Number Of Antigenic Sites Research Articles

Overcoming target epitope masking resistance that can occur on low-antigen-expresser AML blasts after IL-1RAP chimeric antigen receptor T cell therapy using the inducible caspase 9 suicide gene safety switch

Although chimeric antigen receptor CAR) T cell immunotherapies are an undeniable and unequivocal success, knowledge obtained from the monitoring of the first clinical trials targeting the CD19 antigen in B malignancies, either refractory/relapsed acute lymphoid leukemia (ALL) or lymphomas, contributed to the identification of tumor cell escape in about 30–50% of B-ALL. Resistance occurred due to loss of surface expression of the antigen (rCD19−) or to the early disappearance or inactivation of CAR T cells (rCD19+). In a recently reported clinical case, rCD19− relapse resulted from masking of the antigen by the CAR at the surface of B-ALL leukemia cells following the unexpected viral transduction of a leukemic cell present in the cytapheresis sample. The objective of this work was to reproduce this epitope-masking resistance model, in the context of acute myeloid leukemia (AML), based on our immunotherapeutic CAR T cell model targeting the accessory protein of the interleukin-1 receptor (IL-1RAP) expressed by leukemic stem cells. As AML primary blasts express different levels of IL-1RAP, we modeled transduction of different AML tumor cell lines screened for density of antigenic sites with our lentiviral vectors carrying a third-generation IL-1RAP CAR, an iCASP9 suicide gene, and a truncated CD19 surface gene. We demonstrated that primary AML blasts can be easily transduced (74.55 ± 21.29%, n = 4) and that CAR T cytotoxicity to IL-1RAP is inversely correlated with epitope masking in relation to the number of antigenic sites expressed on the surface of IL-1RAP+ lines. Importantly, we showed that, in vitro, a 24-h exposure of IL-1RAP+/CAR+ leukemia lines to Rimiducid eliminated >85% of the cells. We confirmed that the expression of IL-1RAP CAR by an IL-1RAP+ leukemic cell, by decreasing the membrane availability of the targeted antigen, can induce resistance while a high epitope density maintains sensitivity to CAR T cells. Moreover, the presence of the iCASP9/Rimiducid suicide system safety switch makes this immunotherapy approach safe for application in a future phase 1 clinical trial.

A one-step method for determining the maximum number of bound antibodies, and the affinity and association rate constants for antibody binding

A reliable analysis of antibody binding may lead to more successful selection of the optimal antibodies. The most important parameters are affinity (equilibrium dissociation constant, Kd), the number of antigen sites on the cells (Bmax) and the on (ka) and off (kd) rate constants of binding. The affinity and the number of cellular binding sites are usually determined by equilibrium binding experiments and subsequent Scatchard analysis. The on and off rate constants are determined by kinetic binding experiments. However, it is necessary to perform two to three different types of experiment in order to determine these parameters. We have developed an alternative one-step method based on a kinetic binding experiment and a mathematical description of antibody binding to antigen. The method was compared with kinetic and equilibrium binding methods. The results obtained using two different cell lines were in good agreement with results obtained with Scatchard analysis and kinetic binding experiments. An alternative one-step method for determination of parameters describing binding of antibodies to antigens on cells has been developed. The method gives reliable estimates of affinity and number of antigens and in addition gives information on the kinetics of binding.

Administered activity and metastatic cure probability during radioimmunotherapy of ovarian cancer in nude mice with 211At-MX35 F(ab′)2

