Characterization of a human antibody fragment Fab and its calcium phosphate nanoparticles that inhibit rabies virus infection with vaccine

Recombinant antibody phage display technology has been used to mimic many aspects of the processes that govern the generation and selection of high-affinity natural human antibodies in the human immune system, especially for infectious disease prophylaxis. An anti-rabies virus immunized phage-display Fab library was constructed from peripheral blood lymphocytes from vaccinated volunteers. The immunized antibody library, with a diversity of 6.7×10(8), was used to select and produce antibodies that bound to rabies virus glycoprotein. After five rounds of immobilized fixed rabies virion panning, four unique DNA sequences were found in the higher binding clones, and only one, Fab094, showed neutralization activity. Fab094 components were analyzed by ELISA, immunoprecipitation and immunofluorescent staining. ELISA and immunofluorescence showed that Fab094 bound specifically to rabies virions. Immunoprecipitation and mass spectrometry showed that Fab094 reacted with rabies virus glycoprotein. To improve the penetration power of Fab094 antibodies, we developed Fab094 calcium phosphate nanoparticles (Fab094-CPNPs) and tested their efficacy. The rapid fluorescent focus inhibition test indicated that the neutralizing antibody titers of Fab094 and Fab094-CPNPs were reached at 200.17 IU/Kg and 246.12 IU/Kg, respectively. These findings were confirmed in vivo in a Kunming mouse challenge model. Our results demonstrate that human Fab094 and Fab094-CPNPs are efficacious candidate drugs to replace rabies immunoglobulin in post-exposure prophylaxis (PEP).     

Novel human monoclonal antibody combination effectively neutralizing natural rabies virus variants and individual in vitro escape mutants

The need to replace rabies immune globulin (RIG) as an essential component of rabies postexposure prophylaxis is widely acknowledged. We set out to discover a unique combination of human monoclonal antibodies (MAbs) able to replace RIG. Stringent criteria concerning neutralizing potency, affinity, breadth of neutralization, and coverage of natural rabies virus (RV) isolates and in vitro escape mutants were set for each individual antibody, and the complementarities of the two MAbs were defined at the onset. First, we identified and characterized one human MAb (CR57) with high in vitro and in vivo neutralizing potency and a broad neutralization spectrum. The linear antibody binding site was mapped on the RV glycoprotein as antigenic site I by characterizing CR57 escape mutants. Secondly, we selected using phage display a complementing antibody (CR4098) that recognized a distinct, nonoverlapping epitope (antigenic site III), showed similar neutralizing potency and breadth as CR57, and neutralized CR57 escape mutants. Reciprocally, CR57 neutralized RV variants escaping CR4098. Analysis of glycoprotein sequences of natural RV isolates revealed that the majority of strains contain both intact epitopes, and the few remaining strains contain at least one of the two. In vitro exposure of RV to the combination of CR57 and CR4098 yielded no escape mutants. In conclusion, a novel combination of human MAbs was discovered suitable to replace RIG.     

Construction of human Fab library and isolation of monoclonal Fabs with rabies virus-neutralizing ability

A combinatorial human Fab library was constructed using RNAs from peripheral blood lymphocytes of 6 rabies vaccine-boosted volunteers using pComb3X phagemid vector. The size of the constructed library was approximately 7.0 x 10(7) Escherichia coli transformants. The library was selected against purified rabies virus (RV) virion or purified RV glycoprotein for isolation of phages displaying RVneutralizing human Fab antibody. Among 132 selected clones, two Fab preparations revealed neutralizing activities against RV strain CVS when assayed in the rapid fluorescent focus inhibition test (RFFIT). The Fab preparation EP5G3 exhibited neutralizing activity with an infected cell count reduction of 76% at a dilution of 1: 2, and of 20% at a dilution of 1: 4. The Fab preparation GD2D12 also exhibited neutralizing activity with a 57% reduction at 1: 2 and 41% reduction at 1: 4. In the co-immunoprecipitation using strain CVS, the RV glycoprotein was precipitated in reactions with both Fab preparations. The RV neutralizing ability of the Fab preparations described in the study were not directly correlated with their binding specificity for RV antigens detected by ELISA.     

