231 research outputs found
Towards integrated control of East Cost fever, a devastating disease of cattle
No full textInfection of cattle with the tick-borne apicomplexan parasite Theileria parva results in the fatal lympho-proliferative disease known as East Coast fever (ECF). ECF is considered to be one of the most devastating diseases of livestock in sub-Saharan Africa affecting all sectors of livestock production. The three-host brown ear tick, Rhipicephalus appendiculatus is the primary vector of T. parva with all tick life stages adapted to feeding on cattle. Currently, there is no effective vaccine against ECF and control of R. appendiculatus and T. parva occurs primarily through the use of disease tolerant breeds, chemotherapy and acaricide application. Widespread application of acaricides for tick and tick borne-disease control is becoming less viable due to rapid development of acaricide resistance. Alternative measures for tick control based on integrated and sustainable methods are urgently needed.
Control of the one host tick Rhipicephalus microplus through anti-tick vaccination has been shown to be viable culminating in the commercial products GavacTM and TickGardTM. Both vaccines are based on the recombinant protein Bm86 expressed as concealed antigen in the R. microplus midgut. Vaccination elicits humoral immune response targeting Bm86 during tick feeding resulting in extensive gut damage. In this thesis, anti-tick vaccines were evaluated for their ability to control R. appendiculatus tick populations and to interfere with transmission of T. parva in their natural host-pathogen-vector system.
The R. appendiculatus homologue of Bm86, named Ra86, was examined for of its impact on nymphal and adult R. appendiculatus ticks after feeding on Ra86 vaccinated cattle. The molting success of nymphal ticks to the adult stage was significantly reduced in ticks feeding on Ra86 vaccinated animals in comparison control animals. Simulations based on our empirical data suggest that repeated Ra86 vaccinations would reduce tick populations over successive generations. This experiment showed for the first time that Ra86 based vaccination could play a part in integrated pest management and control strategies for R. appendiculatus. Reducing the nymphal population molting to adult instars has implications for
ECF clinical disease severity. T. parva transmission by adult ticks is commonly associated with more severe ECF disease symptoms when compared to nymph mediated transmission. Reduction of the number of adult ticks before they can transmit T. parva may lead to diminution of the negative impact of ECF on animal productivity. Additionally, Ra86 vaccination lowered T. parva infection levels in ticks that fed on vaccinated cattle indicating that targeting the tick gut could affect the uptake of T. parva from infected cattle and/or further development within the vector.
Vaccines that block parasite transmission either to- or from- the vertebrate host have been proposed for the control of numerous vector-borne diseases including ECF. Cattle were vaccinated with a multivalent recombinant antigen cocktail containing R. appendiculatus antigens TRP64, histamine binding protein (male and female variants) and subolesin. Included in this cocktail was the T. parva protein p67, located on the sporozoite surface. Transmission of T. parva sporozoites from infected ticks to the vaccinated cattle was compared to control cattle. Establishment of ECF was 20 % lower in the vaccinated animals indicating that this multivalent vaccine partially prevented disease establishment. Importantly, animals that showed no or only mild ECF symptoms after cocktail vaccination and infected tick exposure were solidly protected from lethal needle challenge with the homologous T. parva stock. This work demonstrated for the first time that transmission of T. parva can be reduced by vaccination resulting in lowering of ECF clinical cases while still advantageously enables establishment of immune protection.
Current experimental infection of animals with T. parva involves the needle administration of cryo-preserved isolated sporozoites. This highly artificial infection method differs from natural tick based T. parva infection in both the delivery route and the number of parasite injected over time. A reliable tick-based infection method for ECF resembling natural field situations is essential for future intervention studies. Here we show for the first time that the R. appendiculatus RAM-L tick line is a suitable tool that reproducibly delivers T. parva infectious sporozoite doses to cattle resembling endemically stable field situations. Our newly developed RAM-L tick based infection model can provide information on the potential protective capacity of experimental subunit T. parva vaccines requiring substantially smaller animal numbers than conventional field trials. Together, this work adds novel information on anti-tick and transmission blocking vaccine testing for control of R. appendiculatus and T. parva
Comorbidity between counnunicable and non-communicable diseases : the example of the dual burden of tuberculosis and diabetes in Dar es Salaam, Tanzania
No full textBackground
Although recognized for centuries, the association between tuberculosis (TB) and diabetes mellitus (DM) was forgotten with the discovery of efficient treatments. In the last decade, the prevalence of DM has dramatically increased, particularly in low- and middle-income countries experiencing a high burden of TB, leading to a new interest in this association. DM increases TB risk while TB, as an infectious disease, leads to hyperglycemia. The relationship between TB and DM has been poorly studied in Sub-Saharan Africa, where the high incidence of TB is associated with HIV infection. Concentration of vitamin D is inversely associated with TB and DM, and it has been suggested that low vitamin D could mediate some of the association between TB and DM. DM affects the immune response to TB, but the precise mechanisms underlying this association are not clear.