To elucidate the therapeutic efficacy of alpha-radioimmunotherapy of ovarian cancer in mice. This study: (i) estimated the minimum required activity (MRA), giving a reasonable high therapeutic efficacy; and (ii) calculated the specific energy to tumor cell nuclei and the metastatic cure probability (MCP) using various assumptions regarding monoclonal-antibody (mAb) distribution in measured tumors. The study was performed using the alpha-particle emitter Astatine-211 (211At) labeled to the mAb MX35 F(ab')2. Animals were inoculated intraperitoneally with approximately 1 x 10(7) cells of the cell line NIH:OVCAR-3. Four weeks later animals were treated with 25, 50, 100, or 200 kBq 211At-MX35 F(ab')2 (n = 74). Another group of animals was treated with a nonspecific mAb: 100 kBq 211At-Rituximab F(ab')2 (n = 18). Eight weeks after treatment the animals were sacrificed and presence of macro- and microscopic tumors and ascites was determined. An MCP model was developed and compared with the experimentally determined tumor-free fraction (TFF). When treatment was given 4 weeks after cell inoculation, the TFFs were 25%, 22%, 50%, and 61% after treatment with 25, 50, 100, or 200 kBq (211)At-MX35 F(ab')2, respectively, the specific energy to irradiated cell nuclei varying between approximately 2 and approximately 400 Gy. As a significant increase in the therapeutic efficacy was observed between the activity levels of 50 and 100 kBq (TFF increase from 22% to 50%), the conclusion was that the MRA is approximately 100 kBq (211)At-MX35 F(ab')2. MCP was most consistent with the TFF when assuming a diffusion depth of 30 mum of the mAbs in the tumors.

PDGF and microvessel wall remodeling in adult rat lung: imaging PDGF-AA and PDGF-Rα molecules in progenitor smooth muscle cells developing in experimental pulmonary hypertension

Smooth muscle cells are mostly absent from the walls of microvessels in the adult lung but develop in large numbers as part of the pathology of human and experimental pulmonary hypertensions (PHs). We have previously shown, in an in vivo model of experimental PH, that mesenchymal (interstitial) fibroblasts and intermediate cells are the progenitors of these cells. Although smooth muscle cell development is a defining pathophysiological feature of human PH, little is known about the angiogenic signaling molecules responsible. Here, we report data for platelet-derived growth factor AA (PDGF-AA) and PDGF-Ralpha, two components of an important signaling pathway for fibroblast and myofibroblast proliferation and migration. Using antibodies linked to protein-A gold and high-resolution imaging techniques, we analyzed the expression of these molecules as smooth muscle cells developed from progenitor cell populations and in endothelial cells of the same microvessels. PDGF-AA was highly expressed by each cell type in control lung. As PH developed, the number of antigenic sites for PDGF-AA decreased with time. PDGF-Ralpha expression levels in the control lung were low, relative to the ligand, and fell in PH. These data show, for the first time, a marked phenotypic shift in expression levels of the PDGF-AA isoform and its receptor tyrosine kinase in the progenitor smooth muscle cells developing in the microvessels of the adult hypertensive lung.

Pharmacokinetics and concentration–effect relationships of therapeutic monoclonal antibodies and fusion proteins

Although monoclonal antibodies (mAbs) constitute a major advance in therapeutics, their pharmacokinetic (PK) and pharmacodynamic (PD) properties are not fully understood. Saturable mechanisms are thought to occur in distribution and elimination of mAbs, which are protected from degradation by the Brambell’s receptor (FcRn). The binding of mAbs to their target antigen explains part of their nonlinear PK and PD properties. The interindividual variability in mAb PK can be explained by several factors, including immune response against the biodrug and differences in the number of antigenic sites. The concentration–effect relationships of mAbs are complex and dependent on their mechanism of action. Interindividual differences in mAb PD can be explained by factors such as genetics and clinical status. PK and concentration–effect studies are necessary to design optimal dosing regimens. Because of their above-mentioned characteristics, the interindividual variability in their dose–response relationships must be studied by PK–PD modelling.

Presence of immunoglobulin M antibodies around the glomerular capillaries and in the mesangium of normal and passive Heymann nephritis rats.

