[Show abstract][Hide abstract] ABSTRACT: Background
M. tuberculosis remains one of the world’s deadliest pathogens in part because of its ability to establish persistent, latent infections, which can later reactivate to cause disease. In regions of the globe where disease is endemic, as much as 50% of the population is thought to be latently infected, complicating diagnosis and tuberculosis control. The tools most commonly used for diagnosis of latent M. tuberculosis infection are the tuberculin skin test and the newer interferon-gamma release assays, both of which rely on an antigen-specific memory response as an indicator of infection. It is clear that the two tests, do not always give concordant results, but the factors leading to this are only partially understood.
In this study we examined 245 healthy school children aged from 12 to 20 years from Addis Ababa, a tuberculosis-endemic region, characterised them with regard to response in the tuberculin skin test and QuantIFERON™ test and assessed factors that might contribute to discordant responses.
Although concordance between the tests was generally fair (90% concordance), there was a subset of children who had a positive QuantIFERON™ result but a negative tuberculin skin test. After analysis of multiple parameters the data suggest that discordance was most strongly associated with the presence of parasites in the stool.
Parasitic gut infections are frequent in most regions where M. tuberculosis is endemic. This study, while preliminary, suggests that the tuberculin skin test should be interpreted with caution where this may be the case.
Full-text · Article · Jun 2013 · BMC Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Background
Despite major public health initiatives and the existence of efficacious treatment regimes, tuberculosis (TB) remains a threat, particularly in resource-limited settings. A significant part of the problem is the difficulty of rapidly identifying infected individuals, and as a result, there has been renewed interest in developing better diagnostics for infection or disease caused by Mycobacterium tuberculosis. Many of the existing tools, however, have limitations such as poor sensitivity or specificity, or the need for well-equipped laboratories to function effectively. Serodiagnostic approaches in particular have long drawn attention, due to their potential utility in large field studies, particularly in resource-poor settings. Unfortunately none of the serodiagnostic approaches have so far proven useful under field conditions.
We screened a large panel of antigens with serodiagnostic potential by ELISA and selected a subpanel that was strongly and broadly recognised by TB patients, but not by controls. These antigens were then formulated into a simple immuno-chromatographic lateral flow assay format, suitable for field use, and tested against panels of plasma and blood samples from individuals with different clinical status (confirmed TB patients, household contacts, and apparently healthy community controls), recruited from Ethiopia (a highly TB-endemic country) and Turkey (a TB meso-endemic country). While specificity was good (97-100% in non TB-endemic controls), the sensitivity was not as high as expected (46-54% in pulmonary TB, 25-29% in extra-pulmonary TB).
Though below the level of sensitivity the consortium had set for commercial development, the assay specifically identified M. tuberculosis-infected individuals, and provides a valuable proof of concept.
Full-text · Article · May 2013 · BMC Research Notes
[Show abstract][Hide abstract] ABSTRACT: Induction of broad T-cell immune responses is regarded as critical for vaccines against the human immunodeficiency virus type 1 (HIV-1) which exhibit high diversity and, therefore, focus has been on inducing cytotoxic CD8 T-cell responses against the more conserved parts of the virus, such as the Gag protein. Herein, we have used the p24 protein which contains a range of conserved T-cell epitopes. We demonstrate that a vaccine of HIV-1 subtype B consensus group-specific antigen (Gag) p24 protein with the CD8-inducing liposomal cationic adjuvant formulation (CAF) 05, induces both CD4 and CD8 T-cell responses in CB6F1 mice. The adjuvanted vaccine also induced functional antigen-specific cytotoxicity in vivo. Furthermore, we found that when fragmenting the Gag p24 protein into overlapping Gag p24 peptides, a broader T-cell epitope specificity was induced in the humanized human leukocyte antigen (HLA)-A2/DR-transgenic mouse model. Thus, combining overlapping Gag p24 peptides with CAF05 appears to be a promising and simple strategy for inducing broader T-cell responses to multiple conserved epitopes which will be relevant for both prophylactic and therapeutic HIV-1 vaccines.
