Takashi Ohashi

Tumor immunity against adult T‐cell leukemia

Human T‐cell leukemia virus type‐I (HTLV‐I) causes adult T‐cell leukemia (ATL) in a small population of infected individuals after a long incubation period. Although the process of clonal evolution of ATL cells may involve multiple steps, ATL cells from half of the ATL cases still retain the ability to express HTLV‐I Tax, a key molecule of HTLV‐I leukemogenesis. A recent finding of reactivation of Tax‐specific cytotoxic T lymphocytes (CTL) in ATL patients after hematopoietic stem cell transplantation suggests the presence of Tax expression in vivo and potential contribution of the CTL to antitumor immunity. This is consistent with the results of a series of animal experiments indicating that Tax‐specific CTL limit the growth of HTLV‐I‐infected cells in vivo, although the animal model mimics only an early phase of HTLV‐I infection and leukemogenesis. Establishment of an insufficient HTLV‐I‐specific T‐cell response and an increased viral load in orally HTLV‐I‐infected rats suggests that host HTLV‐I‐specific T‐cell response at a primary HTLV‐I infection can be a critical determinant of persistent HTLV‐I levels thereafter. These findings indicate that Tax‐targeted vaccines may be effective for prophylaxis of ATL in a high‐risk group, and also for therapy of ATL in at least half the cases. (Cancer Sci 2005; 96: 249u2003–255)

Nuclear and cytoplasmic effects of human CRM1 on HIV-1 production in rat cells

The human immunodeficiency virus type 1 (HIV‐1) regulatory protein, Rev, mediates the nuclear export of unspliced gag and singly spliced env mRNAs by bridging viral RNA and the export receptor, CRM1. Recently, rat CRM1 was found to be less efficient than human CRM1 in supporting Rev function in rats. In this study, to understand the role of CRM1 in HIV propagation, the mechanism underlying the function of human and rat CRM1 in HIV‐1 replication was investigated in rat cells. The production of viral particles, represented by the p24 Gag protein, was greatly enhanced by hCRM1 expression in rat cells; however, this effect was not simply because of the enhanced export of gag mRNA. The translation initiation rate of gag mRNA was not increased, nor was the Gag protein stabilized in the presence of hCRM1. However, the processing of the p55 Gag precursor and the release of viral particles were facilitated. These results indicated that hCRM1 exports gag mRNA to the cytoplasm, not only more efficiently than rCRM1 but also correctly, leading to efficient processing of Gag proteins and particle formation.

Synergistic effect of human CycT1 and CRM1 on HIV-1 propagation in rat T cells and macrophages.

BackgroundIn vivo studies of HIV-1 pathogenesis and testing of antiviral strategies have been hampered by the lack of an immunocompetent small animal model that is highly susceptible to HIV-1 infection. Although transgenic rats that express the HIV-1 receptor complex hCD4 and hCCR5 are susceptible to infection, HIV-1 replicates very poorly in these animals. To demonstrate the molecular basis for developing a better rat model for HIV-1 infection, we evaluated the effect of human CyclinT1 (hCycT1) and CRM1 (hCRM1) on Gag p24 production in rat T cells and macrophages using both established cell lines and primary cells prepared from hCycT1/hCRM1 transgenic rats.ResultsExpression of hCycT1 augmented Gag production 20–50 fold in rat T cells, but had little effect in macrophages. Expression of hCRM1 enhanced Gag production 10–15 fold in macrophages, but only marginally in T cells. Expression of both factors synergistically enhanced p24 production to levels approximately 10–40% of those detected in human cells. R5 viruses produced in rat T cells and macrophages were fully infectious.ConclusionThe expression of both hCycT1 and hCRM1 appears to be fundamental to developing a rat model that supports robust propagation of HIV-1.

