Production and characterization of human monoclonal anti-idiotype antibodies to anti-dsDNA antibodies

Anti-dsDNA autoantibodies are the hallmark of systemic lupus erythematosus (SLE) and frequently correlate with disease activity. In this study we report the isolation and characterization of human anti-Id monoclonal antibody fragments as single-chain Fv fragments (scFv) against anti-dsDNA antibody. The anti-Id monoclonal antibodies, specific for anti-dsDNA antibodies, have been cloned from phage display antibody scFv libraries derived from a patient with SLE. The V gene repertoires were derived from the RNA obtained from the B cells of an SLE patient with anti-Ro/SSA and anti-La/SSB antibodies. Affinity-purified anti-dsDNA antibodies were used for selection of bacterial clones producing specific scFv antibody fragments against anti-dsDNA antibodies and little reactivity with normal IgG and other IgG antibodies by ELISA. The anti-Id antibody recognizes a public idiotope that is broadly cross-reactive with polyclonal and monoclonal anti-dsDNA antibodies. This binding was largely inhibited by dsDNA antigen. The anti-Id antibody inhibited anti-dsDNA binding to dsDNA antigen in immunoassays and in the Crithidia luciliae assay. The anti-Id scFv antibody fragments derived from human genes could modulate the pathogenicity of anti-dsDNA autoantibodies and may have therapeutic implications in SLE. They may also be used as probes in studies of the structure of the idiotype.     

The human antibody response to streptococcal C5a peptidase

An ELISA was developed to measure antibody, both IgG and IgA, against the streptococcal C5a peptidase (SCP), in human sera and saliva. Generally, sera and saliva from young, uninfected children lacked antibody to SCP. In contrast, most sera and saliva specimens from healthy adults had measurable levels of anti-SCP IgG and SCP-specific secretory IgA (anti-SCP sIgA). Paired acute and convalescent sera from patients with streptococcal pharyngitis possessed significantly higher levels of anti-SCP IgG than did sera from healthy individuals. Sera containing high concentrations of anti-SCP immunoglobulin were capable of neutralizing SCP activity. A survey of healthy adults and children also showed that the latter were significantly less likely to have anti-SCP sIgA in their saliva. Detection of this antibody in greater than 90% of the saliva specimens obtained from children who had recently experienced streptococcal pharyngitis demonstrated that children can produce a secretory response. This is thought to be the first report of a secretory IgA response in humans to a somatic antigen of Streptococcus pyogenes.     

Pathological classification and prognosis of renal damage caused by antineutrophil cytoplasmic antibody associated vasculitis

正Objective To analyze the pathological characteristics and prognostic factors of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Methods A retrospective analysis of AAV patients with renal biopsy results admitted to Kidney Disease Center of the First Affiliated Hospital from January 2004 to February     

Circulating cytokine levels and antibody responses to human Schistosoma haematobium: IL-5 and IL-10 levels depend upon age and infection status

Experimental schistosome infections induce strong parasite-specific Th2 responses. This study aims to relate human systemic cytokine and antibody levels to schistosome infection levels and history. Levels of anti-Schistosoma haematobium antibodies (directed against crude cercariae, egg and adult worm antigens) and plasma cytokines (IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-13, IL-17, IL-21, and IL-23) were measured by ELISA in 227 Zimbabweans (6-60?years old) in a schistosome-endemic area and related to age and infection status. Egg-positive people had significantly higher levels of specific antibodies, IL-2, IFN-γ and IL-23. In contrast, egg-negative individuals had significantly higher circulating IL-10, IL-4, IL-13 and IL-21 that were detected with high frequency in all participants. Subjects with detectable plasma IL-17 produced few or no eggs. When analyzed by age, IL-4 and IL-10 increased significantly, as did schistosome-specific antibodies. However, when age was combined with infection status, IL-5 declined over time in egg-positive people, while increased with age in the egg-negative group. Older, lifelong residents had significantly higher IL-4 and IL-5 levels than younger egg-negative people. Thus, a mixed Th1/Th2 systemic environment occurs in people with patent schistosome infection, while a stronger Th2-dominated suite of cytokines is evident in egg-negative individuals.     

