[Treatment of patients with COVID-19 on Hemodialysis: Efficacy of Remdesivir]. / Tratamiento de pacientes con COVID-19 en Hemodiálisis: Eficacia de Remdesivir.

BACKGROUND: There is no standard therapy for hemodialysis (HD) patients with COVID-19. Data on remdesivir in HD patients with COVID-19 are scarce. METHODS: We retrospectively analyzed 25 HD patients with COVID-19 treated with remdesivir. RESULTS: The median age of the patients was 78 years (range, 45-92 years) and was predominantly male (84%). A total of 44% of the patients had mild disease, 36% had moderate-1, and 20% had moderate-2. The most common symptoms were fever (76%) and coughing (44%). The most common comorbidity was renal failure (100%), followed by hypertension (60%) and cardiac disease (44%). The most frequent biomarker was elevated creatinine (100%), followed by C-reactive protein (80%), lymphopenia (76%), and D-dimer (68%). C-reactive protein levels decreased significantly before and after remdesivir administration (p < 0.001). Two patients showed deterioration, but none died. All patients recovered from COVID-19 and no adverse effects of treatment with remdesivir were observed. CONCLUSION: Our study suggests the safe use of remdesivir in HD patients with COVID-19.

Identification of cytotoxic T cells and their T cell receptor sequences targeting COVID-19 using MHC class I-binding peptides.

Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) was first reported in China in December 2019, various variants have been identified in different areas of the world such as United Kingdom (alpha), South Africa (beta and omicron), Brazil (gamma), and India (delta). Some of SARS-CoV-2 variants, each of which is characterized by a unique mutation(s) in spike protein, are concerned due to their high infectivity and the capability to escape from neutralizing antibodies elicited by vaccinations. To identify peptide epitopes that are derived from SARS-CoV-2 viral proteins and possibly induce CD8+ T cell immunity, we investigated SARS-CoV-2-derived peptides that are likely to bind to major histocompatibility complex (MHC) class I molecules. We identified a total of 15 peptides that bind to human leukocyte antigen (HLA)-A*24:02, HLA-A*02:01, or HLA-A*02:06, and possibly induce cytotoxic T lymphocytes (CTLs); thirteen of them corresponded to ORF1ab polyprotein, one peptide to spike protein and the remaining one to membrane glycoprotein. CD8+ T cells that recognize these peptides were detected in peripheral blood samples in three individuals recovered from COVID-19 as well as non-infected individuals. Since most of these peptides are commonly conserved among other coronaviruses including SARS-CoV and/or MERS-CoV, these might be useful to maintain T cell responses to coronaviruses that are pandemic at present and will become the future threat. We could define pairs of TRA and TRB sequences of nine CTL clones that recognize SARS-CoV-2-derived peptides. We might use these SARS-CoV-2-derived peptide-reactive TCR sequences for investigating the history of SARS-CoV-2 infection.

Hiccups as a specific neurological manifestation in males with COVID-19.

Several clinical manifestations of COVID-19 have been reported in the literature since then. In addition to upper respiratory symptoms, dysgeusia and anosmia are relatively common neurological manifestations with COVID-19. We had five cases of hiccups in succession; therefore, we assume that hiccups might be a specific symptom of COVID-19. We retrospectively analyzed 46 patients with COVID-19 diagnosed from February 2021 to May 2021. Among the 46 patients, 5 developed hiccups (11%). All patients were male. The median age of was 56 years. None of the patients were smokers. Further, all patients exhibited pneumonia without dysgeusia or anosmia. The median onset of hiccups was 5 days after diagnosis, with a median duration of 2 days. All patients recovered from hiccups and COVID-19. Hiccups might be a specific neurological symptom in male patients with COVID-19.

Red face may be a specific sign of SARS-CoV-2 alpha variant.

