Independent introduction of transmissible F/D recombinant HIV-1 from Africa into Belgium and The Netherlands

Most HIV-1 subtype F viruses described so far have been isolated from individuals originating in South America, Romania, or Central Africa. Previous studies have shown that subtype F viruses from these three areas can be distinguished by phylogenetic tree analysis of various parts of the HIV genome. Subtype F strains circulating in Central Africa and classified as subgroup F2 and F3 have relatively large nucleotide distances from strains of subgroup F1, which includes some African strains, along with strains from Romania and South America. Subtype F strains have now appeared in Europe. In this study, we analyzed the complete gag gene and a large fragment of the pol gene of seven strains of African origin that represent the three F subgroups. At least five of the seven strains appear to be intersubtype recombinants. Of four strains circulating in Belgium and the Netherlands, three were F/D mosaics and the fourth harboured a G(gag)/GH(pol)/F3(env) recombinant structure. Two of the three F/D mosaics showed identical breakpoints and were independently introduced in Belgium and the Netherlands. At least two of the mosaics were further transmitted. The remaining three strains of the seven we studied were isolated from individuals in Cameroon. Two included large or smaller F1 fragments in gag and pol. The third strain was subtype D along the entire gag and pol fragment. A parental African subtype F that showed no evidence for recombination was not found.     

Molecular cloning and biological characterization of full-length HIV-1 subtype C from Botswana

Human immunodeficiency virus type 1 (HIV-1) subtype C is now responsible for more than half of all HIV-1 infections in the global epidemic and for the high levels of HIV-1 prevalence in southern Africa. To facilitate studies of the biological nature and the underlying molecular determinants of this virus, we constructed eight full-length proviral clones from two asymptomatic and three AIDS patients infected with HIV-1 subtype C from Botswana. Analysis of viral lysates showed that Gag, Pol, and Env structural proteins were present in the virions. In four clones, the analysis suggested inefficient envelope glycoprotein processing. Nucleotide sequence analysis of the eight clones did not reveal frameshifts, deletions, premature truncations, or translational stop codons in any structural, regulatory, or accessory genes. None of the subtype C clones were replication competent in donor peripheral blood mononuclear cells (PBMCs), macrophages, Jurkat(tat) cells, or U87. CD4.CCR5 cells. However, infection by two clones could be rescued by complementation with a functional subtype C envelope clone, resulting in a productive infection of PBMCs, macrophages, and U87. CD4.CCR5 cells.     

中国HIV-1 C/B'重组毒株和B'毒株感染者Nef特异性T细胞免疫应答的研究

目的 研究中国主要流行的HIV-1 C/B'重组毒株和B'亚型毒株感染者Nef特异性T细胞反应特征,确定两种亚型感染者共同识别的免疫优势区.方法 本研究以59名HIV-1 C/B'重组毒株、27名B'亚型毒株感染者为研究对象,用ELISPOT检测针对HIV-1型C/B'Nef重叠多肽产生IFN-γ的特异性T细胞反应.结果 44例(74.58%)HIV-1 C/B'重组毒株感染者产生Nef特异性T细胞反应,主要识别EVA7081.1、5、6、7、43、44、45、47、48、49这10条多肽,氨基酸序列为Nef63～115和117～139的区域.20例(74.07%)的HIV-1 B'毒株感染者产生Nef特异性T细胞反应,主要识别EVA7081.1、2、43、49这4条多肽,氨基酸序列为Nef 63～77和87～119的区域.两种亚型感染者特异性T细胞反应的强度和广度与病毒载量和CIM细胞数不相关.结论 中国HIV-1 C/B'重组毒株和B'亚型毒株感染者共同识别氨基酸序列为Nef63～77和87～115的免疫优势区,提示此区域可用于疫苗的设计.     

Large-scale amplification, cloning and sequencing of near full-length HIV-1 subtype C genomes

Full-length HIV-1 genome sequencing provides important data needed to address several vaccine design, molecular epidemiologic and pathogenesis questions. A protocol is presented for obtaining near full-length genomes (NFLGs) from subjects infected with HIV-1 subtype C. This protocol was used to amplify NFLGs from 244 of 366 (67%) samples collected at two clinics in Durban, South Africa (SK and PS). Viral load was directly associated with frequency of successful NFLG amplification for both cohorts (PS; p = 0.005 and SK; p < 0.001). Seventeen of 38 initially NFLG-negative SK samples had variation within the PCR primer binding sites, however only 3 of these were successfully re-amplified using re-designed primers homologous to the target viruses. NFLGs were obtained from 7 of 24 PBMC samples processed from subjects whose plasma did not yield a NFLG. Stable plasmid clones were obtained from all 244 NFLG-positive PCR products, and both strands of each genome were sequenced, using a primary set of 46 primers. These methods thus allow the large-scale collection of HIV-1 NFLGs from populations infected primarily with subtype C. The methods are readily adaptable to other HIV-1 subtypes, and provide materials for viral functional analyses and population-based molecular epidemiology studies that include analysis of viral genome chimerization.     

