Ignite Creation Date:
2024-05-06 @ 4:14 PM
Last Modification Date:
2024-10-26 @ 2:06 PM
Study NCT ID:
NCT04916704
Status:
ACTIVE_NOT_RECRUITING
Last Update Posted:
2024-07-11
First Post:
2021-05-18
Brief Title:
Subclinical Cytomegalovirus Reactivation in Acute ANCA-associated Vasculitis
Sponsor:
University Hospital Birmingham NHS Foundation Trust
Organization:
University Hospital Birmingham NHS Foundation Trust
Study Overview
Official Title:
Subclinical Cytomegalovirus Reactivation in Patients With Newly Diagnosed or Relapsed ANCA-associated Vasculitis and Adverse Clinical Outcomes
Status:
ACTIVE_NOT_RECRUITING
Status Verified Date:
2024-07
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
REACTIVAS
Brief Summary:
This is a prospective observational study to determine the frequency and magnitude of Cytomegalovirus CMV reactivation in patients with anti-neutrophil cytoplasmic antibody ANCA-associated vasculitides AAV in the acute phase of the disease within 12 months of diagnosis or relapse and commencement of induction of remission therapy and its association with clinical outcomes The investigators will also explore whether CMV reactivation causes an increase in CCR2 expressing monocytes and whether these monocytes cause persistent kidney damage in AAV
The investigators hypothesise that reactivation of CMV during the initial 12 months following diagnosis or relapse of AAV occurs frequently but is generally asymptomatic Based on the investigators preliminary data the investigators further hypothesise that subclinical reactivation of CMV during this period will be associated with adverse clinical outcomes including the severity of vasculitis the response to treatment and the damage caused by vasculitis Finally they hypothesise that subclinical CMV reactivation leads to amplification of renal damage in AAV through a monocyte CCR2CCL2 driven pathway
The investigators research has recently shown that asymptomatic reactivation of CMV is a frequent event in AAV patients occurring in roughly 25 of AAV patients in remission However the frequency of asymptomatic reactivation of CMV during the acute phase of the disease is not known The investigators have previously shown that CMV infection and surrogate markers of CMV reactivation in patients with AAV are associated with worse outcomes such as reduced kidney function increased risk of infection and death increased risk of blood clots and increased stiffness of the blood vessels which is a risk factor for heart disease and stroke The investigators also have preliminary findings suggesting that in patients with AAV and CMV reactivation the more CCR2 expressing monocytes in the blood the worse the kidney function If CMV reactivation during the acute phase of the disease is common and linked with worse outcomes this study may then lead on to future research involving treatment to prevent CMV reactivation aiming to improve patient outcomes
The investigators will be looking to recruit patients under the care of the Queen Elizabeth Hospital with newly diagnosed or recently relapsed AAV in the last 2 weeks who are positive for previous CMV infectionThe investigators will follow these patients up with 10 visits over 12 months where possible these will coincide with participants usual vasculitis clinic appointments At each visit the participants will be required to give blood and urine samples and answer questions related to their vasculitis Kidney biopsy tissue taken at diagnosis will be used to assess mechanisms of injury during CMV reactivation
The investigators hypothesise that reactivation of CMV during the initial 12 months following diagnosis or relapse of AAV occurs frequently but is generally asymptomatic Based on the investigators preliminary data the investigators further hypothesise that subclinical reactivation of CMV during this period will be associated with adverse clinical outcomes including the severity of vasculitis the response to treatment and the damage caused by vasculitis Finally they hypothesise that subclinical CMV reactivation leads to amplification of renal damage in AAV through a monocyte CCR2CCL2 driven pathway
The investigators research has recently shown that asymptomatic reactivation of CMV is a frequent event in AAV patients occurring in roughly 25 of AAV patients in remission However the frequency of asymptomatic reactivation of CMV during the acute phase of the disease is not known The investigators have previously shown that CMV infection and surrogate markers of CMV reactivation in patients with AAV are associated with worse outcomes such as reduced kidney function increased risk of infection and death increased risk of blood clots and increased stiffness of the blood vessels which is a risk factor for heart disease and stroke The investigators also have preliminary findings suggesting that in patients with AAV and CMV reactivation the more CCR2 expressing monocytes in the blood the worse the kidney function If CMV reactivation during the acute phase of the disease is common and linked with worse outcomes this study may then lead on to future research involving treatment to prevent CMV reactivation aiming to improve patient outcomes
The investigators will be looking to recruit patients under the care of the Queen Elizabeth Hospital with newly diagnosed or recently relapsed AAV in the last 2 weeks who are positive for previous CMV infectionThe investigators will follow these patients up with 10 visits over 12 months where possible these will coincide with participants usual vasculitis clinic appointments At each visit the participants will be required to give blood and urine samples and answer questions related to their vasculitis Kidney biopsy tissue taken at diagnosis will be used to assess mechanisms of injury during CMV reactivation
Detailed Description:
INTRODUCTIONBACKGROUND
This is an observational study to determine the frequency and magnitude of CMV reactivation in patients with AAV in the acute phase of the disease within 12 months of diagnosis or relapse and commencement of induction of remission therapy
AAVs are systemic autoimmune inflammatory conditions characterized by necrotising inflammation affecting small to medium blood vessels leading to end-organ damage Without treatment AAV is life-threatening treatment involves powerful immunosuppression to induce remission followed by maintenance treatment to prevent disease relapse Treatment induction for both new-onset AAV and major life-threatening relapses is usually in the form of corticosteroids in combination with either cyclophosphamide or rituximab Whilst prognosis has significantly improved with current treatment options for AAV there remains significant morbidity and mortality associated with these conditions especially during the first 12 months following diagnosis and commencement of induction of remission therapy Infection is the leading cause of death within the first 12 months accounting for approximately 50 of mortality as well as considerable morbidity and hospitalisation