Structures of complexes formed by H5 influenza hemagglutinin with a potent broadly neutralizing human monoclonal antibody

H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer’s formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion.The initial steps in influenza virus infection involve sialic acid receptor binding and membrane fusion, both of which are functions of the hemagglutinin (HA) virus membrane glycoprotein. Anti-HA antibodies that block these functions neutralize virus infectivity. Such antibodies are induced by infection and by vaccination, and the immune pressure that they impose on subsequently infecting viruses is responsible for the antigenic drift for which influenza viruses are notorious. Zoonotic infections, which can lead to new pandemics, occur periodically, and H5N1, H7N9, and H10N8 avian viruses are recent examples of this sort. The threat that zoonotic infections present is based, in part, on the lack of immunity in the human population to the novel HAs that they contain. In attempts to substitute for this deficiency, human immune sera have been used successfully to treat severe infections (1), and monoclonal antibodies have been prepared from mice and from humans for potential use in immunotherapy.Analyses of antibodies produced by cloned immune cells derived from infected patients have revealed that antibodies are induced that are either subtype- or group-specific and others that cross-react with HAs of both groups (2). To date, cross-reactive antibodies have been shown to recognize both membrane-distal and membrane-proximal regions of HA (3). Subtype-specific antibodies, on the other hand, bind to the membrane-distal region, covering the receptor-binding site and, in some cases, inserting into it (4, 5).In the studies reported here, a human monoclonal antibody is described that recognizes the HAs of viruses of all three clades of the H5 subtype that have caused human infection and is shown to be effective in protecting mice from lethal challenge. EM and X-ray crystallography studies of HA-Fab complexes indicate that the antibody binds to a site containing residue 122, located on the membrane-distal surface of the HA trimer. We describe the antibody-binding site in detail to show that binding occurs at a distance from the receptor-binding site. Infectivity neutralization and receptor-binding experiments, together with these observations, lead to the conclusion that the antibody neutralizes viruses by blocking receptor binding in a way that is dependent on the Fc region of the bound antibody. We compare the site with similar sites reported by others (6–9) for antibodies that have not as yet given crystalline HA-Fab complexes.Under the conditions that we obtain crystals of the HA-Fab complex, the HA dissociates and reveals the structure of a monomeric HA. We consider the structure of the monomer in relation to the structure that HA has been shown to assume after exposure to the pH of membrane fusion.     

Progression of autoimmune-mediated hepatic lesions in a murine graft-versus-host reaction by neutralizing IL-10

C57BL/6 (B6) spleen T cells which were injected into major histocompatibility complex (MHC) class II-disparate (B6.C-H-2^bm/2×B6) F1 hybrid mice induced autoimmune graft-versus-host reaction (GVHR). Early production of interferon (IFN)-γ and delayed production of interleukin (IL)-10 might play an important role in the formation of GVHR hepatic lesions. To clarify whether blocking of IL-10 deteriorate autoimmune-mediated hepatic lesions induced by GVHR, and to elucidate the change of the T helper (Th)1/Th2 cytokines in the liver, anti-IL-10 monoclonal antibodies (mAbs, 500 μg) were given 4 h before the induction of GVHR. We evaluated the change of splenomegaly and GVHR-induced hepatic lesions. The changes of the expressions of IFN-γ and IL-4 mRNA isolated from liver-infiltrating lymphocytes were measured by real-time polymerase chain reaction (PCR). In GVHR with anti-IL-10 mAbs mice splenomegaly and periportal cellular infiltration was significantly increased compared with those of GVHR mice. In these mice, both IFN-γ and IL-4 mRNA expression levels were significantly elevated by the neutralization of IL-10. These findings suggest an important role of IL-10 in murine GVHR due to MHC class II disparity. IL-10 may play a crucial role in down-regulating autoimmune-related hepatic lesions.     

Maternal neutralizing antibody and transmission of hepatitis C virus to infants

To determine whether lower levels of hepatitis C virus (HCV)-specific neutralizing antibodies (nAb) are associated with an increased risk of mother-to-child transmission (MTCT) of HCV, HCV nAb titers were assessed in 63 mothers coinfected with HCV and human immunodeficiency virus (HIV) type 1. Of the mothers, 16 transmitted HCV to their infant, but no difference was detected between the ability of maternal plasma from transmitters and nontransmitters to neutralize heterologous HCV pseudoparticles (median nAb titer, 1:125 vs. 1:100; P = .23). In the setting of HIV/HCV coinfection, we found no evidence that HCV nAbs are associated with the prevention of MTCT of HCV.     

Membrane association and epitope recognition by HIV-1 neutralizing anti-gp41 2F5 and 4E10 antibodies

It has been proposed that HIV-1-neutralizing 2F5 and 4E10 monoclonal antibodies recognize gp41 ectodomain pretransmembrane sequences in the context of the membrane interface. With the aim of evaluating lipid-bilayer surface effects on recognition, we use phospholipid model systems to investigate the ability of 2F5 and 4E10 to transfer into membrane interfaces and bind epitope sequences immersed therein. The experimental data support the predicted tendencies for partitioning and recognition of membrane-bound epitopes, albeit with lower affinity in the case of 2F5. Our findings support the existence of two different recognition mechanisms at membrane surfaces: the association of 2F5 with the interface possibly prevents epitope immersion into the bilayer, and 4E10 membrane association might allow recognition of the membrane-bound epitope. We discuss the relevance of these observations for the design of immunogens aimed at eliciting 2F5- and 4E10- like humoral responses.     

