Comprehensive analysis of antibody recognition in convalescent humans from highly pathogenic avian influenza H5N1 infection

Zuo, Teng; Sun, Jianfeng; Wang, Guiqin; Jiang, Liwei; Zuo, Yijun; Li, Danyang; Shi, Xuanling; Xi, Liu; Fan, Shilong; Ren, Huanhuan; Hu, Hongxing; Sun, Li; Zhou, Boping; Liang, Mifang; Zhou, Paul; Wang, Xinquan; Zhang, Linqi

doi:10.1038/ncomms9855

Cited by 38 publications

(62 citation statements)

References 62 publications

(121 reference statements)

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“…27 The key residues of these neutralization epitopes, as well as the AVFluIgG01 neutralization epitope, were mapped by various mutagenesis assays, such as indirect immunofluorescence assay, yeast surface display and PN assay.…”

Section: Comparison Of Amino Acid Conservation Of Neutralization Epitmentioning

confidence: 99%

Cross-protection of newly emerging HPAI H5 viruses by neutralizing human monoclonal antibodies: A viable alternative to oseltamivir

Ren

Wang

et al. 2016

mAbs

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Newly emerging highly pathogenic avian influenza (HPAI) H5N2, H5N3, H5N5, H5N6, H5N8 and H5N9 viruses have been spreading in poultry and wild birds. The H5N6 viruses have also caused 10 human infections with 4 fatal cases in China. Here, we assessed the cross-neutralization and cross-protection of human and mouse monoclonal antibodies against 2 viruses: a HPAI H5N8 virus, A/chicken/Netherlands/14015526/2014 (NE14) and a HPAI H5N6 virus, A/Sichuan/26221/2014 (SC14). The former was isolated from an infected chicken in Netherlands in 2014 and the latter was isolated from an infected human patient in Sichuan, China. We show that antibodies FLA5.10, FLD21.140, 100F4 and 65C6, but not AVFluIgG01, AVFluIgG03, S139/1 and the VRC01 control, potently cross-neutralize the H5N8 NE14 and H5N6 SC14 viruses. Furthermore, we show that a single injection of >1 mg/kg of antibody 100F4 at 4 hours before, or 20 mg/kg antibody 100F4 at 72 hours after, a lethal dose of H5N8 NE14 enables mice to withstand the infection. Finally, we show that a single injection of 0.5 or 1 mg/kg antibody 100F4 prophylactically or 10 mg/kg 100F4 therapeutically outperforms a 5-day course of 10 mg/kg/day oseltamivir treatment against lethal H5N8 NE14 or H5N6 SC14 infection in mice. Our results suggest that further preclinical evaluation of human monoclonal antibodies against newly emerging H5 viruses is warranted.

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Section: Comparison Of Amino Acid Conservation Of Neutralization Epitmentioning

confidence: 99%

Cross-protection of newly emerging HPAI H5 viruses by neutralizing human monoclonal antibodies: A viable alternative to oseltamivir

Ren

Wang

et al. 2016

mAbs

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“…The two pan-H5 Abs that we studied, 65C6 and 100F4, recognize two conformational epitopes in the globular head region but outside the RBS (26,27,30). Since we previously showed that SZ06, SX06, and NE14 pseudotypes exhibit different neutralization sensitivities to 65C6 and 100F4 in vitro (26,39), testing Abs 65C6 and 100F4 with these H5 strains would allow us to determine whether Fc-Fc␥R interactions are required for in vivo protection against different individual virus strains.…”

Section: Discussionmentioning

confidence: 99%

“…The 100F4 epitope is located away from the RBS, while the 65C6 epitope is close in proximity to it. Also, binding of 65C6 to its epitope might slightly hinder the engagement of HA to sialic acid (SA) receptors (30). This may explain why Ab 65C6 exhibits low but measureable inhibition of virus attachment, but Ab 100F4 does not (27).…”

Section: Discussionmentioning

confidence: 99%

“…In addition, Abs against the globular head with different degrees of cross-reactivity have also been isolated (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Many of these Abs are virus strain specific and recognize epitopes located in the receptor binding site (RBS), but some Abs recognize conserved epitopes within or outside the RBS of diverse strains of different subtypes (17)(18)(19) or within a HA subtype (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). The antibody repertoire against epitopes located in the head is more diverse than those Abs targeting epitopes in the stem (31).…”

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confidence: 99%

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Divergent Requirement of Fc-Fcγ Receptor Interactions for In Vivo Protection against Influenza Viruses by Two Pan-H5 Hemagglutinin Antibodies

