Antibody Response to Lipopolysaccharide in Patients Colonized or Infected with an Endemic Strain of Acinetobacter Genomic Species 13 Sensu Tjernberg and Ursing

The levels of antilipopolysaccharide (anti-LPS) antibodies in patients colonized with an endemic Acinetobacter strain were compared to those in patients with bloodstream infections. Seropositivity and seronegativity correlated with positive and negative blood cultures, respectively, indicating that determination of the level of anti-LPS antibodies is useful for diagnosing Acinetobacter infections.     

Antibody response to lipopolysaccharide in patients colonized or infected with an endemic strain of Acinetobacter genomic species 13 sensu Tjernberg and Ursing

The levels of antilipopolysaccharide (anti-LPS) antibodies in patients colonized with an endemic Acinetobacter strain were compared to those in patients with bloodstream infections. Seropositivity and seronegativity correlated with positive and negative blood cultures, respectively, indicating that determination of the level of anti-LPS antibodies is useful for diagnosing Acinetobacter infections.     

Use of Representational Difference Analysis To Identify Genomic Differences between Pathogenic Strains of Vibrio cholerae

Representational difference analysis (RDA) is a recently developed technique used for amplifying genetic differences between two closely related genomes. We compared RDA and a modified version of RDA to examine genomic differences between the two Vibrio cholerae serogroups that cause epidemic cholera, O1 and O139, and between the two biotypes of the O1 serogroup. With both techniques, we recovered several sequences known to be found only in V. cholerae O139 but absent in its presumed progenitor, V. cholerae O1 El Tor. A greater number of unique fragments were generated in comparing the two V. cholerae O1 biotypes, consistent with the probable greater genetic differences between the two biotypes.     

Use of Monoclonal Antibodies To Identify Serotypes of Enterovirus Isolates

Nonpoliovirus enteroviruses cause a variety of diseases that are common in young children and adults. The “gold standard” for laboratory diagnosis of enteroviruses is cell culture isolation, followed by serotype identification by neutralization assay. These procedures are time-consuming and expensive. Rapid serotype identification of enteroviruses is important in differentiating nonpoliovirus enterovirus pathogens from vaccine strain polioviruses that can be shed for some time after vaccination. In the present investigation, we evaluated a rapid method for serotype identification of enteroviruses by indirect immunofluorescence assay (IFA) using commercially available monoclonal antibodies for polioviruses, coxsackieviruses type B, and six serotypes of commonly circulating echoviruses. Of 291 isolates of enteroviruses included in the study, 95 were polioviruses and 196 were nonpoliovirus enteroviruses. Two hundred thirty-four of these (38 polioviruses and 196 nonpoliovirus enteroviruses) were consecutively grown in the laboratory over a 5-year period. IFA identified the serotypes of 74% of the consecutive isolates and 71% of all enterovirus isolates by yielding a positive staining result. The levels of agreement in the identification of the enterovirus group between IFA and neutralization tests were 92% for consecutively grown isolates and 85% for all enterovirus isolates. The sensitivity of the IFA for the detection of viruses for which specific monoclonal antibodies were applied was 73% for polioviruses, 85% for coxsackieviruses type B, and 94% for echoviruses. Specificity was near 100% for polioviruses and coxsackieviruses type B and 94% for echoviruses. We conclude that IFA can be helpful as a preliminary test for serotype identification of enteroviruses. The results are most accurate when the test identifies the isolate as a poliovirus.     

Generation and Serological Characterization of Murine Monoclonal Antibodies against O Antigens from Acinetobacter Reference Strains

O-antigen-specific monoclonal antibodies were generated against Acinetobacter strains from international type culture collections and characterized by enzyme immunoassay and Western and colony blotting. The antibodies aid in the further completion of an O-serotyping scheme for Acinetobacter and, due to their high specificity, are especially useful to all working with these strains.     