[Show abstract][Hide abstract] ABSTRACT: The bacille Calmette-Guérin vaccine provides very efficient protection in standard animal models of Mycobacterium tuberculosis challenge. We show in this article that although bacille Calmette-Guérin controlled M. tuberculosis growth for 7 wk of infection, the protection was gradually lost as the infection entered the chronic phase. The regrowth of M. tuberculosis coincided with an almost complete disappearance of IL-2-producing CD4 T cells. Booster vaccination with a subunit vaccine (Ag85B-ESAT-6+CAF01) expanded IL-2(+) CD4(+) T cell coexpressing either TNF-α or TNF-α/IFN-γ, and the maintenance of this population in the late stage of infection was associated with enhanced control of bacterial growth. The IL-2(+) CD4(+) T cell subsets were KLRG1(-) (nonterminally differentiated), were found to be CD62L(high), and further maintained a pronounced proliferative and cytokine-producing potential in the draining lymph nodes, when the animals were challenged 2 y postvaccination. These results suggest that the CD4(+) KLRG1(-) IL-2-secreting subsets are central memory T cells with the potential to continuously replenish the T cells at the site of infection and prevent attrition and functional exhaustion.
Full-text · Article · May 2013 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Tuberculin skin testing is simple and relatively inexpensive, but the specificity of PPD is affected by BCG vaccination.
Determine optimal dose and specificity of recombinant ESAT-6 and CFP-10 (C-Tb) produced in Lactococcus lactis for diagnosis of M. tuberculosis infection.
In a dose finding phase I trial 0.01 or 0.1 µg preserved and unpreserved C-Tb was injected by Mantoux technique in 38 patients with active tuberculosis and induration responses measured. In a phase II specificity trial in 151 uninfected, BCG vaccinated participants 0.1 µg C-Tb was compared to 2 TU PPD.
0.1 µg C-Tb gave a median induration of 15 mm after 2 days. Phenol preservation did not affect the response. The specificity of C-Tb was 99.3% (95% CI 96-100%) regarding indurations ≥5 mm as a positive outcome. This was higher than the specificity of PPD (63% using a cut-off of 5 mm or 92% using a cut-off of 15 mm to adjust for non-specific BCG responses). Local adverse reactions following C-Tb injection included transient itching and discomfort as expected components of the immune response.
C-Tb offers a simple and convenient skin test to diagnose M. tuberculosis infection using a single, universal cut-off unaffected by BCG vaccination.
ClinicalTrials.gov NCT01033929 and NCT01241188.
[Show abstract][Hide abstract] ABSTRACT: We have designed a therapeutic HIV-1 vaccine concept based on peptides together with the adjuvant CAF01. Peptides represented 15 HLA-supertype-restricted subdominant and conserved CD8 T-cell epitopes and 3 CD4 T-helper cell epitopes. In this phase I clinical trial, safety and immunogenicity were assessed in untreated HIV-1 infected individuals in Guinea-Bissau, West Africa. 23 HIV-1-infected individuals were randomized to receive placebo (n=5) or vaccine (n=18). Safety was appraised by clinical follow-up combined with monitoring of biochemistry, haematology, CD4 T-cell counts and HIV-1 viral loads. T-cell immunogenicity was monitored longitudinally by IFNγ ELISPOT. New vaccine-specific T-cell responses were induced in 6/14 vaccinees for whom ELISPOT data were valid. CD4 T-cell counts and viral loads were stable. The study shows that therapeutic immunization is feasible and safe in Guinea-Bissau and that it is possible to redirect T-cell immunity with CAF01-adjuvanted HIV-1 peptide vaccine during untreated HIV-1 infection in some patients. However, relatively few pre-existing and vaccine-induced HIV-1 T-cell responses to CD8 T cell epitopes were detected against HIV-1 using IFNγ ELISPOT in this chronically infected African population.
