Human monoclonal HLA antibodies reveal interspecies crossreactive swine MHC class I epitopes relevant for xenotransplantation

Crossreactivity of anti-HLA antibodies with SLA alleles may limit the use of pig xenografts in some highly sensitized patients. An understanding of the molecular basis for this crossreactivity may allow better selection of xenograft donors. We have tested 68 human monoclonal HLA class I antibodies (mAbs) for reactivity with pig lymphocytes from SLA defined pigs and found nine to be crossreactive. Eight of nine were broadly HLA reactive IgM-mAbs. The putative HLA epitopes for seven mAbs. were conserved in the aminoacid sequence of the SLA alleles studied. The lack of reactivity of a large number of mAbs largely correlated with the absence of the putative epitopes in the SLA alleles studied. We conclude that most patients with anti-HLA class I antibodies should be able to find pig donors lacking SLA antigens that cross react with their antibodies and that many of the crossreacting epitopes can be defined by analysis of shared epitopes in the aminoacid sequence of human and pig MHC antigens.     

Production and characterization of monoclonal antibodies directed againstEimeria falciformis and cross-reactive with sporozoites from two species of avian coccidia

Monoclonal antibodies (mAbs) were obtained against the surface antigens of theEimeria falciformis sporozoite by immunizing mice with whole homogenized sporozoite. The hybridomas were selected by their reactivities against oocyst extracts, then against glutaraldehyde-treated sporozoites. Three mAbs recognized both the surface ofE. falciformis, E. tenella, andE. acervulina and their refractile bodies, whereas a fourth mAb recognized only one epitope on the refractile bodies. All mAbs bound to the same immunoaffinity-purified antigens in Western-blot analysis (P27 forE. falciformis and P25 forE. tenella andE. acervulina). Thus, the mAbs define at least two shared epitopes between sporozoite antigens from different eimerian species. Two of these mAbs are involved in the in vitro phagocytosis ofE. falciformis sporozoites by macrophages and also in their lysis by neutrophils. Altogether, these properties showed that the four mAbs came from different activated B-cells. The P27 antigen recognized by our mAbs represents a major target of the in vitro destructive immune response.     

HLA class I epitopes: recognition of binding sites by mAbs or eluted alloantibody confirmed with single recombinant antigens

Development of beads coated with single recombinant HLA antigens has permitted the confirmation and further definition of HLA class I epitopes. In this study, monoclonal antibodies (mAbs) or alloantibodies eluted from recombinant cell lines were tested for reactivity with Luminex beads individually coated with 79 recombinant HLA class I single antigen (rHLA SA). Published amino acid sequences were used to map epitopes common to sets of antigens reactive with each antibody. While several epitopes have already been demonstrated, this study confirmed them by adsorption of allosera with transfectants or SA beads having a single HLA antigen and specific binding of the eluted antibody on SA beads. The allosera and mAbs used in this study recognized a total of at least 58 HLA class I epitopes, as demonstrated by their different adsorption/reactivity patterns. Of these, 25 epitopes were characterized by a single unique common amino acid, 30 shared 2 signature amino acids in close proximity, and 3 epitopes involved 3 specific amino acids in a non-linear sequence. Since these epitopes may be targets for antibody-mediated allograft rejection, epitope analysis should complement HLA and CREG assignment for defining complex antibodies and identifying suitable donors for highly sensitized transplant patients.     

Cross-reactive monoclonal antibodies to multiple HIV-1 subtype and SIVcpz envelope glycoproteins

The extraordinarily high level of genetic variation of HIV-1 env genes poses a challenge to obtain antibodies that cross-react with multiple subtype Env glycoproteins. To determine if cross-reactive monoclonal antibodies (mAbs) to highly conserved epitopes in HIV-1 envelope glycoproteins can be induced, we immunized mice with wild-type or consensus HIV-1 Env proteins and characterized a panel of ten mAbs that reacted with varying breadth to subtypes A, B, C, D, F, G, CRF01_AE, and a highly divergent SIVcpzUS Env proteins by ELISA and Western blot analysis. Two mAbs (3B3 and 16H3) cross-reacted with all tested Env proteins, including SIVcpzUS Env. Surface plasmon resonance analyses showed both 3B3 and 16H3 bound Env proteins with high affinity. However, neither neutralized primary HIV-1 pseudoviruses. These data indicate that broadly reactive non-neutralizing monoclonal antibodies can be elicited, but that the conserved epitopes that they recognize are not present on functional virion trimers. Nonetheless, such mAbs represent valuable reagents to study the biochemistry and structural biology of Env protein oligomers.     

