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1.  Generation and application of new rat monoclonal antibodies against synthetic FLAG and OLLAS tags for improved immunodetection 
Journal of immunological methods  2007;331(1-2):27-38.
Previously, we prepared monoclonal antibodies (mAbs) by immunizing rats with the recombinant fusion proteins of mouse Langerin/CD207, which contained a flexible linker sequence from E. coli OmpF and a FLAG epitope. We found many of new rat mAbs were not reactive to mouse Langerin, and here we identify the epitopes of two of these IgG mAbs, L2 and L5, and assess their efficacy in various immunodetection methods. MAb L5 is a rat IgG mAb against the FLAG epitope, which detected both N-terminal and C-terminal FLAG tagged protein 2 to 8 times better than the conventional anti-FLAG mAb M2 by Western blot. For mAb L2, we found its epitope to be a 14 amino acid sequence SGFANELGPRLMGK which consisted of both sequences from the OmpF derived linker and mouse Langerin. This epitope sequence was named OLLAS (E. coli OmpF Linker and mouse Langerin fusion Sequence), and mAb L2 as mAb OLLA-2. When the OLLAS sequence was inserted into recombinant proteins at N-terminal, C-terminal, or internal sites, the OLLAS tag was detected by mAb OLLA-2 with very high sensitivity compared to other conventional epitope tags and anti-tag mAbs. MAb OLLA-2 recognized OLLAS tagged proteins with at least 100-fold more sensitivity than anti-FLAG M2 and anti-V5 mAbs in Western blot analyses. We also find the OLLAS epitope to be superior in immunoprecipitation and other immunodetection methods, such as fluorescent immunohistochemistry and flow cytometry. In the process, we successfully utilized the OLLAS epitope sequence as an internal linker for fusion between the engineered mAb and the antigen, and thus achieved improved immunodetection.
PMCID: PMC2864634  PMID: 18054954
Monoclonal Antibody; Protein Tagging; FLAG Tag; OLLAS Tag
2.  Functional Evaluation of DC-SIGN Monoclonal Antibodies Reveals DC-SIGN Interactions with ICAM-3 Do Not Promote Human Immunodeficiency Virus Type 1 Transmission 
Journal of Virology  2002;76(12):5905-5914.
DC-SIGN, a type II membrane-spanning C-type lectin that is expressed on the surface of dendritic cells (DC), captures and promotes human and simian immunodeficiency virus (HIV and SIV) infection of CD4+ T cells in trans. To better understand the mechanism of DC-SIGN-mediated virus transmission, we generated and functionally evaluated a panel of seven monoclonal antibodies (MAbs) against DC-SIGN family molecules. Six of the MAbs reacted with myeloid-lineage DC, whereas one MAb preferentially bound DC-SIGNR/L-SIGN, a homolog of DC-SIGN. Characterization of hematopoietic cells also revealed that stimulation of monocytes with interleukin-4 (IL-4) or IL-13 was sufficient to induce expression of DC-SIGN. All DC-SIGN-reactive MAbs competed with intercellular adhesion molecule 3 (ICAM-3) for adhesion to DC-SIGN and blocked HIV-1 transmission to T cells that was mediated by THP-1 cells expressing DC-SIGN. Similar but less efficient MAb blocking of DC-mediated HIV-1 transmission was observed, indicating that HIV-1 transmission to target cells via DC may not be dependent solely on DC-SIGN. Attempts to neutralize DC-SIGN capture and transmission of HIV-1 with soluble ICAM-3 prophylaxis were limited in success, with a maximal inhibition of 60%. In addition, disrupting DC-SIGN/ICAM-3 interactions between cells with MAbs did not impair DC-SIGN-mediated HIV-1 transmission. Finally, forced expression of ICAM-3 on target cells did not increase their susceptibility to HIV-1 transmission mediated by DC-SIGN. While these findings do not discount the role of intercellular contact in facilitating HIV-1 transmission, our in vitro data indicate that DC-SIGN interactions with ICAM-3 do not promote DC-SIGN-mediated virus transmission.
PMCID: PMC136240  PMID: 12021323
3.  Functional and Antigenic Characterization of Human, Rhesus Macaque, Pigtailed Macaque, and Murine DC-SIGN 
Journal of Virology  2001;75(21):10281-10289.
DC-SIGN, a type II membrane protein with a C-type lectin binding domain that is highly expressed on mucosal dendritic cells (DCs) and certain macrophages in vivo, binds to ICAM-3, ICAM-2, and human and simian immunodeficiency viruses (HIV and SIV). Virus captured by DC-SIGN can be presented to T cells, resulting in efficient virus infection, perhaps representing a mechanism by which virus can be ferried via normal DC trafficking from mucosal tissues to lymphoid organs in vivo. To develop reagents needed to characterize the expression and in vivo functions of DC-SIGN, we cloned, expressed, and analyzed rhesus macaque, pigtailed macaque, and murine DC-SIGN and made a panel of monoclonal antibodies (MAbs) to human DC-SIGN. Rhesus and pigtailed macaque DC-SIGN proteins were highly similar to human DC-SIGN and bound and transmitted HIV type 1 (HIV-1), HIV-2, and SIV to receptor-positive cells. In contrast, while competent to bind virus, murine DC-SIGN did not transmit virus to receptor-positive cells under the conditions tested. Thus, mere binding of virus to a C-type lectin does not necessarily mean that transmission will occur. The murine and macaque DC-SIGN molecules all bound ICAM-3. We mapped the determinants recognized by a panel of 16 MAbs to the repeat region, the lectin binding domain, and the extreme C terminus of DC-SIGN. One MAb was specific for DC-SIGN, failing to cross-react with DC-SIGNR. Most MAbs cross-reacted with rhesus and pigtailed macaque DC-SIGN, although none recognized murine DC-SIGN. Fifteen of the MAbs recognized DC-SIGN on DCs, with MAbs to the repeat region generally reacting most strongly. We conclude that rhesus and pigtailed macaque DC-SIGN proteins are structurally and functionally similar to human DC-SIGN and that the reagents that we have developed will make it possible to study the expression and function of this molecule in vivo.
PMCID: PMC114602  PMID: 11581396
4.  Protection of rat intestine against cholera toxin challenge by monoclonal anti-idiotypic antibody immunization via enteral and parenteral routes. 
Infection and Immunity  1991;59(10):3651-3658.
