Inactivation of human immunodeficiency virus type 1 in blood samples stored as high-salt lysates

Blood samples to be tested for the presence of parasite DNA by using specific DNA probes are routinely stored in our laboratory as high-salt lysates (HSL). To safeguard against the risk of accidental infection with etiological agents such as the human immunodeficiency virus type 1 (HIV-1) while manipulating large numbers of blood samples in preparation for DNA probing, we determined the residual infectivity of HIV-1 after exposure to HSL components. Both high-titer virus stocks or provirus-carrying cells, suspended either in tissue culture medium or freshly drawn blood, were completely inactivated upon contact with the HSL components. This was verified by the absence of any detectable HIV-1-specific antigen in the supernatants of long-term cultures and the absence of virus-specific DNA fragments after amplification by polymerase chain reaction with DNA from such cultures as target DNA. These results support the conclusion that the virus is in fact completely inactivated by contact with the HSL components, rendering blood specimens stored as HSL noninfectious in regard to HIV-1.     

Selection for specific sequences in the external envelope protein of human immunodeficiency virus type 1 upon primary infection.

Viral RNA was extracted from plasma samples collected from five individuals during the period of viremia before seroconversion in primary infection with human immunodeficiency virus type 1 (HIV-1) and amplified by polymerase chain reaction. Nucleotide sequence analysis of amplified DNA from the V3 and V4 hypervariable regions indicated that the initial virus population of each acutely infected individual was completely homogeneous in sequence. No intrasample variability was found among the 44,090 nucleotides sequenced in this region of env, contrasting with the high degree of variability normally found in seropositive individuals. Paradoxically, substantial sequence variability was found in the normally high conserved gag gene (encoding p17) in most of the preseroconversion samples. The diversity of p17 sequences in samples that were homogeneous in V3 and V4 can most readily be explained by the existence of strong selection for specific env sequences either upon transmission or in the interval between exposure and seroconversion in the exposed individual. Evidence that localizes the selected region upon transmission to V3 is provided by the similarity or identity of V3 loop sequences in five individuals with epidemiologically unrelated HIV-1 infections, while regions flanking the V3 loop and the V4 hypervariable region were highly divergent. The actual V3 sequences were similar to those associated with macrophage tropism in primary isolates of HIV, irrespective of whether infection was acquired by sexual contact or parenterally through transfusion of contaminated factor VIII. Proviral DNA sequences in peripheral blood mononuclear cells remained homogeneous in the V3 and V4 regions (and variable in p17gag) for several months after seroconversion. The persistence of HIV sequences in peripheral blood mononuclear cells identical to those found at primary infection in the absence of continued virus expression provides an explanation for the previously observed differences in the composition of circulating DNA and RNA populations in sequential samples from seropositive individuals.     

Human immunodeficiency virus does not induce interleukin-1, interleukin-6, or tumor necrosis factor in mononuclear cells.

The production of interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) by fresh peripheral blood mononuclear cells was evaluated after exposure to human immunodeficiency virus (HIV) or purified recombinant HIV-1 envelope glycoprotein (rgp120). To exclude the role of contaminating endotoxin in this study, all media were subjected to ultrafiltration and reagents contained less than 25 pg of endotoxin per ml by Limulus assay. Under endotoxin-free conditions, no increases in IL-1 beta, IL-6, or TNF-alpha mRNA or protein were detectable in cell cultures exposed to HIV-1, HIV-2, or rgp120 (0.1 to 10 micrograms/ml), as compared with cytokine levels in mock-exposed cultures. However, concentrations of endotoxin (lipopolysaccharide) as low as 0.5 ng/ml induced significant production of mRNA and protein for these three cytokines. Preincubation of mononuclear cells with "shake" HIV-1 preparations and also mock-infected shake preparations prior to lipopolysaccharide stimulation resulted in a two- to threefold increase in IL-1 beta and TNF-alpha production. This priming effect was not observed with rgp120 (0.1 to 10 micrograms/ml) or standard HIV-1 or mock-infected supernatants, suggesting the presence of biologically active material independent of virus in the shake preparations. Our studies indicate that, in the absence of endotoxin, HIV-1, HIV-2, and HIV gp120 do not induce production of IL-1 beta, IL-6, or TNF-alpha by peripheral blood mononuclear cells.     

Human immunodeficiency virus type 1 tropism for brain microglial cells is determined by a region of the env glycoprotein that also controls macrophage tropism.

