Antigenic variation in Giardia lamblia

Clones of the WB isolate of Giardia lamblia were exposed to cytotoxic mAb 6E7 which reacts with a 170-kDa surface Ag. Surviving progeny occurred at a frequency of about 1 in 1000 and were resistant to the effects of mAb 6E7. Analysis of progeny and clones of these progeny by surface radiolabeling, surface immunofluorescence, and Western blotting failed to detect the 170-kDa Ag. Loss of this Ag was associated with the appearance of a series of new surface Ag. A cytotoxic mAb (5C1) was produced to one of the newly appearing antigens (approximately equal to 64 kDa) and Giardia resistant to the cytotoxic effects of 5C1 isolated. Neither the approximately equal to 64 kDa nor the 170 kDa Ag were present and were replaced by a second series of new Ag. These studies clearly establish the loss and subsequent replacement of two antigenically distinct epitopes on Giardia derived from a single organism.     

Periodic appearance of a predominant variant antigen type during a chronic Giardia lamblia infection in a mouse model

Giardia lamblia (syn. Giardia duodenalis, Giardia intestinalis) infections are associated with continuous antigenic variation of the parasite which is mediated by the parasite's major surface antigen, named variant surface protein. Offspring mice and corresponding mothers were infected with G. lamblia clone GS/M-83-H7 (expressing variant surface protein H7) and various parameters of this infection were assessed in a long-term follow-up investigation. Our experimentation revealed that variant surface protein H7-type trophozoites were replaced by new variant-type trophozoites during the early stage of infection (around day 8 p.i.), but the original variant-type re-emerged at at least two time-points during the later stages of infection (at days 22 and 42 p.i.). Such periods of variant surface protein H7-type trophozoite re-expansion were accompanied by transient production of intestinal IgA against variant-specific epitopes on a 314-aa N-terminal region of variant surface protein H7. At late stages of infection (between days 42 and 200 p.i.), most mice produced intestinal IgA against both variant surface protein H7 and other antigens of the parasite. At these stages, infection seemed to be resolved in most mice, but occasional reappearance of relatively high (at day 64 p.i.) or at least detectable (at days 80 and 120 p.i.) amounts of intestinal parasites indicated that G. lamblia GS/M-83-H7 infections in mice may enter into a latent chronic phase which is interrupted by sporadic breakthroughs of parasite growth.     

Frequency of variant antigens in Giardia lamblia.

Giardia lamblia undergoes antigenic variation. The rate of antigenic variation and the size of the variant antigen repertoire were estimated in clones of Giardia lamblia which reexpresses surface variant antigens that are characteristics of its parent. Calculations were based on determinations of the number of trophozoites expressing defined or nondefined epitopes as well as the total number of trophozoites in newly established clones. The rate of appearance of variant antigens containing defined epitopes was expressed as the number of generations until the first trophozoite expressing a defined epitope appeared. In clones of isolate WB, tested because their major surface variant antigens were largely nondefined, variants expressing epitopes recognized by Mabs 6E7 or 3F6 appeared after approximately 12 generations. Variants expressing epitopes recognized by Mab 5C1 appeared at about 13 generations, significantly greater than for the other epitopes. The rate of antigenic variation was studied in another isolate, GS/M, whose surface epitope repertoire differs from that of isolate WB. A single epitope recognized by Mab G10/4 was tested. Trophozoites reexpressing this epitope first appeared after about 6.5 generations, significantly less than in WB. Therefore, the single epitope studied in isolate GS/M is reexpressed much more frequently than those of WB. In isolate WB, the epitopes recognized by Mab 6E7 and 3F6 tended to appear at the same time. The median number of variant antigens in WB was estimated to lie between 20.5 and 184.     