Ultrastructural colocalization of phosphorylcholine and a phosphorylcholine-associated epitope in first-stage larvae ofTrichinella spiralis

Although the presence of phosphorylcholine (PC) inTrichinella is well established, the structures of the TSL-4 antigens that bear this epitope are unknown. A subset of TSL-4 antigens (TSL-8 antigens) has been reported to be absent from the surface of first-stageT. spiralis larvae. We report experiments with a monoclonal antibody (mAb US2) developed in mice with a relative inability to produce antibodies to PC. In immunoblotting, mAb US2 and anti-PC mAb (BH8) showed apparently identical binding patterns. In addition, we used an immunogold double-labeling technique to study the anatomical distribution of the epitopes recognized by these mAbs; the results obtained indicate close colocalization of epitopes for BH8 and US2 in tissues ofT. spiralis first-stage larvae. On the basis of these results, we suggest that US2 probably binds to allT. spiralis TSL-4 antigens, including TSL-8 antigens. We also clarify some conflicting previous reports on the distribution of PC immunoreactivity in first-stage larvae ofT. spiralis.     

Using monoclonal antibodies to stimulate antitumor cellular immunity

Monoclonal antibodies (mAbs) have an established role in current cancer therapy with seven approved for the treatment of a wide variety of tumors. The approved mAbs directly target tumor cells; however, it is becoming increasingly clear that as well as their direct effects, these mAbs can present antigens to the immune system. This stimulates long-lasting T-cell immunity, which may correlate with long-term survival. A more direct approach is to use mAbs to target antigens directly to antigen-presenting cells. One approach, ImmunoBody, which has just entered the clinic, stimulates antitumor immunity using mAbs genetically engineered to express tumor-specific T-cell epitopes. T cells not only respond via their T-cell receptors recognizing T-cell epitopes presented on MHC but are also influenced by stimulation of a wide variety of costimulatory molecules. mAbs targeting these molecules can also influence antitumor immunity. The main protagonist in this class of mAbs is ipilimumab, which has recently been shown to improve survival at 2 years in 23% of advanced melanoma patients. Combinations of mAbs targeting tumor antigens to activated antigen-presenting cells and mAbs targeting costimulatory receptors may provide effective therapy for a broad range of tumors.     

Variant specific epitopes of Giardia lamblia.

Giardia lamblia undergoes surface antigenic variation. The capability of different isolates to express certain epitopes on the surfaces of trophozoites from different isolates and clones was determined using 4 surface-reacting monoclonal antibodies (mAbs) to variants derived from WB or WB-like Giardia (mAbs 6E7, 5C1, and 3F6) and GS/M (mAb G10/4). Of 28 isolates, 11 possessed trophozoites reactive with mAbs 6E7, 5C1 and 3F6, 6 with mAb 3F6, 2 with Mab G10/4, 1 with mAb 6E7, and 8 showed no reactivity as determined by direct or indirect immunofluorescence. Newly established clones from different isolates generated small numbers of reactive trophozoites similar to their parents. Only one epitope was found on any single trophozoite. Southern blots hybridized to a probe encoding for the epitope recognized by mAb 6E7 revealed that the inability to express the antigen in most isolates was due to lack of the gene. Analysis of the surface antigens of mAb 6E7 reactive clones from 3 isolates revealed that mAb 6E7 reacted with surface antigens of different molecular masses.     

A new antigen ofCryptosporidium parvum micronemes possessing epitopes cross-reactive with macrogamete granules

Three monoclonal antibodies (mAbs) raised against purified excysted oocysts and sporozoites ofCryptosporidium parvum reacted in an immunofluorescent assay with antigens located at the apical pole of the sporozoites. On Western blots of oocyst extract, an antigen of 100 kDa was recognized by the three mAbs. Periodic acid treatment abolished this reactivity, suggesting that the corresponding epitopes are carbohydrates. As determined by immunoelectron microscopy, the three mAbs reacted with micronemes in sporozoites and merozoites and recognized a heterogeneous population of granules in the developing macrogametes. Gold particles were also detected in the parasitophorous vacuole of the macrogametes and sporulating oocysts. The detection of a microneme antigen in a family of macrogamete granules has not previously been reported in coccidia. The significance of this observation and the nature of these macrogamete inclusions are discussed.     

