasite transmitted from a reduviid insect vector to humans by exposure of mucosal surfaces to infected insect excreta. We have used an oral challenge murine model that mimics vector-borne transmission to study T. cruzi mucosal infection. Although gastric secretions have microbicidal activity against most infectious pathogens, we demonstrate that T. cruzi can invade and replicate in the gastric mucosal epithelium. In addition, gastric mucosal invasion appears to be the unique portal of entry for systemic T. cruzi infection after oral challenge. The mucosal immune responses stimulated by T. cruzi gastric infection are protective against a secondary mucosal parasite challenge. This protective mucosal immunity is associated with increased numbers of lymphocytes that secrete parasite-specific immunoglobulin A. Our results document the first example of systemic microbial invasion through gastric mucosa and suggest the feasibility of a mucosal vaccine designed to prevent infection with this important human pathogen.     

Evidence for long-term memory of the mucosal immune system: milk secretory immunoglobulin A against Shigella lipopolysaccharides.

Although the common mucosal immune system has generally been considered to have only short-term memory, recent data suggest that long-term memory exists for Shigella virulence plasmid antigens. Because such antigens might cross-react with environmental antigens, we investigated milk for the persistence of antibodies to the specific Shigella lipopolysaccharide (LPS) antigens. Enzyme-linked immunosorbent assays to detect secretory immunoglobulin A (sIgA) against Shigella flexneri and Shigella sonnei LPS in milk samples were developed; 15 random milk samples tested on different days correlated from one day to the next (P = 0.0001). Of 18 Mexican mothers, 18 (100%) had one or more milk samples positive for anti-S. flexneri LPS, 14 (78%) had one or more milk samples positive for anti-S. sonnei LPS, and 14 (78%) had one or more milk samples positive for both. Of 27 Houston mothers, 16 (59%) had one or more milk samples positive for anti-S. flexneri LPS, 7 (26%) had one or more milk samples positive for anti-S. sonnei LPS, and 5 (19%) had one or more milk samples positive for both. Mexican mothers were significantly more likely than Houston mothers to have at least one sample with a positive titer for anti-S. flexneri LPS (P less than 0.02) and at least one sample with a positive titer for anti-S. sonnei LPS (P less than 0.002). Although the Houston women had a lower rate of titer positivity for both Shigella species, the rate was too high to be consistent with short-lived mucosal immunity. It is unlikely that 18 of the 27 Houston women had shigellosis during or just prior to lactation. The data suggest that there exists a long-term hormonally driven memory in the secretory immune system for Shigella spp.     

Humoral immune responses in periodontal disease may have mucosal and systemic immune features

The humoral immune response, especially IgG and IgA, is considered to be protective in the pathogenesis of periodontal disease, but the precise mechanisms are still unknown. Immunoglobulins arriving at the periodontal lesion are from both systemic and local tissue sources. In order to understand better the local immunoglobulin production, we examined biopsy tissue from periodontitis lesions for the expression of IgM, IgG, IgA, IgE and in addition the IgG and IgA subclasses and J-chain by in situ hybridization. Tissues examined were superficial inflamed gingiva and the deeper granulation tissue from periodontal sites. These data confirm that IgM, and IgG and IgA subclass proteins and J-chain can be locally produced in the periodontitis tissues. IgG1 mRNA-expressing cells were predominant in the granulation tissues and in the gingiva, constituting approx. 65% of the total IgG-expressing plasma cells. There was a significantly increased proportion of IgA-expressing plasma cells in the gingiva compared with the granulation tissue (P < 0.01). Most of the IgA-expressing plasma cells were IgA1, but a greater proportion expressed IgA2 mRNA and J-chain mRNA in the gingival tissues (30.5% and 7.5%, respectively) than in the periodontal granulation tissues (19% and 0-4%, respectively). The J-chain or dimeric IgA2-expressing plasma cells were located adjacent to the epithelial cells, suggesting that this tissue demonstrates features consistent with a mucosal immune response. Furthermore, we were able to detect the secretory component in gingival and junctional epithelial cells, demonstrating that the periodontal epithelium shares features with mucosal epithelium. In contrast, deeper tissues had more plasma cells that expressed IgM, and less expressing IgA, a response which appears more akin to the systemic immune response. In conclusion, this study suggests that immune mechanisms involved in the pathogenesis of periodontitis may involve features of both the mucosal and systemic immune systems, dependent on tissue location.     

Cellular immune responses of hydatid patients to Echinococcus granulosus antigens.