Cross-antigenicity between the major surface proteins (ospA and ospB) and other proteins of Borrelia burgdorferi

Two of the major surface Ag of Borrelia burgdorferi, the 31-kDa OspA and 34-kDa OspB proteins, are encoded by a 49-kb plasmid. In this study, mAb and monospecific polyclonal antibodies were used to define cross-antigenicity of the OspA and OspB protein to each other and to other lower molecular mass proteins by Western blot analysis. Two mAb studied, 105.5 and 184.1, were directed predominantly against the 31-kDa OspA protein. However, each also reacted with other minor bands, though with different specificities. Using V8 protease digestion and cleavage by cyanogen bromide, we demonstrated that each mAb reacted to the 31-kDa protein differently. Monospecific polyclonal rabbit and human antibodies directed against the 34-, 31-, 22-, and 20-kDa proteins were eluted from blots and used to further corroborate the cross-reactivity among these Ag. Rabbit antibodies to the 31- and 22-kDa Ag gave remarkably similar peptide maps after V8 protease digestion of the 31-kDa OspA protein, as did mAb 184.1, suggesting that this mAb recognized an immunodominant epitope common to the 22- and 31-kDa proteins. It seems likely therefore that the humoral immune response to Borrelia surface Ag may be due to a limited number of cross-reactive epitopes on distinct, but related, gene products.     

Acanthamoeba castellanii: characterization of an adhesin molecule.

Acanthamoeba castellanii is a free-living protozoan that causes keratitis in humans and has been associated with pneumonia and granulomatous amebic encephalitis in dogs, sheep, and other species. Adherence of the Acanthamoeba to epithelial cells is critical to the pathogenesis of this disease. In this study, several mouse monoclonal antibodies (MAb) generated to whole Acanthamoeba trophozoites identified surface membrane epitopes by ELISA and IFA. Nine antibodies inhibited adherence of [(35)S]-methionine-labeled Acanthamoeba trophozoites to hamster corneal epithelial cells by 27-90%. Sodium periodate treatment, but not proteinase K digestion, of whole Acanthamoeba destroyed epitopes recognized by adherence-inhibiting antibodies such as MAb 7H6, suggesting that the adherence epitopes are carbohydrates. Other antibodies, MAb 2A8 for example, recognized surface membrane peptide epitopes that were proteinase K sensitive and sodium periodate resistant. Purified MAb 2A8 was used in an antigen-capture ELISA with peroxidase-labeled MAb 7H6 and demonstrated that the carbohydrate adhesion molecule was linked to the peptide recognized by MAb 2A8. Both MAbs 7H6 and 2A8 recognized a >207-kDa band on a Western blot of eluant from a MAb 2A8 immunoaffinity column, confirming that MAb 7H6 and MAb 2A8 recognize different epitopes on the same adherence molecule. MAbs 7H6 and 2A8 also identified the adhesion molecule in soluble Acanthamoeba membrane preparations and MAb 2A8 immunoaffinity column eluant by ELISA and Western blot. Neither of these antibodies were inhibited from binding to whole trophozoites nor membrane extracts by mannose or mannan in competitive binding assays. When our Acanthamoeba membrane preparations were electrophoresed and immunoblotted with alpha-d-mannosylated-biotin albumin, no bands were recognized in the >207 kDa range by our adherence-associated antibodies. These results suggest that the Acanthamoeba adhesin is not identical to the mannose binding protein of Acanthamoeba but rather is a distinct surface membrane glycoprotein.     

Giardia duodenalis: adhesion-deficient clones have reduced ability to establish infection in Mongolian gerbils