To address this issue of high public health relevance, we undertook a project on the association between TB, DM and HIV in Tanzania. The project had three major components:
(1) Assessing the association of TB and its outcome with the presence and persistence of hyperglycemia in Tanzania, using three different DM screening tests.
(2) Describing the association between vitamin D, TB and DM.
(3) Studying the immunological features underlying TB and DM comorbidity in sub-Saharan Africa and testing the hypothesis of delayed adaptive immune response with increasing glycemia.
The overall aim of the project was to improve knowledge on the dynamic interaction between TB and DM in an African setting with high HIV prevalence by integrating a longitudinal component into the case-control study.
Methods
A case-control study with longitudinal follow-up of cases was conducted in Dar es Salaam. Consecutive adults with new active TB were included and followed up for five months after the start of anti-TB treatment. Healthy controls, matched by age and sex to TB cases, were recruited among volunteering adults accompanying patients to the outpatient departments of the same hospitals. Exclusion criteria were a biological relationship to TB case, TB history, symptoms or signs of TB, other acute infection or major trauma within the last three months. All underwent 25-hydroxyvitamin D (25(OH)D) measurement and DM screening tests (fasting glucose (FCG), 2-hour capillary glucose after standard oral glucose tolerance test (2h-CG) and glycated hemoglobin (HbA1c)) at enrolment and TB patients were again tested after five months of TB treatment. Data on the outcome of TB (treatment failure, death, lost to follow-up) were collected.
For the nested immunological study, four groups of HIV negative patients were included: i) active TB without DM, ii) active TB with DM, iii) latent TB patients without DM and iv) latent TB patients with DM. Latent TB patients were selected among the healthy volunteering adults, as well as among diabetic patients attending the DM clinic in the participating hospitals. Exclusion criteria for groups iii and iv were past TB history and symptoms or signs of active TB. Peripheral blood mononuclear cells were stimulated with Mycobacterium tuberculosis (Mtb)-specific peptide pools and live Mycobacterium bovis BCG and then analysed by polychromatic flow cytometry for Th1, Th2, Th9 and Th17 cytokine production. Cell culture supernatants were analysed by Luminex® for 34 cytokines and chemokines.
Findings
At enrolement, DM prevalence was significantly higher among TB patients (N=539; FCG>7mmol/L: 4.5%, 2-hCG>11mmol/L: 6.8% and HbA1c>6.5%: 9.3%) compared to controls (N=496; 1.2%, 3.1% and 2.2%). However, the association between hyperglycemia and TB disappeared after TB treatment (aOR(95% CI) at enrolment vs follow-up: FCG 9.6(3.7-24.7) vs 2.4(0.7-8.7); 2-hCG 6.6(4.0-11.1) vs 1.6(0.8-2.9); HbA1c 4.2(2.9-6.0) vs 1.4(0.9-2.0)). FCG hyperglycemia at enrolment was associated with TB treatment failure or death (aOR(95%CI) 3.3(1.2-9.3).
The prevalence of 25(OH)D insufficiency (25(OH)D<75nmol/l) was not statistically different between TB patients and controls (25.8% versus 31.0%; p=0.22). But the association between 25(OH)D insufficiency and TB was modified by hyperglycemia (pinteraction=0.01). Patient with vitamin D insufficiency were only at higher risk for TB in the presence of underlying hyperglycemia. The OR (95%CI) for TB risk in patients with vitamin D insufficiency and hyperglycemia was 4.94(1.16-21.0) versus 0.68(0.39-1.17) for patients with vitamin D insufficiency and normoglycemia where normoglycemia and normal vitamin D were the reference category.
Patients with active TB and DM had a lower frequency of INF-γ CD4+ T cells and a lower proportion of CD4+ T cells producing both TNF-α and IFN-γ after live M. bovis BCG but not after Mtb-specific peptide pool stimulation, compared to normoglycemic TB patients. A negative correlation between INF-γ or TNF-α CD4+ T cell frequency and increasing glycemia was observed in the context of live M. bovis BCG stimulation only.
Conclusions
Transient hyperglycemia is frequent during TB, and DM needs confirmation after TB treatment. However, DM screening at TB diagnosis gives the opportunity to detect patients at risk of adverse outcome.
