Multiple subsets of HIV-specific cytotoxic T lymphocytes in humans and in mice

The human immunodeficiency virus type 1 (HIV-1) induces a strong cytotoxic T lymphocyte (CTL) response in humans following infection. HIV-specific CTL can be detected directly in the blood and lungs of infected patients, and can be expanded in vitro by stimulation with autologous HIV-infected lymphoblasts. Furthermore, CTL specific for HIV envelope glycoprotein gp160 have been obtained in mice by immunization with recombinant vaccinia virus (VV) that carry the HIV env gene. In this study, we show that mice also produce strong CTL responses to gag and nef proteins following immunization with VV recombinants, thus providing a convenient model system to study T lymphocyte immunity to defined HIV antigens. To determine the specificity of circulating HIV-immune CTL in humans, a panel of doubly transfected mouse P815 tumor cells was produced which express the human HLA-A2 or HLA-A3 transplantation antigen gene and one HIV-1 gene (env, gag or nef). Using these cells as targets to CTL, we show that HIV-infected humans carry co-existing CTL sub-populations of different specificities. Each subpopulation appears to vary in intensity among different individuals. Surprisingly, CTL specific for regulatory, non-structural nef protein appear to be a major constituent of the human immune response to HIV.     

Expression of human immunodeficiency virus type 1 Gag protein precursor and envelope proteins from a vesicular stomatitis virus recombinant: high-level production of virus-like particles containing HIV envelope

Recombinant vesicular stomatitis viruses have been developed as high-level expression vectors which serve as effective vaccine vectors in animals (Roberts et al., 1998, J. Virol. 72, 4704-4711; Roberts et al., 1999, J. Virol. 73, 3723-3732). Here we show that two genes can be expressed simultaneously from a single, live-attenuated VSV recombinant. The genes used encode the Pr55(gag) protein precursor of HIV-1 (1.7-kb gene) and an HIV-1 envelope (Env) protein (2.4 kb gene). Our results show that VSV can accommodate up to a 40% increase in genome size with only a threefold reduction in virus titer. Recombinants expressing the Pr55(gag) protein precursor with or without Env protein produced abundant HIV virus-like particles (VLPs) in addition to bullet-shaped VSV particles. HIV Env protein expressed from a VSV recombinant also expressing Gag was specifically incorporated into the HIV VLPs but not into the VSV particles. In contrast, VSV G protein was found in both VSV particles and in HIV VLPs. Such VSV/HIV recombinants producing HIV VLPs with Env protein could be an effective source of HIV-like particles inducing both cellular and antibody-mediated immunity to HIV-1.     

Comparison of human immunodeficiency virus antigens as stimulants for lymphocyte proliferation assays

CD4 proliferative responses to the human immunodeficiency virus (HIV) type 1 (HIV-1) p24 (gag) antigen inversely correlate with the plasma viral load in HIV-infected subjects who control viral replication without antiretroviral therapy. Use of a single HIV-1 protein to assess CD4 proliferative responses may not reflect the global response to this pathogen. We compared the abilities of HIV p24 and gp120 antigens from two different vendors, an inactivated whole HIV-1 MN virion preparation and an HIV-1E culture supernatant antigen, to elicit proliferative responses in HIV-seropositive and HIV-seronegative donors. Peripheral blood mononuclear cells from 12 HIV-seropositive donors (each with HIV-1 loads <4,000 copies/ml of plasma, >350 CD4 T lymphocytes/mm(3), and no antiretroviral therapy) and 15 HIV-seronegative donors were assessed with multiple concentrations of each stimulant by standard lymphocyte proliferation assays. Wide variations in response rates were found, with zero, three, five, and eight individuals demonstrating stimulation indices of >3 for the HIV culture antigen supernatant, gp120, p24, and inactivated whole-virus preparations, respectively. These results suggest that the use of the inactivated whole virus resulted in a more sensitive assay for detection of CD4 T-lymphocyte function in HIV-infected subjects.     

Acquired resistance to type II collagen-induced arthritis in rhesus monkeys is reflected by a T cell low-responsiveness to the antigen.

