An Improved Strategy for Efficient Expression and Purification of Soluble HIV-1 Tat Protein in E.coli

Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% （14 of 101） rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 （DE3） due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B （DE3）, which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.     

Transmission,acute HIV-1 infection and the quest for strategies to prevent infection

By the acute stage of HIV-1 infection, the immune system already faces daunting challenges. Research on mucosal barriers and the events immediately after heterosexual transmission that precede this acute stage could facilitate the development of effective microbicides and vaccines.     

HIV-1 Diversity,Transmission Dynamics and Primary Drug Resistance in Angola

Objectives

To assess HIV-1 diversity, transmission dynamics and prevalence of transmitted drug resistance (TDR) in Angola, five years after ART scale-up.

Methods

Population sequencing of the pol gene was performed on 139 plasma samples collected in 2009 from drug-naive HIV-1 infected individuals living in Luanda. HIV-1 subtypes were determined using phylogenetic analysis. Drug resistance mutations were identified using the Calibrated Population Resistance Tool (CPR). Transmission networks were determined using phylogenetic analysis of all Angolan sequences present in the databases. Evolutionary trends were determined by comparison with a similar survey performed in 2001.

Results

47.1% of the viruses were pure subtypes (all except B), 47.1% were recombinants and 5.8% were untypable. The prevalence of subtype A decreased significantly from 2001 to 2009 (40.0% to 10.8%, P = 0.0019) while the prevalence of unique recombinant forms (URFs) increased>2-fold (40.0% to 83.1%, P<0.0001). The most frequent URFs comprised untypable sequences with subtypes H (U/H, n = 7, 10.8%), A (U/A, n = 6, 9.2%) and G (G/U, n = 4, 6.2%). Newly identified U/H recombinants formed a highly supported monophyletic cluster suggesting a local and common origin. TDR mutation K103N was found in one (0.7%) patient (1.6% in 2001). Out of the 364 sequences sampled for transmission network analysis, 130 (35.7%) were part of a transmission network. Forty eight transmission clusters were identified; the majority (56.3%) comprised sequences sampled in 2008–2010 in Luanda which is consistent with a locally fuelled epidemic. Very low genetic distance was found in 27 transmission pairs sampled in the same year, suggesting recent transmission events.

Conclusions

Transmission of drug resistant strains was still negligible in Luanda in 2009, five years after the scale-up of ART. The dominance of small and recent transmission clusters and the emergence of new URFs are consistent with a rising HIV-1 epidemics mainly driven by heterosexual transmission.     

实验猴直肠脱的防治

目的探索实验猴直肠脱的理想治疗方案。方法根据本中心治疗成功的经验对实验猴直肠脱的治疗方案、术后护理及预防措施进行了详细论述。结果经临床实践,本防治方案对实验猴直肠脱的治愈率可达90%以上。结论本方案可以在生产中推广应用。     

HIV—1复制抑制剂的新靶标

现在市场上的多种治疗ＡＩＤＳ的药物都是病毒逆转录酶和蛋白水解酶的特异性抑制剂 ,但是发现有 2 0 %以上的病人不能在短期内承受这些药物的治疗 ,而且长期使用蛋白水解酶抑制剂对人体正常新陈代谢的影响也越来越突出 ,现已发现ＨＩＶ 1的耐药变种已经出现并正以很快的速度传播。另外 ,这些药物不能直接清除感染ＨＩＶ 1病人体内的病毒。所以现在需要发现新型的治疗ＡＩＤＳ病的药物 ,这些药物可以长期使用 ,能补充或者部分代替已有的药物 ,而这就需要先找到ＨＩＶ 1复制抑制剂的新靶标。ＨＩＶ 1病毒的生活周期是这样的 :病毒被膜和细胞表…     

Mapping of Epitopes Exposed on Intact Human Immunodeficiency Virus Type 1 (HIV-1) Virions: a New Strategy for Studying the Immunologic Relatedness of HIV-1

