Development of an HIV-1 Subtype Panel in China: Isolation and Characterization of 30 HIV-1 Primary Strains Circulating in China

BackgroundThe complex epidemic and significant diversity of HIV-1 strains in China pose serious challenges for surveillance and diagnostic assays, vaccine development and clinical management. There is a lack of HIV-1 isolates in current canonical HIV-1 subtype panels that can represent HIV-1 diversity in China; an HIV-1 subtype panel for China is urgently needed.MethodsBlood samples were collected from HIV-1 infected patients participating in the drug-resistance surveillance program in China. The samples were isolated, cultured and stored as neat culture supernatant. The HIV-1 isolates were fully characterized. The panel was used to compare 2 viral load assays and 2 p24 assays as the examples of how this panel could be used.ResultsAn HIV-1 subtype panel for China composed of 30 HIV-1 primary strains of four subtypes (B [including Thai-B], CRF01_AE, CRF07_BC and G) was established. The samples were isolated and cultured to a high-titer (10⁶-10⁹ copies/ml)/high-volume (40ml). The HIV-1 isolates were fully characterized by the final viral load, p24 concentration, gag-pol and envC2V3 sequencing, co-receptor prediction, determination of the four amino acids at the tip of the env V3-loop, glycosylation sites in the V3 loop and the drug-resistance mutations. The comparison of two p24 assays and two viral load assays on the isolates illustrated how this panel may be used for the evaluation of diagnostic assay performance. The Pearson value between p24 assays were 0.938. The viral load results showed excellent concordance and agreement for samples of Thai-B, but lower correlations for samples of CRF01_AE.ConclusionThe current panel of 30 HIV-1 isolates served as a basis for the development of a comprehensive panel of fully characterized viral isolates, which could reflect the current dynamic and complex HIV-1 epidemic in China. This panel will be available to support HIV-1 research, assay evaluation, vaccine and drug development.     

Comparison of heterologous neutralizing antibody responses of human immunodeficiency virus type 1 (HIV-1)- and HIV-2-infected Senegalese patients: distinct patterns of breadth and magnitude distinguish HIV-1 and HIV-2 infections

Neutralizing antibody responses against heterologous isolates in human immunodeficiency virus type 1 (HIV-1) and HIV-2 infections were compared, and their relationships with established clinical markers of progression were examined. Neutralizing responses against 7 heterologous primary isolates and 1 laboratory strain were compared between 32 untreated HIV-1-infected subjects and 35 untreated HIV-2-infected subjects using a pseudotyped reporter virus assay. The breadth of the neutralizing response, defined as the proportion of panel viruses positively neutralized by patient plasma, was significantly greater among HIV-2-infected subjects than among HIV-1-infected subjects. Notably, for fully one-third of HIV-2 subjects, all viruses were effectively neutralized in our panel. Magnitudes of responses, defined as reciprocal 50% inhibitory concentration (IC(50)) titers for positive reactions, were significantly greater among HIV-1-infected subjects than among HIV-2-infected subjects. When plasma samples from HIV-1 patients were tested for cross-neutralization of HIV-2 and vice versa, we found that these intertype responses are very rare and their prevalences comparable in both HIV-1 and HIV-2 infection. The significantly higher magnitude of heterologous responses for HIV-1 compared to HIV-2 prompted us to examine associations with viremia, which is known to be significantly higher in HIV-1 infection. Importantly, there was a significant positive correlation between the IC(50) titer and viral load within both the HIV-1 and HIV-2 groups, suggesting heterologous antibodies may be driven by viral replication. We conclude that HIV-2 infection is characterized by a broad, low-magnitude intratype neutralization response, while HIV-1 is characterized by a narrower but higher-magnitude intratype response and that a significant positive association between the IC(50) titer and viremia is common to both HIV-1 and HIV-2 infections.     

