In previous studies, our lab has characterized a number of highly mutated antibodies against structural epitopes of the human immunodeficiency virus (HIV) envelope protein. These antibodies were first isolated from long-term nonprogressors (LTNPs). We have previous mapped 6F5 to a novel structural epitope that encompasses areas in both heptad repeats of GP41, mapping to amino acids of 557, 654 and 657 of reference sequence HXB2. In these studies, three other antibodies that were <90% homologous to 6F5 also resolved amino acid 657. On sequence analysis, 6F5 and its relatives had the same gene usage and general structure. These similarities and the similar epitope mapping implied these were once distantly related to a single B-cell lineage. As fusion of the viral membrane to the target cell depends on these heptad repeat regions associating and forming a six-helix postfusion bundle, antibodies that can interfere in this may be highly useful. See results. Because 6F5 maps to 557 and 654/657 which are widely separated on the primary sequence, we explored if there was differential binding to the postfusion six-helix-bundle form. Two peptides (N36 and C34) each containing one of the heptad repeats can form the post-fusion six-helix-bundle in vitro. On sandwich ELISA testing, 6F11 and 7B6 did not bind any form. Interestingly, 4E4 specifically captured both peptides alone, but not the six-helix-bundle and 6F5 only bound the six-helix-bundle but not the other peptide. A small number of samples were obtained to assess the prevalence of these responses in LTNPs. Antibodies that compete 6F11 are much more prevalent in LTNPs than normal progressors (75% vs. 20%). Functionally, we found that despite being mapped to a similar portion of Gp41 (657), only 6F5 is shown to have significant ADCC activity, however relative 6F11 does not. If targeting these epitopes correlates with the LTNP state, then these sites may be highly significant as targets of therapeutics or in vaccine strategies. Further studies on a larger cohort of LTNPs are ongoing. Additionally, deep sequencing of antibody sequences are being done to explore the development of structural epitope targeting by this family of antibodies. All authors: No reported disclosures.
During HIV infection, fusion of the viral and cellular membranes is dependent on folding of the gp41 trimer into a six-helix bundle. Fusion inhibitors, such as the antiretroviral Enfuvirtide (T20), interfere with the formation of the gp41 six-helix bundle. Recent in vitro studies reveal that the gp41 immunodominant region one targeting antibody 3D6 can block T20 interference, but the clinical and pathophysiologic significance of this finding is unclear.We have previously characterized a number of antibodies that target conformational epitopes on gp41and herein characterized their ability to interfere with T20 in multiple assays and assess their prevalence in HIV infected subjects.The T20 interference by antibody 3D6 was confirmed in a CHO-HXB2 envelope/ HeLaT4+ cell culture assay. Antibodies that target an immunodominant region one epitope, as well as a gp41 discontinuous epitope, also interfered in this assay, however, not all antibodies that targeted these epitopes showed T20 interference. This response was not due to the direct binding of T20 by the antibodies and could not be replicated utilizing TZM-bl and HL2/3 cells. Notably, serum competition studies on a panel of HIV subjects demonstrate that these conformational targeting antibodies are common in the HIV population.The relatively common nature of antibodies targeting these epitopes, the disparate in vitro results, and lack of reported clinical failures ascribed to such antibodies leads us to conclude that antibody interference of T20 is likely not clinically relevant. However, this warrants continued consideration with the advancement of other fusion inhibitors.