The reactivity of peripheral blood mononuclear cells (PBMC) from 40 hydatid patients to hydatid fluid (HF) and to two hydatid fractions (pH5PPT and pH5SUP) was evaluated by the incorporation of 3H-methylthymidine into DNA. Maximal responses were detected using 200 micrograms/ml protein after 7-9 days incubation. The three antigen preparations were inducers of PBMC proliferation, with a good correlation (r2 = 0.87) of the responses induced by HF and by the fraction pH5PPT, which contains the two major hydatid antigens (5 and B). Lymphocytes from healthy donors and non-hydatid patients showed no response to these antigens. Neither direct nor inverse correlation was found between the results of the serological tests and of the PBMC proliferation assays. The majority of the patients (75%) responded in serological and in cellular tests. Of the remaining patients, six showed high antibody response associated with a negative PBMC proliferation assay and conversely four seronegative patients were found to respond positively in the PBMC proliferation assay. No relationship of the pattern of immune responsiveness to the patient's clinical forms could be established. Use of the PBMC proliferation assay with hydatid antigens appears rational in those patients which are low antibody producers, but the test is still not to be considered applicable for routine diagnosis.     

The influence of oral immunization on local and systemic immune responses to heterologous antigens.

Oral immunization with 25 mg/day of ovalbumin resulted in a number of changes in non-specific immune responsiveness, including increases in antibody produced to T-dependent but not T-independent antigens and the abrogation of tolerance induced by both oral and intravenous routes. The effect was transient, sex- and strain-dependent. Evidence suggests that it is a consequence of cell-mediated immunity to the oral immunogen.     

Myosin-cross-reactive epitope of Shigella flexneri invasion plasmid antigen B.

IpaB, invasion plasmid antigen B, of Shigella flexneri is a 62-kDa protein required for invasion of intestinal epithelial cells. IpaB is also one of several major protein antigens recognized by the humoral immune systems of most humans and monkeys after infection with shigellae. Computer analysis of the deduced IpaB amino acid sequence indicates that an alpha-helical structure is likely through much of the molecule. Homology searches with protein data banks show that one alpha-helical domain between amino acid residues 95 and 181 has a moderate level of identity with myosin and streptococcal M protein. By using a monoclonal antibody (2F1) which recognizes an epitope in the amino-terminal third of the IpaB protein, it was possible to demonstrate a cross-reactive epitope(s) on skeletal muscle myosin. Epitope mapping localized the 2F1 epitope to three noncontiguous regions of the IpaB protein within the alpha-helical domain that contains homology with myosin. Antibodies produced in rabbits immunized with synthetic peptides from one of the 2F1 epitope regions (residues 99 to 110) of IpaB were capable of reacting with IpaB as well as myosin. Furthermore, sera from several monkeys previously infected with S. flexneri 2a contained antibodies to IpaB pep 101-116 (IpaB peptide 101-116) and also myosin. Sera from animals with antibodies against other IpaB peptides did not contain antibodies against myosin.     

Inhibition of specific immune responses by feeding protein antigens

A profound and long-lasting state of specific immune unresponsiveness may be induced in adult inbred mice given a single dose of protein immunogens--such as ovalbumin or hemocyanin--by the digestive route. The degree of unresponsiveness induced by intragastric exposure to ovalbumin could not be achieved by intravenous injection of deaggregated ovalbumin solutions across a wide range of doses. Unresponsiveness induced by intragastric exposure to hapten-protein conjugates is specific to the carrier protein.     

Strong adjuvant properties of cholera toxin on gut mucosal immune responses to orally presented antigens.

There is a great need for substances that can act as adjuvants on local mucosal immune responses to perorally (p.o.) administered immunogens and which could be included in future oral vaccines. In this study we show that in mice cholera toxin (CT) is a potent adjuvant on enteric mucosal immune responses to related (cholera B subunit) as well as unrelated (KLH) antigens presented by the p.o. route. The adjuvant action of CT was dose-dependent and was achieved only when CT was given p.o. and together with the antigen. Both priming (memory induction) and boosting of the gut mucosal immune system by the oral route were greatly potentiated by CT. High numbers of specific antibody-producing cells as well as substantial mucosal memory in the lamina propria were stimulated by p.o. priming immunizations if CT adjuvant was included. Anamnestic responses could be elicited by a single p.o. booster immunization for at least 10 weeks and probably much longer. The adjuvant action of CT is suggested to involve activation of adenylate cyclase and cyclic AMP-mediated signals with differential effects on B and regulatory T intestinal lymphocytes. The adjuvant-active dose of CT, 100-500 ng, was lower than the immunogenic dose (2 micrograms) and much below the p.o. dose needed for detectable net fluid secretion in mouse intestine (5-10 micrograms). Cholera B subunit (10 micrograms) administered p.o. together with 500 ng of CT was 50 times more effective in stimulating gut mucosal anti-toxin responses compared with B subunit vaccine alone. Our results suggest that CT or substances that use similar adjuvant mechanisms may substantially increase the mucosal immunogenicity and efficacy of non-replicating oral vaccines.     