Use of an Isogenic Mutant Constructed in Moraxella catarrhalis To Identify a Protective Epitope of Outer Membrane Protein B1 Defined by Monoclonal Antibody 11C6

Moraxella catarrhalis-induced otitis media continues to be a significant cause of infection in young children, prompting increased efforts at identifying effective vaccine antigens. We have previously demonstrated that M. catarrhalis expresses specific outer membrane proteins (OMPs) in response to iron limitation and that this organism can utilize transferrin and lactoferrin for in vitro growth. One of these proteins, which binds human transferrin, is OMP B1. As the human host presents a naturally iron-limited environment, proteins, like OMP B1, which are expressed in response to this nutritional stress are potential vaccine antigens. In this study, we have developed monoclonal antibody (MAb) 11C6, which reacts to a surface-exposed epitope of OMP B1 expressed by M. catarrhalis 7169. This antibody was used to clone ompB1, and sequence analysis suggested that OMP B1 is the M. catarrhalis homologue to the transferrin binding protein B described for pathogenic Neisseriaceae, Haemophilus influenzae, Actinobacillus pleuropneumoniae, and M. catarrhalis. Expression of recombinant OMP B1 on the surface of Escherichia coli confers transferrin binding activity, confirming that this protein is likely involved in iron acquisition. In addition, ompB1 was used to construct an isogenic mutant in M. catarrhalis 7169. This mutant, termed 7169b12, was used as the control in bactericidal assays designed to determine if OMP B1 elicits protective antibodies. In the presence of MAb 11C6 and human complement, wild-type 7169 demonstrated a 99% decline in viability, whereas the ompB1 isogenic mutant was resistant to this bactericidal activity. Further analysis with MAb 11C6 revealed the presence of this OMP B1 epitope on 31% of the clinical isolates tested. These data suggest that OMP B1 is a potential vaccine antigen against M. catarrhalis infections.     

Use of a Monoclonal Antibody against an Escherichia coli O26 Surface Protein for Detection of Enteropathogenic and Enterohemorrhagic Strains

A monoclonal antibody (MAb) was obtained from a mouse immunized with solubilized outer membrane proteins extracted from a bovine enterohemorrhagic strain of Escherichia coli (EHEC), O26. The MAb produced a strong immunoblot reaction at approximately 21 kDa for an O26 strain containing the intimin gene (eae) and verocytotoxin (VT), but not with an O26 eae- and VT-negative strain, or O157 eae- and VT-positive strains. The MAb was used in a sandwich enzyme-linked immunosorbent assay (ELISA) format to screen strains from animal and human sources, and all reactive strains were characterized for the presence of eae and the gene encoding VT factors by PCR. The antigen was detected in a group of strains containing a high proportion of O26, the majority of which were eae positive with or without VT; these were isolated mostly from animal enteritis cases but included a small number of human enteric isolates. Nonreactors included eae-positive (with or without VT) O157 strains and one O26 strain. In a survey of mixed cultures from both animal and human enteric disease, ELISA-positive reactions were obtained from 7.1 to 11.2% of samples from bovine, porcine, ovine, and human sources. The two human O8 and ten animal O26 ELISA-reactive pure strains obtained from these samples contained six eae- and/or VT-positive strains; the other six strains lost their ELISA positivity following storage at −70°C, after which none were found to contain either eae or VT factors. The association of the antigen detected by the MAb with significant enteropathogenic E. coli and EHEC virulence factors in isolates from both animal and human enteric infections indicates a diagnostic potential for the assay developed.     

Production and Characterization of a Novel Monoclonal Antibody Inhibitory for Murine Natural Killer Cell Activity

Natural killer (NK) cells are considered to play an important role in tumor surveillance. The killing of tumor target cells by NK cells is the result of a complex series of sequential binding, signal processing and lytic events. However, the mechanism which NK cells use to recognize tumor targets is poorly understood. To further study the cell-surface molecules involved in tumor recognition, we immunized rats against cloned murine T cells with NK activity (DBA/2.1) and generated rat-mouse hybridomas which were screened for the ability to block lytic activity of DBA/2.1 effector cells. Culture supernatants from one IgM-producing hybridoma, designated S1C4, were found to consistently inhibit DBA/2.1-mediated lysis of YAC-1 target cells. Endogenous splenic NK activity was also diminished in the presence of S1C4 monoclonal antibody (mAb) while alloantigen-specific cytotoxic T lymphocyte (CTL) activity was not affected. S1C4 mAb appears to react with effector cell-surface structures involved in the recognition/adhesion phase of NK activity since pretreatment of effector cells with mAb S1C4 inhibits their ability to bind to YAC-1 target cells. ELISA studies revealed that the S1C4 antigen is expressed by a range of lymphoid cell lines, as well as by DBA/2.1 cells and fresh splenic NK cells. S1C4 mAb were shown to react with 22, 24, 30, and 46 kiloDalton (kDa) DBA/2.1 cell membrane components on immunoblots performed under reducing conditions. These structures do not correspond to any known recognition/adhesion molecules, suggesting that mAb S1C4 defines novel cell membrane components involved in NK cell function.     