A mouse monoclonal anti-idiotypic (anti-id) immunoglobulin M (IgM) antibody, called MAb2, was raised against a mouse monoclonal anti-cholera toxin (anti-CT) antibody (MAb1). The MAb2 was shown, by competition with CT for MAb1, to bear the internal image of an epitope of CT. MAb2 immunization of rats was performed via the intraperitoneal, intragastric, and intrajejunal routes and compared with immunization of rats with either a control, isotype- and allotype-matched MAb or with CT via the same routes. Both serum IgG and bile IgA anti-CT Ab3's were detected by enzyme-linked immunosorbent assay in anti-id MAb2-immunized rats, although their titers were lower than those in CT-immunized rats. No anti-CT antibodies were detected in sera and bile of rats immunized with the control MAb. When tested for degree of gut protection against a CT challenge, rats immunized with MAb2 by the intrajejunal route showed a rather high degree of protection, which was only slightly lower than that of rats immunized with CT via the same route; all rats but one immunized with the control MAb were unprotected. There was, however, no correlation between serum or bile anti-CT titers and degree of gut protection in MAb2-immunized rats. Their serum anti-CT Ab3's were purified by adsorption and elution from a CT immunosorbent and resembled anti-CT MAb1 in their unique reactivity with MAb2. This constitutes to our knowledge the second report of protection against a pathogen by anti-id immunization via the enteric route.
PMCID: PMC258934  PMID: 1894367
5.  Development of a biotin-streptavidin-enhanced enzyme-linked immunosorbent assay which uses monoclonal antibodies for detection of group C rotaviruses. 
Journal of Clinical Microbiology  1992;30(7):1667-1673.
A biotin-streptavidin-enhanced enzyme-linked immunosorbent assay (ELISA) which uses monoclonal antibodies (MAbs) for the detection of group C rotaviruses was developed. An assay in which plates were coated with three pooled MAbs and biotinylated polyclonal immunoglobulin G (IgG) (polyclonal antibody [PAb]) was used as the detector (MAb capture-PAb detector) was found to be the most sensitive and specific of the assays when it was compared with assays in which plates were coated with polyclonal antiserum and detection was done with either biotinylated polyclonal antiserum (PAb capture-PAb detector) or biotinylated pooled MAbs (PAb capture-MAb detector). The MAb capture-PAb detector ELISA detected 83% of samples confirmed to be positive for group C rotaviruses, whereas the PAb capture-PAb detector assay detected 63% of positive samples and the PAb capture-MAb detector assay detected 65% of positive samples. All three procedures detected both of the bovine and the two human group C rotaviruses, but none of the three procedures detected fecal samples containing group A and B rotaviruses or fecal samples negative for group C rotaviruses used in this study. The sensitivity of the MAb capture-PAb detector ELISA was determined by serially diluting fecal group C rotaviruses; antigens were detected in maximal positive dilution ranges of 1:1,000 to 1:3,000 for the samples tested. On the basis of the cell culture immunofluorescence assay infectivity titer of semipurified cell culture-passaged Cowden group C rotavirus, the sensitivity of the MAb capture-PAb detection ELISA for detection of homologous group C rotavirus was 53 fluorescent focus units per ml. Epitope mapping by use of the biotinylated MAbs in competition assay suggested that our MAbs may bind to three different but overlapping epitopes. These results suggest that the MAb capture-PAb detector ELISA can be used to study the epidemiology of group C rotaviruses in humans and animals.
PMCID: PMC265361  PMID: 1321166
6.  Analysis of Epitopes on Dengue Virus Envelope Protein Recognized by Monoclonal Antibodies and Polyclonal Human Sera by a High Throughput Assay 
The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored.
Methodology/Principal Findings
We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC′ loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer.
Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level.
Author Summary
Dengue virus is the leading cause of arboviral diseases worldwide. The envelope protein is the major target of neutralizing antibodies and vaccine development. While previous studies have reported several epitopes on envelope protein, the possibility of interdomain epitopes and the relationship of epitopes to neutralizing potency remain unexplored. We developed a high throughput dot blot assay by using 67 alanine mutants of surface-exposed envelope residues as a systematic approach to identify epitopes recognized by mouse monoclonal antibodies and polyclonal human sera. Our results suggested the presence of interdomain epitopes more frequent than previously appreciated. Compared with monoclonal antibodies generated by traditional protocol, the potent neutralizing monoclonal antibodies generated by a new protocol showed several unique features of their epitopes. Moreover, the predominant epitopes of antibodies against envelope protein in polyclonal sera can be identified by this assay. These findings have implications for future development of epitope-specific diagnostics and epitope-based dengue vaccine, and add to our understanding of humoral immune responses to dengue virus at the epitope level.
PMCID: PMC3250511  PMID: 22235356
7.  Antitumour effects of single or combined monoclonal antibodies directed against membrane antigens expressed by human B cells leukaemia 
Molecular Cancer  2011;10:42.
The increasing availability of different monoclonal antibodies (mAbs) opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL) respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells.
The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to longer mouse survival, as compared to single mAb injection, up to complete inhibition of tumour growth in 100% mice treated with both anti-HLA-DR and anti-CD5.
Altogether these data suggest that the combined use of two mAbs, such as anti-HLA-DR and anti-CD5, may significantly enhance their therapeutic potential.
PMCID: PMC3103468  PMID: 21504579
8.  Prediction of the interacting surfaces in a trimolecular complex formed between the major dust mite allergen Der p 1, a mouse monoclonal anti-Der p 1 antibody, and its anti-idiotype 
Molecular Pathology  2000;53(6):324-332.
Background—Two mouse monoclonal antibodies (mAbs) have been described recently; namely, mAb 2C7 (IgG2bκ), which is directed against the major house dust mite allergen Der p 1, and mAb 2G10 (IgG1κ), which is an anti-idiotypic antibody raised against mAb 2C7. The anti-idiotype mAb 2G10 does not block the binding of mAb 2C7 to Der p 1, which means that mAb 2C7 can simultaneously bind to Der p 1 and to mAb 2G10, thereby generating a trimolecular complex consisting of antigen–idiotype–anti-idiotype.
Aims—To sequence and model the V region of the anti-idiotypic antibody mAb 2G10 to enable the prediction of the interacting surfaces in the trimolecular complex consisting of Der p 1–mAb 2C7–mAb 2G10.
Methods—DNA sequencing of mAb 2G10 was carried out and the Swiss Model and Swiss PDB-Viewer programs were used to build a three dimensional model of the trimolecular complex.
