Optimization of anti-tumor necrosis factor-alpha single chain Fv displayed on phages for creation of functional antibodies

In this study, we converted the immunoglobulin-type anti-human tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody (Mab) to a scFv-type antibody in order to assess its basic properties. The immunoglobulin VH and VL genes were isolated from the hybridoma that produced an anti-TNF-alpha neutralizing Mab, and they were then linked together to create scFvs of the VL-VH or VH-VL-form. The binding affinity to TNF-a was retained in both scFvs. Interestingly, the VL-VH-type scFv effectively inhibited the TNF-alpha-mediated cytotoxicity, while this neutralization activity was dramatically decreased in the VH-VL-type scFv. These results suggest that the VL-VH-type scFv is a suitable template to create improved versions of the anti-TNF-alpha antibody using a phage display system, and they also show that the structural format must be taken into account in manufacturing scFvs.     

Full Neutralization of Centruroides sculpturatus Scorpion Venom by Combining Two Human Antibody Fragments

A fundamental issue of the characterization of single-chain variable fragments (scFvs), capable of neutralizing scorpion toxins, is their cross-neutralizing ability. This aspect is very important in Mexico because all scorpions dangerous to humans belong to the Centruroides genus, where toxin sequences show high identity. Among toxin-neutralizing antibodies that were generated in a previous study, scFv 10FG2 showed a broad cross-reactivity against several Centruroides toxins, while the one of scFv LR is more limited. Both neutralizing scFvs recognize independent epitopes of the toxins. In the present work, the neutralization capacity of these two scFvs against two medically important toxins of the venom of Centruroides sculpturatus Ewing was evaluated. The results showed that these toxins are recognized by both scFvs with affinities between 1.8 × 10⁻⁹ and 6.1 × 10⁻¹¹ M. For this reason, their ability to neutralize the venom was evaluated in mice, where scFv 10FG2 showed a better protective capacity. A combination of both scFvs at a molar ratio of 1:5:5 (toxins: scFv 10FG2: scFv LR) neutralized the venom without the appearance of any signs of intoxication. These results indicate a complementary activity of these two scFvs during venom neutralization.     

A Four-Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotypes C and D

Human botulism can be caused by botulinum neurotoxin (BoNT) serotypes A to G. Here, we present an antibody-based antitoxin composed of four human monoclonal antibodies (mAbs) against BoNT/C, BoNT/D, and their mosaic toxins. This work built on our success in generating protective mAbs to BoNT /A, B and E serotypes. We generated mAbs from human immune single-chain Fv (scFv) yeast-display libraries and isolated scFvs with high affinity for BoNT/C, BoNT/CD, BoNT/DC and BoNT/D serotypes. We identified four mAbs that bound non-overlapping epitopes on multiple serotypes and mosaic BoNTs. Three of the mAbs underwent molecular evolution to increase affinity. A four-mAb combination provided high-affinity binding and BoNT neutralization of both serotypes and their mosaic toxins. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing and neutralizing BoNT/C and BoNT/D serotypes and their mosaic toxins. A derivative of the four-antibody combination (NTM-1634) completed a Phase 1 clinical trial (Snow et al., Antimicrobial Agents and Chemotherapy, 2019) with no drug-related serious adverse events.     

Avian influenza: a review.

PURPOSE: A review of the avian influenza A/H5N1 virus, including human cases, viral transmission, clinical features, vaccines and antivirals, surveillance plans, infection control, and emergency response plans, is presented. SUMMARY: The World Health Organization (WHO) considers the avian influenza A/H5N1 virus a public health risk with pandemic potential. The next human influenza pandemic, if caused by the avian influenza A/H5N1 virus, is estimated to have a potential mortality rate of more than a hundred million. Outbreaks in poultry have been associated with human transmission. WHO has documented 258 confirmed human infections with a mortality rate greater than 50%. Bird-to-human transmission of the avian influenza virus is likely by the oral-fecal route. The most effective defense against an influenza pandemic would be a directed vaccine to elicit a specific immune response toward the strain or strains of the influenza virus. However, until there is an influenza pandemic, there is no evidence that vaccines or antivirals used in the treatment or prevention of such an outbreak would decrease morbidity or mortality. Surveillance of the bird and human populations for the highly pathogenic H5N1 is being conducted. Infection-control measures and an emergency response plan are discussed. CONCLUSION: Avian influenza virus A/H5N1 is a public health threat that has the potential to cause serious illness and death in humans. Understanding its pathology, transmission, clinical features, and pharmacologic treatments and preparing for the prevention and management of its outbreak will help avoid its potentially devastating consequences.     