Attempted Passive Prophylaxis with a Monoclonal Anti-Burkholderia Pseudomallei Exopolysaccharide Antibody in a Murine Model of Melioidosis

Melioidosis is a severe gram-negative infection caused by the facultative intracellular bacterium Burkholderia pseudomallei, which is responsible for a broad spectrum of symptoms in both humans and animals. No licensed vaccine currently exists. This study evaluated the protective effect of a monoclonal antibody (Mab Ps6F6) specific to B. pseudomallei exopolysaccharide in an outbred murine model of sub-acute melioidosis. When administered before the infectious challenge, Ps6F6 significantly increased resistance to infection and restrained bacterial burden in the spleen over a 30-days period. Patterns of IFN-γ production were similar in the treated and non treated groups of mice. However, Ps6F6 lowered IFN-γ levels over the duration of the assay period, except on day 1, suggesting a transient and rapid production of IFN-γ under Ps6F6 control. Minor but persisting increases occurred in IL-12 levels while TNF-α was detected only in the controls at the later stages of infection. No IL-10 secretion was detected in both groups of mice. These data suggest that passive prophylaxis with Mab Ps6F6 provide a moderate and transient induction of inflammatory responses in infected mice but failed to trigger a sterilizing protective immunity.     

A Murine Monoclonal Antibody (928) Recognizing a New Epitope Formed with a Combination of and Gene Products

ABSTRACT: A murine monoclonal antibody (mAb), 928, that recognizes a cell surface antigen (928 Ag) on a human Epstein-Barr virus-transformed fetal liver-derived lymphoid progenitor cell line (FL4.4) was generated. The 928 mAb reacted with only FL4.4; it did not react with any other 57 cell lines tested. Two color flowcytometry analysis of peripheral blood mononuclear cells (PBMC) revealed that the 928 mAb reacted with B cell and monocyte fractions from only two individuals out of 63 unrelated donors. Biochemical analyses showed that the 928 Ag composes of two molecules (33 and 34 K_d) and forms a SDS-resistant, noncovalently linked dimer conformation, the feature being similar to that of peptide-bound MHC class II molecules. Treatment of FL4.4 cells with the 928 mAb significantly facilitated homotypic cell aggregation. In addition, treatment of PBMC of the 928 Ag⁺ donor with recombinant IL-4 augmented the expression of the 928 Ag on CD64⁺ monocytes. Typing of and alleles of the 928 Ag expressing and nonexpressing cells revealed that the 928 Ag is expressed only on PBMC of and positive donors. Finally, anti-DP antibody precleared 928 Ag from the cell lysate. These results demonstrate that the 928 mAb recognizes a polymorphic determinant of - gene products. The possibility that amino acids in the groove of the peptide-binding site of HLA-DP molecules are critical for the 928 epitope is discussed.     

Use of a Single Monoclonal Antibody To Determine the Susceptibilities of Herpes Simplex Virus Type 1 and Type 2 Clinical Isolates to Acyclovir

This report describes a flow cytometry drug susceptibility assay that uses a single fluorochrome-labeled monoclonal antibody to determine the acyclovir susceptibilities of herpes simplex virus (HSV) type 1 or type 2 clinical isolates. This assay yields 50% effective doses (drug concentrations that reduce the number of antigen-positive cells by 50%) for HSV clinical isolates that are equivalent to those obtained with the plaque reduction assay.     