Results—Complementarity of shape and charge was revealed when comparing the protrusion of the previously determined Der p 1 epitope (Leu147–Gln160) with the cavity formed by the complementarity determining regions (CDRs) of mAb 2C7. Such complementarity was also observed between the mAb 2C7 epitope predicted to be recognised by mAb 2G10 (residues Lys19 from framework region 1 (FRW1) and Ser74–Gln81 from FRW3) and residues from the CDRs of mAb 2G10 (a negatively charged patch flanked by the residues Asp55H/Glu58H and Glu27L/Glu27cL). As expected, the location of the mAb 2C7 epitope recognised by mAb 2G10 does not appear to interfere with the binding of Der p 1 to mAb 2C7.
Conclusion—Although the results obtained represent only an approximation, they nevertheless provide a rare insight into how an antigen (Der p 1) might bind to its antibody (mAb 2C7) while in complex with an anti-idiotype (mAb 2G10).
PMCID: PMC1186988  PMID: 11193052
Der p 1; anti-idiotype antibodies; monoclonal antibodies; antigen-antibody binding
9.  Production and Characterization of Mouse Monoclonal Antibodies Recognizing Multiple Subclasses of Human IgG 
Different IgG subclass profiles are produced in response to different antigenic stimuli in a variety of diseases. IgG subclass levels may reflect disease severity. Quantification of IgG subclasses depends on the availability of specific Monoclonal antibodies (MAbs). In the present study seven hybridoma clones producing MAbs reactive with multiple subclasses of human IgG were established. Splenocytes from Balb/c mice immunized with Fc fractions of human IgG1 or IgG2 myeloma proteins were fused with mouse myeloma cells. Fused cells were selected and cloned by limiting dilution assay. Antibody secreting cells were screened by Enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of purified human myeloma paraproteins of different IgG subclasses by ELISA and immunoblotting. Cross-reactivity to immunoglobulins (Igs) of other species was studied by indirect ELISA using serum samples collected from 9 animals. The MAbs were found to react with triple IgG subclasses, including IgG1,2,4 (n=4) and IgG1,2,3 (n=3). Immunoblotting studies revealed recognition of linear (n=4) or conformational (n=3) epitopes by these MAbs. The most abundant cross-reactivity (71.4%) was observed with monkey Ig while no cross-reactivity was detected with hen and cat sera. The MAbs mostly displayed a restricted pattern of cross-reactivity and one of them did not bind to any of the animal sera tested. The affinity constant of 3 MAbs was measured by ELISA. Based on the data obtained from this study, mouse MAbs reactive with multiple human IgG subclasses are directed to a variety of immunogenic epitopes, mostly shared with IgG of other species. These MAbs are valuable tools for purification of non-reactive IgG subclasses through negative affinity chromatography. These MAbs could also provide an opportunity for epitope mapping of the Fc region of IgG, as well as serological phylogenetic studies.
PMCID: PMC3558140  PMID: 23408735
ELISA; Hybridoma; IgG; Isotype; Monoclonal antibody; Subclass
10.  Epitope Mapping of Monoclonal Antibodies Capable of Neutralizing Cytotoxic Necrotizing Factor Type 1 of Uropathogenic Escherichia coli 
Infection and Immunity  2001;69(4):2066-2074.
Cytotoxic necrotizing factor type 1 (CNF1) of uropathogenic Escherichia coli belongs to a family of bacterial toxins that target the small GTP-binding Rho proteins that regulate the actin cytoskeleton. Members of this toxin family typically inactivate Rho; however, CNF1 and the highly related CNF2 activate Rho by deamidation. Other investigators have reported that the first 190 amino acids of CNF1 constitute the cellular binding domain and that the CNF1 enzymatic domain lies within a 300-amino-acid stretch in the C terminus of the toxin. Amino acids 53 to 75 appear to be critical for cell receptor recognition, while amino acids Cys866 and His881 are considered essential for deamidation activity. To delineate further the functional domains of CNF1, we generated 16 monoclonal antibodies (MAbs) against the toxin and used them for epitope mapping studies. Based on Western blot immunoreactivity patterns obtained from a series of truncated CNF1 proteins, this panel of MAbs mapped to epitopes located throughout the toxin, including the binding and enzymatic domains. All MAbs showed reactivity to CNF1 by Western and dot blot analyses. However, only 7 of the 16 MAbs exhibited cross-reactivity with CNF2. Furthermore, only three MAbs demonstrated the capacity to neutralize toxin in either HEp-2 cell assays (inhibition of multinucleation) or 5637 bladder cell assays (inhibition of cytotoxicity). Since CNF1 epitopes recognized by neutralizing MAbs are likely to represent domains or regions necessary for the biological activities of the toxin, the epitopes recognized by these three MAbs, designated JC4 (immunoglobulin G2a [IgG2a]), BF8 (IgA), and NG8 (IgG2a), were more precisely defined. MAbs JC4 and BF8 reacted with epitopes that were common to CNF1 and CNF2 and located within the putative CNF1 binding domain. MAb JC4 recognized an epitope spanning amino acids 169 to 191, whereas MAb BF8 mapped to an epitope between amino acids 135 and 164. Despite the capacity of both MAbs to recognize CNF2 in Western blot analyses, only MAb BF8 neutralized CNF2. MAb NG8 showed reactivity to a CNF1-specific epitope located between amino acids 683 and 730, a region that includes a very small portion of the putative enzymatic domain. Taken together, these findings identify three new regions of the toxin that appear to be critical for the biological activity of CNF1.
PMCID: PMC98131  PMID: 11254559
11.  Antigen-Binding Properties of Monoclonal Antibodies Reactive with EBNA1 and Use in Immunoaffinity Chromatography 
PLoS ONE  2009;4(2):e4614.
Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) was overexpressed and purified from Escherichia coli. Mouse monoclonal antibodies (mAbs) were prepared that react with EBNA1. Eleven high affinity mAbs were recovered. Nine mAbs are isotype IgG (all subisotype IgG1) and two mAbs are isotype IgM. All mAbs react strongly with EBNA1 in an ELISA assay while only one mAb (designated 1EB6) fails to react in a Western blot assay. The epitopes for these mAbs were mapped to seven different regions, providing good coverage of the entire EBNA1 protein. The mAbs had differing affinity for an EBNA1/DNA complex with four mAbs able to supershift the complex completely. All mAbs can immunoprecipitate EBNA1 from E. coli overexpressing EBNA1. A modified ELISA assay, termed ELISA-elution assay, was used to screen for mAbs that release EBNA1 in the presence of a low molecular weight polyhydroxylated compound (polyol) and a nonchaotropic salt. MAbs with this property, termed polyol-responsive (PR)-mAbs, allow gentle elution of labile proteins and protein complexes. Four mAbs are polyol-responsive with two showing usefulness in gentle immunoaffinity chromatography. Purification with these PR-mAbs may be useful in purifying EBNA1 complexes and elucidating EBNA1-associated proteins. This panel of anti-EBNA1 mAbs will advance the study of EBV by providing new tools to detect and purify EBNA1.