Cytomegalovirus CMV is a widely prevalent herpesvirus that is not cleared after primary infection and establishes a state of persistent infection CMV is present in over half the population by middle age and is thought to undergo a state of latency with intermittent periods of viral reactivation This can be a significant clinical problem amongst patients that receive immunosuppression for bone marrow or solid organ transplants Although patients with AAV are heavily immunosuppressed symptomatic CMV disease is uncommon in this patient group 2 However asymptomatic reactivation is not uncommon In a proof-of-concept study the investigators have shown that asymptomatic subclinical reactivation of CMV is a frequent event amongst patients with stable AAV in remission occurring in approximately 1 in 4 CMV seropositive AAV patients in remission over a 12-month period The investigators and others have shown that asymptomatic CMV infection is associated with key adverse clinical outcomes amongst patients with AAV such as reduced kidney function increased risk of infection and mortality increased risk of venous thromboembolism and increased arterial stiffness a marker of cardiovascular mortality
The investigators have previously demonstrated that subclinical reactivation of CMV is associated with the expansion of a cytotoxic T-cell subset known as CD4CD28null T-cells Importantly significant expansion of CD4CD28null T-cells is exclusively seen in CMV seropositive patients The investigators recently demonstrated that CD4CD28null T-cells expansion is linked to a reduced functional capacity of the CD4 compartment and subsequent reduced response to the pneumonia vaccine amongst patients with AAV in remission Importantly the investigators found that participants with evidence of subclinical CMV reactivation during the 6 months preceding the vaccination did not mount a response to the vaccine but treatment with antiviral Valacyclovir suppressed CD4CD28null T-cells expansion and suppression of CD4CD28null T-cells was in itself associated with a better response to pneumococcal vaccination
The investigators anticipate that CMV reactivation will be significantly higher during the acute phase of the disease process both with de-novo disease and major relapses at a time where patients are exposed to intensive immunosuppressive therapy and heightened inflammation However the degree to which subclinical reactivation of CMV occurs during the acute phase of AAV is not currently known
The mechanism of CMV-induced kidney damage is not yet known The investigators have developed preliminary unpublished data that has led them to hypothesise that subclinical CMV reactivation may amplify renal injury in AAV via the expansion of CCR2 expressing pro-inflammatory monocytes These preliminary findings suggest that in these patients the more CCR2 expressing monocytes in the blood the worse the kidney function There is also an increasing amount of evidence now that blocking CCR2 monocytes in mice reduces kidney damage across a wide variety of kidney conditions This suggests that this monocyte-induced kidney damage pathway is not just limited to patients with vasculitis and this research could be relevant to other inflammatory renal conditions
RATIONALE
If this current proposed study confirms that subclinical CMV reactivation occurs frequently during the acute phase of AAV and is linked to worse patient outcomes the investigators would propose that suppression of CMV during the acute phase of the disease would be a potentially valuable therapeutic target to improve patient outcomes and will seek funding for a multi-centre interventional study to test this hypothesis
Another important aim of this study is to investigate the contribution of monocyte induced renal damage in AAV via the CCR2 CCL2 axis and to determine whether repeated rounds of CMV reactivation drive expansions of CCR2 monocytes that amplify renal injury The investigators anticipate that this will be highly relevant to other conditions where there is intra-renal inflammation The findings of this present study may facilitate patient selection for clinical trials to maximise the benefit of CCR2 blockade treatment approaches as well as open new exciting therapeutic opportunities in AAV and other renal conditions including diabetic kidney disease
DESIGN
Up to 50 CMV seropositive and 20 CMV seronegative participants will be recruited within 14 days of disease presentation or major disease relapse and commencement of induction of remission therapy over a period of 18 months For the purposes of this study a major relapse will be defined as a disease relapse that requires re-induction of remission therapy with high dose corticosteroids AND either rituximab or cyclophosphamide
Study participants will be followed up for a total of 12 months during their usual clinical visits at the NIHR Wellcome Trust Clinical Research Facility WTCRF at University Hospitals Birmingham NHS Foundation Trust UHBFT Some study visits may be conducted at a remote offsite location in order to minimize patient hospital visits in light of the current COVID-19 pandemic It is estimated that the study will take 36 months to complete
OBJECTIVES
Aim 1
To determine the frequency and clinical importance of CMV reactivation during the acute phase first 12 months following diagnosis or relapse of AAV and commencement of induction of remission therapy
Blood and urine samples will be drawn at all time points to measure CMV viral DNA copies by quantitative PCR
This will be performed at UHBFT virology laboratory using a validated commercial assay which is routinely utilized for clinical samples As this test does not form part of standard of care for these patients the CMV PCR results will be held back by the Virology Laboratory and released to the research team at the end of the study The investigators will therefore be blind to the frequency of subclinical CMV reactivation over the 12-month study period whilst the clinical outcome data is collected
