Takuro Kimura

Amplification of superoxide anion generation in phagocytic cells by HIV‐1 infection

Amplification of superoxide (O− 2) generation by HIV‐1 infection was examined in two human myeloid‐monocytic cell lines. The level of O− 2 generation in HL‐60 after infection became significantly higher than that of the steady‐state. A similar phenomenon was also shown in U937, but only after acquisition of O− 2 generation ability by differentiation to macrophages. By means of the NADPH oxidase‐coupled response in infected cells, we reconstituted the O− 2‐generating machinery in cell‐free system. The results suggested that cytosolic factor(s) exerted by infection might be responsible for the amplification of O− 2 generation. Thus, HIV‐1 infection could elevate the level of oxidative stress in macrophages which might play an important role in disease progression.

Cytotoxic T lymphocyte response in mice induced by a recombinant BCG vaccination which produces an extracellular α antigen that fused with the human immunodeficiency virus type 1 envelope immunodominant domain in the V3 loop

The host immune response of cell-mediated immunity, particularly that of cytotoxic T lymphocytes (CTLs), is a major immune defence mechanism which may provide resistance to a human immunodeficiency virus type 1 (HIV-1) spread leading to acquired immune deficiency syndrome (AIDS). To prevent the accompanying activity of HIV-1 proteins responsible for the loss of helper T-lymphocyte function, it is crucial to develop a live attenuated recombinant vaccine expressing only T- or both T- and B-cell epitopes. Here, we examined the expression of the HIV-1 Env protein V3 region (15 amino acids from Arg315 to Lys329) in Mycobacterium bovis BCG as a fused form with an extracellular alpha antigen of Mycobacterium kansasii. Balb/c mice inoculated with this recombinant BCG (rBCG), rapidly induced V3 peptide-specific CTLs. Target cell lysis was restricted to the murine class I major histocompatibility complex, H-2d. A similar CTL response was also elicited after Balb/c mice were immunized with the same rBCG even when pre-inoculated with non-recombinant BCG. Thus, the rapid induction of HIV-1-specific CTLs indicates that this vaccine may be a therapeutic approach to preventing progression to AIDS.

Highly conserved epitope domain in major core protein p24 is structurally similar among human, simian and feline immunodeficiency viruses

Linear B cell epitopes were mapped on the major core protein p24 of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIVAGM) and feline immunodeficiency virus (FIV) using a fusion protein-based method and murine monoclonal antibodies reactive against the p24 antigens expressed on the surface of HIV-1- and FIV-infected cells. The results suggest that the sites identified here are encoded at similar positions in the three virus genomes and consist of highly conserved epitopes, which could exhibit immunodominance.

Cells surviving infection by human immunodeficiency virus type 1 : vif or vpu mutants produce non-infectious or markedly less cytopathic viruses

Under conditions in which a clonal cell line (M10) isolated from a human T cell lymphotrophic virus type I-transformed MT-4 cell line was completely killed by infection with wild-type human immunodeficiency virus type 1 (HIV-1), equivalent M10 cells survived infection with HIV-1 vif, vpr or vpu mutant virus after transient cytopathic effects. Several cell clones, which were isolated from the proliferating M10 cells after infection with vif and vpu mutant viruses (M10/vif- and M10/vpu-), had heterogeneous HIV-1 phenotypes in terms of HIV-1 antigen expression, their syncytium forming capacity, reverse transcriptase activity and the infectivity of HIV-1 particles produced. When the replication kinetics of the HIV-1 particles produced were assayed in M10 cells, the clones could be classified into three types, i.e. type I producing non-infectious HIV-1, type II producing infectious HIV-1 with low replicative ability and type III producing infectious HIV-1 with a replicative ability similar to that of wild-type HIV-1. HIV-1 major viral cell proteins and virus particle fractions were almost typical in types II and III but not in type I. Electron microscopic examination of particles released by I, II and III clones revealed rare defective, predominantly defective and essentially normal virions, respectively. Northern and Southern blot analyses revealed no apparent deletion in the proviral DNA and mRNA prepared from these clones, except in the case of type I and II clones isolated from M10/vpu- which contained large deletions in the mRNAs for gag and gag-pol proteins. Thus, M10 cells surviving infection with HIV-1 vif or vpu mutants are heterogeneous, persistently expressing HIV-1 antigens and producing non-infectious or less cytopathic virus.

Superoxide enhances the spread of HIV-1 infection by cell-to-cell transmission

Oxidative stress is thought to be involved in the progression of human immunodeficiency virus type 1 (HIV‐1)‐induced disease. We examined the effect of Superoxide (O2 −) on HIV‐1 spread in cultured human CD4+ cell lines. The O2 − significantly enhanced cell‐to‐cell transmission of HIV‐1, although its effect on HIV‐1 replication was not evident, presumably due to its cytostatic activity. The effect was notable on the HIV‐1 transmission from macrophages to T lymphocytes by endogenous, macrophage‐generated O2 −. This amplification was specifically reduced to the steady‐state level by antioxidants, and further to the basal level by anti‐CD4 antibodies, indicating the specificity of O2 − for enhancing HIV‐1 spread by cell‐to‐cell transmission.

