Kei Ouchi

CD8 Epitope Escape and Reversion in Acute HCV Infection

In the setting of acute hepatitis C virus (HCV) infection, robust HCV-specific CD8+ cytotoxic T lymphocyte (CTL) responses are associated with initial control of viremia. Despite these responses, 70–80% of individuals develop persistent infection. Although viral escape from CD8 responses has been illustrated in the chimpanzee model of HCV infection, the effect of CD8 selection pressure on viral evolution and containment in acute HCV infection in humans remains unclear. Here, we examined viral evolution in an immunodominant human histocompatibility leukocyte antigen (HLA)-B8–restricted NS3 epitope in subjects with acute HCV infection. Development of mutations within the epitope coincided with loss of strong ex vivo tetramer and interferon γ enzyme-linked immunospot responses, and endogenous expression of variant NS3 sequences suggested that the selected mutations altered processing and presentation of the variant epitope. Analysis of NS3 sequences from 30 additional chronic HCV-infected subjects revealed a strong association between sequence variation within this region and expression of HLA-B8, supporting reproducible allele-specific selection pressures at the population level. Interestingly, transmission of an HLA-B8–associated escape mutation to an HLA-B8 negative subject resulted in rapid reversion of the mutation. Together, these data indicate that viral escape from CD8+ T cell responses occurs during human HCV infection and that acute immune selection pressure is of sufficient magnitude to influence HCV evolution.

Comprehensive Analysis of CD8+-T-Cell Responses against Hepatitis C Virus Reveals Multiple Unpredicted Specificities

ABSTRACT The hepatitis C virus (HCV)-specific CD8+-T-cell response is thought to play a critical role in HCV infection. Studies of these responses have largely relied on the analysis of a small number of previously described or predicted HCV epitopes, mostly restricted by HLA A2. In order to determine the actual breadth and magnitude of CD8+-T-cell responses in the context of diverse HLA class I alleles, we performed a comprehensive analysis of responses to all expressed HCV proteins. By using a panel of 301 overlapping peptides, we analyzed peripheral blood mononuclear cells (PBMC) from a cohort of 14 anti-HCV-positive, HLA A2-positive individuals in an enzyme-linked immunospot assay. Only four subjects had detectable HLA A2-restricted responses in PBMC, and only 3 of 19 predicted A2 epitopes were targeted, all of which were confirmed by tetramer analysis. In contrast, 9 of 14 persons showed responses with more comprehensive analyses, with many responses directed against previously unreported epitopes. These results indicate that circulating HCV-specific CD8+-T-cell responses can be detected in PBMC in the majority of infected persons and that these responses are heterogeneous with no immunodominant epitopes consistently recognized. Since responses to epitopes restricted by single HLA alleles such as HLA A2 do not predict the overall response in an individual, more comprehensive approaches, as shown here, should facilitate definition of the role of the CD8+-T-cell response in HCV infection. Moreover, the low level or absence of responses to many predicted epitopes provides a rationale for immunotherapeutic interventions to broaden cytotoxic-T-lymphocyte recognition.

Broad repertoire of the CD4+ Th cell response in spontaneously controlled hepatitis C virus infection includes dominant and highly promiscuous epitopes.

A vigorous hepatitis C virus (HCV)-specific Th cell response is regarded as essential to the immunological control of HCV viremia. The aim of this study was to comprehensively define the breadth and specificity of dominant HCV-specific CD4+ T cell epitopes in large cohorts of subjects with chronic and spontaneously resolved HCV viremia. Following in vitro stimulation of PBMC, HCV-specific cell cultures from each subject were screened with an overlapping panel of synthetic 20-mer peptides spanning the entire HCV polyprotein. Of 22 subjects who spontaneously controlled HCV viremia, all recognized at least one of a group of six epitopes situated within the nonstructural (NS) proteins NS3, NS4, and NS5, each of which was detected by >30% of subjects, but most subjects recognized additional, more heterogeneous specificities. In contrast, none of the most frequently targeted epitopes was detected by >5% of persons with chronic infection. The most frequently recognized peptides showed promiscuous binding to multiple HLA-DR molecules in in vitro binding assays and were restricted by different HLA-DR molecules in functional assays in different persons. These data demonstrate that predominant CD4+ T cell epitopes in persons with resolved HCV infection are preferentially located in the nonstructural proteins and are immunogenic in the context of multiple class II molecules. This comprehensive characterization of CD4+ T cell epitopes in resolved HCV infection provides important information to facilitate studies of immunopathogenesis and HCV vaccine design and evaluation.

