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Last Updated :2024/08/15
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- Cong Thanh Nguyen, Misako Nakayama, Hirohito Ishigaki, Yoshinori Kitagawa, Akemi Kakino, Marumi Ohno, Masashi Shingai, Yasuhiko Suzuki, Tatsuya Sawamura, Hiroshi Kida, Yasushi ItohVirology 110052 - 110052 0042-6822 2024/03
- Chimuka Handabile, Marumi Ohno, Toshiki Sekiya, Naoki Nomura, Tomomi Kawakita, Mamiko Kawahara, Masafumi Endo, Tomohiro Nishimura, Minako Okumura, Shinsuke Toba, Michihito Sasaki, Yasuko Orba, Brendon Y Chua, Louise C Rowntree, Thi H O Nguyen, Masashi Shingai, Akihiko Sato, Hirofumi Sawa, Kazumasa Ogasawara, Katherine Kedzierska, Hiroshi KidaScientific reports 14 (1) 4204 - 4204 2024/02/20Due to the synchronous circulation of seasonal influenza viruses and severe acute respiratory coronavirus 2 (SARS-CoV-2) which causes coronavirus disease 2019 (COVID-19), there is need for routine vaccination for both COVID-19 and influenza to reduce disease severity. Here, we prepared individual WPVs composed of formalin-inactivated SARS-CoV-2 WK 521 (Ancestral strain; Co WPV) or influenza virus [A/California/07/2009 (X-179A) (H1N1) pdm; Flu WPV] to produce a two-in-one Co/Flu WPV. Serum analysis from vaccinated mice revealed that a single dose of Co/Flu WPV induced antigen-specific neutralizing antibodies against both viruses, similar to those induced by either type of WPV alone. Following infection with either virus, mice vaccinated with Co/Flu WPV showed no weight loss, reduced pneumonia and viral titers in the lung, and lower gene expression of proinflammatory cytokines, as observed with individual WPV-vaccinated. Furthermore, a pentavalent vaccine (Co/qFlu WPV) comprising of Co WPV and quadrivalent influenza vaccine (qFlu WPV) was immunogenic and protected animals from severe COVID-19. These results suggest that a single dose of the two-in-one WPV provides efficient protection against SARS-CoV-2 and influenza virus infections with no evidence of vaccine interference in mice. We propose that concomitant vaccination with the two-in-one WPV can be useful for controlling both diseases.
- Masashi Shingai, Sayaka Iida, Naoko Kawai, Mamiko Kawahara, Toshiki Sekiya, Marumi Ohno, Naoki Nomura, Chimuka Handabile, Tomomi Kawakita, Ryosuke Omori, Junya Yamagishi, Kaori Sano, Akira Ainai, Tadaki Suzuki, Kazuo Ohnishi, Kimihito Ito, Hiroshi KidaJournal of Virology 0022-538X 2024/02/07As specific interactions between antigens and cell-surface antibodies trigger the proliferation of B-cell clones, the frequency of each antibody sequence in the samples reflects the size of each clonal population. Nevertheless, it is extremely difficult to extract antigen-specific antibody sequences from the comprehensive bulk antibody sequences obtained from blood samples due to repertoire bias influenced by exposure to dietary antigens and other infectious agents. This issue can be addressed by subtracting the background noise from the post-immunization or post-infection repertoire data. In the present study, we propose a method to quantify repertoire data from comprehensive repertoire data. This method allowed subtraction of the background repertoire, resulting in more accurate extraction of expanded antibody repertoires upon influenza virus infection. This accurate extraction of antigen- or infection-specific repertoire information is a useful tool for vaccine evaluation.
- Marumi Ohno, Siddabasave Gowda B Gowda, Toshiki Sekiya, Naoki Nomura, Masashi Shingai, Shu-Ping Hui, Hiroshi KidaScientific reports 13 (1) 14210 - 14210 2023/08/30Although influenza virus infection has been shown to affect lipid metabolism, details remain unknown. Therefore, we elucidated the kinetic lipid profiles of mice infected with different doses of influenza virus A/Puerto Rico/8/34 (H1N1) (PR8) by measuring multiple lipid molecular species using untargeted lipidomic analysis. C57BL/6 male mice were intranasally infected with PR8 virus at 50 or 500 plaque-forming units to cause sublethal or lethal influenza, respectively. Plasma and tissue samples were collected at 1, 3, and 6 days post-infection (dpi), and comprehensive lipidomic analysis was performed using high-performance liquid chromatography-linear trap quadrupole-Orbitrap mass spectrometry, as well as gene expression analyses. The most prominent feature of the lipid profile in lethally infected mice was the elevated plasma concentrations of phosphatidylethanolamines (PEs) containing polyunsaturated fatty acid (PUFA) at 3 dpi. Furthermore, the facilitation of PUFA-containing phospholipid production in the lungs, but not in the liver, was suggested by gene expression and lipidomic analysis of tissue samples. Given the increased plasma or serum levels of PUFA-containing PEs in patients with other viral infections, especially in severe cases, the elevation of these phospholipids in circulation could be a biomarker of infection and the severity of infectious diseases.
- Tsukasa Seya, Masashi Shingai, Tomomi Kawakita, Misako MatsumotoCells 12 (11) 2023/05/29Viral infections are usually accompanied by systemic cytokinemia. Vaccines need not necessarily mimic infection by inducing cytokinemia, but must induce antiviral-acquired immunity. Virus-derived nucleic acids are potential immune-enhancers and particularly good candidates as adjuvants in vaccines in mouse models. The most important nucleic-acid-sensing process involves the dendritic cell (DC) Toll-like receptor (TLR), which participates in the pattern recognition of foreign DNA/RNA structures. Human CD141+ DCs preferentially express TLR3 in endosomes and recognize double-stranded RNA. Antigen cross-presentation occurs preferentially in this subset of DCs (cDCs) via the TLR3-TICAM-1-IRF3 axis. Another subset, plasmacytoid DCs (pDCs), specifically expresses TLR7/9 in endosomes. They then recruit the MyD88 adaptor, and potently induce type I interferon (IFN-I) and proinflammatory cytokines to eliminate the virus. Notably, this inflammation leads to the secondary activation of antigen-presenting cDCs. Hence, the activation of cDCs via nucleic acids involves two modes: (i) with bystander effect of inflammation and (ii) without inflammation. In either case, the acquired immune response finally occurs with Th1 polarity. The level of inflammation and adverse events depend on the TLR repertoire and the mode of response to their agonists in the relevant DC subsets, and could be predicted by assessing the levels of cytokines/chemokines and T cell proliferation in vaccinated subjects. The main differences in the mode of vaccine sought in infectious diseases and cancer are defined by whether it is prophylactic or therapeutic, whether it can deliver sufficient antigens to cDCs, and how it behaves in the microenvironment of the lesion. Adjuvant can be selected on a case-to-case basis.
- Marumi Ohno, Masataka Sagata, Toshiki Sekiya, Naoki Nomura, Masashi Shingai, Masafumi Endo, Kazuhiko Kimachi, Saori Suzuki, Cong Thanh Nguyen, Misako Nakayama, Hirohito Ishigaki, Kazumasa Ogasawara, Yasushi Itoh, Yoichiro Kino, Hiroshi KidaVaccine 41 (3) 787 - 794 2023/01/16 [Refereed]
Among inactivated influenza vaccines, the whole virus particle vaccine (WPV) elicits superior priming responses to split virus vaccine (SV) in efficiently inducing humoral and cellular immunity. However, there is concern for undesired adverse events such as fever for WPV due to its potent immunogenicity. Therefore, this study investigated the febrile response induced by subcutaneous injection with quadrivalent inactivated influenza vaccines of good manufacturing grade for pharmaceutical or investigational products in cynomolgus macaques. Body temperature was increased by 1 °C-2 °C for 6-12 h after WPV administration at the first vaccination but not at the second shot, whereas SV did not affect body temperature at both points. Given the potent priming ability of WPV, WPV-induced fever may be attributed to immune responses that uniquely occur during priming. Since WPV-induced fever was blunted by pretreatment with indomethacin (a cyclooxygenase inhibitor), the febrile response by WPV is considered to depend on the increase in prostaglandins synthesized by cyclooxygenase. In addition, WPV, but not SV, induced the elevation of type I interferons and monocyte chemotactic protein 1 in the plasma; these factors may be responsible for pyrogenicity caused by WPV, as they can increase prostaglandins in the brain. Notably, sufficient antibody responses were acquired by half the amount of WPV without causing fever, suggesting that excessive immune responses to trigger the febrile response is not required for acquired immunity induction. Thus, we propose that WPV with a reduced antigen dose should be evaluated for potential clinical usage, especially in naïve populations. - Kyoko Hayashida, Alejandro Garcia, Lavel Chinyama Moonga, Tatsuki Sugi, Kodera Takuya, Mitsuo Kawase, Fumihiro Kodama, Atsushi Nagasaka, Nobuhisa Ishiguro, Ayato Takada, Masahiro Kajihara, Naganori Nao, Masashi Shingai, Hiroshi Kida, Yasuhiko Suzuki, William W Hall, Hirofumi Sawa, Junya YamagishiPloS one 18 (5) e0285861 2023 [Refereed]
A novel multiplex loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography was developed for the simultaneous detection of three important respiratory disease-causing viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus. Amplification was performed at a constant temperature, and a positive result was confirmed by a visible colored band. An in-house drying protocol with trehalose was used to prepare the dried format multiplex LAMP test. Using this dried multiplex LAMP test, the analytical sensitivity was determined to be 100 copies for each viral target and 100-1000 copies for the simultaneous detection of mixed targets. The multiplex LAMP system was validated using clinical COVID-19 specimens and compared with the real-time qRT-PCR method as a reference test. The determined sensitivity of the multiplex LAMP system for SARS-CoV-2 was 71% (95% CI: 0.62-0.79) for cycle threshold (Ct) ≤ 35 samples and 61% (95% CI: 0.53-0.69) for Ct ≤40 samples. The specificity was 99% (95%CI: 0.92-1.00) for Ct ≤35 samples and 100% (95%CI: 0.92-1.00) for the Ct ≤40 samples. The developed simple, rapid, low-cost, and laboratory-free multiplex LAMP system for the two major important respiratory viral diseases, COVID-19 and influenza, is a promising field-deployable diagnosis tool for the possible future 'twindemic, ' especially in resource-limited settings. - Brendon Y. Chua, Toshiki Sekiya, Marios Koutsakos, Naoki Nomura, Louise C. Rowntree, Thi H. O. Nguyen, Hayley A. McQuilten, Marumi Ohno, Yuki Ohara, Tomohiro Nishimura, Masafumi Endo, Yasushi Itoh, Jennifer R. Habel, Kevin J. Selva, Adam K. Wheatley, Bruce D. Wines, P. Mark Hogarth, Stephen J. Kent, Amy W. Chung, David C. Jackson, Lorena E. Brown, Masashi Shingai, Katherine Kedzierska, Hiroshi KidaPLOS Pathogens 18 (10) e1010891 - e1010891 2022/10/07 [Refereed]
Although antibody-inducing split virus vaccines (SV) are currently the most effective way to combat seasonal influenza, their efficacy can be modest, especially in immunologically-naïve individuals. We investigated immune responses towards inactivated whole influenza virus particle vaccine (WPV) formulations, predicated to be more immunogenic, in a non-human primate model, as an important step towards clinical testing in humans. Comprehensive analyses were used to capture 46 immune parameters to profile how WPV-induced responses differed to those elicited by antigenically-similar SV formulations. Naïve cynomolgus macaques vaccinated with either monovalent or quadrivalent WPV consistently induced stronger antibody responses and hemagglutination inhibition (HI) antibody titres against vaccine-matched viruses compared to SV formulations, while acute reactogenic effects were similar. Responses in WPV-primed animals were further increased by boosting with the same formulation, conversely to modest responses after priming and boosting with SV. 28-parameter multiplex bead array defined key antibody features and showed that while both WPV and SV induced elevated IgG responses against A/H1N1 nucleoprotein, only WPV increased IgG responses against A/H1N1 hemagglutinin (HA) and HA-Stem, and higher IgA responses to A/H1N1-HA after each vaccine dose. Antibodies to A/H1N1-HA and HA-Stem that could engage FcγR2a and FcγR3a were also present at higher levels after one dose of WPV compared to SV and remained elevated after the second dose. Furthermore, WPV-enhanced antibody responses were associated with higher frequencies of HA-specific B-cells and IFN-γ-producing CD4+ T-cell responses. Our data additionally demonstrate stronger boosting of HI titres by WPV following prior infection and support WPV administered as a priming dose irrespective of the follow up vaccine for the second dose. Our findings thus show that compared to SV vaccination, WPV-induced humoral responses are significantly increased in scope and magnitude, advocating WPV vaccination regimens for priming immunologically-naïve individuals and also in the event of a pandemic outbreak. - Hirotaka Minagawa, Hirofumi Sawa, Tomoko Fujita, Shintaro Kato, Asumi Inaguma, Miwako Hirose, Yasuko Orba, Michihito Sasaki, Koshiro Tabata, Naoki Nomura, Masashi Shingai, Yasuhiko Suzuki, Katsunori HoriiBiochemical and biophysical research communications 614 207 - 212 2022/07/23 [Refereed]
