2021—2023年贵港市HIV-1感染者的流行特征和检测结果分析

目的分析2021—2023年贵港市人类免疫缺陷病毒1型(HIV-1)抗体阳性者的免疫蛋白印迹试验(WB)带型特征,探讨免疫状况及相关指标在获得性免疫缺陷综合征(AIDS)发展中的作用。方法对2021—2023年1719份经贵港市AIDS确证实验室确证为HIV-1抗体阳性且在治疗前开展了CD4^(+)T淋巴细胞检测的样本,用SPSS 25.0分析人口学特征和实验室相关检测结果。结果1719例HIV-1抗体阳性以男性、40岁以上、已婚、农民、文化程度较低、医疗机构临床筛查为主;CD4^(+)T淋巴细胞计数与年龄呈负相关,与WB条带数呈正相关(P<0.05)。带型p55、p39、p17检出阳性率在不同免疫程度和病程间差异有统计学意义(P<0.05)。结论农民仍然是贵港市AIDS宣教、检测工作的重点人群,p55、p39和p17可作为疾病发展和免疫情况的一个潜在判别依据。

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.

164例HIV-1感染者梅毒RPR出现生物学假阳性情况分析

目的了解性病门诊人类免疫缺陷病毒1亚型(HIV-1)感染者梅毒快速血浆反应素试验(RPR)出现生物学假阳性(BFP)情况。方法收集该院性病门诊中已确诊HIV-1感染者,采用梅毒螺旋体抗体吸收试验(FTA-ABS-IgG)、RPR进行检测,对RPR出现BFP的HIV-1感染者,根据抗病毒治疗情况分成已治疗和未治疗两组,检测病毒载量及CD4^(+)T细胞检测并进行比对,观察抗病毒治疗情况对RPR出现BFP有无影响,1年后随访检测RPR,分析RPR出现BFP的临床转归情况。结果2146例已确诊HIV-1感染者中RPR出现BFP的有164例;4072例HIV抗体阴性者中RPR出现BFP的有22例,HIV-1感染者和HIV阴性者BFP发生率差异有统计学意义(P<0.01)。164例RPR出现BFP的患者中未进行抗病毒治疗140例,病毒载量基线HIV RNA 3.50(1.48~6.15)lg copies/mL,CD4^(+)T细胞计数344.50(239.75~480.25)mm^(3);已进行抗病毒治疗24例,未检测到HIV RNA目标基因,CD4^(+)T细胞计数420.00(289.25~611.75)mm^(3)。1年后随访检测RPR,其中109例转为阴性,31例RPR持续阳性,24例FTA-ABS-IgG转为阳性。结论性病门诊HIV-1感染者RPR出现BFP和HIV感染存在一定关系,未进行抗病毒治疗的HIV-1感染者,RPR更容易出现BFP,对HIV-1感染者RPR出现BFP情况,建议临床进行随访追踪,以便及时治疗或排除梅毒感染。

靶向EBV潜伏膜蛋白1的单克隆抗体促进HIV相关EBV阳性伯基特淋巴瘤细胞凋亡的分子机制

目的制备和筛选抑制伯基特淋巴瘤(Burkitt lymphoma,BL)的抗EBV潜伏膜蛋白1(latent membrane protein-1,LMP1)单克隆抗体。方法人工合成EBV潜伏膜蛋白1的跨膜结构域5(transmembrane domain 5,TMD5),并以此为抗原,采用杂交瘤法制备抗TMD5单抗。ELISA法测定小鼠腹水中的抗体效价。7-氨基放线菌素D(7-Aminoactinomycin D,7-AAD)/Annexin V-PE双标记流式细胞术和JC-1染色联合流式细胞术测定线粒体膜电势评估单抗对BL细胞系Daudi细胞的促凋亡活性。蛋白质印迹法测定Daudi细胞内促存活信号通路p38-MAPK/IKK/NF-κB相关蛋白的表达水平。结果经过2轮筛选,获得R2-2-5E-6C和R2-2-8D-3A两种单抗,这2种单抗均能与TMD5发生抗体-抗原特异性结合,而与GAPDH无免疫反应。与NC组相比,R2-2-5E-6C组和R2-2-8D-3A组处理后的Daudi细胞中Annexin V+7-AAD+细胞所占百分比增加;JC-1荧光强度<104的细胞所占百分比增加;胞内p38-MAPK、IKK和NF-κB的表达水平降低(P<0.05)。结论筛选获得的抗TMD5单抗R2-2-5E-6C和R2-2-8D-3A可通过抑制LMP1介导的p38-MAPK/IKK/NF-κB信号通路的活性促进BL凋亡。