大黄素抑制IgG型抗双链DNA抗体诱导的系膜细胞表型转化的研究

目的探讨大黄素对IgG型抗双链DNA（dsDNA）抗体诱导的大鼠系膜细胞（MCs）表型转化的抑制作用。方法利用马疫锥虫动基体DNA诱导大鼠产生IgG型抗dsDNA抗体，小牛胸腺DNA纤维素层析法提纯抗体。体外培养正常MCs（空白组），或在培养上清中分别加入IgG型抗dsDNA抗体、25mg／L大黄素（对照组），或同时加IgG型抗dsDNA抗体与大黄素（实验组），检测MCs合成转化生长因子-β1（TGF-β1）、α-平滑肌肌动蛋白（α-SMA）等水平，并在透视电镜下观察细胞显微结构的变化。结果与空白组比较，抗体对照组合成TGF-β1等水平明显升高（P〈0．05），并出现肌成纤维细胞样结构变化；而大黄素对照组的上述指标下降（P〈0．05），且细胞显微结构基本正常。与抗体对照组比较，实验组合成TGF-β1等水平降低，细胞显微结构大致正常。结论抑制IgG型抗dsDNA抗体诱导的系膜细胞表型转化可能是大黄素治疗狼疮肾炎的药理机制之一。     

Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus

Hepatitis C virus (HCV) infects ～2% of the world's population. It is estimated that there are more than 500,000 new infections annually in Egypt, the country with the highest HCV prevalence. An effective vaccine would help control this expanding global health burden. HCV is highly variable, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human mAbs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies to HCV.     

Identification of human neutralizing antibodies that bind to complex epitopes on dengue virions

Dengue is a mosquito-borne flavivirus that is spreading at an unprecedented rate and has developed into a major health and economic burden in over 50 countries. Even though infected individuals develop potent and long-lasting serotype-specific neutralizing antibodies (Abs), the epitopes engaged by human neutralizing Abs have not been identified. Here, we demonstrate that the dengue virus (DENV)-specific serum Ab response in humans consists of a large fraction of cross-reactive, poorly neutralizing Abs and a small fraction of serotype-specific, potently inhibitory Abs. Although many mouse-generated, strongly neutralizing monoclonal antibodies (mAbs) recognize epitopes that are present on recombinant DENV envelope (E) proteins, unexpectedly, the majority of neutralizing Abs in human immune sera bound to intact virions but not to the ectodomain of purified soluble E proteins. These conclusions with polyclonal Abs were confirmed with newly generated human mAbs derived from DENV-immune individuals. Two of three strongly neutralizing human mAbs bound to E protein epitopes that were preserved on the virion but not on recombinant E (rE) protein. We propose that humans produce Abs that neutralize DENV infection by binding a complex, quaternary structure epitope that is expressed only when E proteins are assembled on a virus particle. Mapping studies indicate that this epitope has a footprint that spans adjacent E protein dimers and includes residues at the hinge between domains I and II of E protein. These results have significant implications for the DENV Ab and vaccine field.     

Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment

The cytokine thrombopoietin (TPO), the ligand for the hematopoietic receptor c-Mpl, acts as a primary regulator of megakaryocytopoiesis and platelet production. We have determined the crystal structure of the receptor-binding domain of human TPO (hTPO(163)) to a 2.5-A resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO(163) has an antiparallel four-helix bundle fold. The neutralizing Fab mainly recognizes the C-D crossover loop containing the species invariant residue Q111. Titration calorimetric experiments show that hTPO(163) interacts with soluble c-Mpl containing the extracellular cytokine receptor homology domains with 1:2 stoichiometry with the binding constants of 3.3 x 10(9) M(-1) and 1.1 x 10(6) M(-1). The presence of the neutralizing Fab did not inhibit binding of hTPO(163) to soluble c-Mpl fragments, but the lower-affinity binding disappeared. Together with prior genetic data, these define the structure-function relationships in TPO and the activation scheme of c-Mpl.     

C5a对人中性粒细胞凋亡影响的实验观察

目的 探讨C5a影响人中性粒细胞(PMN)的凋亡.方法 采用流式细胞术,检测PMN被C5a作用后其凋亡率随浓度及时间的变化.结果 随着C5a浓度增加PMN凋亡率下降;随着C5a刺激时间的延长,其抑制PMN凋亡作用有所增强.结论 C5a抑制中性粒细胞凋亡存在浓度及时间依赖关系.C5a延长中性粒细胞的炎性反应的时间,可能由此导致炎症失控和消散延迟,从而参与了脓毒症及急性肺损伤的发生发展.     