25(OH)D insufficiency seams to increase the risk of TB only if associated with hyperglycemia. DM patients living in high TB burden settings might benefit from preventive vitamin D supplementation.
The immunological findings suggest that DM might affect Mtb-specific CD4+ T cell immune responses at the level of reduced antigen processing and presentation, a defect that could be compensated by metformin.
The results of the study are of public health and clinical utility. First, they lend support to the integration of care between TB and DM programs. Second, they imply that, at the time of TB diagnosis, patients should be screened for hyperglycemia using cost-effective fasting glucose tests. Treatment of hyperglycemia should be initiated to improve TB outcome. Third, before initiation of long-term DM treatment, DM diagnosis must be confirmed after the resolution of TB. Finally, in the absence of evidence for a strong contribution of DM to TB risk in this African setting with high HIV prevalence, DM patients should not be screened for TB with expensive test. DM physicians and patients should rather be trained for recognizing TB symptoms and signs as a cost-effective way to recognize TB early
Analysis of expression of PDCP and MAL13P1.308 of Plasmodium falciparum employing a quantitative proteomics approach based on SILAC
No full textPlasmodium falciparum is a deadly parasite that causes malaria in humans. This disease causes the death of one million people every year. To find new means to fight this parasite, it is important to learn more about its biology. As the pathways of protein expression are better understood, it becomes easier to find out how to block these mechanisms. The completion of the genome sequencing has opened new perspective of genome wide analysis. One of these studies was done by LaCount et al. who used the yeast1two1hybrid system to map the complete interaction network between proteins of P. falciparum. ¨ From this network of interaction an interesting protein was studied further by Daubenberger et al. which is PDCP. This protein is closely related to MSP11 in this protein network, a protein involved in erythrocyte invasion. PDCP is a CCCH1type zinc finger protein, a family of proteins that are involved in protein1protein interaction, nucleic acid binding and binding in small ligands. It was shown that its expression was dependant on the density of the parasites. In this study we used the SILAC technology to confirm these previous results. A culture is grown with isotopically heavy isoleucine in the medium as a control sample. After a few cell cycles, almost all the natural isoleucines are replaced with the labeled ones. Three cultures with parasitemia of 2, 5 and 10% are grown and mixed with an equal amount of the control culture. When the proteins are analyzed by mass spectrometry, the peptides that contained isoleucine will be detected as two separate peaks. After normalization of each peptide area with this internal control, the quantity of PDCP can be compared between cultures of different parasitemias. 11 peptides of PDCP have been detected by mass spectrometry, proving its existence for the first time. These peptides were labeled to more than 95%, allowing the comparison of PDCP expression between the cultures. But because of difficulty of reproducibility in in vitro cultures, the regulation of PDCP by the parasitemia has not been observed by SILAC. We wanted to study further the protein interaction network in which PDCP is involved. MAL13P1.308, a protein directly interacting with PDCP and MSP11 in the protein network map, was analyzed by IFA. It was found to be expressed during all stages of the asexual blood stage cycle and it was not imported in the host cell. The next step will be to see if it interacts with PDCP by co1localization studiesSSVSwiss Tropical Institute, Proteomics group; F. Hoffmann La Roche Ltd; Global Health Institute, EPF
Understanding drivers of experimental malaria sub-unit vaccine induced immunity in Tanzania volunteers
No full textDespite being a preventable and treatable disease, Plasmodium falciparum malaria remains a major threat, especially in children and pregnant women in sub-Saharan Africa. Considerable progress has been achieved during the past decade, however, these positive trends have stalled in 2017. Efforts towards better disease control and focal elimination are hindered by development and spread of insecticide and drug resistance, leaving a malaria vaccine as a required tool to complement these approaches. RTS, S a subunit pre-erythrocyte stage vaccine is the only advanced malaria vaccine that has received approval for pilot administration in three countries in sub-Saharan Africa. This vaccine is however challenged by low efficacy and fast waning of protection. There is, therefore, an urgent need for the development of more potent malaria vaccines. WHO targets malaria elimination by 2030 and achieving this goal will depend on stopping malaria transmission. This goal will largely depend on reducing asexual blood stage Plasmodium parasites – which are not only the cause of morbidity and mortality -but also responsible for the development of gametocytes. Induction of parasite growth inhibitory antibodies has been shown to be key for protection following natural exposure and therefore, many vaccine development approaches try to follow this guidance from nature.
