Repetitive Antigens Research Articles

Antihomotypic affinity maturation improves human B cell responses against a repetitive epitope.

Affinity maturation selects B cells expressing somatically mutated antibody variants with improved antigen-binding properties to protect from invading pathogens.We determined the molecular mechanism underlying the clonal selection and affinity maturation of human B cells expressing protective antibodies against the circumsporozoite protein of the malaria parasite Plasmodium falciparum (PfCSP).We show in molecular detail that the repetitive nature of PfCSP facilitates direct homotypic interactions between two PfCSP repeat-bound monoclonal antibodies, thereby improving antigen affinity and B cell activation. These data provide a mechanistic explanation for the strong selection of somatic mutations that mediate homotypic antibody interactions after repeated parasite exposure in humans. Our findings demonstrate a different mode of antigen-mediated affinity maturation to improve antibody responses to PfCSP and presumably other repetitive antigens.

Heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT), typically occurring in the second week of heparin therapy, is an antibody-mediated adverse drug reaction associated with increased thrombotic risk. The most important antigens are located on platelet factor 4 (PF4)/heparin complexes. HIT is always caused by platelet-activating antibodies, but not all PF4/heparin-reactive antibodies cause HIT. Thus, tests have a high negative, but only a moderate, positive predictive value. Clinical suspicion of HIT requires cessation of heparin and substitution with an alternative anticoagulant. As these drugs have an increased bleeding risk, they should be used in therapeutic doses only if HIT is considered very likely. Avoiding/postponing coumarin is crucial in minimizing microthrombotic complications. Recent studies of HIT immunobiology suggest that HIT mimics immunity against repetitive antigens, as are relevant in microbial defense. Thus, understanding HIT may help unravel why host defenses can trigger autoimmunity.

Heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT), typically occurring in the second week of heparin therapy, is an antibody-mediated adverse drug reaction associated with increased thrombotic risk. The most important antigens are located on platelet factor 4 (PF4)/heparin complexes. All HIT is caused by platelet-activating antibodies, but not all PF4/heparin-reactive antibodies cause HIT. Thus, tests have high negative, but only moderate, positive predictive value. Cessation of heparin requires substitution with an alternative anticoagulant, but as these drugs have increased bleeding risk, they should be used in therapeutic doses only if HIT is considered very likely. Avoiding/postponing coumarin is crucial in minimizing microthrombotic complications. Recent studies of HIT immunobiology suggest that HIT mimics immunity against repetitive antigens, as they are relevant in microbial defense. Thus, understanding HIT may help unravel why host defenses can trigger autoimmunity.

CCR7-deficient mice develop atypically persistent germinal centers in response to thymus-independent type 2 antigens

Thymus-independent type 2 (TI-2) antigens are repetitive antigens capable of eliciting antibody responses without T cell help. They are important in the immune response against encapsulated bacteria and as a rapid first line of defense against pathogens. TI-2 antigens induce strong proliferation in extrafollicular foci. However, any germinal centers forming in response to TI-2 antigens involute synchronously 5 days after immunization. This is thought to be caused by the lack of T cell help. Surprisingly, immunization of mice deficient for the homeostatic chemokine receptor CCR7 with TI-2 antigens resulted not only in the expected, vigorous extrafollicular plasma cell response but also in persisting splenic germinal centers. This was observed for two different TI-2 antigens, heat-killed Streptococcus pneumoniae and (4-hydroxy-3-nitrophenyl)acetyl-Ficoll (NP-Ficoll). Germinal centers induced by TI-2 and thymus-dependent (TD) antigens were located in the periarteriolar area of the white pulp in CCR7 knockout mice, corresponding to the T zone of wild-type (WT) mice. The TI-2-induced germinal centers contained peripheral rings of follicular dendritic cells and unusually for TI-2-induced germinal centers, T cells. The licensing responsible for their atypical persistence did not endow TI-2-induced germinal centers with the full range of characteristics of classic germinal centers induced by TD antigens. Thus, class-switching, affinity maturation, and memory B cell generation were not increased in CCR7-deficient mice. It seems unlikely that a defect in regulatory T cell (Treg) location was responsible for the atypical persistence of TI-2-induced germinal centers, as Tregs were comparably distributed in germinal centers of CCR7-deficient and WT mice.