Construction and characterization of a full-length infectious clone from a fast-replicating, X4-tropic HIV-1 subtype B′ isolate

In HIV-1 epidemics in China, HIV-1 subtype B′ is the most predominant subtype circulating in intravenous drug users. In this study, we constructed an HIV-1 full-length infectious molecular clone based on the primary virus LWJ, which was isolated from an HIV-infected patient in Fujian Province, China. Phylogenetic and bootscanning analysis of the viral sequence revealed that the isolate LWJ belonged to HIV-1 subtype B′. The infectious clone was designated as “pLWJ”. The virus (LWJ-c) produced from this infectious clone by in vitro transfection of 293T cells could infect both human peripheral blood mononuclear cells (PBMCs) and human the T cell line MT4. Interestingly, the cloned LWJ-c virus utilized CXCR4 as its co-receptor and could replicate in vitro with similar efficiency and kinetics compared to its parental primary isolate LWJ as well as the clade B reference virus NL4-3. The LWJ-c virus could also cause cytopathic effects in both PBMCs and MT cells. Sequence analysis of the envelope glycoprotein of pLWJ showed that a conserved GPGR motif and an arginine at position 11 were present in the V3 loop, which was consistent with previous reports regarding CXCR4 co-receptor usage and syncytium-inducing (SI) phenotype. Thus, the infectious clone represents a fast-replicating, high-producing, CXCR4-tropic and syncytium-inducing isolate. Given the prevalence of HIV-1 subtype B′ in China, this infectious clone can be a very useful tool to provide a versatile molecular model for research focusing on the biological properties of this subtype.     

河南省分离的HIV-1 B-Thai亚型流行株全基因组克隆及序列分析

目的 克隆、鉴定自河南省分离的HIV-1 B-Thai亚型流行株并进行系统发育分析。方法收集河南省1份HIV-1感染者全血后分离的淋巴细胞，在植物血凝素存在下与健康献血员淋巴细胞共培养，从培养的淋巴细胞中提取前病毒DNA。在HIV-1基因的长末端重复序列的保守区设计引物，利用LA Tag长链扩增系统扩增HIV-1全长基因，纯化的PCR产物与pWSK29-T载体连接，成功克隆CNHN24株。对鉴定的3个克隆进行全长测序，利用局部同源法计算同源率，同时采用Phylip软件绘制HIV-1 Env、Gag和Pol基因的进化树。结果 V3V4环序列分析表明：本次克隆的CNHN24株病毒为HIV-1 B-Thai亚型，V3环区氨基酸序列比对发现在9个位点发生氨基酸替换。在含有9010bp，全长HIV-1基因的CNHN24株克隆中未发现明显的缺失、插入和重排现象，Gag、Pol、Vpr和Vif基因与RL42株的同源率达到95．42％～97．08％，进化分析显示，CNHN24株与RL42株的遗传距离最近。结论 HIV-1 CNHN24株为国内实验室完成的HIV-1全长基因克隆，为进一步研究HIV-1流行特征提供了基础。     

Genotypic and phenotypic analysis of the env gene from South African HIV-1 subtype B and C isolates

The objective of the study was to assess the genotypic and phenotypic properties of 18 viral strains from human immunodeficiency virus-1 (HIV-1) positive patients and to identify subtype C isolates for vaccine design strategies. All the isolates were non-syncytium-inducing (NSI) in both the primary and MT-2 cell cultures. The amino acid charge of the V3 loop correlated with the NSI phenotype of the strains. The V3 competitive peptide enzyme immunoassay and DNA sequencing of the partial gp120 region gave concordant results on the 15 subtype C strains, whereas the three B genotypes gave a positive to B, a nonreactive to B, and a dual reaction to the B-D peptides, respectively. Sixteen of the isolates used only CCR5 as coreceptor whereas two isolates made use of additional coreceptors including CXCR4. In summary, all our subtype C isolates are NSI phenotypically and almost all of them use CCR5 exclusively as their coreceptor.     