In order to reach this goal of a highly protective vaccine targeting asexual blood stages with acceptable longevity of duration, more research is needed understand mechanisms of optimal induction of long-lived antibody responses in a population that is also affected from other co-infections like helminths or HIV. Therefore, this thesis aimed to 1) investigate a novel blood stage sub-unit malaria vaccine candidate, P27A, for its potential to induce long-lasting antibody responses when formulated in the novel adjuvant GLA-SE in malaria pre-exposed populations, 2) understanding magnitude and cytokine production of the CD4 T cell responses induced by this novel vaccine formulation and the interaction with ongoing helminth co-infections, 3) shed more light on the mechanism of GLA-SE adjuvant being able to induce high and long-lasting antibody responses by studying follicular helper T cells in peripheral blood, 4) implement lymph node excision biopsy in rural Tanzania for detailed investigation of germinal centre responses which are crucial for production of potent antibody response.
The antigen P27A, when formulated with GLA-SE, induced a robust humoral immunity, with enhanced production of cytophilic antibodies, IgG1 and IgG3 and expansion of CD4 Th1 cells producing IL2, TNFa and IFNg, and subsequent memory development. In addition, the adjuvant GLA-SE promoted the expansion of peripheral follicular helper T cells and recruitment of T cells bearing common T cell receptors, which is essential for a vaccine intended for the general population
Developing, testing and implementing novel molecular diagnostic tests and procedures for malaria at the individual and population level in East and West Africa
No full textMalaria is a global public health issue responsible for an estimated 219 million cases and
435’000 deaths in 2017. Although malaria control interventions have led to a significant decrease
in mortality over the past decade, no further progress in reducing global malaria burden
was achieved since 2016. Novel malaria control approaches, such as highly effective
vaccines and improved diagnostic tools are needed to prevent a resurgence of malaria as has
been seen in the 1970s.
The overall goal of this PhD thesis is to contribute to the development, validation and implementation
of novel molecular diagnostic tools in malaria endemic countries targeting all human
infective malaria species. This thesis was conducted within the framework of six clinical
trials evaluating the PfSPZ Vaccine, an experimental malaria vaccine based on the immunization
with radiation-attenuated whole, purified, cryopreserved, metabolically active sporozoites
in Tanzania and Equatorial Guinea. We used controlled human malaria infection (CHMI)
to assess vaccine efficacy in these cohorts. Compared to large field studies, CHMI studies
allow to test various vaccine regimen and dosing in smaller, well defined cohorts in a short
time period.
In part one, we aimed to closely monitor malaria parasites at the individual level by establishing
a robust laboratory qPCR-platform for molecular monitoring of asexual blood stage parasitaemia.
This platform was used to evaluate the efficacy of PfSPZ Vaccine by homologous
CHMI in Tanzanian and Equatorial Guinean adults. Part one was structured around four
manuscripts:
Manuscript 1-3: Safety, immunogenicity and efficacy of attenuated whole sporozoite
vaccines in Tanzanian and Equatorial Guinean volunteers.
In clinical trials conducted in malaria pre-exposed volunteers from Tanzania and Equatorial
Guinea, the PfSPZ Vaccine was found to be well tolerated and safe, even in high-risk populations
such as infants and HIV+ volunteers. The use of CHMI in malaria pre-exposed adults
has been proven to be highly effective. These trials revealed that the degree of immune response
and high level of protection seen in previous studies in malaria-naïve volunteers, was
not reproducible to the same level in Tanzanian and Equato-Guinean populations.
Manuscript 4: Controlled Human Malaria Infections in Tanzania and Equatorial Guinea:
The impact of malaria pre-exposure on diagnosis and multiplication rate of P. falciparum
parasites
We used the parasite multiplication rate (PMR), the fold change in number of parasites in
peripheral blood over one life cycle, to characterize the impact of malaria pre-exposure on the
outcome of CHMI. Compared to malaria-naïve German volunteers, the parasite’s ability to
replicate was significantly reduced in Tanzanian and Equato-Guinean volunteers when challenged
with the identical dose, strain and route of cryopreserved, purified P. falciparum sporozoites.
We used two different approaches to demonstrate the impact of malaria preexposure
on the multiplication rate of P. falciparum parasites. First, we showed that recently
drug-cleared parasitaemia from an ongoing P. falciparum field infection or P. falciparum and
P. malariae co-infection leads to a significantly reduced PMR. Second, PMR is significantly
reduced after two consecutive homologous CHMI conducted in the same volunteer at 7
months interval. This data indicates the rapid acquisition or recall of parasite growth reducing
immune effector mechanisms limiting the growth of asexual blood stages in vivo.