Der p 1 peptide on virus-like particles is safe and highly immunogenic in healthy adults

In mice, highly repetitive antigens, such as those present on bacterial or viral surfaces, efficiently cross-link B-cell receptors and therefore induce strong IgG responses. In this study we covalently coupled a synthetic 16-amino-acid sequence of the allergen Der p 1 to a virus-like particle derived from the bacteriophage Qbeta (Qbeta-Der p 1). We evaluated the safety and immunogenicity of Qbeta-Der p 1 in human subjects and compared different doses and routes of immunization. In a phase I trial 24 healthy volunteers were randomly assigned to one of 4 treatment groups. Group 1 received 50 microg of Qbeta-Der p 1 intramuscularly, group 2 received 50 microg of Qbeta-Der p 1 subcutaneously, group 3 received 10 microg of Qbeta-Der p 1 intramuscularly, and group 4 received 10 microg of Qbeta-Der p 1 subcutaneously. Boosting immunizations with 10 microg were given after 1 and 3 months. Antibody titers were measured after 1, 3, 4, 6, 12, and 18 months. The vaccine Qbeta-Der p 1 was well tolerated. Significant IgG responses were observed 4 weeks after a single injection. Individuals receiving 50 microg of the vaccine had significantly higher IgG titers than those vaccinated with 10 microg. However, the route of immunization (subcutaneous vs intramuscular) had no effect. In the 50-microg dose group, strong antibody responses against Der p 1 with average titers of 1:2000 were obtained. Vaccination with a peptide antigen covalently coupled to highly repetitive virus-like particles represents an adjuvant-free means of rapidly inducing high antibody titers in human subjects. Allergens coupled to virus-like particles can be used to enhance the efficiency of allergen-specific immunotherapy.

Responses to Cohn's Discussion on the Zinkernagel-Hengartner 'Credo 2004'

Responses to Cohn's Discussion on the Zinkernagel-Hengartner 'Credo 2004'

T cell epitope characterization in tandemly repetitive Trypanosoma cruzi B13 protein

Proteins containing tandemly repetitive sequences are present in several immunodominant protein antigens in pathogenic protozoan parasites. The tandemly repetitive Trypanosoma cruzi B13 protein is recognized by IgG antibodies from 98% of Chagas' disease patients. Little is known about the molecular mechanisms that lead to the immunodominance of the repeated sequences, and there is limited information on T cell epitopes in such repetitive antigens. We finely characterized the T cell recognition of the tandemly repetitive, degenerate B13 protein by T cell lines, clones and PBMC from Chagas' disease cardiomyopathy (CCC), asymptomatic T. cruzi infected (ASY) and non-infected individuals (N). PBMC proliferative responses to recombinant B13 protein were restricted to individuals bearing HLA-DQA1*0501(DQ7), -DR1, and -DR2; B13 peptides bound to the same HLA molecules in binding assays. The HLA-DQ7-restricted minimal T cell epitope [FGQAAAG(D/E)KP] was identified with an overlapping combinatorial peptide library including all B13 sequence variants in T. cruzi Y strain B13 protein; the underlined small residues G Q A were the major HLA contact residues. Among natural B13 15-mer variant peptides, molecular modeling showed that several variant positions were solvent (TCR)-exposed, and substitutions at exposed positions abolished recognition. While natural B13 variant peptide S15.9 seems to be the immunodominant epitope for Chagas' disease patients, S15.4 was preferentially recognized by CCC rather than ASY patients, which may be pathogenically relevant. This is the first thorough characterization of T cell epitopes of a tandemly repetitive protozoan antigen and may suggest a role for T cell help in the immunodominance of protozoan repetitive antigens.

Humoral and cellular immune responses in BALB/c and C57BL/6 mice immunized with cytoplasmic (CRA) and flagellar (FRA) recombinant repetitive antigens, in acute experimental Trypanosoma cruzi infection

In previous studies, cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins induced specific humoral and cellular immune responses in susceptible and resistant mice in the absence of Trypanosoma cruzi infection with a significant induction of the Interferon-gamma (IFN-gamma) production in those animals. In this follow-up paper, the immunostimulatory and protective effects of these proteins were evaluated by immunizing with CRA or FRA antigens, BALB/c and C57BL/6 mice and challenging with a T. cruzi (Y strain). Both proteins induced humoral response with high levels of IgG isotypes as well as cellular immunity with high levels of IFN-gamma when compared to controls. However, the lymphocyte proliferative response was minimal. The survival rate at 30 days post-infection was significant in CRA (60%) or FRA (50%)--immunized BALB/c mice and CRA (83.3%)--immunized C57BL/6 mice. Taken as a whole these findings indicate that CRA and FRA are immunogenic and potentially important for protective immunity.

