用于移植患者的巨细胞病毒疫苗(原版) 20110412

Articles 1256 www.thelancet.com Vol 377 April 9, 2011 Lancet 2011; 377: 1256–63 See Comment page 1216 Centre for Virology (Prof P D Griffi ths MD, A Stanton RN, E McCarrell MSc, M Osman PhD, Prof V C Emery PhD, R S B Milne PhD, C E Atkinson MSc, E Rothwell RGN, N Old BA, T Haque FRCPath, S Atabani MRCPath, S Luck MBChB, S Prideaux BSc), Centre for Nephrology (M Harber FRCP, A Davenport FRCP, G Jones MRCP, D C Wheeler FRCP, P Sweny FRCP, R Kinyanjui RN), Sheila Sherlock Liver Centre and University Department of Surgery, Royal Free NHS Trust (J O’Beirne MRCP, D Thorburn FRCP, D Patch FRCP, Prof A K Burroughs FRCP), Department of Infection & Population Health, UCL Medical School, London (C Smith PhD), Pharmacy Department, Royal Free NHS Trust, London (M Lanzman MRPharmS, E Woodford); and Clinical Development, Sanofi Pasteur, Marcy l’Etoile, France (S Pichon MSc) Correspondence to: Prof Paul D Griffi ths, Centre for Virology, UCL Medical School, Rowland Hill Street, London NW3 2PF, UK p.griffi ths@medsch.ucl.ac.uk Cytomegalovirus glycoprotein-B vaccine with MF59 adjuvant in transplant recipients: a phase 2 randomised placebo-controlled trial Paul D Griffi ths, Anna Stanton, Erin McCarrell, Colette Smith, Mohamed Osman, Mark Harber, Andrew Davenport, Gareth Jones, David C Wheeler, James O’Beirne, Douglas Thorburn, David Patch, Claire E Atkinson, Sylvie Pichon, Paul Sweny, Marisa Lanzman, Elizabeth Woodford, Emily Rothwell, Natasha Old, Ruth Kinyanjui, Tanzina Haque, Sowsan Atabani, Suzanne Luck, Steven Prideaux, Richard S B Milne, Vincent C Emery, Andrew K Burroughs Summary Background Cytomegalovirus end-organ disease can be prevented by giving ganciclovir when viraemia is detected in allograft recipients. Values of viral load correlate with development of end-organ disease and are moderated by pre- existing natural immunity. Our aim was to determine whether vaccine-induced immunity could do likewise. Methods We undertook a phase-2 randomised placebo controlled trial in adults awaiting kidney or liver transplantation at the Royal Free Hospital, London, UK. Exclusion criteria were pregnancy, receipt of blood products (except albumin) in the previous 3 months, and simultaneous multiorgan transplantation. 70 patients seronegative and 70 seropositive for cytomegalovirus were randomly assigned from a scratch-off randomisation code in a 1:1 ratio to receive either cytomegalovirus glycoprotein-B vaccine with MF59 adjuvant or placebo, each given at baseline, 1 month and 6 months later. If a patient was transplanted, no further vaccinations were given and serial blood samples were tested for cytomegalovirus DNA by real-time quantitative PCR (rtqPCR). Any patient with one blood sample containing more than 3000 cytomegalovirus genomes per mL received ganciclovir until two consecutive undetectable cytomegalovirus DNA measurements. Safety and immunogenicity were coprimary endpoints and were assessed by intention to treat in patients who received at least one dose of vaccine or placebo. This trial is registered with ClinicalTrials.gov, NCT00299260. Findings 67 patients received vaccine and 73 placebo, all of whom were evaluable. Glycoprotein-B antibody titres were signifi cantly increased in both seronegative (geometric mean titre 12 537 (95% CI 6593–23 840) versus 86 (63–118) in recipients of placebo recipients; p<0·0001) and seropositive (118 395; 64 503–217 272) versus 24 682 (17 909–34 017); p<0·0001) recipients of vaccine. In those who developed viraemia after transplantation, glycoprotein-B antibody titres correlated inversely with duration of viraemia (p=0·0022). In the seronegative patients with seropositive donors, the duration of viraemia (p=0·0480) and number of days of ganciclovir treatment (p=0·0287) were reduced in vaccine recipients. Interpretation Although cytomegalovirus disease occurs in the context of suppressed cell-mediated