Expression of human immunodeficiency virus type 1gag gene using genetically engineered herpes simplex virus type 1 recombinants

Infectious herpes simplex virus type 1 (HSV-1) recombinants were constructed by inserting the cDNA sequence of the human immunodeficiency virus type 1 (HIV-1)gag gene (from nucleotide position 675 [SacI] to 3859 [Asp 718] of the cDNA sequences of HIV-1 strain BH-10) within the DNA sequences of theBamHI DNA fragment B of the genome of an apathogenic HSV-1 strain HFEM. This HSV-1 strain possesses a 4.1-kbp deletion within theBamHI DNA fragment B between 0.762 and 0.789 map units of the viral genome, which allows the insertion of at least 4 kbp of foreign genetic material into this particular region. The DNA sequences of the immediate early promoter (IE4) of HSV-1 that were inserted upstream from thegag gene were used as a promoter. The screening of 205 virus stocks derived from individual plaques revealed that 46 recombinant viruses harbor HIV-1gag-specific DNA sequences. However, it was found that only six of the recombinant viruses are able to express thegag gene product of HIV-1. This indicates that the ratio of the positive recombination events is about 2.9%.     

Human immunodeficiency virus type 1 (HIV-1) and human hematopoietic progenitor cells

Summary Besides a progressive depletion of CD4+ T-lymphocytes, other peripheral blood cytopenias, (granulocytopenia, anemia and thrombocytopenia) are frequently observed in HIV-1 seropositive individuals, especially in patients with overt AIDS. Various experimental evidences suggest that HIV-1 could play a direct role in the pathogenesis of HIV-1 related peripheral blood cytopenias, affecting the survival/proliferation capacity of hematopoietic progenitors. CD34+ human hematopoietic progenitors, however, are substantially not susceptible to HIV-1 infection either in vitro and in vivo and their defects seem rather related to an alteration of bone marrow and peripheral blood microenvironments due to the presence of soluble HIV-1 specific products.     

Quantitative detection of human immunodeficiency virus type 1 (HIV-1) viral load by SYBR green real-time RT-PCR technique in HIV-1 seropositive patients

HIV-1 viral load represents a basic marker for evaluation of the rate and severity of HIV-1 related disease and to monitor the effectiveness of treatment. An SYBR green-based real-time RT-PCR (SYBR green real-time RT-PCR) revealed by Light Cycler technology was evaluated for quantitation of HIV-1 RNA viral load in plasma of HIV-1 seropositive patients. The performance of the SYBR green real-time PCR was assessed on 56 HIV-1 seropositive patients under highly active retroviral therapy (HAART) and 25 blood donors. The results demonstrated that this technique detected 50 HIV-1 RNA copies per millilitre of plasma. Moreover, we compared real-time RT-PCR with the b-DNA technique considered widely a reference technique for HIV-1 RNA viral load measurement. The parallel quantitative analysis of HIV-1 positive samples showed a high correlation (r=0.908) between the two methods. Although b-DNA and the real-time-based method gave similar sensitivity, the assay determined quantitatively HIV-1 RNA copies in 4 out of 16 samples shown as undetectable by b-DNA. The SYBR green real-time RT-PCR represents a good alternative to b-DNA assay in HIV-1 viral load determination especially during the monitoring of HAART treatment.     

An ultrastructural observation of negatively-stained human immunodeficiency virus type 1 (HIV-1) cores

Cores of human immunodeficiency virus type 1 (HIV-1) were observed using negative staining for electron microscopy. On the surfaces of cores isolated from viral particles with a Nonidet P40 and glutaraldehyde mixture, mortar-like units were detected in the wide and narrow margins. In some of the cores which were burst with distilled water, the units were linked together revealing a beaded structure.     

Pretreatment human immunodeficiency virus type 1 (HIV-1) drug resistance in transmission clusters of the Cologne-Bonn region,Germany

Objectives

In Germany, previous reports have demonstrated transmitted human immunodeficiency virus type 1 (HIV-1) drug-resistance mutations (DRM) in 11% of newly diagnosed individuals, highlighting the importance of drug-resistance screening before the initiation of antiretroviral therapy (ART). Here, we sought to understand the molecular epidemiology of HIV DRM transmission in the Cologne-Bonn region of Germany, given one of the highest rates of new HIV diagnoses in western Europe (13.7 per 100 000 habitants).