Development of a Mouse Monoclonal Antibody Cocktail for Post-exposure Rabies Prophylaxis in Humans

As the demand for rabies post-exposure prophylaxis (PEP) treatments has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide the required passive immune component in PEP in countries where canine rabies is endemic. Replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a mouse monoclonal antibody (MoMAb) cocktail with the ultimate goal to develop a product at the lowest possible cost that can be used in developing countries as a replacement for RIG in PEP. Five MoMAbs, E559.9.14, 1112-1, 62-71-3, M727-5-1, and M777-16-3, were selected from available panels based on stringent criteria, such as biological activity, neutralizing potency, binding specificity, spectrum of neutralization of lyssaviruses, and history of each hybridoma. Four of these MoMAbs recognize epitopes in antigenic site II and one recognizes an epitope in antigenic site III on the rabies virus (RABV) glycoprotein, as determined by nucleotide sequence analysis of the glycoprotein gene of unique MoMAb neutralization-escape mutants. The MoMAbs were produced under Good Laboratory Practice (GLP) conditions. Unique combinations (cocktails) were prepared, using different concentrations of the MoMAbs that were capable of targeting non-overlapping epitopes of antigenic sites II and III. Blind in vitro efficacy studies showed the MoMab cocktails neutralized a broad spectrum of lyssaviruses except for lyssaviruses belonging to phylogroups II and III. In vivo, MoMAb cocktails resulted in protection as a component of PEP that was comparable to HRIG. In conclusion, all three novel combinations of MoMAbs were shown to have equal efficacy to HRIG and therefore could be considered a potentially less expensive alternative biological agent for use in PEP and prevention of rabies in humans.     

Recombinant monoclonal antibodies for rabies post-exposure prophylaxis

Rabies virus is a prototypical neurotropic virus that causes one of the most dangerous zoonotic diseases in humans. Humanized or fully human monoclonal antibodies (mAb) that neutralize rabies virus would be the basis for powerful post-exposure prophylaxis of rabies in humans, having several significant benefits in comparison with human or equine rabies polyclonal immunoglobulins. The most advanced antibodies should broadly neutralize natural rabies virus isolates, bind with conserved antigenic determinants of the rabies virus glycoprotein, and show high neutralizing potency in assays in vivo. The antibodies should recognize nonoverlapping epitopes if they are used in combination. This review focuses on basic requirements for anti-rabies therapeutic antibodies. The urgency in the search for novel rabies post-exposure prophylaxis and methods of development of anti-rabies human mAb cocktail are discussed. The rabies virus structure and pathways of its penetration into the nervous system are also briefly described.     

Single Domain Antibody Multimers Confer Protection against Rabies Infection

Post-exposure prophylactic (PEP) neutralizing antibodies against Rabies are the most effective way to prevent infection-related fatality. The outer envelope glycoprotein of the Rabies virus (RABV) is the most significant surface antigen for generating virus-neutralizing antibodies. The small size and uncompromised functional specificity of single domain antibodies (sdAbs) can be exploited in the fields of experimental therapeutic applications for infectious diseases through formatting flexibilities to increase their avidity towards target antigens. In this study, we used phage display technique to select and identify sdAbs that were specific for the RABV glycoprotein from a naïve llama-derived antibody library. To increase their neutralizing potencies, the sdAbs were fused with a coiled-coil peptide derived from the human cartilage oligomeric matrix protein (COMP48) to form homogenous pentavalent multimers, known as combodies. Compared to monovalent sdAbs, the combodies, namely 26424 and 26434, exhibited high avidity and were able to neutralize 85-fold higher input of RABV (CVS-11 strain) pseudotypes in vitro, as a result of multimerization, while retaining their specificities for target antigen. 26424 and 26434 were capable of neutralizing CVS-11 pseudotypes in vitro by 90–95% as compared to human rabies immunoglobulin (HRIG), currently used for PEP in Rabies. The multimeric sdAbs were also demonstrated to be partially protective for mice that were infected with lethal doses of rabies virus in vivo. The results demonstrate that the combodies could be valuable tools in understanding viral mechanisms, diagnosis and possible anti-viral candidate for RABV infection.     