The role of Giardia duodenalis surface molecules in the attachment of trophozoites to epithelial cells has been established through the dual strategies of characterizing G. duodenalis clones with deficient adhesion and blocking experiments with surface-specific monoclonal antibodies. Also, the infectivity of the analyzed clones was tested using Mongolian gerbils as experimental model. Two adhesion-deficient G. duodenalis clones, C6 and C7, were isolated from the wild type C5 clone which in turn was obtained from the WB strain. The adhesion efficiencies of C6 and C7 clones (48.2 ± 4.9 and 32.6 ± 2.4, respectively) were significantly lower as compared with WB strain or C5 clone (82.8 ± 6.4 and 79.9 ± 7.9). Analysis of radiolabel surface proteins by 1D and 2D SDS-PAGE revealed prominently labelled 28 and 88 kDa components in C6 and C7 clones and a major 200 kDa protein in the C5 clone and the WB strain. The 88 and 200 kDa components are acidic proteins by two-dimensional electrophoretic analyses. The most striking difference between wild-type and adhesion-deficient Giardia trophozoites was the reduced expression of a 200 kDa surface protein in the latter. Significantly, a mAb (IG3) specific for the 200 kDa protein that reacted with more than 99% of WB and C5 trophozoites and less than 1% of C6 and C7 trophozoites as determined by indirect immunofluorescence inhibited the adhesion of trophozoites from WB and C5 clone to Madin Darby Canine Kidney cells by 52% and 40.9%, respectively, suggesting a participation of this antigen in adherence. Finally, the functional relevance of trophozoite adhesion to epithelial cells was indicated by the reduced capacity of the adhesion-deficient clones to establish the infection in Mongolian gerbils.     

Generation and characterization of hamster-mouse hybridomas secreting monoclonal antibodies with specificity for lipopolysaccharide receptor.

Experiments are described for the partial purification of the 80-kDa LPS binding protein expressed on macrophages and lymphocytes. This partially purified Ag was used to immunize adult Armenian hamsters and splenocytes from immunized animals were fused with murine myeloma cell lines. Hybridoma cell culture supernatants containing mAb were screened by ELISA for positive binding to the immunizing Ag, murine splenocytes and the murine 70Z/3 pre B cell and for an absence of binding to sheep E. Positive clones were further screened for reciprocal competitive binding with LPS on spleen cells and ability to modulate B lymphocyte mitogenic activity. Two hybridoma cell lines secreting IgM monoclonals, termed mAb3D7 and mAb5D3, were identified that satisfied all of the selection criteria. These hybridoma cell lines were subcloned and expanded. Binding of one (mAb3D7) was abrogated by treatment of Ag with mild periodate; binding of the second (mAb5D3) was destroyed by digestion of Ag with proteinase K. Binding specificity for mAb5D3 has been confirmed by ELISA using highly purified 80-kDa protein. These mAb have been of value in establishing that the 80-kDa LPS binding protein previously identified may serve as a specific functional receptor for LPS.     

Monoclonal Antibodies Specific for the Different Subunits of Asymmetric Acetylcholinesterase from Chick Muscle

The asymmetric (20S) acetylcholinesterase (AChE, EC 3.1.1.7) from 1-day-old chick muscle, purified on a column on which was immobilised a monoclonal antibody (mAb) to chick brain AChE, was used to immunise mice. Eight mAbs against the muscle enzyme were hence isolated and characterised. Five antibodies (4A8, 1C1, 10B7, 7G8, and 8H11) recognise a 110-kilodalton (kDa) subunit with AChE catalytic activity, one antibody (7D11) recognises a 72-kDa subunit with pseudocholinesterase or butyrylcholinesterase (BuChE, EC 3.1.1.8) catalytic activity, and two antibodies (6B6 and 7D7) react with the 58-kDa collagenous tail unit. Those three polypeptides can be recognised together in the 20S enzyme used, which is a hybrid AChE/BuChE oligomer. Antibodies 6B6 and 7D7 are specific for asymmetric AChE. Four of the mAbs recognising the 110-kDa subunit were reactive with it in immunoblots. Sucrose density gradient analysis of the antibody-enzyme complexes showed that the anti-110-kDa subunit mAbs cross-link multiple 20S AChE molecules to form large aggregates. In contrast, there is only a 2-3S increase in the sedimentation constant with the mAbs specific for the 72-kDa or for the 58-kDa subunit, suggesting that those subunits are more inaccessible in the structure to intermolecular cross-linking. The 4A8, 10B7, 7D11, and 7D7 mAbs showed cross-reactivity to the corresponding enzyme from quail muscle; however, none of the eight mAbs reacted with either enzyme type from mammalian muscle or from Torpedo electric organ. All eight antibodies showed immunocytochemical localisation of the AChE form at the neuromuscular junctions of chicken twitch muscles.     