HIV-1 LTR subtype and perinatal transmission

Multiple subtypes of HIV-1 have been identified; however, there is little data on the relative transmissibility of viruses belonging to different subtypes. A matched case-control study addressed whether viruses with different long terminal repeat (LTR) subtypes were transmitted equally from mother to infant. The LTR subtype was determined for 45 matched cases and controls who participated in a clinical trial in Tanzania. HIV-1 subtypes A, C, and D and intersubtype recombinant sequences were identified. Exact matched logistic regression analysis showed that viruses containing subtype A or intersubtype recombinant LTRs were 3.2 and 4.8 times more likely to be transmitted from mother to infant than viruses with subtype D LTRs. Viruses containing subtype C LTRs were 6.1 times more likely to be transmitted than those with subtype D LTRs. These differences in transmission were independent of maternal CD4 at enrollment. Thus, it appears that HIV-1 subtype may be associated with differing rates of perinatal transmission in Tanzania.     

Most env and gag subtype A HIV-1 viruses circulating in West and West Central Africa are similar to the prototype AG recombinant virus IBNG

The genetic subtype was identified in gag and env of 219 HIV-1-positive samples collected in different African countries, 44 from Senegal, 55 from Cameroon, 82 from Gabon, and 38 from Djibouti. In total, 20 (9.1%) samples had discordant subtypes between gag and env, 6 of 44 (13.9%) in Senegal, 4 of 55 (7.2%) in Cameroon, 1 of 38 (2.6%) in Djibouti, and 10 of 82 (12.1%) in Gabon. Subtypes A and G were predominantly involved in the recombination events. Phylogenetic tree analysis of gag showed that an important number of the A sequences form a distinct subcluster with the AG-IBNG prototype strain (a complex A/G mosaic virus): 27 of 32 (84.3%) in Senegal, 12 of 17 (70.6%) in Nigeria, 24 of 39 (61.5%) in Cameroon, and 38 of 70 (54.3%) in Gabon. Full-length genome analysis of 3 and additional sequences in pol for 10 such strains confirmed that they have a similar complex A/G mosaic genomic structure. These data suggest that in West Africa, most probably between 60% and 84% of the subtype A viruses are recombinant AG-IBNG viruses. This finding has potential implications on future vaccine, diagnostic, and treatment strategies. The actual and future role of these viruses in the global pandemic must be monitored in all new molecular epidemiologic studies, a discrimination between subtype A and AG-IBNG-like viruses is necessary.     

HIV-1 subtypes D and F are prevalent in Guinea Conakry

BackgroundLimited data is available upon the distribution of different HIV-1/2 genotypes in the blood donor population from Guinea Conakry.ObjectivesTo investigate the prevalence of HIV-1/2 subtypes in asymptomatic blood donors in Guinea Conakry, in order to update knowledge of HIV-1/2 epidemiology within this country.Study designSamples from 104 blood donors seropositive for HIV-1/2 were tested for HIV-1 by real-time RT-PCR. Those negative for HIV-1 were tested with HIV-2 nested RT-PCR. Positive samples were further amplified in the HIV-1 gag and pol regions and sequenced. Subtypes were determined by phylogenetic analysis on amplicon sequences.Results61 samples were positive by HIV-1 real-time RT-PCR. Of the 43 negative, 2 (4.6%) were positive for HIV-2. 52/61 (85.3%) samples were positive by nested RT-PCR. Of the 52, 43 (70.5%) and 31(59.6%) sequences were obtained in the gag and pol regions, respectively; 23 for both regions. HIV-1 subtype distribution was 1 B (2.1%), 8 F (17%), 8 D (17%) and 28 CRF02_AG (59.6%) with 2 unclassified recombinants (4.3%). Unique clusters for subtype D and F distinguished Guinea from HIV-1 subtype distribution in neighboring countries.ConclusionsSubtype F and subtype D strains, uncommon in West Africa, are a substantial part of HIV-1 epidemiology in Guinea.     