In cases where there is clinical suspicion of symptomatic CMV reactivation or CMV end-organ disease separate relevant samples will be sent as per clinical need Should symptomatic CMV reactivation or CMV end-organ disease be diagnosed or strongly suspected the management will be as per standard clinical practice
Subclinical asymptomatic CMV reactivation will be defined as a detectable viral titre of 20 copies ml in at least one sample of blood or urine in the absence of symptoms or signs in keeping with CMV disease and in the absence of symptoms or signs of what would be consistent with CMV syndrome CMV syndrome as defined in the transplant population Patients with symptomatic CMV reactivation and or CMV end-organ disease would be excluded from these comparisons However based on clinical experience the investigators anticipate that such symptomatic disease will be very uncommon in this population
Associations between subclinical asymptomatic CMV and the following outcomes will be assessed
Clinical vasculitis outcomes Birmingham Vasculitis Activity score BVAS time to achieve disease remission proteinuria urine albumin creatinine ratio kidney function time to renal recovery C-reactive protein CRP concentration and concentration of urinary markers of inflammation MCP-1 CD163
Disease damage Vasculitis Damage Index VDI at 12 months
Patient well-being Patient-reported outcome using AAV-PRO at 12 months
Frailty measures frailty index Short physical performance battery SPPB and hand grip strength at 12 months
The incidence of non-CMV infections over the 12-month study period
The immune response to clinically indicated vaccinations
The concentration of plasma markers of inflammation endothelial damage and pro-coagulant activity
Disease activity will be monitored using the BVAS at all time points Inflammation will be assessed by CRP determination at all time points as per standard of care Disease burden will be assessed using the VDI score at baseline 6 and 12 months Patient well-being will be assessed as a patient-reported outcome using the AAV-PRO tool at 0 3 and 12 months Clinical assessment and standard of care clinical blood and urine test monitoring will take place at all time points to evaluate disease remission ongoing activity and document renal function renal replacement therapy status and albumin creatinine ratio ACR to assess the level of proteinuria Time to remission will be defined as time to attaining a BVAS score of 0 Time to renal recovery will be defined as time to achieving dialysis independence or time to stabilisation of renal function following initial improvement
The concentration of urinary soluble CD163 and MCP-1 has recently been shown to accurately reflect the presence of subtle active renal vasculitis Urinary CD163 and MCP-1 will be assessed at baseline 3 6 10 and 12 months from cryopreserved urine samples
Frailty will be assessed using the SPPB as a global assessment of function and cognition and hand grip strength The SPPB will be performed at study visits at baseline month 1 and month 12 Hand grip strength will be measured using a hand-held dynamometer at the same points A frailty index will be calculated at the same time points using at least 20 different deficit variables including the SPPB hand grip strength and questions and blood tests taken during each study visit
Participants will be assessed at every study visit as per standard of care and any infection experienced by the participants will be documented along with treatment received Participants will be vaccinated with clinically indicated vaccinations as per standard of care during the follow up period A plasma sample will be drawn immediately prior to vaccination and 2 months after vaccination to measure vaccination specific functional antibodies in order to determine the response to vaccination Samples will be tested at the Clinical Immunology Laboratory University of Birmingham using a validated assay utilized for clinical samples
Plasma and peripheral blood mononuclear cells PBMC will be collected from participants at the specified time points outlined in Table 1 and cryopreserved
Whole blood will be collected in EDTA tubes 4mL and centrifuged to yield plasma Citrated whole blood 35mL will also be collected and centrifuged within 1 hour of collection to yield platelet-free plasma before being frozen at -80 degrees Celsius until further use Whole blood will be collected in lithium heparin tubes 42mL and processed within four hours to separate PBMC Whole blood will also be stained with monoclonal antibodies to enumerate peripheral blood immune subsets
Plasma aliquots will subsequently be thawed and batch assayed to measure markers of endothelial damage and markers of inflammation and microparticle tissue factor activity PBMC will be thawed and immunophenotyped to determine the extended immunophenotype and activation status of CD4CD28null T-cells
CMV seronegative patients will be used as controls for comparison of these outcome measures at 3 monthly intervals baseline months 3 6 10 12 The same procedures will be undertaken for CMV seronegative patients apart from assessment of CMV PCR
Aim 2
Characterisation of the local and systemic monocytic repertoire in relation to CMV reactivation
Flow cytometry will be used to determine the extended phenotypic profile of monocytes both in relation to CMV serostatus and viral reactivation Furthermore discrete monocytic subsets initially focussing on CCR2 and CCR2- will be FACS-sorted and CMV viral load quantified through use of droplet digital PCR Single cell RNA-Seq will be applied to appropriate subpopulations identified by CMV carriage The profile and number of virally infected and CCR2 monocytes will then be compared to episodes of CMV reactivation to determine whether repeated rounds of CMV reactivation drives expansion of this cell subset
Aim 3
Assessment of potential mechanisms of renal damage induced by CCR2 monocytes
Kidney tissue from our recruited patients obtained at the time of diagnosis of new or relapsing disease with renal involvement from a standard of care renal biopsy will be used to explore the relationship between CMV reactivation monocytes and kidney damage We anticipate the vast majority of patients with renal involvement will have a renal biopsy 50-70 of total recruited patients Additional biopsy tissue will not be required the cores obtained as per standard of care will be sufficient for research purposes
