PhD Project Summaries
PhD Project I Summary
Title: Vaccine-induced and natural immune responses to AMA1 in Mali
Supervisors: Prof. Alan Thomas (BioMedical Primate Research Center, the Netherlands) and Prof. Ogobara Doumbo (Malaria Research and Training Centre, Mali)
Collaborators: Dr. Ed Remarque (BioMedical Primate Research Center, the Netherlands), Dr. Roma Chilengi (African Malaria Network Trust, Tanzania), Dr. Bourema Kouriba (Malaria Research and Training Centre, Mali), Dr. Daniel Dodoo (Noguchi Memorial Institute for Medical Research, Ghana), Dr. Leonie van Duivenvoorde (BioMedical Primate Research Center, the Netherlands)
Background: Apical Membrane Antigen 1 (AMA1) is a micronemal protein of apicomplexan parasites that appears to be essential during infection of host cells. Immune responses to Plasmodium AMA1 can have profound parasite inhibitory effects both as measured in vitro and in animal challenge models, suggesting AMA1 as a potential vaccine candidate, and phases I and II studies are currently ongoing. However, AMA1 is polymorphic, probably as a result of immune selection operating on an important target of naturally occurring immunity. A thorough understanding of the acquisition and maintenance of AMA1 allele-specific B and T-cell responses in naturally exposed and vaccinated subjects is pivotal for the development of an efficacious vaccine. The ultimate target group for malaria vaccines are young children, and additional information is required on the kinetics of AMA1-allele-specific responses in this group. The PhD programme will focus on the evaluation of natural and vaccine-induced immune responses to AMA1. Both humoral and cellular immunity to AMA1 will be in depth investigated in adults and children.
Aim of the project:
1. Evaluate immune responses in AMA-1 Phase Ib clinical trial (Humoral and Cellular)
2. Describe how AMA1 allele specific immunity develops with age
3. Improve understanding why memory responses to AMA1 are short-lived
4. Strengthen immunology capacity of MRTC.
Methods: Samples from the following sources will be available to the project: 1- Sera and PBMC collected in the currently ongoing AMANET-sponsored Phase Ib clinical trial with AMA1 in Bandiagara. 2- Samples collected in a Bandiagara field study in children. 3- PBMC collected at the Bamako National Center for Blood Transfusion (CNTS). 4- bone marrow samples collected at the Bamako University Hospital (BUH).
Samples collected in the phase Ib trial will yield pivotal information on AMA1 immunity in adults and the following topics will be addressed: 1- Vaccine safety and basic immunology (Elisa and IFA on vaccine Ag). 2- Allelic responses, antibody isotypes and subclasses on a broad panel of AMA1 alleles. 3- Functionality, quality and fine specificity of humoral response (GIA, Avidity and Competition Elisa). 4- T cell immunology: Lymphocyte Stimulation Tests for proliferation and cytokine production, Elispot as well as Intra Cellular Staining with emphasis on memory T cells (CD4 and CD8). 5- B cell immunology with emphasis on memory B-cells: Elispot on the vaccine antigen and FACS analysis, how long-lived are malaria-specific memory B-cells as compared to control antigens (e.g. Tetanus Toxoid and influenza)? The latter question will also be addressed in PBMC samples from the Phase Ib, the CNTS and in bone marrow samples from the BUH.
A prospective field study will be conducted in children aged 1 to 10 years. Morbidity data and blood samples will be collected before, during and at the end of the transmission season. AMA1 alleles will be typed and humoral immune responses to a broad panel of AMA1 alleles will be determined. This will yield insights on the number of circulating AMA1 alleles and the polyclonality of infections as well as on the relation between circulating alleles and AMA1 allele-specific antibody responses in children. The relation between antibody levels and clinical malaria will also be studied in this cohort. The panel of antigens for this study will be extended by the Afro Immuno Assay (AIA) antigens (viz. MSP119, MSP3 and GLURP). MRTC has recently joined AIA.
The PhD programme will be a sandwich type with half of time to be spent in Mali. The PhD will be supervised by senior BPRC and MRTC immunologists and will be embedded in the AIA programme. Both AIA and BPRC will transfer the required methodology and thereby contribute to the immunology capacity at MRTC.