Assessing clinicians’ Post-Exposure Prophylaxis recommendations for rabies virus exposures in Hunan Province,China

BackgroundTimely and appropriate administration of post-exposure prophylaxis (PEP) is an essential component of human rabies prevention programs. We evaluated patient care at rabies clinics in a high-risk county in Hunan Province, China to inform strategies needed to achieve dog-mediated human rabies elimination by 2030.MethodsWe collected information on PEP, staff capacity, and service availability at the 17 rabies clinics in the high-risk county during onsite visits and key staff interviews. Additionally, we conducted observational assessments at five of these clinics, identified through purposive sampling to capture real-time information on patient care during a four-week period. Wound categories assigned by trained observers were considered accurate per national guidelines for comparison purposes. We used the kappa statistic and an alpha level of 0.05 to assess agreement between observers and clinic staff.ResultsIn 2015, the 17 clinics provided PEP to 5,261 patients. Although rabies vaccines were available at all 17 clinics, rabies immune globulin (RIG) was only available at the single urban clinic in the county. During the assessment period in 2016, 196 patients sought care for possible rabies virus exposures. According to observers, 88 (44%) patients had category III wounds, 104 (53%) had category II wounds and 4 (2%) had category I wounds. Observers and PEP clinic staff agreed on approximately half of the assigned wound categories (kappa = 0.55, p-value< 0.001). Agreement for the urban county-level CDC clinic (kappa = 0.93, p-value<0.001) was higher than for the township clinics (kappa = 0.16, p-value = 0.007). Using observer assigned wound categories, 142 (73%) patients received rabies vaccinations and RIG as outlined in the national guidelines.ConclusionRabies PEP services were available at each town of the project county; however, gaps between clinical practice and national rabies guidelines on the use of PEP were identified. We used these findings to develop and implement a training to rabies clinic staff on wound categorization, wound care, and appropriate use of PEP. Additional risk-based approaches for evaluating human rabies virus exposures may be needed as China progresses towards elimination.     

人源中和性抗汉滩病毒单克隆抗体Fab段基因的获得和表达

运用噬菌体表面表达技术,获得人源和中性抗滩滩病毒汉滩型Ｇ１基因工程单克隆抗体Ｆａｂ段基因及其表达,并同时获得抗汉滩病毒核蛋白的Ｆａｂ抗体。从能综合征出血热疫区恢复期病人抗凝血中分离到的外周淋巴细胞中,提取了部细胞ＲＮＡ。通过ＲＴ－ＰＣＲ方法,用一组人ＩｇＧ Ｆａｂ基因特异性引物,从合成了ｃＤＮＡ中经ＰＣＲ扩增了一组轻链和重链Ｆａｂ段基因,将轻链和重链先后插入噬菌体载体ｐＣｏｍｂ３,ｄｎａｌｆ ｖｆ     

A DNA Construct That Encodes the Rabies Virus Consensus Glycoprotein with a Proteasome Degradation Signal Induces Antibody Production with IgG2A Subtype Predominance

The possibility of enhancing the immunogenicity of the rabies virus glycoprotein antigen encoded by a DNA vaccine has been investigated. Ubiquitin-like protein FAT10 has been attached to the N-terminus of the glycoprotein to target it to the proteasome and stimulate its presentation by MHC class I. Two forms of the protein, chimeric and original, have been detected in cells transfected with the DNA construct encoding the chimeric protein. The presence of the glycoprotein on the cell surface has been detected by immunostaining of transfected cells. The production of IgG and IgG2a antibodies has been more efficiently induced in mice immunized with the plasmid that encodes the chimeric protein than in those immunized with the plasmid that encodes unmodified glycoprotein. Moreover, the level of IgG2a antibodies exceeded the level of IgG1 antibodies, which indicates a preferential increase in the Th1 component of the immune response. The proposed DNA construct that encodes a modified glycoprotein with a proteasome degradation signal may be a promising DNA vaccine immunogen for post-exposure prophylaxis of rabies.     