Full-Breadth Analysis of CD8+ T-Cell Responses in Acute Hepatitis C Virus Infection and Early Therapy

ABSTRACT Multispecific CD8+ T-cell responses are thought to be important for the control of acute hepatitis C virus (HCV) infection, but to date little information is actually available on the breadth of responses at early time points. Additionally, the influence of early therapy on these responses and their relationships to outcome are controversial. To investigate this issue, we performed comprehensive analysis of the breadth and frequencies of virus-specific CD8+ T-cell responses on the single epitope level in eight acutely infected individuals who were all started on early therapy. During the acute phase, responses against up to five peptides were identified. During therapy, CD8+ T-cell responses decreased rather than increased as virus was controlled, and no new specificities emerged. A sustained virological response following completion of treatment was independent of CD8+ T-cell responses, as well as CD4+ T-cell responses. Rapid recrudescence also occurred despite broad CD8+ T-cell responses. Importantly, in vivo suppression of CD3+ T cells using OKT3 in one subject did not result in recurrence of viremia. These data suggest that broad CD8+ T-cell responses alone may be insufficient to contain HCV replication, and also that early therapy is effective independent of such responses.

Do antiviral CD8+ T cells select hepatitis C virus escape mutants? Analysis in diverse epitopes targeted by human intrahepatic CD8+ T lymphocytes

Summary.  Hepatitis C virus (HCV) is a variable RNA virus that can readily establish persistent infection. Cellular immune responses are important in the early control of the virus. Evidence from animal models suggests that mutation in epitopes recognized by CD8+ T lymphocytes may play an important role in the establishment of persistence but in human persistent infection, equivalent evidence is lacking. We investigated this by analysing a unique resource: viruses from a set of chronically HCV‐infected individuals in whom the CD8+ T‐cell responses in liver had previously been accurately mapped. Virus was sequenced in seven individuals at 10 epitopes restricted by 10 human leucocyte antigen (HLA) molecules. Two main patterns emerged: in the majority of epitopes sequenced, no variation was seen. In three epitopes, mutations were identified which were compatible with immune escape as assessed using phylogenetic and/or functional studies. These data suggest that – even where specific intrahepatic T cells are detectable – many epitopes do not undergo mutation in chronic human infection. On the contrary, virus may escape from intrahepatic CD8+ T‐cell responses in a ‘patchy’ manner in certain specific epitopes. Furthermore, longitudinal studies to identify the differences between ‘selecting’ and ‘nonselecting’ intrahepatic CD8+ T‐cell responses are needed in HCV infection.

The Prevalence of Inpatients at 33 U.S. Hospitals Appropriate for and Receiving Referral to Palliative Care

BACKGROUND The extent of unmet need for palliative care in U.S. hospitals remains largely unknown. We conducted a multisite cross-sectional, retrospective point prevalence analysis to determine the size and characteristics of the population of inpatients at 33 U.S. hospitals who were appropriate for palliative care referral, as well as the percentage of these patients who were referred for and/or received palliative care services. We also conducted a qualitative assessment of barriers and facilitators to referral, focusing on organizational characteristics that might influence palliative care referral practices. METHODS Patients appropriate for palliative care referral were defined as adult (≥18 years) patients with any diagnosis of a poor-prognosis cancer, New York Heart Association class IV congestive heart failure, or oxygen-dependent chronic obstructive pulmonary disease who had inpatient status in 1 of 33 hospitals on May 13, 2014. Qualitative assessment involved interviews of palliative care team members and nonpalliative care frontline providers. RESULTS Nearly 19% of inpatients on the point prevalence day were deemed appropriate for palliative care referral. Of these, approximately 39% received a palliative care referral or services. Delivery of palliative care services to these patients varied widely among participating hospitals, ranging from approximately 12% to more than 90%. Factors influencing differences in referral practices included nonstandardized perceptions of referral criteria and variation in palliative care service structures. CONCLUSION This study provides useful information to guide providers, administrators, researchers, and policy experts in planning for optimal provision of palliative care services to those in need.