Summary Diffuse distribution of small, faintly staining, beaded deposits of rat immunoglobulin M (IgM) around the glomerular capillary blood vessels, and a more intensely staining larger deposition in the mesangium, were observed on the kidney sections of normal rats. As glomerular-fixed nephritogenic antigens are known to be present on the epithelial aspect of the glomerular basement membrane (GBM), especially at the soles of foot processes and at the slit pores, it was assumed that the IgM antibodies were directed against these antigens. Investigation by immunofluorescent antibody double-staining techniques of rat kidney sections obtained from normal and rabbit anti-FX1A-injected rats stained for the nephritogenic antigen showed that a number of antigenic sites in the glomeruli and in the mesangium shared antibody hits by heterologous rabbit IgG and autologous rat IgM antibodies. Most sites in the glomeruli stained specifically for rat IgM or rabbit IgG, but preferentially for the latter. The intensely fluorescent mesangial deposits stained mainly for rat IgM, indicating that at these sites the antigenic material was virtually saturated, while areas at the entry to the mesangial space also stained for rabbit IgG, indicating that at these locations free nephritogenic epitopes were still available for reaction with the anti-FX1A antibody. Western blot analysis have shown that the rabbit anti-rat FX1A IgG and the rat anti-rat KF3 IgM antibodies are directed against the same renal tubular-derived antigen with a molecular weight of 70,000. These experimental findings collectively demonstrate that the heterologous IgG and autologous IgM antibodies are directed against the same nephritogenic antigen, which is found in the glomeruli, the mesangium and the proximal convoluted tubules. Thus, the IgM autoantibody has a possible physiological role but, in addition, there is evidence of active immunophagocytic events, manifested in a rapid and continuous entrapment and expulsion of macromolecules after their processing by the mesangial cells of normal and passive Heymann nephritis rats.

Measurement of anti-D intrinsic affinity with unlabeled antibodies.

The method most commonly used for measuring the affinity of MoAbs specific for the D antigen is a binding assay using 125I-labeled MoAb. Although the method is relatively simple, there are several drawbacks that can lead to inaccurate results. The objective of the present work was to develop a method for the determination of the affinity of anti-D MoAb using unlabelled antibodies. Rh-positive RBCs were sensitized with varying amounts of unlabeled anti-D MoAb, and, at equilibrium, the amount of anti-D bound to the RBCs was measured by ELISA. The affinity and the number of antigenic sites were determined with the Scatchard and Langmuir equations. The method was applied to determine the affinity of several MoAbs specific for the D antigen on RBCs of different Rh-positive phenotypes. Similar results were observed with both equations. The affinity constant (Ka) for 3 MoAbs specific for the D antigen on group O R1r RBCs ranged from 1.3 to 7.4 108 M-1, depending on the antibody. When measured by a binding assay with 125I-labeled anti-D MoAbs, the Ka were significantly lower, indicating that 125I-labeling diminishes anti-D affinity, even when the labeling is at a low level. A simple ELISA method was developed for the measurement of the affinity of anti-D MoAbs for the D antigen and for the number of antigenic sites per RBC. As a result, the affinity of anti-D can be estimated accurately, thus avoiding the drawbacks inherent in modification of the antibody by 125I-labeling.

Rh-prophylaxis in early abortion

Rh-prophylaxis in early abortion

Ultrastructural distribution of the S100A1 Ca2+-binding protein in the human heart

Impaired calcium homeostasis and altered expression of Ca2+-binding proteins are associated with cardiomyopathies, myocardial hypertrophy, infarction or ischemia. S100A1 protein with its modulatory effect on different target proteins has been proposed as one of potential candidates which could participate in these pathological processes. The exact localization of S100A1 in human heart cells on the ultrastructural level accompanied with biochemical determination of its target proteins may help clarify the role of S100A1 in heart muscle. In the present study the distribution of the S100A1 protein using postembedding (Lowicryl K4M) immunocytochemical method in human heart muscle has been determined quantitatively, relating number of antigen sites to the unit area of a respective structural component. S100A1 antigen sites have been detected in elements of sarcoplasmic reticulum (SR), in myofibrils at all levels of sarcomere and in mitochondria, the density of immunolabeling at Z-lines being about 3 times and at SR more than 5 times higher than immunolabeling of remaining structural components. The presence of the S100A1 in SR and myofibrils may be related to the known target proteins for S100A1 at these sites.

