Lower Humoral and Cellular Immunity Following Asymptomatic SARS-CoV-2 Infection Compared to Symptomatic Infection in Education (The ACE Cohort).

PURPOSE: Asymptomatic SARS-CoV-2 infections were widely reported during the COVID-19 pandemic, acting as a hidden source of infection. Many existing studies investigating asymptomatic immunity failed to recruit true asymptomatic individuals. Thus, we conducted a longitudinal cohort study to evaluate humoral- and cell-mediated responses to infection and vaccination in well-defined asymptomatic young adults (the Asymptomatic COVID-19 in Education [ACE] cohort). METHODS: Asymptomatic testing services located at three UK universities identified asymptomatic young adults who were subsequently recruited with age- and sex-matched symptomatic and uninfected controls. Blood and saliva samples were collected after SARS-CoV-2 Wuhan infection, and again after vaccination. 51 participant's anti-spike antibody titres, neutralizing antibodies, and spike-specific T-cell responses were measured, against both Wuhan and Omicron B.1.1.529.1. RESULTS: Asymptomatic participants exhibited reduced Wuhan-specific neutralization antibodies pre- and post-vaccination, as well as fewer Omicron-specific neutralization antibodies post-vaccination, compared to symptomatic participants. Lower Wuhan and Omicron-specific IgG titres in asymptomatic individuals were also observed pre- and post-vaccination, compared to symptomatic participants. There were no differences in salivary IgA levels. Conventional flow cytometry analysis and multi-dimensional clustering analysis indicated unvaccinated asymptomatic participants had significantly fewer Wuhan-specific IL-2 secreting CD4+ CD45RA+ T cells and activated CD8+ T cells than symptomatic participants, though these differences dissipated after vaccination. CONCLUSIONS: Asymptomatic infection results in decreased antibody and T cell responses to further exposure to SARS-CoV-2 variants, compared to symptomatic infection. Post-vaccination, antibody responses are still inferior, but T cell immunity increases to match symptomatic subjects, emphasising the importance of vaccination to help protect asymptomatic individuals against future variants.

Spontaneous, persistent, T cell-dependent IFN-γ release in patients who progress to Long Covid.

After acute infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a proportion of patients experience persistent symptoms beyond 12 weeks, termed Long Covid. Understanding the mechanisms that cause this debilitating disease and identifying biomarkers for diagnostic, therapeutic, and monitoring purposes are urgently required. We detected persistently high levels of interferon-γ (IFN-γ) from peripheral blood mononuclear cells of patients with Long Covid using highly sensitive FluoroSpot assays. This IFN-γ release was seen in the absence of ex vivo peptide stimulation and remains persistently elevated in patients with Long Covid, unlike the resolution seen in patients recovering from acute SARS-CoV-2 infection. The IFN-γ release was CD8+ T cell-mediated and dependent on antigen presentation by CD14+ cells. Longitudinal follow-up of our study cohort showed that symptom improvement and resolution correlated with a decrease in IFN-γ production to baseline levels. Our study highlights a potential mechanism underlying Long Covid, enabling the search for biomarkers and therapeutics in patients with Long Covid.

Repression of the major immediate early promoter of human cytomegalovirus allows transcription from an alternate promoter.