抗狂犬病毒中和抗体研究进展

狂犬病是一种人兽共患传染病,人和动物一旦发病后死亡率几乎百分之百,而有效的暴露后预防措施可以将死亡风险降至零。根据WHO推荐的狂犬病暴露后预防方案,一般狂犬病暴露者需要进行疫苗注射,严重者则需在进行疫苗注射的同时注射抗狂犬病毒中和抗体。常用的中和抗体有马抗狂犬病毒免疫球蛋白和人抗狂犬病毒免疫球蛋白,然而两者都存在引起过敏反应或血液疾病的风险。人源抗狂犬病毒中和抗体则因为具有安全性高、成本低、可量产等优点有望代替免疫球蛋白用于暴露后预防。基因工程抗体技术的发展加速了抗体人源化的进程。就抗狂犬病毒中和抗体的发展历程,不同类型中和抗体的优缺点以及中和抗体的未来研究方向作了综述及展望,以期为新一代狂犬疫苗的研发提供参考。     

Post-exposure Treatment with Anti-rabies VHH and Vaccine Significantly Improves Protection of Mice from Lethal Rabies Infection

Post-exposure prophylaxis (PEP) against rabies infection consists of a combination of passive immunisation with plasma-derived human or equine immune globulins and active immunisation with vaccine delivered shortly after exposure. Since anti-rabies immune globulins are expensive and scarce, there is a need for cheaper alternatives that can be produced more consistently. Previously, we generated potent virus-neutralising VHH, also called Nanobodies, against the rabies glycoprotein that are effectively preventing lethal disease in an in vivo mouse model. The VHH domain is the smallest antigen-binding functional fragment of camelid heavy chain-only antibodies that can be manufactured in microbial expression systems. In the current study we evaluated the efficacy of half-life extended anti-rabies VHH in combination with vaccine for PEP in an intranasal rabies infection model in mice. The PEP combination therapy of systemic anti-rabies VHH and intramuscular vaccine significantly delayed the onset of disease compared to treatment with anti-rabies VHH alone, prolonged median survival time (35 versus 14 days) and decreased mortality (60% versus 19% survival rate), when treated 24 hours after rabies virus challenge. Vaccine alone was unable to rescue mice from lethal disease. As reported also for immune globulins, some interference of anti-rabies VHH with the antigenicity of the vaccine was observed, but this did not impede the synergistic effect. Post exposure treatment with vaccine and human anti-rabies immune globulins was unable to protect mice from lethal challenge. Anti-rabies VHH and vaccine act synergistically to protect mice after rabies virus exposure, which further validates the possible use of anti-rabies VHH for rabies PEP.     

人源抗狂犬病毒中和性全抗体在昆虫细胞中的表达

将来源于噬菌体抗体库的人源狂犬病毒糖蛋白特异性单抗G10Fab的基因 ,克隆入杆状病毒人源IgG抗体表达载体 ,通过转染将重组质粒导入昆虫细胞 ,以全抗体的形式表达了一株人源抗狂犬病毒基因工程抗体G10。用亲和层析的方法纯化了表达产物 ,经与一株鼠源糖蛋白特异性单抗竞争证实 ,该单克隆抗体特异性识别狂犬病毒糖蛋白 ,亲和力约为 10 -9M。体外中和实验证明 ,该单抗对狂犬病毒aG株具有体外中和活性     

Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1.

Six recombinant human Fab fragments that were derived from the same human immunodeficiency virus type 1 (HIV-1)-infected individual and are directed against the CD4 binding site (CD4bs) of the gp120 envelope glycoprotein were studied. A range of neutralizing activity against the HIV-1 (HXBc2) isolate was observed, with Fab b12 exhibiting the greatest potency among the Fabs tested. The neutralizing potency of Fab b12 was better than that of monoclonal whole antibodies directed against the third variable (V3) region of gp120. To explore the basis for the efficient neutralizing activity of b12, the recognition of a panel of HIV-1 gp120 mutants by the six Fabs was studied. The patterns of sensitivity to particular gp120 amino acid changes were similar for all six Fabs to those seen for anti-CD4bs monoclonal antibodies derived from HIV-1-infected individuals by conventional means. In addition, recognition by Fab b12 demonstrated an atypical sensitivity to changes in the V1 and V2 variable regions. Next, the binding of the Fabs to monomeric gp120 and to the envelope glycoprotein complex was examined. Neither the binding properties of the b12 Fab to monomeric gp120 nor the ability of the Fab to compete with soluble CD4 for monomeric gp120 binding appeared to account for the greater neutralizing potency. However, both quantitative and qualitative differences between the binding of b12 and that of less potent Fabs to the cell surface envelope glycoprotein complex were observed. Relative to less potently neutralizing Fabs, Fab b12 exhibited a higher affinity for a subpopulation of cell surface envelope glycoproteins, the conformation of which was best approximated by the mature gp120 glycoprotein. Apparently, subtle differences in the gp120 epitope recognized allow some members of the group of anti-CD4bs antibodies to bind to the functionally relevant envelope glycoprotein complex and to neutralize virus more efficiently.     

