Challenges on Induction of Broadly Neutralizing Antibodies for Optimization of HIV Vaccines Development and Vectored Immunoprophylaxis

Despite extensive research efforts, a preventive human immunodeficiency virus (HIV) vaccine remains one of the major challenges in the field of AIDS research. Experimental strategies which have been proven successful for other viral vaccines are not enough to tackle HIV-1 and new approaches to design effective preventive AIDS vaccines are of utmost importance. Due to enormous diversity among global circulating HIV strains, an effective HIV vaccine must elicit broadly protective antibodies based responses;therefore discovering new broadly neutralizing antibodies (bNAbs) against HIV has become major focus in HIV vaccine research. However further understanding of the viral targets of such antibodies and mechanisms of action of bNAbs is required for advancement of HIV vaccine research. This technical note discusses our current knowledge on the bNAbs and immunoprophylaxis using viral vectors with their relevance in designing of new candidates to HIV-1 vaccines.

Broadly neutralizing antibodies and vaccine design against HIV-1 infection

Remarkable progress has been achieved for prophylactic and therapeutic interventions against human immunodeficiency virus type I(HIV-1)through antiretroviral therapy.However,vaccine development has remained challenging.Recent discoveries in broadly neutralizing monoclonal antibodies(bNAbs)has led to the development of multiple novel vaccine approaches for inducing bNAbs-like antibody response.Structural and dynamic studies revealed several vulnerable sites and states of the HIV-1 envelop glycoprotein(Env)during infection.Our review aims to highlight these discoveries and rejuvenate our endeavor in HIV-1 vaccine design and development.

含抗HIV广谱中和抗体2F5、4E10靶基因载体的构建及鉴定

目的:构建含HIV gp120,gp41序列中广谱中和抗体2F5,4E10作用靶基因的载体并进行鉴定,为后期重组载体表达产物诱导产生中和抗体及抗HIV亚单位疫苗的研究奠定基础.方法:根据NCBI中HIV gp120,gp41基因序列中可与2F5、4E10结合的区域设计引物并进行PCR反应,将PCR得到的目的片段插入到载体pET28a中,对重组载体进行PCR鉴定、酶切鉴定及DNA测序.结果:PCR鉴定、酶切鉴定及DNA测序结果证实重组载体构建成功.结论:成功构建了含HIV gp120,gp41序列中广谱中和抗体2F5,4E10作用靶基因的载体.

HPV31型L2保守中和表位可诱发广谱中和抗体

目的研究人乳头瘤病毒31型(HPV31)次要外壳蛋白L2保守中和表位的免疫活性及诱发抗体的中和范围。方法合成法获得HPV31 L2 aa.17-40多肽,用EDC法偶联KLH,联合弗氏佐剂免疫新西兰大白兔,用假病毒中和实验检测免疫血清对来自α4、α7、α9、α10及β1亚属的多个HPV型别的中和抗体。结果 HPV31 L2-KLH偶联肽可在新西兰大白兔体内诱发针对至少17种HPV型别的广谱中和抗体,其中HPV31的中和抗体滴度最高,HPV5/45/57的次之。结论首次发现HPV31 L2保守中和表位免疫血清具有广谱中和活性,为基于该表位的广谱HPV疫苗研发奠定了基础。