Simple, highly sensitive detection technologies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are crucial for the effective implementation of public health policies. We used the systematic evolution of ligands by exponential enrichment with a modified DNA library, including a base-appended base (uracil with a guanine base at its fifth position), to create an aptamer with a high affinity for the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. The aptamer had a dissociation constant of 1.2 and < 1 nM for the RBD and spike trimer, respectively. Furthermore, enzyme-linked aptamer assays confirmed that the aptamer binds to isolated authentic SARS-CoV-2 wild-type and B.1.617.2 (delta variant). The binding signal was larger that of commercially available anti-SARS-CoV-2 RBD antibody. Thus, this aptamer as a sensing element will enable the highly sensitive detection of SARS-CoV-2. - Masanori Shiohara, Saori Suzuki, Shintaro Shichinohe, Hirohito Ishigaki, Misako Nakayama, Naoki Nomura, Masashi Shingai, Toshiki Sekiya, Marumi Ohno, Sayaka Iida, Naoko Kawai, Mamiko Kawahara, Junya Yamagishi, Kimihito Ito, Ryotarou Mitsumata, Tomio Ikeda, Kenji Motokawa, Tomoyoshi Sobue, Hiroshi Kida, Kazumasa Ogasawara, Yasushi ItohVaccine 40 (30) 4026 - 4037 2022/06/26 [Refereed][Not invited]
The All-Japan Influenza Vaccine Study Group has been developing a more effective vaccine than the current split vaccines for seasonal influenza virus infection. In the present study, the efficacy of formalin- and/or β-propiolactone-inactivated whole virus particle vaccines for seasonal influenza was compared to that of the current ether-treated split vaccines in a nonhuman primate model. The monovalent whole virus particle vaccines or split vaccines of influenza A virus (H1N1) and influenza B virus (Victoria lineage) were injected subcutaneously into naïve cynomolgus macaques twice. The whole virus particle vaccines induced higher titers of neutralizing antibodies against H1N1 influenza A virus and influenza B virus in the plasma of macaques than did the split vaccines. At challenge with H1N1 influenza A virus or influenza B virus, the virus titers in nasal swabs and the increases in body temperatures were lower in the macaques immunized with the whole virus particle vaccine than in those immunized with the split vaccine. Repertoire analyses of immunoglobulin heavy chain genes demonstrated that the number of B-lymphocyte subclones was increased in macaques after the 1st vaccination with the whole virus particle vaccine, but not with the split vaccine, indicating that the whole virus particle vaccine induced the activation of vaccine antigen-specific B-lymphocytes more vigorously than did the split vaccine at priming. Thus, the present findings suggest that the superior antibody induction ability of the whole virus particle vaccine as compared to the split vaccine is attributable to its stimulatory properties on the subclonal differentiation of antigen-specific B-lymphocytes. - Chimuka Handabile, Toshiki Sekiya, Naoki Nomura, Marumi Ohno, Tomomi Kawakita, Masashi Shingai, Hiroshi KidaVaccines 10 (5) 2022/05/19 [Refereed]
Despite the use of vaccines, seasonal influenza remains a risk to public health. We previously proposed the inactivated whole virus particle vaccine (WPV) as an alternative to the widely used split vaccine (SV) for the control of seasonal and pandemic influenza based on the superior priming potency of WPV to that of SV. In this study, we further examined and compared the immunological potency of monovalent WPV and SV of A/California/7/2009 (X-179A) (H1N1) pdm09 (CA/09) to generate immune responses against heterologous viruses, A/Singapore/GP1908/2015 (IVR-180) (H1N1) pdm09 (SG/15), and A/duck/Hokkaido/Vac-3/2007 (H5N1) (DH/07) in mice. Following challenge with a lethal dose of heterologous SG/15, lower virus titer in the lungs and milder weight loss were observed in WPV-vaccinated mice than in SV-vaccinated ones. To investigate the factors responsible for the differences in the protective effect against SG/15, the sera of vaccinated mice were analyzed by hemagglutination-inhibition (HI) and neuraminidase-inhibition (NI) assays to evaluate the antibodies induced against viral hemagglutinin (HA) and neuraminidase (NA), respectively. While the two vaccines induced similar levels of HI antibodies against SG/15 after the second vaccination, only WPV-vaccinated mice induced significantly higher titers of NI antibodies against the strain. Furthermore, given the significant elevation of NI antibody titers against DH/07, an H5N1 avian influenza virus, WPV was also demonstrated to induce NA-inhibiting antibodies that recognize NA of divergent strains. This could be explained by the higher conservation of epitopes of NA among strains than for HA. Taking these findings together, NA-specific antibodies induced by WPV may have contributed to better protection from infection with heterologous influenza virus SG/15, compared with SV. The present results indicate that WPV is an effective vaccine for inducing antibodies against both HA and NA of heterologous viruses and may be a useful vaccine to conquer vaccine strain mismatch. - Yasutake Yanagihara, Sharon Y A M Villanueva, Naoki Nomura, Marumi Ohno, Toshiki Sekiya, Chimuka Handabile, Masashi Shingai, Hideaki Higashi, Shin-Ichi Yoshida, Toshiyuki Masuzawa, Nina G Gloriani, Mitsumasa Saito, Hiroshi KidaMicrobiology spectrum 10 (2) e0215721 2022/04/27 [Refereed]
Leptospirosis is a zoonotic disease caused by infection with pathogenic leptospires. Consistent with recent studies by other groups, leptospires were isolated from 89 out of 110 (80.9%) soil or water samples from varied locations in the Philippines in our surveillance study, indicating that leptospires might have a life cycle that does not involve animal hosts. However, despite previous work, it has not been confirmed whether leptospires multiply in the soil environment under various experimental conditions. Given the fact that the case number of leptospirosis is increased after flood, we hypothesized that waterlogged soil, which mimics the postflooding environment, could be a suitable condition for growing leptospires. To verify this hypothesis, pathogenic and saprophytic leptospires were seeded in the bottles containing 2.5 times as much water as soil, and bacterial counts in the bottles were measured over time. Pathogenic and saprophytic leptospires were found to increase their number in waterlogged soil but not in water or soil alone. In addition, leptospires were reisolated from soil in closed tubes for as long as 379 days. These results indicate that leptospires are in a resting state in the soil and are able to proliferate with increased water content in the environment. This notion is strongly supported by observations that the case number of leptospirosis is significantly higher in rainy seasons and increased after flood. Therefore, we reached the following conclusion: environmental soil is a potential reservoir of leptospires. IMPORTANCE Since research on Leptospira has focused on pathogenic leptospires, which are supposed to multiply only in animal hosts, the life cycle of saprophytic leptospires has long been a mystery. This study demonstrates that both pathogenic and saprophytic leptospires multiply in the waterlogged soil, which mimics the postflooding environment. The present results potentially explain why leptospirosis frequently occurs after floods. Therefore, environmental soil is a potential reservoir of leptospires and leptospirosis is considered an environment-borne as well as a zoonotic disease. This is a significant report to reveal that leptospires multiply under environmental conditions, and this finding leads us to reconsider the ecology of leptospires. - Marumi Ohno, Michihito Sasaki, Yasuko Orba, Toshiki Sekiya, Md Abdul Masum, Osamu Ichii, Tatsuya Sawamura, Akemi Kakino, Yasuhiko Suzuki, Hiroshi Kida, Hirofumi Sawa, Masashi ShingaiViruses 13 (11) 2021/11 [Refereed]
Systemic symptoms have often been observed in patients with coronavirus disease 2019 (COVID-19) in addition to pneumonia, however, the details are still unclear due to the lack of an appropriate animal model. In this study, we investigated and compared blood coagulation abnormalities and tissue damage between male Syrian hamsters of 9 (young) and over 36 (aged) weeks old after intranasal infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite similar levels of viral replication and inflammatory responses in the lungs of both age groups, aged but not young hamsters showed significant prolongation of prothrombin time and prominent acute kidney damage. Moreover, aged hamsters demonstrated increased intravascular coagulation time-dependently in the lungs, suggesting that consumption of coagulation factors causes prothrombin time prolongation. Furthermore, proximal urinary tract damage and mesangial matrix expansion were observed in the kidneys of the aged hamsters at early and later disease stages, respectively. Given that the severity and mortality of COVID-19 are higher in elderly human patients, the effect of aging on pathogenesis needs to be understood and should be considered for the selection of animal models. We, thus, propose that the aged hamster is a good small animal model for COVID-19 research. - Divyavani Gowda, Marumi Ohno, Siddabasave Gowda B Gowda, Hitoshi Chiba, Masashi Shingai, Hiroshi Kida, Shu-Ping HuiScientific reports 11 (1) 20161 - 20161 2021/10/11 [Refereed]
Influenza remains a world-wide health concern, causing 290,000-600,000 deaths and up to 5 million cases of severe illnesses annually. Noticing the host factors that control biological responses, such as inflammatory cytokine secretion, to influenza virus infection is important for the development of novel drugs. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite and has essential biological functions in inflammation. However, the kinetic effects of influenza virus infection on physiological S1P levels and their signaling in multiple tissues remain unknown. In this study, we utilized a mouse model intranasally infected with 50 or 500 plaque forming units (PFU) of A/Puerto Rico/8/34 (H1N1; PR8) virus to investigate how S1P levels and expression of its regulating factors are affected by influenza virus infection by the liquid-chromatography/mass spectrometry and real-time PCR, respectively. The S1P level was significantly high in the plasma of mice infected with 500 PFU of the virus than that in control mice at 6 day-post-infection (dpi). Elevated gene expression of sphingosine kinase-1 (Sphk1), an S1P synthase, was observed in the liver, lung, white adipose tissue, heart, and aorta of infected mice. This could be responsible for the increased plasma S1P levels as well as the decrease in the hepatic S1P lyase (Sgpl1) gene in the infected mice. These results indicate modulation of S1P-signaling by influenza virus infection. Since S1P regulates inflammation and leukocyte migration, it must be worth trying to target this signaling to control influenza-associated symptoms. - Marumi Ohno, Akemi Kakino, Toshiki Sekiya, Naoki Nomura, Masashi Shingai, Tatsuya Sawamura, Hiroshi KidaScientific reports 11 (1) 15675 - 15675 2021/08/03 [Refereed]