Renal tissue will be analysed to determine expression of markers of interest to explore the mechanisms underlying tissue damage in renal biopsies using scanned biopsy image software The focus will be on i potential anatomical sites of viral replication ii phenotype and infiltration of CD4CD28- T cells and iii detailed transcriptional analysis of CCR2 monocytes These observations will also be correlated with matched urinary MCP-1 and CSF2 concentration to determine the potential value of these as markers of intrarenal monocyte infiltration
To further interrogate the potential mechanisms of monocyte-induced glomerular injury in-vitro add-back experiments will determine whether CCR2 monocytes are capable of damaging glomerular endothelium
SCHEDULE OF ASSESSMENTS
All study participants will have a maximum of 11 interactions with the research team The timing of the study visits has been selected to coincide with clinical visits that patients would be expected to attend as part of their ongoing clinical management
Participants will either be recruited as in-patients or out-patients For in-patients the first contact will be face-to-face during their inpatient stay to provide verbal and written information including a Patient Information Sheet PIS The baseline visit will then occur at least 24 hours later at which the patient will be given the option to be recruited to the study
For out-patients the first contact will be written information in the post PIS and contact details for the research team will be provided This will be followed by a telephone consultation to answer any questions and establish interest For those patients who express interest the third point of contact baseline visit will be face-to-face with the research team at their routine AAV clinic appointment or treatment schedule this will not be an additional hospital visit
A total of 10 study clinic appointments will be held at UHBFT for CMV seropositive and 5 for CMV seronegative subjects During the baseline visit participants will be recruited to the study either during their hospital admission or during a scheduled hospital visit either to the clinic or for treatment After inclusionexclusion criteria are met written informed consent will be obtained for all patients prior to enrolment A clinical assessment with be made BVAS and VDI calculated AAV-PRO completed SPPB and hand grip strength carried out and blood and urine samples collected
The remaining follow-up visits will be scheduled at intervals over the next 12 months In light of the current COVID-19 pandemic patients are only attending face-to-face hospital appointments when necessary with telephone consultations being conducted when appropriate decisions on who is allocated to which appointment type are being made on an individual patient basis The investigators will not bring participants to the hospital for additional study visits where they cannot come through a clean pathway in these circumstances with the agreement of the subject the investigators will arrange for home study visits
At each follow-up visit the research team will complete a clinical assessment calculate BVAS and VDI where applicable conduct SPPB and measure hand grip strength where applicable and further blood and urine samples will be taken The AAV-PRO may be sent out to patients a few days prior to their study visit via the post or email at their preference and patients will be asked to return completed forms at their visit The investigators will aim to give participants clinically indicated vaccinations during follow up as per standard of care at month 10 the investigators will contact the patients GP to clarify previous vaccine history prior to administration Participants will have completed the study when the 12-month follow-up visit has been completed
Renal biopsy tissue obtained at the time of diagnosis of new or relapsing disease with renal involvement from a standard of care renal biopsy will be used to explore the relationship between CMV reactivation monocytes and kidney damage We anticipate the vast majority of patients with renal involvement will have a renal biopsy 50-70 of total recruited patients Additional biopsy tissue will not be required the cores obtained as per standard of care will be sufficient for research purposes
SAMPLE SIZE
This is an observational study with the primary outcome to estimate the rate of subclinical CMV reactivation so there is no formal power calculation There is also a limit to the number of patients that will meet the inclusion criteria during the study timeframe As such no formal power calculation was performed with the aim instead being to recruit as many patients as is feasible
The investigators estimate that they will recruit up to 50 CMV seropositive and 20 CMV seronegative patients over 18 months as they expect this number will be the maximum feasible within a single centre with a population of 250 patients around 40 new diagnoses AAVyear The investigators expect around 70 of this cohort will be CMV seropositive Patients recruited following a major relapse of AAV will be limited to 30 of the total study population Historically the AAV cohort of patients is a very well-motivated group of patients with very high participation in research studies and usually excellent clinic attendance As such the investigators would expect the vast majority of eligible patients to be willing to join the study and for negligible loss to follow up or study withdrawal
STATISTICAL ANALYSIS
Analysis of study data will be conducted by the research team with the assistance of a statistical advisor The main analysis will be conducted on the entire study cohort although planned sub-group analysis will also evaluate potential differences between patients with newly diagnosed AAV and those with a major relapse of previously diagnosed disease
Analysis of the primary outcome of CMV reactivation will be performed using a time-to-event approach in order to account for the time that this was identified and the potential for varying follow up times across the cohort in patients that do not complete the study As such patients will be censored where they are either lost to follow up withdraw from the study or die during the study period In addition to performing this analysis for the cohort as a whole a subgroup analysis will also be performed after excluding those with CMV reactivation at the baseline visit in order to estimate the rate of new-onset CMV reactivation after the commencement of treatment for AAV
The remaining clinical outcomes are based on comparisons between patients with asymptomatic CMV reactivation and those without CMV reactivation as well as between those who are CMV seropositive and those who are seronegative as a control group Analyses will be performed which consider the actual viral titre in those where it is detectable to assess whether there is a dose-response relationship between viral load and patient outcomes