The distribution of viral blips observed in HIV-1 infected patients treated with combination antiretroviral therapy

Human immunodeficiency virus type 1 (HIV-1) infected patients treated with combination antiretroviral therapy frequently have the level of HIV-1 RNA detectable in plasma driven below the lower limit of detection of current assays, 50 copies ml⁻¹. Patients may continue to exhibit viral loads (VLs) below the assay limit for years, yet on some occasions the VL may be above the limit of detection. Whether these ‘blips’ in VL are simply assay errors or are indicative of intermittent episodes of increased viral replication is of great clinical concern. By analyzing the occurrence of viral blips in 123 treated HIV-infected patients, we show that patients do not share a common probability distribution of blip amplitude and thus reject the hypothesis that blips are solely due to assay variation. Work performed under the auspices of the U.S. Department of Energy.     

Role of human immunodeficiency virus (HIV)-specific T-cell immunity in control of dual HIV-1 and HIV-2 infection

Progressive immune dysfunction and AIDS develop in most cases of human immunodeficiency virus type 1 (HIV-1) infection but in only 25 to 30% of persons with HIV-2 infection. However, the natural history and immunologic responses of individuals with dual HIV-1 and HIV-2 infection are largely undefined. Based on our previous findings, we hypothesized that among patients with dual infection the control of HIV-1 is associated with the ability to respond to HIV-2 Gag epitopes and to maintain HIV-specific CD4⁺ T-cell responses. To test this, we compared the HIV-specific ex vivo IFN-γ enzyme-linked immunospot (ELISPOT) assay responses of 19 dually infected individuals to those of persons infected with HIV-1 or HIV-2 only. Further, we assessed the functional profile of HIV Gag-specific CD4⁺ and CD8⁺ T cells from nine HIV dually infected patients by using a multicolor intracellular cytokine staining assay. As determined by ELISPOT assay, the magnitude and frequency of IFN-γ-secreting T-cell responses to gene products of HIV-1 were higher than those to gene products of HIV-2 (2.64 versus 1.53 log₁₀ IFN-γ spot-forming cells/10⁶ cells [90% versus 63%, respectively].) Further, HIV-1 Env-, Gag-, and Nef- and HIV-2 Gag-specific responses were common; HIV-2 Nef-specific responses were rare. HIV-specific CD4⁺ T helper responses were detected in nine of nine dually infected subjects, with the majority of these T cells producing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and, to a lesser extent, interleukin-2. The HIV-1 plasma viral load was inversely correlated with HIV-2 Gag-specific IFN-γ-/TNF-α-secreting CD4⁺ and HIV-2 Gag-specific IFN-γ-secreting CD8⁺ T cells. In conclusion, the T-cell memory responses associated with containment of single HIV-1 and HIV-2 infection play a similar significant role in the immune control of dual HIV-1 and HIV-2 infection.     

应用斑点金免疫渗滤试验快速同步检测抗HIV-1，HIV-2 IgG抗体

应用斑点金免疫渗滤试验(dotimmunogoldfiltrationassay,DIGFA)建立了一种同步快速检测四种抗HIV-1/2IgG抗体的HIV诊断试纸。通过基因工程技术在大肠杆菌中表达了5种HIV抗原蛋白片段(P24,GP41,GP36,GP120V3,GP120C)。这5种抗原蛋白首先被固定在硝酸纤维素膜上,然后滴加待测血清,其中的病毒抗体通过免疫反应与抗原结合,再加胶体金标记的葡萄球菌蛋白A(SPA),待其渗过膜片后,洗涤,即可形成肉眼可见的红色斑点。用已确证的21份HIV阳性血清(其中包括1份HIV-1标准阳性血清和1份HIV-2标准阳性血清)和30份阴性血清进行了试验,结果表明该快速检测方法与ELISA方法无显著差异。该检测方法不需任何仪器,仅凭肉眼即可判定结果,整个检测过程不超过5分钟。与传统的的ELISA法相比,具有方便快速,成本低廉,应用范围广等优点。同时,此HIV快速诊断试纸可以同步检测并区分针对HIV-1和HIV-2感染的不同检测标志物(抗P24、GP41、GP120和GP36抗体),这对提高快速检测的灵敏度和准确性,以及对判断HIV感染者是否临近或已进入AIDS期有着较高的应用价值。     