Although coagulation abnormalities, including microvascular thrombosis, are thought to contribute to tissue injury and single- or multiple-organ dysfunction in severe influenza, the detailed mechanisms have yet been clarified. This study evaluated influenza-associated abnormal blood coagulation utilizing a severe influenza mouse model. After infecting C57BL/6 male mice with intranasal applications of 500 plaque-forming units of influenza virus A/Puerto Rico/8/34 (H1N1; PR8), an elevated serum level of prothrombin fragment 1 + 2, an indicator for activated thrombin generation, was observed. Also, an increased gene expression of oxidized low-density lipoprotein (LDL) receptor-1 (Olr1), a key molecule in endothelial dysfunction in the progression of atherosclerosis, was detected in the aorta of infected mice. Body weight decrease, serum levels of cytokines and chemokines, viral load, and inflammation in the lungs of infected animals were similar between wild-type and Olr1 knockout (KO) mice. In contrast, the elevation of prothrombin fragment 1 + 2 levels in the sera and intravascular thrombosis in the lungs by PR8 virus infection were not induced in KO mice. Collectively, the results indicated that OLR1 is a critical host factor in intravascular thrombosis as a pathogeny of severe influenza. Thus, OLR1 is a promising novel therapeutic target for thrombosis during severe influenza. - Masashi Shingai, Naoki Nomura, Toshiki Sekiya, Marumi Ohno, Daisuke Fujikura, Chimuka Handabile, Ryosuke Omori, Yuki Ohara, Tomohiro Nishimura, Masafumi Endo, Kazuhiko Kimachi, Ryotarou Mitsumata, Tomio Ikeda, Hiroki Kitayama, Hironori Hatanaka, Tomoyoshi Sobue, Fumihito Muro, Saori Suzuki, Cong Thanh Nguyen, Hirohito Ishigaki, Misako Nakayama, Yuya Mori, Yasushi Itoh, Marios Koutsakos, Brendon Y Chua, Katherine Kedzierska, Lorena E Brown, David C Jackson, Kazumasa Ogasawara, Yoichiro Kino, Hiroshi KidaVaccine 39 (29) 3940 - 3951 2021/06/29 [Refereed]
Current detergent or ether-disrupted split vaccines (SVs) for influenza do not always induce adequate immune responses, especially in young children. This contrasts with the whole virus particle vaccines (WPVs) originally used against influenza that were immunogenic in both adults and children but were replaced by SV in the 1970s due to concerns with reactogenicity. In this study, we re-evaluated the immunogenicity of WPV and SV, prepared from the same batch of purified influenza virus, in cynomolgus macaques and confirmed that WPV is superior to SV in priming potency. In addition, we compared the ability of WPV and SV to induce innate immune responses, including the maturation of dendritic cells (DCs) in vitro. WPV stimulated greater production of inflammatory cytokines and type-I interferon in immune cells from mice and macaques compared to SV. Since these innate responses are likely triggered by the activation of pattern recognition receptors (PRRs) by viral RNA, the quantity and quality of viral RNA in each vaccine were assessed. Although the quantity of viral RNA was similar in the two vaccines, the amount of viral RNA of a length that can be recognized by PRRs was over 100-fold greater in WPV than in SV. More importantly, 1000-fold more viral RNA was delivered to DCs by WPV than by SV when exposed to preparations containing the same amount of HA protein. Furthermore, WPV induced up-regulation of the DC maturation marker CD86 on murine DCs, while SV did not. The present results suggest that the activation of antigen-presenting DCs, by PRR-recognizable viral RNA contained in WPV is responsible for the effective priming potency of WPV observed in naïve mice and macaques. WPV is thus recommended as an alternative option for seasonal influenza vaccines, especially for children. - Toshiki Sekiya, Marumi Ohno, Naoki Nomura, Chimuka Handabile, Masashi Shingai, David C Jackson, Lorena E Brown, Hiroshi KidaViruses 13 (6) 2021/05/24 [Refereed]
Despite seasonal influenza vaccines having been routinely used for many decades, influenza A virus continues to pose a global threat to humans, causing high morbidity and mortality each year. The effectiveness of the vaccine is largely dependent on how well matched the vaccine strains are with the circulating influenza virus strains. Furthermore, low vaccine efficacy in naïve populations such as young children, or in the elderly, who possess weakened immune systems, indicates that influenza vaccines need to be more personalized to provide broader community protection. Advances in both vaccine technologies and our understanding of influenza virus infection and immunity have led to the design of a variety of alternate vaccine strategies to extend population protection against influenza, some of which are now in use. In this review, we summarize the progress in the field of influenza vaccines, including the advantages and disadvantages of different strategies, and discuss future prospects. We also highlight some of the challenges to be faced in the ongoing effort to control influenza through vaccination. - Naoki Nomura, Keita Matsuno, Masashi Shingai, Marumi Ohno, Toshiki Sekiya, Ryosuke Omori, Yoshihiro Sakoda, Robert G Webster, Hiroshi KidaVirology 557 55 - 61 2021/05 [Refereed]
Genetic reassortment of influenza A viruses through cross-species transmission contributes to the generation of pandemic influenza viruses. To provide information on the ecology of influenza viruses, we have been conducting a global surveillance of zoonotic influenza and establishing an influenza virus library. Of 4580 influenza virus strains in the library, 3891 have been isolated from over 70 different bird species. The remaining 689 strains were isolated from humans, pigs, horses, seal, whale, and the environment. Phylogenetic analyses of the HA genes of the library isolates demonstrate that the library strains are distributed to all major known clusters of the H1, H2 and H3 subtypes of HA genes that are prevalent in humans. Since past pandemic influenza viruses are most likely genetic reassortants of zoonotic and seasonal influenza viruses, a vast collection of influenza A virus strains from various hosts should be useful for vaccine preparation and diagnosis for future pandemics. - Marios Koutsakos, Toshiki Sekiya, Brendon Y Chua, Thi Hoang Oanh Nguyen, Adam K Wheatley, Jennifer A Juno, Marumi Ohno, Naoki Nomura, Yuki Ohara, Tomohiro Nishimura, Masafumi Endo, Saori Suzuki, Hirohito Ishigaki, Misako Nakayama, Cong T Nguyen, Yasushi Itoh, Masashi Shingai, Kazumasa Ogasawara, Yoichiro Kino, Stephen J Kent, David C Jackson, Lorena E Brown, Hiroshi Kida, Katherine KedzierskaImmunology and cell biology 99 (1) 97 - 106 2021/01 [Refereed]
Influenza remains a significant global public health burden, despite substantial annual vaccination efforts against circulating virus strains. As a result, novel vaccine approaches are needed to generate long-lasting and universal broadly cross-reactive immunity against distinct influenza virus strains and subtypes. Several new vaccine candidates are currently under development and/or in clinical trials. The successful development of new vaccines requires testing in animal models, other than mice, which capture the complexity of the human immune system. Importantly, following vaccination or challenge, the assessment of adaptive immunity at the antigen-specific level is particularly informative. In this study, using peripheral blood mononuclear cells (PBMCs) from cynomolgus macaques, we describe detection methods and in-depth analyses of influenza virus-specific B cells by recombinant hemagglutinin probes and flow cytometry, as well as the detection of influenza virus-specific CD8+ and CD4+ T cells by stimulation with live influenza A virus and intracellular cytokine staining. We highlight the potential of these assays to be used with PBMCs from other macaque species, including rhesus macaques, pigtail macaques and African green monkeys. We also demonstrate the use of a human cytometric bead array kit in detecting inflammatory cytokines and chemokines from cynomolgus macaques to assess cytokine/chemokine milieu. Overall, the detection of influenza virus-specific B and T cells, together with inflammatory responses, as described in our study, provides useful insights for evaluating novel influenza vaccines. Our data deciphering immune responses toward influenza viruses can be also adapted to understanding immunity to other infections or vaccination approaches in macaque models. - Marumi Ohno, Toshiki Sekiya, Naoki Nomura, Taku ji Daito, Masashi Shingai, Hiroshi KidaScientific Reports 10 (1) 2020/12 [Refereed]
- Toshiki Sekiya, Edin J Mifsud, Marumi Ohno, Naoki Nomura, Mayumi Sasada, Daisuke Fujikura, Takuji Daito, Masashi Shingai, Yuki Ohara, Tomohiro Nishimura, Masafumi Endo, Ryotarou Mitsumata, Tomio Ikeda, Hironori Hatanaka, Hiroki Kitayama, Kenji Motokawa, Tomoyoshi Sobue, Saori Suzuki, Yasushi Itoh, Lorena E Brown, Kazumasa Ogasawara, Yoichiro Kino, Hiroshi KidaVaccine 37 (15) 2158 - 2166 0264-410X 2019/03 [Refereed][Not invited]
- Yoshiaki Nishimura, Rajeev Gautam, Tae-Wook Chun, Reza Sadjadpour, Kathryn E. Foulds, Masashi Shingai, Florian Klein, Anna Gazumyan, Jovana Golijanin, Mitzi Donaldson, Olivia K. Donau, Ronald J. Plishka, Alicia Buckler-White, Michael S. Seaman, Jeffrey D. Lifson, Richard A. . Koup, Anthony S. Fauci, Michel C. . Nussenzweig, Malcolm A. MartinNATURE 543 (7646) 559 - + 0028-0836 2017/03 [Refereed][Not invited]
Highly potent and broadly neutralizing anti-HIV-1 antibodies (bNAbs) have been used to prevent and treat lentivirus infections in humanized mice, macaques, and humans(1-12). In immunotherapy experiments, administration of bNAbs to chronically infected animals transiently suppresses virus replication, which invariably returns to pre-treatment levels and results in progression to clinical disease. Here we show that early administration of bNAbs in a macaque simian/human immunodeficiency virus (SHIV) model is associated with very low levels of persistent viraemia, which leads to the establishment of T-cell immunity and resultant longterm infection control. Animals challenged with SHIVAD8-EO by mucosal or intravenous routes received a single 2-week course of two potent passively transferred bNAbs (3BNC117 and 10-1074 (refs 13, 14)). Viraemia remained undetectable for 56-177 days, depending on bNAb half-life in vivo. Moreover, in the 13 treated monkeys, plasma virus loads subsequently declined to undetectable levels in 6 controller macaques. Four additional animals maintained their counts of T cells carrying the CD4 antigen (CD4+) and very low levels of viraemia persisted for over 2 years. The frequency of cells carrying replication-competent virus was less than 1 per 106 circulating CD4(+) T cells in the six controller macaques. Infusion of a T-cell-depleting anti-CD8 beta monoclonal antibody to the controller animals led to a specific decline in levels of CD8(+) T cells and the rapid reappearance of plasma viraemia. In contrast, macaques treated for 15 weeks with combination anti-retroviral therapy, beginning on day 3 after infection, experienced sustained rebound plasma viraemia when treatment was interrupted. Our results show that passive immunotherapy during acute SHIV infection differs from combination anti-retroviral therapy in that it facilitates the emergence of potent CD8(+) T-cell immunity able to durably suppress virus replication. - Hiromi Takaki, Hiroyuki Oshiumi, Masashi Shingai, Misako Matsumoto, Tsukasa SeyaMICROBIOLOGY AND IMMUNOLOGY 61 (3-4) 107 - 113 0385-5600 2017/03 [Refereed][Not invited]
Viruses usually exhibit strict species-specificity as a result of co-evolution with the host. Thus, in mouse models, a great barrier exists for analysis of infections with human-tropic viruses. Mouse models are unlikely to faithfully reproduce the human immune response to viruses or viral compounds and it is difficult to evaluate human therapeutic efficacy with antiviral reagents in mouse models. Humans and mice essentially have different immune systems, which makes it difficult to extrapolate mouse results to humans. In addition, apart from immunological reasons, viruses causing human diseases do not always infect mice because of species tropism. One way to determine tropism would be a virus receptor that is expressed on affected cells. The development of gene-disrupted mice and Tg mice, which express human receptor genes, enables us to analyze several viral infections in mice. Mice are, indeed, susceptible to human viruses when artificially infected in receptor-supplemented mice. Although the mouse cells less efficiently permit viral replication than do human cells, the models for analysis of human viruses have been established in vivo as well as in vitro, and explain viral pathogenesis in the mouse systems. In most systems, however, nucleic acid sensors and type I interferon suppress viral propagation to block the appearance of infectious manifestation. We herein review recent insight into in vivo antiviral responses induced in mouse infection models for typical human viruses. - Chia-Yen Chen, Masashi Shingai, Sarah Welbourn, Malcolm A. Martin, Pedro Borrego, Nuno Taveira, Klaus StrebelJOURNAL OF VIROLOGY 90 (24) 11062 - 11074 0022-538X 2016/12 [Refereed][Not invited]