Human immunodeficiency virus type 1 (HIV-1), the agent of AIDS, frequently infects the central nervous system. We inoculated adult human brain cultures with chimeric viruses containing parts of the env gene of a cloned primary isolate from brain tissue, HIV-1 JRFl, inserted into the cloned DNA of a T-cell-tropic strain. A chimeric virus containing the carboxy-terminal portion of HIV-1 JRFl env did not replicate in these brain tissue cultures, while a chimera expressing an env-encoded protein containing 158 amino acids of HIV-1 JRFl gp120, including the V3 loop, replicated well in brain microglial cells, as it does in blood macrophages. Infection of brain microglial cells with such a chimera was blocked by an antibody to the V3 loop of gp 120. Thus, env determinants in the region of gp120, outside the CD4-binding site and comprising the V3 loop, are critical for efficient viral binding to and/or entry into human brain microglia.     

Relationships between the presence of anti-Tat antibody, DNA and RNA viral load

The possible relationships between the intensity of humoral response to full length Tat protein, the amount of proviral DNA reservoir in peripheral blood mononuclear cells and RNA viral load were analyzed in plasma samples obtained from a group of HIV-1 seropositive subjects, who never received any antiretroviral therapy. All HIV-1 patients showed detectable levels of serum IgG to full-length Tat by immunoenzymatic assay. We found a higher percentage of HIV-1 seropositive subjects with low levels of antibody in the presence of barely detectable proviral DNA copies (< or =10 copies/1.5x10(5) PBMCs) and a high anti-Tat antibody response accompanied by variable (from >10(1) to > or =10(3) copies/1.5x10(5) PBMCs) levels of DNA load (p=0.011). Moreover, an inverse relationship between anti-Tat antibody titers and HIV-1 RNA viral load was demonstrated HIV-1 seropositive patients. In HIV-1-infected patients, a strong humoral immune response against HIV-1 transactivating Tat protein, able to down-modulate viral replication in peripheral blood, does not seem to inhibit the number of proviral DNA molecules in peripheral blood mononuclear cells. Even though our data strongly confirm the "positive" role of anti-Tat antibody on viral replication, the persistence of significant amount of DNA viral load in peripheral blood mononuclear cells, despite high level of anti Tat antibody, suggests a more cautious approach to HIV-1 Tat-containing vaccines, able to stimulate an immune specific response to transactivating Tat protein sufficient in inhibiting circulating virus, but not completely efficient in decreasing proviral DNA integration.     

Components of apoptotic pathways modulate HIV-1 latency in Jurkat cells

The ability of the human immunodeficiency virus type 1 (HIV-1) to establish latent infections serves as a major barrier for its cure. This process could occur when its host cells undergo apoptosis, but it is uncertain whether the components of the apoptotic pathways affect viral latency. Using the susceptible Jurkat cell line, we investigated the relationship of apoptosis-associated components with HIV-1 DNA levels using the sensitive real-time PCR assay. Here, we found that the expression of proapoptotic proteins, including Fas ligand (FasL), FADD, and p53, significantly decreased HIV-1 viral DNA in cells. In contrast, the expression of antiapoptotic molecules, such as FLIP, Bcl2, and XIAP, increased the levels of viral DNA. Furthermore, promoting cellular antiapoptotic state via the knockdown of Bax with siRNA and FADD with antisense mRNA or the treatment with the Caspase-3 inhibitor, Z-DEVD, also raised viral DNA. We also simultaneously measured viral RNA from supernatants of these cell cultures and found that HIV-1 latency is inversely proportional to viral replication. Furthermore, we demonstrated that HIV-1-infected cells that underwent the transient expression of FLIP- or XIAP-induced viral latency would then produce an increased level of viral RNA upon the reversal of these antiapoptotic effects via PMA treatment compared to LacZ control cells. Taken together, these data suggest that HIV-1 infection could be adapted to employ or even manipulate the cellular apoptotic pathway to its advantage: when the host cell remains in a pro-apoptotic state, HIV-1 favors active replication, while when the host cell prefers an anti-apoptotic state, the virus establishes viral latency and promotes latent reservoir seeding in a way which would enhance viral replication and cytopathogenesis when the cellular conditions shift to encourage the productive infection phase.     

