Properties and therapeutic efficacy of broadly reactive chimeric and humanized H5-specific monoclonal antibodies against H5N1 influenza viruses

Highly pathogenic H5N1 virus infection causes severe disease and a high rate of fatality in humans. Development of humanized monoclonal antibodies may provide an efficient therapeutic regime for H5N1 virus infection. In the present study, broadly cross-reactive monoclonal antibodies (MAbs) derived from mice were humanized to minimize immunogenicity. One chimeric antibody (cAb) and seven humanized antibodies (hAbs) were constructed. These antibodies retained broad-spectrum reactivity to H5N1 viruses, binding to recombinant H5-subtype HA1 molecules expressed in CHO cells in a dose-dependent manner and exhibiting similar reactivities against antigenically distinct H5N1 viruses in hemagglutination inhibition (HI) assays. One humanized antibody, 37 hAb, showed HI and neutralization activities comparable to that of the parental murine antibody, 13D4 MAb, while the other six antibodies were less reactive to H5N1 viruses. Analysis of amino acid sequences in the variable region frameworks of the seven humanized antibodies found that Q5 and Y27 in the VH region are highly conserved murine residues. Comparison of the three-dimensional structures derived from the variable regions of MAbs 37 hAb, H1202-34, and 13D4 revealed that residue substitutions at sites 70 and 46 may be the major cause for the observed differences in binding affinity. Examination of the chimeric antibody and one of the humanized antibodies, 37 hAb, showed that both antibodies offered postinfection protection against lethal challenge with antigenically diverse H5N1 viruses in the mouse model. Chimeric and humanized antibodies which retain the broadly reactive and protective properties of murine H5-specific monoclonal antibodies have great potential for use in the treatment of human H5N1 infection.     

Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection

The 2009 pandemic H1N1 influenza pandemic demonstrated the global health threat of reassortant influenza strains. Herein, we report a detailed analysis of plasmablast and monoclonal antibody responses induced by pandemic H1N1 infection in humans. Unlike antibodies elicited by annual influenza vaccinations, most neutralizing antibodies induced by pandemic H1N1 infection were broadly cross-reactive against epitopes in the hemagglutinin (HA) stalk and head domain of multiple influenza strains. The antibodies were from cells that had undergone extensive affinity maturation. Based on these observations, we postulate that the plasmablasts producing these broadly neutralizing antibodies were predominantly derived from activated memory B cells specific for epitopes conserved in several influenza strains. Consequently, most neutralizing antibodies were broadly reactive against divergent H1N1 and H5N1 influenza strains. This suggests that a pan-influenza vaccine may be possible, given the right immunogen. Antibodies generated potently protected and rescued mice from lethal challenge with pandemic H1N1 or antigenically distinct influenza strains, making them excellent therapeutic candidates.     

Demonstration of antibodies to both hemagglutinin and neuraminidase antigens of H3N2 influenza A virus in domestic dogs.

Serologic evidence of infection with human (H3N2) influenza viruses of 6 of 79 dogs sampled in New York City in March-April 1973 was obtained through the use of four different methods for measurement of anti-hemagglutination and anti-neuraminidase antibody.     

Development of influenza vaccines against newly emerging A/H5N1 virus

Emergence of highly virulent influenza A/H5N1 viruses in Hong Kong in 1997 posed a threat of pandemic and brought an urgent need to develop a suitable seed virus for vaccine production. The virulence of the H5N1 viruses to chicken embryos should hamper the efficient production of the vaccine. In addition, potential virulence to humans raised safety issue in manufacturing vaccine. Toward vaccine development, one approach is to use an avirulent avian influenza virus antigenically similar to the virulent ones as a surrogate vaccine strain. The other approach is based on the attenuation of pathogenicity of virulent H5N1 virus by genetic engineering of the hemagglutinin gene and selection of a gene constellation. The reverse genetics technique can make the latter approach possible. Candidate strains suitable for vaccine production could be prepared by using either approach.     