Abstract Kawasaki Disease (KD) is a childhood vasculitis, marked by prolonged fevers and coronary artery inflammation/aneurysms in near one quarter of those untreated. The cause remains unknown; however, epidemiology and demographic data support a single preceding infectious agent may lead to KD. Plasmablasts (PBs) are a stage of transitional B-cells that lead to plasma cells, the long-lived antibody producing cells of the bone marrow. After initial infection, peripherally circulating PB populations are enriched for cells with antibodies against the preceding infection. We have recently published data showing children with KD have similar PB responses to children with infections. We sought to define the antibody characteristics, including clonality, of these PBs during KD. We used antibody repertoire next-generation sequencing to characterize memory and PB populations. Additionally, pairing of heavy and light chains was performed with Chromium Single Cell Gene Expression (10x Genomics, Pleasanton, CA) using the Human B cell Single Cell V(D)J Enrichment Kit. From subject 24, antibody sequences using VH4-34 and a 19 amino acid length complementarity determining region 3 showed a massive expansion between day 4 and 6 of fever. Chromium single cell sequencing produced over 946 heavy and light chain paired sequences. Sequence comparison showed 40% of sequences demonstrated markers of clonal expansion, which represented 100 clonal groups. One clonal group (24-01) reflected the massive clonal expansion (VH4-34, CDR3 19) previously shown. Conclusions This clonal expansion within plasmablast populations supports that Kawasaki disease is caused by an infection. Antigen targeting of these monoclonal antibodies is currently being explored.
Previous studies in our laboratory characterized a panel of highly mutated HIV-specific conformational epitope-targeting antibodies (Abs) from a panel of HIV-infected long-term nonprogressors (LTNPs). Despite binding HIV envelope protein and having a high number of somatic amino acid mutations, these Abs had poor neutralizing activity. Because of the evidence of antigen-driven selection and the long CDR3 region (21 amino acids [aa]), we further characterized the epitope targeting of monoclonal Ab (MAb) 76-Q3-2C6 (2C6). We confirmed that 2C6 binds preferentially to trimeric envelope and recognizes the clades A, B, and C SOSIP trimers. 2C6 binds gp140 constructs of clades A, B, C, and D, suggesting a conserved binding site that we localized to the ectodomain of gp41. Ab competition with MAb 50-69 suggested this epitope localizes near aa 579 to 613 (referenced to HXB2 gp160). Peptide library scanning showed consistent binding in this region but to only a single peptide. Lack of overlapping peptide binding supported a nonlinear epitope structure. The significance of this site is supported by 2C6 having Ab-dependent cell cytotoxicity (ADCC) against envelope proteins from two clades. Using 2C6 and variants, alanine scanning mutagenesis identified three amino acids (aa 592, 595, and 596) in the overlapping region of the previously identified peptide. Additional amino acids at sites 524 and 579 were also identified, helping explain its conformational requirement. The fact that different amino acids were included in the epitope depending on the targeted protein supports the conclusion that 2C6 targets a native conformational epitope. When we mapped these amino acids on the trimerized structure, they spanned across oligomers, supporting the notion that the epitope targeted by 2C6 lies in a recessed pocket between two gp41 oligomers. A complete understanding of the epitope specificity of ADCC-mediating Abs is essential for developing effective immunization strategies that optimize protection by these Abs.IMPORTANCE This paper further defines the function and area of the HIV trimeric envelope protein targeted by the monoclonal antibody 2C6. 2C6 binding is influenced by amino acid mutations across two separate gp41 sections of the envelope trimer. This epitope is recognized on multiple clades (variant groups of circulating viruses) of gp41, gp140 trimers, and SOSIP trimers. For the clades tested, 2C6 has robust ADCC. As the target of 2C6 is available in the major clades of HIV and has robust ADCC activity, further definition and appreciation of targeting of antibodies similar to 2C6 during vaccine development should be considered.
Abstract Kawasaki disease (KD), the leading cause of acquired heart disease in children, primarily affects infants and toddlers. Future studies on KD immune responses are hampered by a limited understanding of normal immune responses in these ages. Plasmablasts are a transitional form of B cells that lead to long-term plasma cells, and their levels rise in the peripheral blood after exposure to a foreign antigen. In adults, these responses are both temporally and functionally well characterized. To date, there have been few studies on plasmablasts in the predominant age range of KD. This study is focused on characterizing the normal plasmablast responses in the predominant age range of KD presentation. Children presenting to an urban pediatric emergency room who had fever and symptoms overlapping those of KD, were recruited. Peripheral blood mononuclear cells were isolated and evaluated utilizing flow cytometry with specific B cell markers from 18 KD subjects and 69 febrile controls. Plasmablast numbers and timing of response are similar between infectious disease control and KD subjects. In both groups, infants have diminished plasmablast responses compared to older children. Using deep sequencing, immunoglobluin variable gene usage and sequences will be compared between infants and older children. In this single-time point survey, we demonstrate that infants have a blunted peripheral plasmablast response. Overall, similar plasmablast responses in KD and controls support an infectious disease relationship to KD. Future time-course studies of plasmablasts in infants are warranted as this phenomenon may contribute to observed poor serum immune responses and maintenance of such responses in this age group.