Expected outcome:
1. Description of B and T-cell immune responses in AMA-1 Phase Ib clinical trial
2. Description of AMA1 allele specific immunity development with age
3. Improved understanding of memory responses to AMA1
4. Scientific rationale for improved AMA1-based vaccines
5. Strengthened immunology capacity of MRTC
PhD Project II Summary
Title: Development of viral vector combination vaccine strategies targeting both pre-erythrocytic and blood-stage malaria
Supervisors: Prof. Adrian Hill (Oxford University, United Kingdom) and Prof. Alan Thomas (BioMedical Primate Research Centre, the Netherlands)
Collaborators: Dr. Simon Draper (Oxford University, United Kingdom)
In recent years, recombinant viral vectors administered in heterologous prime-boost regimes have been developed as approaches to target both the liver- and blood-stages of malaria infection. This work employed various vectors but the most promising regimes involve priming with replication-defective adenoviruses (Ads) and boosting with the poxvirus vector modified virus Ankara (MVA). Priming with a recombinant adenovirus and boosting with recombinant MVA eight weeks later has now been shown to induce remarkably strong T cell and antibody responses in animal models, and a clinical trial of this approach using the TRAP pre-erythrocytic antigen is in progress.
Adenovirus prime-poxvirus boost regimes are the most effective pre-erythrocytic vaccine approach to inducing liver-stage protection with subunit vaccines in both P. berghei and P. yoelii. Recently an adenovirus-MVA prime-boost regime, using vectors expressing the P. yoelii MSP-142 blood-stage antigen, could induce potent antibody and T cell responses, capable of providing partial protection against the late liver-stage parasites and complete protection against blood-stage malaria infection (Draper et al. submitted). Adenovirus and MVA vectors encoding P. falciparum MSP-1 and AMA-1 antigens are also highly immunogenic in mice and strong growth inhibitory activity has been induced.
Extensive studies in the HIV field have shown that vectored vaccines can be used as mixtures and clinical trials of such mixtures are in progress as HIV vaccines. This suggests that evaluating vectored malaria vaccines as mixtures might be an effective approach to generating strong pre-erythrocytic and blood stage protection.
This EMVDA PhD programme would focus on the development of combination vaccine approaches targeting liver- and blood-stage malaria, aiming to maintain maximal immunogenicity and efficacy of each component viral vaccine. Immunological studies into antigenic competition would be required, as well as studies using vaccine mixtures versus bi-cistronic viral vectors, and same versus separate site administration. Our laboratory is well set up to study the immunogenicity of each component vaccine using state of the art assays such as multi-parameter flow cytrometry. Using established P. yoelii CSP and MSP-1 models, vaccine efficacy in mice against the sporozoite, liver-stage and blood-stage forms can be assayed in house. Results can be translated directly to P. falciparum vaccine candidates, using vectors expressing ME-TRAP and PfMSP-1 and/or AMA-1. As well as assessing immunogenicity, the efficacy of these P. falciparum combination vaccines will be studied using i) in vitro growth inhibition assays against blood-stage P. falciparum, and ii) P. berghei sporozoite challenges (due to the presence of the Pb9 CSP epitope in the ME-TRAP multi-epitope string). The findings from this PhD programme could be directly translated into phase I / II clinical trials using existing facilities.
PhD Project III Summary
Title: Statistical and epidemiological methods for assessment of correlates of antibody mediated immunity to P. falciparum malaria in children
Supervisors: Dr. Paul Milligan (Reader in Epidemiology and Medical Statistics, Department of Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, United Kingdom) and Dr. Daniel Dodoo (Senior Scientist, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Ghana)
Collaborators: Dr. Michael Theisen (Statens Serum Institut, Copenhagen, Denmark)
Background: Immuno-epidemiological cohort studies have been used to study the role of malaria antigen-specific antibody in protecting from clinical malaria. Antibody levels are measured at baseline before a period of malaria surveillance, to investigate the association of particular pre-existing antibody responses with clinical immunity. This approach presents several statistical challenges. Estimates of association may be affected by the impact of the relatively short half life of naturally induced malaria antibodies; exposure is an important confounder but it is not clear what the best measures of exposure are or to what extent inadequate adjustment for confounding effects of exposure is responsible for the lack of consistency in published results; statistical analysis of antibody concentrations is complicated by the presence of zero (below detection threshold) values, while analysis of the presence/absence of antibodies is also problematic because the zero group are contaminated by an unknown number of false negatives owing to the limited sensitivity of measurement. Cohorts kept under active surveillance for malaria receive antimalarial treatments but the effects of these treatments are rarely taken into account in analyses. These methodological challenges may partly explain the difficulty in establishing clear independent relationships between antigen specific antibody responses and protection from clinical malaria and may have contributed to the lack of consistency in results from immunoepidemiological studies. This project aims at developing and applying appropriate statistical and epidemiological methods for the analysis of such studies. The work will include meta-analysis of cohort studies completed under the multi-site Afro-Immuno-Assay (AIA) project. It is envisaged the successful candidate will also contribute to statistical work for malaria vaccine trials coordinated by AMANET.