Modulation of immunodominant sites in influenza hemagglutinin compromise antigenic variation and select receptor-binding variant viruses

The regions of antigenic variation in influenza hemagglutinin (HA) are located on surface-accessible regions in the three-dimensional structure of the HA1 monomer. The aim of this study was to establish whether a novel variant virus, IMUT4, in which we had mutated specific amino acid residues (HA1 63, 144, 158, and 193) in these regions, previously shown to be immunodominant for CBA/Ca mice, would either (a) establish holes in the antibody (ab) repertoire or (b) preclude further antigenic variation in IMUT4. CBA/Ca mice were able to mount a neutralizing ab response to IMUT4 infection and molecular recognition sites were established by sequencing of the HA genes of monoclonal antibody (mAb)-selected laboratory variants of wild-type X31 virus (HA1 131, 145, 155, and 196). However, each of these mAbs failed to select further antigenic variants of IMUT4, in ovo, but rather a receptor binding mutant (HA1 190 Glu-->Asp; 226 Leu-->Gln) that was still recognized by the selecting mAb, specific for HA1 155 of X31 virus. The facility for antigenic variation in influenza would appear to be compromised, therefore, by targeted mutation of immunodominant sites, as initially proposed by S. Fazekas de St. Groth (Fazekas de St. Groth, S. 1977. Antigenic, adaptive and adsorptive variants of the influenza haemagglutinin. In Topics in Infectious Diseases. Vol. 3. R.G. Laver, H. Bachmayer, and R. Weil, editors. Springer-Verlag, Vienna. 25-48.). It is interesting to note that recent isolates of the H3 subtype, (e.g., A/Beijing/92) obtained between 1991 and 1993, contain the same substitutions at HA1 190 and 226, which may indicate similar constraints to immune evasion and the relevance of our findings to antigenic variation in the human population.     

Anti-high mobility group box-1 monoclonal antibody treatment provides protection against influenza A virus (H1N1)-induced pneumonia in mice

IntroductionProvision for the emergence of an influenza pandemic is an urgent issue. The discovery of a novel anti-influenza therapeutic approach would increase the effectiveness of traditional virus-based strategies. This study was undertaken to evaluate the therapeutic effects of anti-high mobility group box-1 (HMGB1) monoclonal antibody (mAb) treatment on influenza A virus (H1N1)-induced pneumonia in mice.MethodsNine-week-old male C57BL/6 mice were inoculated with H1N1, then anti-HMGB1 mAb or control mAb were administered intravenously at 1, 24 and 48 hours after H1N1 inoculation and the survival rate was analyzed. Lung lavage and histopathological analysis were performed on days 3, 5, 7 and 10 after inoculation.ResultsAnti-HMGB1 mAb significantly improved the survival rate of H1N1-inoculated mice (1 out of 15 versus 8 out of 15 deaths in the anti-HMGB1 mAb-treated group versus the control mAb-treated group, p < 0.01), although the treatment did not affect virus propagation in the lungs. The treatment also significantly attenuated histological changes and neutrophil infiltration in the lungs of H1N1-inoculated mice. This was associated with inhibition of HMGB1 and suppression of inflammatory cytokine/chemokine expression and oxidative stress enhancement, which were observed in H1N1-inoculated mice. The expression of receptor for advanced glycation end products and nuclear factor κB was attenuated by the treatment.ConclusionsAnti-HMGB1 mAb may provide a novel and effective pharmacological strategy for severe influenza virus infection in humans by reducing the inflammatory responses induced by HMGB1.

Electronic supplementary material

The online version of this article (doi:10.1186/s13054-015-0983-9) contains supplementary material, which is available to authorized users.     

甲型H1N1流感病毒核酸检测的应用及临床相关性的探讨

实验室诊断季节性流感和世界大流行流感的方法有多种,每种方法都有其优缺点[1].病毒培养被视为"金标准"方法,它敏感度特异度较好,但需要特殊的实验条件,费时耗力,不适用常规检测.血清学方法敏感性高,但特异抗体需在感染后1～3周才可产生,因此抗体检测更适合于回顾性诊断或人群感染状况的流行病调查,不适于早期诊断.快速抗原检测方法虽然在15～30 min可完成,特异性强但敏感性低于核酸检测方法.     