Serum immune response to Shigella protein antigens in rhesus monkeys and humans infected with Shigella spp.

The serum antibody response to proteins encoded by the virulence-associated plasmid of Shigella flexneri was determined in monkeys challenged with virulent S. flexneri serotype 2a. With water-extractable antigen in an enzyme-linked immunosorbent assay, a significant increase in antibody titer against proteins from a plasmid-carrying, virulent strain of S. flexneri serotype 5 could be demonstrated in convalescent sera. There were minimal antibody titers against proteins from an avirulent (plasmid-free) organism. Previously identified plasmid-coded polypeptides a, b, c, and d were predominant antigens recognized by a majority of the convalescent sera in immunoblots. An additional 140-megadalton plasmid-coded polypeptide was also recognized by half of the sera. Convalescent serum from an infected monkey recognized antigens on the bacterial surface in several different plasmid-containing Shigella species and in an enteroinvasive Escherichia coli strain. A survey of sera obtained from children 5 to 10 years of age who had been infected with S. flexneri or S. sonnei revealed high enzyme-linked immunosorbent assay titers in both acute and convalescent sera against a water extract from a virulent Shigella strain. In contrast, children under 3 years of age had no antibody titer in either acute or convalescent sera against the virulence-associated shigella proteins, while 3- to 4-year-old children mounted an immune response against these proteins only in convalescence.     

双价痢疾菌苗滴鼻免疫小鼠诱导粘膜与系统免疫反应

目的探讨双价痢疾菌苗滴鼻免疫后对小鼠不同粘膜部位和系统免疫部位的影响.方法BALB/c小鼠随机分为3组,每组20只,FSM-2117或FS-54164×107CFU/只经滴鼻途径免疫小鼠,间隔2w,3次免疫后7d活杀,分离NALT、鼻通道、脾、小肠PP及MLN淋巴细胞,采用流式细胞术检测其淋巴细胞表型的变化;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、宋内氏LPSIgA和IgG.结果两株菌苗经鼻内免疫都诱发了鼻咽、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、宋内氏LPSIgA、IgG的显著增加(P＜0.01);NALT、NP和PP淋巴细胞中CD3+T细胞显著增加,其中以CD4+T细胞增加为主;FSM-2117免疫组的脾细胞中B220+细胞显著增加,而FS-5416免疫组的脾细胞中CD3+T细胞显著增加.结论两株菌苗经鼻粘膜免疫均可诱导不同粘膜部位及系统免疫反应的发生,鼻粘膜是一个安全有效的免疫途径.     

Mucosal and systemic immune responses to plasmid protein pgp3 in patients with genital and ocular Chlamydia trachomatis infection

The circulating and cervical B cell responses to Chlamydia trachomatis plasmid protein pgp3 were characterized in children and adults with ocular or genital chlamydial infection using the enzyme-linked immunospot assay (ELISPOT) and ELISA. No pgp3-specific ASCs were detected in healthy controls, but predominantly IgA ASCs were detected in UK adults with uncomplicated cervicitis or urethritis (P = 0.03, 0.019). In patients with extragenital complications or pelvic inflammatory disease a mixed response with more IgG and IgM ASCs was evident, suggesting a breach of mucosal immune compartmentalization with more extensive infection. In women with chlamydial cervicitis, ASCs secreting predominantly IgA, but also IgG, to pgp3 were present in cervix at presentation, with a frequency 30-50 times higher than blood. Cervical ASC numbers, especially IgG, fell markedly six weeks after antibiotic treatment. We detected principally IgA pgp3-specific antibody secreting cells (ASCs) in children resident in a Gambian endemic area, with a trend towards suppression of IgA responses during intense trachomatous inflammation (P = 0.06), as previously reported for other chlamydial antigens, and in keeping with the findings in genital disease. These data provide a rationale for further studies of immune responses to pgp3 in humans and animal models of chlamydia-induced disease, and its potential use in diagnostic assays and protective immunization strategies.     

Human cell-mediated immune responses to chlamydial antigens.