Although HIV-2 does not encode a vpu gene, the ability to antagonize bone marrow stromal antigen 2 (BST-2) is conserved in some HIV-2 isolates, where it is controlled by the Env glycoprotein. We previously reported that a single-amino-acid difference between the laboratory-adapted ROD10 and ROD14 Envs controlled the enhancement of virus release (referred to here as Vpu-like) activity. Here, we investigated how conserved the Vpu-like activity is in primary HIV-2 isolates. We found that half of the 34 tested primary HIV-2 Env isolates obtained from 7 different patients enhanced virus release. Interestingly, most HIV-2 patients harbored a mixed population of viruses containing or lacking Vpu-like activity. Vpu-like activity and Envelope functionality varied significantly among Env isolates; however, there was no direct correlation between these two functions, suggesting they evolved independently. In comparing the Env sequences from one HIV-2 patient, we found that similar to the ROD10/ROD14 Envs, a single-amino-acid change (T568I) in the ectodomain of the TM subunit was sufficient to confer Vpu-like activity to an inactive Env variant. Surprisingly, however, absence of Vpu-like activity was not correlated with absence of BST-2 interaction. Taken together, our data suggest that maintaining the ability to antagonize BST-2 is of functional relevance not only to HIV-1 but also to HIV-2 as well. Our data show that as with Vpu, binding of HIV-2 Env to BST-2 is important but not sufficient for antagonism. Finally, as observed previously, the Vpu-like activity in HIV-2 Env can be controlled by single-residue changes in the TM subunit. IMPORTANCE Lentiviruses such as HIV-1 and HIV-2 encode accessory proteins whose function is to overcome host restriction mechanisms. Vpu is a well-studied HIV-1 accessory protein that enhances virus release by antagonizing the host restriction factor BST-2. HIV-2 does not encode a vpu gene. Instead, the HIV-2 Env glycoprotein was found to antagonize BST-2 in some isolates. Here, we cloned multiple Env sequences from 7 HIV-2-infected patients and found that about half were able to antagonize BST-2. Importantly, most HIV-2 patients harbored a mixed population of viruses containing or lacking the ability to antagonize BST-2. In fact, in comparing Env sequences from one patient combined with site-directed mutagenesis, we were able to restore BST-2 antagonism to an inactive Env protein by a single-amino-acid change. Our data suggest that targeting BST-2 by HIV-2 Env is a dynamic process that can be regulated by simple changes in the Env sequence. - Masashi Shingai, Sarah Welbourn, Jason M. Brenchley, Priyamvada Acharya, Eri Miyagi, Ronald J. Plishka, Alicia Buckler-White, Peter D. Kwong, Yoshiaki Nishimura, Klaus Strebel, Malcolm A. MartinPLOS PATHOGENS 11 (5) e1004928 1553-7366 2015/05 [Refereed][Not invited]
For nearly 20 years, the principal biological function of the HIV-2/SIV Vpx gene has been thought to be required for optimal virus replication in myeloid cells. Mechanistically, this Vpx activity was recently reported to involve the degradation of Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1) in this cell lineage. Here we show that when macaques were inoculated with either the T cell tropic SIVmac239 or the macrophage tropic SIVmac316 carrying a Vpx point mutation that abrogates the recruitment of DCAF1 and the ensuing degradation of endogenous SAMHD1 in cultured CD4(+) T cells, virus acquisition, progeny virion production in memory CD4(+) T cells during acute infection, and the maintenance of set-point viremia were greatly attenuated. Revertant viruses emerging in two animals exhibited an augmented replication phenotype in memory CD4(+) T lymphocytes both in vitro and in vivo, which was associated with reduced levels of endogenous SAMHD1. These results indicate that a critical role of Vpx in vivo is to promote the degradation of SAMHD1 in memory CD4(+) T lymphocytes, thereby generating high levels of plasma viremia and the induction of immunodeficiency. - Joseph R. Francica, Zizhang Sheng, Zhenhai Zhang, Yoshiaki Nishimura, Masashi Shingai, Akshaya Ramesh, Brandon F. Keele, Stephen D. Schmidt, Barbara J. Flynn, Sam Darko, Rebecca M. Lynch, Takuya Yamamoto, Rodrigo Matus-Nicodemos, David Wolinsky, Martha Nason, Nicholas M. Valiante, Padma Malyala, Ennio De Gregorio, Susan W. Barnett, Manmohan Singh, Derek T. O'Hagan, Richard A. Koup, John R. Mascola, Malcolm A. Martin, Thomas B. Kepler, Daniel C. Douek, Lawrence Shapiro, Robert A. SederNATURE COMMUNICATIONS 6 6565 2041-1723 2015/04 [Refereed][Not invited]
Developing predictive animal models to assess how candidate vaccines and infection influence the ontogenies of Envelope (Env)-specific antibodies is critical for the development of an HIV vaccine. Here we use two nonhuman primate models to compare the roles of antigen persistence, diversity and innate immunity. We perform longitudinal analyses of HIV Env-specific B-cell receptor responses to SHIVAD8 infection and Env protein vaccination with eight different adjuvants. A subset of the SHIVAD8-infected animals with higher viral loads and greater Env diversity show increased neutralization associated with increasing somatic hypermutation (SHM) levels over time. The use of adjuvants results in increased ELISA titres but does not affect the mean SHM levels or CDR H3 lengths. Our study shows how the ontogeny of Env-specific B cells can be tracked, and provides insights into the requirements for developing neutralizing antibodies that should facilitate translation to human vaccine studies. - Florian Klein, Lilian Nogueira, Yoshiaki Nishimura, Ganesh Phad, Anthony P. West, Ariel Halper-Stromberg, Joshua A. Horwitz, Anna Gazumyan, Cassie Liu, Thomas R. Eisenreich, Clara Lehmann, Gerd Faetkenheuer, Constance Williams, Masashi Shingai, Malcolm A. Martin, Pamela J. Bjorkman, Michael S. Seaman, Susan Zolla-Pazner, Gunilla B. Karlsson Hedestam, Michel C. NussenzweigJOURNAL OF EXPERIMENTAL MEDICINE 211 (12) 2361 - 2372 0022-1007 2014/11 [Refereed][Not invited]
Antibody-mediated immunotherapy is effective in humanized mice when combinations of broadly neutralizing antibodies (bNAbs) are used that target nonoverlapping sites on the human immunodeficiency virus type 1 (HIV-1) envelope. In contrast, single bNAbs can control simian-human immunodeficiency virus (SHIV) infection in immune-competent macaques, suggesting that the host immune response might also contribute to the control of viremia. Here, we investigate how the autologous antibody response in intact hosts can contribute to the success of immunotherapy. We find that frequently arising antibodies that normally fail to control HIV-1 infection can synergize with passively administered bNAbs by preventing the emergence of bNAb viral escape variants. - Masashi Shingai, Olivia K. Donau, Ronald J. Plishka, Alicia Buckler-White, John R. Mascola, Gary J. Nabel, Martha C. Nason, David Montefiori, Brian Moldt, Pascal Poignard, Ron Diskin, Pamela J. Bjorkman, Michael A. Eckhaus, Florian Klein, Hugo Mouquet, Julio Cesar Cetrulo Lorenzi, Anna Gazumyan, Dennis R. Burton, Michel C. Nussenzweig, Malcolm A. Martin, Yoshiaki NishimuraJOURNAL OF EXPERIMENTAL MEDICINE 211 (10) 2061 - 2074 0022-1007 2014/09 [Refereed][Not invited]
It is widely appreciated that effective human vaccines directed against viral pathogens elicit neutralizing antibodies (NAbs). The passive transfer of anti-HIV-1 NAbs conferring sterilizing immunity to macaques has been used to determine the plasma neutralization titers, which must be present at the time of exposure, to prevent acquisition of SIV/HIV chimeric virus (SHIV) infections. We administered five recently isolated potent and broadly acting anti-HIV neutralizing monoclonal antibodies (mAbs) to rhesus macaques and challenged them intrarectally 24 h later with either of two different R5-tropic SHIVs. By combining the results obtained from 60 challenged animals, we determined that the protective neutralization titer in plasma preventing virus infection in 50% of the exposed monkeys was relatively modest (similar to 1:100) and potentially achievable by vaccination. - Hiromi Takaki, Kenya Honda, Koji Atarashi, Fukiko Kobayashi, Takashi Ebihara, Hiroyuki Oshiumi, Misako Matsumoto, Masashi Shingai, Tsukasa SeyaMOLECULAR IMMUNOLOGY 57 (2) 100 - 109 0161-5890 2014/02 [Refereed][Not invited]
Measles virus (MV) infects CD150Tg/ifnar (IFN alpha receptor)(-/-) mice but not CD150 (a human MV receptor)-transgenic (Tg) mice. We have shown that bone marrow-derived dendritic cells (BMDCs) from CD150Tg/Ifnar(-/-) mice are permissive to MV in contrast to those from simple CD150Tg mice, which reveals a crucial role of type I interferon (IFN) in natural tropism against MV. Yet, the mechanism whereby BMDCs produce initial type I IFN has not been elucidated in MV infection. RNA virus infection usually allows cells to generate double-stranded RNA and induce activation of IFN regulatory factor (IRF) 3/7 transcription factors, leading to the production of type I IFN through the retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5)-mitochondrial antiviral signaling protein (MAVS) pathway. In mouse experimental BMDCs models, we found CD150Tg/Mavs(-/-)BMDCs, but not CD150Tg/Irf3(-/-)/Irf7-/-BMDCs, permissive to MV. IFN-alpha/beta were not induced in MV-infected CD150Tg/Mavs--/-BMDCs, while IFN-13 was subtly induced in CD150Tg/Irf3(-/-)/Irf7(-/-)BMDCs. In vivo systemic infection was therefore established by transfer of MV-infected CD150Tg/Mavs-/- BMDCs to CD150Tg//fnar-/- mice. These data indicate that MAVS-dependent, IRF3/7-independent IFN-13 induction triggers the activation of the IFNAR pathway so as to restrict the spread of MV by infected BMDCs. Hence, MAVS participates in the initial induction of type I IFN in BMDCs and IFNAR protects against MV spreading. We also showed the importance of IL-10-producing CD4+ T cells induced by MV-infected BMDCs in vitro, which may account for immune modulation due to the functional aberration of DCs. (C) 2013 Elsevier Ltd. All rights reserved. - Hiromi Takaki, Makoto Takeda, Maino Tahara, Masashi Shingai, Hiroyuki Oshiumi, Misako Matsumoto, Tsukasa SeyaJOURNAL OF IMMUNOLOGY 191 (9) 4740 - 4747 0022-1767 2013/11 [Refereed][Not invited]
Infection by measles virus (MV) induces type I IFN via the retinoic acid-inducible gene I/melanoma differentiation-associated gene 5/mitochondrial antiviral signaling protein (MAVS) pathway in human cells. However, the in vivo role of the MAVS pathway in host defense against MV infection remains undetermined. CD150 transgenic (Tg) mice, which express human CD150, an entry receptor for MV, with the disrupting IFNR gene (Ifnar(-/-)), are susceptible to MV and serve as a model for MV infection. In this study, we generated CD150Tg/Mavs(-/-) mice and examined MV permissiveness compared with that in CD150Tg/Ifnar(-/-) mice. MV replicated mostly in the spleen of i.p.-infected CD150Tg/Ifnar(-/-) mice. Strikingly, CD150Tg/Mavs(-/-) mice were not permissive to MV in vivo because of substantial type I IFN induction. MV barely replicated in any other organs tested. When T cells, B cells, and dendritic cells (DCs) isolated from CD150Tg/Mavs(-/-) splenocytes were cultured with MV in vitro, only the DCs produced type I IFN. In vitro infection analysis using CD150Tg/Mavs(-/-) DC subsets revealed that CD4(+) and plasmacytoid DCs, but not CD8 alpha(+) and CD8 alpha(-)CD4(-) double negative DCs, were exclusively involved in type I IFN production in response to MV infection. Because CD150Tg/Mavs(-/-) mice turned permissive to MV by anti-IFNAR Ab, type I IFN produced by CD4(+) DCs and plasmacytoid DCs plays a critical role in antiviral protection for neighboring cells expressing IFNAR. Induction of type I IFN in these DC subsets was abolished by the MyD88 inhibitory peptide. Thus, production of type I IFN occurs via the MyD88-dependent and MAVS-independent signaling pathway during MV infection. - Shingai M, Nishimura Y, Klein F, Mouquet H, Donau OK, Plishka R, Buckler-White A, Seaman M, Piatak M Jr, Lifson JD, Dimitrov DS, Nussenzweig MC, Martin MANature 503 (7475) 277 - 280 0028-0836 2013/11 [Refereed][Not invited]