Use of Amplified Fragment Length Polymorphism To Identify 42 Cladophialophora Strains Related to Cerebral Phaeohyphomycosis with In Vitro Antifungal Susceptibility

The amplified fragment length polymorphism technique has been applied to identify neurotropic chaetothyrialean black yeasts and relatives from clinical sources. Cladophialophora bantiana, C. emmonsii, C. arxii, C. devriesii, and C. modesta, previously identified on the basis of sequencing and phenotypic and physiological criteria, were confirmed by cluster analysis, demonstrating the clear separation of C. bantiana as a rather homogeneous group from the other species. C. bantiana is a neurotropic fungus causing cerebral abscesses with a mortality of up to 70%. Successful therapy consists of neurosurgical intervention and optimal antifungal therapy. Since the latter is not clearly defined in a large series, we tested the in vitro activities of eight antifungal drugs against clinical isolates of C. bantiana (n = 37), C. modesta (n = 2), C. arxii (n = 1), C. emmonsii (n = 1), and C. devriesii (n = 1), all of which had caused invasive infections. The resulting MIC₉₀s for all neurotropic C. bantiana strains were as follows, in increasing order: posaconazole, 0.125 μg/ml; itraconazole, 0.125 μg/ml; isavuconazole, 0.5 μg/ml; amphotericin B, 1 μg/ml; voriconazole, 2 μg/ml; anidulafungin, 2 μg/ml; caspofungin, 4 μg/ml; and fluconazole, 64 μg/ml. On the basis of these in vitro results and the findings of previous clinical and animal studies, posaconazole seems to be a good alternative to the standard treatment, amphotericin B, for C. bantiana cerebral infections. The new agent isavuconazole, which is also available as an intravenous preparation, has adequate activity against C. bantiana.The genus Cladophialophora represents anamorph members of the ascomycetes in the order Chaetothyriales in the family Herpotrichiellaceae comprising the black yeasts and relatives (10). These dematiaceous fungi are normally associated with soil or vegetative matter; however, they are increasingly being seen as causative agents of mycoses in humans (27, 37, 48) and domestic (14, 23) and wild (29) animals. Cladophialophora carrionii is the type species and an agent of chromoblastomycosis, a cutaneous and subcutaneous disease. The genus Cladophialophora encompasses several other clinically significant species which are potentially able to cause severe fungal infections in otherwise immunocompetent patients. In human infections, the brain is frequently involved (27, 37, 48). Within the genus, the majority of brain abscesses with fatal outcomes are associated with Cladophialophora bantiana (formerly Cladosporium bantianum, Cladosporium trichoides, Cladosporium trichoides var. chlamydosporum, Torula bantiana, and Xylohypha bantiana), a neurotropic fungus, although severe phaeohyphomycotic infections are also caused by novel Cladophialophora species like C. modesta (40, 44), C. arxii (53, 56), C. emmonsii (Xylohypha emmonsii) (45), C. devriesii (22, 28, 42) C. saturnica (4), and C. boppii (34). Moreover, Exophiala dermatitidis and Rhinocladiella mackenziei, other members of the black yeast group, are also frequently isolated from cerebral infections (8, 27, 37). Central nervous system infection due to C. bantiana is reported worldwide, though a general preference for warmer climates with high humidity is apparent (27). Indeed, many cases are reported from India (19, 30, 33, 55), as opposed to arid climatic zones (8). The first case of C. bantiana (Cladosporium trichoides) infection was reported in 1952, when the fungus was isolated from a human brain abscess and was demonstrated to be neurotropic in laboratory animals (6). A review of 17 cases of brain abscess, published in the English language literature by the mid-1970s, reported that the majority of patients had no underlying disease (41). The most recent series of 48 patients with brain abscess due to C. bantiana showed that 35 patients (72%) had no risk factors and that only 13 patients (28%) survived the infection, despite combined surgical and antifungal treatment (48). The minority of immunocompromised patients are transplant recipients, intravenous drug abusers, or individuals on steroids (2, 13, 15, 24, 31, 35, 36, 51, 54, 57, 59).The mode of infection is either by hematogenous spread from an unrecognized pulmonary focus, through direct extension from adjacent paranasal sinuses, or by penetrating trauma to the head. However, the majority of patients had had no recent evidence of pulmonary or sinus infections (27). Cladophialophora species are prone to identification problems (3, 20). Due to the high degree of phenotypic similarity between recently described new Cladophialophora species and C. bantiana, identification problems are imminent. For most cases published in the older literature, identification down to the species level cannot be repeated or confirmed by molecular methods due to the absence of the original isolates; hence, the etiological agent described in older publications may often have been misidentified. It is now well established that molecular identification methods, which have driven new developments in fungal taxonomy, are more reliable than classical morphological methods (3, 20). Amplified fragment length polymorphism (AFLP) is a technique based on the detection of genomic restriction fragments by PCR amplification, which can be used with the DNA of any organism (58). The purpose of this study was to study the inter- and intraspecific genomic variations of 42 Cladophialophora isolates stored in the CBS collection and recovered from cases with cerebral phaeohyphomycosis and other infections. Antifungal therapy is mainly based on the experience gathered from and published in isolated case reports and mostly involved amphotericin B, itraconazole, and flucytosine singly or in different combinations (48). Animal studies (1) and human experience suggested that amphotericin B has no value in treating cerebral infections, probably due to poor penetration of the central nervous system (CNS), but that triazoles might be of value (26, 38). A recent study of antifungal therapy in a murine model of disseminated infection by C. bantiana confirmed the poor activity of amphotericin B but found that posaconazole and flucytosine extended survival (39). This suggests that new antifungal drugs with broad-spectrum activity and suitable pharmacokinetic profiles compared with those of conventional antifungal agents might be more effective against C. bantiana (1, 39). Only limited data on in vitro antifungal activities against the neurotropic fungus C. bantiana are available. Therefore, with no standard therapy available, unfavorable results in animal experiments, and only a small published series of susceptibility testing with itraconazole and voriconazole (47), the second objective of this study was in vitro testing of this large collection of Cladophialophora strains for their susceptibilities to eight antifungal drugs, including the new triazole isavuconazole.(Part of this work was presented as a poster at Trends in Medical Mycology, Athens, Greece, October 2009 [4a].)     

