290 www.thelancet.com/infection Vol 12 April 2012

Articles

Lancet Infect Dis 2012; 12: 290–99

Published OnlineJanuary 10, 2012

DOI:10.1016/S1473-3099(11)70344-9

This online publication has been corrected. The corrected

version fi rst appeared at thelancet.com/infection on March 26, and Dec 17, 2012

See Comment page 257

Department of Blood and Marrow Transplantation and

Department of Oncological Sciences, H Lee Moffi tt Cancer Center and Research Institute

and University of South Florida, Tampa, FL, USA

(M A Kharfan-Dabaja MD); American University of Beirut

Medical Center, Division of Hematology-Oncology and

Bone Marrow Transplantation, Beirut, Lebanon

(M A Kharfan-Dabaja); Vaccine and Infectious Disease Division,

Fred Hutchinson Cancer Research Center, Seattle, USA

(Prof M Boeckh MD); Division of Infectious Diseases,

Dana-Farber Cancer Institute, Brigham and Women’s

Hospital, Boston MA, USA (M B Wilck MBChB); Division of

Infectious Diseases, Hospital of the University of Pennsylvania,

Philadelphia, PA, USA (M B Wilck); Bone Marrow and

Stem Cell Transplant Center, Winship Cancer Institute,

Emory University School of Medicine, Atlanta, GA, USA (Prof A A Langston MD); and

Departments of Clinical Development (R T Kenney MD,

A H Chu MS, D F Guterwill BSMT), Vaccinology (L R Smith PhD,

M K Wloch PhD), and Product Development (A P Rolland PhD),

Vical, San Diego, CA, USA

Correspondence to:Dr Richard T Kenney, Department

of Clinical Development, Vical, 10390 Pacifi c Center Court,

San Diego, CA 92121, USArichard.kenney@vical.com

A novel therapeutic cytomegalovirus DNA vaccine in allogeneic haemopoietic stem-cell transplantation: a randomised, double-blind, placebo-controlled, phase 2 trialMohamed A Kharfan-Dabaja, Michael Boeckh, Marissa B Wilck, Amelia A Langston, Alice H Chu, Mary K Wloch, Don F Guterwill, Larry R Smith, Alain P Rolland, Richard T Kenney

SummaryBackground Cytomegalovirus reactivation occurs within 6 months in 60–70% of cytomegalovirus-seropositive patients after allogeneic haemopoietic stem-cell trans plantation (HSCT), mainly due to immunosuppression associated with the procedure. Pre-emptive antiviral therapy reduces incidence of cytomegalovirus disease but can be toxic. To reduce the potential for disease and subsequent need for such antiviral drugs, we aimed to assess safety and effi cacy of a cytomegalovirus therapeutic DNA vaccine compared with placebo.

Methods In this exploratory double-blind, placebo-controlled, parallel group, phase 2 trial, up to 80 donor–recipient pairs and 80 unpaired recipients undergoing allogeneic HSCT were planned for enrolment at 16 transplant centres in the USA. Eligible recipients were cytomegalovirus-seropositive, 18–65 years old, without high-risk primary disease, T-cell depletion, previous vaccination for cytomegalovirus, or autoimmune diseases. We randomly allocated participants in both parallel groups in a 1:1 ratio to receive a cytomegalovirus therapeutic DNA vaccine (TransVax; Vical, San Diego, CA, USA) or placebo before conditioning and at 1, 3, and 6 months after transplantation. The vaccine contains plasmids encoding cytomegalovirus glycoprotein B and phosphoprotein 65 formulated with poloxamer CRL1005 and benzalkonium chloride. Randomisation was done by sequential allocation based on Pocock and Simon’s method, and stratifi ed by site, donor–recipient HLA matching status, and donor’s cytomegalovirus serostatus. The primary outcome was the occurrence rate of clinically signifi cant viraemia resulting in initiation of cytomegalovirus-specifi c antiviral therapy in the per-protocol assessable population. We assessed rates of adverse events in all participants who received at least one dose of vaccine or placebo. This study is registered with ClinicalTrials.gov, number NCT00285259.

Findings We randomly allocated 108 participants (94 HSCT recipients and 14 paired donors) between June 29, 2006, and Dec 11, 2009. Enrolment of the paired arm was halted in February 2008 for logistical reasons. Safety was assessed in all participants; the effi cacy population was restricted to 74 unpaired recipients. Groups were balanced for demographic and clinical variables. 19 (48%) of 40 vaccine recipients required cytomegalovirus-specifi c antiviral therapy, compared with 21 (62%) of 34 controls (p=0·145). However, during follow-up vaccine signifi cantly reduced the occurrence and recurrence of cytomegalovirus viraemia and improved the time-to-event for viraemia episodes compared with placebo. The vaccine was well-tolerated; only one participant discontinued after an allergic reaction. Incidence of common adverse events after HSCT (eg, graft-versus-host disease or secondary infections) did not diff er between groups.

Interpretation We show proof of concept for an immunotherapeutic cytomegalovirus vaccine (TransVax) for clinically significant viraemia in the HSCT setting. The reported safety and efficacy outcomes support further development in a phase 3 trial, notwithstanding a lack of significant reduction in the use of cytomegalovirus-specific antiviral therapy compared with placebo in this phase 2 trial.

Funding Vical and US National Institute of Allergy and Infectious Diseases.