PhD Project IV Summary
Title: Induction and persistence of memory T cells by candidate malaria vaccines
Supervisors: Prof. Robert Sauerwein (Radboud University Nijmegen Medical Centre, the Netherlands) and Prof. Alan Thomas (BioMedical Primate Research Center, the Netherlands)
Collaborators: Dr. Rinaldo Zurbriggen (Pevion Biotech Ltd, Switzerland) and Dr. Roma Chilengi (African Malaria Network Trust, Tanzania)
Background: The essential biological objective of a malaria vaccine is induction of a sustained memory for protective immunity. Malaria vaccine development has so far primarily concentrated on identification and testing of appropriate Plasmodium antigens. There is no doubt that formulation and adjuvants are equally important since they play a key role in the nature and strength of the induced immune response. Adjuvants are compounds that interact with the innate immune system facilitating antigen uptake, processing and presentation by APC. The APC response orchestrates the downstream immune regulatory mechanisms and the specific immunity that develops after the first clonal expansion of T cells. The latter is a complex process that depends on MHC dependent antigen presentation, co-stimulatory molecule expression, and cytokines. Stimulatory pathways may be counter-regulated by suppressive signals from regulatory T cells. The outcome of this cascade of events on T cells is a key factor for the subsequent antibody profile and for protection.
Little is known on how adjuvantia operate at cellular level and such knowledge is important for a rational approach to vaccine formulation. Several formulations of AMA1 and MSP3-GLURP will be developed in the EMVDA program i.e. with IRIV or in combination with Alum hydrogel or DDA/TDB.
Aim of the project: To study the effect of different adjuvants and delivery systems on induction of central and effector T cell memory by AMA1 vaccine candidates. .
Methods: AMA1 formulated with influenza virus type particle (IRIV) or adjuvated with dimethyldioctadecylammonium (DDA)/TDB or Aluminumhydrogel will be studied for their capacity to induce central and effector memory T cells. Upstream immunological events will be included that may influence this induction such as activation of APC and subsets of CD4 cells (Th1, Th2, Tregs). Activation will be assessed by membrane surface marker expression and cytokine synthesis /profile as well as by functional in vitro assays. Studies, which also include longitudinal follow-up, will be conducted in animal models used for preclinical assessment of the vaccine candidates as well as with PBMC´s obtained from Phase Ia or Ib clinical trials.
Expected outcome: Understanding of the role and mechanisms of adjuvants on the induction of sustained memory T cells allowing a rational choice for clinical development of selected malaria vaccine candidates.
PhD Project V Summary
Title: Functional Analysis of Human Monoclonal Antibodies against MSP2
Supervisors: Prof. François Spertini (Hospices-CHUV, Switzerland) and Prof. Klavs Berzins (Stockholm University, Sweden)
Collaborators: Prof.Giampietro Corradin (Biochemistry Dept, Lausanne University, Switzerland), Dr. Michael Theisen (Statens Serum Institut, Denmark), Prof. Alan Thomas (BioMedical Primate Research Center, the Netherlands) and Prof. Antonio Lanzavecchia (Institute for Research in Biomedicine, Switzerland)
Background: The laboratories of Drs. Corradin and Spertini in Lausanne and associates are currently evaluating two long synthetic peptides based on the conserved regions of the two allelic families of MSP2 as malaria vaccine candidate. These two allelic families of MSP2 have been associated with protection against malaria infection.
Human purified antibodies specific for both MSP2 candidate vaccines were active in antibody-dependent cellular activity (ADCI) and but not in the growth inhibition assay (GIA).