Generation and characterization of human monoclonal antibodies to G5, a linear neutralization epitope on glycoprotein of rabies virus, by phage display technology

The aim of the present study was to discover distinct human MAbs to RV with high neutralizing potency and a broad neutralization spectrum. A phage display technology was used to produce human scFv to G5, a conserved linear neutralization epitope on Gp of RV. A phage display scFv library with 6 x 10(7) members was constructed and the phage-scFv with 'antigen-binding' activities were selected with synthetic peptide G5-24. The obtained scFv genes were cloned into pET22b(+)/BL21(DE3) and from this we prepared purified scFv fragments. The assay of the specificity characteristics and neutralization capacity showed that two distinct clones with new human immunoglobulin V genes can recognize G5 specifically as well as neutralize different RV strains. They have potential for inclusion in an antibodies combination aimed for use in rabies PEP.     

Rabies neutralizing antibody in serum of children compared to adults following post-exposure prophylaxis.

Rabies neutralizing antibody concentrations in serum (SRNA) elicited by experimental post-exposure prophylaxis (PEP) regimens are used to assess their potential clinical efficacy. Although PEP with human rabies immunoglobulin (HRIG) plus human diploid-cell culture vaccine (HDCV) is almost completely protective, the limited availability of these components has stimulated the search for alternative regimens. Since 33% to over 60% of PEP is administered to children, a need exists for data on the SRNA response to HRIG plus HDCV in children, to be used to assess the potential value of alternative PEP treatments. We measured SRNA by the rapid fluorescent focus inhibition test on days 0, 1, 3, 7, 14, 28, 56 and 90 after initiation of PEP with HRIG (20 IU/kg) and HDCV (1.0 ml on day 0, 3, 7, 14, 28) in 10 children [8 +/- 4 (mean +/- SD) years of age] and 32 adult control patients (38 +/- 16 years of age). Early, uniform appearance of SRNA was observed with no differences between groups. These data are consistent with the demonstrated efficacy of PEP with HRIG plus HDCV in children as in adults and provide results that can be used for initial evaluation of experimental pediatric PEP regimens in the future.     

高效价冻干人用狂犬病疫苗暴露后免疫程序的研究

初步确定高效价冻干人用狂犬病疫苗（6．0IU／剂）暴露后免疫程序。制备高效价的冻干人用狂犬病疫苗（6．0IU／剂）,以狂犬病街毒CNX8601和BD06分别攻击小鼠和比格犬的咬肌,接种不同效价的狂犬病疫苗,以RFFIT法检测中和抗体,根据动物死亡情况,计算暴露后疫苗保护率,对不同效价的疫苗进行中和抗体测定和保护率统计分析。在以小鼠为实验动物的疫苗保护率研究中,冻干人用狂犬病疫苗（3．1IU／剂）0／3／7／14／28免疫程序的保护率为40．6％,高效价的冻干人用狂犬病疫苗（6．0IU／剂）0／3／14免疫程序的保护率为56．2％,中和抗体比较,P〈0．05,2组间有显著性差异;在以比格犬为实验动物的保护效果研究中,冻干人用狂犬病疫苗的保护率（3．1IU／剂）为70％;高效价的冻干人用狂犬病疫苗（6．0IU／剂）的保护率为80％,中和抗体的比较,P〉0．05,没有显著性差异。高效价冻干人用狂犬病疫苗暴露后免疫程序可初步确定为0、3、14d免疫。     