人用H5N1禽流感病毒疫苗生产工艺的优化

目的 建立优化的人用H5N1禽流感病毒疫苗生产的工艺。方法 在不同的稀释倍数、收获时间及灭活剂添加量下,通过测量收获液的病毒滴度和血凝效价,来确定病毒的最佳生产条件,并对离心法和凝胶过滤层析法的纯化效果进行对比。结果 103~104半数鸡胚感染量(50% egg infective dose,EID50)病毒接种鸡胚,收获的鸡胚尿囊液的病毒滴度和血凝效价最高,分别为10-8.3EID50和1∶480;在56~72 h血凝效价最高。甲醛浓度1∶10 000灭活144 h为灭活最佳条件。两种纯化方法得到的样品纯度和卵清蛋白的去除率相近,但离心纯化法和凝胶过滤层析纯化法病毒回收率有较大的差异,分别为19%和70%。结论 成功建立了高产毒的鸡胚基质H5N1禽流感病毒培养、灭活及纯化工艺。     

The neuraminidase inhibitor GS4104 (oseltamivir phosphate) is efficacious against A/Hong Kong/156/97 (H5N1) and A/Hong Kong/1074/99 (H9N2) influenza viruses

In 1997, an H5N1 avian influenza A/Hong Kong/156/97 virus transmitted directly to humans and killed six of the 18 people infected. In 1999, another avian A/Hong/1074/99 (H9N2) virus caused influenza in two children. In such cases in which vaccines are unavailable, antiviral drugs are crucial for prophylaxis and therapy. Here we demonstrate the efficacy of the neuraminidase inhibitor GS4104 (oseltamivir phosphate) against these H5N1 and H9N2 viruses. GS4071 (the active metabolite of oseltamivir) inhibited viral replication in MDCK cells (EC(50) values, 7.5-12 microM) and neuraminidase activity (IC(50) values, 7.0-15 nM). When orally administered at doses of 1 and 10 mg/kg per day, GS4104 prevented death of mice infected with A/Hong Kong/156/97 (H5N1), mouse-adapted A/Quail/Hong Kong/G1/97 (H9N2), or human A/Hong Kong/1074/99 (H9N2) viruses and reduced virus titers in the lungs and prevented the spread of virus to the brain of mice infected with A/Hong Kong/156/97 (H5N1) and mouse-adapted A/Quail/Hong Kong/G1/97 (H9N2) viruses. When therapy was delayed until 36 h after exposure to the H5N1 virus, GS4104 was still effective and significantly increased the number of survivors as compared with control. Oral administration of GS4104 (0.1 mg/kg per day) in combination with rimantadine (1 mg/kg per day) reduced the number of deaths of mice infected with 100 MLD(50) of H9N2 virus and prevented the deaths of mice infected with 5 MLD(50) of virus. Thus, GS4104 is efficacious in treating infections caused by H5N1 and H9N2 influenza viruses in mice.     