To study the antigenic conservation of epitopes of human immunodeficiency virus type 1 (HIV-1) isolates of different clades, the abilities of human anti-HIV-1 gp120 and gp41 monoclonal antibodies (MAbs) to bind to intact HIV-1 virions were determined by a newly developed virus-binding assay. Eighteen human anti-HIV MAbs, which were directed at the V2, V3 loop, CD4-binding domain (CD4bd), C5, or gp41 regions, were used. Nine HIV-1 isolates from clades A, B, D, F, G, and H were used. Microtiter wells were coated with the MAbs, after which virus was added. Bound virus was detected after lysis by testing for p24 antigen with a noncommercial p24 enzyme-linked immunosorbent assay. The anti-V3 MAbs strongly bound the four clade B viruses and viruses from the non-B clades, although binding was weaker and more sporadic with the latter. The degrees of binding by the anti-V3 MAbs to CXCR4- and CCR5-tropic viruses were similar, suggesting that the V3 loops of these two categories of viruses are similarly exposed. The anti-C5 MAbs bound isolates of clades A, B, and D. Only weak and sporadic binding of all the viruses tested with anti-CD4bd, anti-V2, and anti-gp41 MAbs was detected. These results suggest that V3 and C5 structures are shared and well exposed on intact virions of different clades compared to the CD4bd, V2, and gp41 regions.     

Infectivity and Immunogenicity of SIVmac/HIV-1 Chimeric Viruses (SHIVs) with Deletions in Two or Three Genes (vpr,nef and vpx)

Two SHIVs with two or three genes deleted (SHIV-drn and SHIV-dxrn) were constructed. The inoculation of monkeys with SHIV-drn resulted in short-term viremia, but inoculation with SHIV-dxrn did not. At 68 weeks post-inoculation, the monkeys were reinoculated with a 100-fold higher dose of each SHIV, but none showed viremia. Killer cell activities against HTV-1 Env were detected in the SHIV-drn- and SHIV-dxrn-inoculated monkeys. Cross-reactive killer activity against HIV-1 Gag and SIVmac Gag was observed in one monkey. Antibodies were not detected in the SHIV-dxrn-inoculated monkeys, but the SHIV-drn-inoculated monkeys showed an anamnestic antibody reaction. These data indicate that SHIV-drn is infectious to and immuno-inducible in macaques but SHIV-dxrn is not.     

Partner Notification for Reduction of HIV-1 Transmission and Related Costs among Men Who Have Sex with Men: A Mathematical Modeling Study

Background

Earlier antiretroviral treatment initiation prevents new HIV infections. A key problem in HIV prevention and care is the high number of patients diagnosed late, as these undiagnosed patients can continue forward HIV transmission. We modeled the impact on the Dutch men-who-have-sex-with-men (MSM) HIV epidemic and cost-effectiveness of an existing partner notification process for earlier identification of HIV-infected individuals to reduce HIV transmission.

Methods

Reduction in new infections and cost-effectiveness ratios were obtained for the use of partner notification to identify 5% of all new diagnoses (Scenario 1) and 20% of all new diagnoses (Scenario 2), versus no partner notification. Costs and quality adjusted life years (QALYs) were assigned to each disease state and calculated over 5 year increments for a 20 year period.

Results

Partner notification is predicted to avert 18–69 infections (interquartile range [IQR] 13–24; 51–93) over the course of 5 years countrywide to 221–830 (IQR 140–299; 530–1,127) over 20 years for Scenario 1 and 2 respectively. Partner notification was considered cost-effective in the short term, with increasing cost-effectiveness over time: from €41,476 -€41, 736 (IQR €40,529-€42,147; €40,791-€42,397) to €5,773 -€5,887 (€5,134-€7,196; €5,411-€6,552) per QALY gained over a 5 and 20 year period, respectively. The full monetary benefits of partner notification by preventing new HIV infections become more apparent over time.

Conclusions

Partner notification will not lead to the end of the HIV epidemic, but will prevent new infections and be increasingly cost-effectiveness over time.     

Modeling Social Transmission Dynamics of Unhealthy Behaviors for Evaluating Prevention and Treatment Interventions on Childhood Obesity