广谱性识别H7亚型禽流感病毒HA蛋白的单克隆抗体制备与鉴定

H7N9亚型禽流感病毒(AIV)的快速进化与变异使得其逃脱现有抗体的识别,为提供H7亚型AIV鉴别诊断所需重要材料,制备可识别H7亚型AIV HA蛋白的单克隆抗体(mAb)。采用差速离心法纯化的H7N9亚型AIV(A/Chicken/Guangdong/SW154/2015)免疫BALB/c小鼠,利用杂交瘤技术制备杂交瘤细胞,采用血凝抑制(HI)试验、免疫过氧化物酶单层细胞试验(IPMA)和间接ELISA检测方法筛选阳性杂交瘤细胞株。结果显示,获得5株稳定分泌抗H7N9 AIV HA蛋白mAb的杂交瘤细胞,腹水ELISA效价均为1∶1000000;其中,4株mAb的重链为IgG1,1株mAb的重链为IgG2b,轻链均为κ链。特异性测定结果显示,5株mAb仅与H7亚型AIV反应,不与其他亚型流感病毒发生反应;广谱性测定结果显示,5株mAb均可与不同年份分离出的H7亚型AIV发生反应;HI结果显示,腹水针对H7-Re3抗原株的HI效价为7 log2~12 log2;MDCK细胞微量中和试验结果显示,5株mAb均具有中和活性;Dot blot检测结果显示,5株mAb均可识别H7-Re2抗原株和H7-Re3抗原株;Western blot检测结果表明,5株杂交瘤细胞培养上清均在63 ku附近出现1条特异性的蛋白质条带,说明这5株mAb均识别线性表位。综上,成功制备了5株广谱性识别H7 AIV HA蛋白的单克隆抗体。

Broad strategies for neutralizing SARS-CoV-2 and other human coronaviruses with monoclonal antibodies

Antibody therapeutics and vaccines for coronavirus disease 2019(COVID-19)have been approved in many countries,with most being developed based on the original strain of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).SARS-CoV-2 has an exceptional ability to mutate under the pressure of host immunity,especially the immune-dominant spike protein of the virus,which is the target of both antibody drugs and vaccines.Given the continuous evolution of the virus and the identification of critical mutation sites,the World Health Organization(WHO)has named 5 variants of concern(VOCs):4 are previously circulating VOCs,and 1 is currently circulating(Omicron).Due to multiple mutations in the spike protein,the recently emerged Omicron and descendent lineages have been shown to have the strongest ability to evade the neutralizing antibody(NAb)effects of current antibody drugs and vaccines.The development and characterization of broadly neutralizing antibodies(bNAbs)will provide broad strategies for the control of the sophisticated virus SARS-CoV-2.In this review,we describe how the virus evolves to escape NAbs and the potential neutralization mechanisms that associated with bNAbs.We also summarize progress in the development of bNAbs against SARS-CoV-2,human coronaviruses(CoVs)and other emerging pathogens and highlight their scientific and clinical significance.

Mutational escape prevention by combination of four neutralizing antibodies that target RBD conserved regions and stem helix

New variants of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) appear rapidly every few months.They have showed powerful adaptive ability to circumvent the immune system. To further understand SARS-CoV-2’s adaptability so as to seek for strategies to mitigate the emergence of new variants, herein we investigated the viral adaptation in the presence of broadly neutralizing antibodies and their combinations. First, we selected four broadly neutralizing antibodies, including pan-sarbecovirus and pan-betacoronavirus neutralizing antibodies that recognize distinct conserved regions on receptor-binding domain(RBD) or conserved stem-helix region on S2 subunit.Through binding competition analysis, we demonstrated that they were capable of simultaneously binding.Thereafter, a replication-competent vesicular stomatitis virus pseudotyped with SARS-CoV-2 spike protein was employed to study the viral adaptation. Twenty consecutive passages of the virus under the selective pressure of individual antibodies or their combinations were performed. It was found that it was not hard for the virus to adapt to broadly neutralizing antibodies, even for pan-sarbecovirus and pan-betacoronavirus antibodies. The virus was more and more difficult to escape the combinations of two/three/four antibodies. In addition, mutations in the viral population revealed by high-throughput sequencing showed that under the selective pressure of three/four combinational antibodies, viral mutations were not prone to present in the highly conserved region across betacoronaviruses(stem-helix region), while this was not true under the selective pressure of single/two antibodies.Importantly, combining neutralizing antibodies targeting RBD conserved regions and stem helix synergistically prevented the emergence of escape mutations. These studies will guide future vaccine and therapeutic development efforts and provide a rationale for the design of RBD-stem helix tandem vaccine, which may help to impede the generation of novel variants.

