Prokaryotic Expression and Purification of HIV-1 Vif and hAPOBEC3G, Preparation of Polyclonal Antibodies

To prepare HIV-1 Vif and hAPOBEC3G and to produce their antibodies, the full length gene fragment of HIV-1 vif was amplified by PCR from a plasmid of HIV-1 NL4.3 cDNA, and the APOBEC3G gene was obtained by RT-PCR from the total RNA of H9 cells. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-32a). Recombinant pET-vif and pET-APOBEC3G were expressed respectively in Eserichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine tag at the C-terminus for convenient purification and detection. To express and purify the HIV-1 Vif and hAPOBEC3G in E.coli cells, the accuracy of inserted gene and specificity of proteins were detected by the two enzyme digestion method, SDS-PAGE, and Western blotting. Rabbits were then immunized by Vif or APOBEC3G protein and serum samples were tested by indirect ELISA to determine the level of antibodies. Immunoenzyme and immunofluorescence assays were performed to identify the specificity of polyclonal antibodies. The titer of the anti-Vif antibodies was 1:204800, and that of the anti-APOBEC3G antibodies was 1:102400. Thus the antibodies could detect the antigen expression in the cells, demonstrating that fusion proteins with high purity and their corresponding polyclonal antibodies with high titer and specificity were achieved.

Analysis of Detecting HIV-1 Antibody in Paired Urine and Serum Specimens from Drug Users by ELISA

Objective- To compare the consistency of the results from detecting HIV-1 antibody in the paired urine and serum specimens from drug users by ELISA. Methods: The paired urine and serum specimens from 273 drug users detained at a detoxification unit were collected, and the HIV-1 antibodies in the specimens of them were screened by urine and serum ELISA kits, respectively. Results: Of 273 serum specimens, 94 ones showed positive reaction and among 94 counterpart urine specimens, 93 ones also appeared positive reaction. Taking the results together,the consistent rate of HIV-1 antibody screened by urine and serum ELISA kits was 99.6%. Conclusion: The urine ELISA kit, which screened HIV-1 antibody of urine showing almost the same results tested by serum ELISA kit, is reliable. It is proposed that urine ELISA be introduced in many fields.

HIV-1抗体蛋白印迹确认与核酸检测复核对比研究

应用病毒核酸载量法NASBA和HIV-1 RNA的巢式逆转录PCR(nested RT-PCR)法与HIV抗体蛋白印迹(WB)方法,对经过初筛的44例HIV-1抗体阳性标本进行了对照检测研究。发现了2例(gp160、p24)和1例(gp160g、p120p、66、p24)的特殊阳性样本,经NASBA法和该RT-PCR法核酸检测为阴性;WB确认的4例gp160阳性带、1例p24、p17阳性带和13例p24阳性带,经NASBA法和该RT-PCR法核酸检测也为阴性;而WB确认的其余全部带型的抗体阳性标本经过NASBA法和该RT-PCR法检测均为阳性。该研究表明对只有gp160p、24和gp160、gp120p、66、p24的特殊阳性标本和以p24为主的抗体不确定标本需要用RT-PCR或NASBA方法进行核酸检测,以进一步确认。

1例出境人员HIV抗体WB不确定结果分析及口岸防控思考

目的分析1例出境人员HIV抗体蛋白印迹试验(WB)不确定结果,提出艾滋病相关口岸防控思考。方法按照《全国艾滋病检测技术规范》检测要求,回顾性分析1例HIV抗体WB不确定样本检测流程。结果筛查试验:第一次和第二次体检初筛结果均为有反应,第一次和第二次体检两种抗体试剂复检结果均为有反应,第一次体检随访筛查两种抗体试剂检测结果为一有一无反应;确证试验:第一次体检、第一次随访及第二次体检WB检测条带分别为p24和gp160、gp160、gp160,结果均为HIV-1不确定;核酸检测:第一次随访及第二次体检病毒核酸载量检测结果均为“未检出”。结论针对HIV抗体不确定现状,建议尽快将HIV抗体确证试验和核酸检测作为补充实验在实际工作中联合使用。就此提出口岸艾滋病防控的思考:织牢疫情监测预警网络;完善国际旅行健康服务体系;强化实验室建设;建立联防联控体系,统筹协调口岸新冠疫情与艾滋病防控,做好“多病共防”。

