Structural differences among monoclonal antibodies with distinct fine specificities and kinetic properties.

The mAbs HyHEL-8, HyHEL-26 (HH8, and HH26, respectively) recognize epitopes on hen egg-white lysozyme (HEL) highly overlapping with the structurally defined HH10 epitope, while the structurally related XRPC-25 is specific for DNP and does not bind HEL. All four Abs appear to use the same Vk23 germ line gene, and all but HH8 use the same VH36-60 germ line gene. Of the three anti-HEL Abs, the sequences of HH26 variable regions are closest to those encoded by the respective germ line sequences. HH8 utilizes a different member of the VH36-60 gene family. Thus, the same Vk and VH genes, combined with somatically derived sequence differences, are used to recognize the unrelated Ags HEL and DNP. In contrast, different VH36-60 germ line genes are used to bind the same antigen (e.g. HH8 vs HH10 and HH26). While the affinities of HH10, HH8, and HH26 for HEL vary by less than 10-fold, their affinities for mutated Ag vary over several orders of magnitude. Analyses of Fab binding kinetics with natural species variants and site-directed mutants of lysozyme indicate that these cross-reactivity differences reflect the relative sensitivities of both the association and dissociation rates to antigenic mutation: HH8 has relatively mutation-insensitive association and dissociation rates, HH10 has a relatively mutation-sensitive association rate but more variable dissociation rates, and HH26 has variable association and dissociation rates. Only a few amino acid differences among the antibodies produce the observed differences in the robustness of the association and dissociation rates. Our results suggest that association and dissociation rates and mutation sensitivity of these rates may be independently modulated during antibody repertoire development.

Plasmacytoma-refractory BALB/cAnPt mice have naive T cell and highly specific B cell responses to antigen.

Recently, a reduction in the incidence of pristane-induced plasmacytomas in BALB/cAnPt (BALB/c) mice that were kept in viral specific pathogen-free (SPF) conditions has been reported. Environmentally, these SPF-BALB/c mice differed from conventionally-housed (CON) mice only in viral exposure and diet (i.e. sterilization of mouse chow), since microbial colonization of the intestinal tract was seen to be equivalent. This report assessed the ability of SPF- and CON-BALB/c mice to respond to immunologic challenge with soluble antigen, i.e. hen egg white lyzosyme (HEL), as a means of evaluating differences in T and B cell function and, indirectly, evaluating the possible effects these differences might have on plasmacytoma development. When cultured in vitro for 5 days with HEL, HEL-primed lymph node cells (LNC) from SPF-BALB/c mice proliferated to a significantly lesser extent than HEL-primed CON-BALB/c LNC. Moreover, HEL-induced production of IFN-gamma and IL-5 was significantly lower in SPF LNC. Serum IgG1 levels were 10-fold lower in SPF-BALB/c mice with, or without prior immunization with HEL and were not reconstituted by repeated injections of HEL in adjuvant. Serum IgM levels of SPF- and CON-BALB/c mice were equivalent. This reduction in immune responses could not be attributed to a lack of colonization of secondary lymphoid organs, since flow cytometric analysis of LNC revealed no difference in the number of recoverable cells and the proportion of lymphocyte subsets (CD4+, CD8+ and CD45+ cells) obtained from SPF- and CON-BALB/c mice. However, only CON LNC were induced to increase surface expression of CD44 after antigenic or mitogenic stimulation in vitro. Antibody responsiveness to HEL, as evidenced by serum anti-HEL binding or splenic hybridoma studies, demonstrated higher levels of IgG1 antibodies in CON BALB/c mice than in SPF mice. However, a greater proportion of the SPF IgG1 antibodies present were specifically directed against HEL, so that specific activity was greater in SPF-BALB/c mice. Therefore, while SPF BALB/c mice have a more restricted response to HEL than CON-BALB/c mice, those antibodies that are produced are more specifically directed against HEL with very little apparent bystander/polyclonal activation of multireactive cells. Resistance to plasmacytomas in SPF-BALB/c mice, therefore, may stem from a reduced number of circulating memory T and B cells, which are capable of reacting and/or crossreacting with a chronic inflammatory stimulus.

Specificity of monoclonal antibodies elicited by mucosal infection of BALB/c mice with virulent Shigella flexneri 2a.

Protective immunity against shigellosis is thought to be determined by the O-antigen side chains of the lipopolysaccharide (LPS) molecule. To study possible common protective epitopes, monoclonal antibodies reacting with Shigella flexneri 2a LPS were generated from BALB/c mice infected ocularly with the virulent serotype 2a strain S. flexneri 2457T and tested against a panel of S. flexneri LPSs by enzyme-linked immunosorbent and immunoblot assays. Four monoclonal antibodies were identified, all of which showed restricted specificity patterns. Three different patterns of reactivity to LPS possessing the 3,4 group antigen were seen: (i) 2a only, (ii) 2a and 5a, and (iii) 2a, 4a, 5a, and Y. These results have implications for designing a Shigella vaccine that will be protective against related serotypes. Electron microscopy studies showed that the monoclonal antibodies bind to the bacterial surface in a patchy pattern, suggesting their potential use for examining the LPS distribution on the surface of the bacteria.

Patterns of antibody specificity during the BALB/c immune response to hen eggwhite lysozyme.