Induction of humoral immune responses following vaccination with envelope-containing, formaldehyde-treated, thermally inactivated human immunodeficiency virus type 1

The lack of success of subunit human immunodeficiency virus type 1 (HIV-1) vaccines to date suggests that multiple components or a complex virion structure may be required. We previously demonstrated retention of the major conformational epitopes of HIV-1 envelope following thermal treatment of virions. Moreover, antibody binding to some of these epitopes was significantly enhanced following thermal treatment. These included the neutralizing epitopes identified by monoclonal antibodies 1b12, 2G12, and 17b, some of which have been postulated to be partially occluded or cryptic in native virions. Based upon this finding, we hypothesized that a killed HIV vaccine could be derived to elicit protective humoral immune responses. Shedding of HIV-1 envelope has been described for some strains of HIV-1 and has been cited as one of the major impediments to developing an inactivated HIV-1 vaccine. In the present study, we demonstrate that treatment of virions with low-dose formaldehyde prior to thermal inactivation retains the association of viral envelope with virions. Moreover, mice and nonhuman primates vaccinated with formaldehyde-treated, thermally inactivated virions produce antibodies capable of neutralizing heterologous strains of HIV in peripheral blood mononuclear cell-, MAGI cell-, and U87-based infectivity assays. These data indicate that it is possible to create an immunogen by using formaldehyde-treated, thermally inactivated HIV-1 virions to induce neutralizing antibodies. These findings have broad implications for vaccine development.     

Stability of HCV, HIV-1 and HBV nucleic acids in plasma samples under long-term storage.

The implementation of nucleic acid amplification technology (NAT) for detection of HCV, HIV-1 and HBV has undoubtedly contributed to the viral safety of blood, reducing the window period. One important matter related to the stability of RNA/DNA is the effect of the storage conditions on samples. In a previous work, we studied the stability of HCV RNA in plasma samples after storage at different temperatures. This work is an update on the follow-up of a sample containing 100 IU/ml HCV RNA for 5 years at -20 degrees C, showing no decrease in the initial titre. The nucleic acid stability of other viruses, such as HIV-1 and HBV, has also been studied. At -20 degrees C, samples containing HIV-1 were followed up for approximately 3 years and the results obtained show no decay in HIV-1 RNA detectability. Regardless of the HIV-1 RNA concentration, samples stored at 5 degrees C maintain their titre for at least 14 days. At 25 degrees C, the HIV-1 RNA half-life was determined at nearly 7 days. The HBV DNA, at 5 degrees C and 25 degrees C, is stable for at least 28 days, regardless of the initial titre.     

Resistance against syncytium-inducing human immunodeficiency virus type 1 (HIV-1) in selected CD4(+) T cells from an HIV-1-infected nonprogressor: evidence of a novel pathway of resistance mediated by a soluble factor(s) that acts after virus entry.

A panel of CD4(+) T-cell clones were generated from peripheral blood lymphocytes from a patient with a nonprogressing infection of human immunodeficiency virus type 1 (HIV-1) by using herpesvirus saimiri as described recently. By and large, all of the clones expressed an activated T-cell phenotype (Th class 1) and grew without any further stimulation in interleukin-2-containing medium. None of these clones produced HIV-1, and all clones were negative for HIV-1 DNA. When these clones were infected with primary and laboratory (IIIB) strains of HIV-1 with syncytium-inducing (SI) phenotypes, dramatic variation of virus production was observed. While two clones were highly susceptible, other clones were relatively or completely resistant to infection with SI viruses. The HIV-resistant clones expressed CXCR4 coreceptors and were able to fuse efficiently with SI virus env-expressing cells, indicating that no block to virus entry was present in the resistant clones. Additionally, HIV-1 DNA was detectable after infection of the resistant clones, further suggesting that HIV resistance occurred in these clones after virus entry and probably after integration. We further demonstrate that the resistant clones secrete a factor(s) that can inhibit SI virus production from other infected cells and from a chronically infected producer cell line. Finally, we show that the resistant clones do not express an increased amount of ligands (stromal-derived factor SDF-1) of CXCR4 or other known HIV-inhibitory cytokines. Until now, the ligands of HIV coreceptors were the only natural substances that had been shown to play antiviral roles of any real significance in vivo. Our data from this study show that differential expression of another anti-HIV factor(s) by selected CD4(+) T cells may be responsible for the protection of these cells against SI viruses. Our results also suggest a novel mechanism of inhibition of SI viruses that acts at a stage after virus entry.     