The use of serological tests for the diagnosis of HIV infection has revealed that some non-infected persons have antibodies that react with HIV-1 gag proteins. Here, the sera of three non-infected subjects reacting with p17 and 11 non-infected subjects reacting with p24 were investigated, using an enzyme immunoassay (EIA) with six recombinant gag antigens and Western blot analysis of proteolytic peptides of two of these gag antigens. The results indicate that whereas all p17-reactive sera could react with an unique epitope, individual p24-reactive sera recognize different epitopes. Investigations by EIA also demonstrated the role of sequences located far from the epitopes in making these epitopes accessible to the antibodies or in providing them with an antigenic conformation. In addition to the 14 subjects mentioned above, another subject was shown to have antibodies reacting with the p9 (NC) gag protein. Several proteins are known as having homology with HIV-1 gag proteins. Their possible role in eliciting cross-reactive antibodies is discussed.     

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 gag precursor protein and secretion of virus-like gag particles of HIV-2 from recombinant baculovirus-infected insect cells

A recombinant baculovirus carrying the gag gene but lacking the protease coding sequences of human immunodeficiency virus type 2 (HIV-2) has been constructed. When this recombinant baculovirus is used to infect insect cells, a high level of gag precursor protein, gag pr41, is expressed. Electron microscopy showed that the majority of gag pr41 was budding through the plasma membrane and being released into the culture medium in spherical virus-like particles with a diameter of approximately 100 nm. Metabolic labeling demonstrates that gag pr41 is myristylated. Our results demonstrated that HIV-2 gag pr41 can be assembled into virus-like particles in the absence of other HIV proteins. Rabbits immunized with purified gag pr41 particles produced high-titer antibody and Western blot analysis showed that anti-gag pr41 rabbit sera recognize p17, p24, and p55 gag proteins of HIV-1. These results show that gag pr41 particles are highly immunogenic and that gag proteins of HIV-1 and HIV-2 have similar antigenic epitopes.     

Comparative humoral responses to human immunodeficiency virus type 1-p24gag linear B-cell epitopes among individuals showing atypical western immunoblotting reactions and implications for diagnosis.

Serum specimens from 25 individuals with an isolated human immunodeficiency virus type 1 (HIV-1) core antigen reactivity in a Western immunoblot test were examined for their reactivities with HIV-1 virions, control cellular antigens, HIV-1-Bru p24gag recombinant protein (p24gag), and a panel of 22 p24gag-derived peptides. The results were as follows: (i) serum specimens from eight HIV-1-uninfected subjects did bind to virions but failed to bind to p24gag; (ii) sera from 13 HIV-1-uninfected subjects and from one HIV-2-infected patient reacted with HIV-1 virions and p24gag but failed to bind to any of the peptides expressing major p24gag epitopes, and (iii) 3 serum specimens obtained from one neonate carrying anti-HIV-1 maternal antibody and from two HIV-1-infected subjects who had seroconverted during the study reacted with HIV-1 virions, p24gag, and one or more peptides containing the major p24gag epitopes. Our data suggest that the combination of p24gag and appropriate peptides could be useful for resolution when atypical Western immunoblot results are encountered.     

Expression of antigen reactive with a monoclonal antibody to HTLV-1 P19 in salivary glands in Sjögren''s syndrome.

To examine the possible involvement of retroviruses in Sjögren's syndrome (SS), labial salivary gland sections from 99 individuals were probed with three MoAbs to core (gag) proteins of human T cell leukaemia virus-1 (HTLV-1) and two MoAbs to HIV-1. Sections from 31% of 39 patients with primary SS (pSS) contained an epithelial cytoplasmic protein reactive with a MoAb(197) to the p19 group specific antigen (gag) of HTLV-1. The antigen was also detected in samples from 24% of 17 patients with rheumatoid arthritis (RA) and SS. 21% of 14 patients with sicca symptoms and 12.5% of 16 patients with other connective tissue diseases. It was not found in the salivary glands of 13 normal controls. A second MoAb to p19 gag, a MoAb to the p24 gag of HTLV-1 and MoAbs to HIV-1 p17and p24 gags gave negative reactions. Serum antibodies to HTLV-1 were negative, confirming that the antigen was not part of HTLV-1. The antigen showed properties consistent with an endogenous retrovirus in that it was absent in healthy tissues or resting cells but inducible by stimulation with phytohaemagglutinin (PHA) or interferon-gamma (IFN-γ) It appeared to be distinct from the endogenous retroviral sequence HRES-1. These data suggest the presence of an endogenous retrovirus in salivary gland epithelium which could contribute to the chronic inflammation of SS.     