Two antibodies show broad,synergistic neutralization against SARS-CoV-2 variants by inducing conformational change within the RBD

Continual evolution of the severe acute respiratory syndrome coronavirus(SARS-CoV-2)virus has allowed for its gradual evasion of neutralizing antibodies(nAbs)produced in response to natural infection or vaccination.The rapid nature of these changes has incited a need for the development of superior broad nAbs(bnAbs)and/or the rational design of an antibody cocktail that can protect against the mutated virus strain.Here,we report two angiotensin-converting enzyme 2 competing nAbs—8H12 and 3E2—with synergistic neutralization but evaded by some Omicron subvariants.Cryo-electron microscopy reveals the two nAbs synergistic neutralizing virus through a rigorous pairing permitted by rearrangement of the 472-489 loop in the receptor-binding domain to avoid steric clashing.Bispecific antibodies based on these two nAbs tremendously extend the neutralizing breadth and restore neutralization against recent variants including currently dominant XBB.1.5.Together,these findings expand our understanding of the potential strategies for the neutralization of SARS-CoV-2 variants toward the design of broad-acting antibody therapeutics and vaccines.

Exploration of a Sequential Gp140-Gp145 Immunization Regimen with Heterologous Envs to Induce a Protective Cross-Reactive HIV Neutralizing Antibody Response In Non-human Primates

Raising a heterologous tier 2 neutralizing antibody(nAb)response remains a daunting task for HIV vaccine development.In this study,we explored the utility of diverse HIV-1 envelope(Env)immunogens in a sequential immunization scheme as a solution to this task.This exploration stemmed from the rationale that gp145,a membrane-bound truncation form of HIV Env,may facilitate the focusing of induced antibody response on neutralizing epitopes when sequentially combined with the soluble gp140 form as immunogens in a prime-boost mode.We first showed that gp140 DNA prime-gp145 Tiantan vaccinia(TV)boost likely represents a general format for inducing potent nAb response in mice.However,when examined in rhesus macaque,this modality showed little effectiveness.To improve the efficacy,we extended the original modality by adding a strong protein boost,namely native-like SOSIP.664 trimer displayed on ferritin-based nanoparticle(NP),which was generated by a newly developed click approach.The resulting three-immunization regimen succeeded in eliciting tier-2 nAb response with substantial breadth when implemented in rhesus macaque over a short 8-week schedule.Importantly,the elicited nAb response was able to effectively contain viremia upon a heterologous SHIV challenge.Collectively,our studies highlighted that diversification of Env immunogens,in both types and formulations,under the framework of a sequential immunization scheme might open new opportunity toward HIV vaccine development.

1例慢性HIV-1感染者体内CD4结合位点特异性记忆B细胞分选及抗体表达研究

目的通过抗原特异性记忆B细胞(Memory B)分选、抗体基因克隆表达和鉴定方法,从艾滋病病毒1型(HIV-1)感染者中获得HIV-1中和抗体,并进一步探讨获得抗体的特征。方法通过检测血浆中和抗体活性和表位筛选,挑选出1例含有CD4结合位点(CD4bs)特异性抗体的慢性HIV-1感染者。采用一对含有/缺失CD4-bs的探针RSC3/ΔRSC3进行Memory B分选,获得抗体可变区基因后进行抗体的表达纯化,并检验所得抗体的结合能力和中和能力。结果从9×106外周血单核淋巴细胞(PBMCs)中分离得6个特异性Memory B。通过反转录聚合酶链反应(RT-PCR)分别扩增出3对重链和轻链可变区基因,配对得到3个抗体,其中有一个抗体具有HIV-1CD4bs结合能力。中和实验表明,这个抗体能中和毒株SF162[50%抑制浓度(IC50=0.89μg/mL]。抗体可变区基因家系分析表明,该抗体属于IGHV1-18家系,重链可变区(VH)自体突变率为12%,低于广谱中和抗体VRC01VH的自体突变率(32%)。结论建立的单克隆特异性Memory B分选方法,可以获得有中和能力的抗体。该平台有希望获得具有我国自主知识产权的广谱中和抗体,并为开发抗体药物和设计新型免疫原提供技术支持。

新一代抗HIV-1中和抗体的研究进展

2007年德国Merck公司宣布艾滋病病毒（HIV）疫苗失败后，艾滋病疫苗研究陷入低谷。2011年在泰国进行的RV144疫苗显示受试人群获得了31．2％的保护，这个出人意料的结果让研究者重燃信心，即有效的HIV疫苗是可能的。随后，抗HIV-1中和抗体产生条件、靶点、抗体结构及运用等，成为了近年的研究热点。