IntroductionCytomegalovirus is a β herpesvirus that causes a lifelong latent and asymptomatic infection in most individuals but can become pathogenic in immunocompromised patients such as transplant recipients. Until eff ective antiviral therapy became available, nearly 25% of at-risk recipients of haemopoietic stem cell transplantations died from cytomegalovirus-associated disease in the fi rst year after transplantation.1 Patients who are cytomegalo-virus seropositive can have reactivation of a latent strain or reinfection with a new strain and patients who are

cytomegalovirus seronegative are at risk of primary infection after transplantation.2

Virological surveillance and pre-emptive treatment of cytomegalovirus infections have substantially reduced the incidence and severity of life-threatening illness in recipients of allogeneic haemopoietic stem-cell trans-plants or solid organ transplants. Nonetheless, the rate of cytomegalovirus infection or reactivation is 50–80% after transplantation.2–5 Cytomegalovirus prophylaxis is limited by several shortcomings, which are mainly attributable to toxic eff ects associated with current anti-cytomegalovirus

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therapies.2,6 The fi rst-line antiviral drugs ganciclovir and valganciclovir are generally associated with haematological toxic eff ects (particularly neutro penia) and delayed immune reconstitution. Second-line drugs (ie, foscarnet and cidofovir) can cause serious side-eff ects (especially nephrotoxic ones). Such toxic eff ects have led to the use of a targeted pre-emptive strategy, which allows anti-cytomegalovirus treatment to be given only to patients with clinical evidence of cytomegalovirus viraemia, rather than prophylaxis with existing drugs in the setting of haemopoietic stem-cell transplantation.7,8

Next-generation prophylactic antiviral drugs are undergoing clinical testing and might eff ectively reduce the rate of cytomegalovirus infection, although a phase 3 study assessing maribavir did not show a benefi t in prevention of cytomegalovirus disease when started after engraftment.9 The recent introduction of sirolimus-based regimens for prophylaxis of graft-versus-host disease (GVHD) might also result in lower rates of cytomegalovirus reactivation.10 However, calcineurin inhibitors (tacrolimus or ciclosporin) combined with either methotrexate or mycophenolate mofetil continue to be the most commonly used regimens for GVHD prophylaxis worldwide. In the haemopoietic stem-cell transplantation setting, an eff ective vaccine must overcome the immunosuppressive eff ects of previous chemotherapy and transplant conditioning regimens, as well as GVHD prophylaxis and treatment. By contrast with the number of new antiviral drugs presently undergoing clinical evaluation, there is an unmet need for a vaccine that can exploit natural immune response mechanisms to control viral replication.

T-cell-mediated immunity is key to control of cyto-megalovirus replication;11,12 and its consequent impair-ment resulting from conditioning before allogeneic haemopoietic stem-cell transplantation (and secondary immune suppression from GVHD prophylaxis) increases the risk of cytomegalovirus replication.1 Moreover, the increasing use of haploidentical allogeneic haemopoietic stem-cell transplantations, which can be used for patients who do not have a suitable HLA-matched related or unrelated donor, results in an increased risk of serious infections (including cytomegalovirus) due to delayed or incomplete immune reconstitution.13 Both the initial viral load and the rate of increase in cytomegalovirus load are correlated with development of serious cyto megalovirus disease.14 Furthermore, secondary eff ects of cytomega-lovirus after haemopoietic stem-cell transplant ations can cause poor recovery of cytomegalovirus-specifi c pro-liferative responses and increased relapse of the underlying disease.15

We aimed to assess the safety, immunogenicity, and clinical benefi t of an immunotherapeutic cytomegalovirus vaccine (TransVax; Vical, San Diego, CA, USA) that contains plasmids encoding the surface glycoprotein B and tegument phosphoprotein-65. The vaccine is formulated with CRL1005 poloxamer and benzalkonium

chloride, which is a delivery system that enhances gene expression in vivo16,17 and increases immune responses compared with DNA vaccines formulated in phosphate-buff ered saline alone.17,18 This formulation has been used safely in normal healthy cytomegalovirus-seropositive and seronegative adults.19 We did our study in donors and cytomegalovirus-seropositive recipients undergoing allogeneic haemo poietic stem-cell transplantations for various malignant haematological diseases.

MethodsStudy design and participantsIn our randomised, double-blind, placebo-controlled, parallel-group phase 2 trial, we enrolled donor–recipient pairs and unpaired recipients undergoing allogeneic haemopoietic stem-cell transplantations at 16 participating transplant centres in the USA. Transplant recipients were eligible if they were cytomegalovirus-seropositive, 18–65 years old, and had a suitable HLA-matched (6/6 A, B, and DR alleles) or HLA-mismatched (5/6 alleles) related or unrelated donor willing to undergo peripheral blood stem-cell mobilisation with granulocyte colony-stimulating factor. Use of myeloablative, reduced inten-sity, and non-myeloablative conditioning regimens were permitted before transplantation. Complete ex-vivo T-cell depletion of the stem cell graft was a study exclusion criterion. Donors were excluded if they had received a live-attenuated vaccine within 30 days, previous therapy for cytomegalovirus infection, or evidence of congenital or acquired immune defi ciencies. Recipients were also excluded if their primary disease was deemed as high risk or if they had planned T-cell in-vivo depletion with alemtuzumab or complete allograft T-cell depletion, planned prophylaxis with cytomegalovirus immuno-globulin or anti-cytomegalovirus therapy, or previous diagnoses of autoimmune diseases. We obtained approval from institutional review boards before initiation of enrolment at every site and all participants provided written informed consent.

