Specific immune response and pathological findings in BALB/c mice inoculated with recombinant BCG expressing HIV-1 antigen

Recombinant BCGs (rBCGs) containing extrachromosomal plasmids with different HIV-1 insert sequences: nef, env (V3J1 and E9Q), gag p17 or whole gag p55 were evaluated for their immunogenicity, safety and persistent infection in BALB/c mice. Animal injected with, rBCG-plJKV3J1, rBCG-pSO gag p17 or rBCG-pSO gag p55 could elicit lymphocyte proliferation as tested by specific HIV-1 peptides or protein antigen. Inoculation with various concentration of rBCG-pSO gag p55 generated satisfactory specific lymphocyte proliferation in dose escalation trials. The rBCG-pSO gag p55 recovered from spleen tissues at different time interval post-inoculation could express the HIV protein as determined by ELISA p24 antigen detection kit. This result indicated that the extrachromosomal plasmid was stable and capable to express Gag protein. It was also demonstrated that rBCGs did not cause serious pathological change in the inoculated animals. The present study suggested the role of BCG as a potential vehicle for using in HIV vaccine development.     

Th1 cells specific for HIV-1 gag p24 are less efficient than Th0 cells in supporting HIV replication, and inhibit virus replication in Th0 cells.

This report provides three lines of evidence to suggest that T-helper type 1 (Th1) and type 0 (Th0) cells could play an opposing role in acquired immune deficiency syndrome (AIDS). Using a panel of Th1 and Th0 clones specific for human immunodeficiency virus-1 (HIV-1) gag p24, derived from seronegative volunteers immunized with gag p24: Ty virus-like particles, a Th1 clone specific for tuberculin (PPD), and a Th0 clone derived by random activation from the same volunteer, we have demonstrated the following differences in the capacity of these clones to regulate the in vitro replication of HIV. (1) Th1 clones were less efficient than Th0 clones in supporting HIV replication, both in their resting state (by 10-1000-fold) and after antigen activation (by five to 100-fold). Furthermore, the infectious titre of HIV recovered from the Th0 population was more than 1000-fold higher than virus from the Th1 population, and the number of HIV-infected Th0 cells was five to 16 times higher than the number of infected Th1 cells. (2) Antigen- or mitogen-activated Th1, but not Th0 clones, inhibited HIV in bystander CEM-4 cells. Th1 cells also inhibited HIV in autologous and allogeneic Th0 cells. The level of inhibition in these experiments ranged from 50% to 100% and was three to 10-fold higher and more sustained in the presence of p24-specific clones compared to the PPD-specific Th1 clone. The capacity of Th1 cells to inhibit HIV in neighbouring cells was also reflected in the reduced replication of HIV in the clones immediately after antigen activation compared to unstimulated cells. Kinetic studies of virus production, cytokine release and proliferation showed that inhibition of HIV was associated with peak cytokine release and preceeded proliferation. (3) The Th1 clones had higher cytolytic potential than the Th0 clones. Therefore, the HIV inhibitory activity of Th1 cells could be partly due to cell to cell killing. These data demonstrate the opposing effects of Th1 and Th0 cells on the in vitro replication of HIV, and suggest that Th1 cells might be important in immunity whereas Th0/Th2 cells might lay a role in promoting disease.     

HIV-2 gp105-gag截短体在毕赤酵母中重组表达研究

目的　实现HIV 2 RODgp10 5 gag截短体重组蛋白tgp10 5 gag的高效分泌表达 ,对其表达条件进行研究。方法　用PCR方法获得HIV 2 RODtgp10 5截短基因 ,将其与HIV 2 RODgag构建HIV 2 RODtgp10 5 gag嵌合基因并克隆到毕赤酵母 (Pichiapastoris)分泌型表达载体pPIC9中 ,转化GS115酵母菌 ,经MD/MM表型筛选、PCR扩增筛选阳性克隆 ,以甲醇诱导表达后进行SDS PAGE分析及Westernblot证实。结果　HIV 2 RODtgp10 5 gag嵌合截短基因在Pichiapastoris酵母系统中获得了有效分泌表达 ,相对分子质量 (Mr)约为 14 0× 10 3 ,表达量约为 16 % ,表达产物可被HIV 2阳性血清识别。最佳表达条件是大于 85 %的溶解氧 ,摇瓶转速 2 4 0r/min ,1.0 %～ 1.5 %甲醇诱导 ,培养时间 3d。结论　在Pichiapas toris表达系统中实现了HIV 2 RODtgp10 5 gag蛋白的有效分泌性表达 ,表达产物具有良好的抗原特异性。     