immunity post-transplantation, humoral immunity has a role in reduction of cytomegalovirus viraemia. Vaccines containing cytomegalovirus glycoprotein B merit further assessment in transplant recipients. Funding National Institute of Allergy and Infectious Diseases, Grant R01AI051355 and Wellcome Trust, Grant 078332. Sponsor: University College London (UCL). Introduction Cytomegalovirus is an important pathogen for women of childbearing age and for allograft recipients, two populations in whom development of a vaccine has been rated as high priority.1–3 The life-long latency and ability to reinfect despite pre-existing natural immunity make the production of a vaccine against cytomegalovirus challeng ing.4,5 In the allograft recipient, viraemic dissemin ation can cause end-organ disease, such as hepatitis, pneumonitis, gastroenteritis, and retinitis6,7 and can predispose to transplant rejection. The antiviral drug ganciclovir and its prodrug valganciclovir potently inhibit cytomegalovirus replication. Two strategies can be deployed to control end-organ disease related to the virus: antiviral prophylaxis, in which the drug is given routinely from the time of transplantation; or pre-emptive treatment, in which patients are monitored to detect the virus in blood and treatment is begun once a defi ned quantity of viral load is detected. Both strategies are eff ective in control of such disease.8–13 Cytomegalovirus infection after transplantation might originate from the donor or from reactivation in the recipient. Infection might cause either primary infection in recipients who are initially seronegative for the virus or reinfection with a new strain in seropositive recipients.4 The most serious clinical eff ects result from primary infection, followed by reinfection, with reactivation being the least likely to cause end-organ disease.4 Thus, most end-organ disease arises from donor-derived virus. This hierarchy of risk occurs because natural immunity before Articles www.thelancet.com Vol 377 April 9, 2011 1257 trans plantation provides substantial protection against virus replication after transplantation14–16 and a high viral load is needed to cause end-organ disease.17–19 Given that natural immunity before transplantation can modulate the pathogenicity of cytomegalovirus after transplantation,16 we tested whether vaccine-induced immunity could do likewise. No correlates of protective immunity that defi ne whether a given vaccine is suffi ciently immunogenic exist to justify a phase-3 clinical trial of effi cacy. We therefore designed a phase-2 proof-of-concept study, selecting a group of patients given pre-emptive treatment as standard of care, so that no patient received antiviral prophylaxis. This study focused on pharmacodynamics rather than pharma cokinetics. Methods Patients studied In this phase-2 randomised placebo-controlled trial, patients were recruited from the kidney or liver transplant waiting lists at the Royal Free Hospital, London, UK, between Aug 3, 2006, and Oct 30, 2008. Exclusion criteria included: pregnancy (a negative pregnancy test was required before each vaccine dose); receipt of blood products (except albumin) in the previous 3 months, and Vaccine group Placebo group Cytomegalovirus positive Cytomegalovirus negative Cytomegalovirus positive Cytomegalovirus negative Total number of patients 32 35 38 35 Organ awaiting transplantation Liver 10 (31%) 15 (43%) 13 (34%) 16 (46%) Kidney 22 (69%) 20 (57%) 25 (66%) 19 (54%) Sex Male 16 (50%) 22 (63%) 17 (45%) 27 (77%) Female 16 (50%) 13 (37%) 21 (55%) 8 (23%) Age (years) 55 (12) 49 (12) 52 (12) 48 (13) Race Caucasian 24 (75%) 32 (91%) 22 (58%) 33 (94%) Black 1 (3%) 0 (0%) 7 (18%) 1 (3%) Asian 5 (16%) 3 (9%) 5 (13%) 0 (0%) Other 2 (6%) 0 (0%) 4 (11%) 1 (3%) Number of vaccinations received 1 1 (3%) 1 (3%) 4 (11%) 1 (3%) 2 12 (38%) 18 (51%) 12 (32%) 8 (23%) 3 19 (59%) 16 (46%) 22 (58%) 26 (74%) Days from vaccine 1 to vaccine 2 (median, range) 32 (21–118) n=31 35 (22–274) n=34 30 (21–119) n=34 31 (23–241) n=34 Days from vaccine 1 to vaccine 3 (median, range) 186 (154–416) n=19 188 (147–224) n=16 188 (167–298) n=22 188 (151–375) n=26 Total number of patients who proceeded to transplantation during study period 18 23 22 15 Organ transplanted Liver 8 (44%) 11 (48%) 10 (46%) 10 (67%) Kidney 10 (56%) 12 (52%) 12 (55%) 5 (33%) Sex Male 7 (39%) 15 (65%) 7 (32%) 13 (87%) Female 11 (61%) 8 (35%) 15 (68%) 2 (13%) Age at transplantation (years) 53 (12) 50 (13) 50 (12) 49 (12) Race Caucasian 12 (67%) 22 (96%) 14 (64%) 15 (100%) Black 1 (6%) 0 (0%) 2 (9%) 0 (0%) Asian 4 (22%) 1 (4%) 3 (14%) 0 (0%) Other 1 (6%) 0 (0%) 3 (14%) 0 (0%) Number of doses of vaccine or placebo received before transplantation 1 0 (0%) 1 (4%) 1 (5%) 0 (0%) 2 9 (50%) 16 (70%) 8 (36%) 6 (40%) 3 9 (50%) 6 (26%) 13 (59%) 9 (60%) Days from vaccine 1 to transplantation (median, range) 216 (40–636) 123 (22–604) 199 (8–1134) 262 (36–1231) (Continues on next page) Articles 1258 www.thelancet.com Vol 377 April 9, 2011 simultaneous multiorgan transplantation. The study was approved by the Research Ethics Committee and all patients gave written informed consent. Randomisation and masking After patient consent, a pharmacist allocated placebo or vaccine using a scratch-off randomisation code provided by Sanofi Pasteur. The randomisation (ratio 1:1) was stratifi ed by cytomegalovirus status (seropositive vs sero- negative) and by transplanted organ (renal vs liver). Because the vaccine (white emulsion) and the placebo (colourless liquid) appeared diff erent, a blind-observer procedure was followed for product preparation and administration and safety assessment. Specifi cally, one investigator prepared the vaccine by transferring 0·35 mL of the MF59 emulsion to the 0·35 mL of cytomegalovirus glycoprotein-B antigen vial and then withdrawing 0·5 mL to vaccinate the patient. A second investigator (unaware of whether vaccine or placebo had been given) was then responsible for safety assessment. The material to be injected was obscured from patients who were asked to face away from the injection site. Procedures The fi rst patient was vaccinated on Aug 3, 2006. Vaccine doses containing 20 μg of recombinant cytomegalovirus glycoprotein B plus 9·75 mg of MF59 adjuvant were given intramuscularly at 0, 1, and 6 months, which was a dose schedule that had been previously assessed in healthy volunteers.20–22 The placebo was normal saline. Once a patient was transplanted, which could occur before all three doses were given, no further doses were given. Patients received a diary card to record solicited local (injection site pain, erythema, and swelling) and systemic (headache, fever, and myalgia) symptoms for 7 days after every injection and a thermometer and ruler to help with these measure ments. These adverse events were classifi ed as mild, moderate, or severe by reference to a pre-specifi ed chart. Patients were telephoned 48 h after every injection to remind them to complete the diary cards. Any serious adverse events that occurred within 28 days after an injection were recorded, and so were unexpected serious adverse reactions occurring at any time until the trial ended in September, 2009. The principal investigator was responsible for study design, protocol development and, together with the statis- tician, prepared the prespecifi ed data analysis plan. Adverse and serious adverse events were tabulated and presented to a Data Safety Committee on six occasions. This Com- mittee felt it was necessary to break the code in April, 2009, to ensure that there was no imbalance in the number of deaths occurring in each study group, but all investigators remained unaware of the allocation until the formal breaking of the code in September, 2009, after all partici- pants had completed the vaccination phase of