PMCID: PMC2644765  PMID: 19242546
12.  Fully human IgG and IgM antibodies directed against the carcinoembryonic antigen (CEA) Gold 4 epitope and designed for radioimmunotherapy (RIT) of colorectal cancers 
BMC Cancer  2004;4:75.
Human monoclonal antibodies (MAbs) are needed for colon cancer radioimmunotherapy (RIT) to allow for repeated injections. Carcinoembryonic antigen (CEA) being the reference antigen for immunotargeting of these tumors, we developed human anti-CEA MAbs.
XenoMouse®-G2 animals were immunized with CEA. Among all the antibodies produced, two of them, VG-IgG2κ and VG-IgM, were selected for characterization in vitro in comparison with the human-mouse chimeric anti-CEA MAb X4 using flow cytometry, surface plasmon resonance, and binding to radiolabeled soluble CEA and in vivo in human colon carcinoma LS174T bearing nude mice.
Flow cytometry analysis demonstrated binding of MAbs on CEA-expressing cells without any binding on NCA-expressing human granulocytes. In a competitive binding assay using five reference MAbs, directed against the five Gold CEA epitopes, VG-IgG2κ and VG-IgM were shown to be directed against the Gold 4 epitope. The affinities of purified VG-IgG2κ and VG-IgM were determined to be 0.19 ± 0.06 × 108 M-1 and 1.30 ± 0.06 × 108 M-1, respectively, as compared with 0.61 ± 0.05 × 108 M-1 for the reference MAb X4. In a soluble phase assay, the binding capacities of VG-IgG2κ and VG-IgM to soluble CEA were clearly lower than that of the control chimeric MAb X4. A human MAb concentration of about 10-7 M was needed to precipitate approximatively 1 ng 125I-rhCEA as compared with 10-9 M for MAb X4, suggesting a preferential binding of the human MAbs to solid phase CEA. In vivo, 24 h post-injection, 125I-VG-IgG2κ demonstrated a high tumor uptake (25.4 ± 7.3%ID/g), close to that of 131I-X4 (21.7 ± 7.2%ID/g). At 72 h post-injection, 125I-VG-IgG2κ was still concentrated in the tumor (28.4 ± 11.0%ID/g) whereas the tumor concentration of 131I-X4 was significantly reduced (12.5 ± 4.8%ID/g). At no time after injection was there any accumulation of the radiolabeled MAbs in normal tissues. A pertinent analysis of VG-IgM biodistribution was not possible in this mouse model in which IgM displays a very short half-life due to poly-Ig receptor expression in the liver.
Our human anti-CEA IgG2κ is a promising candidate for radioimmunotherapy in intact form, as F(ab')2 fragments, or as a bispecific antibody.
PMCID: PMC526287  PMID: 15488142
13.  Capture and Transfer of Simian Immunodeficiency Virus by Macaque Dendritic Cells Is Enhanced by DC-SIGN 
Journal of Virology  2002;76(23):11827-11836.
Dendritic cells (DCs) are among the first cells encountered by human and simian immunodeficiency virus (HIV and SIV) following mucosal infection. Because these cells efficiently capture and transmit virus to T cells, they may play a major role in mediating HIV and SIV infection. Recently, a C-type lectin protein present on DCs, DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), was shown to efficiently bind and present HIV and SIV to CD4+, coreceptor-positive cells in trans. However, the significance of DC-SIGN for virus transmission and pathogenesis in vivo remains unclear. Because SIV infection of macaques may represent the best model to study the importance of DC-SIGN in HIV infection, we cloned and characterized pig-tailed macaque DC-SIGN and generated monoclonal antibodies (MAbs) against it. We demonstrate that, like human DC-SIGN, pig-tailed macaque DC-SIGN (ptDC-SIGN) is expressed on DCs and macrophages but not on monocytes, T cells, or B cells. Moderate levels of ptDC-SIGN expression were detected on the surface of DCs, and low-level expression was found on macrophages. Additionally, we show that ptDC-SIGN efficiently binds and transmits replication-competent SIVmne variants to CD4+, coreceptor-positive cells. Moreover, transmission of virus between pig-tailed macaque DCs and CD4+ T cells is largely ptDC-SIGN dependent. Interestingly, MAbs directed against ptDC-SIGN vary in the capacity to block transmission of different SIVmne variants. These data demonstrate that ptDC-SIGN plays a central role in transmitting virus from macaque DCs to T cells, and they suggest that SIVmne variants may differ in their interactions with ptDC-SIGN. Thus, SIVmne infection of pig-tailed macaques may provide an opportunity to investigate the significance of DC-SIGN in primate lentiviral infections.
PMCID: PMC136877  PMID: 12414925
14.  Epitope reactions can be gauged by relative antibody discriminating specificity (RADS) values supported by deletion, substitution and cysteine bridge formation analyses: potential uses in pathogenesis studies 
BMC Research Notes  2012;5:208.
Epitope-mapping of infectious agents is essential for pathogenesis studies. Since polyclonal antibodies (PAbs) and monoclonal antibodies (MAbs) are always polyspecific and can react with multiple epitopes, it is important to distinguish between specific and non-specific reactions. Relative antibody discriminating specificity (RADS) values, obtained from their relative ELISA reactions with L-amino acid peptides prepared in the natural versus reverse orientations (x-fold absorbance natural/absorbance reverse = RADS value) may be valuable for this purpose.
PAbs generated against the dengue type-2 virus (DENV-2) nonstructural-1 (NS1) glycoprotein candidate vaccine also reacted with both DENV envelope (E) glycoproteins and blood-clotting proteins. New xKGSx/xSGKx amino acid motifs were identified on DENV-2 glycoproteins, HIV-1 gp41 and factor IXa. Their potential roles in DENV and HIV-1 antibody-enhanced replication (AER) and auto-immunity were assessed.
