Broadly neutralizing antibodies recognizing different antigenic epitopes act synergistically against the influenza B virus

The discovery of broadly neutralizing monoclonal antibodies against influenza viruses has raised hope for the successful development of new antiviral drugs. However, due to the speed and variety of mutations in influenza viruses, single-component antibodies that recognize specific epitopes are susceptible to viral escape and have limited efficacy when administration is delayed. Hence, it is necessary to develop alternative strategies with better antiviral activity. Influenza B virus infection can cause severe illness in children and the elderly. Commonly used anti-influenza drugs have low clinical efficacy against influenza B virus. In this study, we investigated the antiviral efficacy of combinations of representative monoclonal antibodies targeting different antigenic epitopes against the influenza B virus. We found that combinations of antibodies recognizing the hemagglutinin (HA) head and stem regions showed a stronger neutralizing activity than single antibodies and other antibody combinations in vitro. In addition, we found that pair-wise combinations of antibodies recognizing the HA head region, HA stem region, and neuraminidase enzyme-activated region showed superior antiviral activity than single antibodies in both mouse and ferret in vivo protection assays. Notably, these antibody combinations still displayed good antiviral efficacy when treatment was delayed. Mechanistic studies further revealed that combining antibodies recognizing different epitope regions resulted in extremely strong antibody-dependent cell-mediated cytotoxicity, which may partly explain their superior antiviral effects. Together, the findings of this study provide new avenues for the development of better antiviral drugs and vaccines against influenza viruses.     

Detection of specific IgA antibodies against BK virus by ELISA.

The development and evaluation of an ELISA for analysis of anti-BKV specific IgA antibodies in human sera are described. All children with cancer with a primary BKV infection developed specific IgA antibodies, without any specific symptoms during the infection. Specific IgA was found in 61% of sera from healthy persons containing BKV IgG antibodies, using the chosen cut-off value, and BKV IgM in 4%. These results indicate that IgA production is more persistent than IgM. The high frequency of specific IgA antibodies could either be explained by frequent reactivations or long-lasting persistence of antibodies.     

Secretory IgA as a Measure of Immunocompetence

Abstract

The field of psychoimmunology has rapidly expanded in recent years and various parameters of the immune system have been examined in relation to psychological factors. The secretory immune system is one of the more interesting aspects of the entire immune system because it protects mucosal membranes from invading organisms. Stress-produced changes in secretory immunoglobulin A (s-IgA) as measured by radial immunodiffusion assays have been reported in several studies. We present three reasons why total s-IgA protein, the measure derived from radial immunodiffusion assays, may not be a reasonable measure of immune system functioning, and we suggest an alternative method for examining secretory IgA that focuses on s-IgA antibody response to a novel antigen.     

Secretory immunoglobulins in serum from human immunodeficiency virus (HIV)-infected patients

Infection by the human immunodeficiency virus is associated with polyclonal B cell activation and increased levels of serum IgA. In order to characterize the molecular species of serum IgA, we have measured total IgA, IgA1, and IgA2 in sera from 60 HIV-1-infected patients and 40 healthy controls. In addition, secretory IgA (S-IgA), secretory IgM (S-IgM), free immunoreactive secretory component (SC), and the distribution of monomeric and polymeric IgA were determined. The data confirm the elevation of total serum IgA levels in HIV-1-infected patients, and both IgA1 and IgA2 concentrations are elevated. Furthermore, the data show a substantial increase in serum levels of both monomeric and polymeric IgA. Serum S-IgA levels were significantly increased in CDC group II patients versus controls and more frequently elevated in CDC group IV patients. The highest S-IgA levels were found among patients with the lowest blood CD4+ cell counts. Serum S-IgA levels were not correlated with serum levels of either total IgA or polymeric IgA. Serum S-IgM levels were also increased in HIV-1-infected patients and positively correlated with serum S-IgA levels. Conversely, serum levels of free SC were not altered. An increase in serum S-IgA was not related to human hepatitis B virus infection and/or to hepatic dysfunction or to diarrhea or overt intestinal infection. The data indicate that secretory Ig (S-IgM and S-IgA), which are likely to be produced at mucosal sites, increase in the serum of HIV-1-infected patients.     