Aim of the project: We are collaborating with the laboratory of Dr. Lanzavecchia, who has recently optimized the development of human monoclonal antibodies by immortalizing memory B cells (Barford et al 2007) and Lagos State University Teaching Hospital, Nigeria, to develop human monoclonal antibodies against the two allelic families of MSP2.
We plan to evaluate the biological activity of specific IgG subtypes of human monoclonal antibodies and sera from immunized naive volunteers in IFAT, ADCI and GIA. The possible therapeutic effects of the monoclonal antibodies in primate models of P. falciparum will also be planned.
We envisage close interactions with the laboratories of Drs. M. Theisen , K. Berzin and A. Thomas for some of these activities.
Methods: EBV infection, cloning and production of human B cells from donors withhigh antibody titers against the antigens of interest; immunofluorescence; SDS-PAGE electrophoresis; western blot; biological activity using growth inhibition assay (GIA) and antibody dependent cellular inhibition (ADCI); animal handling.
Expected outcome: Production of human monoclonal antibodies specific for the two MSP2 allelic proteins with ADCI biological activity. Mapping of the relevant epitopes.
PhD Project VI Summary
Title: Immune responses to the erythrocytic stage of Plasmodium falciparum and the development of vaccines against malaria
Supervisors: Dr. David Cavanagh (University of Edinburgh, United Kingdom) and Prof. Anthony Holder (National Institut for Medical Research, United Kingdom)
Collaborators: Dr. Alison Creasey and Dr. Mike Blackman (University of Edinburgh, United Kingdom)
Background: The research interests of the groups centre on the immunobiology of the human malaria parasite, Plasmodium falciparum with a particular focus on the blood stage form that causes the disease. One of the main thrusts of our research at present is on the development of vaccines, based on recombinant surface proteins derived from genes expressed by the merozoite stage of the parasite life cycle. Much of this work is focused on merozoite surface protein 1 (MSP-1), but there are several merozoite proteins that may be the target of protective antibodies, including some previously uncharacterised proteins initially identified from proteome and genomic approaches. We examine the development of antibody responses to specific proteins of the malaria parasite in naturally exposed individuals, including studying the associations between specific responses and protection against clinical malaria episodes. This sero-epidemiological approach is supplemented by examining the functional activity of specific antibodies in parasite growth inhibition assays in vitro and studies on the role of the proteins in the interaction between parasite and host cell.
Aim of the project: The project is to identify and evaluate new and partially characterised proteins that may be the target of protective antibodies.
Methods: The work will include gene cloning and expression in recombinant form of candidate proteins that will be purified and used to develop functional assays. These assays will include measurement and functional analysis of both naturally acquired antibodies and antibodies raised by immunization, as well as binding assays to study the interaction between individual proteins and other parasite or host components. Studies on human parasite proteins may be supplemented with parallel studies on the corresponding protein in a rodent model.
Expected outcome: The intention is to develop new candidates to be evaluated as malaria vaccine components.
References:
1. Cavanagh, DR, Dodoo, D, Hviid, L, Kurtzhals, JA, Theander TG, Akanmori, BD, Polley, SD, Conway, DJ, Koram, K, and McBride, JS. (2004). Antibodies to the N-terminal block 2 of Plasmodium falciparum merozoite surface protein 1 are associated with protection against clinical malaria. Infect Immun72:6492-6502.
2. Conway, D. J., Cavanagh, D. R., Tanabe, K., Roper, C., Mikes, Z. S., Sakihama, N., Bojang, K. A., Oduola, A. M. J., Kremsner, P. G., Arnot, D. E., Greenwood, B. M., and McBride, J. S.(2000)A principal target of immunity to malaria identified by molecular population genetic and immunological analysis. Nature Medicine6: 689-692.
3. Cavanagh, D.R., I.M. Elhassan, C. Roper, V.J. Robinson, H. Giha, A.A. Holder, L. Hviid, T.G. Theander, D.E. Arnot, and J.S. McBride (1998) A longitudinal study of type-specific antibody responses to Plasmodium falciparum merozoite surface protein-1 in an area of unstable malaria in Sudan. Journal of Immunology161: 347-359.