No preview · Article · May 2013 · AIDS research and human retroviruses
[Show abstract][Hide abstract] ABSTRACT: Dry powder vaccine formulations are highly attractive due to improved storage stability and the possibility for particle engineering, as compared to liquid formulations. However, a prerequisite for formulating vaccines into dry formulations is that their physicochemical and adjuvant properties remain unchanged upon rehydration. Thus, we have identified and optimized the parameters of importance for the design of a spray dried powder formulation of the cationic liposomal adjuvant formulation 01 (CAF01) composed of dimethyldioctadecylammonium (DDA) bromide and trehalose 6,6'-dibehenate (TDB) via spray drying. The optimal excipient to stabilize CAF01 during spray drying and for the design of nanocomposite microparticles was identified among mannitol, lactose and trehalose. Trehalose and lactose were promising stabilizers with respect to preserving liposome size, as compared to mannitol. Trehalose and lactose were in the glassy state upon co-spray drying with the liposomes, whereas mannitol appeared crystalline, suggesting that the ability of the stabilizer to form a glassy matrix around the liposomes is one of the prerequisites for stabilization. Systematic studies on the effect of process parameters suggested that a fast drying rate is essential to avoid phase separation and lipid accumulation at the surface of the microparticles during spray drying. Finally, immunization studies in mice with CAF01 in combination with the tuberculosis antigen Ag85B-ESAT6-Rv2660c (H56) demonstrated that spray drying of CAF01 with trehalose under optimal processing conditions resulted in the preservation of the adjuvant activity in vivo. These data demonstrate the importance of liposome stabilization via optimization of formulation and processing conditions in the engineering of dry powder liposome formulations.
Full-text · Article · Feb 2013 · Journal of Controlled Release
[Show abstract][Hide abstract] ABSTRACT: Neonates and juvenile ruminants are very susceptible to paratuberculosis infection. This is likely due to a high degree of exposure from their dams and an immature immune system. To test the influence of age on vaccine induced responses a cocktail of recombinant MAP proteins (MAP0217, MAP1508, MAP3701c, MAP3783 and MAP1609c/Ag85B) was formulated in a cationic liposome adjuvant (CAF01) and used to vaccinate animals of different age. Male jersey calves were divided into 3 groups that were vaccinated at 2, 8 or 16 weeks of age and boosted twice at week 4 and 12 relative to the first vaccination. Vaccine induced immune responses, the IFN-γ cytokine secretion and antibody responses were followed for 20 weeks. In general, the specific responses were significantly elevated in all three vaccination groups after the first booster vaccination with no or only minor effect of the second booster. However, significant differences were observed in the immunogenicity of the different proteins and it appears that the older age group produced a more consistent IFN-γ response. In contrast, the humoral immune response is seemingly independent of the vaccination age as we found no difference in the IgG1 responses when comparing the three vaccination groups. Combined, our results suggest that appropriate age of vaccination should be considered in vaccination protocols and there is a possible interference of vaccine induced immune responses with weaning (week 8).
Full-text · Article · Feb 2013 · Clinical and vaccine Immunology: CVI
[Show abstract][Hide abstract] ABSTRACT: This present study compares the efficacy of microsphere formulations, and their method of antigen presentation, for the delivery of the TB sub-unit vaccine antigen, Ag85B-ESAT-6. Microspheres based on poly(lactide-co-glycolide) (PLGA) and chitosan incorporating dimethyldioctadecylammonium bromide (DDA) were prepared by either the w/o/w double emulsion method (entrapped antigen) or the o/w single emulsion method (surface bound antigen), and characterised for their physico-chemical properties and their ability to promote an immune response to Ag85B-ESAT-6. The method of preparation, and hence method of antigen association, had a pronounced effect on the type of immune response achieved from the microsphere formulations, with surface bound antigen favouring a humoural response, whereas entrapped antigen favoured a cellular response.
No preview · Article · Feb 2013 · Current Drug Delivery
[Show abstract][Hide abstract] ABSTRACT: To successfully enter the latent stage, Mycobacterium tuberculosis must adapt to conditions such as nutrient limitation and hypoxia. In vitro models which mimic latent infection are valuable tools to describe the changes in metabolism which occur when the bacterium exists in a non-growing form. We used two complementary proteomic approaches, label-free LC-MS/MS analysis and two-dimensional DIGE, to determine the proteome profile of extracellular proteins from M. tuberculosis cultured under nutrient starvation. By label-free LC-MS/MS analysis of fractionated samples, 1176 proteins were identified from culture filtrates of log phase and nutrient starved cultures, and the protein levels of 230 proteins were increased in nutrient starved culture filtrates, while 208 were decreased. By Gene Ontology clustering analysis, significant differences in the overall metabolism during nutrient starvation were detected. Notably, members of the toxin-antitoxin systems were present in larger quantities in nutrient starved cultures supporting a role for these global modules as M. tuberculosis switches its metabolism into dormancy. Besides, decreased abundance of proteins involved in amino acid and protein synthesis was apparent as well as changes in the lipid metabolism. Further analysis of the dataset identified increased abundance of lipoproteins and decreased abundance of ESAT-6 family proteins. Results from the two-dimensional DIGE based proteomics demonstrated an overall agreement with the LC-MS/MS data and added complementary insights about protein degradation and modification.
