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HLA-A, -C, -B, and -DRB1 genotypes were analyzed in 178 Japanese COVID-19 patients to investigate the association of HLA with severe COVID-19. Analysis of 32 common HLA alleles at four loci revealed a significant association between HLA-DRB1*09:01 and severe COVID-19 (odds ratio [OR], 3.62; 95% CI, 1.57-8.35; p = 0.00251 [permutation p value = 0.0418]) when age, sex, and other common HLA alleles at the DRB1 locus were adjusted. The DRB1*09:01 allele was more significantly associated with risk for severe COVID-19 compared to preexisting medical conditions such as hypertension, diabetes, and cardiovascular diseases. These results indicate a potential role for HLA in predisposition to severe COVID-19.

Abstract An expanded myeloid cell compartment is a hallmark of severe coronavirus disease 2019 (COVID-19). However, data regarding myeloid cell expansion have been collected in Europe, where the mortality rate by COVID-19 is greater than those in other regions including Japan. Thus, characteristics of COVID-19-induced myeloid cell subsets remain largely unknown in the regions with low mortality rates. Here, we analyzed cellular dynamics of myeloid-derived suppressor cell (MDSC) subsets and examined whether any of them correlate with disease severity and prognosis, using blood samples from Japanese COVID-19 patients. We observed that polymorphonuclear (PMN)-MDSCs, but not other MDSC subsets, transiently expanded in severe cases but not in mild or moderate cases. Contrary to previous studies in Europe, this subset selectively expanded in survivors of severe cases and subsided before discharge, but such transient expansion was not observed in non-survivors in Japanese cohort. Analysis of plasma cytokine/chemokine levels revealed positive correlation of PMN-MDSC frequencies with IL-8 levels, indicating the involvement of IL-8 on recruitment of PMN-MDSCs to peripheral blood following the onset of severe COVID-19. Our data indicate that transient expansion of the PMN-MDSC subset results in improved clinical outcome. Thus, this myeloid cell subset may be a predictor of prognosis in cases of severe COVID-19 in Japan.

Adult T-cell leukemia (ATL) is induced by chronic latent infection with human T-cell leukemia virus type 1 (HTLV-1). HTLV-1 Tax is an oncogenic factor that can be targeted by host T-cell responses. However, the expression of Tax in vivo is little in ATL cells and the impact of Tax-specific T-cell responses on ATL progression remains unclear. In the present study, we examined Tax-specific T-cell responses in C57BL/6 mice after syngeneic transplantation with tax-transgenic mouse-derived ATL (mATL) cells. We first confirmed that cellular tax cDNAs are mostly maintained and detectable in the spleen three weeks after mATL cell transplantation. However, mATL cell transplantation did not induce significant Tax-specific T-cell responses. Mice immunized with DNA and adenovirus vectors expressing Tax exhibited Tax-specific CD4+ T-cell responses but showed no enhancement of the responses or reduction in cellular tax cDNA levels after mATL cell transplantation. This study provides an animal model for analyzing the interaction between ATL cells and host immune responses as well as indicates the limited impact of Tax-specific T-cell responses on the proliferation of ATL cells.

Effective vaccines are essential for the control of the COVID-19 pandemic. Currently-developed vaccines inducing SARS-CoV-2 spike (S) antigen-specific neutralizing antibodies (NAbs) are effective, but the appearance of NAb-resistant S variant viruses is of great concern. A vaccine inducing S-independent or NAb-independent SARS-CoV-2 control may contribute to containment of these variants. Here, we investigate the efficacy of an intranasal vaccine expressing viral non-S antigens against intranasal SARS-CoV-2 challenge in cynomolgus macaques. Seven vaccinated macaques exhibit significantly reduced viral load in nasopharyngeal swabs on day 2 post-challenge compared to nine unvaccinated controls. The viral control in the absence of SARS-CoV-2-specific NAbs is significantly correlated with vaccine-induced viral antigen-specific CD8+ T-cell responses. Our results indicate that CD8+ T-cell induction by intranasal vaccination can result in NAb-independent control of SARS-CoV-2 infection, highlighting a potential of vaccine-induced CD8+ T-cell responses to contribute to COVID-19 containment., Graphical Abstract, Ishii et al. show neutralization-independent SARS-CoV-2 control associated with vaccine-induced CD8+ T-cell responses, indicating that virus-specific CD8+ T-cell induction by intranasal vaccination can result in SARS-CoV-2 control. Results highlight the potential of vaccine-induced CD8+ T-cell responses to contribute to the control of SARS-CoV-2 variants.