HIV-1B/C重组病毒感染者Gag特异性T淋巴细胞免疫应答的研究

目的 研究中国主要流行的HIV-1 B/C重组毒株感染者Gag特异性T淋巴细胞反应特征.方法 本研究以10例感染时间＜1年和25例感染时间＞3年未接受抗病毒治疗的HIV-1 B/C重组毒株感染者为研究对象,以10例HIV-1阴性健康人作为对照,用Elispot方法检测其针对HIV-1B/C同义B Gag重叠多肽产生γ干扰素的特异性T淋巴细胞反应.结果 8例(8/10)感染时间＜1年的HIV-1 B/C重组毒株感染者产生Gag特异性分泌γ干扰素的T淋巴细胞反应,主要识别散在分布的五条多肽;17例(68％)感染时间＞3年的感染者产生反应,主要识别p17区域内的一条和p24区域内六条多肽.感染时间＞3年组产生IFN-γ的特异性T淋巴细胞反应强度与病毒载量呈明显正相关(P =0.0318,r=0.519),感染时间＜1年的感染者反应强度明显高于感染时间＞3年的感染者(P=0.021).健康人对照组无阳性反应.结论 HIV-1 B/C重组病毒感染者在疾病进程不同阶段识别Gag的不同区域.     

The protease and gag gene products of the human immunodeficiency virus: authentic cleavage and post-translational modification in an insect cell expression system

Three recombinant baculoviruses which are capable of expressing human immunodeficiency virus (HIV) protease, p55gag, and both products simultaneously in insect cell culture have been constructed. Upon co-infection of cells with the protease and p55gag-expressing viruses, authentic processing of the gag precursor is observed to take place. This processing could be reproduced in vitro using mixtures of cellular lysates containing the expressed proteins. When expressed alone, uncleaved p55gag precursor appears to form retroviral core-like particles within the cytoplasm of infected cells. Metabolic labeling studies of the baculovirus-expressed gag products have demonstrated that p17 is myristylated at its amino terminus, and that p24 is phosphorylated. In these respects, the insect cell system is evidently capable of carrying out post-translational processing resembling that which occurs in authentic HIV-1 replication.     

HIV-1B／C重组病毒感染者Gag特异性T淋巴细胞免疫应答的研究

目的研究中国主要流行的HIV-1B／C重组毒株感染者Gag特异性T淋巴细胞反应特征。方法本研究以10例感染时间〈1年和25例感染时间〉3年未接受抗病毒治疗的HIV-1B／C重组毒株感染者为研究对象,以10例HIV．1阴性健康人作为对照,用Elispot方法检测其针对HIV-1B／C同义BGag重叠多肽产生1干扰素的特异性T淋巴细胞反应。结果8例（8／10）感染时间〈1年的HIV-1B／C重组毒株感染者产生Gag特异性分泌Y干扰素的T淋巴细胞反应,主要识别散在分布的五条多肽;17例（68％）感染时间〉3年的感染者产生反应,主要识别p17区域内的-条和p24区域内六条多肽。感染时间〉3年组产生IFN-吖的特异性T淋巴细胞反应强度与病毒载量呈明显正相关（P=0．0318,r=0．519）,感染时间〈1年的感染者反应强度明显高于感染时间〉3年的感染者（P=0．021）。健康人对照组无阳性反应。结论HIV-1B／C重组病毒感染者在疾病进程不同阶段识别Gag的不同区域。     

Immunogenicity of the recombinant adenovirus type 5 vector with type 35 fiber containing HIV-1 gag gene