Improving Protein Pharmacokinetics by Genetic Fusion to Simple Amino Acid Sequences

The role of primary amino acid sequences in protein pharmacokinetics, an issue of relevance in both basic knowledge and biotechnology, was addressed here using as a starting point two repetitive antigens from the hemoflagellate Trypanosoma cruzi that are known to stabilize their associated proteins in the bloodstream. A major drawback to their pharmacological application is that these repetitive sequences are highly immunogenic, being therefore the deletion of this characteristic desirable. Based on sequence homology and epitope mapping analyses, an artificial repetitive sequence (PSTAD) was engineered. This motif was tested by genetic fusion to the C terminus of both the trypanosomal trans-sialidase and the rat tyrosine aminotransferase and found to produce a 4.5-6-fold increase in the half-life of the associated proteins in blood while displaying significantly lower immunogenicity. Residues involved in the stabilizing properties of the novel peptide were mapped by a site-directed mutagenesis approach, allowing us to successfully identify another two motifs. Searching databases for sequences displaying some homology, embedded in proline frameworks and associated to shed virulence factors from unrelated microorganisms, resulted in the identification of four other protein extensions. Remarkably, three of them (from Streptococcus pneumoniae, Actinomyces viscosus, and Escherichia coli) revealed similar pharmacokinetic features, suggesting therefore an analogous evolutionarily acquired mechanism to ensure the biodistribution of their corresponding proteins. Our findings indicate that the insertion of defined motifs into a proline-rich framework constitutes a suitable alternative to construct a chimeric protein with extended half-life in blood.

Characterization of cDNA clones encoding ribonucleoprotein antigens expressed in Trypanosoma cruzi amastigotes.

Sera from patients with Chagas' disease were used to screen a Trypanosoma cruzi amastigote cDNA library. Characterization of 50 positive clones showed that 21 (42%) encode previously identified T. cruzi ribosomal and flagellar proteins, heat-shock proteins or proteins with repetitive motifs. Twenty-nine clones (58%) correspond to nine genes not previously described in T. cruzi. Three cDNAs, encoding novel repetitive antigens with homology to ribosomal proteins and to other RNA binding proteins, were further characterized. Patient humoral responses against the recombinant proteins encoded by these cDNAs were evaluated in anticipation that they may constitute potential new targets for serodiagnostic assays.

Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response.

The lymphoid organs contain specialized microanatomic structures composed of lymphoid, myeloid and stromal cells that are vital to the generation of an effective adaptive immune response. Although the existence of these specialized structures has been known for over a century, the developmental signals that generate them and the specific roles of these structures in the immune response have remained largely elusive. Because of their position adjacent to the marginal sinuses, marginal zone B (MZB) cells are amongst the first population of cells seen by blood born antigens and are presumed to have a critical role in host defense against bacterial pathogens. Here we demonstrate that a deficiency of the tyrosine kinase (Pyk-2) results in a cell autonomous defect of MZB cell production. In response to repetitive polysaccharide antigens (T-independent type II (TI-II)) Pyk-2-deficient mice displayed marked suppression of IgM, IgG3 and IgG2a production. Furthermore, complement receptor engagement proved necessary for the specific targeting of polysaccharide antigens to MZB cells. These results suggest how innate immune responses mediated through complement coupling are translated into an adaptive response by MZB cells, and provide a potential mechanism for the T cell independence of humoral responses to polysaccharide antigens.

Localization dose and time of antigens determine immune reactivity

Two models to explain patterns of immune reactivity of T and B cells are compared: the two-signal theories and the antigen-localization-dose-time and structure concept. The two-signal theory states that signal 1 ( = antigen alone signalling via specific T or B cell receptor) turns T and B cells off, signal 1 plus co-stimulatory signals 2 induces them. Our model employs immuno-reactivity antigen parameters, i.e. localization-dose-time kinetics and structure of antigen in determining T and B cell reactivity. Both concepts have in common that immune reactivity is somatically learned and not germline defined and that there is nothing unique to the antigenic structure itself that could distinguish self from nonself antigens. While two-signal theories base positive versus negative reactivity on the presence or absence of co-stimulatory signals anywhere in the body, our alternative model proposes that besides antigen structure, dose and time it is the localization of antigen—vis-à-vis the organized lymphoid tissues—that determines reactivity patterns as follows. First, antigen that does not reach secondary lymphoid organs in minimum doses or for sufficiently long time periods, is immunologically ignored. Second, antigen that either usually exists in the lymphoid system or reaches it and persists in excessive amounts for long periods deletes T cells. Third, antigen that is transported to secondary lymphoid organs in sufficient (but not excessive) amounts and for a sufficient time period (but does not persist) induces an effective immune response. B cell responses are also induced exclusively in lymphoid tissues. Short-term B cell responses are T independent against antigens linked to bacterial lipopolysaccharides or against highly repetitive and strictly ordered antigens; thus, B cells are pattern recognizers (monomeric antigens usually accessible to B cells are in general likely to be self-antigens. Strictly ordered repetitive antigens are virtually by definition infectious agents). Long-term (switched) B cell responses against mono- and polymorphic antigens are T cell dependent regulated by time dose and localization of antigen.