病毒载量检测在渝西四区HIV-1抗体不确定和阴性病例诊断中的应用

目的探讨病毒载量检测在HIV-1抗体不确定和阴性病例诊断中的应用价值。方法对重庆市永川区疾病预防控制中心2023年3-9月HIV-1抗体筛查试验有反应而确证试验结果为HIV-1抗体不确定和阴性的病例样本进行HIV-1病毒载量检测,并对其抗体确证、病毒载量和随访结果进行统计分析。结果103例HIV-1抗体不确定和阴性病例中,34.0%(35例)抗体不确定,66.0%(68例)抗体阴性。35例抗体不确定病例中54.3%(19/35)首次VL高于检测下限且均>1×10^(5)拷贝/mL;68例抗体阴性病例中19.1%(13/68)首次VL高于检测下限且均>1×10^(6)拷贝/mL。含有env带(gp160/gp120/gp41)的不确定病例VL检出率较高,为70.8%(17/24)。13例随访到的不确定病例中,11例抗体转阳且首次VL>1×10^(5)拷贝/mL。5例随访到的阴性病例中,2例抗体转阳,1例转为不确定,此3例首次VL>1×10^(6)拷贝/mL。在随访到的18例不确定和阴性病例中有2例在抗体转阳前用药,VL由>1×10^(5)拷贝/mL下降至<5×10^(3)拷贝/mL。结论HIV-1病毒载量检测与抗体确证试验互为补充,在减少病例漏检的同时,能缩短HIV感染诊断时间。此外,对于发现的高病毒载量不确定和阴性病例及时用药可有效降低其体内病毒载量,减少传播的可能性。

Preparation and Characterization of Three Monoclonal Antibodies against HIV-1 p24 Capsid Protein

HIV-1 p24 detection provides a means to aid the early diagnosis of HIV-1 infection, track the progression of disease and assess the efficacy of antiretroviral therapy. In the present study, three monoclonal antibodies （mAbs） p3JB9, p5F1 and p6F4 against HIV-1 p24 were generated. All mAbs could detect p24 of HIV-1ⅢB, HIV-1Ada-M, HIV-174v mAbs p5F1 and p6F4 could detect HIV-1KM018, while p3JB9 could not. Three mAbs did not react with HIV-2ROD, HIV-2CBL-20 and SIVagmTYO-1. The recognized epitope of p5F1 was located on the Gag amino acid region DCKTILKALGPAATLEEMMTAC. The p5F1 was used to establish a modified sandwich ELISA with rabbit anti-p24 serum and showed good specificity and high sensitivity, which has been used to measure HIV-1 p24 antigen levels in research. Cellular ＆ Molecular Immunology.

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.

Generation of HIY-resistant cells with a single-domain antibody:implications for HIV-1 gene therapy

The cure or functional cure of the"Berlin patient"and"London patient"indicates that infusion of HIV-resistant cells could be a viable treatment strategy.Very recently,we genetically linked a short-peptide fusion inhibitor with a glycosylphosphatidylinositol(GPI)attachment signal,rendering modified cells fully resistant to HIV infection.In this study,GPI-anchored m36.4,a single-domain antibody(nanobody)targeting the coreceptor-binding site of gp120,was constructed with a lentiviral vector.We verified that m36.4 was efficiently expressed on the plasma membrane of transduced TZM-bl cells and targeted lipid raft sites without affecting the expression of HIV receptors(CD4,CCR5,and CXCR4).Significantly,TZM-bl cells expressing GPI-m36.4 were highly resistant to infection with divergent HIV-1 subtypes and potently blocked HIV-1 envelope-mediated cell-cell fusion and cell-cell viral transmission.Furthermore,we showed that GPI-m36.4-modified human CEMss-CCR5 cells were nonpermissive to both CCR5-and CXCR4-tropic HIV-1 isolates and displayed a strong survival advantage over unmodified cells.It was found that GPI-m36.4 could also Impair HIV-1 Env processing and viral infectivity in transduced cells,underlying a multifaceted mechanism of antiviral action.In conclusion,our studies characterize m36.4 as a powerful nanobody that can generate HIV-resistant cells,offering a novel gene therapy approach that can be used alone or in combination.

