Immunotherapy with a candidate for an antileprosy vaccine, My co bacterium W, was given in addition to standard multidrug therapy (MDT) to 53 multibacillary lepromin negative patients belonging to BB, BL and LL types of leprosy (vaccine group).An equal control group received MDT and injections of micronized starch as placebo.Both the vaccine and placebo were administered intradermally every 3 months.The patients were evaluated at determined intervals by clinical, bacteriological and histopathological parameters and lepromin testing.Reactional episodes were analysed with reference to incidence, onset, frequency and severity during and after release from treatment (RFT).Incidence of reversal reaction (RR) was marginally higher in the vaccine group (22'6% vaccine group vs 15% control group).All cases with a history of downgrading type I reaction developed RR during therapy.Most episodes occurred within the I st year of the commencement of therapy-50% developing within 3 months.Late reversal reaction (after RFT) were observed in 3•8% of cases in both groups, and 50% of the reactors in the control group and 33% in the vaccine group had repeated reactional episodes.Incidence of neuritis associated with RR as well as isolated neuritis was similar in both groups.This study forms part of a large-scale immunotherapeutic clinical trial of a candidate for an antileprosy vaccine, My cobacterium w (M.w), which is currently in progress at the Dr. RML and the Safdarjang Hospitals of New Delhi in association with the National
different candidates would be an important asset.Research sponsored three meetings of experts to discuss assay harmonization for new tuberculosis vaccine trials.assay approaches and make specific recommendations for phase I/IIa trials.These include introducing a single and simple harmonised assay for all trials.
Increasing knowledge about DosR regulon-encoded proteins has led us to produce novel Mycobacterium tuberculosis antigens for immunogenicity testing in human populations in three countries in Africa to which tuberculosis (TB) is endemic. A total of 131 tuberculin skin test-positive and/or ESAT-6/CFP10-positive, human immunodeficiency virus-negative adult household contacts of active pulmonary TB cases from South Africa (n = 56), The Gambia (n = 26), and Uganda (n = 49) were tested for gamma interferon responses to 7 classical and 51 DosR regulon-encoded M. tuberculosis recombinant protein antigens. ESAT-6/CFP10 fusion protein evoked responses in >75% of study participants in all three countries. Of the DosR regulon-encoded antigens tested, Rv1733c was the most commonly recognized by participants from both South Africa and Uganda and the third most commonly recognized antigen in The Gambia. The four most frequently recognized DosR regulon-encoded antigens in Uganda (Rv1733c, Rv0081, Rv1735c, and Rv1737c) included the three most immunogenic antigens in South Africa. In contrast, Rv3131 induced the highest percentage of responders in Gambian contacts (38%), compared to only 3.4% of Ugandan contacts and no South African contacts. Appreciable percentages of TB contacts with a high likelihood of latent M. tuberculosis infection responded to several novel DosR regulon-encoded M. tuberculosis proteins. In addition to significant similarities in antigen recognition profiles between the three African population groups, there were also disparities, which may stem from genetic differences between both pathogen and host populations. Our findings have implications for the selection of potential TB vaccine candidates and for determining biosignatures of latent M. tuberculosis infection, active TB disease, and protective immunity.
Although tuberculosis (TB) causes more deaths than any other pathogen, most infected individuals harbor the pathogen without signs of disease. We explored the metabolome of >400 small molecules in serum of uninfected individuals, latently infected healthy individuals and patients with active TB. We identified changes in amino acid, lipid and nucleotide metabolism pathways, providing evidence for anti-inflammatory metabolomic changes in TB. Metabolic profiles indicate increased activity of indoleamine 2,3 dioxygenase 1 (IDO1), decreased phospholipase activity, increased abundance of adenosine metabolism products, as well as indicators of fibrotic lesions in active disease as compared to latent infection. Consistent with our predictions, we experimentally demonstrate TB-induced IDO1 activity. Furthermore, we demonstrate a link between metabolic profiles and cytokine signaling. Finally, we show that 20 metabolites are sufficient for robust discrimination of TB patients from healthy individuals. Our results provide specific insights into the biology of TB and pave the way for the rational development of metabolic biomarkers for TB.