4. Pachebat, J. A., Kadekoppala, M., Grainger, M., Dluzewski, A. R., Gunaratne, R. S., Ogun, S. A., Ling, I. T., Scott-Finnigan, T. J., Bannister, L. H., Taylor, H. M., Mitchell, G. H. and Holder,A.A. (2007) Extensive proteolytic processing of the malaria parasite Merozoite Surface Protein 7 during biosynthesis and parasite release from erythrocytes. Mol Biochem Parasitol., 151(1):59-69. Epub 2006 Nov 2
5. Green, J.L., L. Hinds, M. Grainger, E. Knuepfer and A. A. Holder (2006). Plasmodium thrombospondin related apical merozoite protein (PTRAMP) is shed from the surface of merozoites by PfSUB2 upon invasion of erythrocytes. Mol Biochem Parasitol. 150(1):114-117 Epub 2006 Jul 17
6. O’Keeffe, A. H., J. L. Green, M. Grainger and A. A. Holder (2005) A novel Sushi domain-containing protein of Plasmodium falciparum. Mol. Biochem. Parasitol. 140: 61-68
PhD Project VII Summary
Title: Assessment of the immune responses to a malaria candidate vaccine in immunized volunteers and exposed populations
Supervisors: Dr. Michael Theisen (Statens Serum Institut, Copenhagen, Denmark) and Dr. Benjamin Mordmüller (University of Tübingen, Institut für Tropenmedizin, Germany)
Collaborators: Prof. Peter G. Kremsner (University of Tübingen, Institut für Tropenmedizin, Germany), Dr. Saadou Issifou (Albert Sweitzer Hospital, Lamberene, Gabon), Dr. Rinaldo Zurbriggen (Pevion Biotech Ltd, Switzerland), Dr. Roma Chilengi (African Malaria Network Trust, Tanzania), Dr. Babatunde Imoukhuede and Dr. Odile Leroy (European Malaria Vaccine Initiative, Denmark).
Background: Both the quantitative and the qualitative response to immunizations is of importance. Thus, high titers against the immunogen may not always achieve the protective efficacy aimed at. The switch from IgM to IgG following booster immunizations is of paramount importance for a proper memory function. Besides the ability to recognize important epitopes on the vaccine immunogen the avidity of the antibodies raised seem to be crucially important. Thus, both proper T and B-cell stimulation by the immunogen has to be assured. Aluminium Hydroxide is the preferred adjuvant but it is uncertain whether this adjuvant can provide the required stimulation of the immune system especially when employing subunit vaccines.
Vaccines against infectious diseases are administered to the target population (infants / children) prior to exposure of the pathogen in question. Maternally derived antibodies are known to interfere with the development of proper immune response to measles and possibly also other infectious pathogens. For measles this has implication for the recommended immunization schedule especially in developing countries. Maternally derived antibodies against adenovirus might also influence the window of opportunity of immunization with Ad-vectored vaccines. Immunization against malaria should preferably be started as early as possibly before repeated exposure might possibly modify the immune response.
Aim of the project is: A comparative study of the immune response in non-exposed European volunteers employing different adjuvants and delivery systems, which will not only provide important information about the antigen used but might also provide information of importance for other vaccine constructs. In addition it is essential to compare with a non-adjuvanted delivery system such as the Virosome system. Further, a comparison between the response obtained in non-exposed volunteers and exposed adult volunteers may also provide information whether the desired immune response can be obtained in individuals who are already primed via exposure to the parasite. Finally, a comparison of the immune response in lightly exposed children to that of non-exposed adult volunteers might provide information on whether the reactivity in non-exposed adult individuals is likely to mimic the response in infants/children from malaria endemic areas.
To assess the window of opportunity for immunization with malaria sub-unit vaccine with an aim to investigate the possibility to finally include a malaria vaccine in the EPI programme.
Methods: It will be essential to develop micro assays for detection of specific antibodies in infants. Equipment for micro assays suitable for infant studies is available. IgM, IgG and IgG subclass responses, memory B-cell ELISPOT and IgG avidity kinetics against vaccine-antigens will be assessed. It is not known to what extent maternally derived antibodies against malaria might negatively influence immunizations with sub-unit vaccines.
Mother and cord blood samples will be used to assess the Ab profile and sampling three and 6 month later from the infants combined with clinical examination and microscopy of malaria smears will provide information on specific antibody kinetics and the experience of clinical malaria. Active surveillance and assessment of antibody kinetics in micro assays will be the core activities.