IgA肾病肾组织内乙型肝炎病毒感染的发病机制研究

目的：探讨乙型肝炎病毒感染致IgA肾病肾损伤的发病机制。方法： 随机选取48例IgA肾病肾穿刺组织，参照Meadow病变分级标准分为Ⅰ-Ⅴ级5个实验组，应用Envision免疫组织化学方法检测各级肾组织内HBsAg和HBcAg；同时用直接IS-PCR技术检测其中18例IgA肾病肾组织内HBV DNA。结果： 48例IgA肾病肾组织内HBcAg和HBsAg总的阳性检出率分别为75.00%(36/48)和43.75%(21/48)；18例IgA肾病肾组织内HBV DNA阳性检出率为61.11%(11/18)；3者均表现为肾小管阳性检出率高于肾小球(P＜0.05)，但各级之间，HBcAg、HBsAg和HBV DNA检出率均无显著差异(P＞0.05)。结论： HBV参与了IgA肾病的发生，其导致肾组织损伤的机制可能主要是由细胞免疫或一系列细胞因子介导，并非病毒直接所致；肾小管上皮细胞可能是HBV感染的靶对象。     

Protection against influenza virus infection by intranasal administration of hemagglutinin vaccine with chitin microparticles as an adjuvant

Chitin in the form of microparticles (chitin microparticles, CMP) has been demonstrated to be a potent stimulator of macrophages, promoting T-helper-1 (Th1) activation and cytokine response. In order to examine the mucosal adjuvant effect of CMP co-administered with influenza hemagglutinin (HA) vaccine against influenza infection, CMP were intranasally co-administered with influenza HA vaccine prepared from PR8 (H1N1) virus. Inoculation of the vaccine with CMP induced primary and secondary anti-HA IgA responses in the nasal wash and anti-HA IgG responses in the serum, which were significantly higher than those of nasal vaccination without CMP, and provided a complete protection against a homologous influenza virus challenge in the nasal infection influenza model. In addition, CMP-based immunization using A/Yamagata (H1N1) and A/Guizhou (H3N2) induced PR8 HA-reactive IgA in the nasal washes and specific-IgG in the serum. The immunization with A/Yamagata and CMP resulted in complete protection against a PR8 (H1N1) challenge in A/Yamagata (H1N1)-vaccinated mice, while that with A/Guizhou (H3N2) and CMP exhibited a 100-fold reduction of nasal virus titer, demonstrating the cross-protective effect of CMP and influenza vaccine. It is suggested that CMP provide a safe and effective adjuvant for nasal vaccination with inactivated influenza vaccine.     

IgA nephropathy associated with chronic hepatitis B virus infection in adults: the pathogenetic role of HBsAG

Five adult cases of IgA nephropathy associated with chronic hepatitis B virus infection were studied. Serum HBsAg and anti-HBc were present in five patients and HBeAg in four patients. Glomerular changes were typical of primary IgA nephropathy in four patients, and a mixed picture of IgA and membranous nephropathy was demonstrated in one patient. Immunofluorescence microscopy using polyclonal and monoclonal antibodies against HBsAg, HBcAg, and HBeAg revealed mesangial deposits of HBsAg in renal biopsies from four patients. One renal biopsy showed only mesangial and capillary HBcAg by polyclonal antiserum, and virus-like particles were demonstrated in the intramembranous electron-dense deposits on ultrastructural examination. Mesangial HBeAg was not detected in the renal biopsies from these patients with IgA nephropathy. As for the single patient with a mixed picture of IgA and membranous nephropathy, granular deposits of HBeAg with a distribution similar to IgG were detected in the glomerular capillary walls in addition to the mesangial deposition of HBsAg. These findings suggest that HBsAg rather than HBeAg may play a role of the pathogenesis in some of the adult patients with IgA nephropathy associated with chronic hepatitis B virus infection.     

