Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors

Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.     

Induction of epitope-specific neutralizing antibodies against West Nile virus

Previous studies have established that an epitope on the lateral ridge of domain III (DIII-lr) of West Nile virus (WNV) envelope (E) protein is recognized by strongly neutralizing type-specific antibodies. In contrast, an epitope against the fusion loop in domain II (DII-fl) is recognized by flavivirus cross-reactive antibodies with less neutralizing potential. Using gain- and loss-of-function E proteins and wild-type and variant WNV reporter virus particles, we evaluated the expression pattern and activity of antibodies against the DIII-lr and DII-fl epitopes in mouse and human serum after WNV infection. In mice, immunoglobulin M (IgM) antibodies to the DIII-lr epitope were detected at low levels at day 6 after infection. However, compared to IgG responses against other epitopes in DI and DII, which were readily detected at day 8, the development of IgG against DIII-lr epitope was delayed and did not appear consistently until day 15. This late time point is notable since almost all death after WNV infection in mice occurs by day 12. Nonetheless, at later time points, DIII-lr antibodies accumulated and comprised a significant fraction of the DIII-specific IgG response. In sera from infected humans, DIII-lr antibodies were detected at low levels and did not correlate with clinical outcome. In contrast, antibodies to the DII-fl were detected in all human serum samples and encompassed a significant percentage of the anti-E protein response. Our experiments suggest that the highly neutralizing DIII-lr IgG antibodies have little significant role in primary infection and that the antibody response of humans may be skewed toward the induction of cross-reactive, less-neutralizing antibodies.     

A single serum dilution method for the quantitation of neutralizing antibodies to varicella-zoster virus

A one-point serum dilution method for determination of neutralizing antibody in human sera to varicella-zoster (V-Z) virus instead of the serial serum dilution method was investigated. Focus counting was performed under a microscope on day 5 to 6 after inoculation of V-Z virus into 6-well plastic trays in which human embryonic lung cells were grown. A table was constructed to estimate the ND50 titers by the per cent reduction of the focus count from the control at only one dilution of test sera. The estimated ND50 values agreed well with those determined by the serial serum dilution method. Test sera showed a slight nonspecific reactivity at low serum dilutions, but reliable results could usually be obtained at a serum dilution of 1:8 or more. This method, which saves materials and labor, was applied to the quantification of neutralizing antibody against V-Z virus in human sera with satisfactory accuracy and reproducibility.     

Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge

A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable.     

抗人类免疫缺陷病毒1型广谱中和抗体的研究进展

现行抗反转录病毒治疗药物的联合应用可有效抑制艾滋病进程并显著延长患者寿命,但由于人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)潜伏库的存在,艾滋病迄今尚无法治愈。近年发现抗HIV广谱中和抗体能有效降低患者体内病毒载量并延缓疾病进程,为研发艾滋病疫苗和治愈策略带来了曙光,尤其是序贯免疫策略的使用极大推进了广谱中和抗体的开发和应用进程。2018年,美国食品药品管理局(Food and Drug Administration,FDA)批准了第1个临床应用的广谱中性单克隆和抗体,无疑为抗HIV单克隆抗体药物的研发注入了一支强心剂。本文围绕近年来抗HIV广谱中和抗体的研究进展进行综述,探讨未来广谱中和抗体研发面临的挑战。     

Epitope mapping of neutralizing monoclonal antibodies against duck hepatitis B virus.

In this article we report the first topological mapping of neutralizing epitopes of a hepadnavirus. Duck hepatitis B virus is the only hepadnavirus that can replicate and spread from cell to cell in tissue culture. As a result, it is possible to study hepadnaviral neutralization in vitro with this system. To accomplish this goal, we produced a library of monoclonal antibodies against duck hepatitis B virus and identified 12 neutralizing monoclonal antibodies by using an in vitro neutralization assay. The characteristics of six of the neutralizing monoclonal antibodies were further studied by epitope mapping. From the results of competitive binding studies, three distinct neutralizing epitopes were identified on the pre-S polypeptides and one was identified on the S polypeptide. Our findings suggest that antibodies to both the pre-S and S gene products of duck hepatitis B virus can neutralize viral infection in vitro. The pre-S gene product is at least as important as the S gene product in eliciting neutralizing antibodies.     

Radioimmunoassay for antibodies in varicella-zoster virus serology.

The method of RIA for antibodies was employed with success in VZ virus serology. The method is suitable for testing VZ antibodies in the course of varicella or herpes zoster disease as well as for determining anamnestic titres. Its advantages are stability of antigen, objective reading of results and applicability to testing large serum sets.     