Following infection, the human cytomegalovirus (HCMV) genome becomes rapidly associated with host histones which can contribute to the regulation of viral gene expression. This can be seen clearly during HCMV latency where silencing of the major immediate early promoter (MIEP), normally responsible for expression of the key lytic proteins IE72 and IE86, is mediated by histone methylation and recruitment of heterochromatin protein 1. Crucially, reversal of these histone modifications coupled with histone acetylation drives viral reactivation which can be blocked with specific histone acetyltransferase inhibitors (HATi). In lytic infection, a role for HATi is less clear despite the well-established enhancement of viral replication observed with histone deacetylase inhibitors. Here we report that a number of different broad-acting HATi have a minor impact on viral infection and replication during lytic infection with the more overt phenotypes observed at lower multiplicities of infection. However, specific analyses of the regulation of major immediate early (MIE) gene expression reveal that the HATi C646, which targets p300/CBP, transiently repressed MIE gene expression via inhibition of the MIEP but by 24 h post-infection MIE gene expression was rescued due to compensatory activation of an alternative IE promoter, ip2. This suggested that silencing of the MIEP promoted alternative ip2 promoter activity in lytic infection and, consistent with this, ip2 transcription is impaired in cells infected with a recombinant HCMV that does not auto-repress the MIEP at late times of infection. Furthermore, inhibition of the histone methyltransferases known to be responsible for auto-repression is similarly inhibitory to ip2 transcription in wild-type infected cells. We also observe that these discrete transcriptional activities of the MIEP and ip2 promoter are also reflected in reactivation; essentially in cells where the MIEP is silenced, ip2 activity is easier to detect at very early times post-reactivation whereas in cells where robust activation of the MIEP is observed ip2 transcription is reduced or delayed. Finally, we observe that inhibition of pathways demonstrated to be important for reactivation of HCMV in dendritic cells, e.g. in response to IL-6, are preferentially important for activation of the MIEP and not the ip2 promoter. Together, these data add to the hypothesis that the existence of multiple promoters within the MIE region of HCMV can drive reactivation in a cell type- and ligand-specific manner and also suggest that inter-dependent regulatory activity between the two promoters exists.

Atypical B cells and impaired SARS-CoV-2 neutralization following heterologous vaccination in the elderly.

Suboptimal responses to a primary vaccination course have been reported in the elderly, but there is little information regarding the impact of age on responses to booster third doses. Here, we show that individuals 70 years or older (median age 73, range 70-75) who received a primary two-dose schedule with AZD1222 and booster third dose with mRNA vaccine achieve significantly lower neutralizing antibody responses against SARS-CoV-2 spike pseudotyped virus compared with those younger than 70 (median age 66, range 54-69) at 1 month post booster. Impaired neutralization potency and breadth post third dose in the elderly is associated with circulating "atypical" spike-specific B cells expressing CD11c and FCRL5. However, when considering individuals who received three doses of mRNA vaccine, we did not observe differences in neutralization or enrichment in atypical B cells. This work highlights the finding that AdV and mRNA COVID-19 vaccine formats differentially instruct the memory B cell response.

Long COVID: what is known and what gaps need to be addressed.

INTRODUCTION: Long COVID is a chronic condition that follows after acute COVID-19 and is characterized by a wide range of persistent, cyclic symptoms. SOURCES OF DATA: PubMed search for publications featuring 'Long COVID' or 'post-acute sequelae of COVID-19'. AREAS OF AGREEMENT: Long COVID occurs frequently post-acute COVID-19, with a majority of people experiencing at least one symptom (such as cough, fatigue, myalgia, anosmia and dyspnoea) 4 weeks after infection. AREAS OF CONTROVERSY: The specific symptoms and the minimum duration of symptoms required to be defined as Long COVID. GROWING POINTS: There is a consistent reduction in Long COVID incidence amongst vaccinated individuals, although the extent of this effect remains unclear. AREAS TIMELY FOR DEVELOPING RESEARCH: There is an urgent need to understand the causes of Long COVID, especially extreme fatigue more than 6 months after infection. We must understand who is at risk and whether reinfections similarly risk Long COVID.

Proteomic analysis of circulating immune cells identifies cellular phenotypes associated with COVID-19 severity.

Certain serum proteins, including C-reactive protein (CRP) and D-dimer, have prognostic value in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nonetheless, these factors are non-specific, providing limited mechanistic insight into the peripheral blood mononuclear cell (PBMC) populations that drive the pathogenesis of severe COVID-19. To identify cellular phenotypes associated with disease, we performed a comprehensive, unbiased analysis of total and plasma-membrane PBMC proteomes from 40 unvaccinated individuals with SARS-CoV-2, spanning the whole disease spectrum. Combined with RNA sequencing (RNA-seq) and flow cytometry from the same donors, we define a comprehensive multi-omic profile for each severity level, revealing that immune-cell dysregulation progresses with increasing disease. The cell-surface proteins CEACAMs1, 6, and 8, CD177, CD63, and CD89 are strongly associated with severe COVID-19, corresponding to the emergence of atypical CD3+CD4+CEACAM1/6/8+CD177+CD63+CD89+ and CD16+CEACAM1/6/8+ mononuclear cells. Utilization of these markers may facilitate real-time patient assessment by flow cytometry and identify immune populations that could be targeted to ameliorate immunopathology.