In human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections, host major histocompatibility complex class I (MHC-I) genotypes have a great impact on viral replication and MHC-I-associated viral genome mutations are selected under CD8+ T-cell pressure. Association of MHC-I genotypes with HIV/SIV control has been investigated at MHC-I allele levels but not fully at haplotype levels. We previously established groups of rhesus macaques sharing individual MHC-I haplotypes. In the present study, we compared viral genome diversification after SIV infection in macaques possessing a protective MHC-I haplotype, 90-010-Id, with those possessing a non-protective MHC-I haplotype, 90-010-Ie. These two MHC-I haplotypes are associated with immunodominant CD8+ T-cell responses targeting similar regions of viral Nef antigen. Analyses of viral genome sequences and antigen-specific T-cell responses showed four and two candidates of viral CD8+ T-cell targets associated with 90-010-Id and 90-010-Ie, respectively, in addition to the Nef targets. In these CD8+ T-cell target regions, higher numbers of mutations were detected at the setpoint after SIV infection in macaques possessing 90-010-Id than those possessing 90-010-Ie. These results indicate higher selective pressure on overall CD8+ T-cell targets associated with the protective MHC-I haplotype, suggesting a pattern of HIV/SIV control by multiple target-specific CD8+ T-cell responses.

Induction of antibodies targeting viral glycoproteins is a key for the development of a vaccine against enveloped virus infection. Glycoproteins on the virion exhibiting native multimer structure may be a good immunogen to present antibody epitopes, but it is often difficult to prepare immunogenic inactivated virions. Preparation of soluble glycoprotein multimers has been attempted, while virus-like particles carrying target glycoproteins can be a more immunogenic antigen. In the present study, a target glycoprotein-embedded Sendai virus (SeV) particle was developed for induction of anti-virus antibodies. We constructed a chimeric antigen, HIV-1 EnvF, consisting of HIV-1 Env ectodomain and SeV F transmembrane-cytoplasmic domain, which was shown to be efficiently incorporated into the SeV virion. EnvF was recognized by anti-HIV-1 broadly-neutralizing monoclonal antibodies (bnAbs) including 35O22 that targets an Env trimer-dependent epitope. Analysis revealed that HIV-1

SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8+ T-cell responses in convalescent individuals, the role of virus-specific CD8+ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 105 or 106 TCID50 of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8+ T cells were undetectable on day 7 and thereafter, while virus-specific CD8+ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10-17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8+ T cells, implying that CD8+ T-cell dysfunction may not solely lead to viral control failure.Author SummarySARS-CoV-2 infection presents a wide spectrum of clinical manifestations ranging from asymptomatic to fatal respiratory failure. The determinants for failure in viral control and/or fatal disease progression have not been elucidated fully. Both acquired immune effectors, antibodies and CD8+ T cells, are considered to contribute to viral control. However, it remains unknown whether a deficiency in either of these two arms is directly linked to failure in the control of SARS-CoV-2 replication. In the present study, to know the requirement of CD8+ T cells for viral control after the establishment of infection, we examined the effect of CD8+ cell depletion by monoclonal anti-CD8 antibody administration in the subacute phase on SARS-CoV-2 replication in cynomolgus macaques. Unexpectedly, our analysis revealed no significant impact of CD8+ cell depletion on viral replication, indicating that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but this study suggests that CD8+ T-cell dysfunction may not solely lead to viral control failure or fatal disease progression.

SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8+ T-cell responses in convalescent individuals, the role of virus-specific CD8+ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 105 or 106 TCID50 of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8+ T cells were undetectable on day 7 and thereafter, while virus-specific CD8+ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10–17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8+ T cells, implying that CD8+ T-cell dysfunction may not solely lead to viral control failure., Author summary SARS-CoV-2 infection presents a wide spectrum of clinical manifestations ranging from asymptomatic to fatal respiratory failure. The determinants for failure in viral control and/or fatal disease progression have not been elucidated fully. Both acquired immune effectors, antibodies and CD8+ T cells, are considered to contribute to viral control. However, it remains unknown whether a deficiency in either of these two arms is directly linked to failure in the control of SARS-CoV-2 replication. In the present study, to know the requirement of CD8+ T cells for viral control after the establishment of infection, we examined the effect of CD8+ cell depletion by monoclonal anti-CD8 antibody administration in the subacute phase on SARS-CoV-2 replication in cynomolgus macaques. Unexpectedly, our analysis revealed no significant impact of CD8+ cell depletion on viral replication, indicating that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but this study suggests that CD8+ T-cell dysfunction may not solely lead to viral control failure or fatal disease progression.