Contact with Thymic Epithelial Cells as a Prerequisite for Cytokine-Enhanced Human Immunodeficiency Virus Type 1 Replication in Thymocytes

We report here that human immunodeficiency virus type 1 (HIV-1)-infected human thymocytes, in the absence of any exogenous stimulus but cocultivated with autologous thymic epithelial cells (TEC), obtained shortly (3 days) after thymus excision produce a high and sustained level of HIV-1 particles. The levels and kinetics of HIV-1 replication were similar for seven distinct viral strains irrespective of their phenotypes and genotypes. Contact of thymocytes with TEC is a critical requirement for optimal viral replication. Rather than an inductive signal resulting from the contact itself, soluble factors produced in the mixed culture are responsible for this effect. Specifically, the synergistic effects of tumor necrosis factor, interleukin-1 (IL-1), IL-6, and granulocyte-macrophage colony-stimulating factor may account by themselves for the high level of HIV-1 replication in thymocytes observed in mixed cultures. In conclusion, the microenvironment generated by TEC-thymocyte interaction might greatly favor optimal HIV-1 replication in the thymus.     

Fate of the human immunodeficiency virus type 1 provirus in infected cells: a role for vpr.

We investigated the fate of human immunodeficiency virus type 1 (HIV-1) viral DNA in infected peripheral blood lymphocytes and immortalized T-cell lines by using a replication-defective HIV-1. We observed that integrated HIV-1 DNA and viral gene expression decrease over time. A frameshift mutation in vpr resulted in maintenance of the HIV-1 provirus and stable persistence of viral expression. Transfection of vpr together with the neomycin resistance gene in the absence of other viral genes decreased the formation of geneticin-resistant colonies, indicating either a cytotoxic or a cytostatic effect upon cells. Therefore, maintenance of HIV-1 infection within an infected proliferating population is due to two competing processes, the rate of viral spread and the degree of cell growth inhibition and/or death induced by Vpr.     

AT-2灭活HIV-1病毒及纯化回收鉴定

目的制备具备完整空间构型且纯度在90%以上的灭活HIV-1病毒,用于HIV疫苗研究。方法应用化学制剂2,2’-dithiodipyridine（aldrithiol-2;AT-2）灭活HIV-1病毒,对灭活的病毒超速离心浓缩并洗涤去除灭活用的化学制剂AT-2。采用分子筛技术去除灭活病毒中残存的杂质蛋白质。结果 250μmol/L AT-2与HIV-137℃作用1 h可以彻底灭活病毒的感染性,同时保留病毒的免疫原性。灭活的病毒纯化后检测不到AT-2的残留,检测牛血清蛋白残余量低于50 ng/mL。结论灭活的HIV-1病毒经过纯化后纯度达到95.6%,可以满足作为HIV疫苗研究的免疫刺激剂的使用要求。     

Cytopathic killing of peripheral blood CD4(+) T lymphocytes by human immunodeficiency virus type 1 appears necrotic rather than apoptotic and does not require env

An important unresolved issue of AIDS pathogenesis is the mechanism of human immunodeficiency virus (HIV)-induced CD4(+) T-lymphocyte destruction. We show here that HIV type 1 (HIV-1) exerts a profound cytopathic effect upon peripheral blood CD4(+) T lymphocytes that resembles necrosis rather than apoptosis. Necrotic cytopathology was found with both laboratory-adapted strains and primary isolates of HIV-1. We carefully investigated the role of env, which has been previously implicated in HIV cytopathicity. HIV-1 stocks with equivalent infectivity were prepared from constructs with either an intact or mutated env coding region and pseudotyped with the glycoprotein of vesicular stomatitis virus (VSV-G) so that the HIV envelope was not rate-limiting for infection. Infected Jurkat T cells died whether or not env was intact; however, the expression of env accelerated death significantly. The accelerated death was blocked by protease inhibitors, indicating that it was due to reinfection by newly produced virus in env(+) cultures. Accordingly, we found no disparity in kinetics in CD4(lo) Jurkat cells. In highly infected peripheral blood T cells, profound necrosis occurred equivalently with both env(+) and env(-) stocks of HIV-1. We also found that HIV-1 cytopathicity was undiminished by the absence of nef. However, viral stocks made by complementation or packaging of HIV-1 genomes with the natural protein-coding sequences replaced by the green fluorescent protein were highly infectious but not cytopathic. Thus, env can accelerate cell death chiefly as an entry function, but one or more viral functions other than env or nef is essential for necrosis of CD4(+) T cells induced by HIV-1.     

Integration is not necessary for expression of human immunodeficiency virus type 1 protein products.