A reproducible method was developed to determine the ability of chlamydial antigens to stimulate lymphocytes from volunteers. In tests repeated 4 to 14 times, the cells from a given volunteer gave a relatively narrow range of responses, but there were great differences in the mean response of different volunteers. In the entire group of 52 volunteers, lymphocyte stimulation was significantly associated with the presence of antibody, but in a given individual results of one test did not aid in predicting the results of the other. A majority of persons with either antichlamydial antibody or elevated lymphocyte stimulation, or both, did not have a history of signs or symptoms within a spectrum of chlamydial diseases. This may reflect the great frequency of asymptomatic infection with these organisms. The lymphocytes of some individuals were stimulated to a significantly greater degree by antigens of one chlamydial species (Chlamydia trachomatis or C. psittaci) than by the other. These and other cell-mediated reactions in human chlamydial infections, and their possible medical significance, are under continued study.     

Two novel virulence loci, mxiA and mxiB, in Shigella flexneri 2a facilitate excretion of invasion plasmid antigens.

A bank of over 4,200 lacZ protein fusions in Shigella flexneri 2a was screened for fusions to temperature-regulated promoters. One mutant, BS260, was completely noninvasive on HeLa cells and mapped to a region on the 220-kb virulence plasmid in which we had previously localized several avirulent temperature-regulated operon fusions (A.E. Hromockyj and A.T. Maurelli, Infect. Immun. 57:2963-2970, 1989). The phenotype of BS260 was similar to that of the previously identified mxi (membrane expression of invasion plasmid antigens) mutants, since it made wild-type intracellular levels of the invasion plasmid antigens (Ipa) but was deficient in the surface expression of IpaB and IpaC. Six kilobases of DNA upstream of the BS260 fusion end joint were cloned, but no temperature-regulated promoter was found, whereas the fusion end joint clone of the noninvasive mxi operon fusion mutant BS226 contained a temperature-regulated promoter. The locus defined by BS260 was designated mxiA, and that defined by BS226 was designated mxiB. Closer analysis of the mxiA and mxiB phenotypes by a cell-free enzyme-linked immunosorbent assay revealed that the mutants failed to excrete IpaB and IpaC into the culture medium, whereas wild-type cells actively released these antigens. Excretion of the ipa polypeptides from wild-type bacteria was confirmed by Western blot analysis of culture supernatants. Protease protection experiments revealed that wild-type S. flexneri 2a actually had much lower levels of surface-exposed IpaB and IpaC relative to those in the total antigen pool. In addition, examination of cellular fractions showed that, although there was no IpaB or IpaC in the outer membrane of BS260 and BS226, the antigens did accumulate in the cytoplasmic membrane. A 76-kDa temperature-regulated polypeptide in wild-type S. flexneri was identified as the putative mxiA gene product. These results strongly suggest that IpaB and IpaC represent truly excreted proteins of S. flexneri and that the mxiA and mxiB loci on the plasmid code for accessory proteins required to facilitate their export through the bacterial outer membrane. These data also suggest that mxiA is part of an operon that specifies additional mxi genes. The products of this operon may constitute a unique multicomponent protein secretion apparatus involved in the transport of Shigella virulence determinants.     

Heteropentameric cholera toxin B subunit chimeric molecules genetically fused to a vaccine antigen induce systemic and mucosal immune responses: a potential new strategy to target recombinant vaccine antigens to mucosal immune systems

Noninvasive mucosal vaccines are attractive alternatives to parenteral vaccines. Although the conjugation of vaccine antigens with the B subunit of cholera toxin (CTB) is one of the most promising strategies for vaccine delivery to mucosal immune systems, the molecule cannot tolerate large-protein fusion, as it severely impairs pentamerization and loses affinity for GM1-ganglioside. Here we report a new strategy, in which steric hindrance between CTB-antigen fusion subunits is significantly reduced through the integration of unfused CTB "molecular buffers" into the pentamer unit, making them more efficiently self-assemble into biologically active pentamers. In addition, the chimeric protein took a compact configuration, becoming small enough to be secreted, and one-step affinity-purified proteins, when administered through a mucosal route, induced specific immune responses in mice. Since our results are not dependent on the use of a particular expression system or vaccine antigen, this strategy could be broadly applicable to bacterial enterotoxin-based vaccine design.     

Antibody response of monkeys to invasion plasmid antigen D after infection with Shigella spp.