Immunodiagnostically applicable monoclonal antibodies to the circulating anodic antigen of Schistosoma mansoni bind to small,defined oligosaccharide epitopes

Gut-associated glycoproteins constitute a major group of the circulating excretory antigens produced by human Schistosoma species. The O-glycans of the relatively abundant circulating anodic antigen (CAA) from S. mansoni carry long stretches of unique -->6(GlcA beta 1-->3)GalNAc beta 1--> repeats. Specific anti-carbohydrate monoclonal antibodies (mAbs) are essential tools for the immunodiagnostic detection of CAA in the serum or urine of Schistosoma-infected subjects. In order to define the epitopes recognised by these anti-CAA mAbs, we screened a series of protein-coupled synthetic di- to pentasaccharide building blocks of the CAA polysaccharide for immunoreactivity, using ELISA and surface plasmon resonance spectroscopy. It was shown that anti-CAA IgM mAbs preferentially recognise -->6(GlcA beta 1-->3)GalNAc beta 1--> disaccharide units. Interestingly, no mouse anti-CAA mAbs of the IgG class were found that bind to the synthetic epitopes, although many of the IgG mAbs tested do recognise native CAA in a carbohydrate-dependent manner. In addition, both IgM and IgG class antibodies could be detected in human infection sera using the synthetic CAA fragments. These synthetic schistosome glycan epitopes and their matching set of specific mAbs are useful tools that further the development of diagnostic methods and are helpful in defining the immunological responses of the mammalian hosts to schistosome glycoconjugates.     

Differentiation of Entamoeba histolytica from E. dispar facilitated by monoclonal antibodies against a 150-kDa surface antigen

Murine monoclonal antibodies (mAbs) were produced against an n-octyl-β-D-glucopyranoside- extracted fraction of trophozoites of Entamoeba histolytica HM-1:IMSS. Four of the mAbs were reactive with a 150-kDa surface antigen characterized by Western-immunoblot analysis under nonreducing conditions. When the reactivity of the four mAbs with nine reference strains of E. histolytica was examined by an indirect fluorescence antibody test, two of the mAbs (EH3015 and EH3023) were found to react with all nine strains and the other two mAbs (EH3056 and EH3126) reacted with seven strains. The four mAbs did not react with any E. dispar reference strain or with other enteric protozoan parasites. The reactivity of EH3015 and EH3023 with numerous isolates of E. histolytica and E. dispar collected in our laboratories was also examined. The 2 mAbs reacted with all of the 37 E. histolytica isolates tested but did not react with any of the 33 isolates of E. dispar. These results indicate that common antigenic epitopes of E. histolytica are on the 150-kDa surface molecule and that mAbs can distinguish between E. histolytica and E. dispar. Received: 12 October 1996 / Accepted 13 December 1996     

Mapping of Taenia solium TSOL18 antigenic epitopes by phage display library

Taenia solium is a cestode parasite that causes cysticercosis in humans and pigs. TSOL18 has been identified as a host-protective oncosphere antigen. To obtain mouse monoclonal antibodies (mAbs) against TSOL18 and to map its antigenic epitopes are potentials to develop a vaccine for the prevention of T. solium infection. In this study, mAbs were produced by the hybridoma technique using purified glycosylated TSOL18 produced in Pichia pastoris as the immunogen. mAb was used to define the B-cell epitopes of TSOL18 with phage-displayed random dodecapeptide library (Ph.D.-12), and some of the positive phage clones were sequenced and analyzed. The predominant mimotopes were ETTKLQRFQAML (L1) found in 83%, followed by DHTXF in 15% (L2: DHTLFAASHNHR, DHTLFSTGHSHG, and DHTFMQRYHTHQ). Comparison of the peptide sequences with native TSOL18 protein sequence using Clustal W software showed that they did not completely match, suggesting that the ETTKLQRFQAML and DHTXF sequences should be conformational epitopes. The sera of mice immunized with the selected phage clones obviously recognized the TSOL18 protein. Meanwhile, sera collected from TSOL18-vaccinated pigs reacted to both epitopes in enzyme-linked-immunosorbent serologic assay test. Our work demonstrated that the antigenic epitope could be mapped through screening the phage-displayed peptide libraries with mAbs and a mimotope of TSOL18, which could provide an alternative approach for the diagnosis and development of a vaccine for T. solium.     

