Altered Exosomal RNA Profiles in Bronchoalveolar Lavage from Lung Transplants with Acute Rejection.

RATIONALE: The mechanism by which acute allograft rejection leads to chronic rejection remains poorly understood despite its common occurrence. Exosomes, membrane vesicles released from cells within the lung allograft, contain a diverse array of biomolecules that closely reflect the biologic state of the cell and tissue from which they are released. Exosome transcriptomes may provide a better understanding of the rejection process. Furthermore, biomarkers originating from this transcriptome could provide timely and sensitive detection of acute cellular rejection (AR), reducing the incidence of severe AR and chronic lung allograft dysfunction and improving outcomes. OBJECTIVES: To provide an in-depth analysis of the bronchoalveolar lavage fluid exosomal shuttle RNA population after lung transplantation and evaluate for differential expression between acute AR and quiescence. METHODS: Serial bronchoalveolar lavage specimens were ultracentrifuged to obtain the exosomal pellet for RNA extraction, on which RNA-Seq was performed. MEASUREMENTS AND MAIN RESULTS: AR demonstrates an intense inflammatory environment, skewed toward both innate and adaptive immune responses. Novel, potential upstream regulators identified offer potential therapeutic targets. CONCLUSIONS: Our findings validate bronchoalveolar lavage fluid exosomal shuttle RNA as a source for understanding the pathophysiology of AR and for biomarker discovery in lung transplantation.

Interaction between Pseudomonas and CXC chemokines increases risk of bronchiolitis obliterans syndrome and death in lung transplantation.

RATIONALE: Pseudomonas aeruginosa is the most commonly isolated gram-negative bacterium after lung transplantation and has been shown to up-regulate glutamic acid-leucine-arginine-positive (ELR(+)) CXC chemokines associated with bronchiolitis obliterans syndrome (BOS), but the effect of pseudomonas on BOS and death has not been well defined. OBJECTIVES: To determine if the influence of pseudomonas isolation and ELR(+) CXC chemokines on the subsequent development of BOS and the occurrence of death is time dependent. METHODS: A three-state model was developed to assess the likelihood of transitioning from lung transplant (state 1) to BOS (state 2), from transplant (state 1) to death (state 3), and from BOS (state 2) to death (state 3). This Cox semi-Markovian approach determines state survival rates and cause-specific hazards for movement from one state to another. MEASUREMENTS AND MAIN RESULTS: The likelihood of transition from transplant to BOS was increased by acute rejection, CXCL5, and the interaction between pseudomonas and CXCL1. The pseudomonas effect in this transition was due to infection rather than colonization. Movement from transplant to death was facilitated by pseudomonas infection and single lung transplant. Transition from BOS to death was affected by the length of time in state 1 and by the interactions between any pseudomonas isolation and CXCL5 and aspergillus, either independently or in combination. CONCLUSIONS: Our model demonstrates that common post-transplantation events drive movement from one post-transplantation state to another and influence outcomes differently depending upon when after transplantation they occur. Pseudomonas and the ELR(+) CXC chemokines may interact to negatively influence lung transplant outcomes.

CXCR3 ligands are associated with the continuum of diffuse alveolar damage to chronic lung allograft dysfunction.

RATIONALE: After lung transplantation, insults to the allograft generally result in one of four histopathologic patterns of injury: (1) acute rejection, (2) lymphocytic bronchiolitis, (3) organizing pneumonia, and (4) diffuse alveolar damage (DAD). We hypothesized that DAD, the most severe form of acute lung injury, would lead to the highest risk of chronic lung allograft dysfunction (CLAD) and that a type I immune response would mediate this process. OBJECTIVES: Determine whether DAD is associated with CLAD and explore the potential role of CXCR3/ligand biology. METHODS: Transbronchial biopsies from all lung transplant recipients were reviewed. The association between the four injury patterns and subsequent outcomes were evaluated using proportional hazards models with time-dependent covariates. Bronchoalveolar lavage (BAL) concentrations of the CXCR3 ligands (CXCL9/MIG, CXCL10/IP10, and CXCL11/ITAC) were compared between allograft injury patterns and "healthy" biopsies using linear mixed-effects models. The effect of these chemokine alterations on CLAD risk was assessed using Cox models with serial BAL measurements as time-dependent covariates. MEASUREMENTS AND MAIN RESULTS: There were 1,585 biopsies from 441 recipients with 62 episodes of DAD. An episode of DAD was associated with increased risk of CLAD (hazard ratio, 3.0; 95% confidence interval, 1.9-4.7) and death (hazard ratio, 2.3; 95% confidence interval, 1.7-3.0). There were marked elevations in BAL CXCR3 ligand concentrations during DAD. Furthermore, prolonged elevation of these chemokines in serial BAL fluid measurements predicted the development of CLAD. CONCLUSIONS: DAD is associated with marked increases in the risk of CLAD and death after lung transplantation. This association may be mediated in part by an aberrant type I immune response involving CXCR3/ligands.