We tested 49 BALB/c antilysozyme mAb from seven intervals during the immune response to lysozyme for patterns of specificity and avidity. We found that the antibody epitopes in composite covered at least 80% of the lysozyme surface, and their patterns of overlap suggest a continuum of potential antibody epitopes. Previously observed regional specificities, which emerged at different times in the immune response, were more discretely defined in late response antibodies, when the majority of mAb could be assigned to one of three functionally nonoverlapping complementation groups. The area covered by each antigenic region may be greater than an individual epitope, and may include multiple epitopes that overlap structurally and functionally to varying degrees. Connectivity between antigenic regions was seen in interactions among early and late stage antibodies, and among secondary stage mAb, but not among tertiary stage mAb from hyperimmunized mice. Patterns of overlap of early and late response antibodies suggest that the organization of antibody specificities change during the progression from primary to secondary to tertiary response. Over the same period in the response, the average relative avidity of IgG1 kappa mAb did not increase, suggesting that "affinity maturation" of serum antibodies reflects an increase in the number and diversity of antibodies, rather than an overall increase in the avidity of individual antibodies.

Antigenic regions defined by monoclonal antibodies correspond to structural domains of avian lysozyme.

The epitopes recognized by six new BALB/c hybridomas specific for the protein antigen hen egg-white lysozyme (HEL) were mapped in detail. Although fine specificities of all the antibodies were distinct, many of the epitopes overlap in complex patterns. The antibodies could be grouped into three complementation groups, one of which also included the previously characterized HyHEL -5 which is specific for Arg68 . Complex interactions were observed among the antibodies, both among and within complementation groups, including nonreciprocal competition and enhanced binding. Two of the complementation groups mapped near the catalytic site in a new antigenic region. The antibodies HyHEL -8 and HyHEL -10 had very similar and over-lapping specificities, and may recognize very closely related epitopes. The results suggest that the epitopes may form a continuous antigenic surface, and that antigenic regions correspond to structural domains defined by the tertiary structure of HEL.

A refined model for the variable domains (Fv) of the J539 beta(1,6)-D-galactan-binding immunoglobulin.

A refined protocol for building a hypothetical model of the J539 Fv is described. Computer programs for positioning amino acid side chains and structure energy minimization [CHARMM program of Brooks et al., J. comp. Chem. 4, 187-217 (1983)] were employed. Computer modeling was accomplished on an Evans and Sutherland picture system which permitted structure visualization in three dimensions. Peptide backbone breaksites were rejoined by monitoring for correct distances and torsion angles. A physical model was then constructed and used as a basis for further refinements such as aligning conformations around remodeled sites, adjusting proline substitutions and optimizing hydrogen-bond-forming potentials. This structure (J539-ADO) was energy minimized and the final coordinates were obtained from the energy-refined model. The resulting hypothetical J539 structure can be compared to the structure of J539 now being determined by X-ray crystallography. The procedures described can be used for other Fv fragments.

VL-VH expression by monoclonal antibodies recognizing avian lysozyme.

Seven BALB/c hybridoma antibodies directed against the protein antigen, hen egg-white lysozyme c (HEL), were characterized on the basis of their ability to bind lysozymes from 10 species of birds, and their ability to bind HEL competitively. The hybridomas were separable into three complementation groups based upon competitive interactions. The fine specificities of all antibodies were distinct, but two, HyHEL-8 and HyHEL-10, had very similar and overlapping reactivity patterns. To test the hypothesis that VL-VH pairing correlates with binding specificity, the N-terminal amino acid sequences were determined to identify the VL and VH isotopes (subgroups) of the anti-HEL antibodies. HyHEL-8 and -10 shared the VK23 light chain isotype and nearly identical heavy chains in Kabat subgroup I, whereas the heavy and light chain isotypes of all other antibodies differed from HyHEL-8 and -10 and from each other. The heavy and light chain isotypes expressed by HyHEL-8 and -10 are also expressed by XRPC-25, a DNP-binding myeloma protein that does not bind lysozyme. These results are discussed with respect to the contributions of various genetic sources of structural diversity to antibody functional diversity.

Mapping the antigenic epitope for a monoclonal antibody against lysozyme.

A monoclonal antibody (HyHEL-5), prepared to chicken lysozyme c by the method of Köhler and Milstein, identified an antigenic site (epitope) that was shared by the lysozymes of seven different species of galliform birds. The lysozymes of two galliform species, bobwhite quail and chachalaca, shared only partial antigenic identity with the epitope defined by this antibody. Duck lysozyme did not react with the antibody at all. Amino acids that determined the epitope structure were tentatively identified by comparing the amino acid sequences of these lysozymes and assuming the antigenic changes produced by evolutionary substitutions are not due to long-range conformational changes. Arg 68 was identified as a determining amino acid. Arg 68 is hydrogen-bonded to Arg 45, and together these two amino acids form a basic cluster that may be a subsite of the epitope. The antibody inhibited lysis of Micrococcus lysodeikticus by chicken lysozyme. Additionally, Biebrich Scarlet, a dye that binds to the catalytic site, inhibited antibody binding to this lysozyme, which indicates that the epitope extends into the cleft region between Arg 45 and Arg 114. The epitope was hypothesized to involve a region measuring at least 13 x 6 x 15 A including the Arg 68-Arg 45 complex that borders the enzymatic catalytic site. Four other monoclonal antibodies to lysozyme have been partially characterized; each had a distinct pattern of binding specificity for various species of bird lysozymes.