T-Cell-independent immunoglobulin G responses in vivo are elicited by live-virus infection but not by immunization with viral proteins or virus-like particles.

Immunoglobulin G (IgG) responses to viruses are generally assumed to be T-cell dependent (TD). Recently, however, polyomavirus (PyV) infection of T-cell-deficient (T-cell receptor beta chain [TCR-beta] -/- or TCR-betaxdelta -/-) mice was shown to elicit a protective, T-cell-independent (TI) antiviral IgM and IgG response. A repetitive, highly organized antigenic structure common to many TI antigens is postulated to be important in the induction of antibody responses in the absence of helper T cells. To test whether the repetitive structure of viral antigens is essential and/or sufficient for the induction of TI antibodies, we compared the abilities of three forms of PyV antigens to induce IgM and IgG responses in T-cell-deficient mice: soluble capsid antigens (VP1), repetitive virus-like particles (VLPs), and live PyV. Immunization with each of the viral antigens resulted in IgM production. VLPs and PyV elicited 10-fold-higher IgM titers than VP1, indicating that the highly organized, repetitive antigens are more efficient in IgM induction. Antigen-specific TI IgG responses, however, were detected only in mice infected with live PyV, not in VP1- or VLP-immunized mice. These results suggest that the highly organized, repetitive nature of the viral antigens is insufficient to account for their ability to elicit TI IgG response and that signals generated by live-virus infection may be essential for the switch to IgG production in the absence of T cells. Germinal centers were not observed in T-cell-deficient PyV-infected mice, indicating that the germinal center pathway of B-cell differentiation is TD even in the context of a virus infection.

Repetitive Protein Antigens of Trypanosoma cruzi Have Diverse Intracellular Locations

We previously identified by immunoscreening Trypanosoma cruzi cDNA libraries a group of proteins containing long stretches of tandem repeats. The goal of the current project was to gain insight into the functions of these proteins through ultrastructural analyses consisting of western blotting and electron microscopic localization studies. By comparing western blots of total parasite lysate and different fractions of T. cruzi, we found that 3 of the repetitive antigens are exclusively associated with the parasite membrane, or cytoskeleton, or both. One of the 4 repetitive antigens studied has some association with the membrane or cytoskeleton but also appears to be free in the cytosol. In immunoelectron microscopic studies, the 4 repetitive antigens were detected in different intracellular locations. One of the proteins is located between the flagellum and parasite body, the second has a nuclear distribution, the third is associated with the cell membrane, and the fourth is dispersed throughout the cytoskeletal network. These findings suggest that despite the general structure similarities of these repetitive proteins, they may serve different cellular functions.

Identification and characterisation of a novel repetitive antigen from Onchocerca spp.

A novel repetitive antigen from the cattle parasite Onchocerca gibsoni was shown to be recognised by sera from humans infected with Onchocerca volvulus, Wuchereria bancroftii or Brugia malayi. The O. gibsoni protein was produced in a recombinant form, and antibodies raised to this protein used to screen cDNA libraries for O. volvulus. A series of clones were isolated which encoded repetitive regions very similar to those in O. gibsoni, but interspersed between these were longer repeating units which we have not so far found in O. gibsoni. The repetitive antigen was shown to be of high molecular weight and present only in the insoluble (membrane) fraction of O. gibsoni microfilariae. Immunofluoresence techniques demonstrated that the antigen was associated both with muscle and with specific membrane layers, including a peripheral layer which corresponds to either the outer hypodermis or an inner region of the cuticule in adult female O. gibsoni. In many respects, the proteins encoded by the O. gibsoni and O. volvulus cDNA clones resembled repetitive antigens from several distantly related eukaryotic parasites, and a possible common role in immune evasion is discussed.

Specificity variants in monoclonal antibodies reactive with peptide epitopes of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum.

It has been suggested that repeat sequence antigens of Plasmodium falciparum may serve the parasite in immune evasion by modifying the host antibody response and impairing the development of protective immunity. According to this proposal networks of cross-reactive, repeat sequence malarial antigens have the ability to stimulate a high proportion of all somatically mutated B cells with altered antibody specificity, and thus to hinder the normal process of antibody affinity maturation. To determine the rate at which immunoglobulin mutations produce new reactivities with repeat sequence antigens, hybridoma cell lines specific for the ring-infected erythrocyte surface antigen (RESA) were examined for the incidence of specificity variants that arose naturally or as a result of treatment with the chemical mutagen ethylmethane sulphonate (EMS). From one of the cell lines variants were readily isolated having reactivity towards a very closely related repeat sequence epitope within the same RESA antigen. However, the other hybridoma/antigen combinations revealed no variants. In general, mutations giving rise to antibodies with altered specificity for related repetitive antigens were not readily induced and only limited support of the hypothesis was obtained.