Cytomegalovirus and Cardiovascular Disease: A Hypothetical Role for Viral G-Protein-Coupled Receptors in Hypertension.

Cytomegalovirus (CMV) is a member of the ß-herpesviruses and is ubiquitous, infecting 50%-99% of the human population depending on ethnic and socioeconomic conditions. CMV establishes lifelong, latent infections in their host. Spontaneous reactivation of CMV is usually asymptomatic, but reactivation events in immunocompromised or immunosuppressed individuals can lead to severe morbidity and mortality. Moreover, herpesvirus infections have been associated with several cardiovascular and post-transplant diseases (stroke, atherosclerosis, post-transplant vasculopathy, and hypertension). Herpesviruses, including CMV, encode viral G-protein-coupled receptors (vGPCRs) that alter the host cell by hijacking signaling pathways that play important roles in the viral life cycle and these cardiovascular diseases. In this brief review, we discuss the pharmacology and signaling properties of these vGPCRs, and their contribution to hypertension. Overall, these vGPCRs can be considered attractive targets moving forward in the development of novel hypertensive therapies.

Human Cytomegalovirus Infection of Epithelial Cells Increases SARS-CoV-2 Superinfection by Upregulating the ACE2 Receptor.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused widespread morbidity and mortality since its onset in late 2019. Here, we demonstrate that prior infection with human cytomegalovirus (HCMV) substantially increases infection with SARS-CoV-2 in vitro. HCMV is a common herpesvirus carried by 40%-100% of the population, which can reactivate in the lung under inflammatory conditions, such as those resulting from SARS-CoV-2 infection. We show in both endothelial and epithelial cell types that HCMV infection upregulates ACE2, the SARS-CoV-2 cell entry receptor. These observations suggest that HCMV reactivation events in the lung of healthy HCMV carriers could exacerbate SARS-CoV-2 infection and subsequent COVID-19 symptoms. This effect could contribute to the disparity of disease severity seen in ethnic minorities and those with lower socioeconomic status, due to their higher CMV seroprevalence. Our results warrant further clinical investigation as to whether HCMV infection influences the pathogenesis of SARS-CoV-2.

Reduced Incidence of Long Coronavirus Disease Referrals to the Cambridge University Teaching Hospital Long Coronavirus Disease Clinic.

Long coronavirus disease (COVID [LC]) constitutes a potential health emergency as millions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections lead to chronic symptoms. We must understand whether vaccines reduce LC because this has major implications for health policy. We report a 79% reduction in LC referrals correlating with vaccination in the United Kingdom.

IL-10-Secreting CD8+ T Cells Specific for Human Cytomegalovirus (HCMV): Generation, Maintenance and Phenotype.

HCMV-specific CD8+ T-cells are potent anti-viral effector cells in HCMV infected individuals, but evidence from other viral infections suggests that CD8+ T-cells can also produce the immunomodulatory cytokine IL-10. In this work we show that there are HCMV-specific IL-10 CD8+ T-cell responses in a cohort of individuals aged 23-76 years of age, predominantly directed against the HCMV proteins known to be expressed during latent infections as well as towards the proteins US3 and pp71. The analysis of HCMV-specific responses established during primary infection has shown that the IL-10 responses to US3 and pp71 HCMV proteins are detectable in the first weeks post infection, but not the responses to latency-associated proteins, and this IL-10 response is produced by both CD8+ and CD4+ T-cells. Phenotyping studies of HCMV-specific IL-10+ CD8+ T-cells show that these are CD45RA+ effector memory cells and co-express CD28 and CD57, however, the expression of the inhibitory receptor PD-1 varied from 90% to 30% between donors. In this study we have described for the first time the HCMV-specific IL-10 CD8+ T-cell responses and have demonstrated their broad specificity and the potential immune modulatory role of the immune response to HCMV latent carriage and periodic reactivation.