Randomisation and maskingWe randomly allocated participants in a one-to-one ratio to receive intramuscular injections of vaccine or placebo. We randomly allocated participants dynamically as per Pocock and Simon,20 stratifi ed by site, donor–recipient HLA matching status, and donor’s cytomegalovirus serostatus. Minimisation was accomplished with an algorithm based on stratifi cation factors of patients already enrolled in the trial. Treatment for each participant was assigned and an imbalance score was computed for every available treatment; the score was the imbalance that would be generated across treatments, taking into account stratifi cation factors if that treatment were assigned. In general, the treatment with the lowest imbalance score was then given preference when assigning new participant treatments. Once the donor–recipient pair or unpaired recipient had been fully

For the study protocol see http://www.vical.com/CB01-202/default.aspx

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qualifi ed for the trial and all screening activities had been completed, a central interactive voice response system provided treatment assignment to the site pharmacists, who were unmasked to treatment-group allocation. To assure that the trial remained masked, the person who gave the injections could not be involved in the undertaking of any protocol-required procedures or in medical care of participants for the duration of the trial. Study doctors, staff , and the sponsors were masked until the last patient completed follow-up on Nov 19, 2010, and the database was closed. No evidence of unmasking was noted during monitoring.

ProceduresThe investigational vaccine contained plasmids en-coding cytomegalovirus glycoprotein B and phospho-protein 65, each at 2·5 mg/mL (ie, 5 mg overall per mL), formulated with poloxamer CRL1005 and benzalkonium chloride in phosphate-buff ered saline.17–19 Placebo was phosphate-buff ered saline. Both vaccines were dosed intramuscularly in 1·0 mL. The 5 mg dose was the highest dose studied in the dose-escalating phase 1 trial19 and was restricted by the bulk drug substance con-centration. In the donor–recipient pair group, donors

received vaccine or placebo three times before donation (2–21 days before collection of peripheral-blood stem cells). Recipients of haemopoietic stem-cell transplants received vaccine or placebo once before transplantation (between days –5 to –3) and three times after trans-plantation (at 21–42 days, dependent on platelet recovery, and on days 84 and 196) and were scheduled for follow-up and immunogenicity assessments at 56, 84, 126, 196, 210, and 365 days after transplantation. The dosing schedule was designed to optimise immunogenicity during periods of greatest risk after transplantation. An independent data safety monitoring board assessed the safety of the fi rst 20 recipients after their day 56 visit, and monitored safety on an ongoing basis.

The primary endpoints were the safety of vaccine compared with placebo and rates of cytomegalovirus viraemia resulting in initiation of cytomegalovirus-specifi c antiviral therapy when evidence of viraemia indicated its use. Viraemia was defi ned as 500 viral copies per mL or more (the lower reporting limit), and was assessed with the LightCycler PCR assay (analyte specifi c reagents and instrument manufactured by Roche Applied Science, Indianapolis, IN, USA). This assay was developed and validated in a central laboratory (Mayo

Figure 1: Trial profi le and participants*Paired recipients were only assessed for safety; all 14 donors received three doses.

42 randomly allocated to receive vaccine 38 randomly allocated to receive placebo

80 unpaired recipients

42 recipients assessed 25 received four doses 7 received three doses 4 received two doses 6 received one dose

38 recipients assessed 23 received four doses 6 received three doses 4 received two doses 5 received one dose

2 excluded 1 cytomegalovirus negative 1 died before PCR

4 excluded 2 cytomegalovirus negative 2 exclusion violations

40 analysed 34 analysed

12 randomly allocated to receive vaccine 16 randomly allocated to receive placebo

28 paired participants 14 donors 14 recipients

6 recipients assessed* 2 received four doses 0 received three doses 3 received two doses 1 received one dose

8 recipients assessed* 4 received four doses 3 received three doses 0 received two doses 1 received one dose

119 individuals assessed for eligibility

7 screening failures

112 randomly allocated

4 withdrew before dosing 2 exclusion violations 2 withdrew consent

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Clinical Laboratories, Rochester, MN, USA). The PCR oligo nucleo tides amplify a segment of the UL54 viral polymerase gene. Sites were also allowed to use their local laboratories for monitoring of cytomegalovirus viraemia and for treatment decisions.

Secondary endpoints included the immunogenicity of vaccine compared with placebo by measurement of inter feron-γ enzyme-linked immunosorbent spot (ELISPOT) responses to phosphoprotein 65 and glyco-protein B, and glycoprotein B-specifi c antibody concen-trations measured in an indirect binding IgG ELISA with recombinant full-length glycoprotein B protein as the coating antigen.19 For recipients of haemopoietic stem-cell transplants, we recorded time to engraftment, occurrence of cytomegalovirus reactivation, time-to viraemia (defi ned as number of days from transplantation to the date of a positive result in the central laboratory assay), occurrence rates of cytomegalovirus-associated diseases, acute or chronic GVHD, secondary infections, death, and the cumulative number of cytomegalovirus-specifi c antiviral treatment days. Haemopoietic engraftment was defi ned as occurring on the fi rst of 3 consecutive days when the peripheral blood absolute neutrophil count was more than 500 cells per μL. We also assessed exploratory endpoints related to cytomegalovirus viraemia, such as prevalence of viraemia, time to the composite of viraemia and antiviral therapy, and duration of viraemia.

