Functional Activities and Immunoglobulin Variable Regions of Human and Murine Monoclonal Antibodies Specific for the P1.7 PorA Protein Loop of Neisseria meningitidis

ABSTRACT The meningococcal PorA protein is considered a promising vaccine candidate. Although much is understood regarding the structure of PorA proteins, little is known about the structure-function relationships of PorA antibodies. The aim of this study was to compare the functional and molecular characteristics of a human monoclonal antibody (MAb) and three murine MAbs specific for the PorA P1.7 serosubtype. Murine MAbs 207,B-4 (immunoglobulin G2a [IgG2a]) and MN14C11.6 (IgG2a) were both bactericidal and opsonophagocytic for P1.7-expressing meningococci, whereas human MAb SS269 (IgG3) and murine MAb 208,D-5 (IgA) initiated neither effector function. Epitope mapping with synthetic peptides revealed that MAbs 207,B-4 and 208,D-5 recognized the sequence ASGQ, which is the same specificity motif that a previous study had established for SS269 and MN14C11.6. Nucleotide and amino acid sequence analyses of the variable regions of the four MAbs showed that the SS269 VH region belonged to the VH3 family and was approximately 70% homologous to those of the murine MAbs which were all from the 7183 family, whereas the SS269 VL region belonged to the Vλ1-b family and was less than 40% homologous to those of the murine MAbs which were all members of the Vκ1 family. The Fab fragment of SS269 was cloned and expressed in Escherichia coli and was shown by enzyme-linked immunosorbent assay analyses to bind as well as intact SS269 MAb to P1.7,16 serosubtype group B strain 44/76. We conclude that distinct differences exist in the effector function activities and variable region gene sequences of human and murine P1.7-specific MAbs despite their recognition of similar epitopes.

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Rapid identification of anti-idiotypic mAbs with high affinity and diverse epitopes by rabbit single B-cell sorting-culture and cloning technology

PLoS ONE ◽

10.1371/journal.pone.0244158 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0244158

Author(s):

WeiYu Lin ◽

Wei-Ching Liang ◽

Trung Nguy ◽

Mauricio Maia ◽

Tulika Tyagi ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Sorting ◽

Assay Development ◽

Enzyme Linked Immunosorbent Assay ◽

Fab Fragment ◽

Variable Regions ◽

High Affinity ◽

Clinical Assay ◽

Cloning Technology

The proactive generation of anti-idiotypic antibodies (anti-IDs) against therapeutic antibodies with desirable properties is an important step in pre-clinical and clinical assay development supporting their bioanalytical programs. Here, we describe a robust platform to generate anti-IDs using rabbit single B cell sorting-culture and cloning technology by immunizing rabbits with therapeutic drug Fab fragment and sorting complementarity determining regions (CDRs) specific B cells using designed framework control as a negative gate to exclude non-CDRs-specific B cells. The supernatants of cultured B cells were subsequently screened for binding to drug-molecule by enzyme-linked immunosorbent assay and the positive hits of B cell lysates were selected for cloning of their immunoglobulin G (IgG) variable regions. The recombinant monoclonal anti-IDs generated with this method have high affinity and specificity with broad epitope coverage and different types. The recombinant anti-IDs were available for assay development to support pharmacokinetic (PK) and immunogenicity studies within 12 weeks from the start of rabbit immunization. Using this novel rapid and efficient in-house approach we have generated a large panel of anti-IDs against a series of 11 therapeutic antibody drugs and successfully applied them to the clinical assay development.

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Production of an Anti-Severe Acute Respiratory Syndrome (SARS) Coronavirus Human Monoclonal Antibody Fab Fragment by Using a Combinatorial Immunoglobulin Gene Library Derived from Patients Who Recovered from SARS

Clinical and Vaccine Immunology ◽

10.1128/cvi.13.5.594-597.2006 ◽

2006 ◽

Vol 13 (5) ◽

pp. 594-597 ◽

Cited By ~ 8

Author(s):

Jinye Liu ◽

Hongxia Shao ◽

Yanlin Tao ◽

Bin Yang ◽

Lisheng Qian ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Human Monoclonal Antibody ◽

Antibody Fragments ◽

Spike Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Immunoglobulin Gene ◽

Gene Library ◽

Fab Fragment ◽

Human Antibodies ◽

Infected Cells

ABSTRACT A combinatorial human immunoglobulin gene library was constructed from the peripheral lymphocytes of two patients who recovered from severe acute respiratory syndrome (SARS). The library was screened for the production of Fab antibody fragments to a recombinant spike protein of SARS-associated coronavirus (SARS-CoV). One Fab clone, AS3-3, reacted with the spike protein in an enzyme-linked immunosorbent assay. The dissociation constant of AS3-3 was 1.98 × 10−8 M. Immunofluorescent microscopy revealed that it reacted with SARS-CoV-infected cells. The library seems to be a potent tool for the production of human antibodies to SARS-CoV.