For heterogeneous tissues, such as blood, measurements of gene expression are confounded by relative proportions of cell types involved. Conclusions have to rely on estimation of gene expression signals for homogeneous cell populations, e.g. by applying micro-dissection, fluorescence activated cell sorting, or in-silico deconfounding. We studied feasibility and validity of a non-negative matrix decomposition algorithm using experimental gene expression data for blood and sorted cells from the same donor samples. Our objective was to optimize the algorithm regarding detection of differentially expressed genes and to enable its use for classification in the difficult scenario of reversely regulated genes. This would be of importance for the identification of candidate biomarkers in heterogeneous tissues. Experimental data and simulation studies involving noise parameters estimated from these data revealed that for valid detection of differential gene expression, quantile normalization and use of non-log data are optimal. We demonstrate the feasibility of predicting proportions of constituting cell types from gene expression data of single samples, as a prerequisite for a deconfounding-based classification approach. Classification cross-validation errors with and without using deconfounding results are reported as well as sample-size dependencies. Implementation of the algorithm, simulation and analysis scripts are available. The deconfounding algorithm without decorrelation using quantile normalization on non-log data is proposed for biomarkers that are difficult to detect, and for cases where confounding by varying proportions of cell types is the suspected reason. In this case, a deconfounding ranking approach can be used as a powerful alternative to, or complement of, other statistical learning approaches to define candidate biomarkers for molecular diagnosis and prediction in biomedicine, in realistically noisy conditions and with moderate sample sizes.
A workshop entitled “Hands on laboratory training in immunology of relevance to global health issues in resource-poor settings” was held on 12th and 13th of September 2009 at the Max Planck Institute for Infection Biology in Berlin (MPIIB) as a pre-congress satellite meeting of the European Congress of Immunology (ECI) 2009. Twelve participants from 12 different countries, including 10 originating from resource-poor settings, participated in this “hands on laboratory workshop”, which was directed by the authors of this report. Five members (Kellen Fae, Laura Lozza, Jeroen Maertzdorf, Henrik Mueller and Geraldine Nouailles) of the Immunology Department of MPIIB contributed to the training workshop as facilitators in the laboratories. The rationale behind and the aims for such a workshop are detailed in this report. Global health, in general, considers the health needs of the whole planet, transcending national borders. It includes problems, such as infectious and insect-borne diseases, that can spread from one country to another, as well as health problems of high magnitude, such as tuberculosis, influenza A (H5N1), meningitis and dengue fever, which have a global political and economic impact. Global health also refers to the collective efforts to address the needs/problems by cooperative actions and solutions, and has areas of overlap with the more established disciplines of public and international health. Global, public and international health share population-based approaches with a preventive focus; a focus on poorer and more vulnerable populations; multi- and interdisciplinary approaches; emphasis on health as a public good; the importance of systems and structures to provide health to the population; and the participation of several stakeholders 1. Health is the foundation of a civil society, social and cultural growth, political stability, and economic sustainability. There is a great need to foster an effective global research effort on infectious diseases of poverty in which the disease-endemic countries play a pivotal role. The interdependence of resource-rich and resource-scarce nations and multilateral involvement for innovative solutions needs to be stressed 2, 3. The unprecedented loss of lives due to HIV/AIDS, TB and malaria are humanitarian reasons to solve global health problems and to avert the needless suffering and preventable deaths of millions of adults and children; however, global health efforts also have enormous economic impact in terms of directly reduced health costs, as well as reduced indirect costs due to enhanced productivity. The “90/10 gap” alludes to the disparity of proportional resource allocations of health research and budgets – 90% of resources are allocated to 10% of the global health issues, i.e. the major diseases of the developed nations, whereas 10% of resources are allocated to 90% of the global health issues afflicting the world's impoverished population 4. There are several initiatives worldwide to step-up work on diseases that disproportionately affect the poor, and to measure research success according to its impact on human welfare 5-9. The Global Forum for Health Research is an independent, international organization committed to demonstrate the essential role of research and innovation for health and health equity, benefiting poor and marginalized populations 10. Its central objective is to help correct the 10/90 gap in health research and focus research efforts on the health problems of the poor by bringing together key players and creating a movement for analysis and debate on health research priorities, the allocation of resources, public–private partnerships and the access of all people to the outcomes of health research. There are enormous gaps between the potential of health systems and their actual