HIV-1自然感染中的中和抗体反应

中和抗体（Nab）可以防止I型人类免疫缺陷病毒（HIV-1）侵入靶细胞。HIV-1感染数周后即可诱导产生Nab，这些早期抗体只能特异性地中和自体病毒但不能中和异源性病毒。在一些慢性感染者体内则可以检测到可同时中和同源性和异源性病毒的广谱中和抗体（BNab）。BNab的靶点通常位于包膜蛋白的保守区域。HIV-1BNab的产生还受到病毒变异及结构遮盖等因素的限制，同时Nab的中和广度与病毒载量具有相关性。

抗A型流感病毒广谱中和抗体及广谱通用疫苗研究进展

接种流感疫苗是预防和控制流感流行的最有效手段，可诱导广谱、长期保护作用的通用流感疫苗研究尚处于初级阶段。现已鉴定报道了几个广谱中和抗体可以抵抗多种A型流感病毒，病毒表面抗原血凝素（haemagglutinin，HA）与抗体的复合物品体结构揭示了至少3个高度保守表位。深入了解中和抗体与抗原互作分子机制可以为合理设计通用流感疫苗提供新策略，同时对开发新的抗流感病毒治疗抗体药物也具有重要启示意义。本文综述了A型流感病毒广谱中和抗体和广谱通用疫苗的研究进展。

HIV-1包膜序列变异对疾病进程和包膜免疫原性的影响

I型人免疫缺陷病毒（HIV-1）env基因编码的包膜糖蛋白是该病毒的重要抗原成分，又是HIV-1中最易变异的组分。env基因的变异导致其编码氨基酸的数目和种类改变，由此导致的包膜糖蛋白结构改变会影响包膜中和表位暴露和诱导中和抗体产生的能力。研究表明，HIV-1包膜糖蛋白氨基酸序列、可变区长度以及潜在糖基化位点（PNGS）均会影响疾病进程及病毒的中和敏感性。

Antibody Cocktail Exhibits Broad Neutralization Activity Against SARS-CoV-2 and SARS-CoV-2 Variants

Severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)has precipitated multiple variants resistant to therapeutic antibodies.In this study,12 high-affinity antibodies were generated from convalescent donors in early outbreaks using immune antibody phage display libraries.Of them,two RBD-binding antibodies(F61 and H121)showed high-affinity neutralization against SARS-Co V-2,whereas three S2-target antibodies failed to neutralize SARS-Co V-2.Following structure analysis,F61 identified a linear epitope located in residues G446–S494,which overlapped with angiotensinconverting enzyme 2(ACE2)binding sites,while H121 recognized a conformational epitope located on the side face of RBD,outside from ACE2 binding domain.Hence the cocktail of the two antibodies achieved better performance of neutralization to SARS-Co V-2.Importantly,these two antibodies also showed efficient neutralizing activities to the variants including B.1.1.7 and B.1.351,and reacted with mutations of N501 Y,E484 K,and L452 R,indicated that it may also neutralize the recent India endemic strain B.1.617.The unchanged binding activity of F61 and H121 to RBD with multiple mutations revealed a broad neutralizing activity against variants,which mitigated the risk of viral escape.Our findings revealed the therapeutic basis of cocktail antibodies against constantly emerging SARS-Co V-2 variants and provided promising candidate antibodies to clinical treatment of COVID-19 patients infected with broad SARS-Co V-2 variants.