Evidence of previous SARS-CoV-2 infection in seronegative patients with long COVID.

BACKGROUND: There is currently no consensus on the diagnosis, definition, symptoms, or duration of COVID-19 illness. The diagnostic complexity of Long COVID is compounded in many patients who were or might have been infected with SARS-CoV-2 but not tested during the acute illness and/or are SARS-CoV-2 antibody negative. METHODS: Given the diagnostic conundrum of Long COVID, we set out to investigate SARS-CoV-2-specific T cell responses in patients with confirmed SARS-CoV-2 infection and/or Long COVID from a cohort of mostly non-hospitalised patients. FINDINGS: We discovered that IL-2 release (but not IFN-γ release) from T cells in response to SARS-CoV-2 peptides is both sensitive (75% +/-13%) and specific (88%+/-7%) for previous SARS-CoV-2 infection >6 months after a positive PCR test. We identified that 42-53% of patients with Long COVID, but without detectable SARS-CoV-2 antibodies, nonetheless have detectable SARS-CoV-2 specific T cell responses. INTERPRETATION: Our study reveals evidence (detectable T cell mediated IL-2 release) of previous SARS-CoV-2 infection in seronegative patients with Long COVID. FUNDING: This work was funded by the Addenbrooke's Charitable Trust (900276 to NS), NIHR award (G112259 to NS) and supported by the NIHR Cambridge Biomedical Research Centre. NJM is supported by the MRC (TSF MR/T032413/1) and NHSBT (WPA15-02). PJL is supported by the Wellcome Trust (PRF 210688/Z/18/Z, 084957/Z/08/Z), a Medical Research Council research grant MR/V011561/1 and the United Kingdom Research and a Innovation COVID Immunology Consortium grant (MR/V028448/1).

B cell receptor repertoire kinetics after SARS-CoV-2 infection and vaccination.

B cells are important in immunity to both severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and vaccination, but B cell receptor (BCR) repertoire development in these contexts has not been compared. We analyze serial samples from 171 SARS-CoV-2-infected individuals and 63 vaccine recipients and find the global BCR repertoire differs between them. Following infection, immunoglobulin (Ig)G1/3 and IgA1 BCRs increase, somatic hypermutation (SHM) decreases, and, in severe disease, IgM and IgA clones are expanded. In contrast, after vaccination, the proportion of IgD/M BCRs increase, SHM is unchanged, and expansion of IgG clones is prominent. VH1-24, which targets the N-terminal domain (NTD) and contributes to neutralization, is expanded post infection except in the most severe disease. Infection generates a broad distribution of SARS-CoV-2-specific clones predicted to target the spike protein, while a more focused response after vaccination mainly targets the spike's receptor-binding domain. Thus, the nature of SARS-CoV-2 exposure differentially affects BCR repertoire development, potentially informing vaccine strategies.

Whole blood-based measurement of SARS-CoV-2-specific T cells reveals asymptomatic infection and vaccine immunogenicity in healthy subjects and patients with solid-organ cancers.