HIV-2 antibody testing of blood donors with doubtful immunoblot results for HIV-1

Antibodies against human immunodeficiency virus type-1 (HIV-1) in samples from blood donors are commonly detected by various enzyme-linked immunosorbent assays (ELISA) and by confirmatory tests, e.g., "Western blot" or immunofluorescence tests. Immunoblot reactivity, which is directed only towards the HIV-1 core proteins p 18, p 24 and p 55, may represent false-positive reactions. Out of 125,000 blood donations, 140 were repeatably HIV-1 antibody reactive by ELISA; of these, 20 were doubtful positive sera with isolated p 18 and/or p 24 bands in the HIV-1 confirmatory assay. Antibodies to HIV-2 are known to cross-react with these HIV-1 core proteins. We therefore assayed the 20 sera by immunofluorescence and immunoblotting for the presence of antibodies to HIV-2. None of these doubtful HIV-1 antibody positive blood donor sera was found to have antibodies to HIV-2.     

Four novel bis-(naphtho-gamma-pyrones) isolated from Fusarium species as inhibitors of HIV-1 integrase

Integration of viral DNA into host cell DNA is an essential step in retroviral (HIV-1) replication and is catalyzed by HIV-1 integrase. HIV-1 integrase is a novel therapeutic target and is the focus of efforts to identify effective inhibitors that will prevent/or cure HIV infections. Four novel naphtho-gamma-pyrones, belonging to the chaetochromin and ustilaginoidin family, were discovered as inhibitors of HIV-1 integrase from the screening of fungal extracts using a recombinant in vitro assay. These compounds inhibit both the coupled and strand transfer activity of HIV-1 integrase with IC(50) values of 1-3 and 4-12 microM, respectively. The discovery, structure elucidation, chemical modification and the structure-activity relationship of these compounds are described.     

Molecules from apoptotic pathways modulate HIV-1 replication in Jurkat cells

The replication of viruses involves control of some aspects of host cell homeostasis by modification of target cell metabolism and regulation of the apoptotic machinery. It is not well known whether molecules involved in apoptotic pathways affect human immunodeficiency virus type 1 (HIV-1) replication and regulate viral yields. Using the susceptible Jurkat cell line, we studied the relationship of apoptosis-associated molecules with HIV-1 virus production using a sensitive real-time RT-PCR assay. Here, we found that expression of proapoptotic proteins, including Fas ligand (FasL), FADD, or p53 significantly increased HIV-1 virus production. In contrast, the expression of antiapoptotic molecules, such as FLIP, Bcl-X_L, and XIAP, decreased HIV-1 virus production. Knockdown of Bax with siRNA and FADD with expression of its antisense mRNA also inhibited viral replication and the caspase-3 inhibitor, Z-DEVD, and decreased virus production. These data indicate that HIV-1 infection regulates the apoptosis process to facilitate viral replication and inhibition of apoptosis may inhibit HIV-1 replication and cytopathogenesis. We also discuss the effects of MAPK signaling pathways and apoptosis on HIV-1 replication.     