Respiratory viral infections in hematopoietic stem cell and solid organ transplant recipients.

Respiratory viral infections (RVIs) are common causes of mild illness in immunocompetent children and adults with rare occurrences of significant morbidity or mortality. Complications are more common in the very young, very old, and those with underlying lung diseases. However, RVIs are increasingly recognized as a cause of morbidity and mortality in recipients of hematopoietic stem cell transplants (HSCT) and solid organ transplants (SOTs). Diagnostic techniques for respiratory syncytial virus (RSV), parainfluenza, influenza, and adenovirus have been clinically available for decades, and these infections are known to cause serious disease in transplant recipients. Modern molecular technology has now made it possible to detect other RVIs including human metapneumovirus, coronavirus, and bocavirus, and the role of these viruses in causing serious disease in transplant recipients is still being worked out. This article reviews the current information regarding epidemiology, pathogenesis, clinical presentation, diagnosis, and treatment of these infections, as well as the aspects of clinical significance of RVIs unique to HSCT or SOT.

CCR4 expression on host T cells is a driver for alloreactive responses and lung rejection.

Despite current immunosuppressive strategies, long-term lung transplant outcomes remain poor due to rapid allogenic responses. Using a stringent mouse model of allo-airway transplantation, we identify the CCR4-ligand axis as a central node driving secondary lymphoid tissue homing and activation of the allogeneic T cells that prevent long-term allograft survival. CCR4 deficiency on transplant recipient T cells diminishes allograft injury and when combined with CTLA4-Ig leads to an unprecedented long-term lung allograft accommodation. Thus, we identify CCR4-ligand interactions as a central mechanism driving allogeneic transplant rejection and suggest it as a potential target to enhance long-term lung transplant survival.

Bronchoalveolar immunologic profile of acute human lung transplant allograft rejection.

Bronchoalveolar lavage fluid (BALF) offers a potential means to diagnose acute rejection and could provide insight into the immune mechanisms responsible for lung allograft rejection. Transbronchial biopsies from 29 bronchoscopic procedures were assessed for rejection. Concurrent BALF lymphocyte subsets were examined by flow cytometry, including CD4 and CD8 T cells and their activation status by CD38 expression, natural killer (NK), NK-like T (NT), B, regulatory T, and invariant receptor NK-T cells. Percentages of CD4 were reduced, and CD8 and activation of CD4 T cells correlated with rejection. There were trends for increased NT, reduced NK, and increased B cell percentages with rejection, suggesting potential roles of these cells. Among regulatory cells, the percentages of regulatory T cells decreased and CD4/CD8 invariant NK-T cells increased during rejection, suggesting a proinflammatory profile. A unique BALF lymphocyte profile was associated with rejection and may provide insight into the pathogenesis of allograft rejection.

A Systematic Review and Meta-Analysis of the Data Behind Current Recommendations for Corticosteroids in Non-HIV-Related PCP: Knowing When You Are on Shaky Foundations.

BACKGROUND: Randomized trials show a mortality benefit to adjunctive corticosteroids for human immunodeficiency virus (HIV)-related Pneumocystis jiroveci pneumonia (HIV-PCP). Guidelines for non-HIV PCP (NH-PCP) recommend adjunctive corticosteroids based on expert opinion. We conducted a systematic review and meta-analysis characterizing adjunctive corticosteroids for NH-PCP. METHODS: We searched MEDLINE from 1966 through 2015. Data on clinical outcomes from NH-PCP were extracted with a standardized instrument. Heterogeneity was assessed with the I2 index. Pooled odds ratios and 95% confidence interval were calculated using a fixed effects model. RESULTS: Our search yielded 5044 abstracts, 277 articles were chosen for full review, and 6 articles described outcomes in moderate to severe NH-PCP. Studies were limited by variable definitions, treatment selection bias, concomitant infections and small sample size. Individual studies reported shorter intensive care unit stay and duration of mechanical ventilation of patients given adjunctive corticosteroids. There was no association between corticosteroids and survival in NH-PCP (odds ratio, 0.66; 95% confidence interval, 0.38-1.15; P = 0.14). CONCLUSIONS: The literature does not support an association between adjunctive corticosteroids and survival from NH-PCP but data are limited and findings should not be considered conclusive. Further research with improved methodology is needed to better understand the role of adjunctive corticosteroids for NH-PCP.