Statistical analysisWe calculated the sample size on the basis of the primary endpoint of a reduction of cytomegalovirus viraemia in recipients. With an equal sample size of 40 individuals per group and a historical control reactivation rate of 60%,1,2,11 there was 70% power to detect a decrease in cytomegalovirus infection from 60% to 30%, assuming a two-sided α of 0·05.21 We aimed to enrol up to 80 donor–recipient pairs and 80 unpaired recipients undergoing allogeneic haemo poietic stem-cell trans plantations. An administrative interim analysis by group was planned once all participants had reached day 126, but the study masking was maintained until fi nal analysis. We regarded diff erences between vaccine and placebo groups as signifi cant if p was less than or equal to 0·05 analysed with the Wilcoxon rank sum test, the Cochran-Mantel-Haenszel test (stratifi ed by site), Fisher’s exact test, χ² test, or log-rank test as appropriate. We used the Andersen-Gill generalisation of Cox regression analysis with a sandwich variance estimate22 in a post-hoc analysis to compare the prevalence of episodes of cytomegalovirus viraemia in time between the vaccine and placebo groups. This analysis gives an estimate of effi cacy, and assesses the time to more than one episode of cytomegalovirus viraemia after adjustment for inter-event correlations. The analysis of prevalence and an analysis of the composite of clinically signifi cant viraemia and initiation of cytomegalovirus-specifi c antiviral therapy, requiring

both to be present, were exploratory analyses done to assess effi cacy options for a future pivotal study to license the vaccine. This approach is often used with phase 2 data to develop the best statistical approach for phase 3 trials. All analyses were done with SAS version 9.2.

Vaccine group Placebo group

Haemopoietic stem-cell transplant recipients

n 48 46

Age (years) 49·8 (9·8, 25–63) 48·5 (13·6, 23–72)

Sex (male) 22 (46%) 27 (59%)

Primary diagnosis

Acute myelogenous leukaemia

18 (38%) 12 (26%)

Non-Hodgkin lymphoma 9 (19%) 8 (17%)

Acute lymphoblastic leukaemia

5 (10%) 8 (17%)

Myelodysplastic syndrome 6 (13%) 6 (13%)

Chronic myelogenous leukaemia

3 (6%) 2 (4%)

Hodgkin’s lymphoma 1 (2%) 2 (4%)

Other 6 (13%) 8 (17%)

Karnofsky performance score 88·8 (9·4, 70–100) 86·0 (14·0, 40–100)

Donor cytomegalovirus status

Seropositive 26 (54%) 27 (59%)

Seronegative 22 (46%) 19 (41%)

Donor transplant source

Matched-related donor 29 (60%) 23 (50%)

Matched-unrelated donor 15 (31%) 21 (46%)

Mismatched donor (related or unrelated)

4 (8%) 2 (4%)

CD34 cell dose (×10⁶ cells per kg)

8·4 (4·6, 2–22)* 7·1 (3·4, 1–16)†

Stem cell source

Peripheral blood 47 (98%) 44 (96%)

Bone marrow 1 (2%) 2 (4%)

Conditioning regimen

Myeloablative 36 (75%) 35 (76%)

Reduced intensity or non-myeloablative

12 (25%) 11 (24%)

Anti-thymocyte globulin in conditioning regimen

Yes 4 (8%) 5 (11%)

No 44 (92%) 41 (89%)

Donors

n 6 8

Age (years) 55·2 (9·1, 45–66) 43·9 (13·9, 23–65)

Sex (male) 1 (17%) 2 (25%)

Cytomegalovirus serostatus

Seropositive 6 (100%) 6 (75%)

Seronegative 0 2 (25%)

Data are n, mean (SD, range), or n (%). *Data available for 45 (94%) of 48 patients. †Data available for 45 (98%) of 46 patients.

Table 1: Baseline characteristics of 94 recipients and 14 donors of haemopoietic stem-cell transplants who received at least one dose of vaccine or placebo

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This study is registered with ClinicalTrials.gov, number NCT00285259.

Role of the funding sourceVical sponsored the study, manufactured the investi-gational products, and funded most aspects of this clinical trial. Vical staff developed the study design. All authors contributed to study design, data collection, analysis and interpretation, or writing of the report. All authors had full access to to all the data in the study, reviewed the report, and had fi nal responsibility for the decision to submit for publication.

ResultsWe enrolled and randomly allocated 112 participants to treatment groups between June 29, 2006 and Dec 11, 2009 and 108 (96%) participants received at least one dose of vaccine or placebo (fi gure 1). Although we intended to enrol 80 donor–recipient pairs, enrolment in that treat-ment arm was halted at 14 pairs for logistical reasons in February, 2008, and the number of pairs recruited was too small for meaningful immunogenicity or effi cacy comparisons between groups.

Overall, 94 cytomegalovirus-seropositive recipients of haemopoietic stem-cell transplants and 14 donors received vaccine or placebo (fi gure 1, table 1). We noted no major imbalances in characteristics between the participants randomly assigned to vaccine or to placebo (table 1). 71 (75%) of 94 recipients had myeloablative conditioning, which was evenly distributed between groups, and the remainder had non-myeloablative or reduced-intensity conditioning. 53 (56%) of 94 recipients received transplants from cyto mega lo-virus-seropositive donors. Dosing compliance was high, with most missed doses (77 [20%] of 376 planned doses were missed) attributable to delayed platelet recovery or premature death.

Of the 80 unpaired recipients recruited, we excluded six from further analysis because of inclusion or exclusion violations or premature death before viraemia assessment. Thus, 74 participants were analysed as part of the per-protocol assessable population (40 in the vaccine group and 34 in the placebo group).