Japan is currently suffering the fourth wave of the COVID-19 pandemic, with the dominant type being SARS-CoV-2 alpha variant. Patients with COVID-19 variant types show more aggressive symptoms. In the present study, three patients developed a red face during treatment. Two of them suddenly worsened shortly after. We assumed that the red face reflected a cytokine storm and conjectured that it may be a specific sign of variant type COVID-19, because we have never seen it in patients with non-variant type. Moreover, we believe that red face may be predictive of a sudden deterioration.

Generation of neoantigen-specific T cells for adoptive cell transfer for treating head and neck squamous cell carcinoma.

Adoptive cell therapy using TCR-engineered T cells (TCR-T cells) represents a promising strategy for treating relapsed and metastatic cancers. We previously established methods to identify neoantigen-specific TCRs based on patients' PBMCs. However, in clinical practice isolation of PBMCs from advanced-stage cancer patients proves to be difficult. In this study, we substituted blood-derived T cells for tumor-infiltrating lymphocytes (TILs) and used an HLA-matched cell line of antigen-presenting cells (APCs) to replace autologous dendritic cells. Somatic mutations were determined in head and neck squamous cell carcinoma resected from two patients. HLA-A*02:01-restricted neoantigen libraries were constructed and transferred into HLA-matched APCs for stimulation of patient TILs. TCRs were isolated from reactive TIL cultures and functionality was tested using TCR- T cells in vitro and in vivo. To exemplify the screening approach, we identified the targeted neoantigen leading to recognition of the minigene construct that stimulated the strongest TIL response. Neoantigen peptides were used to load MHC-tetramers for T cell isolation and a TCR was identified targeting the KIAA1429D1358E mutation. TCR-T cells were activated, exhibited cytotoxicity, and secreted cytokines in a dose-dependent manner, and only when stimulated with the mutant peptide. Furthermore, comparable to a neoantigen-specific TCR that was isolated from the patient's PBMCs, KIAA1429D1358E-specific TCR T cells destroyed human tumors in mice. The established protocol provides the required flexibility to methods striving to identify neoantigen-specific TCRs. By using an MHC-matched APC cell line and neoantigen-encoding minigene libraries, autologous TILs can be stimulated and screened when patient PBMCs and/or tumor material are not available anymore. Abbreviations: Head and neck squamous cell carcinoma (HNSCC); adoptive T cell therapy (ACT); T cell receptor (TCR); tumor-infiltrating lymphocytes (TIL); cytotoxic T lymphocyte (CTL); peripheral blood mononuclear cell (PBMC); dendritic cell (DC); antigen-presenting cells (APC).

TCR sequencing analysis of cancer tissues and tumor draining lymph nodes in colorectal cancer patients.

Tumor draining lymph nodes (TDLNs) are located in the routes of lymphatic drainage from a primary tumor and have the highest risk of metastasis in various types of solid tumors. TDLNs are also considered as a tissue to activate the antitumor immunity, where antigen-specific effector T cells are generated. However, T cell receptor (TCR) repertoires in TDLNs have not been well characterized. We collected 23 colorectal cancer tumors with 203 lymph nodes with/without metastatic cancer cells (67 were metastasis-positive and the remaining 136 were metastasis-negative) and performed TCR sequencing. Metastasis-positive TDLNs showed a significantly lower TCR diversity and shared TCR clonotypes more frequently with primary tumor tissues compared to metastasis-negative TDLNs. Principal component analysis indicated that TDLNs with metastasis showed similar TCR repertoires. These findings suggest that cancer-reactive T cell clones could be enriched in the metastasis-positive TDLNs.

Identification of neoantigen-specific T cells and their targets: implications for immunotherapy of head and neck squamous cell carcinoma.