A/H5N1 prepandemic influenza vaccine (whole virion, vero cell-derived, inactivated) [Vepacel®

The influenza A subtype H5N1 virus is a likely causative agent for the next human influenza pandemic. Pandemic influenza vaccine production can begin only after a novel pandemic virus emerges. Cell-based vaccine production has advantages over conventional egg-based methods, allowing more rapid large-scale vaccine production. A reliable Vero cell culture system is available for pandemic and prepandemic influenza vaccine production. Prepandemic influenza vaccines are an important component of influenza pandemic preparedness plans, as their targeted use in the pandemic alert period or early in a pandemic is likely to mitigate the consequences of an influenza outbreak. Vepacel? is a prepandemic influenza vaccine (whole virion, Vero cell-derived, inactivated) containing antigen of H5N1 strain A/Vietnam/1203/2004 and is approved for use in the EU. Clinical immunogenicity studies with the vaccine have demonstrated good rates of functional neutralizing antibody responses against the vaccine strain (A/Vietnam/1203/2004), meeting established immunogenicity criteria for seasonal influenza vaccines, and cross-reactivity against H5N1 strains from other clades. In phase I/II and III studies, a heterologous (A/Indonesia/05/2005) booster vaccine administered to healthy adult and elderly volunteers 6-24 months after the two-dose priming vaccine (A/Vietnam/1203/2004) regimen induced good immunogenic responses against both H5N1 strains, demonstrating strong immunological memory. Broadly similar, albeit less robust, responses were observed in two special risk cohorts of immunocompromised and chronically ill patients. In general, adverse events observed in clinical immunogenicity studies with H5N1 vaccine (A/Vietnam/1203/2004) were similar to those reported with non-adjuvanted, inactivated, seasonal influenza vaccines.     

Biological evaluation of anti-influenza viral activity of semi-synthetic catechin derivatives

Catechin derivatives with different alkyl chain length and aromatic ring substitutions at the 3-hydroxyl group were synthesized from epigallocatechin (EGC) and (+)-catechin (C) and their anti-influenza viral activity were evaluated in vitro and in ovo. Pronounced antiviral activity was observed for derivatives carrying moderate chain length (7-9 carbons) as compared to those with aromatic rings, whereas the 5'-hydroxyl group of the trihydroxy benzyl moiety did not significantly contribute to antiviral activity. The derivatives exerted inhibitory effects for all six influenza subtypes tested including three major types of currently circulating human influenza viruses (A/H1N1, A/H3N2 and B type), H2N2 and H9N2 avian influenza virus. The compounds strongly inhibited adsorption of the viruses on red blood cell (RBC). They also restricted the growth of avian influenza virus in ovo with minimum inhibition concentration (MIC) of 5-10 microM far exceeding the neuraminidase (NA) inhibitor oseltamivir or M2 proton channel inhibitor amantadine. The antiviral activity appears to be mediated by interaction with hemagglutinin (HA)/viral membrane rendering HA less fusogenic at the initial stage of infection. The broad spectrum activity against various subtypes of influenza viruses may complement the limitations of current antivirals and contribute for managing potentially emerging influenza pandemic. The structure-activity data of catechin derivatives may usefully guideline future research endeavors for applying green tea catechins as alternative anti-viral agents.     

Effective small interfering RNAs targeting matrix and nucleocapsid protein gene inhibit influenza A virus replication in cells and mice

RNA interference (RNAi) is a powerful tool to silence gene expression. Small interfering RNA (siRNA)-induced RNA degradation has been recently used as an antivirus agent to inhibit specific virus replication. Here, we showed that several siRNAs specific for conserved regions of influenza virus matrix (M2) and nucleocapsid protein (NP) genes could effectively inhibit expression of the corresponding viral protein. We also evaluated the antiviral potential of these siRNAs targeting M2 and NP of H5N1 avian influenza virus (AIV), which are essential to viral replication. We investigated the inhibitory effect of M2-specific siRNAs and NP-specific siRNAs on influenza A virus (H5N1, H1N1 and H9N2) replication in Madin-Darby canine kidney (MDCK) cells and BALB/c mice. The results showed that treatment with these siRNAs could specifically inhibit influenza A virus replication in MDCK cells (0.51-1.63 TCID(50) reduction in virus titers), and delivery of pS-M48 and pS-NP1383 significantly reduced lung virus titers in the infected mice (16-50-fold reduction in lung virus titers) and partially protected the mice from lethal influenza virus challenge (a survival rate of 4/8 for H1N1 virus-infected mice and 2/8 for H5N1 virus infected mice). Moreover, the treatment of pS-M48 and pS-NP1383 could suppress replication of different subtypes of influenza A viruses, including a H5N1 highly pathogenic avian isolate strain. The results provided a basis for further development of siRNA for prophylaxis and therapy of influenza virus infection in humans and animals.     