[Show abstract][Hide abstract] ABSTRACT: Identification of new vaccine adjuvants with immunopotentiating properties commonly involves in vitro evaluations of candidate compounds for their ability to stimulate cells of the immune system. Subsequent elaborate experiments are then performed on only the positive candidates. Here we show how this strategy may miss good candidates due to context-dependent supramolecular characteristics of the candidate compounds, since both a specific molecular structure and the correct presentation of specific parts of the compounds are required for successful stimulation of the cells. Nevertheless, the supramolecular structure is rarely evaluated although changes in this structure may have a drastic impact on the presentation of the compounds to the cells. Synthetic analogues of the mycobacterial cell wall lipid monomycoloyl glycerol (MMG) possess immunopotentiating properties, but their biophysical characteristics are largely unresolved and the structural features determining their immunoactivating properties have been poorly explored. In the present study, we demonstrate that the immunostimulatory activity in vitro correlates with the supramolecular characteristics of the self-assembled MMG nanostructures. Thus, a series of MMG analogues displaying different stereochemistry in the hydrophobic moiety and the polar headgroup were designed and synthesized with different alkyl chain lengths. Stimulation of human monocyte-derived dendritic cells in vitro was clearly dependent on the stereochemistry of the hydrophobic part and on the alkyl chain length but not on the stereochemistry of the hydrophilic glycerol moiety. Small-angle X-ray scattering (SAXS) analysis showed that the immunoactivating analogues self-assembled into lamellar phases whereas the biologically inert analogues adopted inverse hexagonal phases. Langmuir monolayers confirmed that analogues with opposite lipid acid configurations displayed different packing modes. These data demonstrate that the biophysical properties and the lipid molecular structure are major determinants for the ability of the MMG analogues to activate antigen-presenting cells. Our findings emphasize the importance of investigating the biophysical and structural properties when assessing the effect of adjuvants in vitro.
[Show abstract][Hide abstract] ABSTRACT: We investigated the potential of inducing additional T-cell immunity during chronic HIV-1 infection directed to subdominant HIV-1 epitopes from common HLA-supertypes. Ten treatment-naïve HIV-1-infected individuals were immunized with peptides in the adjuvant CAF01. One individual received placebo. T-cell immunogenicity was examined longitudinally by a flow cytometry (CD107a, IFNγ, TNFα, IL-2 and/or MIP1β expression) as well as IFNγ ELISPOT. Safety was evaluated by clinical follow up combined with monitoring of biochemistry, hematology, CD4 T-cell counts and viral load. New CD4 and CD8 T-cell responses specific for one or more vaccine epitopes were induced in 10/10 vaccinees. The responses were dominated by CD107a and MIP1β expression. There were no significant changes in HIV-1 viral load or CD4 T-cell counts. Our study demonstrates that the peptide/CAF01 vaccine is safe and that it is possible to generate new HIV-1 T-cell responses to defined epitopes in treatment-naïve HIV-1-infected individuals.
No preview · Article · Dec 2012 · Clinical Immunology
[Show abstract][Hide abstract] ABSTRACT: There is a need for reliable markers to diagnose active and latent tuberculosis (TB). The interferon gamma release assays (IGRAs) are compared to the tuberculin skin test (TST) more specific, but cannot discriminate between recent or remote TB infection. Here the Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA), which quantifies expanded T-lymphoblasts by flow-cytometric analysis after long-term antigen stimulation of whole blood, is combined with cytokine/chemokine analysis in the supernatant by multiplex technology for diagnosis of Mycobacterium tuberculosis (Mtb) infection.