Monoclonal Antibodies Recognizing HIV-1 gp41 Could Inhibit Env-Mediated Syncytium Formation

Some monoclonal antibodies （mAbs） could inhibit infection by HIV-1. In this study, four mAbs against HIV-1 gp41 were prepared in mice. All four mAbs could bind to the recombinant soluble gp41 and recognize the native envelope glycoprotein gp160 expressed on the HIV-Env^＋ CHO-WT cell in flow cytometry analysis. Interestingly, the results show that all four mAbs purified by affinity chromatography could inhibit HIV-1 Env-mediated membrane fusion （syncytium formation） by 40%-60% at 10 μg/mL, which implies potential inhibitory activities against HIV-1.

Characterization of Antibody Responses Against the 2F5 Epitope ELDKWA Using HIV-1 Env-Mediated Membrane Fusion and Neutralization Assays

The epitope ELDKWA, which is located in the membrane-proximal external region （MPER） of HIV-1 gp41, is an important neutralizing epitope. The human monoclonal antibody （mAb） 2F5 against this epitope shows broad neutralizing activity toward many HIV strains. However, several reports have shown that the epitope-specific mAbs induced by peptides containing MPER did not exhibit the same neutralizing activities as human mAb 2F5. In this study, four ELDKWA epitope specific mAbs （9E7, 7E10, 6B5, and 2B4） induced by immunization with the ELDKWA epitope in varied molecular contexts, all showed inhibitory activi- ties with different potencies in HIV-1 Env-mediated membrane fusion assays and pseudovirus neutralization assays. This result indicates that though these antibodies recognize the epitope ELDKWA, their characteri- zations differ from that of neutralizing antibodies, implying that the neutralizing mAbs can be induced but also need to be screened, and the protective ability of a related vaccine antigen depends on the concentra- tion of the neutralizing mAbs in the induced polyclonal antibodies.

N-terminal residues of an HIV-1 gp41 membrane-proximal external region antigen influence broadly neutralizing 2F5-like antibodies

The Human immunodeficiency virus type 1(HIV-1) gp41 membrane proximal external region(MPER) is targeted by broadly neutralizing antibodies(e.g. 2F5, 4E10, Z13 e and m66.6), which makes this region a promising target for vaccine design. One strategy to elicit neutralizing antibodies against the MPER epitope is to design peptide immunogens mimicking neutralization structures. To probe 2F5-like neutralizing antibodies, two yeast-displayed antibody libraries from peripheral blood mononuclear cells from a HIV-1 patient were screened against the 2F5 epitope peptide SP62. Two 2F5-like antibodies were identified that specifically recognized SP62. However,these antibodies only weakly neutralized HIV-1 primary isolates. The epitopes recognized by these two 2F5-like antibodies include not only the 2F5 epitope(amino acids(aa) 662–667 in the MPER)but also several other residues(aa 652–655) locating at the N-terminus in SP62. Experimental results suggest that residues of SP62 adjacent to the 2F5 epitope influence the response of broadly neutralizing 2F5-like antibodies in vaccination. Our findings may aid the design of vaccine immunogens and development of therapeutics against HIV-1 infection.

Predefined GPGRAFY-Epitope-Specific Monoclonal Antibodies with Different Activities for Recognizing Native HIV-1 gp120

A seven-amino acid epitope GPGRAFY at the tip of the V3 loop in HIV-1 gp120 is the principal neutralizing epitope, and a subset of anti-V3 antibodies specific for this epitope shows a broad range of neu-tralizing activity. GPGRAFY-epitope-specific neutralizing antibodies were produced using predefined GPGRAFY-epitope-specific peptides instead of a natural or recombinant gp120 bearing this epitope. All six monoclonal antibodies (mAbs) could recognize the GPGRAFY-epitope on peptides and two of the antibod-ies, 9D8 and 2D7, could recognize recombinant gp120 in enzymelinked immunosorkentassy (ELISA) as-says. In the flow cytometry analysis, the mAbs 9D8 and 2D7 could bind to HIV-Env+ CHO-WT cells and the specific bindings could be inhibited by the GPGRAFY-epitope peptide, which suggests that these two mAbs could recognize the native envelope protein gp120 expressed on the cell membrane. However, in syncytium assays, none of the mAbs was capable of inhibiting HIV-Env-mediated cell membrane fusion. The different activities for recognizing native HIV-1 gp120 might be associated with different antibody affinities against the epitopes. The development of conformational mimics of the neutralization epitope in the gp120 V3 loop could elicit neutralizing mAbs with high affinity.

