Infection of human CD4+ rabbit cells with HIV-1: the possibility of the rabbit as a model for HIV-1 infection.

Although human T cell surface glycoprotein CD4 is the cellular receptor for human immunodeficiency virus 1 (HIV-1), the introduction of the human CD4 gene into murine cells does not render them susceptible to HIV-1 infection. Here we have established rabbit transfectant cell lines expressing human CD4 on the cell surface and demonstrated that the CD4+ rabbit transfectants could be readily infected by HIV-1 by co-cultivating with a HIV-1-infected human MOLT-4 T cell line (MOLT-4/HIV). Avid syncytia formation was observed upon co-cultivation and the syncytia abundantly produced HIV-1 mature particles, as revealed by electron microscopy. A significant increase of HIV-1 p24 antigen was also detected in the culture supernatant. The syncytia formation was blocked by pretreating the transfectant with anti-human CD4 or by pretreating the MOLT-4/HIV with anti-HIV-1 serum obtained from an infected individual, indicating that the syncytia formed as a result of the interaction of human CD4 on the rabbit transfectant with the HIV-1 envelope protein expressed on MOLT-4/HIV. In contrast, only a very small proportion of the rabbit transfectants expressed HIV-1-specific antigens upon infection with an HIV-1 stock. This may indicate that, although rabbit cells have partially acquired susceptibility to HIV-1 by transfection of human CD4 gene, rabbit cells may further require such a molecule as might be provided by MOLT-4 to become fully susceptible to HIV-1 infection. The possibility of the rabbit as a model for HIV-1 infection is also discussed.     

Expression of a processed and a non-processed form of the integrase protein of HIV-1 in the baculovirus system

Summary The integrase (IN) protein of HIV-1 was expressed as a processed and a non-processed protein in the eucaryotic baculovirus expression system. In immunoblots we could demonstrate that recombinant baculoviruses containing the completegag andpol reading frames of HIV-1 expressed a gag/pol precursor polyprotein. The specific proteolytic activity of the recombinant protease on the gag and pol precursor proteins was used for the generation of processed gag (p 17, p 24, p 16) and pol (RT/RNaseH, IN) proteins. The non-processed IN protein, expressed as a polyhedrin fusion protein, was produced at much higher level than the processed protein.     

Mapping of B-cell epitopes in rabbits immunised with various gag antigens for the production of HIV-1 gag capture ELISA reagents

An HIV-1 p24 capture enzyme linked immunosorbent assay (ELISA) was developed and used in a study of B-cell epitopes in rabbits immunised with different gag p24 antigens. Rabbits were immunised with virion HIV-1/Lai, baculovirus recombinant p24, Escherichia coli recombinant p24-15 and a mixture of synthetic peptides representing sequences of HIV-1 gag p24 protein, respectively. Five out of nine rabbits developed antibodies that could be used for an antigen capture ELISA. No significant differences in IgG titers to the whole gag protein were seen when comparing rabbits immunised with four different antigens. Three major common linear epitope regions were mapped in the rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24. The rabbit immunised with HIV-1 gag peptides had the broadest linear epitope reactive responses whereas animals immunised with E. coli recombinant antigen had the most restricted linear epitope response. The capture ELISA method thus developed using the different rabbit anti-p24 IgG preparations was shown to capture isolates from HIV-1 subtypes or clades A to G. Only rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24 developed IgG that was capable of efficiently capturing HIV-1 p24 in ELISA, indicating the importance of preparing antibodies able to recognise native or discontinuous and linear antigen configurations.     

Expression of RNA and antigens of human immunodeficiency virus type-1 (HIV-1) in lymph nodes from HIV-1 infected individuals

The presence of proteins (p17 and p24 core proteins, gp41 envelope protein) and mRNA (gag/pol and env gene segments) of human immunodeficiency virus type-1 (HIV-1) was analyzed on frozen tissue sections of lymph nodes from HIV-1 infected individuals. Thirty-one lymph nodes were categorized in the stages of follicle hyperplasia (n = 18), follicle degeneration (n = 5), and total depletion (n = 8). The follicle dendritic cells in germinal centers showed the presence of core proteins and, to a lesser extent, gp41. The staining patterns, being similar to those of immunoglobulins, suggested that they occur in the form of immune complexes. In addition there were solitary cells expressing viral protein, in particular gp41, and mRNA. The number of mRNA-positive cells was very low: about five positive cells were observed in a tissue section with about ten (hyperplastic) follicles. HIV-1-mRNA-positive cells were observed both in follicles and interfollicular areas and showed no differences between various stages. The extent and intensity of distinct HIV-1 proteins and HIV-1-mRNA gene segments in follicles were significantly correlated, as was their presence in interfollicular areas. No significant correlation was found between the presence of HIV-1 components in follicles and in interfollicular areas. This indicates that processes involving HIV-1 components occur in a segregated manner in both lymph node compartments. The presence of HIV-1 components did not correspond to any clinical classification (CDC criteria), nor to other histochemical characteristics. An exception was the correlation between gp41-positive cells and CD1-positive interdigitating cells in the interfollicular areas.     