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Autoantibody-associated cross-reactive idiotypes expressed at high frequency in chronic lymphocytic leukemia relative to B-cell lymphomas of follicular center cell origin

Blood ◽

10.1182/blood.v72.2.422.bloodjournal722422 ◽

1988 ◽

Vol 72 (2) ◽

pp. 422-428 ◽

Cited By ~ 1

Author(s):

TJ Kipps ◽

BA Robbins ◽

P Kuster ◽

DA Carson

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Light Chain ◽

Variable Region ◽

Lymphocytic Leukemia ◽

Kappa Light Chain ◽

Region Gene ◽

Variable Regions ◽

Hodgkin's Lymphomas ◽

Center Cell

Using murine monoclonal antibodies (MoAbs) specific for immunoglobulin (Ig) cross-reactive idiotypes (CRI), we performed immunohistochemical analyses on frozen tissue sections and cytocentrifuge preparations of Ig-expressing malignant cells from patients with chronic lymphocytic leukemia (CLL) and B-cell non-Hodgkin's lymphomas (NHL) of follicular center cell origin. Twenty percent (4/20) of the Ig kappa light chain- expressing CLL cells reacted with 17.109, a MoAb against a major CRI on human IgM autoantibodies that is encoded by a conserved Ig variable- region gene (V gene) of the V kappa IIIb sub-subgroup. Another MoAb specific for V kappa IIIb framework determinant(s) reacted exclusively with all the 17.109-reactive CLL cells. Only one of 20 kappa light- chain-expressing CLL cells reacted with 6B6.6, a monoclonal antibody specific for a CRI commonly found on rheumatoid factor (RF) paraproteins with light-chain variable regions of the V kappa IIIa sub- subgroup. Finally, greater than 20% (8/34) of all CLL reacted with G6, a MoAb specific for an Ig heavy chain-associated CRI present on several RF paraproteins. In contrast, these CRIs were expressed at significantly lower frequencies in NHL of follicular center cell origin. Only one of 30 NHL expressing kappa light chains reacted with the 17.109 MoAb. Also, in contrast to the concordance between the 17.109-CRI and V kappa IIIb framework determinant(s) in CLL, two lymphomas in addition to the 17.109-reactive lymphoma were recognized by the anti-V kappa IIIb framework MoAb. None of the NHL reacted with either the 6B6.6 or the G6 MoAbs. These results are the first to demonstrate that CLL and NHL differ with respect to the expression of autoantibody-associated CRIs. The data support the notion that NHL of follicular center cell origin differs from CLL in its utilization and/or somatic mutation of Ig variable-region genes. The physiological and immunotherapeutic implications of these findings are discussed.

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Construction and Expression of Mouse-human Chimeric Antibody SZ-51 Specific for Activated Platelet P-selectin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656046 ◽

1997 ◽

Vol 77 (04) ◽

pp. 755-759 ◽

Cited By ~ 9

Author(s):

Jianming Gu ◽

Yue Liu ◽

Lijun Xia ◽

Haiying Wan ◽

Peixia Li ◽

...

Keyword(s):