Development and characterization of neutralizing monoclonal antibodies against canine distemper virus hemagglutinin protein

Canine distemper virus (CDV) causes a serious multisystemic disease in dogs and other carnivora. Hemagglutinin (H) protein‐specific antibodies are mainly responsible for protective immunity against CDV infection. In the present study, six neutralizing MAbs to the H protein of CDV were newly obtained and characterized by immunizing BALB/c mice with a recent Chinese field isolate. Competitive binding inhibition assay revealed that they recognized four distinct antigenic regions of the H protein. Immunofluorescence assay and western blotting showed that all MAbs recognize the conformational rather than the linear epitopes of the H protein. Furthermore, in immunofluorescence and virus neutralization assays, two of the MAbs were found to react only with the recent Chinese field isolate and not with older CDV strains, including vaccine strain Onderstepoort, indicating there are neutralization‐related antigenic variations between the recent Chinese field isolate and the older CDV strains examined in this study. The newly established MAbs are useful for differentiating the expanding CDV strains and could be used in immunotherapy and immunodiagnosis against infection with CDV.     

sBAFF mutants induce neutralizing antibodies against BAFF

B cell activating factor belonging to the TNF family (BAFF) is a novel member of the tumor necrosis factor (TNF) ligand family and plays an important role in B lymphocyte maturation and survival. Overexpression of BAFF is closely involved in the pathogenesis and progression of many kinds of autoimmune disorders; therefore, BAFF has been considered as an ideal therapeutic target for these conditions. In this study, we generated several candidate immune inhibitors of human BAFF by conjugating foreign immunodominant T-helper cell (Th) epitopes to the N- or C-terminus of five BAFF mutants. The recombined proteins were successfully expressed in Escherichia coli (E. coli) and purified by Ni-NTA chromatography. BALB/c mice immunized with the recombinant proteins produced high levels of anti-BAFF antibodies, and their sera inhibited the lymphocyte proliferation-inducing activity of recombinant soluble BAFF and natural soluble BAFF. Moreover, antibodies cross-reactive with BAFF were detected in sera from hu-SCID mice immunized with the recombinant proteins. These results indicated that the recombinant BAFF mutants modified with Th epitopes could induce neutralizing antibodies against BAFF in vivo. This study may provide a valuable strategy for treating BAFF-associated autoimmune diseases.     

Human-like antibodies neutralizing Western equine encephalitis virus

This study describes the development of the first neutralizing antibodies against Western equine encephalitis virus (WEEV), a member of the genus Alphavirus. WEEV is transmitted by mosquitoes and can spread to the human central nervous system, causing symptoms ranging from mild febrile reactions to life-threatening encephalitis. WEEV has been classified as a biological warfare agent by the US Centers for Disease Control and Prevention. No anti-WEEV drugs are currently commercially available. Neutralizing antibodies are useful for the pre- and post-exposure treatment of WEEV infections. In this study, two immune antibody gene libraries were constructed from two macaques immunized with inactivated WEEV. Four antibodies were selected from these libraries and recloned as scFv-Fc, with a human Fc part. These antibodies bound WEEV specifically in ELISA with little or no cross-reaction with other alphaviruses. They were further analyzed by immunohistochemistry. All binders were suitable for the intracellular detection of WEEV particles. Neutralizing activity was determined in vitro. Three of the four antibodies were found to be neutralizing; about 1 ng/mL of the best antibody (ToR69–3A2) neutralized 50% of 5x10⁴ TCID₅₀/mL. Due to its human-like nature with a germinality index of 89% (VH) and 91% (VL), the ToR69–3A2 antibody is a promising candidate for future passive vaccine development.     

Isolation and characterization of novel human monoclonal antibodies possessing neutralizing ability against rabies virus

Rabies is a fatal viral encephalitis which is transmitted by exposure to the bite of rabid animals. Human and equine rabies immunoglobulins are indispensable pharmacological agents for severe bite exposure, as is vaccine. However, several disadvantages, including limited supply, adverse reactions, and high cost, hamper their wide application in developing countries. In the present study, two novel huMabs which neutralize rabies virus were established from vaccinated hyperimmune volunteers using the Epstein‐Barr virus transformation method. One MAb (No. 254), which was subclass IgG3, effectively neutralized fixed rabies viruses of CVS, ERA, HEP‐Flury, and Nishigahara strains and recognized a well‐conserved epitope located in antigenic site II of the rabies virus glycoprotein. No. 254 possessed 68 ng/ml of FRNT₅₀ activity against CVS, 3.7 × 10^?7 M of the Kd value, and the enhancing effect of complement‐dependent virolysis. In addition, No. 254 showed effective neutralization potency in vivo in the mouse challenge test. The other MAb, 4D4, was subclass IgM and showed neutralizing activity against CVS and Nishigahara strains. 4D4 recognized a novel antigenic site which is associated with the neurovirulence of rabies, a glycoprotein located between antigenic site I and VI. Both human MAbs against rabies are expected to be utilized as a tool for future post‐exposure prophylaxis.     