Secretory immunoglobulin A (IgA) responses in IgA nephropathy patients after mucosal immunization, as part of a polymeric IgA response

Secretory immunoglobulin A (SIgA), although generated at mucosal surfaces, is also found in low concentrations in the circulation. Recently, SIgA was demonstrated in mesangial deposits of patients with immunoglobulin A nephropathy (IgAN), suggesting a role in the pathogenesis. This finding is in line with the belief that high molecular weight (HMW) immunoglobulin A (IgA) is deposited in the kidney. However, there is little information on the size distribution of antigen-specific IgA in circulation upon mucosal challenge. In this study we measured antigen-specific IgA, including SIgA, in serum following challenge of IgAN patients and controls via intranasal vaccination with a neoantigen, cholera toxin subunit B (CTB). We size-fractionated serum and nasal washes to study the size distribution of total IgA, SIgA and CTB-specific IgA. Finally, we compared the size distribution of antigen-specific IgA after mucosal immunization with the distribution upon systemic immunization. A significant induction of antigen-specific SIgA was detectable in serum of both patients with IgAN and controls after mucosal immunization with CTB. Independent of the route of immunization, in both groups the antigen-specific IgA response was predominantly in the polymeric IgA fractions. This is in contrast to total IgA levels in serum that are predominantly monomeric. We conclude that mucosal challenge results in antigen-specific SIgA in the circulation, and that the antigen-specific IgA response in both IgAN patients and in controls is of predominantly HMW in nature. No differences between IgAN patients and controls were detected, suggesting that the size distribution of antigen-specific IgA in the circulation is not disturbed specifically in IgAN patients.     

Shedding of infectious virus and virus antigen during acute infection with respiratory syncytial virus.

Shedding of respiratory syncytial virus (RSV) in nasopharyngeal aspirates (NPA) of hospitalized children with acute respiratory infection was studied using direct antigen detection by time-resolved fluoroimmunoassay, rapid identification of infectious virus in centrifugally inoculated cell cultures by immunoperoxidase staining and conventional virus culture. Sequential NPAs, in which also local RSV-specific IgA response was measured, were collected from children with proven RSV infection. The shedding pattern was similar for both infectious virus and viral antigen. The overall agreement of the three methods was good (81%) in diagnostic specimens collected on admission, but markedly reduced (46%) in follow-up specimens. Secretory IgA was abundant in specimens giving discrepant or negative results only. The proportion of patients who shed RSV was high (> or = 87%) in the first week after onset of symptoms, and decreased sharply in the second week. An opposite temporal pattern was found in the proportion of patients with detectable RSV-IgA in their secretions. Sequentially isolated strains were antigenically stable as determined by their reactivity with a large panel of monoclonal antibodies. The findings suggest that RSV shedding should be monitored by using more than one method for virus detection.     

Production and diagnostic application of monoclonal antibodies against influenza virus H5

Nine monoclonal antibodies (mAbs) against avian influenza virus (AI) H5 subtype from mice immunized with inactivated virus H5N1 (A/Turkey/ON/6213/66) were produced. Upon testing, the results indicated that the binding epitopes of eight out of the nine mAbs were conformational, while one mAb (#7) reacted with denatured H5N1 only. Two mAbs #10 and #11 reacted with all of the thirteen H5 strains tested indicating that the binding epitopes of these mAbs were conserved among these H5 subtypes.Possible applications of these mAbs in rapid tests for H5 antigen were explored. Double antibody sandwich (DAS) ELISAs were developed using two selected mAbs #10 and #11. This DAS ELISA detects specific H5 viruses and is able to identify all thirteen H5 strains tested. Three mAbs showed reactivity with AI H5 antigen for both immunofluorescence (IF) and immunohistochemistry. A cELISA used to screen chickens that had been infected with an H5 virus was developed with mAb #9 and recombinant H5 antigen. The sera from chickens that have been infected with an H5N1 virus were examined using the cELISA. 80% of the sera from H5 infected chickens showed a positive H5 specific antibody response at 7 days post-infection (dpi) and remained positive until the end of the experiment on day 30 (>40% inhibition). This panel of the AI H5 specific mAbs is valuable for the development of various immunoassays.     