Wang

Ren

Jiang

et al. 2017

J Virol

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Recent studies have shown that Fc-Fc␥ receptor (Fc␥R) interactions are required for in vivo protection against influenza viruses by broadly reactive antihemagglutinin (HA) stem, but not virus strain-specific, anti-receptor binding site (RBS), antibodies (Abs). Since only a few Abs recognizing epitopes in the head region but outside the RBS have been tested against single-challenge virus strains, it remains unknown whether Fc-Fc␥R interactions are required for in vivo protection by Abs recognizing epitopes outside the RBS and whether the requirement is virus strain specific or epitope specific. In the present study, we therefore investigated the requirements for in vivo protection using two pan-H5 Abs, 65C6 and 100F4. We generated chimeric Abs, 65C6/IgG2a and 100F4/IgG2a, which preferentially engage activating Fc␥Rs, and isogenic forms, 65C6/D265A and 100F4/D265A, which do not bind Fc␥R. Virus neutralizing activity, binding, antibody-dependent cellular cytotoxicity (ADCC), and in vivo protection of these Abs were compared using three H5 strains, A/Shenzhen/406H/2006 (SZ06), A/chicken/Shanxi/2/2006 (SX06), and A/chicken/Netherlands/14015526/2014 (NE14). We found that all four chimeric Abs bound and neutralized the SZ06 and NE14 strains but poorly inhibited the SX06 strain. 65C6/IgG2a and 100F4/IgG2a, but not 65C6/D265A and 100F4/D265A, mediated ADCC against target cells expressing HA derived from all three virus strains. Interestingly, both 65C6/IgG2a and 65C6/D265A demonstrated comparable protection against all three virus strains in vivo; however, 100F4/IgG2a, but not 100F4/D265A, showed in vivo protection. Thus, we conclude that Fc-Fc␥R interactions are required for in vivo protection by 100F4, but not by 65C6, and therefore, protection is not virus strain specific but epitope specific.IMPORTANCE Abs play an important role in immune protection against influenza virus infection. Fc-Fc␥R interactions are required for in vivo protection by broadly neutralizing antistem, but not by virus strain-specific, anti-receptor binding site (RBS), Abs. Whether such interactions are necessary for protection by Abs that recognize epitopes outside RBS is not fully understood. In the present study, we investigated in vivo protection mechanisms against three H5 strains by two pan-H5 Abs, 65C6 and 100F4. We show that although these two Abs have similar neutralizing, binding, and ADCC activities against all three H5 strains in vitro, they have divergent requirements for Fc-Fc␥R interactions to protect against the three H5 strains in vivo. The FcFc␥R interactions are required for in vivo protection by 100F4, but not by 65C6. Thus, we conclude that Fc-Fc␥R interactions for in vivo protection by pan-H5 Abs is not strain specific, but epitope specific.KEYWORDS influenza viruses, in vivo protection, monoclonal antibodies

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“…Most of the characterized mAbs against HA from H5N1 virus recognize the epitopes localized in the globular head of HA, most of them overlap the RBS and are isolate-specific, (e.g. Cao et al, 2012b;Du et al, 2013;Wu et al, 2014), while antibodies binding to the stem region have often a broad activity (Tan et al, 2015;Zuo et al, 2015). Extensive review of H5 HA antigenic sites was provided by (Velkov et al, 2013) who described the anti-HA antibodies divided into 3 groups: (i) RBS selective, (ii) non-RBS, membrane-distal globular domain selective and (iii) HA2 selective.…”

Section: Introductionmentioning

confidence: 99%

Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody

Sawicka¹,

Siedlecki²,

Kalenik³

et al. 2016

Acta Biochim Pol

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Hemagglutinin (HA), as a major surface antigen of influenza virus, is widely used as a target for production of neutralizing antibodies. Monoclonal antibody, mAb6-9-1, directed against HA of highly pathogenic avian influenza virus A/swan/Poland/305-135V08/2006(H5N1) was purified from mouse hybridoma cells culture and characterized. The antigenic specificity of mAb6-9-1 was verified by testing its cross-reactivity with several variants of HA. The mimotopes recognized by mAb6-9-1 were selected from two types of phage display peptide libraries. The comparative structural model of the HA variant used for antibody generation was developed to further facilitate epitope mapping. Based on the sequences of the affinity-selected polypeptides and the structural model of HA the epitope was located to the region near the receptor binding site (RBS). Such localization of the epitope recognized by mAb6-9-1 is in concordance with its moderate hemagglutination inhibiting activity and its antigenic specificity. Additionally, total RNA isolated from the hybridoma cell line secreting mAb6-9-1 was used for obtaining two variants of cDNA encoding recombinant single-chain variable fragment (scFv) antibody. To ensure high production level and solubility in bacterial expression system, the scFv fragments were produced as chimeric proteins in fusion with thioredoxin or displayed on a phage surface after cloning into the phagemid vector. Specificity and affinity of the recombinant soluble and phage-bound scFv were assayed by suitable variants of ELISA test. The observed differences in specificity were discussed.

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Comprehensive analysis of antibody recognition in convalescent humans from highly pathogenic avian influenza H5N1 infection

Cited by 38 publications

References 62 publications

Cross-protection of newly emerging HPAI H5 viruses by neutralizing human monoclonal antibodies: A viable alternative to oseltamivir

Cross-protection of newly emerging HPAI H5 viruses by neutralizing human monoclonal antibodies: A viable alternative to oseltamivir

Divergent Requirement of Fc-Fcγ Receptor Interactions for In Vivo Protection against Influenza Viruses by Two Pan-H5 Hemagglutinin Antibodies

Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody

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