Analyses of the secondary and tertiary outcomes will utilise differing methodologies depending on how the outcomes of interest are quantified For outcomes measured at a single time point eg VDI at 12 months comparisons by CMV reactivation status will be performed using independent group comparisons such as independent samples t-tests Outcomes measured at multiple time points eg clinical bloods recorded at every visit will be assessed using repeated-measures analyses in order to account for the correlations between multiple samples contributed by the same patient For time-to-event outcomes eg time to achieve disease remission analyses will be based on Kaplan-Meier curves and Cox regression models Finally count-based outcomes eg development of non-CMV infections will be analysed as an incidence per patient-year in order to account for the follow up time contributed by each patient
In all analyses the asymptomatic CMV reactivation status will initially be considered as a fixed-effects variable such that patients will be assigned to a CMV reactivation group regardless of when this was first diagnosed However analyses will also be performed where this factor is treated as a time-dependent covariate such that all patients are assigned to the negative group at the commencement of follow up before transitioning to the CMV reactivation group at the time that this was diagnosed
Laboratory-based outcomes focused around phenotyping and enumerating monocytes urinary markers of interest and targets within renal tissue will involve comparisons between CMV seropositive and seronegative groups along with CMV reactivation status Comparisons between two groups will be done by unpaired t-tests
QUALITY ASSURANCEMONITORING
The study may be subject to inspection and audit by UHBFT under their remit as sponsor and other regulatory bodies to ensure adherence to GCP and regulations The named investigators will allow study-related monitoring audits Research Ethics Committee review and regulatory inspectors where appropriate by providing direct access to study source data and associated documents eg consent forms UHBFT laboratory results etc
ADVERSE EVENTS
A SAE form should be completed and submitted to the Principal Investigator for all SAEs within 24 hours The Principal Investigators assessment of causality of SAEs ie their relationship to trial treatment will be reported on the Serious Adverse Event form the investigators do not anticipate any related adverse events associated with this observational study Related andor unexpected events will be reported to the main Research Ethics Committee by the Chief Investigator within 15 days of them becoming aware of the event in line with HRA recommendations
This is an observational study to determine the frequency and magnitude of CMV reactivation in patients with AAV in the acute phase of the disease within 12 months of diagnosis or relapse and commencement of induction of remission therapy
AAVs are systemic autoimmune inflammatory conditions characterized by necrotising inflammation affecting small to medium blood vessels leading to end-organ damage Without treatment AAV is life-threatening treatment involves powerful immunosuppression to induce remission followed by maintenance treatment to prevent disease relapse Treatment induction for both new-onset AAV and major life-threatening relapses is usually in the form of corticosteroids in combination with either cyclophosphamide or rituximab Whilst prognosis has significantly improved with current treatment options for AAV there remains significant morbidity and mortality associated with these conditions especially during the first 12 months following diagnosis and commencement of induction of remission therapy Infection is the leading cause of death within the first 12 months accounting for approximately 50 of mortality as well as considerable morbidity and hospitalisation
Cytomegalovirus CMV is a widely prevalent herpesvirus that is not cleared after primary infection and establishes a state of persistent infection CMV is present in over half the population by middle age and is thought to undergo a state of latency with intermittent periods of viral reactivation This can be a significant clinical problem amongst patients that receive immunosuppression for bone marrow or solid organ transplants Although patients with AAV are heavily immunosuppressed symptomatic CMV disease is uncommon in this patient group 2 However asymptomatic reactivation is not uncommon In a proof-of-concept study the investigators have shown that asymptomatic subclinical reactivation of CMV is a frequent event amongst patients with stable AAV in remission occurring in approximately 1 in 4 CMV seropositive AAV patients in remission over a 12-month period The investigators and others have shown that asymptomatic CMV infection is associated with key adverse clinical outcomes amongst patients with AAV such as reduced kidney function increased risk of infection and mortality increased risk of venous thromboembolism and increased arterial stiffness a marker of cardiovascular mortality
The investigators have previously demonstrated that subclinical reactivation of CMV is associated with the expansion of a cytotoxic T-cell subset known as CD4CD28null T-cells Importantly significant expansion of CD4CD28null T-cells is exclusively seen in CMV seropositive patients The investigators recently demonstrated that CD4CD28null T-cells expansion is linked to a reduced functional capacity of the CD4 compartment and subsequent reduced response to the pneumonia vaccine amongst patients with AAV in remission Importantly the investigators found that participants with evidence of subclinical CMV reactivation during the 6 months preceding the vaccination did not mount a response to the vaccine but treatment with antiviral Valacyclovir suppressed CD4CD28null T-cells expansion and suppression of CD4CD28null T-cells was in itself associated with a better response to pneumococcal vaccination
The investigators anticipate that CMV reactivation will be significantly higher during the acute phase of the disease process both with de-novo disease and major relapses at a time where patients are exposed to intensive immunosuppressive therapy and heightened inflammation However the degree to which subclinical reactivation of CMV occurs during the acute phase of AAV is not currently known