Protection against influenza virus infection by intranasal vaccine with surf clam microparticles (SMP) as an adjuvant

A safe and effective adjuvant is necessary to enhance mucosal immune responses for the development of an inactivated intranasal influenza vaccine. The present study demonstrated the effectiveness of surf clam microparticles (SMP) derived from natural surf clams as an adjuvant for an intranasal influenza vaccine. The adjuvant effect of SMP was examined when co-administered intranasally with inactivated A/PR8 (H1N1) influenza virus hemagglutinin vaccine in BALB/c mice. Administration of the vaccine with SMP induced a high anti-PR8 haemagglutinin (HA)-specific immunoglobulin A (IgA) response in the nasal wash and immunoglobulin G (IgG) response in the serum, resulting in protection against both nasal-restricted infection and lethal lung infection by A/PR8 virus. In addition, administration of SMP with A/Yamagata (H1N1), A/Beijing (H1N1), or A/Guizhou (H3N2) vaccine conferred complete protection against A/PR8 virus challenge in the nasal infection model, suggesting that SMP adjuvanted vaccine can confer cross-protection against variant influenza viruses. The use of SMP is suggested as a new safe and effective mucosal adjuvant for nasal vaccination against influenza virus infection.     

Allelic polymorphism controls autoreactivity and vaccine elicitation of human broadly neutralizing antibodies against influenza virus

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Mucosal administration of anti-bacterial antibodies provide long-term cross-protection against Pseudomonas aeruginosa respiratory infection

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Production of IgA monoclonal antibodies against influenza A virus

Nineteen IgA monoclonal antibodies against influenza A virus X-31 were obtained following intranasal infection of mice with influenza A virus X-31. It was demonstrated that specificities of IgA monoclonal antibodies are similar to those of IgG monoclonal antibodies. These IgA antibodies might be useful for the study of mucosal immunity against influenza A viruses. Infection may be an easier and better way of producing monoclonal antibodies against some viral agents.     

抗禽流感病毒H7亚型血凝素特异性单克隆抗体的研制

目的:研制禽流感病毒H7亚型血凝素特异性单克隆抗体(mAb)。方法:以H7亚型禽流感诊断抗原为免疫原免疫6～8周雌性BALB/c小鼠,末次加强免疫后取其脾细胞与骨髓瘤细胞Sp2/0-Ag-14进行融合。通过HA和HI试验筛选阳性克隆。应用HI试验和Western blot试验测定mAb的反应性和特异性。结果:共获得4株分泌抗AIVH7亚型HAmAbs的杂交瘤细胞株,分别命名为2E2、2A4、5F5、7G5。这些mAb的腹水HI效价在5×27～5×211之间,其中2E2属于IgM亚类,2A4属于IgG1亚类,5F5、7G5属于IgG2a亚类。Western blot分析结果显示,4株AIVH7亚型HAmAb能与AIVH7蛋白在Mr75000处反应,但不与新城疫病毒(NDV)蛋白发生反应,表明这些mAb能特异性识别AIVH7亚型HA。mAbHI反应性测定结果表明:4株mAb中,2E2、5F5、7G5只与H7亚型AIV发生特异性HI反应,而不与其他亚型AIV以及NDV、传染性支气管炎病毒(IBV)反应,显示出良好的特异性;而2A4除了与H7亚型AIV反应外,还与H15N8标准株发生低水平交叉反应。结论:这些mAb不仅为H7亚型AIV的HA结构分析提供了工具,而且为建立快速廉价的H7亚型禽流感诊断方法提供了核心试剂。     

Secretory IgA are elevated in both saliva and serum of patients with various types of primary glomerulonephritis.

Secretory immunoglobulin A (IgA) was determined by means of an enzyme-linked immunosorbent assay (using as capture antibody an MoAb specific for secretory component) in saliva and serum from 46 patients with IgA mesangial nephritis (IgAGN), 36 with an idiopathic nephrotic syndrome (INS), 30 with an idiopathic membranous nephropathy (MGN) and 40 healthy controls. Secretory IgA levels were elevated in both saliva and serum of patients with primary glomerulonephritis (P < 0.05; Mann-Whitney test) regardless of the histological type of the primary glomerulonephritis. Salivary IgA1 and IgA2 levels were increased in the saliva of patients with IgAGN, INS and MGN (P < 0.05; Mann-Whitney test). The monomeric/total IgA ratio, and interferon-gamma and soluble IL-2 receptor levels, in saliva did not differ between the patients and controls (P > 0.05; Mann-Whitney test). We conclude that the mucosal immune system is activated in forms of glomerulonephritis other than IgAGN.     