- Reza Sadjadpour, Olivia K. Donau, Masashi Shingai, Alicia Buckler-White, Sandra Kao, Klaus Strebel, Yoshiaki Nishimura, Malcolm A. MartinJOURNAL OF VIROLOGY 87 (15) 8798 - 8804 0022-538X 2013/08 [Refereed][Not invited]
Neutralization-resistant simian-human immunodeficiency virus AD8 (SHIVAD8) variants that emerged in an infected macaque elite neutralizer targeting the human immunodeficiency virus type 1 (HIV-1) gp120 N332 glycan acquired substitutions of critical amino acids in the V3 region rather than losing the N332 glycosylation site. One of these resistant variants, carrying the full complement of gp120 V3 changes, was also resistant to the potent anti-HIV-1 monoclonal neutralizing antibodies PGT121 and 10-1074, both of which are also dependent on the presence of the gp120 N332 glycan. - Masashi Shingai, Olivia K. Donau, Stephen D. Schmidt, Rajeev Gautam, Ronald J. Plishka, Alicia Buckler-White, Reza Sadjadpour, Wendy R. Lee, Celia C. LaBranche, David C. Montefiori, John R. Mascola, Yoshiaki Nishimura, Malcolm A. MartinPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 109 (48) 19769 - 19774 0027-8424 2012/11 [Refereed][Not invited]
The induction of broadly reacting neutralizing antibodies has been a major goal of HIV vaccine research. Characterization of a pathogenic CCR5 (R5)-tropic SIV/HIV chimeric virus (SHIV) molecular clone (SHIVAD8-EO) revealed that eight of eight infected animals developed cross-reactive neutralizing antibodies (NAbs) directed against an envelope glycoprotein derived from the heterologous HIV-1(DH12) strain. A panel of plasmas, collected from monkeys inoculated with either molecularly cloned or uncloned SHIVAD8 stocks, exhibited cross-neutralization against multiple tier 1 and tier 2 HIV-1 clade B isolates. One SHIVAD8-infected animal also developed NAbs against clades A and C HIV-1 strains. In this particular infected macaque, the cross-reacting anti-HIV-1 NAbs produced between weeks 7 and 13 were directed against a neutralization-sensitive virus strain, whereas neutralizing activities emerging at weeks 41-51 targeted more neutralization-resistant HIV-1 isolates. These results indicate that the SHIVAD8 macaque model represents a potentially valuable experimental system for investigating B-cell maturation and the induction of cross-reactive NAbs directed against multiple HIV-1 strains. - Rajeev Gautam, Yoshiaki Nishimura, Wendy R. Lee, Olivia Donau, Alicia Buckler-White, Masashi Shingai, Reza Sadjadpour, Stephen D. Schmidt, Celia C. LaBranche, Brandon F. Keele, David Montefiori, John R. Mascola, Malcolm A. MartinJOURNAL OF VIROLOGY 86 (16) 8516 - 8526 0022-538X 2012/08 [Refereed][Not invited]
There is an urgent need to develop new pathogenic R5 simian/human immunodeficiency viruses (SHIVs) for the evaluation of candidate anti-HIV vaccines in nonhuman primates. Here, we characterize swarm SHIVAD8 stocks, prepared from three infected rhesus macaques with documented immunodeficiency at the time of euthanasia, for their capacity to establish durable infections in macaques following inoculation by the intravenous (i.v.) or intrarectal (i.r.) route. All three viral stocks (SHIVAD8-CE8J SHIVAD8-CK15) and SHIVAD8-CL98) exhibited robust replication in vivo and caused marked depletion of CD4(+) T cells affecting both memory and naive CD4+ T lymphocyte subsets following administration by either route. Eleven of 22 macaques inoculated with the new SHIVAD8 stocks were euthanized with clinical symptoms of immunodeficiency and evidence of opportunistic infections (Pneumocystis, Candida, and Mycobacterium). A single but unique founder virus, also present in the SHIVAD8-CE8J cm swarm stock, was transmitted to two animals following a single i.r. inoculation of approximately 3 50% animal infectious doses, which is close to the threshold required to establish infection in all exposed animals. Because the three new SHIVAD8 viruses are mucosally transmissible, exhibited tier 2 sensitivity to anti-HIV-1 neutralizing antibodies, deplete CD4(+) T lymphocytes in vivo, and induce AIDS in macaques, they are eminently suitable as challenge viruses in vaccine experiments. - Laura M. Walker, Devin Sok, Yoshiaki Nishimura, Olivia Donau, Reza Sadjadpour, Rajeev Gautam, Masashi Shingai, Robert Pejchal, Alejandra Ramos, Melissa D. Simek, Yu Geng, Ian A. Wilson, Pascal Poignard, Malcolm A. Martin, Dennis R. BurtonPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 108 (50) 20125 - 20129 0027-8424 2011/12 [Refereed][Not invited]
It is widely believed that the induction of a broadly neutralizing antibody (bNAb) response will be a critical component of a successful vaccine against HIV. A significant fraction of HIV-infected individuals mount bNAb responses, providing support for the notion that such responses could be elicited through vaccination. Infection of macaques with simian immunodeficiency virus (SIV) or SIV/HIV chimeric virus (SHIV) has been widely used to model aspects of HIV infection, but to date, only limited bNAb responses have been described. Here, we screened plasma from 14 R5-tropic SHIV-infected macaques for broadly neutralizing activity and identified a macaque with highly potent cross-clade plasma NAb response. Longitudinal studies showed that the development of broad and autologous NAb responses occurred coincidentally in this animal. Serum-mapping studies, using pseudovirus point mutants and antigen adsorption assays, indicated that the plasma bNAbs are specific for epitopes that include carbohydrates and are critically dependent on the glycan at position 332 of Env gp120. The results described herein provide insight into the development and evolution of a broad response, suggest that certain bNAb specificities may be more rapidly induced by immunization than others, and provide a potential model for the facile study of the development of bNAb responses. - Masashi Shingai, Takeshi Yoshida, Malcolm A. Martin, Klaus StrebelJOURNAL OF VIROLOGY 85 (19) 9708 - 9715 0022-538X 2011/10 [Refereed][Not invited]
Human immunodeficiency virus type 1 (HIV-1) Vpu enhances the release of viral particles from infected cells by targeting BST-2/tetherin, a cellular protein inhibiting virus release. The widely used HIV-1(NL4-3) Vpu functionally inactivates human BST-2 but not murine or monkey BST-2, leading to the notion that Vpu antagonism is species specific. Here we investigated the properties of the CXCR4-tropic simian-human immunodeficiency virus DH12 (SHIV(DH12)) and the CCR5-tropic SHIV(AD8), each of which carries vpu genes derived from different primary HIV-1 isolates. We found that virion release from infected rhesus peripheral blood mononuclear cells was enhanced to various degrees by the Vpu present in both SHIVs. Transfer of the SHIV(DH12) Vpu transmembrane domain to the HIV-1(NL4-3) Vpu conferred antagonizing activity against macaque BST-2. Inactivation of the SHIV(DH12) and SHIV(AD8) vpu genes impaired virus replication in 6 of 8 inoculated rhesus macaques, resulting in lower plasma viral RNA loads, slower losses of CD4(+) T cells, and delayed disease progression. The expanded host range of the SHIV(DH12) Vpu was not due to adaptation during passage in macaques but was an intrinsic property of the parental HIV-1(DH12) Vpu protein. These results demonstrate that the species-specific inhibition of BST-2 by HIV-1(NL4-3) Vpu is not characteristic of all HIV-1 Vpu proteins; some HIV-1 isolates encode a Vpu with a broader host range. - Yoshiaki Nishimura, Masashi Shingai, Wendy R. Lee, Reza Sadjadpour, Olivia K. Donau, Ronald Willey, Jason M. Brenchley, Ranjini Iyengar, Alicia Buckler-White, Tatsuhiko Igarashi, Malcolm A. MartinJOURNAL OF VIROLOGY 85 (20) 10617 - 10626 0022-538X 2011/10 [Refereed][Not invited]
Evolution of the env gene in transmitted R5-tropic human immunodeficiency virus type 1 (HIV-1) strains is the most widely accepted mechanism driving coreceptor switching. In some infected individuals, however, a shift in coreceptor utilization can occur as a result of the reemergence of a cotransmitted, but rapidly controlled, X4 virus. The latter possibility was studied by dually infecting rhesus macaques with X4 and R5 chimeric simian simian/human immunodeficiency viruses (SHIVs) and monitoring the replication status of each virus using specific primer pairs. In one of the infected monkeys, both SHIVs were potently suppressed by week 12 postinoculation, but a burst of viremia at week 51 was accompanied by an unrelenting loss of total CD4(+) T cells and the development of clinical disease. PCR analyses of plasma viral RNA indicated an env gene segment containing the V3 region from the inoculated X4 SHIV had been transferred into the genetic background of the input R5 SHIV by intergenomic recombination, creating an X4 virus with novel replicative, serological, and pathogenic properties. These results indicate that the effects of retrovirus recombination in vivo can be functionally profound and may even occur when one of the recombination participants is undetectable in the circulation as cell-free virus. - Hiromi Takaki, Yumi Watanabe, Masashi Shingai, Hiroyuki Oshiumi, Misako Matsumoto, Tsukasa SeyaMOLECULAR IMMUNOLOGY 48 (4) 497 - 504 0161-5890 2011/01 [Refereed][Not invited]
Laboratory-adapted and vaccine strains of measles virus (MV) induce type I interferon (IFN) in infected cells to a far greater extent than wild-type strains. We investigated the mechanisms for this differential type I IFN production in cells infected with representative MV strains. The overexpression of the wild-type V protein suppressed melanoma differentiation-associated gene 5 (MDA5)-induced IFN-beta promoter activity, while this was not seen in A549 cells expressing CD150 transfected with the V protein of the vaccine strain. The V proteins of the wild-type also suppressed poly I:C-induced IFN regulatory factor 3 (IRF-3) dimerization. The V proteins of the wild-type and vaccine strain did not affect retinoic acid-inducible gene 1 (RIG-I)- or toll-IL-1R homology domain-containing adaptor molecule 1 (TICAM-1)-induced IFN-beta promoter activation. We identified an amino acid substitution of the cysteine residue at position 272 (which is conserved among paramyxoviruses) to an arginine residue in the V protein of the vaccine strain. Only the V protein possessing the 272C residue binds to MDA5. The mutation introduced into the wild-type V protein (C272R) was unable to suppress MDA5-induced IRF-3 nuclear translocation and IFN-beta promoter activation as seen in the V proteins of the vaccine strain, whereas the mutation introduced in the vaccine strain V protein (R272C) was able to inhibit MDA5-induced IRF-3 and IFN-beta promoter activation. The other 6 residues of the vaccine strain V sequence inconsistent with the authentic sequence of the wild-type V protein barely affected the IRF-3 nuclear translocation. These data suggested that the structural difference of vaccine MV V protein hampers MDA5 blockade and acts as a nidus for the spread/amplification of type I IFN induction. Ultimately, measles vaccine strains have two modes of IFN-beta-induction for their attenuation: V protein mutation and production of defective interference (DI) RNA. (C) 2010 Elsevier Ltd. All rights reserved. - Yoshiaki Nishimura, Masashi Shingai, Ronald Willey, Reza Sadjadpour, Wendy R. Lee, Charles R. Brown, Jason M. Brenchley, Alicia Buckler-White, Rahel Petros, Michael Eckhaus, Victoria Hoffman, Tatsuhiko Igarashi, Malcolm A. MartinJOURNAL OF VIROLOGY 84 (9) 4769 - 4781 0022-538X 2010/05 [Refereed][Not invited]