The cysteine-rich protein gene family of Giardia lamblia: loss of the CRP170 gene in an antigenic variant.

Giardia lamblia trophozoites demonstrate variable expression of a repertoire of cysteine-rich surface antigens in vitro and in vivo. The size of the repertoire has been estimated at 20 to 184, and specific variants can be detected after approximately 12 generations of in vitro growth for the WB isolate. In earlier studies, we cloned a portion of the gene for a 170-kDa surface antigen (CRP170) and demonstrated by DNA sequencing that it was cysteine rich (12%) and contained 2.6 copies of a tandemly repeated 195-bp pair sequence. The clone hybridized to multiple bands on a Southern blot of G. lamblia DNA in a pattern that was variable among the cloned lines but did not correlate with expression of CRP170. We have now cloned a nearly full length cDNA as well as genomic clones for CRP170 from the WBA6 cloned isolate. In addition, we have isolated a cDNA clone from the WB1269 line (expressing CRP72), an antigenic variant which was derived from WBA6. Sequence analysis of the CRP170 and CRP72 genes revealed marked C-terminal amino acid homology, suggesting a conserved functional role such as membrane anchoring. The CRP170 repeat oligonucleotide hybridized to a stairstep of bands approximately 6 kb in size on HindIII-digested WBA6 DNA representing the expressed copy(ies) of CRP170. In contrast, there was no hybridization to a fragment of similar size in WB1269, suggesting that WB1269 trophozoites have lost the expressed copy of the CRP170 gene.     

Monoclonal antibodies against an HLA-B27-derived peptide react with an epitope present on bacterial proteins.

The role of molecular mimicry in the spondyloarthropathies was investigated with respect to the epitopes involved. mAb were produced against a synthetic peptide whose sequence was derived from a polymorphic region of the HLA-B27 molecule (amino acids 63-83). Two antibodies (J7F2 and H2B6) were selected for study on the basis of their ability to react with bacterial envelope proteins (ELISA) and B27-positive cells (immunofluorescence). J7F2 reacted preferentially with B27-positive cells and neither antibody reacted with MHC class I negative cells. Based on SDS-PAGE blot analysis of bacterial envelope proteins, the pattern of reactivity for both antibodies (against 36- and 19-kDa proteins) was the same as that for a third monoclonal produced against bacterial envelope and reactive with B27-positive cells. This apparent epitope similarity was investigated by using synthetic peptides to inhibit binding of the monoclonals. The B27 synthetic peptide and a smaller peptide derived from it were efficient inhibitors of antipeptide and antibacterial antibody binding to bacterial Ag and B27-positive cells. These studies provide insight into the molecular basis of cross-reactivity between bacterial proteins and MHC class I molecules.     

Transfer and co-amplification of a gene encoding a 96-kDa immune IFN-inducible human melanoma-associated antigen. Preferential expression by mouse melanoma host cells

An immune IFN-inducible human melanoma-associated glycoprotein Ag, 96-kDa MAA, having preferential distribution on metastases has been defined by mouse mAb CL203.4. To initiate molecular genetic analysis of 96-kDa MAA, the gene encoding the Ag was transfected into mouse B16 melanoma cell clone B78H1. Formation of B78H1-transfectant colonies expressing a surface Ag reactive with mAb CL203.4 in an immunorosetting assay was dependent on addition of chromosomal DNA from human melanoma cells [primary (1 degree) transfer] or from Ag-expressing transfectant cells (2 degrees, 3 degrees, 4 degrees transfer). The mAb CL203.4-reactive species expressed by the transfectant cells is a glycoprotein with a molecular mass 93-kDa, within the range of 93 to 96-kDa observed for the endogenous human Ag. In the presence of tunicamycin, an inhibitor of N-linked glycosylation, both mouse melanoma transfectants and human melanoma cells express a 50- to 51-kDa antigenic species. Human alu family repeat sequences (h-alu) are present in the genomes of 3 degrees transfectant cells. Continued presence of these h-alu after dilution of extraneous human DNA by three cycles of transfection suggests their association with the transferred 96-kDa MAA gene. Use of a selective co-amplification procedure led to transfectant cells' increased expression of 96-kDa MAA and to commensurate increases in their content of presumed 96-kDa MAA gene-associated h-alu. Preferential DNA-mediated transferability of the 96-kDa MAA+ phenotype into B78H1 cells as compared with LMTK- mouse fibroblasts suggests host cell specificity of 96-kDa MAA gene expression.     