A common feature in the life cycle of cytocidal retroviruses, including human immunodeficiency virus type 1 (HIV-1), is the accumulation of large amounts of unintegrated viral DNA. As yet, the role of unintegrated viral DNA in the cytopathogenesis of cytocidal retrovirus infections remains unresolved. HIV-1 mutants which were deleted in the integrase/endonuclease gene and which were unable to establish an integrated form of the virus were constructed. Despite an inability to integrate, these mutants were fully competent templates for HIV-1 core and envelope antigen production. HIV-1 antigen could be detected in the supernatants of lymphocyte cultures infected with HIV-1 integrase mutants. However, an inability to rescue infectious virus from these cultures indicated that HIV-1 integration was required for the production of infectious HIV-1. On the basis of the ability of unintegrated HIV-1 DNA to serve as a template for HIV-1 antigen production, it is plausible that unintegrated viral DNA can contribute to the HIV-1 antigen pool during HIV-1 replication.     

Accumulation of human immunodeficiency virus type 1 DNA in T cells: results of multiple infection events.

Human immunodeficiency virus type 1 DNA synthesis was followed in a CD4+ line of T cells (C8166) grown in the presence or absence of a monoclonal antibody to CD4 that blocks infection By 48 h after infection, cultures grown in the presence of the antibody contained approximately 4 copies of human immunodeficiency virus type 1 DNA per cell, whereas those grown in the absence of the antibody contained approximately 80 copies of viral DNA per cell. Most of the viral DNA in cultures grown in the absence of the antibody was present in a broad smear of apparently incomplete viral sequences. In cultures grown in the presence or absence of the antibody, the 9.6-kilobase linear duplex of viral DNA appeared to undergo integration within 24 h of its appearance. These results demonstrate that T cells accumulate unintegrated human immunodeficiency virus type 1 DNA as a result of multiple virions entering cells.     

利用鸭乙型肝炎病毒感染模型评价血液成分中病毒的灭活效果

目的 利用鸭乙型肝炎病毒(DHBV)感染动物模型,评价亚甲蓝光化学病毒灭活方法对血液成分中DNA病毒的灭活效果。方法 将超离纯化的DHBV分别加入人血浆或人红细胞,经亚甲蓝光化学灭活病毒,将含不同基因组拷贝数DHBV的血浆成分经静脉感染1 d龄雏鸭。采用放射性核素核酸杂交法对血清中DHBV DNA进行检测,计算病毒灭活处理前、后人血浆及人红细胞中DHBV的半数感染计量(ID50)。结果 结果显示加入DHBV的血浆在未经灭活处理前对1 d龄雏鸭的ID50值为103.33,而经病毒灭活处理后ID50值为1010拷贝,灭活处理可使病毒感染性滴度下降达6个Log;加入DHBV的红细胞灭活前ID50值为103.35,经灭活处理后ID50值为108.35拷贝,灭活处理使病毒感染性滴度下降5个Log。结论 利用DHBV感染动物模型,可以检测到少量病毒在自然感染宿主体内的感染性,可用于评判血液成分中病毒灭活方法的效果,亚甲蓝光化学处理对血浆中DNA病毒的灭活效果较好于对红细胞中DNA病毒的灭活作用。     

Inactivation of viruses with photoactive compounds.

The transmission of human immunodeficiency virus (HIV-1) and other enveloped virus by blood transfusion is a major concern. Photosensitive dyes such as hematoporphyrin derivative (HPD), dihematoporphyrin ether (DHE), benzoporphyrin derivatives (BPD), extended ring porphyrins, sapphyrins and texaphyrins, and various cyanines were used with viral cultures to test the feasibility of using those light-excitable dyes to kill virus. A photodynamic flow cell was used to irradiate viral suspensions or viral infected cells in culture media or in whole blood. Herpes virus (HSV-1) was used to screen compounds. Effective compounds were subsequently tested for their ability to kill HIV-1, CMV, and SIV in culture medium and in blood and proved to effectively kill free virus and infected cells at significant viremias. Irradiation was achieved with a filtered xenon light source and/or tunable dye laser. Concentrations of dyes at 10 times viral kill dose were irradiated in blood which was tested for damage to erythrocytes (RBC), platelets, and blood proteins. No damage to RBC, complement factors, and immunoglobulins was evident immediately after photodynamic treatment. Platelet condition is minimally modified with time. Photodynamic treatment of blood appears to be a feasible means of eradicating virus and some protozoans from blood.