Scientific Resources

UNLABELLED: Due to continuous changes to its antigenic regions,influenza viruses can evade immune detection and cause a significant amount of morbidity and mortality around the world. Influenza vaccinations can protect against disease but must be annually reformulated to match the current circulating strains. In the development of a broad-spectrum influenza vaccine,the elucidation of conserved epitopes is paramount. To this end,we designed an immunization strategy in mice to boost the humoral response against conserved regions of the hemagglutinin (HA) glycoprotein. Of note,generation and identification of broadly neutralizing antibodies that target group 2 HAs are rare and thus far have yielded only a few monoclonal antibodies (MAbs). Here,we demonstrate that mouse MAb 9H10 has broad and potent in vitro neutralizing activity against H3 and H10 group 2 influenza A subtypes. In the mouse model,MAb 9H10 protects mice against two divergent mouse-adapted H3N2 strains,in both pre- and postexposure administration regimens. In vitro and cell-free assays suggest that MAb 9H10 inhibits viral replication by blocking HA-dependent fusion of the viral and endosomal membranes early in the replication cycle and by disrupting viral particle egress in the late stage of infection. Interestingly,electron microscopy reconstructions of MAb 9H10 bound to the HA reveal that it binds a similar binding footprint to MAbs CR8020 and CR8043.backslashnbackslashnIMPORTANCE: The influenza hemagglutinin is the major antigenic target of the humoral immune response. However,due to continuous antigenic changes that occur on the surface of this glycoprotein,influenza viruses can escape the immune system and cause significant disease to the host. Toward the development of broad-spectrum therapeutics and vaccines against influenza virus,elucidation of conserved regions of influenza viruses is crucial. Thus,defining these types of epitopes through the generation and characterization of broadly neutralizing monoclonal antibodies (MAbs) can greatly assist others in highlighting conserved regions of hemagglutinin. Here,we demonstrate that MAb 9H10 that targets the hemagglutinin stalk has broadly neutralizing activity against group 2 influenza A viruses in vitro and in vivo. View Publication

mAbs constitute an important biological tool for influenza virus haemagglutinin (HA) epitope mapping through the generation of escape mutants,which could provide insights into immune evasion mechanisms and may benefit the future development of vaccines. Several influenza A (H1N1) pandemic 2009 (pdm09) HA escape mutants have been recently described. However,the HA antigenic sites of the previous seasonal A/Brisbane/59/2007 (H1N1) (Bris07) virus remain poorly documented. Here,we produced mAbs against pdm09 and Bris07 HA proteins expressed in human HEK293 cells. Escape mutants were generated using mAbs that exhibited HA inhibition and neutralizing activities. The resulting epitope mapping of the pdm09 HA protein revealed 11 escape mutations including three that were previously described (G172E,N173D and K256E) and eight novel ones (T89R,F128L,G157E,K180E,A212E,R269K,N311T and G478E). Among the six HA mutations that were part of predicted antigenic sites (Ca1,Ca2,Cb,Sa or Sb),three (G172E,N173D and K180E) were within the Sa site. Eight escape mutations (H54N,N55D,N55K,L60H,N203D,A231T,V314I and K464E) were obtained for Bris07 HA,and all but one (N203D,Sb site) were outside the predicted antigenic sites. Our results suggest that the Sa antigenic site is immunodominant in pdm09 HA,whereas the N203D mutation (Sb site),present in three different Bris07 escape mutants,appears as the immunodominant epitope in that strain. The fact that some mutations were not part of predicted antigenic sites reinforces the necessity of further characterizing the HA of additional H1N1 strains. View Publication

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However,one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al.,Proc Natl Acad Sci USA,104,6283-6288,2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs,which contained instead nearby single amino acid changes in the HA head. Thus,either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover,this site is relevant for the human antibody response,as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.

A rapid,ultrasensitive,and practical label-free impedimetric immunoassay for measuring trace levels of total polychlorinated biphenyls (PCBs) in insulating oil was developed. First,we developed a novel monoclonal antibody (RU6F9) for PCBs by using a designed immunogen and characterized its binding affinity for a commercial mixtures of PCBs and its main congeners. A micro comblike gold electrode was fabricated,and the antibody was covalently immobilized on the electrode through a self-assembled monolayer formed by dithiobis-N-succinimidyl propionate. The antigen-binding event on the surface of the functionalized electrode was determined as the change in charge transfer resistance by using electrochemical impedance spectroscopy. The resulting impedimetric immunoassay in aqueous solution achieved a wide determination range (0.01-10 μg/L) and a low detection limit (LOD) of 0.001 μg/L,which was 100-fold more sensitive than a conventional flow-based immunoassay for PCBs. By combining the impedimetric immunoassay with a cleanup procedure for insulating oil utilizing a multilayer cleanup column followed by DMSO partitioning,an LOD of 0.052 mg/kg-oil was achieved,which satisfied the Japanese regulation criterion of 0.5 mg/kg-oil. Finally,the immunoassay was employed to determine total PCB levels in actual used insulating oils (n = 33) sampled from a used transformer containing trace levels of PCBs,and the results agreed well with the Japanese official method (HRGC/HRMS).