A new pathogenic R5-tropic simian/human immunodeficiency virus (SHIV) was generated following serial passaging in rhesus macaques. All 13 animals inoculated with SHIVAD8 passaged lineages experienced marked depletions of CD4(+) T cells. Ten of these infected monkeys became normal progressors (NPs) and had gradual losses of both memory and naive CD4(+) T lymphocytes, generated antiviral CD4(+) and CD8(+) T cell responses, and sustained chronic immune activation while maintaining variable levels of plasma viremia (10(2) to 10(5) RNA copies/ml for up to 3 years postinfection [p.i.]). To date, five NPs developed AIDS associated with opportunistic infections caused by Pneumocystis carinii, Mycobacterium avium, and Campylobacter coli that required euthanasia between weeks 100 and 199 p.i. Three other NPs have experienced marked depletions of circulating CD4(+) T lymphocytes (92 to 154 cells/mu l) following 1 to 2 years of infection. When tested for coreceptor usage, the viruses isolated from four NPs at the time of their euthanasia remained R5 tropic. Three of the 13 SHIVAD8-inoculated macaques experienced a rapid-progressor syndrome characterized by sustained plasma viremia of >1 x 107 RNA copies/ml and rapid irreversible loss of memory CD4(+) T cells that required euthanasia between weeks 19 and 23 postinfection. The sustained viremia, associated depletion of CD4(+) T lymphocytes, and induction of AIDS make the SHIVAD8 lineage of viruses a potentially valuable reagent for vaccine studies. - Makoto Kubo, Yoshiaki Nishimura, Masashi Shingai, Wendy Lee, Jason Brenchley, Bernard Lafont, Alicia Buckler-White, Tatsuhiko Igarashi, Malcolm A. MartinJOURNAL OF VIROLOGY 83 (14) 7099 - 7108 0022-538X 2009/07 [Refereed][Not invited]
We investigated whether a 28-day course of potent antiretroviral therapy, initiated at a time point ( 48 h postinoculation) following simian immunodeficiency virus (SIV) inoculation when the acquisition of a viral infection was virtually assured, would sufficiently sensitize the immune system and result in controlled virus replication when treatment was stopped. The administration of tenofovir 48 h after SIV inoculation to six Mamu-A*01-negative rhesus macaques did, in fact, potently suppress virus replication in all of the treated rhesus macaques, but plasma viral RNA rapidly became detectable in all six animals following its cessation. Unexpectedly, the viral set points in the treated monkeys became established at two distinct levels. Three controller macaques had chronic phase virus loads in the range of 1 x 10(3) RNA copies/ml, whereas three noncontroller animals had set points of 2 x 10(5) to 8 x 10(5) RNA copies/ml. All of the noncontroller monkeys died with symptoms of immunodeficiency by week 60 postinfection, whereas two of the three controller animals were alive at week 80. Interestingly, the three controller macaques each carried major histocompatibility complex class I alleles that previously were reported to confer protection against SIV, and two of these animals generated cytotoxic T-lymphocyte escape viral variants during the course of their infections. - Masashi Shingai, Masahiro Azuma, Takashi Ebihara, Miwa Sasai, Kenji Funami, Minoru Ayata, Hisashi Ogura, Hiroyuki Tsutsumi, Misako Matsumoto, Tsukasa SeyaINTERNATIONAL IMMUNOLOGY 20 (9) 1169 - 1180 0953-8178 2008/09 [Refereed][Not invited]
Monocyte-derived dendritic cells (mDCs) recognize viral RNA extrinsically by Toll-like receptor (TLR)3 on the membrane and intrinsically retinoic acid-inducible on the membrane and intrinsically retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm to induce type I IFNs and mDC maturation. When mDCs were treated with live or UV-irradiated respiratory syncytial virus (RSV), early (similar to 4h) induction of IFN-beta usually occurs in other virus infections was barely observed. Live RSV subsequently replicated to activate the cytoplasmic IFN-inducing pathway leading to robust type I IFN induction. We found that RSV initial attachment to cells blocked polyI:C-mediated IFN-beta induction, and this early IFN-beta-modulating event was abrogated by antibodies against envelope proteins of RSV, demonstrating the presence of a IFN-regulatory mode by early RSV attachment to host cells. By IFN-stimulated response element (ISRE) receptor analysis in HEK293 cells, polyI:C- LPS-mediated ISRE activation was dose dependently inhibited by live and inactive RSV to a similar extent. Of the RSV envelope proteins, simultaneously expressed or exogenously added RSV G or soluble G (sG) proteins inhibited TLR/4-mediated ISRE activation in HEK293 cells. sG proteins expressed in cells did not affect the RIG-1/MDA5 pathway but inhibited the TLR adaptor TRIF/TICAM-1 pathway for ISRE activation. Finally, extrinsically added sG protein suppressed the production of IFN-beta in mDCs. Although the molecular mechanism of this extrinsic functional mode of the RSV G glycoprotein (G protein) remains undetermined, G proteins may neutralize the fusion glycoprotein function that promotes IFN-mediated mDC modulation via TCR4 and may cause insufficient raising cell-mediated immunity against RSV. - Takashi Ebihara, Masashi Shingai, Misako Matsumoto, Takaji Wakita, Tsukasa SeyaHEPATOLOGY 48 (1) 48 - 58 0270-9139 2008/07 [Refereed][Not invited]
Dendritic cell maturation critically modulates antiviral immune responses, and facilitates viral clearance. Hepatitis C virus (HCV) is characterized by its high predisposition to persistent infection. Here, we examined the immune response of human monocyte-derived dendritic cells (MODCs) to the JFH1 strain of HCV, which can efficiently replicate in cell culture. However, neither HCV RNA replication nor antigen production was detected in MoDCs inoculated with JFH1. None of the indicators of HCV interacting with MoDCs we evaluated were affected, including expression of maturation markers (CD80, 83, 86), cytokines (interleukin-6 and interferon-beta), the mixed lymphocyte reaction, and natural killer (NK) cell cytotoxicity. Strikingly, MoDCs matured by phagocytosing extrinsically-infected vesicles containing HCV-derived double-stranded RNA (dsRNA). When MoDCs were cocultured with HCV-infected apoptotic Huh7.5.1 hepatic cells, there was increased CD86 expression and interleukin-6 and interferon-beta production in MoDCs, which were characterized by the potential to activate NK cells and induce CD4(+) T cells into the T helper 1 type. Lipid raft-dependent phagocytosis of HCV-infected apoptotic vesicles containing dsRNA was indispensable to MoDC maturation. Colocalization of dsRNA with Toll-like receptor 3 (TLR3) in phagosomes suggested the importance of TLR3 signaling in the MoDC response against HCV. Conclusion: The JFH1 strain does not directly stimulate MoDCs to activate T cells and NK cells, but phagocytosing HCV-infected apoptotic cells and their interaction with the TLR3 pathway in MoDCs plays a critical role in MoDC maturation and reciprocal activation of T and NK cells. - Megumi Higuchi, Aya Matsuo, Masashi Shingai, Kyoko Shida, Akihiro Ishii, Kenji Funami, Yasuhiko Suzuki, Hiroyuki Oshiumi, Misako Matsumoto, Tsukasa SeyaDEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 32 (2) 147 - 155 0145-305X 2008 [Refereed][Not invited]
Human Toll-like receptor 2 (TLR2) subfamily recognizes bacterial lipoproteins (BLP) and peptidoglycan (PGN). According to the genome information, chicken has structural orthologs of TLRs1 and 2, in addition to TLRs3, 4, 5 and 7. Chicken has two additional TLRs, TLR15 and TLR21, whose orthologs human lacks. The chicken (ch)TLR1 and 2 genes are individually duplicated to encode for four different proteins, chTLR1-1, 1-2, 2-1 and 2-2, of the TLR2 subfamily. Here we investigated the functional profile of these TLR2 subfamily proteins of chicken. By NF-kappa B reporter assay using HEK293 cells, we found that chTLR2-1 and chTLR1-2 cooperatively signal the presence of PGN. A combination of chTLR2-1 and chTLR1-2 also most efficiently recognized diacylated BLP, macrophage-activating lipopeptide 2 kDa (Malp-2), while the combination of chTLR2-1 and chTLR1-1 failed to recognize Malp-2. All combinations, however, recognized triacylated BLP, Pamm3. Consistent with these results, human TLR2-stimulating mycobacteria preparations, BCG-cell wall and cell lysate of Mycobacterium avium, induced activation of NF-kappa B in cells expressing chTLR2-1 and 1-2 and to lesser extents, cells with chTLR2-2 and either of chTLR1. Strikingly, expression of either of these alone did not activate the reporter for NF-kappa B. These chTLRs are likely to have the combination functional feature as in the human TLR2 subfamily. Confocal and immunoprecipitation analyses of human cell transfectants showed that they cluster on the cell surface by a physical molecular association, causing all of them to merge and coprecipitate. These results suggest that chTLR2 subfamily members discriminate between their ligands by combinational events. (C) 2007 Elsevier Ltd. All rights reserved. - Minoru Ayata, Masashi Shingai, Xiaojun Ning, Misako Matsumoto, Tsukasa Seya, Sanae Otani, Toshiyuki Seto, Shinji Ohgimoto, Hisashi OguraVIRUS RESEARCH 130 (1-2) 260 - 268 0168-1702 2007/12 [Refereed][Not invited]
Measles virus (MV) is the causative agent of subacute sclerosing panencephalitis (SSPE) and viruses isolated from brains of the patients contain numerous mutations. We have previously demonstrated that the hemagglutinin (H) protein of MV SSPE strains can interact with the signaling lymphocyte activation molecule (SLAM) and an unidentified molecule on Vero cells, but not with CD46, as a receptor. The mechanism by which MV SSPE strains can induce cell-cell fusion in SLAM-negative Vero cells is not understood. We report here on the effect of mutations in the fusion (F) proteins of three MV SSPE strains on syncytium formation. The F proteins of the three SSPE strains were functional and co-expression with H protein from the MV wild-type or SSPE strains in this study induced formation of large syncytia in Vero cells as well as in cell lines expressing SLAM or CD46. Expression of chimeric F proteins of SSPE strains showed that amino acid substitutions in the F protein extracellular as well as cytoplasmic domain contributed to enhanced cell-cell fusion in Vero cells. These findings suggest a common molecular mechanism and a key role of the F protein for syncytium formation in cells expressing an unidentified third receptor for MV. (c) 2007 Elsevier B.V. All rights reserved. - Masashi Shingai, Takashi Ebihara, Nasim A. Begum, Atsushi Kato, Toshiki Honma, Kenji Matsumoto, Hirohisa Saito, Hisashi Ogura, Misako Matsumoto, Tsukasa SeyaJOURNAL OF IMMUNOLOGY 179 (9) 6123 - 6133 0022-1767 2007/11 [Refereed][Not invited]
Laboratory adapted and vaccine strains of measles virus (MV) induced type I IFN in infected cells. The wild-type strains in contrast induced it to a far lesser extent. We have investigated the mechanism for this differential type I IFN induction in monocyte-derived dendritic cells infected with representative MV strains. Laboratory adapted strains Nagahata and Edmonston infected monocyte-derived dendritic cells and activated IRF-3 followed by IFN-,6 production, while wild-type MS failed to activate IRF-3. The viral IRF-3 activation is induced within 2 h, an early response occurring before protein synthesis. Receptor usage of CD46 or CD150 and nucleocapsid (N) protein variations barely affected the strain-to-strain difference in IFN-inducing abilities. Strikingly, most of the IFN-inducing strains possessed defective interference (DI) RNAs of varying sizes. In addition, an artificially produced DI RNA consisting of stem (the leader and trailer of MV) and loop (the GFP sequence) exhibited potential IFN-inducing ability. In this case, however, cytoplasmic introduction was needed for DI RNA to induce type I IFN in target cells. By genesilencing analysis, DI RNA activated the RIG-IIMDA5-mitochondria antiviral signaling pathway, but not the TLR3-TICAM-1 pathway. DI RNA-containing strains induced IFN-beta mRNA within 2 h while the same recombinant strains with no DI RNA required >12 h postinfection to attain similar levels of IFN-beta mRNA. Thus, the stem-loop structure, rather than full genome replication or specific internal sequences of the MV genome, is required for an early phase of type I IFN induction by MV in host cells. - Takashi Ebihara, Hisayo Masuda, Takashi Akazawa, Masashi Shingai, Hideaki Kikuta, Tadashi Ariga, Misako Matsumoto, Tsukasa SeyaINTERNATIONAL IMMUNOLOGY 19 (10) 1145 - 1155 0953-8178 2007/10 [Refereed][Not invited]