中国HIV-1 B/C重组毒株不同阶段感染者多聚酶蛋白特异性T淋巴细胞反应的研究

目的 研究中国主要流行的HIV-1 B/C重组毒株不同时期感染者多聚酶蛋白(Pol)特异性T淋巴细胞反应特征并确定主要识别的免疫优势区域.方法 本研究以11例感染时间<18个月和25例感染时间>3年的HIV-1 B/C重组毒株感染者为研究对象,以10例HIV-1阴性健康人作为对照,应用酶联免疫斑点检测技术综合测定了针对覆盖HIV-1 pol基因的249条重叠多肽产生IFN-γ的特异性T淋巴细胞免疫反应.结果 感染时间<18个月感染者中有8(72.73%)名检测到了分泌IUN-γ的HIV-1特异性T淋巴细胞免疫反应,主要识别位于逆转录酶区、氨基酸位置为Pol 481～631内的Pol5581、Pol5582、Pol5587、Pol5609、Pol5610和Pol5615六条多肽,分泌IFN-γ的特异性T淋巴细胞反应广度与外周血CD4+T细胞数呈现明显负相关(P=0.0212,r=-0.762);感染时间>3年感染者中有15(60%)名检测到了分泌IFN-γ的HIV-1特异性T淋巴细胞免疫反应,主要识别位于逆转录酶区、氨基酸位置为Pol241～295内的Pol5521、Pol5525、Pol5526、Pol5531四条多肽和Pol 708～722内的Pol5638多肽,分泌IFN-γ的特异性T淋巴细胞反应强度与病毒载量呈现明显正相关(P=0.006 95,r=0.660);健康人对照组无阳性反应.结论 中国HIV-1 B/C重组毒株不同阶段感染者主要识别多聚酶蛋白的不同区域.     

Evaluation of Chiron HIV-1/HIV-2 recombinant immunoblot assay.

In a study to determine the reliability, sensitivity, and specificity of the Chiron RIBA HIV-1/HIV-2 Strip Immunoblot Assay (RIBA HIV-1/2 SIA) for confirmation of human immunodeficiency virus type 1 (HIV-1) and HIV-2 antibodies, 1,263 serum samples from various populations in the United States, Caribbean, Africa, India, and Thailand were evaluated by RIBA HIV-1/2 SIA, and the results were compared with those obtained by an HIV-1 Western blot (immunoblot) assay. All sera were tested by HIV enzyme immunoassay, RIBA HIV-1/2 SIA, and Western blotting. Samples with discrepant results were further tested by an HIV-1 and/or HIV-2 immunofluorescent-antibody assay and HIV-1 p24 antigen assay. The RIBA HIV-1/2 SIA detected all 17 HIV-1 and HIV-2 dually reactive serum samples, all 215 HIV-2-positive serum samples, and 480 of 481 HIV-1-positive serum samples for a sensitivity of 99.8%. Of 548 negative samples, 523 were RIBA HIV-1/2 SIA negative, for a specificity of 95.4%, with 22 (4%) samples interpreted as indeterminate and 3 (0.6%) interpreted as falsely positive. Western blotting detected 391 of 548 negative samples (specificity, 71.4%), with 152 (27.7%) samples interpreted as indeterminate and 5 (0.9%) interpreted as falsely positive. In conclusion, the RIBA HIV-1/2 SIA had a sensitivity comparable to that of Western blotting and could discriminate HIV-1 from HIV-2 in one blot, providing a cost advantage. Because of its high degree of specificity, the RIBA HIV-1/2 SIA further reduced the number of indeterminate results found by Western blotting, providing a more accurate means of assessing seronegative individuals.     

Genetic and neutralization properties of HIV-1 env clones from subtype B/BC/AE infections in China

BACKGROUND: As HIV vaccines move into preclinical and clinical trials in China, pseudovirion-based neutralization assays, especially those using env genes of Chinese origin, are widely required to evaluate the ability of HIV vaccines to induce neutralizing antibody (nAb) responses. MATERIALS AND METHODS: Functional gp160 genes from plasma samples from Chinese HIV-infected patients were cloned and sequenced and then used to establish a pseudovirus-based neutralization assay. The neutralization phenotypes of the Env-pseudotyped viruses were characterized with known nAbs (4E10, 2F5, IgG1b12, and 2G12) and 43 plasma samples from patients infected with different HIV subtypes. RESULTS: Overall, 27 functional gp160 genes (18 subtype BC, 3 subtype AE, and 6 subtype B) of HIV-1 were obtained, and their full-length nucleotide sequences were analyzed. The results confirmed the presence of significant genetic diversity among the clones. 4E10 neutralized all 27 Env-pseudotyped viruses, whereas IgG1b12 neutralized 44% of them. 2F5 neutralized 67% and 100% of subtype B and AE clones, respectively, but not subtype BC clones, whereas 2G12 neutralized 33% of subtype B viruses but not subtype BC and AE viruses. There were significant differences in the cross-neutralization activities when the neutralization phenotypes of the 27 Env-pseudotyped viruses were characterized using 43 HIV-positive plasma samples. CONCLUSIONS: These characterized functional HIV-1 env clones should be useful for standardizing neutralization assays in China.     