Immune Recognition of Swine Vesicular Disease Virus Structural Proteins: Novel Antigenic Regions That Are Not Exposed in the Capsid

Swine vesicular disease virus (SVDV) is an enterovirus of the Picornaviridae family that belongs to the coxsackievirus B group. A number of antigenic sites have been identified in SVDV by analysis of neutralizing monoclonal antibody-resistant mutants and shown to be exposed on the surface of the capsid. In this paper we have identified seven new immunodominant antigenic regions in SVDV capsid proteins by a peptide scanning method, using a panel of sera from infected pigs. When these antigenic regions were located in the capsid by using a computer-generated three-dimensional model of the virion, one was readily exposed on the surface of the virus and the remaining sites were located facing the inner side of the capsid shell, at subunit contacts, or in the interior of the subunit structure.

Modification of HLA expression on peripheral lymphocytes and monocytes during ageing

Immunosenescence involves modifications of humoral and cellular immunity. Here we report the analysis of human leukocyte antigen (HLA) expression on T lymphocytes, B lymphocytes and monocytes of 58 healthy subjects aged 23–95 years old. Using a double staining immunofluorescence and flow cytometry analysis, we have determined the percentages of cells expressing HLA class-I and HLA-DR antigens. The number of antigenic sites expressed per cell were evaluated for HLA-ABCw, HLA-A, HLA-B, HLA-DR locus with a flow cytometry quantification technique. With advancing age, we observed: (i) a significant decrease of the percentage of T cells and B cells expressing HLA-A products; (ii) a decrease of the number of HLA class-I antigenic sites expressed per cell on the three populations tested, predominantly on B cells and in a locus-dependent fashion; (iii) a decrease of the number of HLA-DR molecules expressed per T cell, although the percentage of T cells expressing DR products was increased; (iv) a significant diminution of the percentage of B cells expressing HLA-DR molecules, without changes of the number of HLA-DR antigenic sites per cells. These changes in HLA expression with increasing age could contribute to the decreased level of immunologic responsiveness observed with ageing and contribute to the modification of antigen recognition.

Lipid matrix-based vaccines for mucosal and systemic immunization.

For more than a decade our laboratories have been combining concepts in biochemistry, virology, and immunology in order to develop a conceptual basis for vaccine design. Our long-term goals have been to construct simple and well-defined immunogens that would stimulate specific immune responses in vivo. Using this approach, we hypothesized that it should be possible to define the structural and biochemical parameters of an immunogen that are necessary and sufficient to stimulate designated effector arms of the immune system. Through the use of covalently coupled peptide complexes, we have been able to define minimal requirements for the induction of humoral immune responses (Mannino et al., 1992). This represents a significant advance in eliciting an immune response to peptides, because it requires only peptides and phospholipids in the absence of additional adjuvants. It is different from the previous use of peptides and liposomes since here the peptides are covalently linked to a hydrophobic anchor and integrated into the phospholipid complex, rather than passively adsorbed or encapsulated. The presentation of peptide as part of a peptide-phospholipid complex (in contrast to encapsulation or nonspecific absorption) may be more similar to the natural presentation of an epitope in the context of an in vivo antigenic challenge. This technology also allows us to incorporate B and Th epitopes in a number of forms--as a single peptide, as two peptides in the same liposome, or as a peptide with viral glycoproteins in the same liposome. These data also demonstrate that Th epitopes do not have to be covalently linked to the B-cell epitope in order to provide help for that epitope. The implications of the data reported here are significant for both basic science and applied technologies. In basic science, the peptide-phospholipid complexes are potentially useful for analyzing the cooperative effects of B- and T-cell epitopes in the in vivo immune response. Since the peptide-phospholipid complexes are totally synthetic and highly immunogenic, they may be constructed in any formulation required to answer questions on the roles of B and T cells in promoting an immune response. Furthermore, since the number of antigenic sites is limited only by the number of peptides included in the peptide-phospholipid complexes, these constructs may be useful in producing antisera or monoclonal antibodies to weakly antigenic regions of a large protein, since the lack of antigenic competition should enhance the immunogenicity of these regions. Clinically, this technology will expand the potential for subunit vaccines.(ABSTRACT TRUNCATED AT 400 WORDS)