The mechanism of CMV-induced kidney damage is not yet known The investigators have developed preliminary unpublished data that has led them to hypothesise that subclinical CMV reactivation may amplify renal injury in AAV via the expansion of CCR2 expressing pro-inflammatory monocytes These preliminary findings suggest that in these patients the more CCR2 expressing monocytes in the blood the worse the kidney function There is also an increasing amount of evidence now that blocking CCR2 monocytes in mice reduces kidney damage across a wide variety of kidney conditions This suggests that this monocyte-induced kidney damage pathway is not just limited to patients with vasculitis and this research could be relevant to other inflammatory renal conditions
RATIONALE
If this current proposed study confirms that subclinical CMV reactivation occurs frequently during the acute phase of AAV and is linked to worse patient outcomes the investigators would propose that suppression of CMV during the acute phase of the disease would be a potentially valuable therapeutic target to improve patient outcomes and will seek funding for a multi-centre interventional study to test this hypothesis
Another important aim of this study is to investigate the contribution of monocyte induced renal damage in AAV via the CCR2 CCL2 axis and to determine whether repeated rounds of CMV reactivation drive expansions of CCR2 monocytes that amplify renal injury The investigators anticipate that this will be highly relevant to other conditions where there is intra-renal inflammation The findings of this present study may facilitate patient selection for clinical trials to maximise the benefit of CCR2 blockade treatment approaches as well as open new exciting therapeutic opportunities in AAV and other renal conditions including diabetic kidney disease
DESIGN
Up to 50 CMV seropositive and 20 CMV seronegative participants will be recruited within 14 days of disease presentation or major disease relapse and commencement of induction of remission therapy over a period of 18 months For the purposes of this study a major relapse will be defined as a disease relapse that requires re-induction of remission therapy with high dose corticosteroids AND either rituximab or cyclophosphamide
Study participants will be followed up for a total of 12 months during their usual clinical visits at the NIHR Wellcome Trust Clinical Research Facility WTCRF at University Hospitals Birmingham NHS Foundation Trust UHBFT Some study visits may be conducted at a remote offsite location in order to minimize patient hospital visits in light of the current COVID-19 pandemic It is estimated that the study will take 36 months to complete
OBJECTIVES
Aim 1
To determine the frequency and clinical importance of CMV reactivation during the acute phase first 12 months following diagnosis or relapse of AAV and commencement of induction of remission therapy
Blood and urine samples will be drawn at all time points to measure CMV viral DNA copies by quantitative PCR
This will be performed at UHBFT virology laboratory using a validated commercial assay which is routinely utilized for clinical samples As this test does not form part of standard of care for these patients the CMV PCR results will be held back by the Virology Laboratory and released to the research team at the end of the study The investigators will therefore be blind to the frequency of subclinical CMV reactivation over the 12-month study period whilst the clinical outcome data is collected
In cases where there is clinical suspicion of symptomatic CMV reactivation or CMV end-organ disease separate relevant samples will be sent as per clinical need Should symptomatic CMV reactivation or CMV end-organ disease be diagnosed or strongly suspected the management will be as per standard clinical practice
Subclinical asymptomatic CMV reactivation will be defined as a detectable viral titre of 20 copies ml in at least one sample of blood or urine in the absence of symptoms or signs in keeping with CMV disease and in the absence of symptoms or signs of what would be consistent with CMV syndrome CMV syndrome as defined in the transplant population Patients with symptomatic CMV reactivation and or CMV end-organ disease would be excluded from these comparisons However based on clinical experience the investigators anticipate that such symptomatic disease will be very uncommon in this population
Associations between subclinical asymptomatic CMV and the following outcomes will be assessed
Clinical vasculitis outcomes Birmingham Vasculitis Activity score BVAS time to achieve disease remission proteinuria urine albumin creatinine ratio kidney function time to renal recovery C-reactive protein CRP concentration and concentration of urinary markers of inflammation MCP-1 CD163
Disease damage Vasculitis Damage Index VDI at 12 months
Patient well-being Patient-reported outcome using AAV-PRO at 12 months
Frailty measures frailty index Short physical performance battery SPPB and hand grip strength at 12 months
The incidence of non-CMV infections over the 12-month study period
The immune response to clinically indicated vaccinations
The concentration of plasma markers of inflammation endothelial damage and pro-coagulant activity
Disease activity will be monitored using the BVAS at all time points Inflammation will be assessed by CRP determination at all time points as per standard of care Disease burden will be assessed using the VDI score at baseline 6 and 12 months Patient well-being will be assessed as a patient-reported outcome using the AAV-PRO tool at 0 3 and 12 months Clinical assessment and standard of care clinical blood and urine test monitoring will take place at all time points to evaluate disease remission ongoing activity and document renal function renal replacement therapy status and albumin creatinine ratio ACR to assess the level of proteinuria Time to remission will be defined as time to attaining a BVAS score of 0 Time to renal recovery will be defined as time to achieving dialysis independence or time to stabilisation of renal function following initial improvement
The concentration of urinary soluble CD163 and MCP-1 has recently been shown to accurately reflect the presence of subtle active renal vasculitis Urinary CD163 and MCP-1 will be assessed at baseline 3 6 10 and 12 months from cryopreserved urine samples