To develop a practically applicable method for T-cell receptor (TCR)-engineered T cell immunotherapy targeting neoantigens, we have been attempting to identify neoantigen-specific T cell receptors (TCRs) and establish TCR-engineered T cells in a 3-4-month period. In this study, we report the characterization of T cell repertoires in tumor microenvironment (TME) and identification of neoantigen-specific TCRs after stimulation of patient-derived T cells. We screened 15 potential neoantigen peptides and successfully identified two CD8+HLA-dextramer+ T cells, which recognized MAGOHBG17A and ZCCHC14P368L. All three dominant TCR clonotypes from MAGOHBG17A-HLA dextramer-sorted CD8+ T cells were also found in T cells in TME, while none of dominant TCR clonotypes from ZCCHC14P368L-HLA dextramer-sorted CD8+ T cells was found in the corresponding TME. The most dominant TCRA/TCRB pairs for these two neoantigens were cloned into HLA-matched healthy donors' T lymphocytes to generate TCR-engineered T cells. The functional assay showed MAGOHBG17A TCR-engineered T cells could be significantly activated in a mutation-specific, HLA-restricted and peptide-dose-dependent manner while ZCCHC14P368L TCR-engineered T cells could not. Our data showed neoantigen-reactive T cell clonotypes that were identified in the patient's peripheral blood could be present in the corresponding TME and might be good TCRs targeting neoantigens.

The era of immunogenomics/immunopharmacogenomics.

Although germline alterations and somatic mutations in disease cells have been extensively analyzed, molecular changes in immune cells associated with disease conditions have not been characterized in depth. It is clear that our immune system has a critical role in various biological and pathological conditions, such as infectious diseases, autoimmune diseases, drug-induced skin and liver toxicity, food allergy, and rejection of transplanted organs. The recent development of cancer immunotherapies, particularly drugs modulating the immune checkpoint molecules, has clearly demonstrated the importance of host immune cells in cancer treatments. However, the molecular mechanisms by which these new therapies kill tumor cells are still not fully understood. In this regard, we have begun to explore the role of newly developed tools such as next-generation sequencing in the genetic characterization of both cancer cells and host immune cells, a field that is called immunogenomics/ immunopharmacogenomics. This new field has enormous potential to help us better understand changes in our immune system during the course of various disease conditions. Here we report the potential of deep sequencing of T-cell and B-cell receptors in capturing the molecular contribution of the immune system, which we believe plays critical roles in the pathogenesis of various human diseases.

Induction of Neoantigen-Specific Cytotoxic T Cells and Construction of T-cell Receptor-Engineered T Cells for Ovarian Cancer.

Purpose: Current evolution of cancer immunotherapies, such as immune checkpoint blockade, has implicated neoantigens as major targets of anticancer cytotoxic T cells. Adoptive T-cell therapy with neoantigen-specific T-cell receptor (TCR)-engineered T cells would be an attractive therapeutic option for advanced cancers where the host antitumor immune function is strongly inhibited. We previously developed a rapid and efficient pipeline for production of neoantigen-specific TCR-engineered T cells using peripheral blood from an HLA-matched healthy donor. Our protocol required only 2 weeks from stimulation of T cells with neoantigen-loaded dendritic cells to the identification of neoantigen-specific TCRs. We conducted the pilot study to validate our protocol.Experimental Design: We used tumors from 7 ovarian cancer patients to validate our protocol.Results: We chose 14 candidate neoantigens from 7 ovarian tumors (1-3 candidates for each patient) and then successfully induced three neoantigen-specific T cells from 1 healthy donor and identified their TCR sequences. Moreover, we validated functional activity of the three identified TCRs by generating TCR-engineered T cells that recognized the corresponding neoantigens and showed cytotoxic activity in an antigen dose-dependent manner. However, one case of neoantigen-specific TCR-engineered T cells showed cross-reactivity against the corresponding wild-type peptide.Conclusions: This pilot study demonstrated the feasibility of our efficient process from identification of neoantigen to production of the neoantigen-targeting cytotoxic TCR-engineered T cells for ovarian cancer and revealed the importance of careful validation of neoantigen-specific TCR-engineered T cells to avoid severe immune-related adverse events. Clin Cancer Res; 24(21); 5357-67. ©2018 AACR See related commentary by Anczurowski and Hirano, p. 5195.