Large Scale ◽

Variable Region ◽

Expression Vectors ◽

Chimeric Antibody ◽

Scale Production ◽

Fab Fragment ◽

Variable Regions ◽

E Coli ◽

Large Scale Production

SummaryA murine monoclonal (mAb) SZ-51 specific for human P-selectin may be used for in vivo thrombus imaging and for the targeting of fibrinolytic agents to thrombi. In order to reduce the immunogenicity of the murine mAb SZ-51 in humans, we cloned and sequenced the cDNAs encoding the variable region of mAb SZ-51 in order to develop mouse/human chimeric reagents. The E. coli expression vector. pHENl-SZ51 Fab/Hu was constructed by fusing the variable regions of mAb SZ-51 with human IgG γICHI and Cκ genes. The constructs were introduced into E. coli HB2151 for expression of soluble chimeric Fab fragment. We also constructed two fusion products by joining the variable regions of mouse antibody to the appropriate constant regions of human Igγl and κ. These chimeras were cloned into two eukaryotic selectable expression vectors separately, which were then cotransfected into a non-Ig secreting murine myeloma line SP2/0 with lipofectin reagent. Six cell lines remained positive for Ig secretion. The highest producing cell line, which showed stable integration and expression at 5 mg/1 of culture, was selected for the large scale production of chimeric antibody. Immunoblotting analysis demonstrated that both of the chimeric antibodies (SZ51Fab/Hu, SZ51/Hu) in the culture supernatants, like the native mAb SZ-51, bind P-selectin. In addition, the whole chimeric antibody can compete for binding to activated platelets with murine SZ-51. Therefore, the SZ-51 chimeric antibody may be a potential agent for diagnosis and treatment of thrombotic diseases in the future.

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Characterization of a Human Monoclonal Antibody against Shiga Toxin 2 Expressed in Chinese Hamster Ovary Cells

Infection and Immunity ◽

10.1128/iai.73.7.4054-4061.2005 ◽

2005 ◽

Vol 73 (7) ◽

pp. 4054-4061 ◽

Cited By ~ 9

Author(s):

D. E. Akiyoshi ◽

C. M. Rich ◽

S. O'Sullivan-Murphy ◽

L. Richard ◽

J. Dilo ◽

...

Keyword(s):

Hemolytic Uremic Syndrome ◽

Shiga Toxin ◽

Chinese Hamster Ovary ◽

Cho Cells ◽

Chinese Hamster Ovary Cells ◽

Human Monoclonal Antibody ◽

Chinese Hamster ◽

Enzyme Linked Immunosorbent Assay ◽

Variable Regions ◽

Uremic Syndrome

ABSTRACT Shiga toxin-producing Escherichia coli infections can often lead to the development of hemolytic-uremic syndrome (HUS) in a small percentage of infected humans. Patients with HUS receive only supportive treatment as the benefit of antibiotic therapy remains uncertain. We have previously reported the generation and preclinical evaluation of neutralizing human monoclonal antibodies (HuMAbs) against the Shiga toxins (Stx). In this paper, we describe the expression in Chinese hamster ovary (CHO) cells of 5C12 HuMAb, which is directed against the A subunit of Stx2. The cDNAs of the light and heavy chain immunoglobulin (Ig) variable regions of 5C12 HuMAb were isolated and cloned into an expression vector containing human IgG1 constant regions. The vector was transfected into CHO cells, and transfectants secreting Stx2-specific antibody were screened by an Stx2-specific enzyme-linked immunosorbent assay. The CHO-produced recombinant 5C12 (r5C12) showed similar specificity and binding affinity to Stx2 as the parent hybridoma-produced 5C12. More significantly, the r5C12 displayed the same neutralizing activity as the parent 5C12 in vitro and in vivo. In the mouse toxicity model, both antibodies significantly and equally prolonged survival at a dose of 0.312 μg/mouse. The data showed that since r5C12, produced in CHO cells, was equally effective as the parent 5C12, it is our choice candidate as a potential prophylactic or therapeutic agent against hemolytic-uremic syndrome.

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Sequence of a cDNA encoding Basilea kappa light chains (K2 isotype) suggests a possible relationship of protein structure to limited expression.

Journal of Experimental Medicine ◽

10.1084/jem.159.2.635 ◽

1984 ◽

Vol 159 (2) ◽

pp. 635-640 ◽

Cited By ~ 16

Author(s):

K E Bernstein ◽

E Lamoyi ◽

N McCartney-Francis ◽

R G Mage

Keyword(s):