肝癌单抗—蓖麻毒素A链结合物的制备及其细胞毒性质

本文应用异型双功能基化合物SPDP与单克隆抗体反应,产生单克隆抗体-PDP,然后再与暴露巯基的RTA结合,制备肝癌单抗-RTA结合物。ELISA及毒素重组试验鉴定表明:结合物在10~(-9)mol/L浓度时与靶细胞仍有较好的结合能力,结合物中RTA保持良好活力。体外细胞毒结果表明:结合物对靶细胞具有良好的导向杀伤作用,在10~(-9)mol/L浓度时对人肝癌细胞株BEL7404的杀伤率为41.2％,对正常细胞SL_7的杀伤力很弱。     

Identification of a Human Monoclonal Antibody To Replace Equine Diphtheria Antitoxin for Treatment of Diphtheria Intoxication

Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 μg of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic.     

Identification,Genotypic Relation,and Clinical Features of Colistin-Resistant Isolates of Acinetobacter Genomic Species 13BJ/14TU from Bloodstreams of Patients in a University Hospital

Colistin resistance remains rare among clinical isolates of Acinetobacter species. We noted the emergence of colistin-resistant bloodstream isolates of the Acinetobacter genomic species (GS) 13BJ/14TU from patients at a university hospital between 2003 and 2011. We report here, for the first time, the microbiological and molecular characteristics of these isolates, with clinical features of Acinetobacter GS 13BJ/14TU bacteremia. All 11 available patient isolates were correctly identified as Acinetobacter GS 13BJ/14TU using partial rpoB gene sequencing but were misidentified using the phenotypic methods Vitek 2 (mostly as Acinetobacter baumannii), MicroScan (mostly as A. baumannii/Acinetobacter haemolyticus), and the API 20 NE system (all as A. haemolyticus). Most isolates were susceptible to commonly used antibiotics, including carbapenems, but all were resistant to colistin, for which it is unknown whether the resistance is acquired or intrinsic. However, the fact that none of the patients had a history of colistin therapy strongly suggests that Acinetobacter GS 13BJ/14TU is innately resistant to colistin. The phylogenetic tree of multilocus sequence typing (MLST) showed that all 11 isolates formed a separate cluster from other Acinetobacter species and yielded five sequence types. However, pulsed-field gel electrophoresis (PFGE) revealed 11 distinct patterns, suggesting that the bacteremia had occurred sporadically. Four patients showed persistent bacteremia (6 to 17 days), and all 11 patients had excellent outcomes with cleared bacteremia, suggesting that patients with Acinetobacter GS 13BJ/14TU-associated bacteremia show a favorable outcome. These results emphasize the importance of precise species identification, especially regarding colistin resistance in Acinetobacter species. In addition, MLST offers another approach to the identification of Acinetobacter GS 13BJ/14TU, whereas PFGE is useful for genotyping for this species.     