Human monoclonal anti-idiotypic antibodies. I. Establishment of immortalized cell lines from a tumor patient treated with mouse monoclonal antibodies

Patients who undergo immunotherapy with a murine anti-colon carcinoma mAb (mAb17-1A) generate high titers of anti-idiotype and anti-isotype antibodies. Specifically selected anti-idiotypic antibodies that elicit in vivo a humoral and a cellular immune response against the nominal Ag can be used as surrogate Ag for immunization. We established from the B lymphocytes of a treated patient a series of EBV-transformed cell lines. Three weeks after immortalization, the cells were selected for production of antibodies (Ab2) against the Fab fragment of the murine mAb17-1A. The selected cells were cloned and screened by ELISA for specific anti-mAb17-1A idiotypic antibodies. Thirty-six out of 89 clones were anti-idiotypes. Cell culture supernatants and the purified Ig derived from 10 clones completely inhibited the specific binding of radiolabeled mAb17-1A to HT-29 colon carcinoma cells thus resembling Ab2-gamma anti-idiotypes. These cell lines which grow now in culture for 18 mo, continuously secrete IgG,K anti-Ab1-idiotype mAb. Human anti-idiotypic mAb might be candidates for vaccines when the nominal Ag itself is not available or cannot be used as such.     

An in vitro model for Toxoplasma infection in man. Interaction between CD4+ monoclonal T cells and macrophages results in killing of trophozoites

The cell interactions that take place between Toxoplasma gondii trophozoites and the human immune system have been investigated by using an in vitro model of infection. PBMC were co-cultured with live, appropriately attenuated, trophozoites. When cells from immune (seropositive) donors were used, a proliferative response was observed. At the same time, the proliferating T cells proved capable of controlling the growth of live trophozoites. By contrast, cells from seronegative donors failed to mount a proliferative response and intracellular overgrowth of trophozoites with subsequent cell injury occurred. Actively proliferating T cells were expanded in continuous cell lines with IL-2 and periodical restimulation with Ag in the presence of autologous irradiated mononuclear cells. From some of the lines obtained, clones were also derived. Ten clones were selected for further studies. They proliferated in response to trophozoites but not to unrelated Ag. Their response required the presence of autologous monocytes-macrophages isolated from peripheral blood on Percoll density gradients. B cells that were obtained from the same donors and immortalized by EBV infection proved inefficient as APC. These data suggest that live trophozoites have to be processed by macrophages in order to be presented to T cells. Upon appropriate antigen stimulation, all of the clones produced IL-2 and IFN-gamma, a finding that was consistent with both their CD4+ surface phenotype and their helper capacity on B cell proliferation and differentiation in vitro. The supernatants of all of the stimulated clones released a factor that activated macrophages to kill intracellular trophozoites as well as an unrelated pathogen, Listeria monocytogenes. This factor was identified as IFN-gamma because it was neutralized by specific anti-IFN-gamma antibodies. The present in vitro model of response to live protozoa may prove suitable to assess the role of both T lymphocytes and macrophages in intracellular parasite infections in man. Furthermore, this experimental system may be applied to detect specific lesions of cell mediated immunity in a number of immunodeficiency syndromes.     