In this study, a) RADS values were determined for MAbs and PAbs, generated in congeneic (H2: class II) mice against DENV NS1 glycoprotein epitopes, to account for their cross-reaction patterns, and b) MAb 1G5.3 reactions with xKGSx/xSGKx motifs present in the DENV-4 NS1, E and HIV-1 glycoproteins and factor IXa were assessed after the introduction of amino acid substitutions, deletions, or intra-/inter-cysteine (C-C) bridges.
MAbs 1H7.4, 5H4.3, 3D1.4 and 1G5.3 had high (4.23- to 16.83-fold) RADS values against single epitopes on the DENV-2 NS1 glycoprotein, and MAb 3D1.4 defined the DENV complex-conserved LX1 epitope. In contrast, MAbs 1G5.4-A1-C3 and 1C6.3 had low (0.47- to 1.67-fold) RADS values against multiple epitopes. PAb DENV complex-reactions occurred through moderately-high (2.77- and 3.11-fold) RADS values against the LX1 epitope. MAb 1G5.3 reacted with xSGKx motifs present in DENV-4 NS1 and E glycoproteins, HIV-1 gp41 and factor IXa, while natural C-C bridge formations or certain amino acid substitutions increased its binding activity.
These results: i) were readily obtained using a standard 96-well ELISA format, ii) showed the LX1 epitope to be the immuno-dominant DENV complex determinant in the NS1 glycoprotein, iii) supported an antigenic co-evolution of the DENV NS1 and E glycoproteins, and iv) identified methods that made it possible to determine the role of anti-DENV PAb reactions in viral pathogenesis.
PMCID: PMC3392722  PMID: 22546090
Epitope; Mapping; Synthetic peptide; Monoclonal antibody; Relative antibody discriminating specificity value
15.  Monoclonal Antibodies to Escherichia coli-Expressed P46 and P65 Membranous Proteins for Specific Immunodetection of Mycoplasma hyopneumoniae in Lungs of Infected Pigs 
The P46 and P65 proteins of Mycoplasma hyopneumoniae are two membranous proteins carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the genes encoding entire P46 (1,260 bp) and P65 (1,803 bp) and N-terminally truncated P65c (1,200 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species that commonly colonize the porcine respiratory tract. Both amplified genes were then cloned into the pGEX-4T-1 vector to be expressed in Escherichia coli cells as recombinant fusion proteins with glutathione S-transferase (GST). Prior to generation of expression constructs, TGA nonsense codons, exceptionally used for tryptophan residues by M. hyopneumoniae, had been converted to TGG codons by PCR-directed mutagenesis. Following induction by IPTG (isopropyl-β-d-thiogalactopyranoside), both GST-P46 and GST-P65c recombinant fusion proteins were recovered by disrupting transformed cells by sonication, purified by affinity chromatography, and then cut with thrombin to release the P46 and P65c moieties. The enriched E. coli-expressed P46 and P65c proteins were used to immunize female BALB/c mice for the generation of anti-P46 and anti-P65c monoclonal antibodies (MAbs). The polypeptide specificities of MAbs obtained was confirmed by Western blotting with cell lysates prepared from the homologous strain. Cross-reactivity study of the anti-P46 and anti-P65c MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture, suggested that the MAbs obtained against both membranous proteins were directed against highly conserved species-specific epitopes. No reactivity to other mycoplasma species tested was demonstrated. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs that had been inoculated intratracheally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. Both anti-P46 and anti-P65c MAbs permitted effective detection by indirect immunofluorescence and indirect immunoperoxidase assay of M. hyopneumoniae in, respectively, frozen and formalin-fixed, paraffin-embedded lung sections from pigs that were killed after the 6- to 7-week observation period.
PMCID: PMC154955  PMID: 12738649
16.  Characterization of Entamoeba histolytica Intermediate Subunit Lectin-Specific Human Monoclonal Antibodies Generated in Transgenic Mice Expressing Human Immunoglobulin Loci ▿  
Infection and Immunity  2008;77(1):549-556.
Four fully human monoclonal antibodies (MAbs) to Entamoeba histolytica intermediate subunit lectin (Igl) were prepared in XenoMouse mice, which are transgenic mice expressing human immunoglobulin loci. Examination of the reactivities of these MAbs to recombinant Igl1 and Igl2 of E. histolytica showed that XEhI-20 {immunoglobulin G2(κ) [IgG2(κ)]} and XEhI-28 [IgG2(κ)] were specific to Igl1, XEhI-B5 [IgG2(κ)] was specific to Igl2, and XEhI-H2 [IgM(κ)] was reactive with both Igls. Gene analyses revealed that the VH and VL germ lines were VH3-48 and L2 for XEhI-20, VH3-21 and L2 for XEhI-28, VH3-33 and B3 for XEhI-B5, and VH4-4 and A19 for XEhI-H2, respectively. Flow cytometry analyses showed that the epitopes recognized by all of these MAbs were located on the surfaces of living trophozoites. Confocal microscopy demonstrated that most Igl1 and Igl2 proteins were colocalized on the surface and in the cytoplasm, but different localization patterns in intracellular vacuoles were also present. The preincubation of trophozoites with XEhI-20, XEhI-B5, and XEhI-H2 caused significant inhibition of the adherence of trophozoites to Chinese hamster ovary cells, whereas preincubation with XEhI-28 did not do so. XEhI-20, XEhI-B5, and XEhI-H2 were injected intraperitoneally into hamsters 24 h prior to intrahepatic challenge with E. histolytica trophozoites. One week later, the mean abscess size in groups injected with one of the three MAbs was significantly smaller than that in controls injected with polyclonal IgG or IgM isolated from healthy humans. These results demonstrate that human MAbs to Igls may be applicable for immunoprophylaxis of amebiasis.