Monoclonal antibodies, interacting with antigenic determinants of the human immunodeficiency virus

Twenty-five hybridomas of CMK series were generated which produced monoclonal antibodies (MCA) to human immunodeficiency virus (HIV). The MCA were shown to react with HIV antigenic determinants in enzyme-immunoassays to titres 5 X 10(3)--10(5). It was established by immunoblot that some MCA interact with HIV proteins having molecular weights of 60-80 KD, and other MCA with proteins of low molecular weight (9-17 KD). Using recombinant proteins--products of env and gag genes, the immunoblot showed 5 MCA to be specific for proteins of the env gene and 5 others for proteins of the gag gene. Comparative enzyme immunoassays of interaction of MCA of the CMK series with viruses of infectious anemia of horses and HIV led to the conclusion that 4 MCA recognize common determinants of the lentiviruses under study whereas 5 other MCA react specifically with HIV alone.     

Enhanced activation of human T cell clones specific for virus-like particles expressing the HIV V3 loop in the presence of HIV V3 loop-specific polyclonal antibodies

Recombinant virus-like particles (VLP), formed by the yeast Ty p1 protein, carrying the HIV gp120 V3 loop on their surface (V3-VLP) have been tested in vitro for immunogenicity and antigenicity by using VLP p1-specific human CD4⁺ T cell lines and clones. VLP-specific human T cell lines and clones were generated from normal individuals, indicating that VLP-specific precursor cells present in the peripheral lymphocyte pool can be induced to expand clonally upon antigen challenge in vitro, in the absence of previous immunization. It was also shown that V3-specific polyclonal antibodies enhance V3-VLP-induced activation of VLP-specific T cell clones. Antibody-dependent potentiation has been shown previously in other antigen systems, and it depends on enhanced uptake of complexed antigen by Fc receptor-positive antigen-presenting cells. Since in this case antigen is internalized by presenting cells as a complex, it can be inferred that a similar event of antibody-mediated antigen uptake can take place with V3-specific B cells, resulting in presentation by the B cells of T helper epitopes derived from processing of the VLP p1 moiety. This suggests that T helper cells specific for the carrier VLP p1 protein can be activated to provide help to V3-specific B cells in the presence of the appropriate antigen construct.     

人免疫缺陷病毒Ⅱ型(HIV-2)gag蛋白基因在毕赤酵母中的克隆表达

目的：实现HIV-2HCOgag重组蛋白的分泌表达，为研究HIV—2HCO gag蛋白结构与功能及亚单位蛋白疫苗研究打下基础。方法：将HIV-2HCOgag蛋白基因片段，与分泌型毕赤酵母表达载体pPIC9重组，构建了相应的重组表达质粒pPIC9-gag，转化GS115酵母菌，经MD／MM表型筛选、PCR扩增筛选阳性克隆，以甲醇诱导表达后进行SDS-PAGE分析及Wgtem blot证实。结果：获得了HIV—2HCOgag重组蛋白在巴斯德毕赤酵母系统中的分泌表达，表达产物可被HIV—2抗血清识别，分子量约为57kD。结论：pPIC9—gag重组质粒在甲醇营养型酵母菌中可经甲醇诱导产生HIV—2HCOgag蛋白，该蛋白具有强免疫学活性。     

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.     

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.     

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.     

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.     

The immunoenzyme detection of the HIV-1 antigen by using monoclonal antibodies to protein p24]

A test system using monoclonal antibodies to HIV p24 was developed for solid-phase ELISA for detection of HIV antigen (AG) which helped detect the content of AG in samples with trace concentrations, less than 25 pg/ml. The test system can be used for AG monitoring in natural specimens (serum of HIV-infected patients and culture medium of infected cells) and for determination of antigen-recombinant product of HIV gag gene.     