Sulfhydryl Group ◽

Variable Region ◽

Complete Sequence ◽

Light Chains ◽

Small Subset ◽

Constant Region ◽

Region Gene ◽

Variable Regions ◽

Immunoglobulin Kappa ◽

Kappa Light Chains

We present the complete sequence of a cDNA encoding rabbit immunoglobulin kappa light chains of the Basilea isotype (K2). Although all rabbits seem to possess a K2 constant region gene, expression of this gene in most rabbits is minimal if present at all. Even in Basilea rabbits the majority of expressed immunoglobulins are of lambda type. We find that the sequence of our Basilea cDNA constant region and the sequence of a "silent" K2 gene from b4 rabbits (bas-N4) are almost identical. The bas (K2) isotype lacks cysteine at position 171 in the constant region that is present in all K1 constant regions and usually forms an interdomain disulfide bond, with a cysteine at position 80 of the variable region. We postulate that one factor contributing to the low expression of the bas (K2) isotype could be a paucity of V kappa regions lacking cysteine at position 80. If a typical rabbit V kappa encoding Cys at position 80 is rearranged and expressed with th K2 isotype. B cells with mRNAs encoding light chains with free sulfhydryl groups would result. These cells may fail to form functional immunoglobulin receptors. Only a small subset of rabbit variable regions that lack the cysteine at position 80 would rearrange and encode K2 light chains lacking a free sulfhydryl group.

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Complete amino acid sequences of variable regions of two human IgM rheumatoid factors, BOR and KAS of the Wa idiotypic family, reveal restricted use of heavy and light chain variable and joining region gene segments.

Journal of Experimental Medicine ◽

10.1084/jem.166.2.550 ◽

1987 ◽

Vol 166 (2) ◽

pp. 550-564 ◽

Cited By ~ 80

Author(s):

M M Newkirk ◽

R A Mageed ◽

R Jefferis ◽

P P Chen ◽

J D Capra

Keyword(s):

Amino Acid ◽

Gene Segment ◽

Variable Region ◽

Amino Acid Sequences ◽

Light Chains ◽

Rheumatoid Factors ◽

Region Gene ◽

Variable Regions ◽

D Region ◽

Restricted Use

Evidence derived from the complete amino acid sequences of the variable regions of both the heavy and light chains of two members (BOR and KAS) of the Wa idiotypic family of human rheumatoid factors suggests that not only are the light chains of these molecules derived from possibly one variable region gene segment, but the heavy chain variable regions are all derived from the VHI subgroup of human V region genes. These molecules exhibit a surprising conservation in the size of D region, and all use the JH4 gene element. This restriction in use of VL, VH, D, and JH suggests all of these elements may play a crucial role in either antigen binding and/or expression of the crossreactive idiotype.

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Autoantibody-associated cross-reactive idiotypes expressed at high frequency in chronic lymphocytic leukemia relative to B-cell lymphomas of follicular center cell origin

Blood ◽

10.1182/blood.v72.2.422.422 ◽

1988 ◽

Vol 72 (2) ◽

pp. 422-428 ◽

Cited By ~ 34

Author(s):

TJ Kipps ◽

BA Robbins ◽

P Kuster ◽

DA Carson

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Light Chain ◽

Variable Region ◽

Lymphocytic Leukemia ◽

Kappa Light Chain ◽

Region Gene ◽

Variable Regions ◽

Hodgkin's Lymphomas ◽

Center Cell

Abstract Using murine monoclonal antibodies (MoAbs) specific for immunoglobulin (Ig) cross-reactive idiotypes (CRI), we performed immunohistochemical analyses on frozen tissue sections and cytocentrifuge preparations of Ig-expressing malignant cells from patients with chronic lymphocytic leukemia (CLL) and B-cell non-Hodgkin's lymphomas (NHL) of follicular center cell origin. Twenty percent (4/20) of the Ig kappa light chain- expressing CLL cells reacted with 17.109, a MoAb against a major CRI on human IgM autoantibodies that is encoded by a conserved Ig variable- region gene (V gene) of the V kappa IIIb sub-subgroup. Another MoAb specific for V kappa IIIb framework determinant(s) reacted exclusively with all the 17.109-reactive CLL cells. Only one of 20 kappa light- chain-expressing CLL cells reacted with 6B6.6, a monoclonal antibody specific for a CRI commonly found on rheumatoid factor (RF) paraproteins with light-chain variable regions of the V kappa IIIa sub- subgroup. Finally, greater than 20% (8/34) of all CLL reacted with G6, a MoAb specific for an Ig heavy chain-associated CRI present on several RF paraproteins. In contrast, these CRIs were expressed at significantly lower frequencies in NHL of follicular center cell origin. Only one of 30 NHL expressing kappa light chains reacted with the 17.109 MoAb. Also, in contrast to the concordance between the 17.109-CRI and V kappa IIIb framework determinant(s) in CLL, two lymphomas in addition to the 17.109-reactive lymphoma were recognized by the anti-V kappa IIIb framework MoAb. None of the NHL reacted with either the 6B6.6 or the G6 MoAbs. These results are the first to demonstrate that CLL and NHL differ with respect to the expression of autoantibody-associated CRIs. The data support the notion that NHL of follicular center cell origin differs from CLL in its utilization and/or somatic mutation of Ig variable-region genes. The physiological and immunotherapeutic implications of these findings are discussed.