Research evidence indicates that obesity has spread through social networks, but lever points for interventions based on overlapping networks are not well studied. The objective of our research was to construct and parameterize a system dynamics model of the social transmission of behaviors through adult and youth influence in order to explore hypotheses and identify plausible lever points for future childhood obesity intervention research. Our objectives were: (1) to assess the sensitivity of childhood overweight and obesity prevalence to peer and adult social transmission rates, and (2) to test the effect of combinations of prevention and treatment interventions on the prevalence of childhood overweight and obesity. To address the first objective, we conducted two-way sensitivity analyses of adult-to-child and child-to-child social transmission in relation to childhood overweight and obesity prevalence. For the second objective, alternative combinations of prevention and treatment interventions were tested by varying model parameters of social transmission and weight loss behavior rates. Our results indicated child overweight and obesity prevalence might be slightly more sensitive to the same relative change in the adult-to-child compared to the child-to-child social transmission rate. In our simulations, alternatives with treatment alone, compared to prevention alone, reduced the prevalence of childhood overweight and obesity more after 10 years (1.2–1.8% and 0.2–1.0% greater reduction when targeted at children and adults respectively). Also, as the impact of adult interventions on children was increased, the rank of six alternatives that included adults became better (i.e., resulting in lower 10 year childhood overweight and obesity prevalence) than alternatives that only involved children. The findings imply that social transmission dynamics should be considered when designing both prevention and treatment intervention approaches. Finally, targeting adults may be more efficient, and research should strengthen and expand adult-focused interventions that have a high residual impact on children.     

Mother-to-Child HIV-1 Transmission Events Are Differentially Impacted by Breast Milk and Its Components from HIV-1-Infected Women

Breast milk is a vehicle of infection and source of protection in post-natal mother-to-child HIV-1 transmission (MTCT). Understanding the mechanism by which breast milk limits vertical transmission will provide critical insight into the design of preventive and therapeutic approaches to interrupt HIV-1 mucosal transmission. However, characterization of the inhibitory activity of breast milk in human intestinal mucosa, the portal of entry in postnatal MTCT, has been constrained by the limited availability of primary mucosal target cells and tissues to recapitulate mucosal transmission ex vivo. Here, we characterized the impact of skimmed breast milk, breast milk antibodies (Igs) and non-Ig components from HIV-1-infected Ugandan women on the major events of HIV-1 mucosal transmission using primary human intestinal cells and tissues. HIV-1-specific IgG antibodies and non-Ig components in breast milk inhibited the uptake of Ugandan HIV-1 isolates by primary human intestinal epithelial cells, viral replication in and transport of HIV-1- bearing dendritic cells through the human intestinal mucosa. Breast milk HIV-1-specific IgG and IgA, as well as innate factors, blocked the uptake and transport of HIV-1 through intestinal mucosa. Thus, breast milk components have distinct and complementary effects in reducing HIV-1 uptake, transport through and replication in the intestinal mucosa and, therefore, likely contribute to preventing postnatal HIV-1 transmission. Our data suggests that a successful preventive or therapeutic approach would require multiple immune factors acting at multiple steps in the HIV-1 mucosal transmission process.     

Th1 and th2 responses, HIV-1 coreceptors, and HIV-1 infection.

The Th1/Th2 model provides an interesting paradigm for understanding several pathophysiological processes and possibly for developing new immunotherapeutical strategies. In HIV-1 infection the interaction between the type of HIV-1 strain and the pathway of the ongoing T-cell effector response, despite its complexity, may represent one of the crucial mechanisms in determining the outcome of virus infection. While the possibility of an HIV-1-driven Th1 to Th2 switch of the immune response is still debated, evidence is accumulating to suggest that cytokines produced during an immune response can contribute to promote a selective pressure toward the evolution of HIV-1 viral strains with different tropism. This article summarizes the results of our recent studies in which the expression of CCR5 and CXCR4 HIV-1 co-receptors, as well as the activity of R5- or X4- tropic strains of HIV-1 in different in vitro models of Th1/Th2 polarization was analyzed.     

Transmission selects for HIV-1 strains of intermediate virulence: a modelling approach

Recent data shows that HIV-1 is characterised by variation in viral virulence factors that is heritable between infections, which suggests that viral virulence can be naturally selected at the population level. A trade-off between transmissibility and duration of infection appears to favour viruses of intermediate virulence. We developed a mathematical model to simulate the dynamics of putative viral genotypes that differ in their virulence. As a proxy for virulence, we use set-point viral load (SPVL), which is the steady density of viral particles in blood during asymptomatic infection. Mutation, the dependency of survival and transmissibility on SPVL, and host effects were incorporated into the model. The model was fitted to data to estimate unknown parameters, and was found to fit existing data well. The maximum likelihood estimates of the parameters produced a model in which SPVL converged from any initial conditions to observed values within 100-150 years of first emergence of HIV-1. We estimated the 1) host effect and 2) the extent to which the viral virulence genotype mutates from one infection to the next, and found a trade-off between these two parameters in explaining the variation in SPVL. The model confirms that evolution of virulence towards intermediate levels is sufficiently rapid for it to have happened in the early stages of the HIV epidemic, and confirms that existing viral loads are nearly optimal given the assumed constraints on evolution. The model provides a useful framework under which to examine the future evolution of HIV-1 virulence.     