Rates of initiation of cytomegalovirus-specifi c antiviral therapy did not diff er between groups in the per-protocol assessable population (p=0·145), and treatments lasted much the same amount of time (table 2). The rates may have been aff ected by the widespread use of results from various local laboratories for treatment decisions rather than the central laboratory. Conversely, rates of cytomegalovirus viraemia on central laboratory measure-ments were lower in the vaccine group than the placebo group (p=0·008) and fewer participants in the vaccine group had more than one episode of cytomegalovirus viraemia (table 2). On Kaplan-Meier analysis, the time to fi rst cyto mega lovirus viraemia was longer in the vaccine group than it was in the placebo group, and there was

Vaccine group (n=40) Placebo group (n=34) p value

Days to haemopoietic engraftment 15 (12–16) 16 (14–18) 0·170

Acute graft-versus host disease ·· ·· 0·152

Grade 0 14 (35%) 7 (21%) ··

Grade I 0 3 (9%) ··

Grade II 24 (60%) 19 (56%) ··

Grade III 1 (3%) 3 (9%) ··

Grade IV 1 (3%) 2 (6%) ··

Chronic graft-versus host disease ·· ·· 0·538

None 12 (30%) 9 (26%) ··

Mild 10 (30%) 6 (18%) ··

Moderate 9 (23%) 10 (29%) ··

Severe 4 (10%) 7 (21%) ··

Not available 5 (13%) 2 (6%) ··

Secondary infection* 34 (85%) 33 (97%) 0·116

Bacterial 28 (70%) 29 (85%) 0·119

Fungal 14 (35%) 14 (41%) 0·585

Viral (excluding cytomegalovirus) 11 (28%) 15 (44%) 0·136

Protozoal 4 (10%) 0 0·120

Other 7 (18%) 10 (29%) 0·225

Data are median (95% CI) or n (%). *Participants might have had more than one occurrence of the same secondary infection, but only one event is counted.

Table 3: Selected outcomes in the per-protocol assessable population of unpaired recipients of haemopoietic stem-cell transplants

Vaccine group (n=40) Placebo group (n=34) p value

Initiation of pre-emptive CMV-specifi c antiviral therapy

19 (47·5%, 32·0–63·0) 21 (61·8%, 45·4–78·1) 0·145

CMV viraemia (≥500 copies per mL) 13 (32·5%, 18·0–47·0) 21 (61·8%, 45·4–78·1) 0·008

CMV episodes ·· ·· 0·017

0 27 (68%) 13 (38%) ··

1 8 (20%) 14 (41%) ··

2 4 (10%) 3 (9%) ··

3 1 (3%) 3 (9%) ··

4 0 1 (3%) ··

Median 0 (1) 1 (1) ··

Median number of days to initial CMV viraemia Not reached 109·5 0·003

Viraemia-free rate at 1 year, % (95% CI) 65·2% (49·7–80·6) 36·8% (20·2–53·5) 0·014*

Mean duration of viraemia in all participants, days

10·6 (18·3, 0–68) 19·5 (35·6, 0–181) 0·071

Normalised as percentage time on study 4·9% ( 12·4, 0–63) 7·7% (11·8, 0–49) 0·042

Mean duration of CMV-specifi c antiviral therapy 33·7 (64·1, 0–316) 40·4 (57·7, 0–212) 0·450

Median number of days to a clinical composite event†

Not reached 106 0·016

Event-free rate at 1 year, % (95% CI) 71·0% (56·5–85·5) 43·5% (26·7–60·4) 0·015*

CMV-associated disease 3 (7·5%)‡ 3 (8·8%) 1·000

Gastrointestinal 3 (7·5%) 2 (5·9%) ··

CMV pneumonia 1 (2·5%) 1 (2·9%) ··

Data are n (%, 95% CI), n (%), or mean (SD, range), unless otherwise stated. CMV=cytomegalovirus. *Based on a Greenwood method.23 †Composite event of CMV viraemia and antiviral therapy. ‡One participant had gastrointestinal disease and CMV pneumonia during follow-up.

Table 2: Cytomegalovirus viraemia and cytomegalovirus-specifi c antiviral treatment endpoints in the per-protocol assessable population of unpaired recipients of haemopoietic stem-cell transplants after 1 year follow-up

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evidence to suggest mean duration of viraemia was shorter in the vaccine group than the placebo group when the data were normalised by percentage time on study (p=0·042; table 2). Incidence of biopsy-confi rmed cytomegalovirus-associated disease did not diff er between groups (table 2).

We assessed safety in 108 participants who received at least one dose of vaccine or placebo, although 94 reci-pients were assessed separately from the 14 donors. All recipients developed at least one treatment-emergent adverse event during the study. The most frequently reported events (in >50% of participants) in both treat-ment groups were thrombocytopenia, nausea, diarrhoea, vomiting, stomatitis, peripheral oedema, rash, headache, insomnia, hypomagnesaemia, decreased appetite, fatigue, pyrexia, or cough (appendix). Ten (21%) of 48 vaccine recipients and fi ve (11%) of 46 placebo recipients had injection-site pain (p=0·26). Incidence of treatment-emergent adverse events was much the same between treatment groups. We noted no between-group diff erences in the assessments of laboratory tests, vital signs, or physical examination results.