A multiplexed immunoassay for detection of antibodies against avian influenza virus

Avian influenza (AI) is a highly contagious disease in poultry and outbreaks can have dramatic economic and health implications. For effective disease surveillance, rapid and sensitive assays are needed to detect antibodies against AI virus (AIV) proteins. In this study, we report the development of a multiplexed fluorescence microsphere immunoassay (FMIA) for detection of antibodies against AIV proteins in poultry. Recombinant nucleoprotein (NP), matrix protein (M1), and non-structural protein 1 (NS1) were expressed using a baculovirus expression system, purified and covalently coupled to fluorescent xMAP microspheres. Using these reagents, a triplex bead assay was developed for the Luminex platform. The assay displayed minimal cross reactivity when screened against a panel of reference sera raised against common avian viruses. For detection of anti-NP antibodies, the sensitivity and specificity of the assay were comparable to a commercially available ELISA. The assay was also employed to investigate the early kinetics of antibody response in chickens infected with AIV. Our results suggest that NP should be the protein of choice when detecting AI infections in commercial chickens, as the immune response was higher and persisted longer than that of M1 and NS1 proteins. This report provides a framework from which a more robust assay could be developed to profile exposure to many AIV subtypes in a single test.     

Establishment of a cynomolgus macaque model of influenza B virus infection

Pathogenicity of influenza B virus was examined in cynomolgus macaques to establish a macaque model suitable for vaccine and antiviral drug development. We prepared influenza B viruses for inoculation with minimal passages after isolation from patients. Macaques inoculated with influenza B virus showed higher body temperature than that before infection for 6 to 12 days. Virus was detected in nasal, tracheal, and bronchial samples until 6 days after inoculation followed by an increase in neutralizing antibody. High levels of IL-6 and TNF-α in nasal swabs from the infected macaques were correlated with fever. Symptoms and duration of the viral replication would be sufficient to evaluate efficacy of vaccines and antiviral agents. In addition, measurement of immune responses including antibody and cytokine production would provide an immunological rationale in efficacy of vaccines and antiviral agents. The results suggest that cynomolgus macaques are appropriate model animals for research of influenza B virus.     

Detection by ELISA of IgA and IgM antibodies in secretion and IgM antibodies in serum in primary lower respiratory syncytial virus infection

Twenty-six infants and children with primary lower RS virus infection, diagnosed by the detection of RS virus in nasopharyngeal secretion (NPS) by use of immunofluorescent antibody (FA) technique, were studied with respect to the presence of IgA and IgM antibodies. Samples of NPS and serum obtained during the first 3-4 months following the beginning of illness, were investigated. Employing a reverse ELISA technique, we found IgM antibodies in the acute, but not during the convalescent, phase of illness in NPS from 20 of the patients and in serum from 21 of the patients. The majority of the IgM antibody conversions observed occurred in NPS as well as in serum on days 5-8 following the illness. RS virus IgA antibodies, also detected by a reverse ELISA technique, were demonstrated in NPS in 22 of the patients, with antibody conversions being found in 19 of the patients on days 5-8 following the beginning of the illness. Two patients still had IgA antibodies in NPS approximately 3 months FSOI. By comparison, RS virus was detected in acute-phase NPS by double-antibody sandwich ELISA in 25 of the 26 patients investigated.     

A latex agglutination test using a recombinant nucleoprotein for detection of antibodies against avian influenza virus

A latex agglutination test (LAT) was developed for detecting antibodies against avian influenza virus. The recombinant avian influenza virus nucleoprotein expressed in Escherichia coli was purified, coupled with latex beads, and used as an antigen for the LAT. The LAT was capable of detecting anti-avian influenza virus antibodies irrespective of the avian-influenza subtype, and in most cases, the results correlated with the results of an agar gel precipitation test (AGPT). However, in comparison with the AGPT, the LAT could detect the anti-avian influenza virus antibodies for a longer period of time after the infection. The nonspecific agglutination observed in uninfected chicken sera was resolved by pretreating the sera with dried chicken-liver powder for 1 h. The LAT is easy to perform, and even after considering the time required for pretreatment of the serum, the total time required for obtaining the results is reduced in comparison to the time required in the case of the AGPT. This easy and rapid LAT is considered to be useful for monitoring avian influenza virus infection in the field.