The prognostic importance of CXCR3 chemokine during organizing pneumonia on the risk of chronic lung allograft dysfunction after lung transplantation.

RATIONALE: Since the pathogenesis of chronic lung allograft dysfunction (CLAD) remains poorly defined with no known effective therapies, the identification and study of key events which increase CLAD risk is a critical step towards improving outcomes. We hypothesized that bronchoalveolar lavage fluid (BALF) CXCR3 ligand concentrations would be augmented during organizing pneumonia (OP) and that episodes of OP with marked chemokine elevations would be associated with significantly higher CLAD risk. METHODS: All transbronchial biopsies (TBBX) from patients who received lung transplantation between 2000 to 2010 were reviewed. BALF concentrations of the CXCR3 ligands (CXCL9, CXCL10 and CXCL11) were compared between episodes of OP and "healthy" biopsies using linear mixed-effects models. The association between CXCR3 ligand concentrations during OP and CLAD risk was evaluated using proportional hazards models with time-dependent covariates. RESULTS: There were 1894 bronchoscopies with TBBX evaluated from 441 lung transplant recipients with 169 (9%) episodes of OP and 907 (49%) non-OP histopathologic injuries. 62 (37%) episodes of OP were observed during routine surveillance bronchoscopy. Eight hundred thirty-eight (44%) TBBXs had no histopathology and were classified as "healthy" biopsies. There were marked elevations in BALF CXCR3 ligand concentrations during OP compared with "healthy" biopsies. In multivariable models adjusted for other injury patterns, OP did not significantly increase the risk of CLAD when BAL CXCR3 chemokine concentrations were not taken into account. However, OP with elevated CXCR3 ligands markedly increased CLAD risk in a dose-response manner. An episode of OP with CXCR3 concentrations greater than the 25th, 50th and 75th percentiles had HRs for CLAD of 1.5 (95% CI 1.0-2.3), 1.9 (95% CI 1.2-2.8) and 2.2 (95% CI 1.4-3.4), respectively. CONCLUSIONS: This study identifies OP, a relatively uncommon histopathologic finding after lung transplantation, as a major risk factor for CLAD development when considered in the context of increased allograft expression of interferon-γ inducible ELR- CXC chemokines. We further demonstrate for the first time, the prognostic importance of BALF CXCR3 ligand concentrations during OP on subsequent CLAD risk.

Gene Expression Profiling of Bronchoalveolar Lavage Cells Preceding a Clinical Diagnosis of Chronic Lung Allograft Dysfunction.

BACKGROUND: Chronic Lung Allograft Dysfunction (CLAD) is the main limitation to long-term survival after lung transplantation. Although CLAD is usually not responsive to treatment, earlier identification may improve treatment prospects. METHODS: In a nested case control study, 1-year post transplant surveillance bronchoalveolar lavage (BAL) fluid samples were obtained from incipient CLAD (n = 9) and CLAD free (n = 8) lung transplant recipients. Incipient CLAD cases were diagnosed with CLAD within 2 years, while controls were free from CLAD for at least 4 years following bronchoscopy. Transcription profiles in the BAL cell pellets were assayed with the HG-U133 Plus 2.0 microarray (Affymetrix). Differential gene expression analysis, based on an absolute fold change (incipient CLAD vs no CLAD) >2.0 and an unadjusted p-value ≤0.05, generated a candidate list containing 55 differentially expressed probe sets (51 up-regulated, 4 down-regulated). RESULTS: The cell pellets in incipient CLAD cases were skewed toward immune response pathways, dominated by genes related to recruitment, retention, activation and proliferation of cytotoxic lymphocytes (CD8+ T-cells and natural killer cells). Both hierarchical clustering and a supervised machine learning tool were able to correctly categorize most samples (82.3% and 94.1% respectively) into incipient CLAD and CLAD-free categories. CONCLUSIONS: These findings suggest that a pathobiology, similar to AR, precedes a clinical diagnosis of CLAD. A larger prospective investigation of the BAL cell pellet transcriptome as a biomarker for CLAD risk stratification is warranted.