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Trends in molecular characteristics and antimicrobial resistance of group B streptococci: a multicenter study in Serbia, 2015–2020

Scientific Reports ◽

10.1038/s41598-020-79354-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Dusan Kekic ◽

Ina Gajic ◽

Natasa Opavski ◽

Milan Kojic ◽

Goran Vukotic ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Late Onset ◽

Group B Streptococcus ◽

Neonatal Morbidity ◽

Disease Rate ◽

Molecular Characteristics ◽

Group B Streptococci ◽

Live Births ◽

Invasive Infections ◽

Group B

AbstractGroup B Streptococcus (GBS) is a major cause of neonatal morbidity and mortality. Serbia has not fully implemented preventive measures against GBS neonatal diseases. Therefore, we aimed to assess the maternal GBS colonisation and invasive neonatal disease rate, to reveal the trends of antimicrobial resistance and serotype distribution of GBS from various patient groups. Randomly selected non-invasive (n = 991) and all invasive GBS (n = 80) collected throughout Serbia from 2015 to 2020 were tested for antimicrobial susceptibility, capsular typing, and hvgA detection. Overall, 877/5621 (15.6%) pregnant women were colonised with GBS. Invasive GBS infections incidence in infants (0.18/1000 live births) showed a decreasing trend (0.3 to 0.1/1000 live births). Type III was overrepresented in infants with invasive infections (n = 35, 58.3%), whereas type V predominated among colonised adults (n = 224, 25.5%) and those with noninvasive (n = 37, 32.5%) and invasive infections (n = 8, 40%). The hypervirulent clone III/ST17 was highly associated with invasive infections (n = 28, 35%), particularly late-onset disease (n = 9, 47.4%), showing an increase from 12.3 to 14.8%. The GBS resistance to erythromycin and clindamycin was 26.7% and 22.1%, respectively, with an upward trend. The emergence of the hypervirulent clone III/ST17 and the escalation in GBS resistance highlight an urgent need for continuous monitoring of GBS infections.

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Molecular characteristics of antibodies bearing an anti-DNA-associated idiotype.

Journal of Experimental Medicine ◽

10.1084/jem.174.6.1639 ◽

1991 ◽

Vol 174 (6) ◽

pp. 1639-1652 ◽

Cited By ~ 53

Author(s):

A Manheimer-Lory ◽

J B Katz ◽

M Pillinger ◽

C Ghossein ◽

A Smith ◽

...

Keyword(s):

B Cells ◽

Dna Binding ◽

Lupus Erythematosus ◽

Germ Line ◽

High Titer ◽

Variable Region ◽

Amino Acid Sequences ◽

Molecular Characteristics ◽

Systemic Lupus ◽

Dna Antibodies

Anti-double-stranded DNA antibodies are the hallmark of the disease systemic lupus erythematosus and are believed to contribute to pathogenesis. While a large number of anti-DNA antibodies from mice with lupus-like syndromes have been characterized and their variable region genes sequenced, few human anti-DNA antibodies have been reported. We describe here the variable region gene sequences of eight antibodies produced by Epstein-Barr virus (EBV)-transformed B cells that bear the 3I idiotype, an idiotype expressed on anti-DNA antibodies and present in high titer in patients with systemic lupus. The comparison of these antibodies to the light chains of 3I+ myeloma proteins and serum antibodies reveals that EBV transformation yields B cells producing antibodies representative of the expressed antibody repertoire. The analysis of nucleotide and amino acid sequences of these antibodies suggests the first complementarity determining region of the light chain may be important in DNA binding and that paradigms previously generated to account for DNA binding require modification. The understanding of the molecular genetics of the anti-DNA response requires a more complete description of the immunoglobulin germ line repertoire, but data reported here suggest that somatic diversification is a characteristic of the anti-DNA response.

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