HLAMatchmaker-based analysis of human monoclonal antibody reactivity demonstrates the importance of an additional contact site for specific recognition of triplet-defined epitopes

Five HLA-A3 reactive human monoclonal antibodies (mAbs) originating from a parous woman were screened against HLA-typed panels by means of complement-dependent lymphocytotoxicity, high-definition ELISA, and flow cytometry with single antigen beads. Antibody reactivity profiles were compared with triplet amino acid sequence polymorphisms identified by HLAMatchmaker, and a three-dimensional structural modeling program (Cn3D of the National Center for Biotechnology Information) was used to determine the topography of epitopes recognized by each mAb. These mAbs originated from a woman who during pregnancy developed antibodies to the paternal HLA-A3 antigen of her child. Each mAb was specific for one mismatched triplet on HLA-A3, and the reactivity patterns of these IgM-type mAbs were practically the same in lymphocytotoxicity and antigen-binding assays. One mAb was specific for 163dT, a unique triplet present only on A3. The other mAbs reacted with 62Qe, 142mI, or 144tKr; these triplets are present on different groups of HLA-A alleles, some of which, however, did not react. Topographic modeling of triplet-defined epitopes identified clusters of polymorphic surface residues that were shared between reactive alleles. These clusters may serve as primary contact sites for the specificity-determining complementarity-determining region (CDR) loops of antibody. The reactivity with these mAbs required also the presence of self-sequence elsewhere on the HLA molecular surface as a critical secondary contact site for antibody, likely through another CDR loop. For instance, the reactivity of the 62Qe-specific mAb required the presence of a glycine residue in position 56 and the reactivity of the 142mI-specific mAb required the presence of the GTLRG sequence in positions 79-83. Conversely, there were many other amino acid differences between the mAb-reactive alleles and HLA-A3 that did not prevent antibody binding. For instance, the 62Qe-specific mAb-reactive alleles had 35 and the 142mI-reactive alleles had 50 of such "permissive" residue differences. An HLAMatchmaker-based analysis of the reactivity of human mAbs will increase our understanding of the structural definition of HLA epitopes and their reactivity with alloantibodies.     

Epitope analysis of the allergen ovalbumin (Gal d II) with monoclonal antibodies and patients' IgE.

Ovalbumin (OVA) is a major allergen (Gal d II) of hen egg white and is often the cause of hypersensitivity reactions to food. Further knowledge of the antigenic and allergenic epitopes of allergens will provide better treatment of this disease. To analyse these epitopes we produced a panel of monoclonal antibodies (mAbs) against native OVA. The initial information about the epitopes was obtained with the binding patterns of these mAbs in IEF-immunoprints and western blots of OVA under reducing and non-reducing conditions. It was possible to demonstrate that the different conformations of OVA exhibit different epitopes, and that there are other epitopes which are shared by each conformation. Seven different, although sometimes overlapping epitopes, could be determined on native OVA; four different epitopes on denaturated non-reduced OVA by means of immunoblots of the intact molecule. The number of epitopes which could be differentiated by the mAbs was increased by the use of peptide blots after CNBr fragmentation of the molecule. IgE binding to different OVA conformations and to CNBr-fragments of OVA was also detectable and appears in the same regions as the reactivity of some mAbs. Western blots of OVA and CNBr-peptides demonstrate that some antigenic/allergenic binding sites seem at least partly to be continuous epitopes. The identification of the CNBr-fragments was performed by a microsequence analysis of blotted CNBr-fragments after a 2-dimensional electrophoresis. IgE was found to bind the two largest CNBr-fragments (residues 41-172 and 301-385), but not the fragment corresponding to residues 173-196. A number of monoclonal antibodies also reacted with the two large fragments, especially with fragment 301-385, and some bind also to shorter peptides, such as fragment 173-196, which were not reactive to patients' IgE. Most of the monoclonal antibodies and patients' IgE bind to the fragments 41-172 and 301-385 in 2D-PAGE blots suggesting that these fragments are involved in an immunogenic structure.     