Effective screening of T cells recognizing neoantigens and construction of T-cell receptor-engineered T cells.

Neoantigens are the main targets of tumor-specific T cells reactivated by immune checkpoint-blocking antibodies or when using tumor-infiltrating T cells for adoptive therapy. While cancers often accumulate hundreds of mutations and harbor several immunogenic neoantigens, the repertoire of mutation-specific T cells in patients might be restricted. To bypass suboptimal conditions, which impede the reactivation of existing T cells or the priming of neoantigen-specific T cells in a patient, we employ T cells of healthy donors with an overlapping HLA repertoire to target cancer neoantigens. In this study, we focus on streamlining the process of in vitro-induction of neoantigen-specific T cells and isolating their T cell receptors (TCRs) to establish a time-efficient protocol that will allow the patient to benefit from subsequent therapy. We first optimized the priming of T cells to omit multiple restimulations and extended culturing. Neoantigen-specific T cells were enriched using specific dextramers and next-generation sequencing was applied to determine the TCR repertoire. This allowed us to circumvent the laborious process of expanding T cell clones. Using this protocol, we successfully identified HLA-A-restricted TCRs specific for neoantigens found in an esophageal cancer cell line (TE-8) and a primary ovarian cancer. To verify TCR specificity, we generated TCR-engineered T cells and confirmed recognition of the tumor-derived neoantigens. Our results also emphasize the importance of neoepitope selection in order to avoid cross-reactivity to corresponding wild-type peptide sequences. In conclusion, we established a 2-week protocol for generating and identifying neoantigen-specific TCRs from third-party donors making this strategy applicable for clinical use.

Identification of an HLA-A2-restricted epitope peptide derived from hypoxia-inducible protein 2 (HIG2).

We herein report the identification of an HLA-A2 supertype-restricted epitope peptide derived from hypoxia-inducible protein 2 (HIG2), which is known to be a diagnostic marker and a potential therapeutic target for renal cell carcinoma. Among several candidate peptides predicted by the HLA-binding prediction algorithm, HIG2-9-4 peptide (VLNLYLLGV) was able to effectively induce peptide-specific cytotoxic T lymphocytes (CTLs). The established HIG2-9-4 peptide-specific CTL clone produced interferon-γ (IFN-γ) in response to HIG2-9-4 peptide-pulsed HLA-A*02:01-positive cells, as well as to cells in which HLA-A*02:01 and HIG2 were exogenously introduced. Moreover, the HIG2-9-4 peptide-specific CTL clone exerted cytotoxic activity against HIG2-expressing HLA-A*02:01-positive renal cancer cells, thus suggesting that the HIG2-9-4 peptide is naturally presented on HLA-A*02:01 of HIG-2-expressing cancer cells and is recognized by CTLs. Furthermore, we found that the HIG2-9-4 peptide could also induce CTLs under HLA-A*02:06 restriction. Taken together, these findings indicate that the HIG2-9-4 peptide is a novel HLA-A2 supertype-restricted epitope peptide that could be useful for peptide-based immunotherapy against cancer cells with HIG2 expression.

Serological studies of infectious bronchitis vaccines against Japanese field isolates of homologous and heterologous genotypes.

The genetic diversity of the partial S1 gene involving the hyper variable region for infectious bronchitis (IB) vaccine strains in Japan were compared with those of IB virus isolated from the field in Japan. Field isolates have mainly been classified into three major genotypes, JP-I, JP-II and JP-III, since 2003; however, the 4/91 genotype was detected from recent field isolates in Japan. The virus neutralization (VN) activity with vaccine immunized serum was investigated to evaluate the protective effects of vaccines against Japanese field isolates. In the results of the VN test, antiserum immunized with the GN and C78 (JP-I), TM-86w and Miyazaki (JP-II) and 4/91 (793B) vaccine strains could neutralize a high rate of field isolates of homologous genotype (75% of field isolates of JP-I, 100% of that of JP-II and 100% of that of 793B, respectively). For field isolates of JP-III, even though there are no homologous genotype vaccine strain, some strains of JP-III were neutralized with immune serum from vaccine strains of the heterologous genotype. In this study, a correlation between serological property and genotype was found for JP-I, JP-II and 793B. Our results suggested that an effective vaccine could be predicted in accordance with the genotype of field isolates.