Periodic appearance of a predominant variant antigen type during a chronic Giardia lamblia infection in a mouse model

Giardia lamblia (syn. Giardia duodenalis, Giardia intestinalis) infections are associated with continuous antigenic variation of the parasite which is mediated by the parasite's major surface antigen, named variant surface protein. Offspring mice and corresponding mothers were infected with G. lamblia clone GS/M-83-H7 (expressing variant surface protein H7) and various parameters of this infection were assessed in a long-term follow-up investigation. Our experimentation revealed that variant surface protein H7-type trophozoites were replaced by new variant-type trophozoites during the early stage of infection (around day 8 p.i.), but the original variant-type re-emerged at at least two time-points during the later stages of infection (at days 22 and 42 p.i.). Such periods of variant surface protein H7-type trophozoite re-expansion were accompanied by transient production of intestinal IgA against variant-specific epitopes on a 314-aa N-terminal region of variant surface protein H7. At late stages of infection (between days 42 and 200 p.i.), most mice produced intestinal IgA against both variant surface protein H7 and other antigens of the parasite. At these stages, infection seemed to be resolved in most mice, but occasional reappearance of relatively high (at day 64 p.i.) or at least detectable (at days 80 and 120 p.i.) amounts of intestinal parasites indicated that G. lamblia GS/M-83-H7 infections in mice may enter into a latent chronic phase which is interrupted by sporadic breakthroughs of parasite growth.     

Antigenic variation in Giardia lamblia

Giardia lamblia undergo surface antigenic variation in vitro and in vivo. The presence of variant trophozoites can be detected in clones after exposure to cytotoxic monoclonal antibodies. Surviving Giardia (progeny) no longer possess the initial major surface antigen which is replaced by new antigens. Exposure of a clone from one progeny to another cytotoxic mAb specific to one newly appearing surface antigen resulted in the loss of this antigen and replacement by another set of antigens. The frequency of change was rapid (1:100-1:1000) and was dependent upon the isolate. The presence of variant populations in clones was confirmed by direct and indirect immunofluorescence using mAbs to major surface antigens of subsequent progeny. The putative amino acid sequence of a portion of one antigen revealed a cysteine-rich composition which was confirmed in this variant protein as well as others by preferential uptake of [35S]cysteine. The mechanism(s) responsible most likely involves genomic rearrangements since Southern blots revealed a family of related genes which changed frequently compared to other areas of the genome. However, expression-linked copies have not been detected. Loss and gain of surface antigens have also been found in gerbils and humans infected with defined clones, but there does not appear to be cyclical appearance of variant populations. The biological importance of antigenic variation is not known but it may contribute to chronic and/or repeated infections.     

Identification and immunological characterization of a novel 40-kDa protein linked to CD98 antigen.

Monoclonal antibodies (mAbs) were obtained from hybridoma clones established by cell fusion between mouse myeloma cells and spleen cells from a mouse immunized against an affinity-purified 40-kDa component of rat 125-kDa glycoprotein (GP125). Two mAbs designated as 3F2 and 6B4 detected a 40-kDa and a 125-kDa band under reducing and nonreducing conditions, respectively, in extracts prepared from rat, mouse and human tumor cells. Association of the 40-kDa protein with CD98 was revealed by sandwich-type enzyme-linked immunosorbent assay. The two mAbs were strongly reactive with various tumor cells and activated lymphocytes, but were only weakly reactive with resting lymphocytes. Confocal microscopy indicated colocalization of CD98 and the 40-kDa protein defined with 3F2 and 6B4 at the cell surface and perinuclear regions. On immunohistochemical analysis of frozen sections of rat tongue, the anti-rat CD98 mAb B3 selectively stained the basal layer and 3F2 stained the upper epithelial part in addition to the basal layer, indicating the existence of CD98-unlinked 40-kDa protein.     

Protection from experimental autoimmune thyroiditis conferred by a monoclonal antibody to T cell receptor from a cytotoxic hybridoma specific for thyroglobulin.

