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Characterization of structurally defined epitopes recognized by monoclonal antibodies produced by chronic lymphocytic leukemia B cells.

ABSTRACT: Despite a wealth of information about the structure of surface membrane immunoglobulin (smIg) on chronic lymphocytic leukemia (CLL) cells, little is known about epitopes reacting with their binding sites. Probing phage-displayed peptide libraries, we identified and characterized mimetopes for Igs of 4 patients with IGHV mutated CLL (M-CLL) and 4 with IGHV unmutated CLL (U-CLL). Six of these mAbs were representatives of stereotyped B-cell receptors characteristic of CLL. We found that mimetic epitopes for U- and M-CLL Igs differed significantly. M-CLL-derived peptides exhibited better amino acid motifs, were more similar to each other, aligned more easily, and formed tighter clusters than U-CLL-derived peptides. Mono-, oligo-, and polyreactivity of peptides correlated with structural changes within antigen-binding sites of selecting M-CLL mAbs. Although M-CLL-isolated peptides and certain U-CLL mAbs bound more effectively to the selecting mAb, others were not as specific, reacting with M-CLL and U-CLL mAbs; these data suggest that in vivo structurally diverse epitopes could bind smIgs of distinct CLL clones, thereby altering survival and growth. Finally, an M-CLL-derived peptide inhibited, in a dose-dependent manner, binding of its homologous mAb to human B lymphocytes; therefore peptides that inhibit or alter the consequences of antigen-smIg interactions may represent therapeutic modalities in CLL.

SUBMITTER: Seiler T

PROVIDER: S-EPMC2858644 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Characterization of structurally defined epitopes recognized by monoclonal antibodies produced by chronic lymphocytic leukemia B cells.

Seiler Till T Woelfle Manuela M Yancopoulos Sophia S Catera Rosa R Li Wentian W Hatzi Katerina K Moreno Carol C Torres Marcela M Paul Santanu S Dohner Hartmut H Stilgenbauer Stephan S Kaufman Matthew S MS Kolitz Jonathan E JE Allen Steven L SL Rai Kanti R KR Chu Charles C CC Chiorazzi Nicholas N

Blood 20090818 17

Despite a wealth of information about the structure of surface membrane immunoglobulin (smIg) on chronic lymphocytic leukemia (CLL) cells, little is known about epitopes reacting with their binding sites. Probing phage-displayed peptide libraries, we identified and characterized mimetopes for Igs of 4 patients with IGHV mutated CLL (M-CLL) and 4 with IGHV unmutated CLL (U-CLL). Six of these mAbs were representatives of stereotyped B-cell receptors characteristic of CLL. We found that mimetic epi ...[more]

PMID: 19690339

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Project description:BackgroundPotato virus Y (PVY, genus Potyvirus) causes substantial economic losses in solanaceous plants. Routine screening for PVY is an essential part of seed potato certification, and serological assays are often used. The commercial, commonly used monoclonal antibodies, MAb1128, MAb1129, and MAb1130, recognize the viral coat protein (CP) of PVY and distinguish PVYN strains from PVYO and PVYC strains, or detect all PVY strains, respectively. However, the minimal epitopes recognized by these antibodies have not been identified.Methodology/principal findingsSPOT peptide array was used to map the epitopes in CP recognized by MAb1128, MAb1129, and MAb1130. Then alanine replacement as well as N- and C-terminal deletion analysis of the identified peptide epitopes was done to determine critical amino acids for antibody recognition and the respective minimal epitopes. The epitopes of all antibodies were located within the 30 N-terminal-most residues. The minimal epitope of MAb1128 was 25NLNKEK30. Replacement of 25N or 27N with alanine weakened the recognition by MAb1128, and replacement of 26L, 29E, or 30K nearly precluded recognition. The minimal epitope for MAb1129 was 16RPEQGSIQSNP26 and the most critical residues for recognition were 22I and 23Q. The epitope of MAb1130 was defined by residues 5IDAGGS10. Mutation of residue 6D abrogated and mutation of 9G strongly reduced recognition of the peptide by MAb1130. Amino acid sequence alignment demonstrated that these epitopes are relatively conserved among PVY strains. Finally, recombinant CPs were produced to demonstrate that mutations in the variable positions of the epitope regions can affect detection with the MAbs.Conclusions/significanceThe epitope data acquired can be compared with data on PVY CP-encoding sequences produced by laboratories worldwide and utilized to monitor how widely the new variants of PVY can be detected with current seed potato certification schemes or during the inspection of imported seed potatoes as conducted with these MAbs.