39 (81%) of 48 vaccine recipients and 34 (74%) of 46 placebo recipients had serious adverse events (p=0·393), most of which were regarded as unrelated to study product. However, site investigators regarded the following serious adverse events as possibly related to the investigational product: one allergic reaction in a patient also exposed to a drug in the same class as a known drug allergen (1 h after last dose), one patient with worsening pericardial eff usion with cardiac tamponade (1 month after last dose), one patient with a subarachnoid haemorrhage from a known aneurysm (3 months after last dose), and one patient with cytomegalovirus colitis (2 months after the last dose). The fi rst three events occurred in participants in the vaccine group and the fourth occurred in a participant in the control group. After review, the data and safety monitoring board regarded only the allergic reaction as possibly related to vaccine treatment and that was the only patient in the trial who was discontinued due to a treatment-emergent adverse event. Ten (21%) of 48 vaccine recipients and 15 (33%) of 46 placebo recipients died by 1 year after transplantation, but the diff erence did not reach signifi cance (p=0·196), perhaps because of the small sample size. The vaccine safety profi le was therefore much the same as that of placebo in transplant recipients and the vaccine was well tolerated.

All six donors in the vaccine group and seven of eight donors in the placebo group had at least one treatment-emergent adverse event (injection-site pain in three donors in the vaccine group and one donor in the placebo group). Nausea, which was reported in three donors in the placebo group but no donors in the vaccine group, was the only other symptom present in more than two donors. Incidence of treatment-emergent adverse events were much the same between treatment groups and there were no deaths, serious adverse events, or dis-continuations due to adverse events in donors.

Receipt of vaccine had no eff ect on usual transplant-related outcomes. The median time to engraftment did not diff er between vaccine and placebo groups in the per See Online for appendix

Figure 2: Immunogenicity of vaccine and placeboMean (standard error) T-cell responses to overlapping 15-mer phosphoprotein 65 (A) and glycoprotein B (B) peptides assessed by interferon γ enzyme-linked immunosorbent spot assay. (C) Geometric mean (95% CI) IgG concentrations measured by ELISA, showing glycoprotein B specifi c antibody concentrations. (A and B) Y-axes shown on linear scales, although the statistic uses a logarithmic transformation to help normalise the ELISPOT data before the analysis. (C) Y-axis shown on a logarithmic scale. SFU=spot-forming units. PBMC=peripheral blood mononuclear cells. EU=ELISA units.

–10 29021514065 36529021514065 365Time (days)

29021514065 365

–10 29021514065 365

Mea

n T-

cell

resp

onse

(SFU

/PBM

C×10

6 )

5000

4000

3000

2000

1000

Mea

n T-

cell

resp

onse

(SFU

/PBM

C×10

6 )

2500

2000

1500

1000

0

500

Geom

etric

mea

n Ig

G co

ncen

trat

ion

(EU/

mL)

364 316

100 000

6515

10 000

Vaccine group (n=37)Placebo group (n=34)p<0·232

p=0·204

p=0·749

B

A

C–10

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protocol analysis (table 3); only one participant in each group had graft failure during the trial. Incidences of acute or chronic GVHD were much the same between groups. Occurrence of secondary infections did not diff er between groups. No infection types (other than cytomegalovirus) were signifi cantly more common in either group. A much larger study than ours would be needed to show signifi cant diff erences in endpoints related to the secondary eff ects of cytomegalovirus infection in recipients of haemopoietic stem-cell transplants.

We did the immunogenicity analysis in the per-protocol assessable population. The number of phosphoprotein 65 interferon-γ-producing T cells was increased, although not signifi cantly in a repeated measurements ANOVA, in the vaccine group compared with the placebo group at all time points after transplantation (fi gure 2).

The number of glycoprotein B interferon-γ-producing T cells was much the same at all timepoints (fi gure 2). Although not signifi cantly diff erent, the geometric mean glycoprotein B antibody concentrations were numerically higher in the vaccine group than they were in the placebo group at all timepoints after trans plantation (fi gure 2). The comparison of the overall curves over time between the two groups was made by the F test (an ANOVA test). This strategy employs a linear curve model for each treatment group, as well as a variance-covariance model that incorporates correl ations for all of the observations arising from the same participant.

We did exploratory analyses to assess effi cacy options for a pivotal study in the per-protocol assessable popu-lation. Figure 3 shows the assessment of the prevalence of episodes of viraemia every month after trans plan-tation. The hazard (probability of having events) was reduced 53·6% for vaccine compared with placebo (fi gure 3). Finally, an analysis of the composite of clinically signifi cant viraemia and initiation of cytomegalovirus-specifi c antiviral therapy, requiring both to be present, showed a reduction in the vaccine group compared with placebo after about 6 weeks (p=0·016; fi gure 3). Event-free rates were estimated at 71·0% vs 43·5% at 1 year for the combined endpoint, or an effi cacy of 48·7% (95% CI 8·1–71·3).23,24

DiscussionCompared with placebo, the therapeutic cytomegalovirus DNA vaccine TransVax was well tolerated in the assessable population in our study, and reduced the occurrence, recurrence, duration of episodes of cyto-megalovirus, viraemia and improved time-to-event at 1 year follow-up (table 2). However, the rates of clinically signifi cant viraemia requiring cytomegalovirus-specifi c antiviral therapy after vaccine did not diff er from those noted for placebo.