Standardization of the Flow Cytometric Determination of HLA Class I Antigens, 'Platelet-Specific' Glycoproteins and Activation Markers

Flow cytometry (FC) provides a reproducible investigation of cell surface antigens on platelets. The aim of this study was to elaborate appropriate protocols and to compare them with other techniques that have already been published. (1) Venipuncture with tubes containing citrate was better for the preservation of the antigenicity than using ACD tubes. The isolated platelets could not be completely distinguished from detritus and protein aggregates. Therefore a platelet concentration between 10^7 and 10^8/ml measurement buffer was necessary to obtain a sufficient resolution by FC. (2) Isolation methods using either differential centrifugation or diluted Ficoll- Hypaque as a flotation medium provided platelets of equal purity. The method with Ficoll- Hypaque resulted in a higher number of isolated platelets than differential centrifugation. The demonstration of platelets and their antigens in whole blood without isolation gave good results provided the platelets were not activated. Activation of platelets with 1 NIH-U thrombin/1 resulted in the loss of a part of the highly activated platelets because of their aggregation. (3) Comparing different concentrations of paraformaldehyde in PBS, fixation with 1% for 15 min provided the best antigen preservation for most of the antigens investigated. Isolation induced platelet activation. In order to avoid this effect, the whole anticoagulated blood was fixed with 1% paraformaldehyde for 15 min immediately after venipuncture. Then the platelets were isolated using diluted Ficoll-Hypaque. In this way, systemic activation of platelets can be detected with antibodies against glycoproteins which are translocated from the a-granules or lysosomes to the cell membrane. These activation markers can be determined on immediately fixed platelets (already in the whole blood) without any interference due to unspecific activation caused by the isolation procedure. (4) Platelet treatment with citric acid at pH 3, in order to remove the antigenicity of HLA-class I molecules, was sensitive to immediate fixation with paraformaldehyde in the whole blood. Fixation after isolating the platelets made it possible to demonstrate antigen stripping, and the free heavy chain, devoid of the β(2)-microglobulin, could be clearly demonstrated. (5) Using standardization beads, the average number of antigenic sites per platelet could be determined for the investigated specificities. It was shown that antibodies which have been directly conjugated or biotinylated and combined with streptavidin-phycoerythrin yielded similar results in terms of the number of antigenic binding sites while unconjugated antibodies in combination with FITC-conjugated anti-mouse-IgG led to overestimation of antigenic binding sites.

Ultrastructural and immunocytochemical study of skin fibroblasts from normal and sialidosis patients.

The objectives of this study were to analyze morphologically, morphometrically and immunocytochemically the lysosomal compartment of normal fibroblasts and of fibroblasts with neuraminidase deficiency. The immunocytochemical analyses consisted of quantifying the distribution of saposins and beta-galactosidase in the lysosomes of these cells to test the hypothesis that neuraminidase deficiency is associated with an impairment in the transport of these proteins to the lysosomal compartment. To test this idea, cultured skin fibroblasts of patients with or without sialidosis were prepared for electron microscopy and probed with antibodies against lysosomal beta-galactosidase and lysosomal saposins. The lysosomes of the affected cells had an abnormal accumulation of incompletely digested membranes which was associated with a significant lowering in the density of antigenic sites per lysosome. However, due to a significant increase in the number of lysosomes per affected cell, the total number of antigenic sites in control and neuraminidase deficient cells was similar. This presumably compensatory effect indicates that although the rate of production of beta-galactosidase and saposins remains unchanged, the transport of these molecules to the lysosomes is somehow affected. Our data also indicate that in the fibroblasts, lysosomes require a normal concentration of the three enzymes to maintain neuraminidase activity and sphingolipid degradation.

Optimal visualization of immunogold-silver staining of hepatic PEPCK with epipolarized light microscopy.