The immune efficiency of a recombinant adenovirus type 5 with type 35 fiber containing HIV-1 gag gene (rAd5/F35-mod.gag) was investigated in BALB/c mice, in which the rAd5/F35-mod.gag was firstly identified with PCR, then transfected to 293 cells and the in vitro expression level of Gag protein was determined by Western blotting and indirect immuno-fluorescent assay. Mice were immunized with intramuscular injections of rAd5/F35-mod.gag, rAd5-mod.gag or DNA and were boosted after 3 weeks. To test the effect of pre-existing anti-viral immunity on immunization, mice were also injected with Ad5-GFP vector and then immunized 4 and 7 weeks later with Ad5/F35-mod. gag vector. The P24-specific IgG antibody in sera of immunized mice was determined by ELISA and the specific cytotoxic T lymphocyte (CTL) response was assayed by intracellular cytokine staining. It was demonstrated that the rAd5/F35-mod. gag vector could express efficiently the HIV Gag protein in 293 cells in vitro and induce strong HIV-specific immune responses in vivo. The strongest CTL and serum IgG response occurred when mice were immunized twice with injection of rAd5/F35 alone, but the anti-Ad5 antibody after primary infection with adenovirus could inhibit the specific immune responses induced by rAd5/F35 vector. It is concluded that single immunization with recombinant adenovirus rAd5/F35-mod. gag can induce specific CTL and serum IgG antibody responses in mice, but the immunogenicity of rAd5/F35 is comparably weaker than that of rAd5.     

河南地区HIV-1毒株Gag基因抗原表位变异特征及准种特点研究

目的 探讨中国河南地区人免疫缺陷病毒Ⅰ型(HIV-1)毒株GAG蛋白抗原表位变异特征,并对其准种特点加以分析.方法 套式聚合酶链反应(Nested-PCR)扩增确认HIV阳性样本gagp17～p24基因区段并测序,PCR产物纯化后克隆,挑选克隆株鉴定为阳性后测序,以MEGA(version 3.0)等软件进行分析.结果 河南HIV毒株为B'亚型;gag基因p17区段抗原表位突变有E62G(55.80%),Y79F(48.90%),T84V(48.90%),144V(44.20%),gag基因p24区段抗原表位未见明显变异.结论 HIV-1 B'亚型毒株gag基因p17区段的4个抗原表位,存在较大变异,p24区段较为保守,适合抗原表位疫苗的研制.     

The SIVmac239 Pr55Gag isoform, SIV p43, suppresses proteolytic cleavage of Pr55Gag

In human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) the gag gene encodes the precursor polyprotein Pr55Gag, which is cleaved by the viral protease to produce the major structural proteins. Recently, it has been shown that HIV and SIV gag RNAs contain internal ribosome entry sites (IRESs) that mediate translation of Pr55Gag [Pr57Gag in HIV type 2 (HIV-2)] isoforms. Previously, we demonstrated that SIVmac239 p43(-), a mutant that does not express the Pr55Gag isoform, SIV p43, replicates more efficiently than wild-type (WT) SIVmac239 in cell culture. In this study, we characterize SIVmac239 p43(-) virion production and demonstrate that, in the absence of SIV p43, cleavage of Pr55Gag is increased in budded virions, resulting in a higher percentage of mature particles. Additionally, intracellular cleavage of Pr55Gag is increased in SIVmac239 p43(-), suggesting that SIV p43 suppresses premature cleavage of Pr55Gag by the viral protease.     

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.     

The properties of monoclonal antibodies to HIV gag gene proteins]

Immunization of BALB/c mice with natural purified HIV antigen, fusion of spleen cells with myeloma cells and subsequent selection of hybrid clones using recombinant gag antigen of HIV gave hybridomas producing monoclonal antibodies (MCA) to HIV. The immune blotting method demonstrated that 3 clones interacted with protein p24 and 4 clones with protein p17 of HIV. Competitive EIA led to a conclusion that the resulting MCA detected at least 3 antigenic determinants in proteins, products of gag gene of HIV. The potentials of using these MCA for the detection of viral antigen in HIV-infected continuous cell lines were demonstrated.