Major core proteins, p24s, of human, simian, and feline immunodeficiency viruses are partly expressed on the surface of the virus-infected cells

We have previously shown the expression of human immunodeficiency virus type 1 (HIV-1) major gag protein, p24, on the surface of persistently HIV-1-infected cells by using murine monoclonal antibodies (mAb). We now report that the cell surface gag p24 antigen expression is a universal phenomenon among HIV-1, simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). The mAbs prepared by immunization with purified HIV-1 particles were used as antibodies cross-reactive to HIV-1 and SIVagmp24 antigens. The mAbs to FIV p24 were raised against the gag precursor 50 kDa protein of FIV, which was expressed by Baculovirus vector. The p24 antigen expression on the cell surface was detectable in certain combinations of virus-host cell systems in all of these viruses. Since these p24 regions of the animal viruses seem to play as important a role in cell-mediated immunity as that of HIV-1, the p24 applicability as a candidate epitope for vaccine development could be evaluated in those animals.

Suppression of delayed-type hypersensitivity in mice pretreated with diethylstilbesterol: involvement of sex hormones in immunomodulation.

Delayed‐type hypersensitivity (DTH) responses were suppressed in female mice treated with diethylstilbesterol (DES), a synthetic nonsteroidal compound possessing estrogenic activity, but not in male mice. Upon analysis of this DTH‐suppression in DES‐treated female mice by use of the macrophage migration inhibition (MI) assay, an in vitro correlate of DTH, suppressor adherent cells (i.e., macrophages) in the peritoneal cavity were found to play an important role in this DTH suppression. On the other hand, DES‐induced suppression was observed in surgically castrated male mice with depressed plasma testosterone (TS) levels, but not in TS‐treated female mice or in castrated male mice, which suggests that TS inhibited the DES suppression. These results provide evidence that DTH response may be modulated by sex hormones.

Dependence on host cell cycle for activation of human immunodeficiency virus type 1 gene expression from latency

Human immunodeficiency virus type 1 (HIV-1) establishes latent infection of a certain population of CD4+ host cells which could be long-term reservoirs for HIV-1. The expression of viral genes in such long-term infected cells is strongly regulated by cellular status, such as the phase of the cell cycle or stage of cell differentiation. Here, viral gene expression in synchronized U1 cells, a monocytic cell clone latently infected with HIV-1, was characterized. The expression of HIV-1 antigens was detected exclusively at G2/M phase in U1 cells, irrespective of phorbol myristate acetate (PMA) treatment. The induction of HIV-1 gene expression in PMA-treated cells was due to the recruitment of NF-kappaB with DNA-binding activity at G2/M phase. Activated NF-kappaB was induced only by PMA treatment during the late G1 to S, but not after entering G2 phase, indicating that the transcriptional factor(s) involved in viral gene expression is also largely regulated by the host cell cycle. In contrast, the enhancement of antigen expression by treatment with tumour necrosis factor-alpha (TNF-alpha) was cell cycle-independent. In fact, NF-kappaB was activated 2 h after TNF-alpha treatment at all stages of the cell cycle. Thus, the mechanisms of HIV-1 activation from latency in U1 cells by PMA and TNF-alpha treatment are different. The model system using U1 cells shown here may provide insight into the mechanisms responsible for HIV-1 gene expression from latency.

In vivo induction of human immunodeficiency virus type 1-specific cytotoxic T lymphocytes and delayed-type hypersensitivity by a 23-amino acid peptide from the highly conserved region in major core protein p24

Cell-mediated immune responses are a major immune defence mechanism against the spread of human immunodeficiency virus type 1 (HIV-1) which may lead to acquired immune deficiency syndrome (AIDS). Therefore, the best candidate for a peptide vaccine preventive from the onset of the disease might be a chain section containing both B- and T-cell epitopes in regions of conserved sequences between the different HIV-1 isolates. We previously identified the highly conserved linear B-cell epitope (23 amino acids in the major core protein p24). Since the epitopes of cytotoxic T lymphocytes (CTLs) can be defined by short synthetic peptides, we examined whether this highly conserved region can elicit viral-specific, cell-mediated immune responses. The results showed specific induction of CD8+ CTLs in mice by immunization with the Gag 13-mer peptide. Lysis of targets is specific since unpulsed cells with the same MHC haplotype or cells with a different MHC haplotype pulsed with the peptide were resistant to lysis. This in vivo response induced by the Gag 23-mer peptide was almost the same as that induced by the 15-amino acid peptide from the HIV-1 Env gp120 which is an immunodominant domain in the V3 loop. Lymphocyte proliferation of T-cell fraction from immune spleen cells was observed after in vitro stimulation with the Gag 23-mer peptide, whereas there was no apparent lymphocyte proliferation with the Env 15-mer peptide. In addition, specific antibodies were raised against Gag p24 in mice immunized with the Gag 23-mer peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Superoxide generation by monocytes following infection with human cytomegalovirus.

A significant level of superoxide (O2-) generation was observed in a U937-derived subclone following infection with human cytomegalovirus (HCMV). Although there were no detectable levels of viral mRNA and/or protein expression, HCMV DNA content transiently increased immediately before O2- generation. Similarly, O2- generation was also observed in peripheral blood monocytes derived from healthy donors.