We used immunogold-silver staining to localize phosphoenolpyruvate carboxykinase in 10 microns cryosections of 4% paraformaldehyde perfusion-fixed normal male rat liver. The resolution and sensitivity of detection were improved by epipolarized light microscopy of 0.5 microns semi-thin plastic sections prepared from these pre-embedding immunogold-silver-enhanced 10-microns thick cryosections. Epipolarized light combined with transmitted light simultaneously demonstrated antigenic sites (visualized with epipolarized light illumination) and tissue morphology (revealed by transmitted light). To optimize the conditions for high resolution, an oil immersion objective lens (x 100) with adjustable iris diaphragm was used with different intensity settings for both light sources. Our observations indicate that if the intensity of the transmitted light is too high, the visibility of the gold-cored silver grains by epipolarized illumination is decreased; if the intensity of epipolarized light is too strong, haloes appear around the gold-cored silver particles. By adjusting the aperture in the objective lens and the neutral density filter in the transmitted light pathway to balance the intensities of transmitted and epipolarized light, an optimal image is obtained that shows the maximal number of antigenic sites and excellent morphology.

Imaging and dosimetry determinations using radiolabeled antibodies.

Numerous studies using radiolabeled antibodies for imaging and therapy of lymphoma have been reported (Table 4). The targeting of lymphoma associated antigens with MoAb appears to be more favorable than the targeting of antigens on epithelial tumor. Antigen abundance may not be the overriding factor in this favorable targeting, since the number of antigenic sites per cell are often in the same range or lower than those targeted in epithelial tumors. This improved targeting is likely related to the greater access of antibody to the target antigen in lymph nodes, bone marrow, circulation, and other sites. With certain antibodies, trafficking of the cells targeted with the radiolabeled antibody may also result in favorable localization [19]. While the most frequently used isotope for imaging and therapy has been 131I, certain limitations have been observed, including its high-energy gamma rays and resulting lower resolution, and the frequent occurrence of dehalogenation [21,25,98]. Many of the antigens expressed by lymphomas undergo antigenic modulation. Antigens that undergo modulation may be targeted successfully, but once modulation occurs the antibody is broken down and the iodine is rapidly excreted from the cells. While this rapid release from normal organs is an advantage, it is an undesirable event at the tumor site. In contrast to the case of 131I MoAb, modulation may be an advantage for targeting with 111In labeled antibodies, since the radioactive metals are retained for longer periods at the tumor sites; even if the antibody is broken down, the 111In is not easily excreted from the cells [52]. Among the most consistent and favorable targeting observed to date is that seen with 111In T101 in CTCL. These studies have shown concentration of 111In in tumor of 10-100 times that seen in other tumor systems using iodinated antibodies. Unfortunately no studies have followed this lead and performed the necessary comparisons between 111In and 131I MoAb to determine if this is a consistent finding. The use of 99mTc labeled MoAb for imaging lymphomas is in its infancy, although preliminary reports appear promising [71]. While in epithelial tumors preferential tumor targeting may take more than 48 hours in lymphomas, targeting is usually seen within the first 24 hours, which is within the window of imaging time for 99mTc. Therefore, further evaluation of 99mTc antibodies should be performed. Determination of the optimum dose of antibody for imaging has been attempted. Studies using various anti-lymphoma directed antibodies have shown widely varying biodistribution and variable dose-response curves.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative study of chimpanzee and gorilla counterparts of the human D antigen

The reactivities of three human anti-D monoclonal antibodies (mAbs) with human, chimpanzee, and gorilla red blood cells (RBCs) were compared by quantitative radioimmunology and indirect immunofluorescence methods. The number of antigenic sites varies widely in gorillas (from 48,000-283,000), while in chimpanzees this number is very close to that observed in human R1R2 RBCs. The affinity of the anti-D antibodies was slightly lower with ape RBCs than with D-positive human RBCs. In chimpanzee, the D-like epitopes recognition is enhanced by papain while the gorilla D-like epitopes are destroyed by enzyme treatment.