Osteopontin-integrin interaction as a novel molecular target for antibody-mediated immunotherapy in adult T-cell leukemia

BackgroundAdult T-cell leukemia (ATL) is a CD4+ T-cell neoplasm with a poor prognosis. A previous study has shown that there is a strong correlation between the secreted matricellular protein osteopontin (OPN) level and disease severity in ATL patients. Here, we investigated the role of OPN in ATL pathogenesis and the possible application of anti-OPN monoclonal antibody (mAb) for ATL immunotherapy in NOD/Shi-scid,IL-2Rgnull (NOG) mice.ResultsSubcutaneous inoculation of ATL cell lines into NOG mice increased the plasma level of OPN, which significantly correlated with metastasis of the inoculated cells and survival time. Administration of an SVVYGLR motif-recognizing anti-OPN mAb resulted in inhibition not only of tumor growth but also of tumor invasion and metastasis. The number of fibroblast activating protein-positive fibroblasts was also reduced by this mAb. We then co-inoculated mouse embryonic fibroblasts (MEFs) isolated from wild-type (WT) or OPN knockout mice together with ATL-derived TL-OmI cells into the NOG mice. The mice co-inoculated with WT MEFs displayed a significant decrease in survival relative to those injected with TL-OmI cells alone and the absence of OPN in MEFs markedly improved the survival rate of TL-OmI-inoculated mice. In addition, tumor volume and metastasis were also reduced in the absence of OPN.ConclusionWe showed that the xenograft NOG mice model can be a useful system for assessment of the physiological role of OPN in ATL pathogenesis. Using this xenograft model, we found that fibroblast-derived OPN was involved in tumor growth and metastasis, and that this tumor growth and metastasis was significantly suppressed by administration of the anti-OPN mAbs. Our findings will lead to a novel mAb-mediated immunotherapeutic strategy targeting against the interaction of OPN with integrins on the tumor of ATL patients.

Elicitation of Both Anti HIV-1 Env Humoral and Cellular Immunities by Replicating Vaccinia Prime Sendai Virus Boost Regimen and Boosting by CD40Lm

For protection from HIV-1 infection, a vaccine should elicit both humoral and cell-mediated immune responses. A novel vaccine regimen and adjuvant that induce high levels of HIV-1 Env-specific T cell and antibody (Ab) responses was developed in this study. The prime-boost regimen that used combinations of replication-competent vaccinia LC16m8Δ (m8Δ) and Sendai virus (SeV) vectors expressing HIV-1 Env efficiently produced both Env-specific CD8+ T cells and anti-Env antibodies, including neutralizing antibodies (nAbs). These results sharply contrast with vaccine regimens that prime with an Env expressing plasmid and boost with the m8Δ or SeV vector that mainly elicited cellular immunities. Moreover, co-priming with combinations of m8Δs expressing Env or a membrane-bound human CD40 ligand mutant (CD40Lm) enhanced Env-specific CD8+ T cell production, but not anti-Env antibody production. In contrast, priming with an m8Δ that coexpresses CD40Lm and Env elicited more anti-Env Abs with higher avidity, but did not promote T cell responses. These results suggest that the m8Δ prime/SeV boost regimen in conjunction with CD40Lm expression could be used as an immunization platform for driving both potent cellular and humoral immunities against pathogens such as HIV-1.

Inhibitory effect of human TRIM5α on HIV-1 production

Tripartite motif-containing 5 isoform-alpha (TRIM5alpha), a host restriction factor, blocks infection of some retroviruses at a post-entry, pre-integration stage in a species-specific manner. A recent report by Sakuma et al. describes a second antiretroviral activity of rhesus macaque TRIM5alpha, which blocks HIV-1 production through rapid degradation of HIV-1 Gag polyproteins. Here, we find that human TRIM5alpha limits HIV-1 production. Transient expression of TRIM5alpha decreased HIV-1 production, whereas knockdown of TRIM5alpha in human cells increased virion release. A single amino acid substitution (R437C) in the SPRY domain diminished the restriction effect. Moderate levels of human wild-type TRIM5alpha and a little amount of R437C mutant were incorporated into HIV-1 virions. The R437C mutant also lost restriction activity against N-tropic murine leukemia virus infection. However, the corresponding R to C mutation in rhesus macaque TRIM5alpha had no effect on the restriction ability. Our findings suggest human TRIM5alpha is an intrinsic immunity factor against HIV-1 infection. The importance of arginine at 437 aa in SPRY domain for the late restriction is species-specific.