Spread of predominant neuraminidase and hemagglutinin co-mutations in the influenza A/H3N2 virus genome

Genetic variation of influenza neuraminidase (NA), unlike for hemagglutinin (HA), has not been fully characterized. Therefore, we determined the relation between mutations in the NA and HA genome segments of 205 influenza A/H3N2 viruses isolated from patients in Japan during the five seasons from 2010 to 2015. The amino acid (AA) sequences of the NA and HA proteins in these isolates were then determined. In the 2011–2012 season, there was the emergence of isolates with NA and HA sequences containing AA93G (NA93G) and AA278K (HA278K), respectively (24/48 isolates, 50.0%). This was in contrast to NA93D-HA278N being detected exclusively in the previous 2010–2011 season (24/24 isolates, 100.0%). The isolates with the NA93G-HA278K substitutions became predominant in the following 2012–2013 season (95.8%, 46/48 isolates). The NA and HA phylogenetic trees of the 2011–2012 and 2012–2013 seasons were segregated by clades with NA93D-HA278N or NA93G-HA278K. In the subsequent 2013–2014 and 2014–2015 seasons, the strong relationship between NA93D-HA278N and NA93G-HA278K observed in the previous seasons, was no longer present and NA93G-HA278N (33/52 isolates, 63.5% in the 2014–2015 season) became predominant. In addition, the clades within the NA and HA trees could no longer be segregated based on NA AA93 and HA AA278. These findings suggest that the co-mutation of NA and HA AA sequences is present and may contribute to the formation of an epidemic lineage.     

甲型H1N1流感防治进展

2009年3月18日在墨西哥发现首例甲型H1N1流感病例。2009年11月6日世界卫生组织于公布了全球甲型H1N1流感疫情的最新报告。报告中指出,截至11月1日,全球甲型H1N1流感死亡病例至少已达6071例,而出现甲型H1N1流感疫情的国家和地区达到了199个。目前疫情仍在全球范围迅速蔓延。2009年5月11日我国内地出现首例输入型甲型H1N1流感病例。     

Characterization of new murine monoclonal antibodies directed against glycophorins C and D

SUMMARY. Six new murine monoclonal antibodies (mAbs) directed to the erythrocyte membrane glycophorins C (GPC) and D (GPD) were obtained from splenocytes of different BALB/c mice immunized with human red blood cells, and fully characterized. The mAbs were selected by agglutination tests with control and Gerbich-negative cells, and by immunoblotting analysis. They showed specificity for the N-terminal domain(s) of GPC (and GPD) and were classified into three categories by competitive analysis using ¹²⁵Ilabelled antibodies and real-time biospecific interaction. The first group (NaM10-7G11, NaM70-1G4 and NaM77-7B6) compete for epitope(s) located at the N-terminal portion of GPC. Agglutination-inhibition tests revealed that the 7G11 epitope involves the amino group of Met¹ and sialic acid residue(s) whereas the 1G4 and 7B6 epitopes contain O-glycans. NaM89-2G11 belongs to a second group; its epitope is located in a region including Glu¹⁷, Asp¹⁹ and (an) O-glycan(s). The third group comprises mAbs NaM19-3C4 and NaM98-3Cl which bind to both GPC and GPD in proximity of the binding site of human anti-Ge:3 antibodies.
In addition, mAb 3C4 (anti-GPC/GPD) was found to bind to approximately 125 000 sites per red cell. Considering that the ratio of the GPC to GPD is about 3–4 to 1, the number of GPC and GPD molecules was estimated as 95 000 and 35 000, respectively.     

Monoclonal antibodies directed against the rev protein of human immunodeficiency virus type 1

The rev (art/trs) protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 K apparent molecular weight, is essential to target the mRNA for virion polypeptides into the cytoplasm. The rev protein was expressed in Escherichia coli as a beta-galactosidase fusion protein with a cleavage site for proteinase factor Xa. The rev-specific fragment was isolated to immunize mice. Five stable hybridoma cell lines were obtained producing monoclonal antibodies that reacted with rev protein in Western blot and ELISA. Using the monoclonal antibodies in indirect immunofluorescence, the rev protein could be localized in the nucleus, mostly in the nucleoli, of Hela cells that were transfected with a eukaryotic rev expression plasmid.     