performance and, more strikingly, there are too many inequities in the distribution of health workers between and within countries. The Americas (mainly USA and Canada) are home to 14% of the world's population, bear only 10% of the world's disease burden, have 37% of the global health workforce and spend about 50% of the world's financial health resources. Conversely, sub-Saharan Africa, with about 11% of the world's population bears over 24% of the global disease burden, is home to only 3% of the global health workforce, and spends less than 1% of the world's financial health resources 11. In most developing countries, the health workforce is concentrated in the major towns and cities, while rural areas can only boast about 23 and 38% of the countries' doctors and nurses, respectively. The imbalances exist not only in the total numbers and geographical distribution of the health workers, but also in the skills' mix of the available health workers. The WHO estimates that 57 countries worldwide have a critical shortage of health workers, equivalent to a global deficit of about 2.4 million doctors, nurses and midwives. Thirty six of these countries are in sub-Saharan Africa. The European and Developing Countries Clinical Trials Partnership (EDCTP), a partnership between 47 sub-Saharan African countries and 14 European Union member states plus Norway and Switzerland, was formed to fund the acceleration of new clinical trial interventions to fight HIV/AIDS, malaria and TB in the sub-Saharan African region 12. It has established four regional “networks of excellence” in central, western, eastern and southern Africa bringing together research institutes in the region to lay the groundwork for conducting Phase 2 and 3 clinical trials in HIV/AIDS, TB and malaria 13. As we immunologists move on to harvest the fruits of our efforts in basic and translational research, we have to embark upon phased clinical trials. Although the research is carried out mostly in westernized countries, translation requires testing in human subjects in the relevant endemic countries which, for infectious diseases, often equates with resource-poor settings. We, in resource-rich countries, have the onus to create the intellectual capacities and skills in the resource-poor regions to empower them to solve their own public health problems. Access to basic levels of health care is considered a fundamental component of human rights; yet, one-third of the world's population, from the rural and remote villages in sub-Saharan Africa, Asia and Latin America to the urban centers of America, is still deprived of this essential element of life. Every year, 10 million lives could be saved through the expansion of existing and effective medical interventions. The lack of adequate research in the area of neglected diseases as well as the lack of resource allocations for these activities are the major hindrances in saving these precious lives. The training workshop, which was supported by the European Federation of Immunological Societies (EFIS), was intended to empower researchers from resource-poor settings with the knowledge and know–how to deal with the immunological research, such as cellular assays, required to assess vaccines or intervention strategies in GCP clinical trials. It was a true reflection of global efforts involving 22 different nationalities, as can be seen in Fig. 1, with 10 participants from resource-poor settings. The workshop was an interactive training exercise in which each participant performed hands-on immunological assays. Lectures and discussions explained the assay principles and provided the tools for analyzing the results and generating interpretable data. Such initiatives need to be sustained over time to build on the knowledge and scientific expertise developed, enabling the participants to fully engage in and drive research/clinical trials in their countries. Furthermore, similar efforts executed in the resource-poor settings could also stimulate young researchers to take on the challenges of such research in their own countries, eventually stopping the exodus and brain-drain to the ivory towers in the industrialized world. Photograph of all participants, facilitators and faculty of the workshop. Vaccination remains one of the most effective approaches to control the spread of infectious disease. Most of the vaccines currently approved for use in human target diseases for which protection is mostly mediated by antibodies; however, new vaccines, for which we currently have no correlates of protection or adequate challenge models, are under development. For a subset of target diseases, protection is thought to be mediated, at least in part, by effector T-cell responses. These include tuberculosis, leprosy, HIV, leishmania and malaria. For these indications, the decision to pursue clinical development of novel vaccine formulations relies almost entirely on assessing the T-cell immunogenicity of different candidate vaccines in early clinical-phase testing. In this context, a number of requirements, such as the identification and development of immunological assays, that are perceived to be relevant to protection, the coordination of these assays and the training of staff to perform the assays have become a critical part of vaccine selection and development 14. In resource-poor countries where the infrastructure and the availability of local, trained personnel are often insufficient to enable immunogenicity assessments to be performed proficiently and with consistency, the upgrading of facilities and significant training of personnel are essential to conduct the required immunological studies at the clinical trial sites. The goal of establishing a network of field sites with qualified operators is a long-term effort, and the training