Use of Monoclonal Antibodies To Identify Phospholipase C as the Enterotoxic Factor of the Bifunctional Hemolysin-Phospholipase C Molecule of Vibrio cholerae O139

Two hybrid clones producing monoclonal antibodies (MAbs) raised against the purified enterotoxic hemolysin-phospholipase C (HlyPC) bifunctional molecule of a Vibrio cholerae O139 strain were used to study its enterotoxicity in relation to its hemolytic and enzymatic activities. Fab fragments of MAbs from ascites produced by the two hybrids neutralized the hemolytic activity of HlyPC, leaving the enzymatic activity unaffected. In ligated rabbit ileal loop and infant mouse intestine, the Fab fragments of the MAbs were not able to neutralize the enterotoxicity of HlyPC, suggesting that PC rather than Hly is the enterotoxic moiety of the molecule. The enterotoxicity of the purified PC molecule isolated from an Hly⁻ spontaneous mutant of the HlyPC-producing parent strain further confirms this contention. The Hly molecule isolated from a PC⁻ mutant was not diarrheagenic.     

胃癌单克隆抗体MGb1在肿瘤患者血清诊断中的初步应用

作者应用刀豆凝集素与单克隆抗体MGb1 ELISA夹心法对238例肿瘤患者、良性疾病患者及正常人的血清进行检测,以探讨检测MGb1相应抗原在肿瘤诊断中的意义。结果显示:单抗MGb1相应抗原在多种肿瘤患者血清中均可检出,其阳性率如下:胃癌患者为48.5％,结肠癌为70.0％,直肠癌为38.1％;卵巢癌为30.0％,肺癌为29.6％,肝癌为35.3％;51例良性疾病患者假谢性率为13.7％;正常人为6.3％。其中胃癌、结肠癌患者与良性疾病患者比较,相差显著,对诊断具有一定帮助。     

A Murine Monoclonal Anti-Idiotypic Antibody Detects a Common Idiotope on Human, Mouse and Rabbit Antibodies to Allergen Lol p IV

A syngeneic mouse monoclonal anti-idiotypic antibody (anti-Id), designated as B1/1, was generated against a monoclonal antibody (MoAb 91) specific for Ryegrass pollen allergen Lol p IV. This anti-Id recognized an idiotope (Id) that was also present on other monoclonal antibodies with the same specificity as MoAb 91. Observations that (i) the anti-Id inhibited the binding of MoAb 91 to Lol p IV and (ii) the Id-anti-Id interaction could be inhibited by Lol p IV indicated that the Id was located within or near the antigen combining site. These properties served to characterize B1/1 as an internal image anti-Id. Evidence that an immune response in different species to Lol p IV elicits the formation of antibodies which express a common Id was provided by the observations that (i) the Id-anti-Id interactions could be inhibited by mouse, human and rabbit antisera to Lol p IV and (ii) the binding of these antisera to Lol p IV could be inhibited by the anti-Id. Interestingly, the internal image anti-Id B1/1 also recognized an Id on a monoclonal antibody which was directed to an epitope of Lol p IV, different from that recognized by MoAb 91.     

Use of Toll-Like Receptor Assays To Detect and Identify Microbial Contaminants in Biological Products