Genetic analysis of the S1 gene of 4/91 type infectious bronchitis virus isolated in Japan.

S1 gene sequences for infectious bronchitis virus (IBV) strains of the 4/91 genotype (commonly called 793B) isolated from field outbreaks in Japan were analyzed to ascertain the relationship to 4/91 vaccine strain. Three field isolates (JP/Wakayama/2003, JP/Iwate/2005 and JP/Saitama/2006) from flocks not immunized with a 4/91 type live IBV vaccine and one isolate (JP/Wakayama-2/2004) from a flock immunized with a 4/91 type live vaccine were examined. The amino acid identities among JP/Wakayama/2003, JP/Iwate/2005 and JP/Saitama/2006 were about 98%, whereas the identities to the 4/91 vaccine strain and JP/Wakayama-2/2004 were about 90%. Three of the field isolates, JP/Wakayama/2003, JP/Iwate/2005 and JP/Saitama/2006, were classified into a cluster closely related to French and Spanish isolates, but different from the cluster including the vaccine and JP/Wakayama-2/2004. These results indicate that JP/Wakayama/2003, JP/Iwate/2005 and JP/Saitama/2006 were derived from foreign field isolates, but not from the vaccine strain. On the other hand, the S1 gene of JP/Wakayama-2/2004 revealed high sequence similarity with that of the 4/91 vaccine strain and appeared to be a vaccine-like virus derived from a vaccine. The field isolates of 4/91 genotype IBV could be distinguished from other genotypes by using the BalI and Pst I enzymes in addition to the polymerase chain reaction (PCR) -restriction fragment length polymorphism (RFLP) methods of Mase et al. [16] using Hae II and EcoR I enzymes. Furthermore, the 4/91 vaccine strain and vaccine-like isolate (JP/Wakayama-2/2004) could be differentiated from the other field isolates by Bgl II digestion. This method, therefore, would assist in identification of field isolates of the 4/91 genotype as outbreaks of IBV in vaccinated flocks.

Isolation of 4/91 type of infectious bronchitis virus as a new variant in Japan and efficacy of vaccination against 4/91 type field isolate.

Among field isolates of infectious bronchitis virus (IBV) from recent outbreaks, some isolates that were classified into the 4/91 genotype, by analysis of the S1 gene, have been confirmed to be a new variant in Japan. To elucidate the characteristics of these isolates, pathogenicity in chicks and the efficacy of vaccines against this new variant were examined. Severe respiratory symptoms were observed in 4-day-old specific pathogen free chicks inoculated with a 4/91 genotype isolate, either JP/Wakayama/2003 or JP/ Iwate/2005; body weights 3 wk after inoculation were significantly lower than those of chicks inoculated with a 4/91 vaccine strain. These 4/91 isolates were neutralized with serum from birds immunized with 4/91 vaccine. In a challenge-protection test, five groups of chicks were immunized with C78, TM-86w, H120, Kita-1, or 4/91 vaccines and then challenged with JP/Iwate/2005 4 wk after vaccination. A protective effect in the 4/91 and TM-86w vaccine groups was indicated by evaluation of the ciliostasis score of the trachea, the respiratory symptom score, and virus isolation from trachea swab samples after challenge. The results of this study suggested that the 4/91 type of IBV, which is virulent to chicks when compared to vaccine strains, has emerged as a new variant in Japan, and vaccines containing the 4/91 strain or the TM-86w strain could be effective for this variant.