activities undertaken as part of the ECI 2009 meeting satellite workshop constitute a first step in acquainting scientists from these resource-poor countries with the detection of T-cell response monitoring technology. When assessing vaccine-induced immune responses during clinical trials, it is essential that the immunogenicity results generated by multiple sites fulfill essential criteria, such as comparable performance and consistency across all participating laboratories. Thus, training must be complemented by a range of activities, including harmonization of procedures, assessment of operator performance by participation in proficiency panel testing, comparison of performance within and across different laboratories, and documenting reproducibility 15. The certification of operators and the maintenance of certified status by regular testing of reference preparations and/or standards are essential to ensure the quality, consistency and comparability of the experimental data. As shown by other large networks, such undertakings can be successful 16. To provide long-lasting protective immune responses, the establishment of a memory compartment (after natural infection or vaccination) is essential. Clearly, the ability to measure vaccine-induced memory responses is crucial to guide the development of new vaccines. In order to provide the most efficient protection, the T-cell memory subsets that should be preferentially expanded by vaccination need to be identified. The T-cell memory compartment has been extensively studied but many questions remain. The quality of the memory response needed to confer protection depends on the site of infection and the pathogen, as shown in a recent mouse model using two Plasmodium species 17. This study revealed differences in the nature of the memory CD8+ T-cell response needed to mediate protection against liver stage Plasmodium infection, with perforin expression by memory CD8+ T cells being necessary for protection against one, but not the other, species. Precursor (naïve) and the different subsets of antigen-experienced memory (central and effector memory) T cells are characterized by different properties and expression of cell-surface markers. For instance, markers such as the tyrosine phosphatase CD45RA and the lymphoid homing receptor CCR7 are used to identify central (CD45RA−CCR7+) and effector (CD45RA+CCR7−) memory T cells. Central memory T cells circulate in secondary lymphoid organs, proliferate rapidly and have reduced immediate effector functions, while effector memory T cells reside in non-lymphatic organs have a reduced proliferative capacity but are able to exert immediate effector functions upon recognition of cognate antigens. Interestingly, the effector cells derived from these two well-characterized T-cell memory subsets may also be different depending on the circumstances such as the nature of the pathogen and site of infection triggering the effector response. As an example, the persistence and differentiation into memory cells of in vitro expanded antigen-specific CD8+ effector T cells was recently demonstrated to have derived from central but not effector memory T cells after adoptive transfer, even though the effector cells derived from the two memory subsets showed a similar phenotype and functional properties before transfer 18. Another example of the heterogeneity of the T-cell memory compartment is illustrated in a study, where multiple cell subsets were detected within the effector memory T-cell subset 19; the analysis of the expression of the costimulatory molecules CD27 and CD28 revealed that CD27+CD28+ effector memory (CD45RA+CCR7−) CD8+ T cells had characteristics (measured by telomerase activity, perforin, granzyme B, IFN-γ, IL-7Rα expression) closely related to those of central memory T cells. Flow cytometers with multiple lasers and detectors allow the simultaneous identification of multiple cell surface, as well as intracellular, markers and enable the simultaneous assessment of a multitude of T-cell subsets characterized by a combination of certain markers or complex phenotypes 20. Nevertheless, converging phenotypical patterns are emerging, and efforts to harmonize assays, nomenclature and the choice of markers to detect defined subsets are necessary to help data interpretation and to compare different studies 21. It should also be noted that, although the study of peripheral blood lymphocytes provides great insights into the T-cell memory compartment, it does not enable tissue resident, e.g. bone marrow 22, intestinal mucosa 23 and memory T-cells, which may not be accessible in human studies, to be studied. Despite its limitations and inherent complexity, the analysis of the peripheral blood T-cell compartment by flow cytometry (in combination with functional assays) in vaccine trials is crucial to understand the vaccine-induced immune response, and also hopefully to identify surrogate markers for vaccine efficacy. Furthermore, the longitudinal analysis of the peripheral T-cell compartment conducted in the context of vaccine trials offers the opportunity to better understand T-cell differentiation dynamics and pathways whose importance, in terms of protection, remain to a large extent under debate. The assessment of immunological responses to vaccination could provide early biomarker information about the potency of the vaccine-induced immunogenicity and first action of immunotherapeutic vaccines 24. It has recently been recommended that proof of biological activity has to be established before initiating clinical efficacy trials 25. There are many assays available for immune monitoring, but reports about early biomarker identification are sparse, and the assays are not necessarily affordable, robust or fast. We are currently at a stage