Accurate assessment of SARS-CoV-2 immunity is critical in evaluating vaccine efficacy and devising public health policies. Whilst the exact nature of effective immunity remains incompletely defined, SARS-CoV-2-specific T-cell responses are a critical feature that will likely form a key correlate of protection against COVID-19. Here, we developed and optimized a high-throughput whole blood-based assay to determine the T-cell response associated with prior SARS-CoV-2 infection and/or vaccination amongst 231 healthy donors and 68 cancer patients. Following overnight in vitro stimulation with SARS-CoV-2-specific peptides, blood plasma samples were analysed for TH 1-type cytokines. Highly significant differential IFN-γ+ /IL-2+ SARS-CoV-2-specific T-cell responses were seen amongst previously infected COVID-19-positive healthy donors in comparison with unknown / naïve individuals (p < 0·0001). IFN-γ production was more effective at identifying asymptomatic donors, demonstrating higher sensitivity (96·0% vs. 83·3%) but lower specificity (84·4% vs. 92·5%) than measurement of IL-2. A single COVID-19 vaccine dose induced IFN-γ and/or IL-2 SARS-CoV-2-specific T-cell responses in 116 of 128 (90·6%) healthy donors, reducing significantly to 27 of 56 (48·2%) when measured in cancer patients (p < 0·0001). A second dose was sufficient to boost T-cell responses in the majority (90·6%) of cancer patients, albeit IFN-γ+ responses were still significantly lower overall than those induced in healthy donors (p = 0·034). Three-month post-vaccination T-cell responses also declined at a faster rate in cancer patients. Overall, this cost-effective standardizable test ensures accurate and comparable assessments of SARS-CoV-2-specific T-cell responses amenable to widespread population immunity testing, and identifies individuals at greater need of booster vaccinations.

HCMV carriage in the elderly diminishes anti-viral functionality of the adaptive immune response resulting in virus replication at peripheral sites.

Human cytomegalovirus (HCMV) infection and periodic reactivation is, generally, well controlled by adaptative immune responses in the healthy. In older people, overt HCMV disease is rarely seen despite the association of HCMV with increased risk of mortality; evidence from studies of unwell aged populations suggest that HCMV seropositivity is an important co-morbidity factor. HCMV genomes have been detected in urine from older donors, suggesting that the immune response prevents systemic disease but possibly immunomodulation due to lifelong viral carriage may alter its efficacy at peripheral tissue sites. Previously we have demonstrated that there were no age-related expansions of T cell responses to HCMV or increase in latent viral carriage with age and these T cells produced anti-viral cytokines and viremia was very rarely detected. To investigate the efficacy of anti-HCMV responses with increasing age, we used an in vitro Viral Dissemination Assay (VDA) using autologous dermal fibroblasts to determine the anti-viral effector capacity of total PBMC, as well as important subsets (T cells, NK cells). In parallel we assessed components of the humoral response (antibody neutralization) and combined this with qPCR detection of HCMV in blood, saliva and urine in a cohort of young and old donors. Consistent with previous studies, we again show HCMV specific cIL-10, IFNγ and TNFα T cell responses to peptides did not show an age-related defect. However, assessment of direct anti-viral cellular and antibody-mediated adaptive immune responses using the VDA shows that older donors are significantly less able to control viral dissemination in an in vitro assay compared to young donors. Corroborating this observation, we detected viral genomes in saliva samples only from older donors, these donors had a defect in cellular control of viral spread in our in vitro assay. Phenotyping of fibroblasts used in this study shows expression of a number of checkpoint inhibitor ligands which may contribute to the defects observed. The potential to therapeutically intervene in checkpoint inhibitor pathways to prevent HCMV reactivation in the unwell aged is an exciting avenue to explore.

Targeting the latent human cytomegalovirus reservoir for T-cell-mediated killing with virus-specific nanobodies.

Latent human cytomegalovirus (HCMV) infection is characterized by limited gene expression, making latent HCMV infections refractory to current treatments targeting viral replication. However, reactivation of latent HCMV in immunosuppressed solid organ and stem cell transplant patients often results in morbidity. Here, we report the killing of latently infected cells via a virus-specific nanobody (VUN100bv) that partially inhibits signaling of the viral receptor US28. VUN100bv reactivates immediate early gene expression in latently infected cells without inducing virus production. This allows recognition and killing of latently infected monocytes by autologous cytotoxic T lymphocytes from HCMV-seropositive individuals, which could serve as a therapy to reduce the HCMV latent reservoir of transplant patients.

A BMPR2/YY1 Signaling Axis Is Required for Human Cytomegalovirus Latency in Undifferentiated Myeloid Cells.