PMCID: PMC2612286  PMID: 19001071
17.  O-Polysaccharide Epitopic Heterogeneity at the Surface of Brucella spp. Studied by Enzyme-Linked Immunosorbent Assay and Flow Cytometry 
Smooth Brucella strains are classified into three serotypes, i.e., A+M−, A−M+, and A+M+, according to slide agglutination with A and M monospecific polyclonal sera. The epitopes involved have been located on the O-polysaccharide (O-PS) moiety of the smooth lipopolysaccharide (S-LPS), which represents the most exposed antigenic structure on the surface of Brucella spp. By use of monoclonal antibodies (MAbs) a number of epitope specificities on the O-PS have been reported: A, M, and epitopes shared by both A and M dominant strains, which have been named common (C) epitopes. The latter have been further subdivided, according to relative MAb binding in enzyme-linked immunosorbent assays (ELISA) to A- and M-dominant Brucella strains and to cross-reacting Yersinia enterocolitica O:9, into five epitopic specificities: C (M>A), C (M=A), C/Y (M>A), C/Y (M=A), and C/Y (A>M). In the present study, we studied the occurrence of these epitopes at the surface of representatives of all Brucella species and biovars including the live vaccine strains by analyzing the levels of MAb binding to whole Brucella cells in ELISA and flow cytometry assays. In ELISA, the level of MAb binding correlated well with the previously defined epitope specificity and the serotype defined by polyclonal sera for each Brucella species, biovar, or strain. However, MAbs to the C (M=A) and C (M>A) epitopes showed insignificant binding to B. suis biovar 2 strains and bound at lower titers to B. suis biovar 3 and B. neotomae than to the other Brucella strains. Some of the flow cytometry results were contradictory to those obtained by ELISA. In fact, it appeared by flow cytometry that all O-PS epitopes, including the A and M epitopes, are shared to different degrees by Brucella spp. which nevertheless show a high degree of O-PS heterogeneity according to MAb binding intensities. The subdivision of MAb specificities and Brucella serotypes was therefore less evident by flow cytometry than by ELISA. Whereas in ELISA the MAb specific for the A epitope showed insignificant binding to Y. enterocolitica O:9, this MAb bound strongly to Y. enterocolitica O:9 in flow cytometry. One of the two MAbs specific to the C (M=A) epitope also bound at a low but significant level to B. suis biovar 2 strains. However, as in ELISA the MAb specific for the C (M>A) epitope did not bind at all to B. suis biovar 2 strains in flow cytometry. Flow cytometry provided new information regarding specificity of the MAbs and may further explain some aspects of the capacity of passive protection of some MAbs against smooth Brucella infection in mice. As shown in the present study the occurrence of Brucella strains apparently completely devoid of one specific C O-PS epitope (e.g., B. suis biovar 2 devoid of the C [M>A] epitope) offers the possibility of obtaining vaccine strains devoid of a diagnostic O-PS epitope, which could further help to resolve the problem of discriminating infected from vaccinated animals that remains a major goal in brucellosis research.
PMCID: PMC96216  PMID: 9801349
18.  Platelet receptors for the Streptococcus sanguis adhesin and aggregation-associated antigens are distinguished by anti-idiotypical monoclonal antibodies. 
Infection and Immunity  1995;63(9):3628-3633.
Platelets aggregate in response to an adhesin and the platelet aggregation-associated protein (PAAP) expressed on the cell surfaces of certain strains of Streptococcus sanguis. We sought to identify the corresponding PAAP receptor and accessory adhesin binding sites on platelets. Since the adhesion(s) of S. sanguis for platelets has not been characterized, an anti-idiotype (anti-id) murine monoclonal antibody (MAb2) strategy was developed. First, MAb1s that distinguished the adhesin and PAAP antigens on the surface of S. sanguis I 133-79 were selected. Fab fragments of MAb1.2 (immunoglobulin G2b [IgG2b]; 70 pmol) reacted with 5 x 10(7) cells of S. sanguis to completely inhibit the aggregation of human platelets in plasma. Under similar conditions, MAb1.1 (IgG1) inhibited the adhesion of S. sanguis cells to platelets by a maximum of 34%, with a comparatively small effect on platelet aggregation. Together, these two MAb1s inhibited S. sanguis-platelet adhesion by 63%. In Western immunoblots, both MAb1s reacted with S. sanguis 133-79 87- and 150-kDa surface proteins and MAb1.2 also reacted with purified type I collagen. The hybridomas producing MAb1.1 and MAb1.2 were then injected into BALB/c mice. Enlarged spleens were harvested, and a panel of MAb2 hybridomas was prepared. To identify anti-ids against the specific MAb1s, the MAb2 panel was screened by enzyme-linked immunosorbent assay for reaction with rabbit polyclonal IgG antibodies against the 87- and 150-kDa antigens. The reactions between the specific rabbit antibodies and anti-ids were inhibited by the 87- and 150-kDa antigens. When preincubated with platelets, MAb2.1 (counterpart of MAb1.1) inhibited adhesion to platelets maximally by 46% and MAb2.2 (anti-MAb1.2) inhibited adhesion to platelets maximally by 35%. Together, both MAb2s inhibited the adhesion of S. sanguis to platelets by 81%. MAb2.2 also inhibited induction of platelet aggregation. MAb2.2 immunoprecipitated a biotinylated platelet membrane antigen of 170 kDa (unreduced); MAb2.1 precipitated membrane antigens of 175- and 230-kDa (unreduced). Therefore, platelet binding sites and the receptor for the S. sanguis adhesin and PAAP, respectively, are distinguished by the anti-id MAb2s.
PMCID: PMC173503  PMID: 7642300
19.  Pathogenic and nonpathogenic strains of Entamoeba histolytica can be differentiated by monoclonal antibodies to the galactose-specific adherence lectin. 
Infection and Immunity  1990;58(6):1802-1806.
Entamoeba histolytica infection results in either asymptomatic colonization or invasive colitis and liver abscess. E. histolytica isolates from patients with invasive disease have characteristic isoenzyme profiles (pathogenic zymodemes), suggesting a role for parasite factors in determining the severity of infection. A galactose-specific cell surface lectin from a pathogenic zymodeme was shown to mediate in vitro adherence to human colonic mucins and contact-dependent killing of target cells. Six nonoverlapping antigenic determinants were identified on the 170-kilodalton heavy subunit of the pathogenic lectin. Anti-lectin monoclonal antibodies (MAb) directed against epitopes 1 and 2 enhanced adherence whereas MAb to epitopes 3 through 6 either inhibited or had no effect on adherence. We tested 50 pathogenic and nonpathogenic strains for reactivity to these anti-lectin MAb by radioimmunoassay. MAb to epitopes 1 through 6 reacted in the radioimmunoassay with all 16 pathogenic zymodeme strains tested. In contrast, only MAb to epitopes 1 and 2 bound to the lectin from nonpathogenic strains. Western immunoblots with anti-lectin antibodies showed that the 170-kilodalton heavy subunit was present in the nonpathogenic amebae. Adherence of the nonpathogenic SAW 760 strain to human erythrocytes was enhanced by MAb to epitope 1 and blocked by galactose, confirming the presence of a functionally active lectin. A lectin radioimmunoassay based on MAb to epitopes 1 and 3 proved to be a simple and rapid method to distinguish pathogenic from nonpathogenic amebae in culture. Further exploration of the functional consequences of the antigenic differences demonstrated for the lectin may lead to a better understanding of its role in pathogenesis.