Strategy for AIDS Prevention and Treatment

HIV/AIDS has been circulating in China for over 25 year. While making progress and achievements on HIV/AIDS prevention,there still are great challenge and difficulties such as HIV epidemic controlling and vaccine research.     

Design and synthesis of sulfoximine based inhibitors for HIV-1 protease

A new class of potent sulfoximine inhibitors for HIV-1 protease has been designed and synthesized. Substitution of the sulfoximine moiety into different parent compounds yields different inhibition effects. While our previously studied sulfoximine-based inhibitors display potency of 2.5 nM (IC(50)) against HIV-1 protease, introduction of the sulfoximine moiety into the asymmetric Indinavir yielded only micromolar inhibition. Docking studies showed structural variations in their modes of binding which explains this unexpected observation. The implication of these observations in the development of other sulfoximine inhibitors is discussed.     

A New Approach to AIDS Research and Prevention: The Use of Gene-Mutated HIV-1/SIV Chimeric Viruses for Anti-HIV-1 Live-Attenuated Vaccines

The lack of a suitable animal model is a major obstacle to developing anti-HIV-1 vaccines. We successfully generated an SIVmac/HIV-1 chimeric virus (SHIV) (designated as NM-3rN) that contains the HIV-1 env gene and is infectious to macaque monkeys. Challenging the vaccinated macaque monkeys with NM-3rN, we developed an evaluation system for anti-HIV-1 Env-targeted vaccines. For the purpose of making the vaccine, a series of gene-mutated SHIVs were constructed. The monkeys vaccinated with these SHIVs had long-term anti-virus immunities without manifesting the disease, and became resistant to a challenge inoculation with NM-3rN. The sera from a monkey showed that, after the vaccination, the neutralizing antibodies not only against the parental HIV-1 but also against an antigenically different HIV-1 were raised. In vivo experiments confirmed that the vaccinated monkeys were protected from the challenge inoculum of an antigenically different SHIV-MN. Vaccination of monkeys with the attenuated SHIVs showed that further gene-deletion of the SHIV resulted in less immunogenicity. Nevertheless, the attenuated SHIVs had a vaccine effect against the challenge inoculation. In addition to specific immunities including neutralizing antibodies and cytotoxic T cells, a more complicated immune mechanism induced by live vaccine appears to play a role in this protection. Our data suggest that the live vaccine can induce strong and wide-range immunity against HIV-1. These SHIVs should contribute to understanding the pathogenicity of AIDS and to the development of future anti-HIV-1 live vaccines for humans.     

Design and evaluation of sifuvirtide, a novel HIV-1 fusion inhibitor

Enfuvirtide (T20) is the first and only HIV-1 fusion inhibitor approved for clinical use, but it can easily induce drug resistance limiting its practical application. A novel anti-HIV peptide, termed sifuvirtide, was designed based on the three-dimensional structure of the HIV-1 gp41 fusogenic core conformation. Here we report its in vitro anti-HIV potency, its mechanism of action, as well as the results from Phase Ia clinical studies. We demonstrated that sifuvirtide inhibited HIV-1-mediated cell-cell fusion in a dose-dependent manner and exhibited high potency against infections by a wide range of primary and laboratory-adapted HIV-1 isolates from multiple genotypes with R5 or X4 phenotypes. Notably, sifuvirtide was also highly effective against T20-resistant strains. Unlike T20, sifuvirtide could efficiently block six-helix bundle formation in a dominant negative fashion. These results suggest that sifuvirtide has a different mechanism of action from that of T20. Phase Ia clinical studies of sifuvirtide (FS0101) in 60 healthy individuals demonstrated good safety, tolerability, and pharmacokinetic profiles. A single dose regimen (5, 10, 20, 30, and 40 mg) by subcutaneous injection once daily at abdominal sites was well tolerated without serious adverse events. Pharmacokinetic studies of single and multiple administration of sifuvirtide showed that its decay half-lives were 20.0 +/- 8.6 h and 26.0 +/- 7.9 h, respectively. In summary, sifuvirtide has potential to become an ideal fusion inhibitor for treatment of HIV/AIDS patients, including those with HIV-1 strains resistant to T20.