Immunogenicity and safety of the vaccinia virus LC16m8Δ vector expressing SIV Gag under a strong or moderate promoter in a recombinant BCG prime-recombinant vaccinia virus boost protocol

We compared the effect of the very strong pSFJ1-10 and moderately strong p7.5 promoters on the immunogenicity and pathogenicity of the replication-competent vaccinia virus (VV) LC16m8Δ (m8Δ) vector harboring the SIV gag gene in a vaccination regimen consisting of a recombinant BCG-SIVGag (rBCG-SIVGag) prime followed by a recombinant vaccinia boost. m8Δ/pSFJ/SIVGag synthesized more Gag protein than m8Δ/p7.5/SIVGag but replicated less efficiently in vitro. In addition, m8Δ/pSFJ/SIVGag was less pathogenic and elicited Gag-specific IFN-γ(+), CD107a(+), CD8(+) cells more efficiently than m8Δ/p7.5/SIVGag. Vaccination by this regimen elicited long-lasting Gag-specific CD8(+) T cells, the majority of which showed a CCR7(-) phenotype at over 8 weeks post-boost. Tetramer staining analyses revealed maintenance of Gag specific tetramer(+), CD62L(-), CD8(+) T cells for long time in vaccinated mice. However, Gag expression increased the neurotoxicity of the vaccinia vector, indicating the necessity of safety testing for each recombinant VV. We propose that this recombinant BCG prime-m8Δ/pSFJ/HIVGag boost regimen would be a promising vaccination procedure for preventing HIV infection.

Functional analysis of Foxp3 and CTLA-4 expressing HTLV 1-infected cells in a rat model

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL). Some ATL cells express Foxp3, which is known as regulatory T cell (Treg cell) specific transcription factor. It is suggested that Treg cell like suppressive activity of Foxp3 expressing ATL cells is associated to ATL development and related immunodeficiency. To develop an HTLV-1 model system that enables to investigate the association of Treg function in ATL progression, we examined the expression of Foxp3 and CTLA-4, Treg cell-associated factor, in established HTLV-1-infected rat cell lines and their regulatory function. We found the expression of Foxp3 in 10 of 22 and CTLA-4 in 10 of 19 HTLV-1-infected rat cell lines. Moreover, some of the Foxp3 and/or CTLA-4 expressing cell lines suppressed proliferation of naïve T cells that were stimulated with anti-CD3 antibody. Particularly all Foxp3(+) CTLA-4(+) cells showed the suppressive activity. Our data suggest the usefulness of our rat model systems for further analysis of the role of Treg cell-associated factors on the development of ATL and related immunodeficiency in vivo.

Effects of different promoters on the virulence and immunogenicity of a HIV-1 Env-expressing recombinant vaccinia vaccine

Previously, we developed a vaccination regimen that involves priming with recombinant vaccinia virus LC16m8Δ (rm8Δ) strain followed by boosting with a Sendai virus-containing vector. This protocol induced both humoral and cellular immune responses against the HIV-1 envelope protein. The current study aims to optimize this regimen by comparing the immunogenicity and safety of two rm8Δ strains that express HIV-1 Env under the control of a moderate promoter, p7.5, or a strong promoter, pSFJ1-10. m8Δ-p7.5-JRCSFenv synthesized less gp160 but showed significantly higher growth potential than m8Δ-pSFJ-JRCSFenv. The two different rm8Δ strains induced antigen-specific immunity; however, m8Δ-pSFJ-JRCSFenv elicited a stronger anti-Env antibody response whereas m8Δ-p7.5-JRCSFenv induced a stronger Env-specific cytotoxic T lymphocyte response. Both strains were less virulent than the parental m8Δ strain, suggesting that they would be safe for use in humans. These findings indicate the vaccine can be optimized to induce favorable immune responses (either cellular or humoral), and forms the basis for the rational design of an AIDS vaccine using recombinant vaccinia as the delivery vector.

Immunogenicity of newly constructed attenuated vaccinia strain LC16m8Δ that expresses SIV Gag protein.

We developed the method to efficiently construct recombinant vaccinia viruses based on LC16m8Delta strain that can replicate in mammalian cells but is still safe in human. Immunization in a prime-boost strategy using DNA and LC16m8Delta expressing SIV Gag elicited 7-30-fold more IFN-gamma-producing T cells in mice than that using DNA and non-replicating vaccinia DIs recombinant strain. As the previous study on the DNA-prime and recombinant DIs-boost anti-SIV vaccine showed protective efficacy in the macaque model [Someya K, Ami Y, Nakasone T, Izumi Y, Matsuo K, Horibata S, et al. Induction of positive cellular and humoral responses by a prime-boost vaccine encoded with simian immunodeficiency virus gag/pol. J Immunol 2006;176(3):1784-95], LC16m8Delta would have potential as a better recombinant viral vector for HIV vaccine.