HPV18 L2保守中和表位肽协同MF59与CpG-ODN免疫可诱发广谱中和抗体反应

目的:人乳头瘤病毒(HPV)次要外壳蛋白L2保守中和表位肽可诱发交叉中和抗体,研究L2保守中和表位肽免疫原性的特点利于HPV通用疫苗的研发。HPV18是第二常见的优势流行高危型,但18L2保守表位肽的免疫活性未见报道。方法:本研究采用化学法合成HPV18 L2N端多肽(18RG-1)并偶联KLH获得18RG1-KLH肽;联合MF59/CpG-ODN复合佐剂或弗氏佐剂皮下免疫BALB/c小鼠5次,用假病毒中和实验检测抗血清针对α6、α7、α9及α11亚属中多个致癌型HPV的中和抗体。结果:MF59/CpG-ODN复合佐剂多肽组抗血清对所有6种检测型别的中和活性与弗氏佐剂多肽组的相当。MF59/CpG-ODN佐剂多肽组抗血清具有广谱中和活性,中和范围至少包括14种致癌型HPV,中和抗体滴度最高的为HPV45(438)和HPV18(325),其次为HPV68(163)和HPV70(150),这四种优势中和型别均为α7亚属。结论:首次发现HPV18 L2 RG1保守中和表位免疫血清可诱发广谱中和抗体反应(其中对α7亚属的HPV中和活性最强,为优势中和型别)。研究结果为基于L2保守表位的广谱HPV疫苗研发奠定基础。

艾滋病抗病毒治疗现状及其研究进展

抗逆转录病毒治疗是艾滋病(acquired immunodeficiency syndrome,AIDS)防治的主要手段,可以减少艾滋病相关疾病发病率、延长生存期和防止艾滋病病毒传播。当前抗病毒药物发展迅速,一些新的治疗策略不断出现,包括抗病毒方案的简化以及新的药物、治疗手段的推陈出新。本文重点对艾滋病治疗现状、近年来进行的主要简化治疗方案试验总结及治疗时机的改进、抗病毒化学药物、广谱中和抗体的最新研究进展和未来的治疗思路进行概述。

艾滋病广谱中和抗体专利分析

利用Derwent Innovation平台,以(broadly neutralizing antibod*or broadly-neutralizing antibod*or bNAb*) and (HIV or human immunodeficiency virus or AIDS or acquired immune deficiency syndrome) and vaccin*为检索词,检索条件是在标题或摘要中出现检索词,检索时间截至2018年8月7日,筛选出309件广谱中和抗体的专利申请,同族专利45件。该领域自1993年出现专利申请,2011年达到峰值后呈下降趋势。该领域最重要的目标市场是美国和欧洲。美国的国际艾滋病疫苗行动组织、Scripps研究所、美国国家健康研究院、杜克大学、加州大学Santa Cruz分校、儿童医学中心和纽约大学是领先的研究机构,前3位的发明人分别为杜克大学的Haynes Barton F、国际疫苗行动组织的Phogat Sanjay K和Hoffenberg Simon。该领域已具有一定的技术基础并处于改进中且与治疗有联系。

新型冠状病毒及泛β冠状病毒广谱中和单抗的研究进展

新型冠状病毒的全球大流行,给人类的生命健康和社会秩序带来了巨大的危害。疫苗、小分子药物及各类抗体药物的研发在遏制新型冠状病毒感染传播、降低重症率和死亡风险上发挥了积极的作用。然而,由于新冠病毒庞大的感染基数及自身易突变的特征,当前已经演化出多种能逃逸疫苗及中和抗体的变异株,显著削弱了抗体的保护效果。研发新型冠状病毒广谱甚至泛β冠状病毒广谱的中和抗体对于未来新冠变异株及其他高致病性β冠状病毒的防治具有重要意义。本文从新型冠状病毒中和抗体的筛选制备策略、作用机制、中和效果及广谱性等方面进行了系统综述,并对当前面临的挑战和未来的发展方向进行了讨论和展望,以期为后续相关研究提供参考。

以流感病毒血凝素为靶标的抗病毒防治策略研究进展

流感是由流感病毒引起的一种在全球范围内流行的传染病,从1918年追溯至今,已给公共健康和社会稳定带来了巨大的冲击。目前,接种流感疫苗是预防和控制流感流行的最有效方式。近几年,对广谱抗病毒药物和中和抗体的研究不断深入。流感病毒表面血凝素在病毒侵入宿主细胞阶段发挥重要作用,是现有流感疫苗的主要有效抗原成分,也是广谱中和抗体、广谱抗病毒药物的主要靶标。本文就近年来基于流感病毒血凝素的新型流感疫苗、中和抗体以及抗病毒药物等防治策略的研究进展进行综述,为后续流感的预防和控制提供新的思路。