Potent intratype neutralizing activity distinguishes human immunodeficiency virus type 2 (HIV-2) from HIV-1

HIV-2 has a lower pathogenicity and transmission rate than HIV-1. Neutralizing antibodies could be contributing to these observations. Here we explored side by side the potency and breadth of intratype and intertype neutralizing activity (NAc) in plasma of 20 HIV-1-, 20 HIV-2-, and 11 dually HIV-1/2 (HIV-D)-seropositive individuals from Guinea-Bissau, West Africa. Panels of primary isolates, five HIV-1 and five HIV-2 isolates, were tested in a plaque reduction assay using U87.CD4-CCR5 cells as targets. Intratype NAc in HIV-2 plasma was found to be considerably more potent and also broader than intratype NAc in HIV-1 plasma. This indicates that HIV-2-infected individuals display potent type-specific neutralizing antibodies, whereas such strong type-specific antibodies are absent in HIV-1 infection. Furthermore, the potency of intratype NAc was positively associated with the viral load of HIV-1 but not HIV-2, suggesting that NAc in HIV-1 infection is more antigen stimulation dependent than in HIV-2 infection, where plasma viral loads typically are at least 10-fold lower than in HIV-1 infection. Intertype NAc of both HIV-1 and HIV-2 infections was, instead, of low potency. HIV-D subjects had NAc to HIV-2 with similar high potency as singly HIV-2-infected individuals, whereas neutralization of HIV-1 remained poor, indicating that the difference in NAc between HIV-1 and HIV-2 infections depends on the virus itself. We suggest that immunogenicity and/or antigenicity, meaning the neutralization phenotype, of HIV-2 is distinct from that of HIV-1 and that HIV-2 may display structures that favor triggering of potent neutralizing antibody responses.     

Higher homologous and lower cross-reactive Gag-specific T-cell responses in human immunodeficiency virus type 2 (HIV-2) than in HIV-1 infection

Human immunodeficiency virus type 2 (HIV-2) infection results in slower CD4⁺ T-cell decline, lower plasma viral load levels, and hence slower progression of the disease than does HIV-1 infection. Although the reasons for this are not clear, it is possible that HIV-2 replication is more effectively controlled by host responses. We used aligned pools of overlapping HIV-1 and HIV-2 Gag peptides in an enhanced gamma interferon enzyme-linked immunospot assay to compare the levels of homologous and cross-reactive Gag-specific T-cell responses between HIV-1- and HIV-2-infected patients. HIV-2-infected patients showed broader and stronger homologous Gag-specific T-cell responses than HIV-1-infected patients. In contrast, the cross-reactive T-cell responses in HIV-2-infected patients were both narrower and weaker than those in HIV-1-infected patients, in line with overall weaker correlations between homologous and heterologous T-cell responses among HIV-2-infected patients than among HIV-1-infected patients. Cross-reactive responses in HIV-2-infected patients tended to correlate directly with HIV-1/HIV-2 Gag sequence similarities; this was not found in HIV-1-infected patients. The CD4⁺ T-cell counts of HIV-2-infected patients correlated directly with homologous responses and inversely with cross-reactive responses; this was not found in HIV-1-infected patients. Our data support a model whereby high-level HIV-2-specific T-cell responses control the replication of HIV-2, thus limiting viral diversification and priming of HIV-1 cross-reactive T-cell responses over time. However, we cannot exclude the possibility that HIV-2 replication is controlled by other host factors and that HIV-2-specific T-cell responses are better maintained in the context of slow viral divergence and a less damaged immune system. Understanding the nature of immune control of HIV-2 infection could be crucial for HIV vaccine design.     

A radiometric assay for HIV-1 protease

A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.     

HIV-1 dynamics in vivo: implications for therapy

The advent of potent combination antiretroviral therapy has been an important breakthrough in the treatment of HIV-1 infection, resulting in marked reductions in HIV-1-related morbidity and mortality. Antiretroviral therapy has also provided researchers with a powerful tool to perturb the equilibrium of viral production and viral clearance, allowing them to dissect the underlying dynamics that control the pathogenesis of AIDS. Here, we review our current understanding of the sources of HIV-1 production, the estimates for the virion and the host-cell half-lives, and the pathways of virion trafficking and clearance. We also discuss the obstacles that result from the ability of HIV-1 to remain dormant for a prolonged period of time in a subset of long-lived cells, despite an apparently effective antiretroviral treatment.