PMCID: PMC258726  PMID: 1692809
20.  Engineering and Characterization of a Mouse/Human Chimeric Anti-Phencyclidine Monoclonal Antibody 
Previously, our laboratory produced a high affinity, anti-phencyclidine (PCP) murine monoclonal antibody (mAb6B5) that also binds other PCP-like arylcyclohexylamines. In this project, mAb6B5 is engineered into a mouse/human chimera (ch-mAb6B5) to assess the feasibility of developing it into a medication for PCP and PCP-like drug abuse. To create ch-mAb6B5, the light and heavy chain constant regions of mAb6B5 were replaced with human κ and IgG2 constant regions in order to decrease its potential immunogenicity in humans. To be an effective anti-PCP medication, ch-mAb6B5 must retain the critical immunochemical binding properties of mAb6B5. Expression vectors containing ch-mAb6B5 light chain and heavy chain cDNA were constructed and expressed in the murine myeloma cell line P3X63-Ag8.653. Immunoassays confirm that ch-mAb6B5 is indeed a chimera, composed of mAb6B5’s PCP-binding variable domains and human κ and IgG constant regions. Radioimmunoassays show that ch-mAb6B5 has the same drug-binding profile as mAb6B5. Ch-mAb6B5 and mAb6B5 bind PCP with a KD of 0.67 nM and 1.17 nM (respectively) and bind PCP-like arylcyclohexylamines 1-[1-(2-thienyl)cyclohexyl]piperidine and N-ethyl-1-phenylcyclohexylamine with similar specificity. Additionally, ch-mAb6B5 and mAb6B5 have the same calculated isoelectric points and molecular weights, critical properties in antigen-antibody interactions. These data demonstrate that mouse/human ch-mAb6B5, a “more human” version of murine mAb6B5, retains mAb6B5’s unique drug-binding properties. This work supports our continued efforts to develop ch-mAb6B5 into a medication for PCP and PCP-like drug abuse – introducing the intriguing possibility of using a single therapeutic mAb for treating a class of abused drugs.
PMCID: PMC2238695  PMID: 18068094
Phencyclidine; Arylcyclohexylamines; Substance abuse; Therapeutic antibody; Chimeric antibody
21.  Overcoming Antigenic Diversity by Enhancing the Immunogenicity of Conserved Epitopes on the Malaria Vaccine Candidate Apical Membrane Antigen-1 
PLoS Pathogens  2013;9(12):e1003840.
Malaria vaccine candidate Apical Membrane Antigen-1 (AMA1) induces protection, but only against parasite strains that are closely related to the vaccine. Overcoming the AMA1 diversity problem will require an understanding of the structural basis of cross-strain invasion inhibition. A vaccine containing four diverse allelic proteins 3D7, FVO, HB3 and W2mef (AMA1 Quadvax or QV) elicited polyclonal rabbit antibodies that similarly inhibited the invasion of four vaccine and 22 non-vaccine strains of P. falciparum. Comparing polyclonal anti-QV with antibodies against a strain-specific, monovalent, 3D7 AMA1 vaccine revealed that QV induced higher levels of broadly inhibitory antibodies which were associated with increased conserved face and domain-3 responses and reduced domain-2 response. Inhibitory monoclonal antibodies (mAb) raised against the QV reacted with a novel cross-reactive epitope at the rim of the hydrophobic trough on domain-1; this epitope mapped to the conserved face of AMA1 and it encompassed the 1e-loop. MAbs binding to the 1e-loop region (1B10, 4E8 and 4E11) were ∼10-fold more potent than previously characterized AMA1-inhibitory mAbs and a mode of action of these 1e-loop mAbs was the inhibition of AMA1 binding to its ligand RON2. Unlike the epitope of a previously characterized 3D7-specific mAb, 1F9, the 1e-loop inhibitory epitope was partially conserved across strains. Another novel mAb, 1E10, which bound to domain-3, was broadly inhibitory and it blocked the proteolytic processing of AMA1. By itself mAb 1E10 was weakly inhibitory but it synergized with a previously characterized, strain-transcending mAb, 4G2, which binds close to the hydrophobic trough on the conserved face and inhibits RON2 binding to AMA1. Novel inhibition susceptible regions and epitopes, identified here, can form the basis for improving the antigenic breadth and inhibitory response of AMA1 vaccines. Vaccination with a few diverse antigenic proteins could provide universal coverage by redirecting the immune response towards conserved epitopes.
Author Summary
Numerous reports of vaccine failure are attributed to a mismatch between the genotype of the vaccine and the circulating target strains. This observation is congruent to the view that polyvalent vaccines protect broadly by inducing a multitude of type-specific antibodies. Polyvalent vaccines that can overcome antigenic diversity by refocusing antibody responses towards conserved functional epitopes are highly desirable. Development of an Apical Membrane Antigen-1 (AMA1) malaria vaccine has been impeded by extreme antigenic diversity in the field. We present here a solution to the AMA1 diversity problem. Antibodies against a mixture of only four naturally occurring AMA1 allelic proteins “Quadvax” inhibited invasion of red blood cells by a diverse panel of malaria parasites that represented the global diversity of AMA1 in the field. Competition experiments suggested that in addition to improving the diversity of strain-specific antibodies, the mechanism of broadened inhibition involved an increase in responses against conserved inhibitory epitopes. Monoclonal antibodies against the Quadvax inhibited invasion either by blocking the binding of AMA1 to its receptor RON2 or by blocking a crucial proteolytic processing event. Some mixtures of these antibodies were much more effective than expected and were shown to act synergistically. Together these two classes of functional invasion inhibitory epitopes can be targeted to engineer a more potent AMA1 vaccine.
PMCID: PMC3873463  PMID: 24385910
22.  Fc-Dependent and Fc-Independent Opsonization of Cryptococcus neoformans by Anticapsular Monoclonal Antibodies: Importance of Epitope Specificity  
Infection and Immunity  2002;70(6):2812-2819.