抗HIV-1核心抗原p24单克隆抗体的制备及特性的初步鉴定

目的 :建立分泌抗HIV 1p2 4单克隆抗体 (mAb)的杂交瘤细胞株 ,并对其特性进行初步鉴定。方法 :以纯化的基因工程制备的p2 4抗原免疫BALB/c小鼠 ,取免疫小鼠的脾细胞与Sp2 /0骨髓瘤细胞融合 ,经HAT、HT选择培养及有限稀释法进行克隆化后 ,用间接ELISA法及Dotblot对其进行筛选和特性鉴定。结果 :筛选到 2株可分泌抗HIV 1p2 4mAb的杂交瘤细胞 ,其腹水效价为 1×10 -5,亲和力为 1.7× 10 4～ 1.8×10 4mol/L ,mAb的Ig亚类均为IgG1。两株mAb与HBcAg、HCVRNA阳性血清及HIVgp4 1等均无交叉反应 ,只与HIV 1p2 4抗原阳性血清产生特异反应。结论 :成功地建立了 2株可分泌抗HIV 1p2 4mAb的杂交瘤细胞 ,为进一步研制HIV 1p2 4抗原的ELISA检测试剂盒奠定了基础     

Processing, assembly, and immunogenicity of human immunodeficiency virus core antigens expressed by recombinant vaccinia virus

Recombinant vaccinia viruses that contained regions of the gag-pol open reading frames of human immunodeficiency virus type 1 (HIV-1) were constructed. Cells infected with recombinants containing both gag and protease genes expressed and processed HIV gag antigens efficiently. Processing was much reduced in cells infected with recombinants containing only gag, but not the protease gene. However, significant amounts of p41 were produced by protease-defective recombinants. This protein was immunoreactive with p24-specific monoclonal antibodies and was produced in a truncated form by a recombinant containing a 3' deletion in the p15 coding region of gag ORF. These results indicate that p41 could represent an alternative gag precursor with N-terminal sequences derived from p24 and C-terminal from p15. Ultrastructural analysis of recombinant-infected cells revealed that the gag antigens expressed were assembled into retrovirus-like particles and were secreted into culture medium. This assembly process was not dependent on HIV protease function, because immature core particles were produced by recombinants lacking HIV-1 protease functions. Immunization of mice and chimpanzees with vaccinia-HIVgag recombinant viruses generated both antibody and cell-mediated immune responses to HIV gag antigens. These recombinants are therefore useful not only for studying HIV virion processing and assembly, but also for designing immunogens for the prophylaxis and immunotherapy against AIDS.     

A new unique HIV-1 recombinant form detected in Belarus

Republican Research-and-Practical Center for Epidemiology and Microbiology, Ministry of Health of Belarus, Minsk The paper presents data on the molecular genetic characteristics of a new HIV-1 recombinant form. The study has shown that the virus is referred to as HIV-1 subtype B in terms of the gag gene and HIV-1 subtype A in terms of the pol and env genes. At the same time the new isolate is closer, in terms of the gag gene, to the HIV-1 DQ207943 strain isolated in Georgia, in terms of the pol gene, to the HIV-1 AF413987.1 strain isolated in Ukraine and, in terms of the env gene to the HIV-1 AY500393 strain isolated in Russia. Thus, the described new HIV-1 recombinant form has the following structure: BgagApolAenv. The gag, pol, and env gene sequences from the new unique HIV-1 recombinant form have been registered in the international database EMBL/Genbank/DDBJ under accession numbers FR775442.1, FN995656.1, and FR775443.1.     

Study of antigenic structure of HIV-1 protein p24 using monoclonal antibodies]

During the experiments 4 murine and 3 rat hybridomas producing monoclonal antibodies (MAb) against the protein p24 of human immunodeficiency virus type 1 (HIV-1) have been obtained. Using the immunoblotting technique, it was established that all the species of MAb reacted with the same viral proteins which are derivatives of gag gene--p24 and p55. The properties of MAb have been studied in competitive binding. Their ability of binding to different fragments of the gag protein produced by the recombinant plasmids in E. coli cells have been investigated in ELISA. The analysis of the findings suggests that the HIV-1 protein p24 contains at least 3 antigenic epitopes. All species of MAb reacted with 3 different HIV-1 strains and 2 HIV-1 isolates, but failed with 2 different HIV-2 strains. The only MAb NS5E4 can be used as an immunosorbent in the antigenic capture reaction.     

Isotypic distribution of HIV-1-specific antibodies in individuals from central Africa.