The antigen preparation most often used for determining the levels of antibodies to virulence-associated proteins of Shigella spp. consists of a mixture of proteins (including IpaB, IpaC, IpaD, and VirG*) extracted from virulent shigellae with water (water extract). To overcome the lack of specificity for individual antigens in the water-extract enzyme-linked immunosorbent assay (ELISA), the ipaD gene from S. flexneri has been cloned, expressed to a high level, and purified for use in a new ELISA for the determination of the levels of antibody against IpaD in monkeys and humans challenged with shigellae. The IpaD ELISA for serum immunoglobulins G and A correlated well with the water-extract ELISA in that monkeys infected with S. flexneri or S. sonnei responded with high serum antibody titers in both assays. The IpaD assay required less antigen per well, had much lower background levels, and did not require correction with antigens from an avirulent organism. In conjunction with the water-extract ELISA, it was possible to identify infected animals that did not respond to IpaD but did produce antibodies that reacted in the water-extract ELISA. This indicates that even though IpaB, IpaC, and IpaD are essential for the invasiveness phenotype, the infected host does not always produce antibodies against all components of the invasiveness apparatus.     

Autonomous histopathological regression of primary tumours associated with specific immune responses to cancer antigens

Spontaneous histopathological regression of cancer has been reported. The involvement of the immune system in such regression has been advocated, leading to the theory of immunological surveillance against cancer. A prediction of this theory is that common tumour antigens can be recognized upon repeated exposure by cell-mediated immunity, which leads to tumour regression and the subsequent appearance of tumour antigen-loss variants. However, no direct evidence has been provided in non-viral-induced experimental animal models of primary malignancy or in human primary cancer. This study examined two groups of melanoma patients where histopathological regression of the primary tumour was observed. Many of the 23 patients with multiple (> or =3) primary melanomas showed significant regression of their last melanoma (median 33%, mean 40) compared with matched melanomas from patients with a single primary melanoma (median 0%, mean 12) (p=0.0080), or compared with their first primary melanoma (p=0.0013). Regression was consistent with an 'immunization effect' seen in murine tumour transplantation studies, where inoculation with > or =3 asynchronous tumours induces transplantation rejection on subsequent challenge. A significant decrease in the expression of the melanoma common tumour antigen MART-1 in the last primary tumour from multiple melanoma patients (median 8%, mean 24) versus matched single melanoma patients (median 79%, mean 68) (p=0.0041) and in the last versus first tumour in multiple primary patients was found (p=0.0083). Metastases from 17 patients whose primary skin melanomas had completely regressed (occult primary melanoma) also showed significant MART-1 loss (median 0%, mean 11) compared with matched metastases from patients with non-regressing primary melanoma (median 51%, mean 50) (p=0.0013). MART-1 antigen-loss variants observed in the multiple primary and occult primary patients correlated with the presence of peripheral blood MART-1-specific cytotoxic T lymphocytes (CTLs) (p=0.03). No similar effects were observed with two other melanoma antigens, gp100 and CD63. Thus, in two groups of human melanoma patients, evidence is provided for histopathological tumour regression associated with cancer immune surveillance.     

Monoclonal antibodies specific for Shigella flexneri lipopolysaccharides: clones binding to type IV, V, and VI antigens, group 3,4 antigen, and an epitope common to all Shigella flexneri and Shigella dysenteriae type 1 stains.

Monoclonal antibodies reactive with Shigella flexneri O antigens were generated in both mouse and rat systems. Antibody-producing hybridomas were screened in an enzyme-linked immunosorbent assay using chemically defined lipopolysaccharides as antigens, and the epitope specificities were determined with a panel of lipopolysaccharides and synthetic O-antigen-specific glycoconjugates as antigens. To verify the specificity seen in the enzyme-linked immunosorbent assay, the antibodies were used in agglutination against a large number of S. flexneri strains. Monoclonal antibodies with the following specificities were identified: type, antigen IV (reactive with serotype 4a and 4b bacteria); type antigen V (reactive with serotype 5a and 5b bacteria); type antigen VI (reactive with serotype 6 bacteria); group antigen 3,4(reactive with serotype 1a, 2a, 3b, 4a, 5a, and Y bacteria); and group antigen 1 (reactive with an epitope present on all S. flexneri and Shigella dysenteriae type 1 bacteria). Furthermore, a monoclonal antibody defining a new O-antigenic epitope present on some S. flexneri strains of serotypes 4a, X, and Y was characterized (4X). The monoclonal antibodies analyzed in this study define epitopes described by polyclonal antisera (type antigens IV, V, and VI), define a hitherto uncharacterized epitope (group antigen 1), and finally identify new epitopes in what has previously been considered as one epitope (group antigen 3,4 and type antigen IV). These immunochemically characterized monoclonal antibodies may have a powerful potential in studies of the importance of humoral immunity in shigellosis.