the study. Antibodies against glycoprotein B were measured by enzyme immunoassay with a method similar to that described in detail elsewhere23 and expressed as geometric mean titres. Patients were managed according to routine clinical standard of care at this institute. Whole blood samples were requested twice a week from inpatients and at all subsequent outpatient visits, which were typically scheduled every week for 4 weeks then every 2 weeks until day 90. If cytomegalovirus viraemia was detected, patients Vaccine group Placebo group Cytomegalovirus positive Cytomegalovirus negative Cytomegalovirus positive Cytomegalovirus negative (Continued from previous page) Immunosuppressive drugs administered* Basiliximab 5 (28%) 9 (39%) 7 (32%) 3 (20%) Tacrolimus 14 (78%) 23 (100%) 22 (100%) 13 (87%) Azathioprine 5 (28%) 6 (26%) 7 (32%) 4 (27%) Mycophenolate mofetil 10 (56%) 17 (74%) 16 (73%) 10 (67%) Prednisolone 13 (72%) 21 (91%) 19 (86%) 11 (73%) Methylprednisolone 10 (56%) 16 (70%) 18 (82%) 9 (60%) Available follow-up by PCR since transplantation (days) Median (range) 93 (89-228) 97 (15–138) 95 (45–173) 95 (20–278) Cytomegalovirus status of donor Positive 7 (39%) 11 (48%) 15 (68%) 5 (33%) Negative 11 (61%) 12 (52%) 7 (32%) 10 (67%) Transplantation type Cadaver 13 (72%) 17 (74%) 16 (73%) 14 (93%) Live person 5 (28%) 6 (26%) 6 (27%) 1 (7%) Data are number (%), mean (SD) unless otherwise indicated. *If drugs known, otherwise we assumed that no drugs were administered. Table 1: Baseline characteristics according to patients’ cytomegalovirus status and randomisation group Articles www.thelancet.com Vol 377 April 9, 2011 1259 were monitored closely until PCR-negative results were obtained, and testing reverted to twice weekly in inpatients and at all subsequent outpatient visits. Each sample was tested by real-time quantitative PCR (rtqPCR) for cytomegalovirus DNA as described elsewhere.24 Viraemia was defi ned as a blood sample that was PCR positive (cutoff 200 genomes per mL whole blood). If viraemia higher than 3000 genomes per mL was detected, the patient was treated with twice daily intravenous ganciclovir 5 mg/kg (or twice daily oral valganciclovir 900 mg), with dose adjustment for renal function, until cytomegalovirus DNA was undetectable in two consecu- tive blood samples. The time from the fi rst PCR-positive sample until the last PCR-positive sample defi ned duration of viraemia, which therefore included days with and without pre-emptive treatment. Previous comparisons showed that changes in viral load values were indistinguishable when patients were treated with ganciclovir or valganciclovir.24 Cytomegalovirus end- organ disease was diagnosed by histopathological demon- stration of inclusion bodies in aff ected organs25 and, from our natural history data,26 was associated with a median viral load of 175 500 genomes per mL in blood with 37 000 genomes per mL as the lower limit of the 95% CI. With the 1 day average doubling-time14,15 of cytomegalovirus and the timing of sampling twice weekly, we aimed to initiate pre-emptive treatment once the viral load increased above 3000 genomes per mL to prevent viral load reaching 37 000 genomes per mL. For the studies of immunogenicity, serum samples were requested at the time of fi rst injection and 28, 56, 180, and 208 days later in those who received all three injections. In the subset of patients who received transplants, additional samples were requested at time of transplantation and 7, 35, 63, and 90 days later. The geometric mean titre and 95% CI of antibodies measured against glycoprotein B was calculated at each timepoint and plotted according to patient cytomegalovirus serostatus and randomisation group. Neutralising antibodies were measured with Towne RC256 (β-galactosidase marker virus) and human fi broblast target cells. Safety and immunogenicity were co-primary endpoints. For secondary endpoints, we