Identification and characterization of two repetitive non-variable antigens from African trypanosomes which are recognized early during infection.

The present paper describes two repetitive proteins representing common antigens of African trypanosomes which are non-variant and which are recognized early in infection by the host immune system. These antigens were identified by their ability to immunoreact with bovine serum taken during the early phase of a cyclic trypanosomal infection. Screening of a cDNA library from T. b. gambiense with such early infection serum identified a protein which contains a repetitive motif consisting of 68 amino acid repeat units (GM6). Immunofluorescence and immunogold electron microscopy revealed that GM6 is located on fibres which connect the microtubules of the membrane skeleton with the flagellum. A second repetitive antigen detected by this serum is MARP1 (microtubule-associated repetitive protein 1), a protein previously characterized in this laboratory as a high-molecular weight component of the membrane skeleton, which consists of more than 50 tandemly repeated, near-identical 38 amino acid repeat units. Beta-galactosidase fusion products of both proteins demonstrated a strong immunoreactivity with sera from T. b. brucei and T. congolense-infected cattle. The result from this preliminary immunological evaluation indicates a high immunodiagnostic sensitivity (90%) of the two recombinant antigens which make them interesting candidates for immunodiagnosis of trypanosomiasis in cattle.

Proteins of unusual sequence composition from the malarial parasite Plasmodium falciparum

Over the last decade many malarial antigens have been cloned and sequenced [ 1-31. Initial sequences were intriguing as many antigens appeared to contain multiple repeats of peptide subunits which ranged from 3 to 80 amino acids in length. These repeat units can occupy between 2 and 90% of the total protein sequence and occur in proteins ranging from 30 to 300 kDa (Fig. 1). It has been postnlated that the large number of repetitive antigens in malaria, some of which contain cross-reactive epitopes and many of which are immunodominant, may play a significant part in protecting the parasite from the host's immune system [2, 31. Before proceeding with this discussion, however, it is necessary to describe the life cycle of the parasite so that the immune response to malaria may be better understood. The most virulent of the human malaria parasites Plasmodium falciparum, like other members of the genus Plamodium, undergoes a complex life

Cloning and expression of a membrane antigen of Entamoeba histolytica possessing multiple tandem repeats.

Entamoeba histolytica causes amebic dysentery and amebic liver abscess, major causes of morbidity and mortality worldwide. We have used differential hybridization screening to isolate an E. histolytica-specific cDNA clone. The cDNA was found to encode a serine-rich E. histolytica protein (SREHP) containing multiple tandem repeats. The structural motif of SREHP resembles some of the repetitive antigens of malarial species, especially the circumsporozoite proteins. A recombinant trpE fusion protein containing the tandem repeats of SREHP was recognized by immune serum from a patient with amebiasis, demonstrating that SREHP is a naturally immunogenic protein. An antiserum raised against the recombinant fusion protein specifically bound to two distinct bands with apparent molecular masses of 46 and 52 kDa in a crude preparation of E. histolytica trophozoite membranes. This antiserum also inhibited E. histolytica trophozoite adhesion to Chinese hamster ovary cells in vitro. The ability to isolate E. histolytica-specific genes, and to express those genes in Escherichia coli, may be important in studying the molecular basis of E. histolytica pathogenesis and for the future development of vaccines.

Homologous recombination within subtelomeric repeat sequences generates chromosome size polymorphisms in P. falciparum

We present restriction maps for chromosomes 1 and 2 of six cloned lines of P. falciparum. These delineate the locations of eight genetic markers, including genes for five antigens. In parasites from diverse areas, chromosome structure is conserved in central regions but is polymorphic both in length and sequence near the telomeres. The telomeres and adjacent sequences comprise a conserved structure at the ends of most P. falciparum chromosomes. However, the subtelomeric zones are polymorphic and coincide with the locations of a repetitive element (rep20). Deletions of rep20 generate clones of P. falciparum that lack rep20 on one or both ends of chromosomes 1 or 2, and larger deletions remove telomere-proximal genes as well. The chromosome length polymorphisms can therefore be largely explained by recombination within these blocks of repeats, a mechanism that is also important in the generation of diversity in genes for repetitive antigens of P. falciparum.