Yohei Okada

Long‐Lasting Immune Response Induced by Recombinant Bacillus Calmette–Guérin (BCG) Secretion System

The recombinant bacillus Calmette–Guérin (rBCG) secretion system utilizing an extracellular α antigen of Mycobacterium kansasii (α‐K) was characterized biochemically and immunologically. The human immunodeficiency virus type1 (HIV‐1) p17gag B cell epitope fused to α‐K was secreted in extremely large amounts. At least three mice out of seven inoculated with rBCG generated high titres of antibody to the epitope. The long‐lasting antibody production persisted more than 14 months.

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)

High susceptibility of U937-derived subclones to human immunodeficiency virus type 1 infection correlates with accumulation of unintegrated circular viral DNA

Our previous report showed that U937-derived subclones were differentiated into at least three types (high, middle, and low types), even in the subclones expressing similar levels of surface CD4, in terms of the kinetics of the appearance of viral antigens and virus production after infection with human immunodeficiency virus type 1 (HIV-1). Here we showed the evidence that high susceptibility to HIV-1 infection, which was confirmed by the profound expression of viral messages and antigens, was exclusively associated with a high number of the unintegrated extrachromosomal form of viral DNA, but not with the amounts of adsorbed virus RNA nor those of integrated DNA form. The difference in the amounts of extrachromosomal form of viral DNA was also observed in the culture with 3′-azido-3′-deoxythymidine (AZT), indicating that the susceptibility is essentially unrelated to multiple infection events. Thus, the susceptibility of U937-derived subclones to HIV-1 infection seems to be affected by the occurrence of specific events involved in the accumulation of unintegrated viral DNA after viral adsorption.

Viral activation from latency during retrodifferentiation of U937 cells exposed to phorbol ester followed by infection with human immunodeficiency virus type 1

To determine the mechanism underlying the human immunodeficiency virus type 1 (HIV-1) latency and its activation in monocyte/macrophage lineage, the human promonocytic cell line U937 was infected with HIV-1 after differentiation with varied doses of phorbol 12-myristate 13-acetate (PMA). Variously differentiated intermediate stages were generated in U937 cells in a dose-dependent manner. When these cells were infected with lymphotropic HIV-1, the kinetics of the production of HIV-1 DNA, the appearance of HIV-1 antigen-positive cells, and viral production in the conditioned media were slower at higher doses of PMA. This different susceptibility to the infection was not due to the rate of HIV-1 adsorption. Viral replication from latency in the differentiated cells was activated in proportion with the retrodifferentiation observed in long-term cultures of the host cells. Thus, our data demonstrate the close correlation between the regulation of HIV-1 replication and the differentiation stage of monocyte/macrophage lineage cells at the time of HIV-1 infection. The retrodifferentiation phenomenon in infected cells seems to be particularly important for understanding the mechanisms for HIV-1 activation from latency.

High susceptibility of U937 -derived subclones to infection with human immunodeficiency virus type 1 is correlated with virus-induced cell differentiation and superoxide generation

The promonocytic human leukemic cell line U937, when infected with lymphotropic human immunodeficiency virus type 1 (HIV-1), becomes a continuous virus producer. A total of 46 U937-derived subclones in suspension was isolated and classified into three (2 high, 42 middle, and 2 low) types based on their susceptibility to the infection. By analyzing subclones before infection, we found that the high-type subclones expressed LFA-1 antigens at a relatively low level. In addition, the ability of these subclones to induce adherence after exposure to phorbol 12-myristate 13-acetate (PMA) was reduced. In contrast, a transition by HIV-1 infection to adherent macrophage-like cells was induced only in the high-type, but not in the low-type subclones. The high-type adherent cells obtained by HIV-1 infection were followed by further lineage to become retrodifferentiated suspension cells showing reduced syncytia formation ability. Superoxide was generated in the high-type subclones, without PMA-mediated differentiation, from the early stage of infection before HIV-1 replication, as well as during undifferentiated, differentiated and retrodifferentiated stages. In contrast, it was only transiently generated at acute phase of HIV-1 replication in low-type subclones. Long-term culture of the low-type subclones decreased the expression of major structural viral protein Gag and also virus production. Thus, the mechanism by which PMA differentiates U937 cells is not the same as that induced by HIV-1 infection. The latter mechanism results in high susceptibility to infection. The HIV-1 phenotypes of finally obtained persistently infected cells were also affected by the cell stages at the time of infection.

Stimulation of human immunodeficiency virus type 1 infected cells with superoxide enhances the chemotactic motile response of CD4+ human T cells: implication for virus transmission by cell-to-cell interaction.

We previously showed that superoxide (O2-) significantly enhanced human immunodeficiency virus type 1 (HIV-1)-induced syncytia formation in co-cultured infected and uninfected human T cells. In this study, we describe a novel chemotactic response of uninfected CD4+ T cells by stimulating infected T cells with O2-. Syncytia formation was amplified only when persistently infected cells were stimulated by O2-. When the infected cells in lower well of microplate were cultured with uninfected cells in the upper well of a Boyden chamber with 8.0 microns pores, uninfected cell migration to the porous membrane was significantly amplified by stimulating infected cells with O2-. In contrast, similar functions were slight under the same assay conditions in the presence of known chemokines such as human RANTES and macrophage inflammatory protein 1 (MIP-1 alpha and beta), which all activate T lymphocytes. In addition, it is unlikely that the O2(-)-induced chemotactic response is due to soluble HIV-1 proteins from infected cells or to amplified expression levels of cell surface functional molecules such as CD4 and LFA-1 (CD11a and CD18) as well as HIV-1 Env gp120 on uninfected and/or infected cells. Thus, an unknown chemotactic factor could be generated from infected T cells by stimulation with O2- and it might contribute to viral transmission by activating cell-to-cell interactions.