产妇血清ACA和抗β2-GP1抗体与不良妊娠结局的相关分析

目的:探讨产妇血清抗心磷脂抗体(ACA)和抗β2-糖蛋白1(β2-GP1)抗体与不良妊娠结局的关系。方法:选取不良妊娠产妇130例作为研究组,同期正常分娩产妇130例作为对照组;采用酶联免疫吸附法(ELISA)检测2组产妇血清ACA及各亚型(ACA-IgA、ACA-IgG、ACA-IgM)、β2-GP1抗体及各亚型(抗β2-GP1-IgA、抗β2-GP1-IgG、抗β2-GP1-IgM)水平,并分析这些指标与围产儿死亡的相关性。结果:研究组产妇血清ACA-IgG与ACA-IgM阳性率明显高于对照组(P<0.01),抗β2-GP1-IgG及β2-GP1-IgM阳性率明显高于对照组(P<0.01);研究组产妇血清ACA、抗β2-GP1抗体及联合检查阳性率均明显高于对照组,差异均具有统计学意义(P<0.01);研究组产妇血清抗ACA-IgA、抗β2-GP1-IgA、抗β2-GP1-IgM抗体水平与围产儿死亡具有相关性(r=0.3126、P=0.018,r=0.3078、P=0.007,r=0.4012、P=0.021)。结论:血清ACA各亚型及抗β2-GP1各亚型抗体检测可用于评估不良妊娠结局。

Complex positive selection pressures drive the evolution of HIV-1 with different co-receptor tropisms

HIV-1 co-receptor tropism is central for understanding the transmission and pathogenesis of HIV-1 infection. We performed a genome-wide comparison between the adaptive evolution of R5 and X4 variants from HIV-1 subtypes B and C. The results showed that R5 and X4 variants experienced differential evolutionary patterns and different HIV-1 genes encountered various positive selection pressures, suggesting that complex selection pressures are driving HIV-1 evolution. Compared with other hypervariable regions of Gp120, significantly more positively selected sites were detected in the V3 region of subtype B X4 variants, V2 region of subtype B R5 variants, and V1 and V4 regions of subtype C X4 variants, indicating an association of positive selection with co-receptor recognition/binding. Intriguingly, a significantly higher proportion (33.3% and 55.6%, P【0.05) of positively selected sites were identified in the C3 region than other conserved regions of Gp120 in all the analyzed HIV-1 variants, indicating that the C3 region might be more important to HIV-1 adaptation than previously thought. Approximately half of the positively selected sites identified in the env gene were identical between R5 and X4 variants. There were three common positively selected sites (96, 113 and 281) identified in Gp41 of all X4 and R5 variants from subtypes B and C. These sites might not only suggest a functional importance in viral survival and adaptation, but also imply a potential cross-immunogenicity between HIV-1 R5 and X4 variants, which has important implications for AIDS vaccine development.

1型糖尿病患者血清自身抗体联合检测的临床意义

目的:分析血清自身抗体联合检测评估1型糖尿病的价值。方法:选取1型糖尿病患者100例作为观察组;选取同期健康体检者100例作为对照组。两组均进行糖尿病抗体检测。比较两组自身抗体阳性率、各种组合形式抗体检测阳性率与4种抗体联合检测阳性率。结果:观察组谷氨酸脱羧酶自身抗体(GADA)、胰岛细胞抗体(ICA)、蛋白酪氨酸磷酸酶IA-2自身抗体(ⅠA-2A)及胰岛素自身抗体(IAA)检测阳性率均高于对照组,差异有统计学意义(P<0.05)。4种抗体联合检测阳性率均高于各抗体单项检测阳性率、任意2种抗体联合检测阳性率及任意3种抗体检测阳性率,差异有统计学意义(P<0.05)。结论:血清IAA对1型糖尿病的判断有重要意义,在多种抗体联合检测作用下能提高对疾病的检出阳性率。