Design, construction, and characterization of a dual-promoter multigenic DNA vaccine directed against an HIV-1 subtype C/B' recombinant

An effective vaccine against HIV-1 is generally considered the best hope for controlling the raging AIDS pandemic. As a part of our AIDS vaccine development effort, we constructed a dual-promoter plasmid capable of high-level expression of 2 independent transgenes. HIV-1 gag, pol, env, nef, and tat from a primary subtype C/B' CCR5-tropic HIV-1 were "codon" optimized, modified to eliminate known functional activity, and assembled using an overlapping polymerase chain reaction into 2 plasmids: ADVAX-I (containing env and gag) and ADVAX-II (containing pol and nef-tat). These 2 dual-promoter candidate vaccines showed levels of HIV-1 gene expression comparable to those observed with single-gene plasmids in vitro. Importantly, immunization of mice with these vaccine constructs resulted in dose-dependent multigenic CD4 and CD8 T-cell responses equivalent to those provided by vaccination with single-gene plasmids. With input from the US Food and Drug Administration, ADVAX-I and ADVAX-II have since been combined as a single candidate DNA vaccine, ADVAX. A phase 1 clinical trial of this product has been successfully completed, and its use in prime-boost studies is now underway.     

基于中国HIV-1 CRF01_AE重组亚型gp41 NHR结构域亚单位疫苗的研究

目的:构建基于中国HIV-1 CRF01_AE重组亚型包膜糖蛋白gp41 NHR结构域N51的亚单位疫苗,并进行免疫原性研究.方法:设计4条引物,运用重叠延伸PCR方法扩增出N51Fd基因,将其插入真核表达载体pFUSE-hIgG1-Fc2,构建重组质粒pFUSE/N51Fd并进行序列测定.Western blot 法检测N51FdFc-AE重组蛋白的表达.用纯化蛋白免疫BALB/c小鼠后,ELISA法检测小鼠的抗体反应.结果:成功构建了pFUSE/N51Fd重组质粒,N51FdFc-AE重组蛋白在真核体系获得了高效表达,Western blot结果显示在相对分子质量(Mr)35000处有目的蛋白条带.小鼠抗血清能特异性识别源于gp41 NHR的抗原,效价高达1∶102400,平均效价为1∶51200.结论:改造后的亚单位疫苗能有效激活机体的免疫响应,可用于HIV候选疫苗的研发.     

Molecular characterization of human immunodeficiency virus (HIV)-1 and -2 in individuals from guinea-bissau with single or dual infections: predominance of a distinct HIV-1 subtype A/G recombinant in West Africa.

Guinea-Bissau in West Africa has the highest prevalence of human immunodeficiency virus (HIV)-2 infection in the world, but recently the HIV-1 prevalence increased rapidly with the subsequent appearance of HIV-1 and HIV-2 dual infections. Information about the genetic subtypes of HIV in the region is limited. Therefore, we characterized the env V3 region of HIV-1 and HIV-2 variants through direct DNA sequencing of peripheral blood mononuclear cell samples from 18 individuals with HIV-1 only and 9 individuals with dual infection. Phylogenetic analyses of these new sequences and database sequences from other West African countries showed that all HIV-1 and HIV-2 sequences from singly as well as dually infected individuals, except one, clustered among HIV-1 subtype A and HIV-2 subtype A, respectively. Importantly, a majority of the HIV-1 sequences from Guinea-Bissau and neighbouring countries were closely related with the isolates IbNG, DJ263, and DJ264, which share a common subtype A/G recombination pattern. Analysis of pol gene sequences from selected HIV-1 variants showed that "IbNG-like" viruses in Guinea-Bissau are also recombinant, indicating that the HIV-1 epidemic in Guinea-Bissau and neighbouring countries is dominated by an epidemic spread of a distinct subtype A/G recombinant, which is strikingly similar to the epidemic spread of a subtype A/E recombinant in Southeast Asia. Furthermore, the HIV-1 and HIV-2 variants carried by individuals with dual infection were intermixed with variants from singly infected individuals, indicating that variants involved in dual and single infections have common epidemiological histories.