Despite advances in development of antiviral drugs and the substantial reduction of disease with pre-emptive treatment, cytomegalovirus remains one of the most diffi cult-to-treat infections associated with haemopoietic stem-cell transplantation. This trial was designed to assess a novel vaccine approach in seropositive recipients, who are at greatest risk for cytomegalovirus recurrence (panel).2 Our inclusion and exclusion criteria were chosen to allow enrolment of the most representative patients who would provide uniform groups; generalisation to larger populations will need additional studies. Thera peutic vaccination is particularly challenging in recipients of haemopoietic stem-cell transplants, whose immune systems are purposefully ablated before donor cells are transplanted and who remain functionally immuno-compromised for 6 months or more afterwards. Ideally, cytomegalovirus-specifi c immune cells could be trans-planted directly from a vaccinated donor and the recipient would be immediately protected. However, we established that donors could not be identifi ed quickly and vaccinated

Figure 3: (A) Prevalence of cytomegalovirus viraemia at 4 week intervals22 and (B) Kaplan-Meier analysis of time to the composite endpoint of viraemia and cytomegalovirus-specifi c antiviral therapy (circles are censored events)HR=hazard ratio.

0 3628168 44

Time since transplantation (weeks)

48403224 56124 20 52

0 3628168 44 48403224 56124 20 52

Part

icipa

nts w

ith ≥

500

copi

es p

er m

L (%

)

35

30

25

15

5

0

20

10

Vaccine group (n=40)Placebo group (n=34)

B

A

Recip

ient

s with

out v

iraem

ia a

nd a

ntiv

iral t

hera

py (%

)

100

80

60

30

10

0

40

20

90

70

50

p=0·036, HR 0·464 (95% CI 0·226–0·953)

p=0·016, HR 0·362 (95% CI 0·154–0·850)

Number at riskVaccine group 28 23 21 21 21 21 Placebo group 18 16 14 14 13 13

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early enough before transplantation; thus, we abandoned the matched-pair strategy in favour of vaccination and close follow-up of unpaired vaccine recipients. Other genetic vaccine approaches, such as with recombinant alphaviruses or poxviruses, have shown immunogenicity against the cytomegalovirus glycoprotein B or phos-pho protein 65 antigens in healthy participants.28–30 Our vaccine was the fi rst molecular approach tested in recipients of haemopoietic stem-cell transplants and might have important advantages compared with genetic vaccine approaches, including the absence of concerns about the potential for spread of replication competent viruses in immunosuppressed individuals and reduced potency from antivector immune responses after repeated dosing.

Griffi ths and colleagues26 assessed a cytomegalovirus vaccine containing recombinant glycoprotein B protein and an MF59 adjuvant in adults awaiting kidney or liver transplantation. Vaccine reduced duration of viraemia and days of ganciclovir treatment compared with placebo only in cytomegalovirus-seronegative recipients of organs from seropositive donors, despite signifi cant increases in glycoprotein B antibodies after vaccination of seronegative and seropositive recipients.26 Although humoral immunity might have an important role in the seronegative-recipient subgroup (which is ~20% of all patients who have solid-organ transplantations), cell-mediated immunity is crucial for protection of transplant recipients from early and late reactivation of cyto megalovirus.11,12 The TransVax vaccine was designed to not only stimulate antibodies to glycoprotein B, which were present at enrolment by defi nition in our population, but also T cells to glycoprotein B and phosphoprotein 65. Both antigens encoded by TransVax (but especially phosphoprotein 65) are among the most frequently recognised of all cytomegalovirus antigens by both CD4+ and CD8+ T cells in healthy cytomegalovirus-seropositive adults who maintain lifelong control of the virus.31 In our study, vaccine seemed to enhance phosphoprotein 65 cellu lar immune responses in these immunosuppressed recipients of transplants during the period when they were at highest risk for cytomegalovirus disease and thus were exposed to the toxicities associated with antiviral therapy. Even though the glycoprotein B T-cell assay used in our study did not show a signifi cant diff erence between groups, cellular responses to glycoprotein B may have an important role in protection as well. Results from our study complement the fi ndings in the glycoprotein B-MF59 study26 and suggest that immunotherapeutic vaccines might be useful in all patients at risk of cytomegalovirus infection in both the haemopoietic stem cell and solid organ transplants settings.

Several outcomes in this trial suggested that the vaccine has a notable eff ect in recipients of haemopoietic stem-cell transplants, primarily in the various measures of cytomegalovirus in the blood. Cytomegalovirus infection was historically documented by virus isolation and cytomegalovirus viraemia is a strong predictor of end-

organ disease and subsequent mortality.32 Increasingly sensitive and rapid tests have been developed that form the basis for pre-emptive care, which is the most common clinical approach to protection from cytomegalovirus disease.7 Quantitative establishment of cytomegalovirus in the blood precedes the diagnosis of cytomegalovirus disease in most patients by several days.33 Even though cytomegalovirus disease might no longer be a viable endpoint in this population because of the number of participants needed to prove effi cacy,9 the relation between initial viral load and risk of cytomegalovirus disease shows the integral role that viraemia plays in pathogenesis.14,34

The decision to initiate cytomegalovirus-specifi c antiviral therapy in the context of viraemia is a strong indicator of clinical relevance for this vaccine, particularly because of the associated toxic eff ects with use of these drugs. However, despite a signifi cant reduction in cytomegalo virus viraemia noted in our study, we were unable to show a reduction in incidence of initiation of antiviral therapy, which we believe was a discrepancy caused by the wide variation in institution-specifi c algorithms as the basis for antiviral treatment decisions and the use of several diff erent local laboratory assays. The selection of a consistent viral load that indicates the need for antiviral therapy is challenging because of the complicated clinical picture, but improvements in assays might make this selection possible in the future. Central laboratory determination of the onset of viraemia