[Show abstract][Hide abstract] ABSTRACT: The ectodomain of the matrix 2 protein (M2e) of influenza A virus represents an attractive target for developing a universal influenza A vaccine, with its sequence being highly conserved amongst human variants of this virus. With the aim of targeting conformational epitopes presumably shared by diverse influenza A viruses, a vaccine (M2e-NSP4) was constructed linking M2e (in its consensus sequence) to the rotavirus fragment NSP4(98-135); due to its coiled-coil region this fragment is known to form tetramers in aqueous solution and in this manner we hoped to mimick the natural configuration of M2e as presented in membranes. M2e-NSP4 was then evaluated side-by-side with synthetic M2e peptide for its immunogenicity and protective efficacy in a murine influenza challenge model. Here we demonstrate that M2e fused to the tetramerizing protein induces an accelerated, augmented and more broadly reactive antibody response than does M2e peptide as measured in two different assays. Most importantly, vaccination with M2e-NSP4 caused a significant decrease in lung virus load early after challenge with influenza A virus and maintained its efficacy against a lethal challenge even at very low vaccine doses. Based on the results presented in this study M2e-NSP4 merits further investigation as a candidate for or as a component of a universal influenza A vaccine.
[Show abstract][Hide abstract] ABSTRACT: Th17 cells are increasingly being recognized as an important T helper subset for immune-mediated protection, especially against pathogens at mucosal ports of entry. In several cases, it would thus be highly relevant to induce Th17 memory by vaccination. Th17 cells are reported to exhibit high plasticity and may not stably maintain their differentiation program once induced, questioning the possibility of inducing durable Th17 memory. Accordingly, there is no consensus as to whether Th17 memory can be established unless influenced by continuous Th17 polarizing conditions. We have previously reported (T. Lindenstrøm, et al., J. Immunol. 182:8047-8055, 2009) that the cationic liposome adjuvant CAF01 can prime both Th1 and Th17 responses and promote robust, long-lived Th1 memory. Here, we demonstrate that subunit vaccination in mice with CAF01 leads to establishment of bona fide Th17 memory cells. Accordingly, Th17 memory cells exhibited lineage stability by retaining both phenotypic and functional properties for nearly 2 years. Antigen-specific, long-term Th17 memory cells were found to be mobilized from lung-draining lymph nodes to the lung following an aerosol challenge by Mycobacterium tuberculosis nearly 2 years after their induction and proliferated at levels comparable to those of Th1 memory cells. During the infection, the vaccine-induced Th17 memory cells expanded in the lungs and adapted Th1 characteristics, implying that they represent a metastable population which exhibits plasticity when exposed to prolonged Th1 polarizing, inflammatory conditions such as those found in the M. tuberculosis-infected lung. In the absence of overt inflammation, however, stable bona fide Th17 memory can indeed be induced by parenteral immunization.
Full-text · Article · Jul 2012 · Infection and immunity
[Show abstract][Hide abstract] ABSTRACT: Adjuvants are often composed of different constituents that can be divided into two groups based on their primary activity: the delivery system which carries and presents the vaccine antigen to antigen-presenting cells, and the immunostimulator that activates and modulates the ensuing immune response. Herein, we have investigated the importance of the delivery system and in particular its physical characteristics by comparing the delivery properties of two lipids which differ only in the degree of saturation of the acyl chains, rendering the liposomes either rigid (DDA, dimethyldioctadecylammonium) or highly fluid (DODA, dimethyldioleoylammonium) at physiological temperature. We show that these delivery systems are remarkably different in their ability to prime a Th1-directed immune response with the rigid DDA-based liposomes inducing a response more than 100 times higher compared to that obtained with the fluid DODA-based liposomes. Upon injection with a vaccine antigen, DDA-based liposomes form a vaccine depot that results in a continuous attraction of antigen-presenting cells that engulf a high amount of adjuvant and are subsequently efficiently activated as measured by an elevated expression of the co-stimulatory molecules CD40 and CD86. In contrast, the fluid DODA-based liposomes are more rapidly removed from the site of injection resulting in a lower up-regulation of co-stimulatory CD40 and CD86 molecules on adjuvant-positive antigen-presenting cells. Additionally, the vaccine antigen is readily dissociated from the DODA-based liposomes leading to a population of antigen-presenting cells that are antigen-positive but adjuvant-negative and consequently are not activated. These studies demonstrate the importance of studying in vivo characteristics of the vaccine components and furthermore show that physicochemical properties of the delivery system have a major impact on the vaccine-induced immune response.