高致病性H7N9禽流感病毒研究进展

摘要：H7N9禽流感病毒在中国出现以来共造成5次流行。在第5次流行中出现了高致病性H7N9变异株,该病 毒株的HA链接肽位置发生了基因插入性突变,导致该病毒对家禽毒力的增强。同时在人感染H7N9禽流感病例中 也相继分离到了该病毒。因此,对高致病性H7N9禽流感病毒病原学及流行病学研究对于该疾病的预防和控制具有 重要意义。本文从高致病性H7N9禽流感病毒的变异来源、流行病学特征及防治措施等方面进行综述,为高致病性 H7N9禽流感的有效防治提供科学策略。     

Antiviral activity of the proteasome inhibitor VL-01 against influenza A viruses

The appearance of highly pathogenic avian influenza A viruses of the H5N1 subtype being able to infect humans and the 2009 H1N1 pandemic reveals the urgent need for new and efficient countermeasures against these viruses. The long-term efficacy of current antivirals is often limited, because of the emergence of drug-resistant virus mutants. A growing understanding of the virus-host interaction raises the possibility to explore alternative targets involved in the viral replication. In the present study we show that the proteasome inhibitor VL-01 leads to reduction of influenza virus replication in human lung adenocarcinoma epithelial cells (A549) as demonstrated with three different influenza virus strains, A/Puerto Rico/8/34 (H1N1) (EC₅₀ value of 1.7 μM), A/Regensburg/D6/09 (H1N1v) (EC₅₀ value of 2.4 μM) and A/Mallard/Bavaria/1/2006 (H5N1) (EC₅₀ value of 0.8 μM). In in vivo experiments we could demonstrate that VL-01-aerosol-treatment of BALB/c mice with 14.1 mg/kg results in no toxic side effects, reduced progeny virus titers in the lung (1.1 ± 0.3 log₁₀ pfu) and enhanced survival of mice after infection with a 5-fold MLD₅₀ of the human influenza A virus strain A/Puerto Rico/8/34 (H1N1) up to 50%. Furthermore, treatment of mice with VL-01 reduced the cytokine release of IL-α/β, IL-6, MIP-1β, RANTES and TNF-α induced by LPS or highly pathogen avian H5N1 influenza A virus. The present data demonstrates an antiviral effect of VL-01 in vitro and in vivo and the ability to reduce influenza virus induced cytokines and chemokines.     

Protective immunity elicited by a pseudotyped baculovirus-mediated bivalent H5N1 influenza vaccine

The development of novel H5N1 influenza vaccines to elicit a broad immune response is a priority in veterinary and human public health. In this report, a baculovirus vector was used to construct bivalent recombinant baculovirus vaccine encoding H5N1 influenza virus hemagglutinin proteins (BV-HAs) from clade 2.3.4 and clade 9 influenza viruses. Mice immunized with 5 × 10⁷ IFU BV-HAs developed significantly high levels of H5-specific neutralizing antibodies and cellular immunity that conferred 100% protection against infection with H5N1 influenza viruses. This study suggests that baculovirus-delivered multi-hemagglutinin proteins might serve as a candidate vaccine for the prevention of pre-pandemic and pandemic H5N1 influenza viruses.     

Avian influenza A (H5N1) infection: targets and strategies for chemotherapeutic intervention

In an avian flu pandemic, which drugs could be used to treat or prevent infection with influenza A (H5N1) virus? Foremost are the viral neuraminidase inhibitors oseltamivir and zanamivir, which have already been used to treat human influenza A (H1N1 and H3N2) and B virus infections. The use of the M2 ion channel blockers amantadine and rimantadine is compounded by the rapid development of drug resistance. Although formally approved for other indications (i.e. treatment of hepatitis C), ribavirin and pegylated interferon might also be useful for controlling avian flu. Combined use of the currently available drugs should be taken into account and attempts should be made to develop new strategies directed at unexplored targets such as the viral proteins hemagglutinin, the viral polymerase (and endonuclease) and the non-structural protein NS1. As has been shown for other viral infections, RNA interference could be a powerful means with which to suppress the replication of avian H5N1.     

