Variable and conserved structural elements of trypanosome variant surface glycoproteins.

The characterization of B cell epitopes on the trypanosome variant surface glycoprotein (VSG) rests on elucidation of variant specific amino acid sequences that may be exposed or buried as a result of the natural conformation of these molecules in the surface coat. Despite the fact that different VSGs have heterogeneous primary sequences and unique antigenic characteristics, recent high resolution X-ray crystallographic analyses of VSGs have revealed a conserved 3-dimensional structure common to these surface proteins [19]. We took advantage of this conserved structural conformation to help predict which variant subregions of VSG molecules may contain exposed or buried variant specific B cell epitopes. Using Staden data tables, we aligned the deduced amino acid sequence of Trypanosoma brucei rhodesiense LouTat 1 VSG, a molecule that has been characterized immunologically in this laboratory, with 12 other complete VSG sequences including the T. b. brucei MiTat 1.2 VSG that has been characterized in crystallographic studies. Results of this analysis predict that there are eight defined clusters of variant amino acids which may contribute to exposed B cell epitopes, and ten defined clusters of variant amino acids which may contribute to buried B cell epitopes, on all VSG molecules. Interestingly, this analysis also revealed a VSG consensus sequence in which certain conserved motifs are present in all VSGs. The shared elements of VSG sequences corresponded to known secondary structures present in MiTat 1.2, and included groups of conserved amino acids responsible for turns in subregions of the protein, for structural positioning of the variable residues on the exposed surface, and for the dimerization of VSG monomers. Overall, these observations may aid in the targeting and mapping of exposed and buried VSG specific B cell epitopes, and also may offer clues as to elements of the primary sequence that are important for the conserved 3-dimensional structure of antigenically distinct VSG molecules.

T-cell-independent and T-cell-dependent B-cell responses to exposed variant surface glycoprotein epitopes in trypanosome-infected mice.

The T-cell dependency of B-cell responses to variant surface glycoprotein (VSG) epitopes exposed in their native surface conformation on Trypanosoma brucei rhodesiense clone LouTat 1 was investigated. T-cell requirements were examined by analyses of gamma globulin preparations derived from trypanosome-infected BALB/c nude (nu/nu) and thymus-intact (nu/+) mice. A radioimmunoassay was used to selectively quantitate antibody binding to native VSG 1 epitopes present on the surface of viable trypanosomes. Such analyses of VSG-specific antibody in infected mice demonstrated that in the absence of T cells there was a significant B-cell response to exposed VSG epitopes; however, in the presence of T cells these surface epitope-specific responses were greatly enhanced. In contrast to infection, immunization of mice with purified VSG 1 or paraformaldehyde-fixed parasites elicited significant VSG surface epitope-specific responses only in the presence of T cells (i.e., in nu/+ mice only). VSG-specific antibody responses in mice infected with three other clonal T. brucei rhodesiense populations (LouTat 1.2, 1.5, and 1.9) were found to be similar in this pattern, although not identical, to the anti-LouTat 1 responses. An important exception was that mice infected with LouTat 1.8 required T cells to produce VSG surface-specific antibody. Thus, the VSG surface epitope-specific B-cell responses in trypanosome-infected mice represent composite T-cell-independent and T-cell-dependent processes, and a significantly stronger response is made in the presence of T cells. However, immunization with VSG in the absence of infection elicited only T-cell-dependent responses. Since the relative contribution of T-cell-independent and T-cell-dependent processes to the total VSG-specific antibody produced during infection was variable (as seen with the absence of a T-cell-independent response to LouTat 1.8), this may reflect differences in the primary structure or display of VSG molecules on the trypanosome membrane or may represent active parasite interference with some epitope-specific B-cell responses.

Regulation of B cell responses to the variant surface glycoprotein (VSG) molecule in trypanosomiasis. I. Epitope specificity and idiotypic profile of monoclonal antibodies to the VSG of Trypanosoma brucei rhodesiense.

Regulatory mechanisms governing B cell responses to the trypanosome variant surface glycoprotein (VSG) molecule currently are being studied. As a fundamental basis for examining such regulation, the epitope specificities and idiotypic profiles of murine mAb produced to the VSG of Trypanosoma brucei rhodesiense clone LouTat 1.5 were determined. Variant specific mAb were used to probe VSG proteolytic peptides in Western blot analysis, to serve as competitive inhibitors in RIA analyses with purified VSG molecules, and to examine membrane-binding patterns of labeled trypanosome cells in order to evaluate epitope specificities. By using these approaches, a conformational epitope expressed only on the VSG 1.5 surface coat of viable trypanosomes was detected, and two nonconformationally determined epitope clusters were recognized within the subsurface V region of the VSG 1.5 molecule. The subsurface epitope clusters may be repeated on the VSG molecule because each was present on more than one proteolytic VSG peptide fragment. Idiotypic profiles of selected VSG-specific mAb subsequently were determined with xenogeneic antiidiotypic typing sera. Results from competitive inhibition RIA analyses using these reagents demonstrated that varying levels of idiotypic cross-reactivity exist among the subsurface VSG epitope-specific mAb; this cross-reactivity extended to idiotope(s) expressed by a mAb recognizing a surface conformational epitope of the VSG 1.5 molecule. Analysis of complementary idiotypic/antiidiotypic antibody pairs revealed that these specific interactions were inhibited by purified VSG 1.5 but not by purified VSG 1.9, which was derived from a heterologous variant antigenic type. The model mAb described here, and reagents recognizing their idiotypic markers, comprise a foundation for analysis of idiotypic regulation of VSG-specific B cell responses during infection.