Preliminary Report:Using Simpli RED HIV-1 Antibody Test to Detect the Samples from Population of Entering Country

Simpli RED HIV-1 antibody test was used to detect 17,378 samples from people of entering coutry. 10 samples were positive. Using Western Blot as the cofirmatory test, 8/10 was positive. The advantages of this test was very sensitive, quick and easy. 20 ul whole blood could be used as sample and got the results in two minutes.

病毒载量试验对“HIV-1抗体不确定”病例诊断的效果评价

目的评价病毒载量（VL）试验对＂艾滋病病毒Ⅰ型（HIV-1）抗体不确定＂病例诊断的价值。方法对一组106例＂HIV-1抗体不确定＂病例进行定期随访,检测其VL与抗体,比较两种检测策略对这类病例的诊断效果。结果共106例HIV-1抗体不确定病例,均在一周内进行了COBAS TaqMan HIV-1Test v2.0病毒载量检测,56例≥100 000拷贝/mL,31例在5 000~100 000拷贝/mL之间,6例在20~5 000拷贝/mL之间,13例核酸未检出。常规随访抗体检测中,27例（25.5%）在4周内明确诊断,59例（55.7%）超过4周复查明确诊断,12例（11.3%）失访,5例（4.7%）死亡,3例多次随访结果仍为＂HIV-1抗体不确定＂。结论 VL试验能快速准确鉴别不确定结果,缩短随访复查的时间,是对现有抗体补充试验复查检测策略的重要补充和完善。

The good and evil of complement activation in HIV-1 infection

The complement system,a key component of innate immunity,is a first-line defender against foreign pathogens such as HIV-1.The role of the complement system in HIV-1 pathogenesis appears to be multifaceted.Although the complement system plays critical roles in clearing and neutralizing HIV-1 virions,it also represents a critical factor for the spread and maintenance of the virus in the infected host.In addition,complement regulators such as human CD59 present in the envelope of HIV-1 prevent complement-mediated lysis of HIV-1.Some novel approaches are proposed to combat HIV-1 infection through the enhancement of antibody-dependent complement activity against HIV-1.In this paper,we will review these diverse roles of complement in HIV-1 infection.

Persistence of VRC01-resistant HIV-1 during antiretroviral therapy

VRC01, a broadly neutralizing monoclonal antibody （bnmAb）, can neutralize a diverse array of HIV-1 isolates by mimicking CD4 binding to the envelope glycoprotein gpl20. We have previously demonstrated the presence of VRC01-resistant strains in an HIV-1 infected patient during antiretroviral therapy. Here, we report follow-up studies of two subsequent samples from the same patient. With genetic and phenotypic analysis of over 70 full-length molecular clones of the HIV-1 envelope, we show that VRC01-resistant HIV-1 continued to exist and change in its proportion of the infecting virus during treatment with a highly active antiretroviral therapy. Consistent with our previous observation, the resistant phenotype was associated with a single asparagine residue at position 460 （N460）, a potential N-linked glycosylation site in the V5 region. The persistence and continuing evolution of VRC01-resistant HIV-1 in vivo presents a great challenge to our future preventative and therapeutic interventions based on VRC01.