Infectious Triggers of Chronic Lung Allograft Dysfunction.

Survival after lung transplantation is limited in large part due to the high incidence of chronic rejection, known as chronic lung allograft dysfunction (CLAD). Pulmonary infections are a frequent complication in lung transplant recipients, due both to immunosuppressive medications and constant exposure of the lung allograft to the external environment via the airways. Infection is a recognized risk factor for the development of CLAD, and both acute infection and chronic lung allograft colonization with microorganisms increase the risk for CLAD. Acute infection by community acquired respiratory viruses, and the bacteria Pseudomonas aeruginosa and Staphylococcus aureus are increasingly recognized as important risk factors for CLAD. Colonization by the fungus Aspergillus may also augment the risk of CLAD. Fostering this transition from healthy lung to CLAD in each of these infectious episodes is the persistence of an inflammatory lung allograft environment.

Graft Loss and CLAD-Onset Is Hastened by Viral Pneumonia After Lung Transplantation.

BACKGROUND: Community-acquired respiratory virus (CARV) infections occur frequently after lung transplantation and may adversely impact outcomes. We hypothesized that while asymptomatic carriage would not increase the risk of chronic lung allograft dysfunction (CLAD) and graft loss, severe infection would. METHODS: All lung transplant cases between January 2000 and July 2013 performed at our center were reviewed for respiratory viral samples. Each isolation of virus was classified according to clinical level of severity: asymptomatic, symptomatic without pneumonia, and viral pneumonia. Multivariate Cox modeling was used to assess the impact of CARV isolation on progression to CLAD and graft loss. RESULTS: Four thousand four hundred eight specimens were collected from 563 total patients, with 139 patients producing 324 virus-positive specimens in 245 episodes of CARV infection. Overall, the risk of CLAD was elevated by viral infection (hazard ratio [HR], 1.64; P < 0.01). This risk, however, was due to viral pneumonia alone (HR, 3.94; P < 0.01), without significant impact from symptomatic viral infection (HR, 0.97; P = 0.94) nor from asymptomatic viral infection (HR, 0.99; P = 0.98). The risk of graft loss was not increased by asymptomatic CARV infection (HR, 0.74; P = 0.37) nor symptomatic CARV infection (HR, 1.39; P = 0.41). Viral pneumonia did, however, significantly increase the risk of graft loss (HR, 2.78; P < 0.01). CONCLUSIONS: With respect to CARV, only viral pneumonia increased the risk of both CLAD and graft loss after lung transplantation. In the absence of pneumonia, respiratory viruses had no impact on measured outcomes.

Does antigenic overload exist? The role of multiple immunizations in infants.

There is no evidence that currently recommended vaccines overload or weaken the infant immune system. Infants have an enormous capacity to respond safely and effectively to multiple vaccines. The schedule for the administration of childhood vaccines is tailored to the unique developmental pattern of the infant immune system. Childhood vaccines provide immediate protection from common childhood illness and establish the foundation for lifelong immunity that develops with subsequent vaccination or infection. Widespread vaccination of infants and children represents a public health triumph of the 20th century. This fact must be reinforced continually by health care workers and parent education to help maintain progress in the 21st century.

Usefulness of immune monitoring in lung transplantation using adenosine triphosphate production in activated lymphocytes.