Toll-like receptor (TLR)-expressing cells, for the first time, detected and identified a microbial contaminant in a product made in Escherichia coli using an old manufacturing process. It was suspected of having a microbial contaminant(s) because, although it tested negative by standard pyrogen assays, it was associated with adverse events in early clinical trials. The assay readout is the induction of NF-κB and/or cytokines in response to TLR activation. Four coded samples, labeled A to D, including a sample prepared by the older manufacturing process, were submitted. The cell lines were activated only by samples B and D. Sample D stimulated only Mono-Mac 6 and HEK-human TLR4 (hTLR4) cells and was later identified as lipopolysaccharide. Except for TLR3 cells, sample B stimulated cells bearing the different TLRs (TLRs 2, 4, 5, 7, 8, and 9) and nontransfected HEK293 cells. These data suggested that flagellin was the microbial contaminant, since TLR5, the receptor for flagellin, is known to be expressed constitutively on HEK293 cells. Moreover, purified flagellin from Salmonella enterica serovar Typhimurium behaved like sample B, stimulating HEK293 and HEK-hTLR5 cells but not HEK-hTLR3 cells, and this stimulation by flagellin and sample B was blocked by an anti-hTLR5 neutralizing antibody. Western blots showed bands positive for flagellin and sample B with the molecular sizes expected for the flagellins from S. Typhimurium and E. coli, respectively. Mass spectrometry data were consistent with the presence of flagellin in the manufacturer''s sample B. Taken together, these data indicate that the microbial contaminant in sample B was flagellin and may have been associated with adverse events when the recombinant product was administered.Biological products, including cellular and acellular vaccines, cells used in gene therapy, and plasma-derived and recombinant proteins, can become contaminated with many different types of organisms, e.g., gram-positive and gram-negative bacteria, fungi, viruses, parasites, and their by-products, during manufacturing. When the product is administered to a patient, these microbial contaminants may cause unwanted side effects, such as by inducing inflammation due to the release of cytokines or by acting as adjuvants that can potentially enhance the immunogenicity of a therapeutic protein.Traditionally, biological products are tested during the manufacturing process and at the time of lot release by the in vivo rabbit pyrogen test (RPT) and by the in vitro bacteria endotoxin test, commonly referred to as the Limulus amebocyte lysate (LAL) test. The requirement for final container product testing is stated in the U.S. Code of Federal Regulations, Title 21 (2, 3). These tests are designed to detect lipopolysaccharide (LPS), which is a constituent of gram-negative bacteria, or endotoxin and rely on nonhuman systems to predict human responses. Plasma-derived products and acellular vaccines can be sterile filtered before they are filled, and therefore, intact microorganisms can be removed. However, other microbial constituents, such as those derived from cell walls and nucleic acids (DNA and RNA), can evade filters and still end up in the final product. Recombinant proteins made in Escherichia coli, yeasts, or cell lines may also contain trace levels of host impurities, such that the final product may contain microbial components. These constituents may be difficult to detect by traditional methods.Recent studies have revealed that microbial pathogens possess specific pathogen-associated molecular patterns (13). The host innate immune system recognizes these pathogen-associated molecular patterns by using germ line-encoded pattern recognition receptors to elicit immune responses. Toll-like receptors (TLRs) are well-known pattern recognition receptors. Cells of the innate immune system utilize TLRs to detect cell wall components of bacteria, mycoplasma, fungi, and protozoa at the cell surface, whereas bacterial and viral nucleic acids are recognized by TLRs in a specialized intracellular endosomal compartment (12, 15, 26).Recent efforts have focused on the development of an in vitro test system that combines the sensitivity of the LAL test with the wide range of pyrogens detectable by the in vivo RPT. The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) evaluated the validation status of five in vitro test methods for assessment of the potential pyrogenicity of pharmaceuticals and other products proposed as potential replacements for the in vivo RPT (10). The ICCVAM proposed in vitro pyrogen tests that use enzyme-linked immunosorbent assays (ELISAs) for interleukin-1β (IL-1β) or IL-6 to measure increased levels of cytokine release when human blood cells, i.e., whole blood (WB), isolated monocytes, and a Mono-Mac 6 (MM6) cell line, are stimulated by endotoxin. On the basis of the findings of two published international validation studies, these five proposed in vitro pyrogenicity test methods are WB/IL-1β, WB/IL-6, peripheral blood mononuclear cell/IL-6, MM6/IL-6, and cryopreserved WB/IL-1β (4, 9, 11, 19).Armed with the knowledge of the critical role of TLRs in microbial detection as well as the need to avoid such issues as donor variability and the hazards associated with human blood products, our approach was to develop an assay that utilized cell lines expressing different TLRs. This was accomplished by using a panel of HEK293 cells transfected with different human TLRs. Initially, a screening test is performed by using the MM6 cell line. Previously, it was shown that MM6 cells respond to ligands to TLR2 and TLR4 (27). On the basis of the data presented in that paper, MM6 cells also respond to the TLR5 ligand flagellin but not to the TLR3, TLR7, TLR8, or TLR9 ligand. If a product sample tests positive with MM6 cells, then cell lines with more restricted TLR expression are used as detector cells to characterize the microbial ligand and, in turn, its microbial origin.Here we show the utility of this approach for the detection of a microbial contaminant in a sample obtained from a process used to make a recombinant product that had passed the standard lot release testing but that was associated with adverse events in humans. Not only was the panel of TLR-bearing cell lines able to detect a strong proinflammatory signal in the product, but also its use facilitated the identification of the microbial constituent at the molecular level.