Monocyte-derived dendritic cells (mDCs) and NK cells are reciprocally activate via cytokines and cell-cell contact. Although seven human NKG2D ligands (NKG2DLs), UL16-binding proteins (ULBP) 1, 2, 3 and 4, retinoic acid early transcript 1G (RAET1G) and MHC class I-related chains A and B, have been reported, the differential distribution and roles of these ligands in the maturation of human mDCs have not been elucidated. In the present study, we produced polyclonal antibodies (pAbs) directed against human ULBP1, 2 and 3. All these ULBPs were detected on human mDCs when probed by the pAbs, although their expression profiles were different. We next investigated what kinds of Toll-like receptor agonists and RNA viruses [influenza virus, human respiratory syncytial virus (RSV), measles virus and hepatitis C virus (HCV)] induced the expression of NKG2DLs on mDCs. ULBP1 was up-regulated on mDCs in response to LPS or infection with RSV. The expression of ULBP2 was induced by LPS and poly I:C, indicating that the TIR-containing adapter molecule-1 (TIR domain-containing adaptor-inducing IFN) pathway is associated with ULBP2 induction. Although infection with HCV did not cause up-regulation of NKG2DLs, other RNA virus infections and poly I:C promoted expression of ULBP2 and RAET1G in an IFN-alpha/beta-independent manner. Finally, the over-expression of ULBP1 and 2 on mDCs facilitated NK cell proliferation and IFN-gamma production through a mDC-NK cell interaction in the presence of IL-2. Hence, the results reflect the important role of NKG2DLs on human mDCs in mDC-mediated NK cell activation. - Takashi Akazawa, Masashi Shingai, Mwa Sasai, Takashi Ebihara, Norimitsu Inoue, Misako Matsumoto, Tsukasa SeyaFEBS LETTERS 581 (18) 3334 - 3340 0014-5793 2007/07 [Refereed][Not invited]
Myeloid dendritic cells (mDCs) play an important role in the initiation of immune responses to cancer and infectious diseases. Toll-like receptors (TLRs) expressed on mDCs recognize microbial products to elicit signals for mDC maturation, including cytokine production, antigen-presentation and induction of effector cells. TLR agonists work as adjuvants to modulate the function of mDCs. In TLR signaling, MyD88 and TRIF/TICAM-1 are major TLR adaptor molecules, which when overexpressed are able to transduce downstream signals without TLR stimuli. We successfully introduced the adaptors into mouse bone marrowderived mDCs using lentiviral vectors. Introduction of MyD88 into mDCs in vitro led to the production of IL-6 and IL-12p40 while introduction of TICAM-1 stimulated interferon (IFN)-alpha production. Expression of TICAM-1, but not MyD88, in mDCs slightly induced the co-stimulatory molecule CD86, while significant upregulation of CD86 was observed in response to other TLR stimuli. Both MyD88 and TICAM-1 augmented allogeneic mixed lymphocyte reaction (MLR). Ex vivo mouse spleen cells pre-exposed to tumor antigen exhibited antitumor cytotoxicity when incubated with MyD88- or TICAM-1 -expressing mDCs. Using mDC adoptive transfer and a syngeneic mouse tumor implant model, we established an antitumor immunotherapy whereby tumor growth is retarded by adaptor-manipulated mDCs. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. - Takashi Akazawa, Takashi Ebihara, Manabu Okuno, Yu Okuda, Masashi Shingai, Kunio Tsujimura, Toshitada Takahashi, Masahito Ikawa, Masaru Okabe, Norimitsu Inoue, Miki Okamoto-Tanaka, Hiroyoshi Ishizaki, Jun Miyoshi, Misako Matsumoto, Tsukasa SeyaPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 104 (1) 252 - 257 0027-8424 2007/01 [Refereed][Not invited]
Myeloid dendritic cells (mDCs) recognize and respond to polyl:C, an analog of dsRNA, by endosomal Toll-like receptor (TLR) 3 and cytoplasmic receptors. Natural killer (NK) cells are activated in vivo by the administration of polyl:C to mice and in vitro are reciprocally activated by mDCs, although the molecular mechanisms are as yet undetermined. Here, we show that the TLR adaptor TICAM-1 (TRIF) participates in mDC-derived antitumor INK activation. In a syngeneic mouse tumor implant model (C57BL/6 vs. B16 melanoma with low H-2 expresser), i.p. administration of polyl:C led to the retardation of tumor growth, an effect relied on by INK activation. This NK-dependent tumor regression did not occur in TICAM-1(-/-) or IFNAR(-/-) mice, whereas a normal NK antitumor response was induced in PKR-/-, MyD88(-/-), IFN-beta(-/-), and wild-type mice. IFNAR was a prerequisite for the induction of IFN-alpha/beta and TLR3. The lack of TICAM-1 did not affect IFN production but resulted in unresponsiveness to IL-12 production, mDC maturation, and polyl:C-mediated NK-antitumor activity. This NK activation required NK-mDC contact but not IL-12 function in in vitro transwell analysis. Implanted tumor growth in IFNAR(-/-) mice was retarded by adoptively transferring polyl:C-treated TICACM-1-positive mDCs but not TICAM-1(-/-) mDCs. Thus, TICAM-1 in mDCs critically facilitated mDC-NK contact and activation of antitumor INK, resulting in the regression of low MHC-expressing tumors. - Miwa Sasai, Masashi Shingai, Kenji Funami, Mitsutoshi Yoneyama, Takashi Fujita, Misako Matsumoto, Tsukasa SeyaJOURNAL OF IMMUNOLOGY 177 (12) 8676 - 8683 0022-1767 2006/12 [Refereed][Not invited]
TLR3 and the cytoplasmic helicase family proteins (retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5)) serve as dsRNA pattern-recognition receptors. In response to poly(I:C), a representative of dsRNA, and viral infection, they have been shown to activate the transcription factor IFN regulatory factor (IRF)-3, which in turn induces activation of the IFN-beta promoter. RIG-I/MDA5 recognizes dsRNA in the cytoplasm, whereas TLR3 resides in the cell surface membrane or endosomes to engage in extracytoplasmic recognition of dsRNA. Recent reports suggest that TLR3 induces cellular responses in epithelial cells in response to respiratory syncytial virus (RSV). The modus for TLR3 activation by RSV, however, remains unresolved. By small interference RNA gene-silencing technology and human cell transfectants, we have revealed that knockdown of NAK-associated protein 1 (NAP1) leads to the down-regulation of IFN-beta promoter activation > 24 h after poly(I:Q or virus (RSV and vesicular stomatitis virus) treatment. NAP1 is located downstream of the adapter Toll-IL-1R homology domain-containing adapter molecule (TICAM)-1 (Toll/IL-IR domain-containing adapter-inducing IFN-beta) in the TLR3 pathway, but TICAM-1 and TLR3 did not participate in the IRF-3 and IFN-beta promoter activation by RSV infection. Virus-mediated activation of the IFN-beta promoter was largely abrogated by the gene silencing of IFN-beta promoter stimulator-1 (mitochondria antiviral signaling (MAVS), VISA, Cardif), the adapter of the RIG-I/MDA5 dsRNA-recognition proteins. In both the TLR and virus-mediated IFN-inducing pathways, I kappa B kinase-related kinase epsilon and TANK-binding kinase I participated in IFN-beta induction. Thus, RSV as well as other viruses induces replication-mediated activation of the IFN-beta promoter, which is intracellularly initiated by the RIG-I/MDA5 but not the TLR3 pathway. Both the cytoplasmic and TLR3-mediated dsRNA recognition pathways converge upon NAP1 for the activation of the IRF-3 and IFN-beta promoter. - ウイルス感染と宿主応答・宿主側の要因 感染樹状細胞における麻疹ウイルスによるI型IFN誘導メカニズム(A mechanism by which measles virus induces Type-I IFN in infected dendritic cells)Shingai Masashi, Ebihara Takashi, Matsumoto Misako, Seya Tsukasa日本免疫学会総会・学術集会記録 36 207 - 207 0919-1984 2006/11
- NK細胞とMHC受容体 TLRリガンド刺激及びRNAウイルス感染によるヒト樹状細胞上のNKG2Dリガンドの誘導(NKG2D ligands are induced on human dendritic cells by TLR ligand stimulation and RNA virus infection)Ebihara Takashi, Shingai Masashi, Akazawa Takashi, Matsumoto Misako, Seya Tsukasa日本免疫学会総会・学術集会記録 36 253 - 253 0919-1984 2006/11
- M Shingai, N Inoue, T Okuno, M Okabe, T Akazawa, Y Miyamoto, M Ayata, K Honda, M Kurita-Taniguchi, M Matsumoto, H Ogura, T Taniguchi, T SeyaJOURNAL OF IMMUNOLOGY 175 (5) 3252 - 3261 0022-1767 2005/09 [Refereed][Not invited]
We generated transgenic (TG) mice that constitutively express human CD46 (huCD46) and/or TLR-inducible CD150 (huCD150), which serve as receptors for measles virus (MV). These mice were used to study the spreading and pathogenicity of GFPexpressing or intact laboratory-adapted Edmonston and wild-type Ichinose (IC) strains of MV. Irrespective of the route of administration, neither type of MV was pathogenic to these TG mice. However, in ex vivo, limited replication of IC was observed in the spleen lymphocytes from huCD46/buCD150 TG and huCD150 TG, but not in huCD46 TG and non-TG mice. In huCD150-positive TG mouse cells, CD11c-positive bone marrow-derived myeloid dendritic cells (mDC) participated in MV-mediated type I IFN induction. The level and induction profile of IFN-beta was higher in mDC than the profile of IFN-alpha. Wild-type IC induced markedly high levels of IFN-beta compared with Edmonston in mDC, as opposed to human dendritic cells. We then generated huCD46/huCD150 TG mice with type I IFN receptor (IFNAR1)-/- mice. MV-bearing mDCs spreading to draining lymph nodes were clearly observed in these triple mutant mice in vivo by i.p. MV injection. Infectious lymph nodes were also detected in the double TG mice into which MV-infected CD11c-positive mDCs were i.v. transferred. This finding suggests that in the double TG mouse model mDCs once infected facilitate systemic MV spreading and infection, which depend on mDC MV permissiveness determined by the level of type I IFN generated via IFNAR1. Although these results may not simply reflect human MV infection, the huCD150/huCD46 TG mice may serve as a useful model for the analysis of MV-dependent modulation of mDC response. - Shingai M, Seya TNihon rinsho. Japanese journal of clinical medicine 63 Suppl 7 95 - 97 0047-1852 2005/07 [Refereed][Not invited]
- J Uehori, K Fukase, T Akazawa, S Uematsu, S Akira, K Funami, M Shingai, M Matsumoto, Azuma, I, K Toyoshima, S Kusumoto, T SeyaJOURNAL OF IMMUNOLOGY 174 (11) 7096 - 7103 0022-1767 2005/06 [Refereed][Not invited]
6-O-acyl-muramyldipeptides (MDP) with various lengths of fatty acid chains were examined for their dendritic cell (DC) maturation activity expressed through TLRs. Judging from anti-TLR mAb/inhibitor-blocking analysis, MDP derivatives with a single octanoyl or stearoyl fatty acid chain were found to activate TLR2 and TLR4 on human DCs, although intact and diacylated MDP expressed no ability to activate TLRs. Human DC activation profiles by the monoacylated MDP were essentially similar to those by Calmette-Guerin (BCG)-cell wall skeleton (CWS) and BCG-peptidoglycan (PGN) based on their ability to up-regulate costimulators, HLA-DR, beta(2)-microglobulin, and allostimulatory MLR. Monoacylated MDP induced cytokines with similar profiles to BCG-CWS or -PGN, although their potency for induction of TNF-alpha, IL-12p40, and IL-6 was less than that of BCG-CWS or -PGN. The MDP derivatives initiated similar activation in normal mouse macrophages, but exhibited no effect on TLR2/4-deficient or MyD88-deficient mouse macrophages. Mutation Of D-isoGln to L-isoGln in monoacylated MDP did not result in loss of the DC maturation activity, suggesting marginal participation of nucleotide-binding oligomerization domain 2, if any, in monoacyl MDP-dependent DC maturation. These results define the adjuvant activity of 6-O-acyl MDP compounds at the molecular level. They target TLR2/TLR4 and act through the MyD88-dependent pathway in DCs and macrophages. Hence, the unusual combined activation of TLR2 and TLR4 observed with Mycobacterium tuberculosis is in part reflected in the functional properties of monoacylated MDP compounds. These findings infer that the essential minimal requirement for TLR2/4-mediated adjuvancy of BCG lies within a modified MDP. - Seya T, Shingai M, Matsumoto MUirusu 日本ウイルス学会 54 (1) 1 - 8 0042-6857 2004/06 [Refereed][Not invited]
Anti-viral host defense harbors a variety of strategies to coup with viral infection. Recent findings suggested that Toll-like receptors (TLRs) and their signaling pathways involve type I IFN induction in response to virus-specific molecular patterns. TLR 3 and TLR 4 in myeloid dendritic cells (mDCs) recognize viral dsRNA and putative viral products, respectively, to induce IFN-β via IRF-3 activation. On the other hand, TLR 7 and TLR 9 in plasmacytoid DCs (pDCs) induce IFN-α in response to their ligands, U/G-rich ssRNA and non-methylated CpG DNA. We identified TICAM-1 which is recruited to the cytoplasmic domain (designated TIR) of TLR 3 and allows to select the pathway to activation of IRF-3. We also identified TICAM-2 which binds TLR 4 and together with TICAM-1 activates IRF-3. TICAM-1 knockdown by RNAi supported the key role of TICAM-1 in IFN-β induction. Hence, the IFN-β induction in mDCs appears in part due to the function of TICAM-1. Viruses are known to activate kinases that directly activate IRF-3 inside the cells, and this pathway may merge with the TLR 3-TICAM-1 pathway. Here we review the relationship between the TLR 3-TICAM-1 pathway and viral infection. - H Ishida, M Ayata, M Shingai, Matsunaga, I, Y Seto, Y Katayama, N Iritani, T Seya, Y Yanagi, O Matsuoka, T Yamano, H OguraMICROBIOLOGY AND IMMUNOLOGY 48 (4) 277 - 287 0385-5600 2004 [Refereed][Not invited]