BACKGROUND: The ImmuKnow (Cylex Inc, Columbia, MD) assay measures the amount of adenosine triphosphate (ATP) produced by helper CD4(+) cells after stimulation with a T-cell mitogen. We hypothesized that this assay can be used to assess the immune function of lung transplant recipients and identify those at risk of developing acute cellular rejection and respiratory infection. METHODS: Lung transplant recipients at University of California Los Angeles between January 1, 2006 and December 31, 2009 received a bronchoscopy with broncheoalveolar lavage, transbronchial biopsy and ImmuKnow values drawn at regular intervals as well as during episodes of clinical deterioration. The recipient's clinical condition at each time-point was classified as healthy, acute cellular rejection, or respiratory infection. Mixed-effects models were used to compare the ATP levels among these groups, and odds ratios for rejection and infection were calculated. RESULTS: The mean ATP level was 431 ± 189 ng/ml for the rejection group vs 377 ± 187 ng/ml for the healthy group (p = 0.10). A recipient with an ATP level > 525 ng/ml was 2.1 times more likely to have acute cellular rejection (95% confidence interval [CI] 1.1-3.8). Similarly, the mean ATP level was 323 ± 169 ng/ml for the infection group vs 377 ± 187 ng/ml for the healthy group (p = 0.03). A recipient with an ATP level < 225 ng/ml was 1.9 times more likely to have respiratory infection (95% CI, 1.1-3.3). However, the test was associated with poor performance characteristics. It had low sensitivity, specificity with an area under the receiver operating characteristic curve of only 0.61 to diagnose rejection and 0.59 to diagnose infection. CONCLUSIONS: The ImmuKnow assay appears to have some ability to assess the overall immune function of lung transplant recipients. However, this study does not support its use as a reliable predictor of episodes of acute cellular rejection or respiratory infection.

Protection against bronchiolitis obliterans syndrome is associated with allograft CCR7+ CD45RA- T regulatory cells.

Bronchiolitis obliterans syndrome (BOS) is the major obstacle to long-term survival after lung transplantation, yet markers for early detection and intervention are currently lacking. Given the role of regulatory T cells (Treg) in modulation of immunity, we hypothesized that frequencies of Treg in bronchoalveolar lavage fluid (BALF) after lung transplantation would predict subsequent development of BOS. Seventy BALF specimens obtained from 47 lung transplant recipients were analyzed for Treg lymphocyte subsets by flow cytometry, in parallel with ELISA measurements of chemokines. Allograft biopsy tissue was stained for chemokines of interest. Treg were essentially all CD45RA(-), and total Treg frequency did not correlate to BOS outcome. The majority of Treg were CCR4(+) and CD103(-) and neither of these subsets correlated to risk for BOS. In contrast, higher percentages of CCR7(+) Treg correlated to reduced risk of BOS. Additionally, the CCR7 ligand CCL21 correlated with CCR7(+) Treg frequency and inversely with BOS. Higher frequencies of CCR7(+) CD3(+)CD4(+)CD25(hi)Foxp3(+)CD45RA(-) lymphocytes in lung allografts is associated with protection against subsequent development of BOS, suggesting that this subset of putative Treg may down-modulate alloimmunity. CCL21 may be pivotal for the recruitment of this distinct subset to the lung allograft and thereby decrease the risk for chronic rejection.

Quantification of cytokeratin 5 mRNA expression in the circulation of healthy human subjects and after lung transplantation.

BACKGROUND: Circulating epithelial progenitor cells are important for repair of the airway epithelium in a mouse model of tracheal transplantation. We therefore hypothesized that circulating epithelial progenitor cells would also be present in normal human subjects and could be important for repair of the airway after lung injury. As lung transplantation is associated with lung injury, which is severe early on and exacerbated during episodes of infection and rejection, we hypothesized that circulating epithelial progenitor cell levels could predict clinical outcome following lung transplantation. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative Real Time PCR was performed to determine peripheral blood mRNA levels of cytokeratin 5, a previously characterized marker of circulating epithelial progenitor cells. Cytokeratin 5 levels were evaluated in healthy human subjects, in lung transplant recipients immediately post-transplant and serially thereafter, and in heart transplant recipients. All normal human subjects examined expressed cytokeratin 5 in their buffy coat in amounts that were not significantly influenced by age or gender. There was a profound, statistically significant decrease in cytokeratin 5 mRNA expression levels in lung transplant patients compared to healthy human subjects (p = 3.1x10(-13)) and to heart transplant recipients. There was a moderate negative correlation between improved circulating cytokeratin 5 mRNA levels in lung transplant recipients with recovering lung function, as measured by improved FEV1 values (rho = -0.39). CONCLUSIONS/SIGNIFICANCE: Levels of cytokeratin 5 mRNA, a proxy marker for circulating epithelial progenitor cells, inversely correlated with disease status in lung transplant recipients. It may therefore serve as a biomarker of the clinical outcome of lung transplant patients and potentially other patients with airway injury.