Secretory IgA as a vaccine carrier for delivery of HIV antigen to M cells

HIV transmission and spread in the host are based on the survival of the virus or infected cells present in mucosal secretions, and the virus' ability to cross the epithelial barrier and access immune target cells, which leads to systemic infection. Therefore, HIV‐specific immunity at mucosal sites is critical for control of infection. Although mucosal delivery would ensure the best onset of protective immunity, most candidate vaccines are administered through the parenteral route. Remarkably, secretory IgA (SIgA) interacts specifically with mucosal microfold (M) cells present in gut‐associated lymphoid tissues. Here we evaluate the feasibility of delivering chemically bound p24HIV antigen via SIgA into the intestinal mucosae in mice. After oral administration, p24–SIgA complexes are quickly delivered into the tissue and selectively captured by CX3CR1⁺ dendritic cells. Oral immunization with p24gag linked to SIgA (p24–SIgA) adjuvanted with E. coli heat labile enterotoxin (HLT) elicits both humoral and cellular immune responses against p24 at the systemic and mucosal levels and induces efficient protection against rectal challenge with a recombinant vaccinia virus encoding gag. This is the first study which underscores the remarkable potential of SIgA to serve as a vaccine carrier for an HIV antigen in mucosal administration targeting the gastrointestinal environment.     

Detection of human immunodeficiency virus (HIV) in serum and body fluids by sequential competition ELISA

A micro-ELISA based on competition with the biotin-labeled 25 kDa gag (p25gag) recombinant protein of the human immunodeficiency virus (HIV) was compared to commercial antigen capture ELISAs for the detection of viral antigens in a variety of body fluids including serum, cerebro-spinal fluid (CSF), sputum, saliva, milk, semen, vaginal and bronchial fluids, as well as earwash fluid. Two-thirds (24/30) of these specimens contained IgG and/or IgA antibodies to HIV. The results were correlated with the recovery of infectious HIV in culture. The competition ELISA detected the presence of HIV antigen in 4 out of 8 sera, 5 out of 6 CSF and 6 out of 15 other body fluids that were found to contain infectious virus. Comparatively, 5 of the 8 sera, 3 of the 6 CSF, and 2 of the 15 body fluids tested positive for HIV antigen by capture ELISA. The data suggest that the competition test is more effective than the capture method in detecting antigen in CSF and body secretions, which might be due to the presence of immune complexes. However, both ELISA methods showed similar susceptibility to antibody interference in spiked specimens. The results confirm that antigenemia status can be of value in assessing HIV infection when used in combination with other clinical and laboratory data.     

The presence of prolines in the flanking region of an immunodominant HIV‐2 gag epitope influences the quality and quantity of the epitope generated

Both the recognition of HIV‐infected cells and the immunogenicity of candidate CTL vaccines depend on the presentation of a peptide epitope at the cell surface, which in turn depends on intracellular antigen processing. Differential antigen processing maybe responsible for the differences in both the quality and the quantity of epitopes produced, influencing the immunodominance hierarchy of viral epitopes. Previously, we showed that the magnitude of the HIV‐2 gag‐specific T‐cell response is inversely correlated with plasma viral load, particularly when responses are directed against an epitope, ₁₆₅DRFYKSLRA₁₇₃, within the highly conserved Major Homology Region of gag‐p26. We also showed that the presence of three proline residues, at positions 119, 159 and 178 of gag‐p26, was significantly correlated with low viral load. Since this proline motif was also associated with stronger gag‐specific CTL responses, we investigated the impact of these prolines on proteasomal processing of the protective ₁₆₅DRFYKSLRA₁₇₃ epitope. Our data demonstrate that the ₁₆₅DRFYKSLRA₁₇₃ epitope is most efficiently processed from precursors that contain two flanking proline residues, found naturally in low viral‐load patients. Superior antigen processing and enhanced presentation may account for the link between infection with HIV‐2 encoding the “PPP‐gag” sequence and both strong gag‐specific CTL responses as well as lower viral load.     

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.     

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.     

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.