H5N1 influenza virus and the safety of plasma products

BACKGROUND: The ever-increasing number of human H5N1 influenza virus infections may enable these viruses to acquire the ability to spread effectively among humans and potentially to cause a pandemic. Recently, more systemic virus dissemination was reported during H5N1 virus infection of humans, resulting in significant virus concentrations also in the blood. The observation has raised concerns about the safety of labile blood products for transfusion and consequentially also for plasma derivatives. To confirm the safety margins of plasma products, dedicated virus inactivation processes used during their production were investigated for their effectiveness in inactivating this virus of recent concern. STUDY DESIGN AND METHODS: Virus inactivation by steps commonly used during the manufacture of plasma derivatives, such as pasteurization for human albumin, solvent/detergent treatment for intravenous immunoglobulin (IVIG), vapor heating for factor VIII inhibitor bypassing activity, and incubation at low pH for IVIG, were investigated with a reassortant strain of H5N1 influenza virus. RESULTS: The results show that H5N1 influenza behaves as expected for lipid-enveloped viruses; that is, the virus is effectively inactivated by all the commonly used virus inactivation procedures tested. CONCLUSION: The safety margins of plasma derivatives against the theoretical transmission of H5N1 influenza virus are very substantial.     

一例人感染高致病性禽流感病毒(H5N1)的流行病学调查

目的通过对1例人感染高致病性禽流感病毒(H5N1)的调查分析,为制定人禽流感预防控制措施提供科学依据。方法采用流行病学调查、临床检查、血清学检查和 RT-PCR、 Real-time PCR 法进行分析和诊断。结果发现1例人感染高致病性禽流感病毒(H5N1),经救治无效因呼吸衰竭死亡。患者有明确的病、死禽接触史。采用隔离治疗、个人防护、医学观察、消毒等措施,疫情得到控制,未出现二代病例。结论需加强禽流感疫情监测,采取综合防控措施,预防疫情再次发生。     

New monoclonal antibodies directed against human renin. Powerful tools for the investigation of the renin system.

Monoclonal antibodies directed against human renin were obtained by the fusing of myeloma cells with spleen cells from Balb/c or high-responder Biozzi mice injected with pure tumoral or highly purified renal renin. These procedures resulted in the production of seven stable monoclonal antibodies to human renin. Antibodies in the hybridoma culture medium were screened by binding to pure iodinated renin or insolubilized renin in a solid phase assay. The concentration of purified antibodies that provided a 50% binding to iodinated renin varied from 1 X 10(-10) to 1 X 10(-7) M. Two monoclonal antibodies were found to be potent inhibitors of renin enzymatic activity in vitro, behaving as noncompetitive inhibitors (Ki, 1 to 4 X 10(-10) M). They were specific for primate renin. Three monoclonal antibodies provided suitable immunoadsorbants for renin purification. One of these immunoadsorbants was used for large-scale purification of the renal enzyme, resulting in an 825-fold renin enrichment in a single step. Two antibodies were able to distinguish between active and inactive renin and enabled concomitant separation and purification of the two enzyme forms in various biological fluids. Monoclonal antibodies also stained human and monkey renal renin when indirect immunofluorescence and peroxidase-antiperoxidase techniques were used. A highly sensitive radioimmunometric assay of renin was constructed with two monoclonal antibodies. The sensitivity of this improved assay should permit the detection of renin in normal human plasma. Monoclonal antibodies have been shown to be superior to polyclonal antibodies in the following areas: the separation of active from inactive renin, the purification of renin from biological fluids, and the setting up of a direct assay of plasma renin.     

2009年甲型H1N1流感研究近况

根据2009年甲型H1N1流感疫情及相关研究资料,回顾近代重大流感疫情的流行病学资料,介绍甲型H1N1流感病毒的结构及抗原特征、甲型H1N1流感流行病学、临床和放射学表现、并发症及目前推荐的诊断治疗策略.