Use of hearts transplanted from donors with severe sepsis and infectious deaths.

BACKGROUND: The reluctance to use organs from donors who have died from severe infections is based on the potential transmission of an infectious agent to the recipient and on the uncertainty about allograft function in the setting of severe donor sepsis. METHODS: From 1999 to 2007, donor hospital records were reviewed which focused on microbiology cultures and sensitivity results; type and duration of antimicrobial therapy; hemodynamic data, results of echocardiogram, and imaging studies. Preliminary positive and negative results from pre-harvest blood, respiratory, urine, and cerebrospinal fluid cultures were verified with the procurement agency. The harvesting surgeon performed gross inspection of donor valvular structures. RESULTS: Nine donor hearts were transplanted from patients who expired from community onset infections with severe septic shock, meningitis, and/or pneumonia caused by Streptococcus pneumoniae (n = 4), Streptococcus milleri (n = 2), Neisseria meningitidis (n = 2), and unidentified gram- positive cocci (n = 1). Four donors had probable infection-induced intracranial hemorrhage, and all donors were vasopressor-dependent before organ procurement. No evidence of donor-transmitted infection, sepsis, or rejection was observed, and long-term function remained excellent; allograft dysfunction in three patients resolved after transplant. Our series of nine donors represents approximately 1.3% of successfully transplanted cardiac allografts during the respective period of review. CONCLUSIONS: Patients succumbing to severe infections (meningitis, pneumonia, and septic shock) should not be arbitrarily excluded for possible heart donation. Assessing the suitability of donors with severe infections requires flawless communication between the donor and transplant facility, including a comprehensive evaluation of donor infection and pathogen(s), severity of sepsis, adequacy of antimicrobial treatment, and the degree of sepsis-induced myocardial dysfunction.

Phase I trial of an alhydrogel adjuvanted hepatitis B core virus-like particle containing epitopes of Plasmodium falciparum circumsporozoite protein.

UNLABELLED: The objectives of this non-randomized, non-blinded, dose-escalating Phase I clinical trial were to assess the safety, reactogenicity and immunogenicity of ICC-1132 formulated with Alhydrogel (aluminum hydroxide) in 51 healthy, malaria-naive adults aged 18 to 45 years. ICC-1132 (Malariavax) is a recombinant, virus-like particle malaria vaccine comprised of hepatitis core antigen engineered to express the central repeat regions from Plasmodium falciparum circumsporozoite protein containing an immunodominant B [(NANP)(3)] epitope, an HLA-restricted CD4 (NANPNVDPNANP) epitope and a universal T cell epitope (T*) (amino acids 326-345, NF54 isolate). We assessed an Alhydrogel (aluminum hydroxide)-adjuvanted vaccine formulation at three ICC-1132 dose levels, each injected intramuscularly (1.0 mL) on study days 0, 56 and 168. A saline vaccine formulation was found to be unstable after prolonged storage and this formulation was subsequently removed from the study. Thirty-two volunteers were followed for one year. Local and systemic adverse clinical events were measured and immune responses to P. falciparum and hepatitis B virus core antigens were determined utilizing the following assays: IgG and IgM ELISA, indirect immunofluorescence against P. falciparum sporozoites, circumsporozoite precipitin (CSP) and transgenic sporozoite neutralization assays. Cellular responses were measured by proliferation and IL-2 assays. Local and systemic reactions were similarly mild and well tolerated between dose cohorts. Depending on the ICC-1132 vaccine concentration, 95 to 100% of volunteers developed antibody responses to the ICC-1132 immunogen and HBc after two injections; however, only 29-75% and 29-63% of volunteers, respectively, developed malaria-specific responses measured by the malaria repeat synthetic peptide ELISA and IFA; 2 of 8 volunteers had positive reactions in the CSP assay. Maximal transgenic sporozoite neutralization assay inhibition was 54%. Forty-seven to seventy-five percent demonstrated T cell proliferation in response to ICC-1132 or to recombinant circumsporozoite protein (rCS) NF-54 isolate. This candidate malaria vaccine was well tolerated, but the vaccine formulation was poorly immunogenic. The vaccine may benefit from a more powerful adjuvant to improve immunogenicity. TRIAL REGISTRATION: ClinicalTrials.gov NCT00587249.