Shared Decision Making to Support the Provision of Palliative and End‐of‐Life Care in the Emergency Department: A Consensus Statement and Research Agenda

BACKGROUND Little is known about the optimal use of shared decision making (SDM) to guide palliative and end-of-life decisions in the emergency department (ED). OBJECTIVE The objective was to convene a working group to develop a set of research questions that, when answered, will substantially advance the ability of clinicians to use SDM to guide palliative and end-of-life care decisions in the ED. METHODS Participants were identified based on expertise in emergency, palliative, or geriatrics care; policy or patient-advocacy; and spanned physician, nursing, social work, legal, and patient perspectives. Input from the group was elicited using a time-staggered Delphi process including three teleconferences, an open platform for asynchronous input, and an in-person meeting to obtain a final round of input from all members and to identify and resolve or describe areas of disagreement. CONCLUSION Key research questions identified by the group related to which ED patients are likely to benefit from palliative care (PC), what interventions can most effectively promote PC in the ED, what outcomes are most appropriate to assess the impact of these interventions, what is the potential for initiating advance care planning in the ED to help patients define long-term goals of care, and what policies influence palliative and end-of-life care decision making in the ED. Answers to these questions have the potential to substantially improve the quality of care for ED patients with advanced illness.

Hospital-Level Care at Home for Acutely Ill Adults: a Pilot Randomized Controlled Trial

BackgroundHospitals are standard of care for acute illness, but hospitals can be unsafe, uncomfortable, and expensive. Providing substitutive hospital-level care in a patient’s home potentially reduces cost while maintaining or improving quality, safety, and patient experience, although evidence from randomized controlled trials in the US is lacking.ObjectiveDetermine if home hospital care reduces cost while maintaining quality, safety, and patient experience.DesignRandomized controlled trial.ParticipantsAdults admitted via the emergency department with any infection or exacerbation of heart failure, chronic obstructive pulmonary disease, or asthma.InterventionHome hospital care, including nurse and physician home visits, intravenous medications, continuous monitoring, video communication, and point-of-care testing.Main MeasuresPrimary outcome was direct cost of the acute care episode. Secondary outcomes included utilization, 30-day cost, physical activity, and patient experience.Key ResultsNine patients were randomized to home, 11 to usual care. Median direct cost of the acute care episode for home patients was 52% (IQR, 28%; p = 0.05) lower than for control patients. During the care episode, home patients had fewer laboratory orders (median per admission: 6 vs. 19; p < 0.01) and less often received consultations (0% vs. 27%; p = 0.04). Home patients were more physically active (median minutes, 209 vs. 78; p < 0.01), with a trend toward more sleep. No adverse events occurred in home patients, one occurred in control patients. Median direct cost for the acute care plus 30-day post-discharge period for home patients was 67% (IQR, 77%; p < 0.01) lower, with trends toward less use of home-care services (22% vs. 55%; p = 0.08) and fewer readmissions (11% vs. 36%; p = 0.32). Patient experience was similar in both groups.ConclusionsThe use of substitutive home-hospitalization compared to in-hospital usual care reduced cost and utilization and improved physical activity. No significant differences in quality, safety, and patient experience were noted, with more definitive results awaiting a larger trial.Trial Registration NCT02864420.

Prognosis After Emergency Department Intubation to Inform Shared Decision-Making.

To inform the shared decision‐making process between clinicians and older adults and their surrogates regarding emergency intubation.

Index to Predict In-hospital Mortality in Older Adults after Non-traumatic Emergency Department Intubations

Introduction Our goal was to develop and validate an index to predict in-hospital mortality in older adults after non-traumatic emergency department (ED) intubations. Methods We used Vizient administrative data from hospitalizations of 22,374 adults ≥75 years who underwent non-traumatic ED intubation from 2008–2015 at nearly 300 U.S. hospitals to develop and validate an index to predict in-hospital mortality. We randomly selected one half of participants for the development cohort and one half for the validation cohort. Considering 25 potential predictors, we developed a multivariable logistic regression model using least absolute shrinkage and selection operator method to determine factors associated with in-hospital mortality. We calculated risk scores using points derived from the final model’s beta coefficients. To evaluate calibration and discrimination of the final model, we used Hosmer-Lemeshow chi-square test and receiver-operating characteristic analysis and compared mortality by risk groups in the development and validation cohorts. Results Death during the index hospitalization occurred in 40% of cases. The final model included six variables: history of myocardial infarction, history of cerebrovascular disease, history of metastatic cancer, age, admission diagnosis of sepsis, and admission diagnosis of stroke/ intracranial hemorrhage. Those with low-risk scores (<6) had 31% risk of in-hospital mortality while those with high-risk scores (>10) had 58% risk of in-hospital mortality. The Hosmer-Lemeshow chi-square of the model was 6.47 (p=0.09), and the c-statistic was 0.62 in the validation cohort. Conclusion The model may be useful in identifying older adults at high risk of death after ED intubation.