where as much information as possible in a given surrounding should be collected so as to pinpoint clinically relevant markers, which can guide further vaccine development 26. A variety of assays are well established and regularly used in the context of monitoring immune responses in resource-rich countries. For the assessment of T-cell responses, Elispot- and flow-cytometry-based assays (such as intracellular cytokine staining (ICS) and HLA-peptide multimer staining) are widely used to evaluate vaccine immunogenicity. Improvements to these technologies are constantly being reported, and harmonization guidelines detailing strict protocol standardization across laboratories have recently been published 15, 27-30. Bearing in mind the wealth of information that can be obtained from vaccine trial immune-response monitoring and its associated pitfalls, lending a hand to scientists in resource-poor settings by providing hands-on training together with freely available, accessible community-based knowledge can go a long way. This realization drove the enthusiasm of the workshop's organizers and supporters. During the 2 days of the workshop, participants received take-home information and hands-on training of how to set-up, run and analyze Elispot and four-color ICS experiments. In addition, a polyfunctional ICS assay focusing on the markers associated with memory was demonstrated. After receiving instructions about appropriate biosafety practices, lectures about cell-mediated immunology and the principles of measuring immune responses using Elispot- and flow-cytometry-based assays were given. The underlying principles of each experiment were outlined, and the protocols were detailed and discussed. The participants were fully and actively involved in each step of the experimental procedures, from actual assay conduct to final acquisition and analysis of data using an Elispot reader or flow cytometer. The participants were divided into small groups, allowing each participant the opportunity to take turns along the various assay steps, after proper demonstration. Despite several participants being novices at the techniques involved and the general pressure and constraints of a workshop setting, all assays were conducted successfully; two healthy donors were tested for reactivity against the CEF peptide pool which is commonly applied in control measurements when assessing CD8-restricted immune responses 31. In addition, the PPD proteins and a peptide pool covering the ESAT-6 antigen were used as stimulatory antigens for monitoring T-cell responses. Figures 2 and 3 demonstrate the successful outcome of the Elispot and ICS experiments, respectively. The results and options for refinement were discussed; protocol recommendations were handed out and sources of available protocols and supporting documentation relating to the assays were indicated. Elispot results. A low PBMC background reactivity (cells+medium) can be seen for both donors A and B, with an expected high reactivity against both TBC peptide pools (cells+CFP-10 and cells+ESAT-6) in donor B, but only against one of the TBC peptide pools (cells+ESAT-6) in donor A. The CEF peptide pool reactivity that has been reported in other experiments was confirmed in one of the donors (donor A, cells+CEF), and the mitogenic PHA control worked well in both the donors (cells+PHA). No false-positive spots were reported (medium only). The intra-assay variability was low and the occurrence of artifacts or technical problems was negligible. The same conclusion applied to the conduct of four-color flow cytometry detection of antigen-activated CD3+CD4+ T cells expressing IL-2 and/or INF-γ (data not shown). Intracellular cytokine staining of cells from one donor stimulated for 18 h in the presence of brefeldin A added 2 h after the onset of cultures. The results show cytokine expression in over 22 000 CD3+ T cells. The response to PPD is significantly higher than that of the control stimulated with antibodies to CD28 and CD49d (p<0.005). Those working in resource-poor settings often require guidance, training and support for the assessment of vaccine-induced immune responses. Many vaccine trials are being conducted in these countries, enthusiastic young scientists are available in these locations, and successful assay performances across continents have been reported. Setting up dedicated workshops is feasible as demonstrated by the ECI workshop, and they can be successful and highly rewarding. The organizers of this ECI workshop hope to encourage the support of organized efforts to train scientists in resource- and support-poor settings, and enable them to perform successful immune monitoring in their home location. The authors thank Prof. Reinhold Schmidt, President of ECI 2009; Prof. Roland Jacobs, General Secretary of ECI 2009 and Prof. Stefan Kaufmann, President of EFIS (2006–2009), for the initiative and support for holding this workshop. The authors acknowledge logistical support from Ms. Riikka Lauhkonen, the MPIIB GC6 Coordination office, and Ms. Lore Lütge-Stork, ECI Secretariat, and Ms. Kerstin Mordhorst, KIT for organizing the event. The authors acknowledge constructive comments of Dr. Christian Ganoza on this article. Finally, they thank Becton Dickinson, CTL, JPT, Mabtech, Millipore and Zellnet Consulting for their generous support by providing technical assistance, equipment, materials and reagents, hence making the workshop a success. They also acknowledge support from the Max Planck Institute for Infection Biology for hosting the laboratory activities and thank Dr. Sabine Englich and Mr. Karl Ebermayer for facilitating the workshop. Conflict of interest: The authors declare no financial or commercial conflict of interest.
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