Measles virus is the causative agent of subacute sclerosing panencephalitis (SSPE). The viruses isolated from brain cells of patients with SSPE (called SSPE viruses) are defective in cell-free virus production in vitro. To investigate the cell tropism of three strains of SSPE virus (Osaka-1, Osaka-2, Osaka-3), SSPE virus-infected cell cultures were treated with cytochalasin D to prepare virus-like particles (CD-VLPs). All CD-VLPs formed syncytia after infection in CHO cells expressing CD150 but not in those expressing CD46. In addition, an antibody to CD46 did not block the infection of Vero cells by SSPE CD-VLPs. The results were consistent with our previous suggestion that one or more unidentified receptors might be involved in the entry process. Infection with the CD-VLPs from three SSPE strains was further examined in different human cell lines, including those of neural origin, and was found to induce syncytia in epithelial cells (HeLa and 293T) as well as neuroblastoma cells (IMR-32 and SK-N-SH) with varying efficiency. SSPE CD-VLPs also infected glioblastoma cells (A172) and astrocytoma cells (U-251) but syncytial formation was rarely induced. These epithelial and neural cell lines were not permissive for the replication of wild-type MV. Together with our previous observations, these results suggest that the cell entry receptor is the major factor determining the cell tropism of SSPE viruses. Further studies are necessary to identify other viral and/or cellular factors that might be involved in the replication of SSPE virus in specific neural cells and in the brain. - M Matsumoto, K Funami, M Tanabe, H Oshiumi, M Shingai, Y Seto, A Yamamoto, T SeyaJOURNAL OF IMMUNOLOGY 171 (6) 3154 - 3162 0022-1767 2003/09 [Refereed][Not invited]
Toll-like receptor (TLR)3 recognizes dsRNA and transduces signals to activate NF-kappaB and IFN-beta promoter. Type I IFNs (IFN-alpha/beta) function as key cytokines in anti-viral host defense. Human fibroblasts express TLR3 on the cell surface, and anti-TLR3 mAb inhibits dsRNA-induced IFN-beta secretion by fibroblasts, suggesting that TLR3 acts on the cell surface to sense viral infection. In this study, we examined the expression and localization of human TLR3 in various DC subsets using anti-TLR3 mAb. In monocyte-derived immature dendritic cells (iDCs), TLR3 predominantly resided inside the cells but not on the cell surface. iDCs produced IL-12p70 and IFN-alpha and -beta in response to poly(I:C). Similar response was observed in iDCs treated with rotavirus-derived dsRNA. These responses could not be blocked by pretreatment of the cells with anti-TLR3 mAb. In CD11c(+) blood DCs, cytoplasmic retention of TLR3 was also observed as in monocyte-derived iDCs, again endorsing a different TLR3 distribution profile from fibroblasts. In precursor DC2, however, TLR3 could not be detected inside or outside the cells. Of note, there was a putative centrosomal protein that shared an epitope with TLR3 in myeloid DCs and precursor DC2, but not peripheral blood monocytes. Immunoelectron microscopic analysis revealed that TLR3, when stably expressed in the murine B cell line Ba/F3, was specifically accumulated in multivesicular bodies, a subcellular compartment situated in endocytic trafficking pathways. Thus, regulation and localization of TLR3 are different in each cell type, which may reflect participation of cell type-specific multiple pathways in antiviral IFN induction via TLR3. - M Shingai, M Ayata, H Ishida, Matsunaga, I, Y Katayama, T Seya, H Tatsuo, Y Yanagi, H OguraJOURNAL OF GENERAL VIROLOGY 84 (Pt 8) 2133 - 2143 0022-1317 2003/08 [Refereed][Not invited]
The vaccine or Vero cell-adapted strains of measles virus (MV) have been reported to use CD46 as a cell entry receptor, while lymphotropic MVs preferentially use the signalling lymphocyte activation molecule (SLAM or CD150). In contrast to the virus obtained from patients with acute measles, little is known about the receptor that is used by defective variants of MV isolated from patients with subacute sclerosing panencephalitis (SSPE), The receptor-binding properties of SSPE strains of MV were analysed using vesicular stomatitis virus pseudotypes expressing the envelope glycoproteins; of SSPE strains of MV. Such pseudotype viruses could use SLAM but not CD46 for entry. The pseudotype viruses with SSPE envelope glycoproteins could enter Vero cells, which do not express SLAM. In addition, their entry was not blocked by the monoclonal antibody to CD46, pointing to another entry receptor for SSPE strains on Vero cells. Furthermore, the unknown receptor(s), distinct from SLAM and CD46, may be present on cell lines derived from lymphoid and neural cells. Biochemical characterization of the receptor present on Vero cells and SK-N-SH neuroblastoma cells was consistent with a glycoprotein. Identification of additional entry receptors for MV will provide new insights into the mechanism of spread of MV in the central nervous system and possible reasons for differences between MVs isolated from patients with acute measles and SSPE. - M Ayata, K Komase, M Shingai, Matsunaga, I, Y Katayama, H OguraJOURNAL OF VIROLOGY 76 (24) 13062 - 13068 0022-538X 2002/12 [Refereed][Not invited]
Numerous mutations are found in subacute sclerosing panencephalitis (SSPE) viruses, and the M gene is the gene most commonly affected. In some SSPE viruses, such as the MF, Osaka-1, Osaka-2, and Yamagata-1 strains, translation of the M protein is complicated by a transcriptional defect that leads to an almost exclusive synthesis of dicistronic P-M mRNA. To understand the molecular mechanisms of this defect, we sequenced the P gene at the P-M gene junction for several virus strains and probed the involvement of several mutations in the readthrough region via their expression in measles virus minigenomes containing different sequences of the P-M gene junction and flanking reporter genes. The deletion of a single U residue in the U tract of the Osaka-1 strain (3'-UAAUAUUUUU-5') compared with the consensus sequence resulted in a marked reduction of the expression of the downstream reporter gene. In addition, the expression of the downstream gene was markedly decreased by (i) the substitution of a C residue in the U tract of the P gene end of the OSA-2/Fr/B strain of the Osaka-2 virus (3'-UGAUAUUCUU-5' compared with the sequence 3'-UGAUAUUUUU-5' from a sibling virus of the same strain, OSA-2/Fr/V), and (ii) the substitution of a G in the sequence of the P gene end of the Yamagata-1 strain at a variable site immediately upstream from the six-U tract (3'-UGAUGUUUUUU-5' instead of 3'-UGAUUUUUUUU-5'). Mutations at the P gene end can account for the readthrough transcription variation at the P-M gene junction, which directly affects M protein expression. - Ning, X, M Ayata, M Kimura, K Komase, K Furukawa, T Seto, N Ito, M Shingai, Matsunaga, I, T Yamano, H OguraVIRUS RESEARCH 86 (1-2) 123 - 131 0168-1702 2002/06 [Refereed][Not invited]
We determined the nucleotide sequence of the fusion (F) gene of three strains (Osaka-1, -2, and -3) of nonproductive variants of measles virus (MV). These viral strains were isolated in Osaka, Japan. from brain tissues of patients with subacute sclerosing panencephalitis (SSPE). Phylogenetic analysis revealed a close relationship among the three strains of SSPE virus. The cytoplasmic tail of the F protein, predicted from sequence analysis of the gene, is altered in all three SSPE strains when compared to the MV field strains. However, the extent and mode of alteration are different in each strain. The F protein of the Osaka-1 strain has six nonconservative amino acid substitutions and a 29-residue elongation of its cytoplasmic tail. The F protein of the Osaka-3 strain has two nonconservative substitutions and a 5-residue truncation of its C-terminus. Although the termination codon is not altered in the F protein of the Osaka-2 strain. five or six amino acids are changed in the cytoplasmic tail of the F protein of the two sibling viruses of this strain. The significance of the altered cytoplasmic domain of the SSPE viruses in the SSPE pathogenesis is discussed. (C) 2002 Elsevier Science B.V. All rights reserved. - N Ito, M Ayata, M Shingai, K Furukawa, T Seto, Matsunaga, I, M Muraoka, H OguraJOURNAL OF NEUROVIROLOGY 8 (1) 6 - 13 1355-0284 2002/02 [Refereed][Not invited]
Two sibling viruses, Fr/V and Fr/B, of the subacute sclerosing panencephalitis (SSPE) virus Osaka-2 strain were isolated from a small biopsy specimen of the brain of an SSPE patient by cocultivation with two different cell lines, Vero and B95a cells, respectively. These two sibling viruses differ from each other in their molecular mechanisms of defective M protein expression. In this study, we found that the Fr/B virus could scarcely form syncytium foci on Vero cells, although the Fr/V virus could do so on both Vero and B95a cells, showing a similar relation of cell tropism between recent field isolates and laboratory strains of the measles virus. Severe neurovirulence of both F/V and Fr/B viruses was observed in hamsters inoculated intracerebrally with less than 100 PFU, in contrast to the negative neurological and pathological findings in hamsters inoculated even with more than 101 PFU of their possible progenitor measles virus. Comparative sequence analysis of inoculated viruses and reisolated viruses from diseased hamster brains showed few variations at a region containing the P-NI gene junction, indicating that the inoculated viruses propagated in the brains and induced neurovirulence. All these results suggest that SSPE virus isolated with a lymphoid cell line is similar in neuropathogenicity to that isolated with a nonlymphoid cell lines, irrespective of differences in the molecular mechanism of M protein defectiveness. - Ogura H, Matsunaga I, Takano Y, Ning XJ, Ayata M, Tanaka K, Seto T, Furukawa K, Ito N, Shingai M, Kimura T, Ichihara K, Kubo H, Murakami TVIRUS RESEARCH 66 (2) 187 - 196 0168-1702 2000/02 [Refereed][Not invited]
MISC
- 野村直樹, 新開大史, 松野啓太, 関屋俊輝, 関屋俊輝, 大野円実, 磯田典和, 迫田義博, 喜田宏, 喜田宏 日本獣医学会学術集会講演要旨集 164th (CD-ROM)- 2021
- SHINGAI Masashi, MATSUMOTO Misako, SEYA Tsukasa 臨床とウイルス 35- (1) 21 -29 2007/03/31
- SHINGAI M. Int. J. Mol. Med. 2005
Research Projects
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2018/04 -2021/03Author : SHINGAI MasashiUsing a mouse model of influenza virus infection, we confirmed that the original antigenic sin does indeed occur. Repertoire analysis of immunoglobulins showed that the PR8-infected group had a higher diversity of repertoires than the non-infected group. This suggests that the infection induced a greater diversity of immunoglobulins. They also analyzed the CDRH3 region of immunoglobulins and found an amino acid sequence of CDRH3 that is specific to influenza viruses. We are now analyzing the differences in the CDRH3 sequence patterns of antibodies induced in control mice and the original antigenic sin mice by immunoglobulin repertoire analysis.
- 日本学術振興会：科学研究費助成事業Date (from‐to) : 2006 -2007Author : 新開 大史本研究は麻疹ウイルス感染モデルマウスを用い、麻疹ウイルス感染時に起こる免疫抑制のメカニズムの解明を目指す。 リンパ球側の要因:各種Tgマウス(hCD46/hCD150 tg,hCD46 tg,hCD150 tg,non-tg,hCD46/hCD150 tg IFNARko,hCD46 tg IFNARko,hCD150 tg IFNARko,non tg IFNARko)に麻疹ウイルス(MV323)株を4×105 PFU静脈内投与し、4日後、マウスの脾臓よりリンパ球を回収、PHAにて刺激した。3H-チミジンの取り込みにてリンパ球の増殖を測定したところ、IFNARkoマウスにて有意にマイトージェン刺激(PHA)に対する応答性が低下した。同様にCD4陽性、CD8陽性細胞それぞれもIFNAR依存性に増殖抑制が確認できた。野外ウイルス株MV323GFP(GFP発現麻疹ウイルス)の蛍光探索から、免疫抑制が起こっているTリンパ球には麻疹ウイルスが感染していないことを明らかにした。以上より、現在麻疹ウイルス感染モデルマウスにおいて、T細胞の免疫抑制がヒトと同様に起こるが、T細胞にはウイルスが直接感染していないことが明らかとなった。 樹上細胞側の要因:各種Tgマウスから、骨髄性樹状細胞を既報によって調整し、抗原提示機能、サイトカイン誘導能、CTL活性化能、NK活性可能などを個別に検定した。IFN誘導が感染応答に重要であったが、これらのマウスではIFNが殆ど誘導されなかった。そこで、CD150陽性でIRF-3,IRF-7の欠損マウスで免疫応答を調べた。結果はIRF-3がIFN誘導のみならず抗ウイルスNK活性化の誘導にも必須の役割を果たしていることが判明した。IRF3欠損マウスでIFNAR欠損マウスと同様の易感染性が見られることも判明した。このマウスにおける抗原提示細胞の機能を検討する予定である。