Ongoing clinical trials as well as planned investigations within the European Malaria Vaccine Initiative (EMVI) and African Malaria Network Trust (AMANET) framework hold unique possibilities to systematically investigate immune responses against the same candidate antigen in different adjuvant formulations and delivery systems in exposed and non-exposed populations in different age groups. The project is embedded in current and future clinical trials in Tübingen and Lambaréné. Collaboration with other clinical trial sites will be solicited.
Expected outcome:
-To develop micro assay for detection of specific antibodies and /or cytokines.
-To contribute to the evaluation of malaria clinical trials.
PhD Project VIII Summary
Title: Development of humoral immune responses and immunological memory against malaria vaccine-candidates in humans.
Supervisors: Dr. Benjamin Mordmüller (University of Tübingen, Institut für Tropenmedizin, Germany) and Dr. Michael Theisen (Statens Serum Institut, Denmark)
Collaborators: Prof. Peter G. Kremsner (University of Tübingen, Institut für Tropenmedizin, Germany) and Prof. Robert Sauerwein (Radboud University Medical Centre, the Netherlands)
Background: Transition from first-time-in-man studies in unexposed volunteers to clinical trials in exposed populations is a critical step in vaccine development. This is especially true for malaria vaccines because encounter with the pathogen starts early in life and reaches high levels in certain populations. In addition, co-infections, administration of other vaccines at the same time, and a continuous and high exposure to a variety of pathogens complicates the immunological response pattern, and affects long-term vaccination-success. So far, only very little is known about the effect of these co-variates on immune responses and B-cell memory to vaccines. Ongoing clinical trials as well as planned investigations within the European Malaria Vaccine Development Association (EMVDA) framework bear the unique possibility to do systematic side by side investigations on the same vaccines (antigens and adjuvants) in exposed and non-exposed populations in different age groups and with different vaccination schemes. The project is embedded in current and future clinical trials in Tübingen, Lambaréné, and Nijmegen. Collaboration with all other clinical trial sites is highly desirable. Our main focus is the humoral and memory B-cell response to erythrocytic asexual-stage-vaccines, but the infrastructure allows synergistic interactions with other immunologically centred projects.
Aim of the project: The projects aim is to compare the development of humoral immunity and immunological memory against different eythrocytic stage malaria vaccine-candidates in non-exposed volunteers, exposed adults, children, and infants.
Methods: Total immunoglobulin G (IgG)-, IgG-subclass-ELISA, and memory B-cell ELISPOT against vaccine-antigens represent the core-experiments. They will be performed in all study populations using the same techniques before and at different time-points after immunization. Furthermore, extended Th1/Th2 profiling with and without stimulation will be done to assess the cytokine and T-helper cell environment of B-cell maturation. These core-techniques are established, validated, and used in two current GMZ2-trials (GMZ2 is a MSP3-GLURP fusion protein). In heavily exposed populations, reactivity against vaccine-antigens is common and complicates the distinction between natural and vaccine-induced effects. Therefore the core-protocol is extended by techniques to investigate the evolution of antibody responses against recombinant as well as crude antigens, which rely on different conformations of the protein (such as ELISA, Western Blot, and immunofluorescence). Furthermore a correlate of individual memory B-cell clones will be analysed by repertoire-cloning of antibody fragments from mRNA of in-vitro generated antibody-secreting cells. Repertoire-cloning can be integrated into already established protocols for memory B-cell ELISPOT. B-cell reactivity provides an internal control for the outcome of the cloning. Additionally, subtractive hybridisation of samples obtained during the course of vaccination can give us information of the plasticity of B-cell memory over time.
Functional antibody-assays (growth inhibition with and without monocytes; GI/GIM) will be compared between different clinical settings within the consortium.
Expected outcome: We will characterise the development of circulating antibody- and B-cell memory-responses in clinical vaccine trials in Europe and Africa by complementing immunological techniques. Established and validated protocols are compared with newly developed methods. All techniques are tested as surrogate marker for efficacy. A spin-off of our work is a set of standard tools, including experimental procedures and software for ELISA, ELISPOT, and colony-counting that will be provided to the scientific community under a general public license.