Panel: Research in context

Systematic reviewWe searched PubMed for randomised controlled trials published in English up to Nov 13, 2011, that showed proof of concept with a cytomegalovirus vaccine in recipients of transplants with the search terms “CMV”, “vaccine”, and “transplant” or any plasmid DNA vaccine against any infectious disease target with the search terms “plasmid DNA vaccine” and “randomised controlled phase 2 trial”. We did not identify any randomised controlled trials on cytomegalovirus vaccine in recipients of haemopoietic stem-cell transplants. In 1994, the Towne-strain live-attenuated cytomegalovirus vaccine was studied in cytomegalovirus-seronegative transplant recipients of seropositive renal allografts and signifi cantly reduced severe cytomegalovirus disease without changing the incidence of cytomegalovirus infection.25 We identifi ed one study26 of the glycoprotein B-MF59 vaccine, which was tested in liver and renal transplants. We identifi ed one phase 2 randomised controlled trial27 testing a plasmid DNA vaccine encoding human papillomavirus antigens for effi cacy against cervical dysplasia. The vaccine promoted cervical intraepithelial neoplasia 2/3 lesion resolution in a prospectively defi ned population of young women.

InterpretationThe plasmid DNA vaccine TransVax, which includes both glycoprotein B and phosphoprotein 65, might provide protection for all cytomegalovirus infections in the haemopoietic stem-cell transplantation setting. Our trial is the fi rst proof of concept for a cytomegalovirus vaccine in a haemopoietic stem-cell transplantation setting, the third to show proof of concept in any transplantation setting, and is the second plasmid DNA vaccine to show proof of concept against an infectious disease pathogen.

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combined with the initiation of antiviral therapy in a composite endpoint showed a strong vaccine eff ect and nearly 50% vaccine effi cacy; this could be a practical clinical endpoint in a pre-emptive antiviral setting for the pivotal study. The benefi cial eff ects of TransVax compared to placebo, combined with an acceptable safety profi le, suggest that this vaccine might fi ll the unmet need for an eff ective therapeutic cytomegalovirus vaccine to aid clinical care in haemopoietic stem-cell trans plantation and warrants further investigation in solid organ transplantations.

ContributorsVical staff developed the study design. MAK-D, MB, MBW, AAL, AHC,

MKW, DFG, LRS, APR, and RTK contributed to the clinical

implementation of the study and supervision of the patients. AHC

designed and did the fi nal statistical analysis and verifi ed its accuracy.

AHC, MKW, LRS, APR, and RTK contributed to the planning and

interpretation of exploratory analyses. MKW and LRS did the laboratory

analyses. DFG and RTK monitored the conduct of the trial. MAK-D and

RTK drafted the report. All authors critically revised, reviewed, and

approved the fi nal version of this report.

Confl icts of interestMAK-D has been a member of advisory boards for Vical and has received

speaker’s fees from Genzyme and Bristol-Myers Squibb. MB has been a

consultant or received research funding from Boehringer Ingelheim,

Chimerix, Genentech/Roche, Vical, Novartis, Astellas, Merck,

GlaxoSmithKline, and Theraclone. MBW has received research funding

from Vical and GlaxoSmithKline. AAL has been a member of advisory

boards for Merck, Schering-Plough, and Genzyme and received research

funding from Vical, Pfi zer, Chimerix, and Wyeth. AHC, MKW, DFG,

LRS, APR, and RTK are employees of and own stock in Vical.

AcknowledgmentsThe study was fi nanced in part by the National Institute of Allergy and

Infectious Diseases (grant 5R44AI58386-04) and was otherwise funded

by Vical. We thank the recipients of haemopoietic stem-cell transplants

and the healthy donors who volunteered for the study; the data and

safety monitoring board members D Winston (UCLA Medical Center,

CA, USA), G Schiller (UCLA School of Medicine, CA, USA), J Wingard

(University of Florida College of Medicine, FL, USA), and C Davis

(CSD Biostatistics, CA, USA) for their safety oversight; the former Vical

employees D Kaslow (Merck Research Laboratories, PA, USA) and

T Evans (Aeras, MD, USA) for their contributions to the design and

conduct of the trial; and J Schulte (Vical, CA, USA) for his expert data

analysis. We thank all of the staff at each institution for their skilful care

of participants and careful recording of results, along with the following

investigators for their participation in this trial: C Anasetti (H Lee Moffi tt

Cancer Center, FL, USA), R Betts (University of Rochester Medical

Center, NY, USA), P Chandrasekar (Barbara Ann Karmanos Cancer

Institute, MI, USA), A Freifeld (Nebraska Medical Center, NE, USA),

R Herzig (James Graham Brown Cancer Center, KY, USA), F Marty

(Brigham and Women’s Hospital, MA, USA), P McCarthy (Roswell Park

Cancer Institute, NY, USA), J McGuirk (University of Kansas Cancer

Center, KS, USA), R Nakamura (City of Hope Medical Center, CA,

USA), G Papanicolaou (Memorial Sloan-Kettering Cancer Center, NY,

USA), S Pergam (Fred Hutchinson Cancer Research Center, WA, USA),

K Rados (Winship Cancer Institute, GA, USA), S Rowley (Hackensack

University Medical Center, NJ, USA), E Vance (Baylor University Medical

Center, TX, USA), and A Yeager (Arizona Cancer Center, AZ, USA); and

the staff at the Montefi ore Medical Center (NY, USA).

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