Flow‐Cytometric Analysis of Erythrocytic Blood Group A Antigen Density Profile

Blood group A antigen density on red blood cells (RBC) was studied using flow cytometry (FCM) and fluoresceinated polyclonal and monoclonal IgG anti-A antisera. Agglutination was a problem, which could only be solved by prefixation of the RBC with glutaraldehyde or formaldehyde. However, this treatment resulted in a significant reduction of the number of antigen sites as compared to the native (i.e. nonfixed) RBC. Two major new findings came out of this study: (1) A antigen density on native RBC seems to be higher than previously recognized, and (2) A antigen density distribution is probably non-Gaussian. The absolute number of A antigen sites was determined, using a human polyclonal IgG antiserum and commercially available absolute fluorescence standards. The site numbers on fixed RBC were comparable to those found by earlier radioimmunological studies (x 10(6)/RBC): A1, 1.07 +/- 0.28; A2, 0.21 +/- 0.09; A1B, 0.79 +/- 0.26 sites (mean +/- SD). The values found for native RBC were considerably higher (x 10(6)/RBC): A1, 2.86 +/- 0.95; A2, 0.47 +/- 0.29; A1B, 1.98 +/- 0.58 sites (mean +/- SD). With the 1 monoclonal and the 3 polyclonal antisera used in this study, and in contrast to Rh D, the erythrocytic A antigen density distribution of a given sample is highly asymmetrical. This non-Gaussian distribution profile does not seem to be affected by such factors as antibody heterogeneity, variability in antibody fluoresceination range, RBC density and reticulocyte content. This suggests that the asymmetrical A antigen distribution may be an intrinsic property of the RBC population.

Antigenicity of rabies virus glycoprotein

Although the number of antigenic sites on the rabies virus glycoprotein that have been described regularly increases with time, no attempt has been made to carefully evaluate the relative importance of each of these sites. Here we provide a more precise description of the antigenicity of the protein in mice of the H-2d haplotype; we developed this description by using 264 newly isolated monoclonal antibodies (MAbs) and a collection of neutralization-resistant (MAR) mutants. Most of the MAbs (97%) recognized antigenic sites previously described as II and III. One minor antigenic site separated from site III by three amino acids, including a proline, was identified (minor site a). Despite their proximity, there is no overlap between site III and minor site a; i.e., site III-specific MAR mutants were neutralized by the six MAbs defining minor site a, and vice versa. One of our MAbs, 1D1, reacted with sodium dodecyl sulfate-treated glycoprotein in Western blots (immunoblots) under reducing conditions and was therefore probably directed against a linear epitope, A MAR mutant selected with this MAb was still neutralized by MAbs of other specificities. This linear epitope was called G1 (G, Gif). As a general rule, we propose to reserve the term "antigenic site" (either major or minor) for regions of the protein which are defined by several MAbs originating from different fusions and to describe regions of the protein which are defined by a single MAb as epitopes. It would be interesting to test whether the same regions of the rabies virus glycoprotein are antigenic in mice of different haplotypes or in other species.

Immunodominance is altered in T cell receptor (beta-chain) transgenic mice without the generation of a hole in the repertoire.

Despite the tremendous plasticity of the TCR repertoire, T cells recognize a limited number of antigenic sites (frequently a single site, or immunodominant epitope) on a complex protein Ag. Current models suggest that the immunodominant epitope of a complex protein is the processed peptide that binds to the MHC molecule with the highest affinity. Conversely, the inability of the T cell population to recognize a specific epitope, termed a "hole" in the repertoire, can prevent the immunodominance of a peptide despite efficient processing and MHC binding of the peptide. The role of specific TCR alpha- or beta-chains in determining MHC restriction and recognizing specific epitopes is complex and incompletely understood. To evaluate the contribution of each TCR chain to the functional diversity of the T cell repertoire, we investigated in vivo the T cell response to phage lambda-repressor protein in transgenic mice expressing a single rearranged beta-chain gene (C57L beta mice) in association with the complete germline alpha-chain repertoire. Our results demonstrate that expression of the TCR beta-chain transgene alters the immunodominant epitope recognized by T cells. However, after immunization with the appropriate peptide the transgenic mice can also respond to the nonimmunodominant epitope; thus, the expression of the TCR beta-chain transgene does not create a hole in the repertoire. These data indicate that the primary site, or immunodominant epitope, of an Ag recognized by T cells can be altered by the preimmune TCR repertoire independent of antigen processing and MHC affinity.