Individuals infected with human immunodeficiency virus type 1 (HIV-1) develop a humoral immune response to the virus's major structural gene products env, gag, and pol. The distribution of antibodies to env, gag, and pol proteins in Central African populations is of interest as they have a high level of immune system activation compared to non-African populations. Using the Western blot technique, we analyzed the isotypic distribution of anti-HIV antibodies in 45 HIV-1-infected individuals from Central Africa that were either symptomatic or asymptomatic. We observed two basic differences between the isotypic profile of individuals from Central Africa and non-African populations. Central African individuals had a strong polyisotypic response to gag and pol, which has only been observed for gag in American and European populations. In addition, individuals from Central Africa had a high frequency of IgG4 to gag and pol, 75 and 51%, respectively, as compared to 29 and 6% in a non-African population. The elevated IgG4 response may result from the high basal level of immune stimulation seen in Africans due to multiple and frequent exposures to viral, bacterial, and parasitic antigens.     

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     

Fine specificity of IgG subclass response to group antigens in HIV-1-infected patients

There is an association between the clinical stage of HIV-1 infection and the presence of antibodies against viral gag proteins (p17 and p24). The IgG subclass (G1 and G3) pattern against these antigens was analysed in stable patients and HIV patients progressing to AIDS. Antibodies were analysed with whole viral or peptide ELISA (using sequentially overlapping peptides) and Western Blots. IgG1 was found to be the dominant anti-HIV-1 IgG subclass and IgG1 antibodies declined in progressing patients against all HIV antigens evaluated in Western blot, including p17, p24, p31, gp41, p64, gp120 and gp160. In contrast IgG3 antibodies, which were found to be predominantly directed against gag proteins, and which could be detected in almost all patients, remained in the circulation during disease progression. By peptide assays distinct immunogenic regions were found in p17 in contrast to more evenly distributed epitopes in p24. A decreased divergence of antibody reactivity to both p17 and p24 peptides in the group of patients who developed AIDS was seen. No reaction to any single gag epitope related to disease progression. The difference between IgG1 and IgG3 anti-gag antibodies in relation to clearance during disease progression may depend on different properties of immune complexes formed by these two IgG subclasses.     

Detection of HIV-1 DNA in microglia/macrophages,astrocytes and neurons isolated from brain tissue with HIV-1 encephalitis by laser capture microdissection

In HIV-1 encephalitis, HIV-1 replicates predominantly in macrophages and microglia. Astrocytes also carry HIV-1, but the infection of oligodendrocytes and neurons is debated. In this study we examined the presence of HIV-1 DNA in different brain cell types in 6 paraffin embedded, archival post-mortem pediatric and adult brain tissues with HIV-1 encephalitis by Laser Capture Microdissection (LCM). Sections from frontal cortex and basal ganglia were stained by immunohistochemistry for CD68 (microglia), GFAP (astrocytes), MAP2 (neurons), and p24 (HIV-1 positive cells) and different cell types were microdissected by LCM. Individual cells or pools of same type of cells were lysed, the cell lysates were subjected to PCR using HIV-1 gag SK38/SK39 primers, and presence of HIV-1 DNA was confirmed by Southern blotting. HIV-1 gag DNA was consistently detected by this procedure in the frontal cortex and basal ganglia in 1 to 20 p24 HIV-1 capsid positive cells, and in pools of 50 to 100 microglia/macrophage cells, 100 to 200 astrocytes, and 100 to 200 neurons in HIV-1 positive cases but not in HIV-1 negative controls. These findings suggest that in addition to microglia, the infection of astrocytes and neurons by HIV-1 may contribute to the development of HIV-1 disease in the brain.     

Natural controlled HIV infection: preserved HIV-specific immunity despite undetectable replication competent virus

Long-term non-progressive HIV infection, characterized by low but detectable viral load and stable CD4 counts in the absence of antiviral therapy, is observed in about 5% of HIV-infected patients. Here we identified four therapy na?ve individuals who are strongly seropositive for HIV-1 but who lack evidence of detectable HIV p24 antigen, plasma RNA, and proviral DNA in routine diagnostic testing. With an ultrasensitive PCR, we established that frequencies of pol proviral DNA sequences were as low as 0.2-0.5 copies/10(6) PBMC. HIV could not be isolated using up to 30x10(6) patient PBMC. One individual was heterozygous for CCR5 Delta32, but CCR5 expression on CD4+ T cells was normal to high in all four individuals. In vitro R5 and X4 HIV-1 susceptibility of CD8-depleted PBMC of all study subjects was significantly lower than the susceptibility of CD8-depleted PBMC of healthy blood donors. All individuals expressed protective HLA-B*58s alleles and showed evidence of HIV-specific cellular immunity either by staining with HLA-B*57 tetramers folded with an HIV RT or gag peptide or after stimulation with HIV-1 p24 gag, RT, or nef peptides in ELIspot analysis. HIV-specific CD4+ T helper cells were demonstrated by proliferation of CD4+ T cells and intracellular staining for IL-2 and IFNgamma after stimulation with an HIV-gag peptide pool. Sera of all individuals showed antibody-mediated neutralization of both R5 and X4 HIV-1 variants. These data implicate that very low-level antigen exposure is sufficient for sustained HIV-specific immunity and suggest the possibility of a multi-factorial control of HIV infection.