Characterization of human IgM and IgG repertoires in individuals with chronic HIV-1 infection

Advancements in high-throughput sequencing(HTS)of antibody repertoires(Ig-Seq)have unprecedentedly improved our ability to characterize the antibody repertoires on a large scale.However,currently,only a few studies explored the influence of chronic HIV-1 infection on human antibody repertoires and many of them reached contradictory conclusions,possibly limited by inadequate sequencing depth and throughput.To better understand how HIV-1 infection would impact humoral immune system,in this study,we systematically analyzed the differences between the IgM(HIV-IgM)and IgG(HIV-IgG)heavy chain repertoires of HIV-1 infected patients,as well as between antibody repertoires of HIV-1 patients and healthy donors(HH).Notably,the public unique clones accounted for only a negligible proportion between the HIV-IgM and HIV-IgG repertoires libraries,and the diversity of unique clones in HIV-IgG remarkably reduced.In aspect of somatic mutation rates of CDR1 and CDR2,the HIV-IgG repertoire was higher than HIV-IgM.Besides,the average length of CDR3 region in HIV-IgM was significant longer than that in the HH repertoire,presumably caused by the great number of novel VDJ rearrangement patterns,especially a massive use of IGHJ6.Moreover,some of the B cell clonotypes had numerous clones,and somatic variants were detected within the clonotype lineage in HIV-IgG,indicating HIV-1 neutralizing activities.The in-depth characterization of HIV-IgG and HIV-IgM repertoires enriches our knowledge in the profound effect of HIV-1 infection on human antibody repertoires and may have practical value for the discovery of therapeutic antibodies.

In vitro inhibition of HIV-1 replication in autologous CD4*T cells indicates viral containment by multifactorial mechanisms

HIV-1-specific cytotoxic T lymphocytes(CTLs) and neutralizing antibodies(NAbs) are present during chronic infection, but the relative contributions of these effector mechanisms to viral containment remain unclear. Here, using an in vitro model involving autologous CD4+ T cells,primary HIV-1 isolates, HIV-1-specific CTLs, and neutralizing monoclonal antibodies, we show that b12, a potent and broadly neutralizing monoclonal antibody to HIV-1, was able to block viral infection when preincubated with virus prior to infection, but was much less effective than CTLs at limiting virus replication when added to infected cell cultures. However, the same neutralizing antibody was able to contain viruses by antibody-dependent cell-mediated virus inhibition in vitro,which was mediated by natural killer cells(NKs) and dependent on an Fc-Fc receptor interaction.Meanwhile, bulk CTLs from HIV-1 controllers were more effective in suppression of virus replication than those from progressors. These findings indicate that control of HIV-1 replication in activated CD4^+ T cells is ineffectively mediated by neutralizing antibodies alone, but that both CTLs and antibody-dependent NK-mediated immune mechanisms contribute to viral containment. Our study systemically compared three major players in controlling HIV-1 infection, CTLs, NAbs, and NKs, in an autologous system and highlighted the multifactorial mechanisms for viral containment and vaccine success.

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.

广西玉林市2015—2020年HIV抗体不确定结果的随访分析

目的 探讨人类免疫缺陷病毒(human immunodeficiency virus, HIV)抗体不确定结果转归特点及HIV-1病毒载量检测在HIV抗体不确定诊断中的应用。方法 收集2015—2020年广西壮族自治区玉林市HIV抗体筛查为HIV抗体有反应的样本,使用胶体金和胶体硒两种快速法检测试剂复核,出现单反应或双反应的标本使用蛋白免疫印迹法(Western-blot, WB)进行确证试验,WB结果为不确定的样本检测其HIV-1病毒载量作为补充试验并在1~4周后进行随访。结果 2015—2020年共收到HIV抗体确证检测样本4 906人份,不确定结果 153人份(3.12%),最终完成随访并有检测结果 91例。在91例中有59例阳转(64.84%),32例阴转(35.16%);复核双反应和单反应的阳转率分别为94.74%(54/57)和14.71%(5/34);WB结果为2条带以上的阳转率为89.80%(44/49),WB结果仅有1条带的阳转率为35.71%。91例随访中病毒载量>5 000 CPs/mL的有59例,阳性率为90%,病毒载量≤5 000 CPs/mL的有32例,阳性率为0。结论HIV抗体复核出现双反应且WB至少2条带型的样本阳转率比较高;HIV抗体复核仅单反应且WB仅有1条带型的也有感染的高风险,可检测HIV-1病毒载量以尽早确定患者是否感染HIV病毒。