Full-text · Article · Jun 2012 · Journal of Controlled Release
[Show abstract][Hide abstract] ABSTRACT: Background
The self-sufficient autotransporter (AT) pathway, ubiquitous in Gram-negative bacteria, combines a relatively simple protein secretion mechanism with a high transport capacity. ATs consist of a secreted passenger domain and a β-domain that facilitates transfer of the passenger across the cell-envelope. They have a great potential for the extracellular expression of recombinant proteins but their exploitation has suffered from the limited structural knowledge of carrier ATs. Capitalizing on its crystal structure, we have engineered the Escherichia coli AT Hemoglobin protease (Hbp) into a platform for the secretion and surface display of heterologous proteins, using the Mycobacterium tuberculosis vaccine target ESAT6 as a model protein.
Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a β-helical core structure (β-stem) was left intact. The resulting Hbp-ESAT6 chimeras were efficiently and stably secreted into the culture medium of E. coli. On the other hand, Hbp-ESAT6 fusions containing a truncated β-stem appeared unstable after translocation, demonstrating the importance of an intact β-stem. By interrupting the cleavage site between passenger and β-domain, Hbp-ESAT6 display variants were constructed that remain cell associated and facilitate efficient surface exposure of ESAT6 as judged by proteinase K accessibility and whole cell immuno-EM analysis. Upon replacement of the passenger side domain of an alternative AT, EspC, ESAT6 was also efficiently secreted, showing the approach is more generally applicable to ATs. Furthermore, Hbp-ESAT6 was efficiently displayed in an attenuated Salmonella typhimurium strain upon chromosomal integration of a single encoding gene copy, demonstrating the potential of the Hbp platform for live vaccine development.
We developed the first structurally informed AT platform for efficient secretion and surface display of heterologous proteins. The platform has potential with regard to the development of recombinant live vaccines and may be useful for other biotechnological applications that require high-level secretion or display of recombinant proteins by bacteria.
Full-text · Article · Jun 2012 · Microbial Cell Factories
[Show abstract][Hide abstract] ABSTRACT: Molecular genotyping of Mycobacterium tuberculosis has proved to be a powerful tool in tuberculosis surveillance, epidemiology, and control. Based on results obtained through 15 years of nationwide IS6110 restriction fragment length polymorphism (RFLP) genotyping of M. tuberculosis cases in Denmark, a country on the way toward tuberculosis elimination, we discuss M. tuberculosis transmission dynamics and point to areas for control interventions. Cases with 100% identical genotypes (RFLP patterns) were defined as clustered, and a cluster was defined as cases with an identical genotype. Of 4,601 included cases, corresponding to 76% of reported and 97% of culture-verified tuberculosis cases in the country, 56% were clustered, of which 69% were Danes. Generally, Danes were more often in large clusters (≥ 50 persons), older (mean age, 45 years), and male (male/female ratio, 2.5). Also, Danes had a higher cluster frequency within a 2-year observation window (60.8%), and higher clustering rate of new patterns over time, compared to immigrants. A dominant genotype, cluster 2, constituted 44% of all clustered and 35% of all genotyped cases. This cluster was primarily found among Danish males, 30 to 59 years of age, often socially marginalized, and with records of alcohol abuse. In Danes, cluster 2 alone was responsible for the high cluster frequency level. Immigrants had a higher incidence of clustered tuberculosis at a younger age (0 to 39 years). To achieve tuberculosis elimination in Denmark, high-risk transmission environments, like the cluster 2 environment in Danes, and specific transmission chains in immigrants in the capital area, e.g., homeless/socially marginalized Somalis/Greenlanders, often with alcohol abuse, must be targeted, including groups with a high risk of reactivation.
Full-text · Article · Jun 2012 · Journal of clinical microbiology
[Show abstract][Hide abstract] ABSTRACT: As a veterinary student I always felt misplaced. Not until I attended a course in immunology did I really feel the urge and drive to dive into the matter. Almost by coincidence, as a brand new candidate, I had the fortune to get a position in Tuberculosis vaccine research, and never since have I doubted that it was the right choice for me. Since then I have been driven by scientific curiosity and the prospects of generating results that can be used for products for which there is a real need.
Preview · Article · May 2012 · Human Vaccines & Immunotherapeutics