postulated that receipt of vaccine would decrease the duration or quantity, or both, of viraemia when compared with that of placebo. A correlate of protective immunity was the tertiary endpoint. Statistical analysis Safety and immunogenicity was assessed by intention to treat in patients who received at least one dose of vaccine or placebo (intention to treat–exposed analysis). No interim analyses were planned and no post-hoc analyses are presented. The percentage of patients reporting any pain (regardless of severity) within a week of fi rst injection was compared in the two groups with a χ² test. Tests were two-sided and a p value of less than 0·05 was regarded as signifi cant. Patients who did not complete a diary card were judged to have had pain (missing-equals-failure analysis). This analysis was repeated for the occurrence of other solicited adverse events (myalgia, redness, site swelling, headaches, or fever) within 1 week of the fi rst injection. The missing-equals-failure analysis was repeated for the percentage of patients reporting pain within a week of the second and third doses, but excluding those who proceeded to transplantation since this was a random event (modifi ed missing-equals-failure approach). Next, the results from all three injections were summarised descrip tively according to severity (and thus each individual could have up to three measurements reported for each adverse event). Those who did not complete a diary card were excluded from the combined analysis of degree of severity. For the studies of immunogenicity, because the outcome was numerical, a missing-equals-excluded approach was taken. The main timepoint of interest was decided a priori to be 1 month after the second dose of vaccine (day 56) and diff erences between groups were compared by a 2-sample t test (with log-transformed data to ensure normality) stratifi ed by the patients’ cytomegalovirus status. The investigation was powered so that about 30 patients with cytomegalovirus viraemia after trans- plantation would be expected, on the basis of our previous natural history data.16 A total sample size of 140 patients was required to expect about 30 patients to develop viraemia. Analyses were done with SAS version 9.2 (SAS Institute Inc, Cary, NC). This trial is registered with ClinicalTrials.gov, NCT00299260. 490 patients assessed for eligibility 140 randomised 67 assigned to receive cytomegalovirus vaccine 67 assessed 35 received 3 doses 30 received 2 doses 2 received 1 dose 12 excluded 4 died 4 at patient’s request 2 referred elsewhere 2 ill health 14 awaiting transplantation 16 excluded 6 died 3 at patient’s request 2 referred elsewhere 4 ill health 1 lost to follow-up 20 awaiting transplantation 73 assessed 48 received 3 doses 20 received 2 doses 5 received 1 dose 41 proceeded to transplantation 37 proceeded to transplantation 41 evaluable 37 evaluable 73 assigned to receive placebo Figure 1: Trial profi le at the time of analysis Articles 1260 www.thelancet.com Vol 377 April 9, 2011 Role of the funding source The sponsors of the study and the funding source had no role in the study design, data collection, data analysis, data interpretation, writing of the manuscript, or in the decision to submit to publication. PG, CS, VE, RM had full access to all the data. All authors reviewed the report and had fi nal responsibility for the decision to submit for publication. Results No major imbalances in demographic or clinical features were noted between seropositive or seronegative patients randomly assigned to vaccine or placebo (table 1). Figure 1 shows the trial profi le at Dec 31, 2009. All 140 randomised patients were evaluable for the two co-primary endpoints of safety and immunogenicity. Of the 67 patients randomly assigned to vaccine, all 67 (100%) had not proceeded to transplantation or death before the fi rst schedule dose, 66 (98·5%) had not done so before t