Human immunodeficiency virus type-1 (HIV-1) Pr55gag virus-like particles are potent activators of human monocytes

Human immunodeficiency virus type-1 (HIV-1) Pr55^Gag virus-like particles (VLP) represent an interesting HIV vaccine component since they stimulate strong humoral and cellular immune responses. We demonstrated that VLP expressed by recombinant baculoviruses activate human PBMC to release pro-inflammatory (lL-6, TNF-α), anti-inflammatory (IL-10) and Th1-polarizing (IFN-γ) cytokines as well as GM-CSF and MIP-1α in a dose-and time-dependent manner. Herein, residual baculoviruses within the VLP preparations showed no or minor effects. Monocytes could be identified as a main target for VLP to induce cytokine production. Furthermore, VLP-induced monocyte activation was shown by upregulation of molecules involved in antigen presentation (MHC II, CD80, CD86) and cell adhesion (CD54). Exposure of VLP to serum inactivates its capacity to stimulate cytokine production. In summary, these investigations establish VLP as strong activators of PBMC and monocytes therein, potently enhancing their functionality and potency to promote an efficient immune response. This capacity makes VLP an interesting component of combination vaccines.     

Production of human immunodeficiency virus type 1 (HIV-1) pseudoviruses using linear HIV-1 envelope expression cassettes

HIV-1 pseudoviruses constitute an important tool in HIV-1 vaccine and entry inhibitor research. Single-cycle pseudoviruses carrying functional envelopes are generated by co-transfecting HEK293T cells with pNL4-3.LucR(-)E(-) and Env expression plasmids. However, cloning of Env genes is time consuming and single Env clones are not representative of the diversity of HIV-1 in a patient's blood sample. A new method to construct Env expression cassettes is proposed which can be used for the rapid generation of heterogeneous HIV-1 pseudoviruses without a cloning step. The linear Env expression cassettes are constructed by ligating PCR amplified Env genes between a 5' CMV promoter and 3' SV40 polyadenylation element. The resulting cassettes generate pseudoviruses carrying heterogeneous Env variants of a primary HIV-1 isolate derived from viral RNA or proviral DNA. The influence of cis-acting sequences upstream of the Env gene on infectivity was compared between pseudoviruses generated from plasmids and linear expression cassettes. The results suggest that the presence of these upstream sequences tends to result in higher infectivity of pseudoviruses when present in heterogeneous Env expression cassettes, but they do not enhance infectivity of pseudoviruses generated with homogeneous Env expression constructs. Using linear expression cassettes allows for the rapid production of heterogeneous patient-derived functional Env genes.     

Chemokine receptors as fusion cofactors for human immunodeficiency virus type 1 (HIV-1)

CD4 is the primary cellular receptor for human immunodeficiency virus type 1 (HIV-1), but is not sufficient for entry of HIV-1 into cells. After a decade-long search, the cellular coreceptors that HIV-1 requires in conjunction with CD4 have been identified as members of the chemokine receptor family of seven-transmembrane G-protein coupled receptors. The discovery of distinct chemokine receptors that support entry of T-cell tropic (CXCR-4) and macrophage tropic HIV-1 strains (CCR-5) explains the differences in cell tropism between viral strains, the inability of HIV-1 to infect most nonprimate cells, and the resistance of a small percentage of the population to HIV-1 infection. Further understanding of the role of chemokine receptors in viral entry may also help explain the evolution of more pathogenic forms of the virus, viral transmission, and HIV-induced pathogenesis. These recent discoveries will aid the development of strategies for combating HIV-1 transmission and spread, the understanding of HIV-1 fusion mechanisms, and the possible development of small animal models for HIV-1 drug and vaccine testing.     