Monoclonal antibodies (mAbs) have proven to be instrumental in the advancement of research,diagnostic,industrial vaccine,and therapeutic applications. The use of mAbs in laboratory protocols has been growing in an exponential fashion for the last four decades. Described herein are methods for the development of highly specific mAbs through traditional hybridoma fusion. For ultimate success,a series of simultaneously initiated protocols are to be undertaken with careful attention to cell health of both the myeloma fusion partner and immune splenocytes. Coordination and attention to detail will enable a researcher with basic tissue culture skills to generate mAbs from immunized rodents to a variety of antigens (including proteins,carbohydrates,DNA,and haptens) (see Note 1). Furthermore,in vivo and in vitro methods used for antigen sensitization of splenocytes prior to somatic fusion are described herein. View Publication

In February 2013,zoonotic transmission of a novel influenza A virus of the H7N9 subtype was reported in China. Although at present no sustained human-to-human transmission has been reported,a pandemic outbreak of this H7N9 virus is feared. Since neutralizing antibodies to the hemagglutinin (HA) globular head domain of the virus are virtually absent in the human population,there is interest in identifying other correlates of protection,such as cross-reactive CD8(+) T cells (cytotoxic T lymphocytes [CTLs]) elicited during seasonal influenza A virus infections. These virus-specific CD8(+) T cells are known to recognize conserved internal proteins of influenza A viruses predominantly,but it is unknown to what extent they cross-react with the newly emerging H7N9 virus. Here,we assessed the cross-reactivity of seasonal H3N2 and H1N1 and pandemic H1N1 influenza A virus-specific polyclonal CD8(+) T cells,obtained from HLA-typed study subjects,with the novel H7N9 virus. The cross-reactivity of CD8(+) T cells to H7N9 variants of known influenza A virus epitopes and H7N9 virus-infected cells was determined by their gamma interferon (IFN-γ) response and lytic activity. It was concluded that,apart from recognition of individual H7N9 variant epitopes,CD8(+) T cells to seasonal influenza viruses display considerable cross-reactivity with the novel H7N9 virus. The presence of these cross-reactive CD8(+) T cells may afford some protection against infection with the new virus. View Publication

Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover,these practices are molecule-specific and so only support one assay for one program at a time. Here,we describe a strategy to generate a unique assay reagent,10C4,that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This panel-specific" feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational� View Publication

OBJECTIVE: The S100A9 and S100A8 proteins are highly expressed by neutrophils and monocytes and are part of a group of damage-associated molecular pattern molecules that trigger inflammatory responses. Sera and synovial fluids of patients with rheumatoid arthritis (RA) contain high concentrations of S100A8/A9 that correlate with disease activity.backslashnbackslashnMETHODS: In this study,we investigated the importance of S100A9 in RA by using neutralizing antibodies in a murine lipopolysaccharide-synchronized collagen-induced arthritis model. We also used an in vitro model of stimulation of human immune cells to decipher the role played by S100A9 in leukocyte migration and pro-inflammatory cytokine secretion.backslashnbackslashnRESULTS: Treatment with anti-S100A9 antibodies improved the clinical score by 50%,diminished immune cell infiltration,reduced inflammatory cytokines,both in serum and in the joints,and preserved bone/collagen integrity. Stimulation of neutrophils with S100A9 protein led to the enhancement of neutrophil transendothelial migration. S100A9 protein also induced the secretion by monocytes of proinflammatory cytokines like TNFα,IL-1β and IL-6,and of chemokines like MIP-1α and MCP-1.backslashnbackslashnCONCLUSION: The effects of anti-S100A9 treatment are likely direct consequences of inhibiting the S100A9-mediated promotion of neutrophil transmigration and secretion of pro-inflammatory cytokines from monocytes. Collectively,our results show that treatment with anti-S100A9 may inhibit amplification of the immune response and help preserve tissue integrity. Therefore,S100A9 is a promising potential therapeutic target for inflammatory diseases like rheumatoid arthritis for which alternative therapeutic strategies are needed. View Publication