Anti-fluorescein antibody 3-13 VH gene rearrangement in idiotypically cross-reactive hybridomas.

The deduced amino acid sequence of anti-fluorescein (F1) antibody 3-13 VH region (residues 1-95) was 78% homologous to the alpha-1----3-dextran binding myeloma protein J558 VH region and was in the Wu-Kabat Subgroup II or Dildrop Group I classifications. The 3-13 VH region was rearranged to a D segment with only 8 of 30 bp in common with DFL16.1 germ line D gene and less homologous to all other previously identified D sequences. This sequence was joined to the third codon of JH4. The sequence encoding VH residues 5-91 was subcloned into pSP65 and used as a probe in Southern analyses to monitor 3-13 VH gene rearrangements in 12 other anti-F1 hybridomas differentially expressing (or not at all) the 3-13 idiotype. Three clones which inhibited the 3-13 idiotype-anti-idiotype interaction as effectively as 3-13 (3-12, 3-17 and 3-35), all had rearranged a gene which hybridized to the cloned 3-13 fragment, however, each was contained on a different size restriction fragment. Analyses of five other idiotypically related (but not identical) hybridomas indicated that four had rearranged a cross hybridizing VH gene while no such rearrangements were detected among four idiotypically negative cell lines. A restriction site assay indicated five clones examined had all rearranged a Vk gene to the Jk1 or Jk2 gene segment. The sequence of the antibody 3-13 VH gene and its use in hybridization studies represent the first molecular analysis of a recurrently expressed repertoire specific idiotype within an unrestricted immune response.

Independent regulation of B cell responses to surface and subsurface epitopes of African trypanosome variable surface glycoproteins.

Regulation of B cell responses to the trypanosome surface Ag was examined in H-2k compatible "responder" B10.BR and "nonresponder" C3H mice after infection with two variant clones of Trypanosoma brucei rhodesiense. Development of a selective RIA for independent detection of antibody binding to surface (exposed) and subsurface (buried) epitopes of the trypanosome variable surface glycoprotein (VSG) molecule permitted sensitive quantitation and kinetic characterization of immune responses to these epitopes. The infected B10.BR mice responded to both exposed and buried VSG epitopes of clone LouTat 1 trypanosomes, whereas a B cell response by C3H mice to exposed VSG epitopes was not detected by RIA analyses at any time. However, VSG-specific IgM and IgG responses were produced to buried VSG epitopes, demonstrating that LouTat 1 induced immunoregulation was specific only for the B cell responses to exposed VSG epitopes. Subsequently, comparisons of B10.BR and C3H B cell responses to a heterologous variant, LouTat 1.5, were made. The results revealed that both infected mouse strains produced VSG 1.5-specific antibody to exposed and buried epitopes with different kinetics and maximal sera concentrations, showing, therefore, that these responses are not coordinately regulated. In addition, it was clear that the observed immunosuppression to exposed LouTat 1 VSG epitopes in C3H mice could be regulated by the parasite since functional C3H B cell responses were mounted against exposed VSG epitopes of a closely related variant (LouTat 1.5) after infection.

Linkage of low and high affinity anti-fluorescein idiotype families.