Restriction fragment length polymorphism analysis for rapid gag subtype determination of human immunodeficiency virus type 1 in South Africa

A rapid method for identification of human immunodeficiency virus Type 1 (HIV-1) gag subtypes was developed based on restriction fragment length polymorphism (RFLP) analysis of 400 or 650 bp long polymerase chain reaction (PCR) fragments encompassing the start of the p17 (400 bp) and part of the p24 (650bp) regions. The consensus sequences of subtypes A-D, the only subtypes identified in South Africa, were analyzed to detect restriction endonucleases which generate unique patterns for each subtype. Four restriction endonucleases were identified: AluI, AccI, SwaI and XmnI. Digestion of a 400 bp fragment with AluI allowed identification of subtype C. Samples not identified were then reamplified, and a 650 bp fragment digested with AccI to identify subtype B, followed by SwaI and XmnI to distinguish between subtypes A and D. This strategy was applied to 87 samples previously subtyped by either sequence analysis of the gag p17 region (n = 33); or heteroduplex mobility assay (HMA) based on the env gene (n = 75); or both (n = 21). Out of the 87 samples, RFLP identified two samples as subtype A, 28 as subtype B, 56 as subtype C and one as a subtype D virus. No discrepancies were found between RFLP gag subtypes and gag sequence subtypes demonstrating the reliability of this method. There was also no discordance between gag RFLP subtypes and env HMA subtypes, suggesting that there were no recombinant viruses detected relating to the genomic regions analyzed. RFLP is an effective technique for the rapid screening in an HIV epidemic of limited diversity, such as in South Africa.     

硫酸酯化箬叶多糖抗HIV-1机制的初步研究

目的 探讨硫酸脂化箬液多糖（Ｓ－ＩＴＰＳ）抗ＨＩＶ的机制,为进一步开发该药提供理论依据。方法 于病毒接种前,接种同时及接种后分别在培养细胞（ＭＴ－４）中加入硫酸脂化箬叶多糖（Ｓ－ＩＴＰＳ）,在Ｓ－ＩＴＰＳ存在或不存在的条件下培养１￣４周。终点以病毒半数感染量（ＴＣＩＤ５０法）,细胞病变（ＣＰＥ）,ＭＴＴ染色细胞保护率（ＭＴＴ法）及培养上清中的ｐ２４抗原滴度９ＥＬＩＳＡ法）作为评价指标,判断药效,分     

Interactions of single and combined human immunodeficiency virus type 1 (HIV-1) DNA vaccines

DNA immunization permits evaluation of possible antagonistic or synergistic effects between the encoded components. The protein expression capacity in vitro was related to the immunogenicity in vivo of plasmids encoding the HIV-1 regulatory genes tat rev, and nef. Neither Tat nor Rev expression was influenced by co-expression in vitro of all three proteins, while Nef expression was slightly inhibited. With the combination of genes, the T-cellular responses of mice against Rev and Nef were inhibited compared with those when single gene immunization was used. No interference was detected for the Tat T-cell response. Thus, co-immunization with certain genes may result in inhibition of specific immune responses.     

T-helper reactivity to simian immunodeficiency virus gag synthetic peptides in human immunodeficiency virus type 2 infected individuals

West African populations are infected with divergent strains of human immunodeficiency virus type 2 (HIV21, some of which are closely related to simian immunodeficiency virus (SIV) and it has been postulated that the HIV2 epidemic might have arisen by cross-species spread of SIV into the human population in West Africa. To gain some insight into the possible basis for cross protection between these two closely related viruses, the T-helper responses to 15 synthetic peptides from SIV gag synthetic peptides were investigated in seven HIV2-infected subjects and in seven healthy controls. Significant reactivity to at least one of the synthetic peptides tested was found in all patients and a statistically significant correlation between CD4+ lymphocyte absolute numbers and the number of reacting peptides was observed. A marginal lymphocyte reactivity was found in two of the healthy controls studied. In conclusion, this preliminary evidence that HIV2-infected patients exhibit T-cell responses to SIV gag peptides suggests that both viruses share t-helper epitopes in the gag viral region and raises the possibility of cross protection between SIV and HIV2 which may be relevant for HIV2 vaccine research based on closely related retroviruses. © 1995 WiIey-Liss, Inc.     