Analysis of taeniid antigens using monoclonal antibodies toEchinococcus granulosus antigen 5 and antigen B

Antigens derived fromEchinococcus granulosus, Taenia hydatigena andT. pisiformis cyst fluids,T. solium cysticerci,E. multilocularis protoscoleces andE. vogeli cyst membranes were examined by enzyme-linked immunosorbent assay (ELISA) and immunoelectrophoresis (IEP) using four monoclonal antibodies (mAb) toE. granulosus antigen 5 (Ag5) and antigen B (AgB). Anti-Ag5 mAbs 24.14 and 61A12 reacted strongly withT. hydatigena andT. pisiformis cyst fluids and, to a lesser degree, anti-AgB MAbs 31.15 and 39B3 also displayed some reaction with these antigens in ELISA. The formation of a modified arc 5 band between Anti-Ag5 mAbs andT. hydatigena cyst fluid (THCF) in IEP further confirms the existence of Ag5 inT. hydatigena cyst fluid. However, the inability of THCF andT. pisiformis cyst fluid (TPCF) to form an AgB band as well as that of TPCF to form an arc 5 band with mAbs in IEP does not exclusively prove the lack of AgB in THCF and TPCF or the lack of Ag5 in TPCF. The absence of a reaction of mAbs withT. solium, E. multilocularis andE. vogeli antigen preparations in ELISA or IEP would suggest that these mAbs may recognise epitopes different from those ofT. solium, E. multilocularis andE. vogeli parasites; this might be exploited for specific differentiation ofE. granulosus.     

Monoclonal antibodies raised in Btk(xid) mice reveal new antigenic relationships and molecular interactions among gp53 and other Trichinella glycoproteins

Tyvelose-bearing glycoproteins or Trichinella spiralis Group 1 antigens (TSL-1 antigens) are thought to be key molecules in the immunobiology of Trichinella. In the present study, we investigated the binding characteristics of several mAbs produced in Btk(xid) immunodeficient mice that recognise gp53 and some other minor glycoproteins of this parasite. The data obtained reveal the existence of an O-glycan/peptide epitope (recognised by mAb US8) common to all TSL-1 glycoproteins, as well as a specific interaction between the TSL-1 antigen gp53 and other unknown Trichinella glycoproteins in the 35-40 kDa range (these latter react with mAbs US8 and US9, but not with mAb US5). Some of the epitopes recognised by our mAbs are differentially expressed in Trichinella species: the epitope recognised by mAb US5 on gp53 (another O-glycan/peptide epitope) is present only in T. spiralis, whereas those recognised by mAbs US8 and US9 (peptide epitopes) are present in encapsulated Trichinella species. The data obtained also reveal that gp53 is synthesised and glycosylated in beta-stichocytes only. The possible relevance of these findings is discussed.     

A study of the effect of chemical inactivants on the epitopes of Rift Valley fever virus glycoproteins using monoclonal antibodies.

A panel of 23 monoclonal antibodies (mAbs) was used to study the effect of formalin, beta propriolactone (BPL) and binary ethylenimine (BEI) on the epitopes of the Rift Valley fever virus glycoproteins. After the initial inactivation period BEI had very little adverse affect on the epitopes whereas BPL significantly altered seven and formalin partially changed the conformation or accessibility of most of the epitopes. The epitopes of all of the inactivated antigens showed a reduced activity against the specific mAbs over a six month storage period.     

SHARED EPITOPES OF THE HLA-DR10 MOLECULE RECOGNIZED BY MURINE AND HUMAN mAbs

In order to produce mAbs directed specifically against HLA-DR10 molecule, transfected mouse L cells, expressing the DRB 1*1001 allele, were used to immunize C3H mice over a period of 4 weeks. Two mAbs, 2C12 and 4B6, derived from this fusion were found to recognize, with different affinity, polymorphic epitopes of DR10 that are shared with DR1, 3, 7, and 9. These mAbs were screened on a large panel of homozygous B lymphoblastoid cell lines using microlymphocytotoxicity and the results were confirmed by flow cytometry. The reactive pattern of 2C12 and 4B6 was compared to that of MP10 human mAb also recognizing the DR10 specificity in addition to DR1, 2 and 9. Based on serologic specificity and cellular absorption experiments, we conclude that the epitopes the murine and human mAbs respectively recognize on the DR10 molecule, are probably different.     