Characteristics of a neutralizing monoclonal antibody to the HIV envelope glycoprotein

We have studied the biologic and physical properties of a monoclonal antibody that binds to gp120, the exterior envelope glycoprotein of the human immunodeficiency virus (HIV) strain HTLV-IIIB. Designated 9284, the antibody possesses viral neutralizing activity and inhibits syncytium formation by infected cells. The antibody recognized a region of the polypeptide backbone previously described as an important neutralizing epitope. This region lies 307-330 residues from amino terminus of the glycoprotein. We have compared the biologic and physical properties of this antibody to those of the recently described 0.5 beta monoclonal antibody to gp120. The 0.5 beta antibody was biologically more potent and bound an epitope slightly downstream to that of the 9284 antibody. The antibodies did not differ significantly in their affinity for gp120. In competition studies, the 0.5 beta antibody was displaced by the 9284 antibody, but the binding of the latter was unaffected by 0.5 beta.     

囊尾蚴病患者IL-4、IL-5和IL-10水平检测

目的探讨白细胞介素4(IL-4)、白细胞介素5(IL-5)和白细胞介素10(IL-10)在囊尾蚴病发病中的免疫学作用。方法用双抗体夹心(ELISA)法检测囊尾蚴病患者血清中IL-4、IL-5和IL-10水平。结果囊尾蚴病患者血清中IL-4、IL-5和IL-10水平分别为(152.3±31.2)、(256.4±23.3)和(343.9±20.8)ng/L,正常对照组血清中IL-4、IL-5和IL-10水平分别为(75.0±28.5)、(119.5±17.6)和(106.7±19.6)ng/L,2组比较差异均有统计学意义(t值分别为10.6、27.1和48.4,P<0.001)。结论囊尾蚴感染患者Th2型细胞因子表达水平失常,体液免疫功能升高,说明囊尾蚴感染可致宿主免疫功能紊乱。     

C5a对人中性粒细胞凋亡的影响

目的探讨C5a影响人中性粒细胞的凋亡。方法采用流式细胞术，检测PMN被C5a作用后其凋亡率随浓度及时间的变化。结果随着C5a浓度增加PMN凋亡率下降，随着C5a和刺激时间延长，其抑制作用有所增强。结论C5a依浓度及时间抑制中性粒细胞的凋亡。     

In vitro fixation of C3d and C5b-9 on platelets by human platelet reactive antibodies

Summary Most platelet-reactive autoantibodies and alloantibodies are not able to fix complement in vitro. However, exceptions have been found. These antibodies are usually characterized by the conventional platelet complement fixation test. A recently developed competitive enzyme immunoassay for quantitation of platelet-associated immunoglobulins and a modification thereof allowed the quantitative study of fixation of C3d and the membrane attack complex (C5b-9) on platelets by HLA antibodies, human platelet autoantibodies, and drug-dependent antibodies (ddab). The highest amounts of both complement products were fixed through ddabs, whereas autoantibodies only showed moderate complement fixation. This enzyme immunoassay is a valuable tool for the characterization of the complement-fixing properties of platelet-reactive antibodies.     

Characterization of a mouse/human chimeric monoclonal antibody (C beta 1) to a principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein.

A chimeric mouse-human antibody (C beta 1) was constructed that recognized the principal neutralizing domain (PND) of human immunodeficiency virus type 1 (HIV-1) gp120. The constant (C) immunoglobulin regions (C gamma 1 and C kappa) of a mouse monoclonal antibody, 0.5 beta, were substituted for the human C gamma 1 and C kappa by recombining the DNA modules encoding variable or C regions. The DNA constructs were then transfected into X63 Ag8.653 myeloma cells. A clone with a high production of the chimeric antibody (C beta 1) was selected. This antibody was tested for its biological activity against HIV-1. It bound to the surface of HTLV-IIIB-infected cells and reacted with gp120/160 with equal affinity and specificity to that of the parental 0.5 beta murine monoclonal antibody in a Western blot assay. Neutralization and/or enhancement of HIV infection were evaluated with C beta 1 and 0.5 beta. Both C beta 1 and 0.5 beta neutralized cell-to-cell infection and cell-free virus infection by HTLV-IIIB. Antibody-dependence enhancement of HIV infection was not observed with either C beta 1 or 0.5 beta in the presence or absence of human complement. Antibody-dependent cell-mediated cytolysis (ADCC) and antibody-dependent complement-mediated cytolysis (ACC) were observed with C beta 1 but not with the parental 0.5 beta. These findings suggest that the neutralizing antibodies to PND may neutralize but not enhance HIV infection. Furthermore, the high levels of ACC and ADCC shown against HIV-infected cells by C beta 1 indicate that the clinical application of such monoclonal antibodies may be possible.