Effective vaccines are essential for the control of the COVID-19 pandemic. Currently-developed vaccines inducing SARS-CoV-2 spike (S) antigen-specific neutralizing antibodies (NAbs) are effective, but the appearance of NAb-resistant S variant viruses is of great concern. A vaccine inducing S-independent or NAb-independent SARS-CoV-2 control may contribute to containment of these variants. Here, we investigated the efficacy of an intranasal vaccine expressing viral non-S antigens against intranasal SARS-CoV-2 challenge in cynomolgus macaques. Nine vaccinated macaques exhibited significantly reduced viral load in nasopharyngeal swabs on day 2 post-challenge compared to nine unvaccinated controls. The viral control in the absence of SARS-CoV-2-specific NAbs was significantly correlated with vaccine-induced viral antigen-specific CD8+ T-cell responses. Our results indicate that CD8+ T-cell induction by intranasal vaccination can result in NAb-independent control of SARS-CoV-2 infection, highlighting a potential of vaccine-induced CD8+ T-cell responses to contribute to COVID-19 containment. Funding: This work was supported by Japan Agency for Medical Research and Development (AMED [JP19fk0108104 to A.K.-T. and JP20nk0101601, JP20jm0110012, JP21fk0410035, JP21fk0108125, and JP21jk0210002 to T.M.]) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) in Japan (JSPS Grants-in-Aid for Scientific Research [21H02745 to T.M.]). Declaration of Interests: H.I., K.K., R.S., and T.M are the inventors on Patent Cooperation Treaty (PCT) application for SeV-NME vaccine. Authors have no other conflicts of interest to declare. Ethics Approval Statement: Approval by the Committee on the Ethics of Animal Experiments in NIID (permission number: 520001) under the guidelines for animal experiments in accordance with the Guidelines for Proper Conduct of Animal Experiments established by the Science Council of Japan.

An expanded myeloid cell compartment is a hallmark of severe coronavirus disease 2019 (COVID-19); however, it remains unclear whether myeloid cells are beneficial or detrimental to the clinical outcome. Here, we tracked cellular dynamics of myeloid-derived suppressor cell (MDSC) subsets and examined whether any of them correlate with disease severity and prognosis by flow cytometric analysis of blood samples from COVID-19 patients. We observed that polymorphonuclear (PMN)-MDSCs, rather than other MDSC subsets, transiently expanded in severe cases but not in mild or moderate cases. Notably, this subset was selectively expanded in survivors of severe cases and diminished during recovery. Analysis of plasma cytokines/chemokines revealed that interleukin-8 increased prior to PMN-MDSC expansion in survivors and returned to basal levels during the recovery phase. In contrast, interleukin-6 and interferon--induced protein 10 were abundantly induced in non-survivors, suggesting possible downstream targets for the immunosuppressive effects of the MDSC subset. Our data indicate that increased cellularity of PMN-MDSCs might be beneficial for the clinical outcome and could be useful as a possible predictor of prognosis in cases of severe COVID-19.

Anti-retroviral therapy (ART) can inhibit HIV proliferation but not achieve virus eradication from HIV-infected individuals. Under ART-based HIV control, virus-specific CD8+ T-cell responses are often reduced. Here, we investigated the impact of therapeutic vaccination inducing virus-specific CD8+ T-cell responses under ART on viral control in a macaque AIDS model. Twelve rhesus macaques received ART from week 12 to 32 after simian immunodeficiency virus (SIV) infection. Six of them were vaccinated with Sendai virus vectors expressing SIV Gag and Vif at weeks 26 and 32, and Gag/Vif-specific CD8+ T-cell responses were enhanced and became predominant. All macaques controlled viremia during ART but showed viremia rebound after ART cessation. Analysis of in vitro CD8+ cell ability to suppress replication of autologous lymphocytes-derived SIVs found augmentation of anti-SIV efficacy of CD8+ cells after vaccination. In the vaccinated animals, the anti-SIV efficacy of CD8+ cells at week 34 was correlated positively with Gag-specific CD8+ T-cell frequencies and inversely with rebound viral loads at week 34. These results indicate that Gag-specific CD8+ T-cell induction by therapeutic vaccination can augment anti-virus efficacy of CD8+ cells, which may be insufficient for functional cure but contribute to more stable viral control under ART.