Flow cytometric analysis reveals unexpected shared antigens between histologically defined populations of thymic stromal cells

Utilizing flow cytometry, the expression of antigens recognizedby six thymic stromal cell (TSC) reactive mAbs was investigatedon fresh TSCs and TSC lines. It was found that some thymic epithelialcells and dendritic cells share antlgenlc phenotypes, and thatmost TSC reactive mAbs have a more extensive distribution thanwould have been predicted from immunohlstology. While thesefindings illustrate the higher sensitivity of flow cytometricanalysis, they more Importantly emphasize the great complexityof TSC that direct T cell development. In order to Identifythe molecular parameters that define the various steps involvedin T cell differentiation, TSC antigens (non-TCR/MHC/co-receptor)that are functional will have to be Identified. This study representsthe initial steps in characterizing such antigens.     

Identification of epitopes recognized by a panel of six anti-human IgG2 monoclonal antibodies.

Human IgG2 contains several subclass specific amino acid residues or deletions in the CH1 and CH2 domains and also in the hinge region. These substituted residues are the structural correlates for IgG2 specific epitopes. Since human IgG2 has different biological properties from other subclasses, some IgG2 epitopes may be located in regions correlating with sites determining the biological functions. Previously, we produced three anti-IgG2 monoclonal antibodies (mAbs) with highly specific and interesting reactivities using improved immunization protocols. However, it has been almost impossible to identify epitopes conventionally, because human IgG2 is so resistant to proteolysis that various proteolytic fragments could not be isolated. In this study, we identified the epitopes recognized by anti-IgG2 mAbs by SDS-PAGE, Western blotting, amino acid sequence analysis and peptide/mAb binding ELISA, thus overcoming the need for fragment isolation. A panel of six anti-human IgG2 mAbs, including the current WHO/IUIS specificity standards (HP6002, HP6008, HP6014) and our own (HG2-6A, HG2-30F, HG2-56F), reacted with distinct epitopes. The residues essential to expression of the epitopes recognized by the mAbs were: Pro234, Val235 and Val309 for HG2-56F, HG2-30F and HP6008, respectively. HP6014 reacted with the epitope expressed by Thr214 and its neighboring residues. HG2-6A was reactive with the hinge region, and HP6002 was assumed to be directed against discontinuous epitopes requiring intact Fc for expression. Through these studies, the pepsin and papain cleavage sites of human IgG2 were also clarified.     

Antigenic cross‐reactivity between Schistosoma mansoni and peanut: a role for cross‐reactive carbohydrate determinants (CCDs) and implications for the hygiene hypothesis

The antigenic reactivity of constituents of Schistosoma mansoni and peanut (Arachis hypogaea) was investigated to determine whether identical antigenic epitopes possessed by both organisms provided a possible explanation for the negative correlation between chronic schistosome infection and atopy to allergens. Aqueous extracts of peanuts were probed in Western immunoblots with rabbit IgG antibodies raised against the egg, cercarial and adult worm stages of S. mansoni. Several molecules in the peanut extract were antigenically reactive with antibodies from the various rabbit anti‐schistosome sera. A pair of cross‐reactive peanut molecules at ~30 000–33 000 molecular weight was purified and both proteins were identified by mass spectrometric analysis as the peanut allergen Ara h 1. Anti‐S. mansoni soluble egg antigen antibodies that were eluted off the peanut molecules reacted with two S. mansoni egg antigens identified by mass spectrometry as IPSE/α‐1 and κ‐5. Alignments of the amino acid sequences of Ara h 1 and either IPSE/α‐1 or κ‐5 revealed a low level of peptide sequence identity. Incubation of nitrocellulose paper carrying electrophoresed peanut molecules, six constituents of other allergic plants and S. mansoni egg antigens in a mild solution of sodium metaperiodate before probing with antibodies, inhibited most of the cross‐reactivities. The results are consistent with the antigenic cross‐reactive epitopes of S. mansoni egg antigens, peanut and other allergic plants being cross‐reactive carbohydrate determinants (CCDs). These findings are novel and an explanation based on ‘blocking antibodies’ could provide an insight for the inverse relationship observed between schistosome infection and allergies.