Sialobiology of influenza: molecular mechanism of host range variation of influenza viruses

The gene pool of influenza A viruses in aquatic birds provides all of the genetic diversity required for human and lower animals. Host range selection of the receptor binding specificity of the influenza virus hemagglutinin occurs during maintenance of the virus in different host cells that express different receptor sialo-sugar chains. In this paper, functional roles of the hemagglutinin and neuraminidase spikes of influenza viruses are described in the relation to 1) host range of influenza viruses, 2) receptor binding specificity of human and other animal influenza viruses, 3) recognition of sialyl sugar chains by Spanish influenza virus hemagglutinin, 4) highly pathogenic and potentially pandemic H5N1, H9N2, and H7N7 avian influenza viruses and molecular mechanism of host range variation of influenza viruses, 5) role of the neuraminidase spike for the host range of influenza viruses, and 6) Development of anti-influenza drugs.     

Characterization of a trypsin-dependent avian influenza H5N1-pseudotyped HIV vector system for high throughput screening of inhibitory molecules

In this study, we have generated and characterized an avian influenza H5N1 hemagglutinin (HA), neuraminidase (NA) and M2 ion channel pseudotyped HIV-based vector system (HaNaM-pseudotyped HIV vector). The cleavage site of the HA protein was modified to necessitate trypsin-dependent maturation of the glycoprotein. HA, NA and M2 were efficiently incorporated in HIV vector particles which could transduce different cell lines in a trypsin-dependent manner. Results also showed that the presence of avian influenza M2 and NA proteins maximized both vector production and transduction and that transduction was highly sensitive to the specific NA inhibitor oseltamivir (Tamiflu). H5N1 HaNaM-pseudotyped HIV vector system was also adapted for cell-based high throughput screening of drug candidates against influenza virus infection, and its high sensitivity to the specific oseltamivir validates its potential utility in the identification of new influenza inhibitors. Overall, the trypsin-dependent H5N1-pseudotyped HIV vector can mimic avian influenza virus infection processes with sufficient precision to allow for the identification of new antivirals and to study avian influenza virus biology in a lower biosafety level laboratory environment.     

抗H7N9流感病毒中和抗体快速检测方法的建立及应用

目的：对建立的抗H7N9流感病毒中和抗体快速检测方法进行方法学验证及初步应用。方法：分别采用不同代次细胞对高、中、低不同滴度的阳性血清进行多次平行检测,考察细胞代次对检验结果的影响;采用NIBSC提供的参考品对方法学的特异性进行验证;同时应用抗H7N9的阳性血清检测进一步评估方法的准确性和精密性;采用ELISA-MNT法和血凝抑制（hemagglutination inhibition,HI）试验分别接种H7N9灭活流感疫苗免疫的小鼠血清样本20份,分析两种方法检测结果的相关性。结果：采用ELISA-MNT中和法,使用不同代次MDCK细胞（25、30和35代）检测相同的血清样本的中和抗体滴度结果相同;该方法只对羊抗H7N9的血清具有较高保护力,对其他血清基本没有交叉反应;该方法准确性良好;该方法组间、组内精密性的平均变异系数分别为4%和11%。该方法测定H7N9型流感疫苗免疫后的小鼠血清抗体效价,其结果与HI抗体的相关系数为0.61,表明两种方法的检测结果之间呈良好的正相关性。结论：建立的微量病毒中和法能够满足H7N9流感病毒中和抗体效价检测的要求,可用于H7N9新型大流行流感疫苗的免疫评价。     