Data presented in this study describes the isolation and characterization of two anti-fluorescein (F1) hybridoma proteins 3-24 and 12-40, both IgG1, kappa with a Ka = 2.8 and 3.4 X 10(6) M-1, respectively, at 37 degrees C. These clones inhibited (6.8 +/- 2.8 -20.8 +/- 0.6% at 1 microgram/well) the idiotype-anti-idiotype interactions (IAII) of anti-F1 clones 3-13 and 3-17, which define a previously described low affinity idiotype family. Antibodies 3-24 and 12-40 also inhibited (45.0 +/- 3.0 and 61.3 +/- 5.6%, respectively, at 1 microgram/well) an IAII defined by a high affinity (Ka = 5.2 +/- 1.5 X 10(9) M-1 at 37 degrees C) anti-F1 clone, 4-4. Hybridoma proteins 3-13 and 3-17 possess similar affinities for F1 (Ka = 3.8 +/- 5.1 and 5.9 +/- 4.0 X 10(4) M-1) and are known to be idiotypically unrelated to clone 4-4. While 3-24 and 12-40 appeared very similar, non-identity of their active sites was established by heterologous idiotypic inhibitions, fine specificity of binding and spectral measurements (Qmax and lambda max) of bound F1. All IAII (3-13, 3-17, 9-40 and 4-4) were inhibited greater than 80% by the presence of 10(-4) M F1 or F1-BSA. In addition, four intermediate affinity (6.0 X 10(6) less than or equal to Ka less than or equal to 5.3 X 10(8) M-1) anti-F1 clones, comprising a second previously described idiotype family (designated the 9-40 family) were further analyzed. Inhibition of the 9-40 IAII by all heterologous proteins in the 9-40 family (except clone 5-27), and clones 3-24, 12-40 and 4-4 ranged from 87.7 +/- 1.3 to 95.4 +/- 1.0% at 1 microgram/well. Titration of the 9-40 IAII inhibition by antibodies 9-40, 3-24, 12-40 or 4-4 generated essentially superimposable profiles. In reciprocal inhibition experiments, using the 4-4 IAII, clones 3-24, 12-40, 9-40 and 4-4 gave distinct idiotypic titration patterns. Thus, members of the 9-40 family, 3-24 and 12-40 were more closely related to intermediate affinity clone 9-40 than high affinity clone 4-4.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of recurrent idiotypes within the unrestricted anti-fluorescein immune response.

The BALB/cV immune response to fluorescein (F1) was analyzed for expression of serologically defined idiotypes (Id) present on two anti-F1 hybridoma proteins, 3-13 and 3-17. After a I degree, II degree, or III degree immunization with F1-KLH, Id 3-13 and 3-17 were expressed in 15 of 28 and 22 of 28 mice, respectively. Solid-phase Id inhibition assays, performed by using pooled or individual anti-F1 antibody, indicated Id 3-13 and 3-17 represented less than 1.0% of the I degree, II degree, or III degree serum anti-F1 repertoire. Hybridoma proteins 3-13 and 3-17 had similar but distinct active sites as indicated by similar patterns of Id inhibition, fine specificity of binding, and affinity for F1. Analyses of 11 anti-F1-AECM-Ficoll hybridoma proteins (predominantly mu, lambda) indicated 4 of 11 clones were weakly cross-reactive with 3-13 and 3-17. Anti-F1-KLH II degree antibody from mice of various Igh allotypes was also screened and indicated these Id were expressed in each strain but at lower levels than in BALB/cV mice and were not strictly allotype associated. Id 3-13 and 3-17 represent the first demonstration of repertoire-specific recurrent Id expressed during a T-dependent immune response lacking a dominant Id.

Idiotypic cross-reactivity of low-affinity anti-fluorescyl monoclonal antibodies.

Previous studies concerning structure-function relationships of anti-fluorescyl hybridoma proteins utilized primarily high-affinity proteins (Ka greater than 5.0 X 10(7) M-1) possessing distinct idiotypes. Low-affinity anti-fluorescyl monoclonal antibodies, predominantly IgGl or IgG2a possessing kappa light chains were analyzed. Two fusions produced 18 monoclonals, 13 binding fluorescein with a low affinity (less than or equal to 3.0 X 10(6) M-1) and five possessing high affinities (greater than or equal to 5.3 X 10(8) M-1). Solid-phase idiotype assays, utilizing rabbit anti-idiotype reagents against two low-affinity proteins (3-13 and 3-17), showed that all the low-affinity clones (except 2-9 and 2-21) were capable of inhibiting (40-100%) these two idiotype-anti-idiotype interactions while no high-affinity proteins inhibited them. The interactions with 3-13 and 3-17 were inhibited 100 and 88%, respectively, by free fluorescein. When these idiotype-anti-idiotype interactions were inhibited with increasing concns of heterologous hybridoma proteins, three clones inhibited both interactions as effectively as the homologous proteins at all concns tested and inhibition reached 100%. These three clones appeared to possess all the idiotopes that the anti-3-13 and anti-3-17 reagents detected on 3-13 and 3-17. Screening of eight high-affinity anti-fluorescyl proteins previously produced [Kranz and Voss, Molec. Immun. 18, 889-898 (1981a)] identified a single clone [20-4-4 (Ka = 5.0 X 10(7) M-1)] significantly inhibiting the 3-13 and 3-17 interactions (71.0 and 63.6%, respectively). In addition, recombination experiments utilizing H- and L-chains derived from three low-affinity and three high-affinity antibodies resulted in reformation of active sites in all six heterologous combinations when both chains were derived from low-affinity antibodies, and in only one of six combinations when both chains were derived from high-affinity molecules. Thus, the apparent lack of private idiotopes on clones 3-13 and 3-17 and the presence of these idiotopes (or cross-reactive ones) on 11 of 13 low-affinity antibodies and on one of 13 high-affinity antibodies may indicate that clones 3-13 and 3-17 are encoded by germline genes. The H- and L-chain recombination experiments indicated that the idiotype and affinity of parental molecules may be involved in H- and L-chain association.