Frailty will be assessed using the SPPB as a global assessment of function and cognition and hand grip strength The SPPB will be performed at study visits at baseline month 1 and month 12 Hand grip strength will be measured using a hand-held dynamometer at the same points A frailty index will be calculated at the same time points using at least 20 different deficit variables including the SPPB hand grip strength and questions and blood tests taken during each study visit
Participants will be assessed at every study visit as per standard of care and any infection experienced by the participants will be documented along with treatment received Participants will be vaccinated with clinically indicated vaccinations as per standard of care during the follow up period A plasma sample will be drawn immediately prior to vaccination and 2 months after vaccination to measure vaccination specific functional antibodies in order to determine the response to vaccination Samples will be tested at the Clinical Immunology Laboratory University of Birmingham using a validated assay utilized for clinical samples
Plasma and peripheral blood mononuclear cells PBMC will be collected from participants at the specified time points outlined in Table 1 and cryopreserved
Whole blood will be collected in EDTA tubes 4mL and centrifuged to yield plasma Citrated whole blood 35mL will also be collected and centrifuged within 1 hour of collection to yield platelet-free plasma before being frozen at -80 degrees Celsius until further use Whole blood will be collected in lithium heparin tubes 42mL and processed within four hours to separate PBMC Whole blood will also be stained with monoclonal antibodies to enumerate peripheral blood immune subsets
Plasma aliquots will subsequently be thawed and batch assayed to measure markers of endothelial damage and markers of inflammation and microparticle tissue factor activity PBMC will be thawed and immunophenotyped to determine the extended immunophenotype and activation status of CD4CD28null T-cells
CMV seronegative patients will be used as controls for comparison of these outcome measures at 3 monthly intervals baseline months 3 6 10 12 The same procedures will be undertaken for CMV seronegative patients apart from assessment of CMV PCR
Aim 2
Characterisation of the local and systemic monocytic repertoire in relation to CMV reactivation
Flow cytometry will be used to determine the extended phenotypic profile of monocytes both in relation to CMV serostatus and viral reactivation Furthermore discrete monocytic subsets initially focussing on CCR2 and CCR2- will be FACS-sorted and CMV viral load quantified through use of droplet digital PCR Single cell RNA-Seq will be applied to appropriate subpopulations identified by CMV carriage The profile and number of virally infected and CCR2 monocytes will then be compared to episodes of CMV reactivation to determine whether repeated rounds of CMV reactivation drives expansion of this cell subset
Aim 3
Assessment of potential mechanisms of renal damage induced by CCR2 monocytes
Kidney tissue from our recruited patients obtained at the time of diagnosis of new or relapsing disease with renal involvement from a standard of care renal biopsy will be used to explore the relationship between CMV reactivation monocytes and kidney damage We anticipate the vast majority of patients with renal involvement will have a renal biopsy 50-70 of total recruited patients Additional biopsy tissue will not be required the cores obtained as per standard of care will be sufficient for research purposes
Renal tissue will be analysed to determine expression of markers of interest to explore the mechanisms underlying tissue damage in renal biopsies using scanned biopsy image software The focus will be on i potential anatomical sites of viral replication ii phenotype and infiltration of CD4CD28- T cells and iii detailed transcriptional analysis of CCR2 monocytes These observations will also be correlated with matched urinary MCP-1 and CSF2 concentration to determine the potential value of these as markers of intrarenal monocyte infiltration
To further interrogate the potential mechanisms of monocyte-induced glomerular injury in-vitro add-back experiments will determine whether CCR2 monocytes are capable of damaging glomerular endothelium
SCHEDULE OF ASSESSMENTS
All study participants will have a maximum of 11 interactions with the research team The timing of the study visits has been selected to coincide with clinical visits that patients would be expected to attend as part of their ongoing clinical management
Participants will either be recruited as in-patients or out-patients For in-patients the first contact will be face-to-face during their inpatient stay to provide verbal and written information including a Patient Information Sheet PIS The baseline visit will then occur at least 24 hours later at which the patient will be given the option to be recruited to the study
For out-patients the first contact will be written information in the post PIS and contact details for the research team will be provided This will be followed by a telephone consultation to answer any questions and establish interest For those patients who express interest the third point of contact baseline visit will be face-to-face with the research team at their routine AAV clinic appointment or treatment schedule this will not be an additional hospital visit
A total of 10 study clinic appointments will be held at UHBFT for CMV seropositive and 5 for CMV seronegative subjects During the baseline visit participants will be recruited to the study either during their hospital admission or during a scheduled hospital visit either to the clinic or for treatment After inclusionexclusion criteria are met written informed consent will be obtained for all patients prior to enrolment A clinical assessment with be made BVAS and VDI calculated AAV-PRO completed SPPB and hand grip strength carried out and blood and urine samples collected
The remaining follow-up visits will be scheduled at intervals over the next 12 months In light of the current COVID-19 pandemic patients are only attending face-to-face hospital appointments when necessary with telephone consultations being conducted when appropriate decisions on who is allocated to which appointment type are being made on an individual patient basis The investigators will not bring participants to the hospital for additional study visits where they cannot come through a clean pathway in these circumstances with the agreement of the subject the investigators will arrange for home study visits