2018-2021年广元市HIV抗体筛查有反应性样本确证检测结果分析

目的 分析人类免疫缺陷病毒(Human immunodeficiency virus, HIV)抗体筛查有反应性样本的确证检测结果,为HIV检测及艾滋病防控工作提供依据。方法 对广元市2018-2021年各HIV初筛实验室检测出的HIV抗体有反应性样本,采用蛋白免疫印迹法进行确证试验,并用WPS 11.1等软件进行统计学分析。结果 2018-2021年广元市筛查的1 057份HIV抗体有反应性样本中,经过确证试验,被判定为HIV-1抗体阳性770份(72.85%),HIV-1抗体不确定120份(11.35%),HIV抗体阴性167份(15.80%),2018年确证阳性率最高为81.90%。男性确证阳性率(84.44%)高于女性(53.42%);确证阳性年龄以>40岁为主,构成比为69.87%;门诊和住院病例为样本主要来源,构成比为64.81%;阳性配偶的确证阳性率最高(94.29%),婚检和孕检的确证阳性率最低(40.74%);不同筛查HIV检测方法确证检测结果差异有统计学意义(χ^(2)=15.679,P<0.05),快速检测试验确证阳性率最高(78.35%),化学发光试验确证阳性率最低(68.05%)。结论 为提高广元市HIV抗体有反应性样本确证阳性率,应提高HIV抗体初筛的检测质量和能力。

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.

Improving the positional adaptability:structurebased design of biphenyl-substituted diaryltriazines as novel non-nucleoside HIV-1 reverse transcriptase inhibitors

In order to improve the positional adaptability of our previously reported naphthyl diaryltriazines(NP-DATAs),synthesis of a series of novel biphenyl-substituted diaryltriazines(BP-DATAs)with a flexible side chain attached at the C-6 position is presented.These compounds exhibited excellent potency against wild-type(WT)HIV-1 with EC50 values ranging from 2.6 to 39 nmol/L and most of them showed low nanomolar anti-viral potency against a panel of HIV-1 mutant strains.Compounds 5 j and 6 k had the best activity against WT,single and double HIV-1 mutants and reverse transcriptase(RT)enzyme comparable to two reference drugs(EFV and ETR)and our lead compound NP-DATA(1).Molecular modeling disclosed that the side chain at the C-6 position of DATAs occupied the entrance channel of the HIV-1 reverse transcriptase non-nucleoside binding pocket(NNIBP)attributing to the improved activity.The preliminary structure-activity relationship and PK profiles were also discussed.

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.

广西VCT门诊HIV-1抗体阳性人群WB条带分析

目的分析2012-2016年,广西自愿咨询检测门诊人群艾滋病病毒1型（HIV-1）抗体阳性样本的蛋白印迹试验（WB）条带,为艾滋病的预防和控制提供科学依据。方法对筛查有反应性的样本经WB进行确证检测,数据用SPSS 17.0进行统计分析。结果 1 018例确证阳性样本中,WB条带6~9条的占62.48%（636例）;各WB条带中均有gp160带型;反应性率〉95.00%的带型有gp120、gp41和p24,反应性率〉80.00%的带型有p66、p51和p31,有p55、p39和p17的,分别占41.45%（422例）、39.69%（404例）和53.93%（549例）。带型p51、p55和p39的反应性率在不同性别间比较,差异有统计学意义（P〈0.05）,带型p66、p51、p31和p55的反应性率在不同年龄组间比较,差异有统计学意义（P〈0.05）,带型p31、p55和p39的反应性率在不同感染途径间比较,差异有统计学意义（P〈0.05）。结论广西地区某些WB带型反应性率在不同感染人群中存在不同程度的差异,并且与其他地区的报道存在差异,其原因有待进一步研究和探讨。