2014～2015年南充市禽流感职业暴露人群及环境监测结果分析

目的 了解南充市禽流感职业暴露人群H5N1感染及环境禽流感病毒污染状况.方法 2014～2015年采集南充市禽类职业暴露人群血清标本,用红细胞凝集抑制试验(HI)检测H5N1抗体;对城乡活禽市场、家禽规模养殖、野鸟禽鸟栖息地以及禽类宰杀市场等场所的笼子、粪便以及污水标本采用实时荧光定量PCR法检测禽流感病毒(甲型流感病毒,即FluA)核酸.结果 共采集职业暴露人群160份血清标本,H5N1抗体结果均为阴性采集环境标本160份进行禽流感病毒核酸检测,FluA核酸阳性31份,总检出率为16.32％.其中H5、H7、H9核酸阳性分别是18份、0份、10份,H5、H7、H9检出率分别是9.47％、0％、5.26％,FluA核酸阳性未分型3份,未分型检出率为1.58％,不同类型标本、不同监测点环境、不同监测场所及不同季节监测结果比较差异有统计学意义(x2值分别是37.43、8.67、30.57和30.44,P＜0.05).结论 禽流感H5亚型在南充市职业暴露人群中未发现隐性感染,南充市各类型标本、各监测场所、各监测点环境及各季节均存在禽流感病毒,建议各相关政府部门加强对禽流感相关知识的宣传,加强交易以及宰杀市场等的监管,加强对外环境的监测,必要时采取短期关闭活禽交易、禽类宰杀市场,季节性休市等措施,降低禽流感病毒感染的风险.     

Antiviral agents active against influenza A viruses

The recent outbreaks of avian influenza A (H5N1) virus, its expanding geographic distribution and its ability to transfer to humans and cause severe infection have raised serious concerns about the measures available to control an avian or human pandemic of influenza A. In anticipation of such a pandemic, several preventive and therapeutic strategies have been proposed, including the stockpiling of antiviral drugs, in particular the neuraminidase inhibitors oseltamivir (Tamiflu; Roche) and zanamivir (Relenza; GlaxoSmithKline). This article reviews agents that have been shown to have activity against influenza A viruses and discusses their therapeutic potential, and also describes emerging strategies for targeting these viruses.     

芦丁对流感病毒的体外抑制作用及其机制研究

目的研究芦丁对流感病毒的体外抑制作用并探讨其机制。方法利用流感病毒感染MDCK细胞模型,观察芦丁对流感病毒的体外抑制作用;结合流感病毒感染前、感染同时及感染后加药的不同方式探索芦丁作用于流感病毒生命周期的具体阶段;采用荧光底物法考察芦丁对流感病毒神经氨酸酶的抑制作用。结果芦丁对A/Puerto Rico/8/1934(H1N1)、A/FM1/1/47(H1N1)、A/Human/Hubei/3/2005(H3N2)、A/Beijing/32/92(H3N2)4株流感病毒均具有体外抑制作用,其中对A/Puerto Rico/8/1934(H1N1)株的半数有效浓度(EC50)最小,选择指数(SI)最大,体外药效最好。芦丁在流感病毒感染后给药的效果最为明显,对流感病毒A/FM1/1/47(H1N1)的IC50最小,对神经氨酸酶活性抑制作用相对最好。结论芦丁抑制了流感病毒神经氨酸酶活性,具有较好的体外抗流感病毒作用。     

人感染甲型H7N9禽流感病毒致病性研究进展

人感染甲型H7N9禽流感病毒（human-infecting H7N9 avian influenza A virus，hH7N9 AIAV）已跨越禽类与人类之间的宿主屏障而感染人类。2013年至今，hH7N9 AIAV已在中国引起5次大的流行。虽然到目前为止仅存在有限的hH7N9 AIAV感染人际传播病例，但hH7N9 AIAV的高突变率特征似存在引发大流行的可能。此文就hH7N9 AIAV的来源、致病性和抗原分子基础，以及人感染H7N9禽流感的临床特征进行综述，以为hH7N9 AIAV防控提供参考。