Project description:Human cytomegalovirus (HCMV) infects 36% to almost 100% of adults and causes severe complications only in immunocompromised individuals. HCMV viral surface trimeric (gH/gL/gO) and pentameric (gH/gL/UL128/UL130/UL131A) complexes play important roles in HCMV infection and tropism. Here, we isolated and identified a total of four neutralizing monoclonal antibodies (MAbs) derived from HCMV-seropositive blood donors. Based on their reactivity to HCMV trimer and pentamer, these MAbs can be divided into two groups. MAbs PC0012, PC0014, and PC0035 in group 1 bind both trimer and pentamer and neutralize CMV by interfering with the postattachment steps of CMV entering into cells. These three antibodies recognize antigenic epitopes clustered in a similar area, which are overlapped by the epitope recognized by the known neutralizing antibody MSL-109. MAb PC0034 in group 2 binds only to pentamer and neutralizes CMV by blocking the binding of pentamer to cells. Epitope mapping using pentamer mutants showed that amino acid T94 of the subunit UL128 and K27 of UL131A on the pentamer are key epitope-associated residues recognized by PC0034. This study provides new evidence and insight information on the importance of the development of the CMV pentamer as a CMV vaccine. In addition, these newly identified potent CMV MAbs can be attractive candidates for development as antibody therapeutics for the prevention and treatment of HCMV infection. IMPORTANCE The majority of the global population is infected with HCMV, but severe complications occur only in immunocompromised individuals. In addition, CMV infection is a major cause of birth defects in newborns. Currently, there are still no approved prophylactic vaccines or therapeutic monoclonal antibodies (MAbs) for clinical use against HCMV infection. This study identified and characterized a panel of four neutralizing MAbs targeting the HCMV pentamer complex with specific aims to identify a key protein(s) and antigenic epitopes in the HCMV pentamer complex. The study also explored the mechanism by which these newly identified antibodies neutralize HCMV in order to design better HCMV vaccines focusing on the pentamer and to provide attractive candidates for the development of effective cocktail therapeutics for the prevention and treatment of HCMV infection.

Project description:Several studies in chronic lymphocytic leukemia (CLL) patients have reported impaired immune cell functions, which contribute to tumor evasion and disease progression. However, studies on CLL-like monoclonal B-cell lymphocytosis (MBL) are scarce. In the study described here, we characterized the immune environment in 62 individuals with clinical MBL, 56 patients with early-stage CLL, and 31 healthy controls. Gene expression arrays and quantitative reverse transcription polymerase chain reaction were performed on RNA from CD4+ peripheral blood cells; serum cytokines were measured with immunoassays; and HLA-DR expression on circulating monocytes, as well as the percentages of Th1, cytotoxic, exhausted, and effector CD4+ T cells, were evaluated by flow cytometry. In addition, cell cultures of clonal B cells and CD14-enriched or -depleted cell fractions were performed. Strikingly, MBL and early-stage CLL differed in pro-inflammatory signatures. An increased inflammatory drive orchestrated mainly by monocytes was identified in MBL, which exhibited enhanced phagocytosis, pattern recognition receptors, interleukin-8 (IL8), HMGB1, and acute response signaling pathways and increased pro-inflammatory cytokines (in particular IL8, interferon γ [IFNγ], and tumor necrosis factor α). This inflammatory signature was diminished in early-stage CLL (reduced IL8 and IFNγ levels, IL8 signaling pathway, and monocytic HLA-DR expression compared with MBL), especially in those patients with mutations in IGHV genes. Additionally, CD4+ T cells of MBL and early-stage CLL exhibited a similar upregulation of Th1 and cytotoxic genes and expanded CXCR3+ and perforin+ CD4+ T cells, as well as PD1+ CD4+ T cells, compared with controls. Cell culture assays disclosed tumor-supporting effects of monocytes similarly observed in MBL and early-stage CLL. These novel findings reveal differences in the inflammatory environment between MBL and CLL, highlighting an active role for antigen stimulation in the very early stages of the disease, potentially related to malignant B-cell transformation.

Dataset Information

Characterization of structurally defined epitopes recognized by monoclonal antibodies produced by chronic lymphocytic leukemia B cells.

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Characterization of structurally defined epitopes recognized by monoclonal antibodies produced by chronic lymphocytic leukemia B cells.

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