AIMS--To produce and characterise a monoclonal antibody specific for O-acetylated sialomucin and to assess its use in immunohistochemistry on a panel of normal and diseased intestinal tissue samples. METHODS--Mouse monoclonal antibodies were developed following immunisation with highly purified human colonic mucin. One of these (MMM-17) showed strong binding to mucin throughout the normal colon with relative lack of binding to colon cancer tissue. The binding epitope of MMM-17 was then characterised by screening for agglutination activity against a panel of human and animal erythrocytes and by assessment of its binding to a range of normal and chemically treated slot blotted mucins. Further immunohistochemical studies were then performed on formalin fixed, normal, and diseased human intestinal samples. RESULTS--Binding of MMM-17 to slot blotted human colonic mucin was reduced by 38 (SD 14%) (n = 4) by alkali treatment of the mucin, sequential alkali and sialidase treatment completely abolished binding. Sialidase treatment alone, however, caused only an 11 (11%) reduction in binding. MMM-17 failed to agglutinate any human, rabbit, rat or mouse erythrocytes. These findings were compatible with specificity of MMM-17 for sialomucins O-acetylated at the C-7 or C-8 positions on the sialic acid. Strong staining by MMM-17 was found in all goblet cells throughout all 40 normal colonic and rectal samples studied, but staining was absent in seven of 13 colorectal carcinomas. Normal duodenum (n = 16) and normal ileum (n = 3) all showed occasional positive goblet cells. The normal gastric antral mucosa was generally negative B MMM-17, but in all of 15 cases of gastritis with intestinal metaplasia the metaplastic glands were strongly positive for MMM-17. CONCLUSION--Monoclonal antibody MMM-17 has specificity for O-acetylated sialomucins and its binding depends both on the position of O-acetylation and on the adjacent oligosaccharide structure. Preliminary studies using the antibody on archival tissue samples support the previous reports of reduced O-acetylation in colon cancer demonstrated by indirect histochemistry and show the neo-formation of O-acetylated sialomucin in intestinal metaplasia in the stomach.
CAM 17.1 is an antimucin monoclonal antibody which has recently been proven valuable as a reagent for serological diagnosis of pancreatic cancer. A series of studies have been performed to characterise its epitope. First it was screened immunohistochemically against a wide range of formalin-fixed normal and neoplastic human tissues and showed widespread binding to mucin throughout the gastro-intestinal tract, in both normal and malignant tissues. In pancreas, strong intracellular staining of acinar and ductal cells was found in normal tissue and in carcinoma cells in tumours. Normal stomach showed only weak staining (n = 6), but gastritis with metaplasia showed strong staining (n = 4). Staining of colonic mucosa from patients of known Lewis phenotype showed Le(a+b–) (7/8) and Le(a–b+) (4/6) samples to be positive, but not Le(a–b–) (0/3) samples. CAM 17.1 agglutinated all donor erythrocytes tested at 4°C regardless of blood group, whereas cord blood red cells were not agglutinated. Since I antigen is the only antigen known to be present on all adult red blood cells but absent from cord blood, this suggests probable involvement of this antigen in the binding site. The agglutination was abolished by sialidase treatment of the red cells and immunoblotting with slot-blotted mucin showed that binding was both acid and sialidase sensitive indicating the involvement of sialic acid in the binding site. These studies show that CAM 17.1 binds to a sialic-acid-containing determinant of mucin, probably sialyl-I, which epitope shows wide distribution throughout the gastro-intestinal tract.
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