Monoclonal antibodies (MAbs) reactive with glucuronoxylomannan (GXM), the major capsular polysaccharide of the yeast Cryptococcus neoformans, produce distinct capsular reactions when viewed by differential interference contrast microscopy. These reactions depend on the epitope specificity of the antibody. Opsonic activities of immunoglobulin G1 (IgG1) MAbs that produce patterns termed rim and puffy were examined. Rim-pattern MAbs are reactive with an epitope shared by GXM serotypes A, B, C, and D. Puffy-pattern MAbs are reactive only with serotypes A and D. In phagocytosis assays, using serotype A cells and resident murine peritoneal macrophages, rim-pattern MAbs were markedly more opsonic than puffy-pattern MAbs. F(ab′)2 fragments of rim-pattern MAbs were synergistic with heat-labile factors in normal human serum for opsonization of the yeast. F(ab′)2 fragments of puffy-pattern MAbs were also synergistic with normal serum in opsonization but at a much lower level than fragments of rim-pattern MAbs. Normal serum alone was not opsonic. F(ab′)2 fragments of rim-pattern MAbs, but not puffy-pattern MAbs, stimulated phagocytosis of encapsulated cryptococci in the absence of serum. This serum-independent opsonic action of F(ab′)2 fragments was abrogated by pretreatment of macrophages with purified GXM, suggesting the involvement of a phagocyte GXM receptor. The results indicate that (i) there are multiple mechanisms by which anticapsular IgG MAbs facilitate phagocytosis of encapsulated cryptococci, (ii) some anti-GXM antibodies are opsonic in an Fc-independent manner, and (iii) opsonic activity correlates with the capsular reaction and occurs in an epitope-specific manner.
PMCID: PMC127994  PMID: 12010967
23.  Combined Roles of Human IgG Subclass, Alternative Complement Pathway Activation, and Epitope Density in the Bactericidal Activity of Antibodies to Meningococcal Factor H Binding Protein 
Infection and Immunity  2012;80(1):187-194.
Meningococcal vaccines containing factor H binding protein (fHbp) are in clinical development. fHbp binds human fH, which enables the meningococcus to resist complement-mediated bacteriolysis. Previously, we found that chimeric human IgG1 mouse anti-fHbp monoclonal antibodies (MAbs) had human complement-mediated bactericidal activity only if the MAb inhibited fH binding. Since IgG subclasses differ in their ability to activate complement, we investigated the role of human IgG subclasses on antibody functional activity. We constructed chimeric MAbs in which three different murine fHbp-specific binding domains were each paired with human IgG1, IgG2, or IgG3. Against a wild-type group B isolate, all three IgG3 MAbs, irrespective of their ability to inhibit fH binding, had bactericidal activity that was >5-fold higher than the respective IgG1 MAbs, while the IgG2 MAbs had the least activity. Against a mutant with increased fHbp expression, the anti-fHbp MAbs elicited greater C4b deposition (classical pathway) and greater bactericidal activity than against the wild-type strain, and the IgG1 MAbs had similar or greater activity than the respective IgG3 MAbs. The bactericidal activity against both wild-type and mutant strains also was dependent, in part, on activation of the alternative complement pathway. Thus, at lower epitope density in the wild-type strain, the IgG3 anti-fHbp MAbs had the greatest bactericidal activity. At a higher epitope density in the mutant, the IgG1 MAbs had similar or greater bactericidal activity than the IgG3 MAbs, and the activity was less dependent on the inhibition of fH binding than at a lower epitope density.
PMCID: PMC3255668  PMID: 22064712
24.  High-Avidity and Potently Neutralizing Cross-Reactive Human Monoclonal Antibodies Derived from Secondary Dengue Virus Infection 
Journal of Virology  2013;87(23):12562-12575.
The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies (Abs) and vaccine development. Previous studies of human dengue-immune sera reported that a significant proportion of anti-E Abs, known as group-reactive (GR) Abs, were cross-reactive to all four DENV serotypes and to one or more other flaviviruses. Based on studies of mouse anti-E monoclonal antibodies (MAbs), GR MAbs were nonneutralizing or weakly neutralizing compared with type-specific MAbs; a GR response was thus not regarded as important for vaccine strategy. We investigated the epitopes, binding avidities, and neutralization potencies of 32 human GR anti-E MAbs. In addition to fusion loop (FL) residues in E protein domain II, human GR MAbs recognized an epitope involving both FL and bc loop residues in domain II. The neutralization potencies and binding avidities of GR MAbs derived from secondary DENV infection were stronger than those derived from primary infection. GR MAbs derived from primary DENV infection primarily blocked attachment, whereas those derived from secondary infection blocked DENV postattachment. Analysis of the repertoire of anti-E MAbs derived from patients with primary DENV infection revealed that the majority were GR, low-avidity, and weakly neutralizing MAbs, whereas those from secondary infection were primarily GR, high-avidity, and potently neutralizing MAbs. Our findings suggest that the weakly neutralizing GR anti-E Abs generated from primary DENV infection become potently neutralizing MAbs against the four serotypes after secondary infection. The observation that the dengue immune status of the host affects the quality of the cross-reactive Abs generated has implications for new strategies for DENV vaccination.
PMCID: PMC3838129  PMID: 24027331
25.  The First Human Epitope Map of the Alphaviral E1 and E2 Proteins Reveals a New E2 Epitope with Significant Virus Neutralizing Activity 
Venezuelan equine encephalitis virus (VEEV) is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion) and E2 (binds receptor and elicits virus neutralizing antibodies). Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs). Six E2 epitopes (E2c,d,e,f,g,h) bound VEEV-neutralizing antibody and mapped to amino acids (aa) 182–207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs) with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.
We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants.
Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115–119. Using a 9 Å resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope.
The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo.
Author Summary
Although the murine immune response to Venezuelan equine encephalitis virus (VEEV) is well-characterized, little is known about the human antibody response to VEEV. In this study we used phage display technology to isolate a panel of 11 VEEV-specfic Fabs from two human donors. Seven E2-specific and four E1-specific Fabs were identified and mapped to five E2 epitopes and three E1 epitopes. Two neutralizing Fabs were isolated, E2-specific F5 and E1-specific L1A7, although the neutralizing capacity of L1A7 was 300-fold lower than F5. F5 Fab was expressed as a complete IgG1 molecule, F5 native (n) IgG. Neutralization-escape VEEV variants for F5 nIgG were isolated and their structural genes were sequenced to determine the theoretical binding site of F5. Based on this sequence analysis as well as the ability of F5 to neutralize four neutralization-escape variants of anti-VEEV murine monoclonal antibodies (mapped to E2 amino acids 182–207), a unique neutralization domain on E2 was identified and mapped to E2 amino acids 115–119.
PMCID: PMC2903468  PMID: 20644615

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