At each follow-up visit the research team will complete a clinical assessment calculate BVAS and VDI where applicable conduct SPPB and measure hand grip strength where applicable and further blood and urine samples will be taken The AAV-PRO may be sent out to patients a few days prior to their study visit via the post or email at their preference and patients will be asked to return completed forms at their visit The investigators will aim to give participants clinically indicated vaccinations during follow up as per standard of care at month 10 the investigators will contact the patients GP to clarify previous vaccine history prior to administration Participants will have completed the study when the 12-month follow-up visit has been completed
Renal biopsy tissue obtained at the time of diagnosis of new or relapsing disease with renal involvement from a standard of care renal biopsy will be used to explore the relationship between CMV reactivation monocytes and kidney damage We anticipate the vast majority of patients with renal involvement will have a renal biopsy 50-70 of total recruited patients Additional biopsy tissue will not be required the cores obtained as per standard of care will be sufficient for research purposes
SAMPLE SIZE
This is an observational study with the primary outcome to estimate the rate of subclinical CMV reactivation so there is no formal power calculation There is also a limit to the number of patients that will meet the inclusion criteria during the study timeframe As such no formal power calculation was performed with the aim instead being to recruit as many patients as is feasible
The investigators estimate that they will recruit up to 50 CMV seropositive and 20 CMV seronegative patients over 18 months as they expect this number will be the maximum feasible within a single centre with a population of 250 patients around 40 new diagnoses AAVyear The investigators expect around 70 of this cohort will be CMV seropositive Patients recruited following a major relapse of AAV will be limited to 30 of the total study population Historically the AAV cohort of patients is a very well-motivated group of patients with very high participation in research studies and usually excellent clinic attendance As such the investigators would expect the vast majority of eligible patients to be willing to join the study and for negligible loss to follow up or study withdrawal
STATISTICAL ANALYSIS
Analysis of study data will be conducted by the research team with the assistance of a statistical advisor The main analysis will be conducted on the entire study cohort although planned sub-group analysis will also evaluate potential differences between patients with newly diagnosed AAV and those with a major relapse of previously diagnosed disease
Analysis of the primary outcome of CMV reactivation will be performed using a time-to-event approach in order to account for the time that this was identified and the potential for varying follow up times across the cohort in patients that do not complete the study As such patients will be censored where they are either lost to follow up withdraw from the study or die during the study period In addition to performing this analysis for the cohort as a whole a subgroup analysis will also be performed after excluding those with CMV reactivation at the baseline visit in order to estimate the rate of new-onset CMV reactivation after the commencement of treatment for AAV
The remaining clinical outcomes are based on comparisons between patients with asymptomatic CMV reactivation and those without CMV reactivation as well as between those who are CMV seropositive and those who are seronegative as a control group Analyses will be performed which consider the actual viral titre in those where it is detectable to assess whether there is a dose-response relationship between viral load and patient outcomes
Analyses of the secondary and tertiary outcomes will utilise differing methodologies depending on how the outcomes of interest are quantified For outcomes measured at a single time point eg VDI at 12 months comparisons by CMV reactivation status will be performed using independent group comparisons such as independent samples t-tests Outcomes measured at multiple time points eg clinical bloods recorded at every visit will be assessed using repeated-measures analyses in order to account for the correlations between multiple samples contributed by the same patient For time-to-event outcomes eg time to achieve disease remission analyses will be based on Kaplan-Meier curves and Cox regression models Finally count-based outcomes eg development of non-CMV infections will be analysed as an incidence per patient-year in order to account for the follow up time contributed by each patient
In all analyses the asymptomatic CMV reactivation status will initially be considered as a fixed-effects variable such that patients will be assigned to a CMV reactivation group regardless of when this was first diagnosed However analyses will also be performed where this factor is treated as a time-dependent covariate such that all patients are assigned to the negative group at the commencement of follow up before transitioning to the CMV reactivation group at the time that this was diagnosed
Laboratory-based outcomes focused around phenotyping and enumerating monocytes urinary markers of interest and targets within renal tissue will involve comparisons between CMV seropositive and seronegative groups along with CMV reactivation status Comparisons between two groups will be done by unpaired t-tests
QUALITY ASSURANCEMONITORING
The study may be subject to inspection and audit by UHBFT under their remit as sponsor and other regulatory bodies to ensure adherence to GCP and regulations The named investigators will allow study-related monitoring audits Research Ethics Committee review and regulatory inspectors where appropriate by providing direct access to study source data and associated documents eg consent forms UHBFT laboratory results etc
ADVERSE EVENTS
A SAE form should be completed and submitted to the Principal Investigator for all SAEs within 24 hours The Principal Investigators assessment of causality of SAEs ie their relationship to trial treatment will be reported on the Serious Adverse Event form the investigators do not anticipate any related adverse events associated with this observational study Related andor unexpected events will be reported to the main Research Ethics Committee by the Chief Investigator within 15 days of them becoming aware of the event in line with HRA recommendations
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None