Human-like antibodies neutralizing Western equine encephalitis virus

This study describes the development of the first neutralizing antibodies against Western equine encephalitis virus (WEEV), a member of the genus Alphavirus. WEEV is transmitted by mosquitoes and can spread to the human central nervous system, causing symptoms ranging from mild febrile reactions to life-threatening encephalitis. WEEV has been classified as a biological warfare agent by the US Centers for Disease Control and Prevention. No anti-WEEV drugs are currently commercially available. Neutralizing antibodies are useful for the pre- and post-exposure treatment of WEEV infections. In this study, two immune antibody gene libraries were constructed from two macaques immunized with inactivated WEEV. Four antibodies were selected from these libraries and recloned as scFv-Fc, with a human Fc part. These antibodies bound WEEV specifically in ELISA with little or no cross-reaction with other alphaviruses. They were further analyzed by immunohistochemistry. All binders were suitable for the intracellular detection of WEEV particles. Neutralizing activity was determined in vitro. Three of the four antibodies were found to be neutralizing; about 1 ng/mL of the best antibody (ToR69–3A2) neutralized 50% of 5x10⁴ TCID₅₀/mL. Due to its human-like nature with a germinality index of 89% (VH) and 91% (VL), the ToR69–3A2 antibody is a promising candidate for future passive vaccine development.     

抗新型冠状病毒中和抗体药物临床研究进展北大核心CSCD

自1998年预防呼吸合胞病毒的帕利珠单抗药物上市以来,多种靶向病毒的治疗性抗体药物已成功用于感染性疾病的临床治疗。新型冠状病毒肺炎疫情暴发后,多种中和抗体药物快速进入临床研究阶段,展现出积极的治疗及预防效果,并以紧急使用授权的方式用于疫情防控。本文对抗新型冠状病毒中和抗体药物的临床进展和主要临床试验结果进行总结,以期为包括新型冠状病毒肺炎在内的新发、突发传染病中和抗体药物研发提供参考。     

Anti-idiotypic antibodies induce neutralizing antibodies to rabies virus glycoprotein.

Rabbit anti-idiotypic antibodies (alpha Id Ab) were prepared against five murine monoclonal antibodies (mAb) specific for the rabies virus glycoprotein. Four of the mAb were directed against three known, type-specific, neutralizing sites on the glycoprotein, and the other mAb was directed against a topographically uncharacterized, nonneutralizing epitope. An absence of significant cross-reactivity among the alpha Id Ab for heterologous mAb suggested that the alpha Id Ab were highly specific for unique variable region determinants. The binding of three of the five alpha Id Ab to their homologous mAb could be inhibited by rabies virus-soluble glycoprotein, suggesting that the alpha Id Ab possessed subpopulations similar or adjacent to the antigen-binding site of the mAb. Two of the five alpha Id Ab injected into mice elicited a specific virus-neutralizing antibody response. Mechanisms to account for the induction of the virus-neutralizing antibody by alpha Id Ab are discussed.     

Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type 1.

Expression vectors based on DNA or plus-stranded RNA viruses are being developed as vaccine carriers directed against various pathogens. Less is known about the use of negative-stranded RNA viruses, whose genomes have been refractory to direct genetic manipulation. Using a recently described reverse genetics method, we investigated whether influenza virus is able to present antigenic structures from other infectious agents. We engineered a chimeric influenza virus which expresses a 12-amino-acid peptide derived from the V3 loop of gp120 of human immunodeficiency virus type 1 (HIV-1) MN. This peptide was inserted into the loop of antigenic site B of the influenza A/WSN/33 virus hemagglutinin (HA). The resulting chimeric virus was recognized by specific anti-V3 peptide antibodies and a human anti-gp120 monoclonal antibody in both hemagglutination inhibition and neutralization assays. Mice immunized with the chimeric influenza virus produced anti-HIV antibodies which were able to bind to synthetic V3 peptide, to precipitate gp120, and to neutralize MN virus in human T-cell culture system. In addition, the chimeric virus was also capable of inducing cytotoxic T cells which specifically recognize the HIV sequence. These results suggest that influenza virus can be used as an expression vector for inducing both B- and T-cell-mediated immunity against other infectious agents.     

Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes

The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV) antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs) was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies. DENV RVPs used against human sera yielded serotype-specific responses and reproducible neutralization titers that were in statistical agreement with Plaque Reduction Neutralization Test (PRNT) results. DENV RVPs were also used to measure neutralization titers against the four DENV serotypes in a panel of human sera from a clinical study of dengue patients. The high-throughput capability, stability, rapidity, and reproducibility of assays using DENV RVPs offer advantages for detecting immune responses that can be applied to large-scale clinical studies of DENV infection and vaccination.