The 2009 H1N1 influenza pandemic was a major international public health crisis which caused considerable morbidity and mortality worldwide. The goal of this study was to produce anti-H1 monoclonal antibodies (MAbs) for improving diagnostic immunological assays and to develop potential immunotherapeutics. Nine MAbs were produced after immunizing mice with recombinant hemagglutinin (HA) protein from A/California/06/09. Two spleenocyte myeloma fusions yielded 1588 hybridoma cultures. After screening the hybridoma culture supernatants for antibody reactivity to rHA,nine clones were selected for further characterization. Cross-reactivity studies of the anti-rHA antibodies against a panel of influenza viruses (H1-H16) revealed eight out of nine MAbs were specific to the pandemic H1 subtype,except for MAb F256G2sc1 which also cross-reacted with H5 subtype virus. All MAbs were of the IgG1κ isotype,except F256G2sc1 which was IgG2aκ. The anti-rHA MAbs had binding affinities to rHA that ranged from a K(D) (disassociation constant) of 1.34×10(-9)M (F255G7sc1) to the weakest affinity of 4.60×10(-8)M (F255G4sc1). Interestingly,in a plaque reduction neutralization assay,all MAbs except F255G3sc1 demonstrated neutralizing ability. Furthermore,all MAbs except F255G3sc1 and F255G9sc1 exhibited anti-hemagglutinin activity against pandemic H1N1 viruses,but not against classical North American swine influenza viruses of the same subtype. Immunofluorescence assay (IFA) demonstrated that all MAbs except F255G1sc1 and F255G3sc1 were able to detect 2009 pandemic H1N1 (2009) virus- infected MDCK cells. The MAbs were also evaluated for potential use in competitive ELISA (cELISA),and with the exception of F255G3sc1,all MAbs showed competitive activity with serum collected from pigs infected with pandemic H1N1 virus (2009). The developed MAbs have demonstrated utility as immunodiagnostic and research reagents,and their neutralizing capabilities also hold potential for designing antiviral drugs against pandemic influenza. View Publication

Tools permitting the isolation of live pancreatic cell subsets for culture and/or molecular analysis are limited. To address this,we developed a collection of monoclonal antibodies with selective surface labeling of endocrine and exocrine pancreatic cell types. Cell type labeling specificity and cell surface reactivity were validated on mouse pancreatic sections and by gene expression analysis of cells isolated using FACS. Five antibodies which marked populations of particular interest were used to isolate and study viable populations of purified pancreatic ducts,acinar cells,and subsets of acinar cells from whole pancreatic tissue or of alpha or beta cells from isolated mouse islets. Gene expression analysis showed the presence of known endocrine markers in alpha and beta cell populations and revealed that TTR and DPPIV are primarily expressed in alpha cells whereas DGKB and GPM6A have a beta cell specific expression profile. View Publication

Trypanosoma congolense is an African trypanosome that causes serious disease in cattle in Sub-Saharan Africa. The four major life cycle stages of T. congolense can be grown in vitro,which has led to the identification of several cell-surface molecules expressed on the parasite during its transit through the tsetse vector. One of these,glutamic acid/alanine-rich protein (GARP),is the first expressed on procyclic forms in the tsetse midgut and is of particular interest because it replaces the major surface coat molecule of bloodstream forms,the variant surface glycoprotein (VSG) that protects the parasite membrane,and is involved in antigenic variation. Unlike VSG,however,the function of GARP is not known,which necessarily limits our understanding of parasite survival in the tsetse. Toward establishing the function of GARP,we report its three-dimensional structure solved by iodide phasing to a resolution of 1.65 Å. An extended helical bundle structure displays an unexpected and significant degree of homology to the core structure of VSG,the only other major surface molecule of trypanosomes to be structurally characterized. Immunofluorescence microscopy and immunoaffinity-tandem mass spectrometry were used in conjunction with monoclonal antibodies to map both non-surface-disposed and surface epitopes. Collectively,these studies enabled us to derive a model describing the orientation and assembly of GARP on the surface of trypanosomes. The data presented here suggest the possible structure-function relationships involved in replacement of the bloodstream form VSG by GARP as trypanosomes differentiate in the tsetse vector after a blood meal. View Publication

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia in the United States and globally. Despite the availability of pneumococcal capsular polysaccharide (PPS) and protein conjugate-based vaccines,the prevalence of antibiotic-resistant pneumococcal strains,serotype (ST) replacement in nonconjugate vaccine strains,and uncertainty as to whether the PPS vaccine that is used in adults protects against pneumonia emphasize the need for continued efforts to understand the nature of protective PPS antibody responses. In this study,we generated mouse monoclonal antibodies (MAbs) to a conjugate consisting of the PPS of serotype 8 (PPS8) S. pneumoniae and tetanus toxoid. Thirteen MAbs,including four IgMs that bound to PPS8 and phosphorylcholine (PC) and five IgMs and four IgG1s that bound to PPS8 but not PC,were produced,and their nucleotide sequences,epitope and fine specificity,and efficacy against lethal challenge with ST8 S. pneumoniae were determined. MAbs that bound to PPS8 exhibited gene use that was distinct from that exhibited by MAbs that bound to PC. Only PPS8-binding MAbs that did not bind PC were protective in mice. All 13 MAbs used germ line variable-region heavy (V(H)) and light (V(L)) chain genes,with no evidence of somatic hypermutation. Our data reveal a relationship between PPS specificity and V(H) gene use and MAb efficacy in mice. These findings provide insight into the relationship between antibody molecular structure and function and hold promise for the development of novel surrogates for pneumococcal vaccine efficacy. View Publication