CD4+ T cell targeting of human immunodeficiency virus type 1 (HIV-1) peptide sequences present in vivo during chronic, progressive HIV-1 disease

We previously detected HIV-1 Gag-specific CD4+ T cells recognizing reference strain viral epitopes in subjects with progressive, chronic infection. To test whether these CD4+ T cells persist in vivo by failing to recognize autologous HIV-1 epitopes, we compared autologous plasma HIV-1 p24 nucleotide sequences with targeted HXB.2 strain Gag p24 CD4+ T cell epitopes in nine chronically infected, untreated subjects. In five responding subjects, 10 of 26 HXB.2 strain p24 peptides targeted by CD4+ T cells exactly matched autologous plasma viral sequences. Four subjects with plasma viral loads >100,000 copies/mL had no measurable p24-specific CD4+ T cell responses despite carrying HIV-1 strains that matched HXB.2 sequences at predicted epitopes. These results show that HIV-1-specific CD4+ T cells can persist in chronic HIV-1 infection despite recognition of epitopes present in vivo. However, with high-level in vivo HIV-1 replication, CD4+ T cells targeting autologous HIV-1 may be non-responsive or absent.     

Evolution of R5 and X4 human immunodeficiency virus type 1 gag sequences in vivo: evidence for recombination

Human immunodeficiency virus type 1 (HIV-1) infection is in general established by CCR5-utilizing (R5) virus variants, which persist throughout the course of infection. R5 HIV-1 variants evolve into CXCR4-utilizing (X4) HIV-1 variants in approximately half of the infected individuals. We have previously observed an ongoing genetic evolution with a continuous divergence of envelope gp120 sequences of coexisting R5 and X4 virus variants over time. Here, we studied evolution of gag p17 sequences in two patients who developed X4 variants in the course of infection. In contrast to the envelope gp120 sequences, gag p17 sequences of R5 and X4 virus populations intermingled in phylogenetic trees and did not diverge from each other over time. Statistical evaluation using the Shimodaira-Hasegawa test indicated that the different genomic regions evolved along different topologies, supporting the hypothesis of recombination. Therefore, our data imply that recombination between R5 and X4 HIV-1 variants occurs in vivo.     

Detection of human immunodeficiency virus type 1 (HIV-1) DNA and RNA sequences in HIV-1 antibody-positive blood donors in Uganda by the Roche AMPLICOR assay.

The ability of commercially available PCR-based assays to accurately detect or quantitate human immunodeficiency virus type 1 (HIV-1) DNA or RNA in individuals predominantly infected with HIV-1 subtypes A and D is not known. Therefore, peripheral leukocytes from 43 individuals in Kampala, Uganda, positive for HIV by the Western blot (immunoblot) assay were tested by using the Roche AMPLICOR HIV-1 assay for the detection of DNA gag sequences. Plasma from these same individuals was tested by using the Roche HIV-1 AMPLICOR MONITOR HIV-1 assay for the quantitation of HIV-1 RNA gag sequences. In addition, peripheral leukocytes were tested for HIV-1 DNA by using a lower annealing temperature or a different primer pair for the HIV-1 pol region. The proportions of individuals with detectable HIV-1 DNA and RNA gag sequences by the Roche assays were 74 and 90%, respectively. The proportions positive for HIV-1 DNA sequences by using a 50 degrees C annealing temperature or the pol primer pair were 71 and 98%, respectively. In summary, the standard Roche assay did not detect HIV-1 DNA sequences in a significant number of HIV-1-infected individuals in Uganda. However, use of a pol primer pair increased the sensitivity of the assay to 98%. The sensitivity of the Roche AMPLICOR MONITOR assay for the detection and quantitation of HIV-1 RNA sequences was significantly higher than that of the DNA-based assay, but the efficiency of the assay, and hence, the accuracy of the values obtained with RNA, is not known. Modifications to existing assays are needed to enhance the sensitivities and accuracies of these commercially available assays for use in developing countries where non-B HIV-1 subtypes predominate.