Anti-retroviral therapy (ART) can inhibit HIV proliferation but not achieve virus eradication from HIV-infected individuals. Under ART-based HIV control, virus-specific CD8+ T-cell responses are often reduced. Here, we investigated the impact of therapeutic vaccination inducing virus-specific CD8+ T-cell responses under ART on viral control in a macaque AIDS model. Twelve rhesus macaques received ART from week 12 to 32 after simian immunodeficiency virus (SIV) infection. Six of them were vaccinated with Sendai virus vectors expressing SIV Gag and Vif at weeks 26 and 32, and Gag/Vif-specific CD8+ T-cell responses were enhanced and became predominant. All macaques controlled viremia during ART but showed viremia rebound after ART cessation. Analysis of in vitro CD8+ cell ability to suppress replication of autologous lymphocytes-derived SIVs found augmentation of anti-SIV efficacy of CD8+ cells after vaccination. In the vaccinated animals, the anti-SIV efficacy of CD8+ cells at week 34 was correlated positively with Gag-specific CD8+ T-cell frequencies and inversely with rebound viral loads at week 34. These results indicate that Gag-specific CD8+ T-cell induction by therapeutic vaccination can augment anti-virus efficacy of CD8+ cells, which may be insufficient for functional cure but contribute to more stable viral control under ART.

Adult T-cell leukemia (ATL) is induced by chronic latent infection with human T-cell leukemia virus type 1 (HTLV-1). HTLV-1 Tax is an oncogenic factor that can be targeted by host T-cell responses. However, the expression of Tax in vivo is little in ATL cells and the impact of Tax-specific T-cell responses on ATL progression remains unclear. In the present study, we examined Tax-specific T-cell responses in C57BL/6 mice after syngeneic transplantation with tax-transgenic mouse-derived ATL (mATL) cells. We first confirmed that cellular tax cDNAs are mostly maintained and detectable in the spleen three weeks after mATL cell transplantation. However, mATL cell transplantation did not induce significant Tax-specific T-cell responses. Mice immunized with DNA and adenovirus vectors expressing Tax exhibited Tax-specific CD4

An expanded myeloid cell compartment is a hallmark of severe coronavirus disease 2019 (COVID-19). However, data regarding myeloid cell expansion have been collected in Europe, where the mortality rate by COVID-19 is greater than those in other regions including Japan. Thus, characteristics of COVID-19-induced myeloid cell subsets remain largely unknown in the regions with low mortality rates. Here, we analyzed cellular dynamics of myeloid-derived suppressor cell (MDSC) subsets and examined whether any of them correlate with disease severity and prognosis, using blood samples from Japanese COVID-19 patients. We observed that polymorphonuclear (PMN)-MDSCs, but not other MDSC subsets, transiently expanded in severe cases but not in mild or moderate cases. Contrary to previous studies in Europe, this subset selectively expanded in survivors of severe cases and subsided before discharge, but such transient expansion was not observed in non-survivors in Japanese cohort. Analysis of plasma cytokine/chemokine levels revealed positive correlation of PMN-MDSC frequencies with interleukin 8 (IL-8) levels prior to the cell expansion, indicating the involvement of IL-8 on recruitment of PMN-MDSCs to peripheral blood following the onset of severe COVID-19. Thus, our data indicates that transient expansion of the PMN-MDSC subset results in improved clinical outcome. Thus, this myeloid cell subset may be a predictor of prognosis in cases of severe COVID-19 in Japan.

In human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections, host major histocompatibility complex class I (MHC-I) genotypes have a great impact on viral replication and MHC-I-associated viral genome mutations are selected under CD8

BackgroundAn outbreak of novel coronavirus (SARS-CoV-2)-associated respiratory infectious diseases (COVID-19) emerged in 2019 and has spread rapidly in humans around the world. The demonstration of in vitro infectiousness of respiratory specimens is an informative surrogate for SARS-CoV-2 transmission from patients with COVID-19; accordingly, viral isolation assays in cell culture are an important aspect of laboratory diagnostics for COVID-19.MethodsWe developed a simple and rapid protocol for isolating SARS-CoV-2 from respiratory specimens using VeroE6/TMPRSS2 cells, a cell line that is highly susceptible to the virus. We also investigated a correlation between isolation of SARS-CoV-2 and viral load detected by real-time RT-PCR (rRT-PCR) using N2 primer/probe set that has been developed for testing of COVID-19 in Japan.ResultsThe SARS-CoV-2 isolation protocol did not require blind passage of inoculated cells and yielded the results of viral isolation within 7 days after inoculation. Specimens with cycle threshold (Ct) values of 35 were virus isolation-positive, indicating that low viral loads (high Ct values) in upper respiratory specimens do not always indicate no risk of containing transmissible virus.ConclusionIn combination with rRT-PCR, the SARS-CoV-2 isolation protocol provides a means for assessing the potential risk of transmissible virus in upper respiratory specimens.