A clonotypic mAb, AG7, has been prepared from splenocytes of CBA/J mice immunized with a cytotoxic T cell hybridoma, HTC2, specific for a pathogenic epitope of the thyroglobulin molecule in association with class I MHC Ag. AG7 binds to HTC2 cells but not to the other T cell hybridomas tested. Moreover, when used in functional studies, AG7 blocks the HTC2 capacity of specific target lysis. It also reacts with a determinant that comodulates with the CD3 Ag present on the surface of HTC2 cells. Immunoprecipitation of 125I-labeled solubilized HTC2 membranes demonstrated two bands located at 90 and 72 kDa under nonreducing conditions, which became a 46-kDa band under reducing conditions. Finally, when AG7 is injected into CBA/J mice, on day -1 before immunization with the pathogenic tryptic fragments of the thyroglobulin molecule, experimental autoimmune thyroiditis is abrogated. Thus, one of the multiple potential mechanisms of the protective immunity against EAT induced by HTC2 cells that we previously proposed, i.e., the generation of anti-clonotypic antibodies to HTC2 TCR, seems apparent.     

Characterization of an immunoreactive 93-kDa core protein of Borrelia burgdorferi with a human IgG monoclonal antibody

Lyme borreliosis is an infectious disease caused by the tick-borne spirochete Borrelia burgdorferi, which carries the potential for chronic infection. Ag on the etiologic Borrelia are currently being defined structurally and their ability to elicit immune responses delineated. EBV can be used to immortalize human B. burgdorferi-specific B cells from infected donors and generate antibodies against antigenic epitopes encountered in natural infection. A human mAb secreting EBV-transformed B cell line, D7, has been developed that is specific for a 93-kDa B. burgdorferi protein and has been used to characterize this potentially important Ag. D7 produces an IgG3 antibody that detects the 93-kDa Ag as well as smaller fragments at 46 kDa and lower molecular mass. The antibody detects similar epitopes on all B. burgdorferi isolates tested and on a Borrelia hermsii protein with molecular mass greater than 100 kDa but binds poorly to Treponema species. In contrast, polyclonal sera from Lyme disease patients show little binding to the homologous Ag in B. hermsii. Structurally, the 93-kDa protein is associated with the flagellum and may be firmly anchored in the protoplasmic cylinder. It is not solubilized by nonionic detergent treatment of the whole Borrelia. Antibodies against a comparable m.w. protein are present in sera from patients with both early and late infection. Thus, antibodies against this Ag are a sensitive and specific marker of Borrelia infection. This Ag is likely of structural importance and may represent a target of host defenses.     

Protective immunity against Brugia malayi infective larvae in mice. II. Induction by a T cell-dependent antigen isolated by monoclonal antibody affinity chromatography and SDS-PAGE

A mAb directed against filarial worm secretory/excretory product and reactive with Brugia malayi larval worm surface was used in conjunction with preparative SDS-PAGE to isolate protective Ag from extracts of adult B. malayi. The IgM mAb OVH bound to a repeating carbohydrate epitope present in adult, infective, and fourth stage larvae and microfilariae of B. malayi, and on the surface of fourth stage larvae. Ag bearing this epitope were also present in the sera of hosts infected with a variety of helminths, including Brugia, Onchocerca, Dirofilaria, and Paragonimus. Affinity chromatography of SDS extract of adult Brugia, using mAb OVH immobilized on agarose beads, isolated several Ag that separated into multiple protein staining bands on SDS-PAGE. In comparing SDS-PAGE-fractionated Ag from the crude SDS extract with fractionated mAb OVH-isolated Ag for the ability to protect BALB/c mice from challenge with B. malayi-infective larvae, it was found that of the mAb OVH-isolated Ag only those at a molecular mass of 26 to 32 kDa were protective while the original SDS extract yielded protective Ag at the following molecular mass: greater than 200, 170 to 200, 40 to 44, 33 to 36, 23 to 28, 20 to 22, and 17 to 19 kDa. Although Ag isolated by mAb OVH were highly protective, they failed to induce high antibody levels against the immunogen or SDS extracts compared to crude SDS extract immunized mouse sera, as determined by immunoblot and ELISA. Transfer of nylon wool non-adherent T cells from BALB/c mice immunized with the 26- to 28-kDa fraction of mAb OVH-isolated Ag to naive mice just before challenge with infective larvae of B. malayi resulted in a 70% reduction in larvae recovered 14 days after challenge.