Human cytomegalovirus (HCMV) presents a major health burden in the immunocompromised and in stem cell transplant medicine. A lack of understanding about the mechanisms of HCMV latency in undifferentiated CD34+ stem cells, and how latency is broken for the virus to enter the lytic phase of its infective cycle, has hampered the development of essential therapeutics. Using a human induced pluripotent stem cell (iPSC) model of HCMV latency and patient-derived myeloid cell progenitors, we demonstrate that bone morphogenetic protein receptor type 2 (BMPR2) is necessary for HCMV latency. In addition, we define a crucial role for the transcription factor Yin Yang 1 (YY1) in HCMV latency; high levels of YY1 are maintained in latently infected cells as a result of BMPR2 signaling through the SMAD4/SMAD6 axis. Activation of SMAD4/6, through BMPR2, inhibits TGFbeta receptor signaling, which leads to the degradation of YY1 via induction of a cellular microRNA (miRNA), hsa-miR-29a. Pharmacological targeting of BMPR2 in progenitor cells results in the degradation of YY1 and an inability to maintain latency and renders cells susceptible to T cell killing. These data argue that BMPR2 plays a role in HCMV latency and is a new potential therapeutic target for maintaining or disrupting HCMV latency in myeloid progenitors. IMPORTANCE Understanding the mechanisms which regulate HCMV latency could allow therapeutic targeting of the latent virus reservoir from where virus reactivation can cause severe disease. We show that the BMPR2/TGFbeta receptor/YY1 signaling axis is crucial to maintain HCMV latency in undifferentiated cells and that pharmacological reduction of BMPR2 in latently infected cells leads to reactivation of the viral lytic transcription program, which renders the infected cell open to immune detection and clearance in infected individuals. Therefore, this work identifies key host-virus interactions which regulate HCMV latent infection. It also demonstrates a potential new therapeutic approach to reduce HCMV reactivation-mediated disease by the treatment of donor stem cells/organs prior to transplantation, which could have a major impact in the transplant disease setting.

HCMV Antivirals and Strategies to Target the Latent Reservoir.

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus. In healthy people, primary infection is generally asymptomatic, and the virus can go on to establish lifelong latency in cells of the myeloid lineage. However, HCMV often causes severe disease in the immunosuppressed: transplant recipients and people living with AIDS, and also in the immunonaive foetus. At present, there are several antiviral drugs licensed to control HCMV disease. However, these are all faced with problems of poor bioavailability, toxicity and rapidly emerging viral resistance. Furthermore, none of them are capable of fully clearing the virus from the host, as they do not target latent infection. Consequently, reactivation from latency is a significant source of disease, and there remains an unmet need for treatments that also target latent infection. This review briefly summarises the most common HCMV antivirals used in clinic at present and discusses current research into targeting the latent HCMV reservoir.

Age-related immune response heterogeneity to SARS-CoV-2 vaccine BNT162b2.

Although two-dose mRNA vaccination provides excellent protection against SARS-CoV-2, there is little information about vaccine efficacy against variants of concern (VOC) in individuals above eighty years of age1. Here we analysed immune responses following vaccination with the BNT162b2 mRNA vaccine2 in elderly participants and younger healthcare workers. Serum neutralization and levels of binding IgG or IgA after the first vaccine dose were lower in older individuals, with a marked drop in participants over eighty years old. Sera from participants above eighty showed lower neutralization potency against the B.1.1.7 (Alpha), B.1.351 (Beta) and P.1. (Gamma) VOC than against the wild-type virus and were more likely to lack any neutralization against VOC following the first dose. However, following the second dose, neutralization against VOC was detectable regardless of age. The frequency of SARS-CoV-2 spike-specific memory B cells was higher in elderly responders (whose serum showed neutralization activity) than in non-responders after the first dose. Elderly participants showed a clear reduction in somatic hypermutation of class-switched cells. The production of interferon-γ and interleukin-2 by SARS-CoV-2 spike-specific T cells was lower in older participants, and both cytokines were secreted primarily by CD4 T cells. We conclude that the elderly are a high-risk population and that specific measures to boost vaccine responses in this population are warranted, particularly where variants of concern are circulating.

Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease.

The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing, and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications.