Regional spread of HIV-1 M subtype B in middle-aged patients by random env-C2V4 region sequencing

A transmission cluster of HIV-1 M:B was identified in 11 patients with a median age of 52 (range 26–65) in North-East Germany by C2V4 region sequencing of the env gene of HIV-1, who—except of one—were not aware of any risky behaviour. The 10 male and 1 female patients deteriorated immunologically, according to their information made available, within 4 years after a putative HIV acquisition. Nucleic acid sequence analysis showed a R5 virus in all patients and in 7 of 11 a crown motif of the V3 loop, GPGSALFTT, which is found rarely. Analysis of formation of this cluster showed that there is still a huge discrepancy between awareness and behaviour regarding HIV transmission in middle-aged patients, and that a local outbreak can be detected by nucleic acid analysis of the hypervariable env region.     

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.     

The spread of HIV-1 subtypes B and CRF01_AE among injecting drug users in Bangkok, Thailand

The HIV epidemic among injecting drug users (IDUs) in Bangkok was initially dominated by HIV subtype B and later by the recombinant CRF01_AE. The present study investigates the distribution of the 2 variants in time and how it is affected by changes in injecting risk behavior and treatment. A mathematic model describing the spread of HIV subtype B and CRF01_AE among IDUs was developed, and data from the AIDSVAX B/E cohort of IDUs in Bangkok were used. From the model, it was calculated that during 1999 to 2003, the annual incidence of HIV was around 0.6 and 2.7 to 3.9 infections per 100 person-years for subtype B and CRF01_AE, respectively. Of the new infections, 18% and 72% are first infections with subtype B and CRF01_AE, respectively, and 9% are superinfections. With increases in risk behavior, the fraction of superinfections rises. If treatment reduces the infectivity of CRF01_AE more than that of subtype B, the fraction of subtype B infections should increase. Subtype B should remain prevalent in a small but considerable fraction of the population for a long time. Changes in risk behavior and the introduction of treatment may alter the distribution of subtypes, but CRF01_AE should remain dominant.     

Selective regimes and evolutionary rates of HIV-1 subtype B V3 variants in the Brazilian epidemic

Half of subtype B Brazilian HIV-1 harbors the V3 tip GWGR instead of the GPGR. To investigate the evolution of GW variants, we analyzed 81 env sequences and 5 full-length GW genomes from antiretroviral-naïve individuals sampled between 1983 and 1999. Phylogenetic analysis indicated that GW strains intermingle in the tree with other subtype B sequences. The mean d_N/d_S values of GW strains were proximal to those of the other sequences, regardless of sampling years or clinical status. In sequences from patients with CD4+ T cell counts ≥ 200 cells/μL, the mean d_N/d_S ratio was greater than one, suggesting a positive selection. The prevalence of GW variants was lower among individuals in whom disease progressed. This is probably attributable to the fact that tryptophan is replaced by other amino acids over time, whereas the GP motif does not evolve as rapidly.     

The Honduran human immunodeficiency virus type 1 (HIV-1) epidemic is dominated by HIV-1 subtype B as determined by V3 domain sero- and genotyping.

The distribution of subtypes A through F of human immunodeficiency virus type 1 (HIV-1) in Honduras was analyzed in 120 HIV-1 positive serum samples by V3 peptide serotyping and HIV-1 cDNA sequencing. In the Honduran HIV-1 epidemic, subtype B was detected in 98 of 99 subtyped samples.     

Genetic characteristics of the V3 region associated with CXCR4 usage in HIV-1 subtype C isolates

CXCR4 coreceptor usage appears to occur less frequently among HIV-1 subtype C viruses. The aim of this study was to investigate the genetic determinants within the V3 region of subtype C isolates able to use CXCR4. Thirty-two subtype C isolates with known phenotypes (16 R5, 8 R5X4 and 8 X4 isolates) were assessed. A subtype C-specific V3 heteroduplex tracking assay (HTA) was used to determine sample complexity, and nucleotide sequencing analysis was used to compare characteristics associated with CCR5 and CXCR4-using isolates. There were sufficient genetic differences to discriminate between R5 viruses and those able to use CXCR4. In general, R5 isolates had an HTA mobility ratio >0.9 whereas CXCR4-using isolates were usually <0.9. Multiple bands were more frequently seen among the dualtropic isolates. Sequence analysis of the V3 region showed that CXCR4-using viruses were often associated with an increased positive amino acid charge, insertions and loss of a glycosylation site, similar to HIV-1 subtype B. In contrast, where subtype B consensus V3 has a GPGR crown motif irrespective of coreceptor usage, all 16 subtype C R5 viruses had a conserved GPGQ sequence at the tip of the loop, while 12 of the 16 (75%) CXCR4-using viruses had substitutions in this motif, most commonly arginine (R). These findings were confirmed using a larger published data set. We therefore suggest that changes within the crown motif of subtype C viruses might be an additional pathway to utilise CXCR4 and thus GPGQ may limit the potential for the development of X4 viruses.     

Maternal neutralizing antibodies against a CRF01_AE primary isolate are associated with a low rate of intrapartum HIV-1 transmission

Mother-to-child transmission (MTCT) of HIV-1 provides a model for studying the role of passively acquired antibodies in preventing HIV infection. We determined the titers of neutralizing antibodies (NAbs) against six primary isolates of clades B and CRF01_AE in sera from 45 transmitting and 45 nontransmitting mothers matched for the main independent factors associated with MTCT in Thailand. A lower risk of MTCT, particularly for intrapartum transmission, was associated only with higher NAb titers against the CRF01_AE strain, MBA. The envelope glycoprotein of this strain showed an unusually long V2 domain of 63 amino acids, encoding six potential N-linked glycosylation sites. We provided experimental data indicating that the extended V2 domain contributed to the higher level of resistance to neutralization by mothers' sera in this strain. Taken together the data suggest that some primary isolates with specific properties may be useful indicators for identifying protective antibodies.     

HIV-1中国流行株CRF01_AE env基因改造及其重组DNA疫苗的构建

目的 构建表达含有密码子优化的HIV-1 CRF01_AE亚型env基因的DNA疫苗,为多载体艾滋病治疗性疫苗的应用提供候选疫苗.方法 对HIV-1感染者血液样本进行型别分析,分型得到的AE亚型标本采用亚型特异性引物克隆env基因,通过序列比对获得其共有序列,对该序列按照哺乳动物密码子使用的偏嗜性进行优化,将人工合成的优化基因克隆至pVR载体,构建DNA疫苗.通过Western Blot方法比较优化前后env基因的表达.结果 成功获得32条具有完整的开放性阅读框的env克隆,型别分析确认均为CRF01_AE亚型.成功对其共有序列进行了优化、合成并构建了DNA疫苗pVR-mod.AE env,该疫苗与野生型的env基因（wt.AE env）相比可以高水平表达env基因,且不依赖Rev的存在.结论 对HIV-1中国流行株CRF01_AE env基因的优化改造及重组DNA疫苗的构建是成功的.     

Soluble CD4 broadens neutralization of V3-directed monoclonal antibodies and guinea pig vaccine sera against HIV-1 subtype B and C reference viruses

To better understand the limits of antigenic reactivity and epitope accessibility of the V3 domain of primary HIV-1 isolates, we evaluated three human anti-V3 monoclonal antibodies (mAbs) and selected guinea pig vaccine sera for neutralization against reference panels of subtype B and C pseudoviruses derived from early stage infections. The mAbs and vaccine sera potently neutralized several prototype viruses, but displayed substantially less neutralization of most reference strains. In the presence of soluble CD4 (sCD4), the breadth of V3-mediated neutralization was increased; up to 80% and 77% of the subtype B and C viruses respectively were sensitive to V3-mediated neutralization. Unlike sCD4, the reaction of CD4-binding site mAbs b12 and F105 with native virus did not lead to full exposure of the V3 domain. These findings confirm that V3 antibodies recognize most primary viral strains, but that the epitope often has limited accessibility in the context of native envelope spike.     

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.     

The evolutionary and transmission characteristic of HIV-1 CRF07_BC in Nanjing,Jiangsu

To understand the epidemiology, evolutionary and transmission characteristics of HIV-1 CRF07_BC in Nanjing, China. One hundred and fifty-nine patients with HIV-1 CRF07_BC were recruited. DNA sequencing, phylogenetic analysis, and molecular transmission cluster analysis were conducted to determine the molecular epidemiology and evolutionary characteristics. Of these HIV-1-infected patients, 95.6% were male, and men who sex with men (76.7%) were the main transmission route. Only 34.0% of these cases were born in Nanjing, and most of them (64.8%) reported having multiple sex partners in the last 6 months. The maximum likelihood phylogenetic analyses of HIV-1 CRF07_BC revealed two lineages. Overall, 67.3% of Nanjing sequences were connected to at least one other individual distributed in 11 clusters, and the average degree was 21.2 with range (1-178). The clustered patients were more likely to be male. The time to a most recent common ancestor for the early HIV-1 CRF07_BC circulating in Nanjing was estimated to be 1998.71[1997.36-2001.07]. The mean estimated evolutionary rate for the epidemic cluster was slightly lower at 2.38[2.12-2.65] × 10⁻³ per site per year with the relaxed exponential clock model. HIV-1 CRF07_BC was transmitted into Nanjing more than 20 years ago from Yunnan and has become one of the most predominant subtypes with a higher evolutionary rate than before.     

Functional complementation of the envelope hypervariable V3 loop of human immunodeficiency virus type 1 subtype B by the subtype E V3 loop.

The hypervariable V3 loop within gp120 of human immunodeficiency virus type 1 (HIV-1) is the major determinant of cell tropism and the entry coreceptor usage of the virus. However, the information obtained thus far has been from only subtype B from North America and Europe, and little is known about other subtypes whose V3 amino acids differ by as much as 50% from subtype B V3. In this study, we examined the functional potential of the V3 element of the HIV-1 subtype E, the most crucial variant causing the AIDS epidemic throughout southeast Asia. A panel of HIV-1LAI recombinants was constructed by the overlap extension method, by which the LAI V3 loop was precisely replaced by that of the subtype E nonsyncytium-inducing (NSI) or syncytium-inducing (SI) variant. All of the recombinant viruses infected peripheral blood mononuclear cells, whereas only those with SI V3 infected MT2 cells, a CD4(+) T cell line. Consistently, the SI V3 recombinants used CXCR4, while the NSI V3 recombinants used CCR5 for infection of HOS-CD4(+) cells. Finally, only the NSI V3 sequence conferred CC-chemokine sensitivity on the parental virus. The data support the notion that the HIV-1 V3 loop consists of a relatively independent domain in gp120 and suggest that the subtype E V3 loop indeed contains the functional element to dictate the cell tropism, coreceptor preference, and chemokine sensitivity of the virus. These findings are of immediate importance in understanding V3 structure-function relationship and for examining phenotypic evolution of HIV-1 subtype E.     

广西HIV-1重组毒株env区快速基因分型方法的建立

目的建立一种快速简便的基因分型方法，对广西HIV-1重组毒株env基因区进行亚型鉴定。方法从HIV阳性样品中提取核酸，使用HIV-1M组通用引物对env区进行第一轮扩增，第二轮则使用分别检测B′/C或C亚型和CRF01-AE亚型的二套特异性引物放入同一反应管中进行扩增，根据不同亚型扩增的目的带位置不同来判断亚型。将通用引物扩增出的所有样本均进行基因测序和系统树分析以验证结果。结果50份样本中，经基因测序和系统树分析证实CRF08-BC样本3份（6％），CRF01-AE样本43份（86％），4份（8％）样本无法确定亚型。经亚型特异性引物PCR法检测得出B′/C或C亚型样本3份（100％），CRF01-AE样本39份（90．7％），灵敏度为91．3％，特异度为100％。两种方法检测结果经差异性检验显示X^2=2．25，P〉0.05，差异无统计学意义，结果一致者占92％。与基因分析结果吻合。重复实验显示CRF08-BC平均重复性为100％（10／10），CRF01．AE为93．8％（61／65）。结论该方法是一种简便、快速、低成本，具有高度灵敏性和特异性的HIV-1毒株env基因区分型法，能够直接对广西HIV-1 CRF01-AE重组毒株进行鉴定。     

Molecular and structural characterization of HIV-1 subtype B Brazilian isolates with GWGR tetramer at the tip of the V3-loop

One of most intriguing features of the HIV-1 subtype B epidemic in Brazil is the high frequency of isolates exhibiting tryptophan (W) in the tetramer (GWGR) at the tip of the V3 loop. We observed that the frequencies of glutamic and aspartic acids at site 25 of the V3 loop are quite distinct in GWGR isolates compared with viruses with other tetramers. The basic amino acids at sites 11 and 25 of V3 are strongly linked with CCR5-to-CXCR4 coreceptor shift. We therefore predicted phenotype usage and found that GWGR isolates are exclusively CCR5-using. Further evidence of this came from intrahost sequences, where basic amino acid substitutions at sites 11 and 25 emerged only in isolates presenting a tryptophan-to-glycine replacement at the tetramer of the V3. In addition, modeled 3D-structures of the V3 loop of GWGR and GGGR in intrahost viruses differ essentially in the binding region of the coreceptor.     

广西HIV-1 CRF01-AE重组毒株env基因V3环序列变异及其与生物表型的关系

目的研究广西HIV-1 CRF01-AE重组毒株env基因V3环序列变异及其与生物表型间的关系。方法从广西主要流行区收集来的50份HIV-1感染者血液样本中提取前病毒DNA，使用巢式聚合酶链反应（nested-PCR）扩增HIV-1 env基因片段并进行亚型鉴定，选择38份CRF01-AE重组型HIV-1毒株env，基因V3环及邻近区域的序列进行系统树和氨基酸变异分析。结果38份CBF01-AE重组毒株中36份与分离于广西地区的CRF01-AE.97CNGX2f和泰国代表毒株THCM240接近，另外2份与中非共和国代表株90CF402聚成一簇；CRF01-AE重组毒株V3环顶端四肽存在着4种类型：CPCQ、GPGR、GPGH和GPGA；根据V3环关键氨基酸推测辅助受体使用情况，结果显示：71．05％的CRF01-AE重组毒株可能使用CCR5作为辅助受体，28．95％不能对其辅助受体的使用情况做出预测。结论广西HIV-1 CRF01-AE重组毒株V3顶端四肽变异较大，而且大部分毒株可能为NSI型。这可为广西该毒株的防治和诊断试剂的更新提供参考。