人源抗狂犬病毒单克隆抗体Fab段基因的获得和表达

运用噬菌体表面呈现（phage display）技术获得了人源抗狂犬病毒糖蛋白基因工程单克隆抗体Fab段基因及其表达。从狂犬病毒PM株Vero细胞疫苗免疫的人抗凝血中分离获得外周淋巴细胞,提取细胞总RNA,通过RTPCR方法,用一组人IgG Fab基因4特异性引物,从合成的cDNA中扩增了一组轻链和重链Fab段基因,将轻链和重链Fab段基因,将轻链和重链先后克隆入噬菌体载体pComb3,成功地建立了抗狂犬病毒抗原的方法,对此抗体库进行富积筛选表达,成功地获得了抗狂犬病毒的人源单抗Fab段基因及其在大肠杆菌中的有效表达,对其中一株单抗G10进行了较为系统的分析,发现它与一株鼠源中和性狂犬病毒糖蛋白特异性单抗存在竞争,证实该单抗能识别狂犬病毒糖蛋白,其序列资料分析表明,该单抗为一株新的抗狂犬病毒人源基因工程抗体。     

Identification of chimpanzee Fab fragments by repertoire cloning and production of a full-length humanized immunoglobulin G1 antibody that is highly efficient for neutralization of dengue type 4 virus

A safe and effective dengue vaccine is still not available. Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative for the prevention of dengue virus infection. Fab monoclonal antibodies to dengue type 4 virus (DENV-4) were recovered by repertoire cloning of bone marrow mRNAs from an immune chimpanzee and analyzed for antigen binding specificity, V(H) and V(L) sequences, and neutralizing activity against DENV-4 in vitro. Fabs 5A7, 3C1, 3E4, and 7G4 were isolated from a library constructed from a chimpanzee following intrahepatic transfection with infectious DENV-4 RNA. Fabs 5H2 and 5D9, which had nearly identical V(H) sequences but varied in their V(L) sequences, were recovered from a library constructed from the same chimpanzee after superinfection with a mixture of DENV-1, DENV-2, and DENV-3. In radioimmunoprecipitation, Fab 5A7 precipitated only DENV-4 prM, and Fabs 3E4, 7G4, 5D9, and 5H2 precipitated DENV-4 E but little or no prM. Fab 3E4 and Fab 7G4 competed with each other for binding to DENV-4 in an enzyme-linked immunosorbent assay, as did Fab 3C1 and Fab 5A7. Fab 5H2 recognized an epitope on DENV-4 that was separate from the epitope(s) recognized by other Fabs. Both Fab 5H2 and Fab 5D9 neutralized DENV-4 efficiently with a titer of 0.24 to 0.58 micro g/ml by plaque reduction neutralization test (PRNT), whereas DENV-4-neutralizing activity of other Fabs was low or not detected. Fab 5H2 was converted to full-length immunoglobulin G1 (IgG1) by combining it with human sequences. The humanized chimpanzee antibody IgG1 5H2 produced in CHO cells neutralized DENV-4 strains from different geographical origins at a similar 50% plaque reduction (PRNT(50)) titer of 0.03 to 0.05 micro g/ml. The DENV-4 binding affinities were 0.42 nM for Fab 5H2 and 0.24 nM for full-length IgG1 5H2. Monoclonal antibody IgG1 5H2 may prove valuable for passive immunoprophylaxis against dengue virus in humans.     

The mouse neutralization test in comparison with the rapid fluorescent focus inhibition test: differences in the results in rabies antibody determinations

Sixteen lots of rabies immune globulin (RIG) and six lots of rabies immune horse serum (RIS) from different producers were examined for rabies antibody by the standard mouse neutralization test (MNT) and the rapid fluorescent focus inhibition test (RFFIT). An equine rabies standard serum was assayed in parallel. In comparisons of RIS with this standard the MNT and RFFIT gave comparable results. In comparisons of RIG the antibody values in the MNT was two to ten times higher than that in the RFFIT in 15 out of 16 lots. The MNT and RFFIT are thus not fully comparable when measuring rabies antibodies in RIG. The choice of the titration method is obviously important in the measurement of the antibody concentration in RIG or RIS in IU against an equine rabies reference preparation. The described differences could have consequences for the use of RIG.     

Characterization of Two Human Monoclonal Antibodies Neutralizing Influenza A H7N9 Viruses

H7N9 was a cause of significant global health concern due to its severe infection and approximately 35% mortality in humans. By screening a Fab antibody phage library derived from patients who recovered from H7N9 infections, we characterized two human monoclonal antibodies (HuMAbs), HNIgGD5 and HNIgGH8. The epitope of these two antibodies was dependent on two residues in the receptor binding site at positions V186 and L226 of the hemagglutinin glycoprotein. Both antibodies possessed high neutralizing activity.