In part two, we aimed to adapt and extend molecular diagnostic tools for improvement of
malaria surveillance at the population level by developing novel high-throughput, fielddeployable
and highly sensitive tools and approaches. Part two was structured around five
manuscripts:
Manuscript 5: A multiplex qPCR approach for detection of pfhrp2 and pfhrp3 gene deletions
in multiple strain infections of Plasmodium falciparum
In response to the emerging threat of P. falciparum strains lacking pfhrp2 and/or pfhrp3
genes and therefore evading detection by PfHRP2-based RDTs, we developed a qPCR-based
assay well suited for high-throughput identification of pfhrp2 and pfhrp3 gene deletions in P.
falciparum isolates. The ability to detect “masked” pfhrp2/3 deletions is probably the most
interesting feature of our approach, because it will allow to study the epidemiology of
pfhrp2/3 deletions in malaria endemic regions where a high proportion of the population carries
multiple strain P. falciparum co-infections.
Manuscript 6: High Throughput Extraction and Analysis of Nucleic Acids from Rapid
Diagnostic Tests for Molecular Surveillance of Malaria
This manuscript describes our attempt to improve extraction of nucleic acids from used malaria
RDTs. We used an approach which enabled us to extract total nucleic acids, including
DNA and RNA from these RDT strips. This approach increased the overall sensitivity for the
detection of Plasmodium spp. parasites in the small volume of blood which is available on an
RDT stored at room temperature. We used samples collected during CHMI as a platform to
evaluate and compare the ability of our protocol to detect and quantify P. falciparum parasites
with microscopy and qPCR.
Manuscript 7: ELIMU-MDx: A Web-Based, Open-Source Platform for Storage, Management
and Analysis of Diagnostic qPCR Data
We developed a web-based and open-source software for storage, management and analysis
of diagnostic qPCR data. In response to the vast amount of qPCR data generated during a
series of CHMIs and other epidemiological studies conducted in Tanzania and Equatorial
Guinea, we decided to design and build this platform which facilities the larger scale analysis
and interpretation of diagnostic qPCR data.
Manuscript 8: Molecular monitoring of the diversity of human pathogenic malaria species
in blood donations on Bioko Island, Equatorial Guinea
In collaboration with the central blood bank in Malabo, Equatorial Guinea we analysed 200
blood donations for the presence of Plasmodium spp. parasites by a novel, multiplex qPCR
monitoring all human infective malaria species in a single reaction. We found that more than
one quarter of the blood donations contained malaria parasites and that 75% of P. falciparum
and 100% of P. malariae and P. ovale spp. infections were missed by routinely performed
RDT and microscopy.
Manuscript 9: Two cases of long-lasting, sub-microscopic Plasmodium malariae infections
in adults from Coastal Tanzania
In this report we describe two cases of P. malariae infections that were identified during a
study evaluating the safety, tolerability and efficacy of the PfSPZ Vaccine in Bagamoyo,
Tanzania. Since these two adult participants were enrolled into a clinical trial, we were provided
with a unique opportunity to study clinical manifestations of P. malariae over a follow
up period of four months
Cellular and humoral immunity in malaria pre-exposed Tanzanian children and adults following vaccination with RTS,S, the most advanced malaria vaccine, and after whole sporozoite based controlled human malaria infections
No full textIntroduction
Malaria is caused by intracellular organisms that belong to the genus Plasmodium. In 2015, there were an estimated 438,000 deaths and 214 million clinical illnesses due to malaria infection, of which the majority were in sub-Saharan African children below five years of age. Amongst the five species that are known to infect humans, Plasmodium falciparum causes the most severe disease, mostly in children and pregnant women in sub-Saharan Africa. Despite malaria control programs being operational for many years, malaria elimination in most endemic regions is far from being achieved. Vaccination is considered the most cost effective method of preventing infectious diseases. To date, there are no effective vaccines available for parasitic infections, despite the existence of strong evidence of acquired immunity in most parasitic infections studied. It is therefore highly likely that the addition of an effective tool such as a vaccine to the current malaria control strategy would have a strong positive impact on our ability to control this disease. In the first part of this thesis, we aimed to investigate the vaccine efficacy as well as the cellular and humoral immunity of African paediatric volunteers vaccinated with the most clinically advanced malaria vaccine; the RTS, S/AS01.
Meanwhile, novel vaccination and testing approaches are being pursued to improve or replace the recombinant subunit malaria vaccine approach to meet the goals formulated in the Malaria Vaccine Roadmap of WHO (http://www.who.int/immunization/topics/malaria/ vaccine_roadmap/en). These goals strategized that by 2030, licensed vaccines targeting Plasmodium falciparum and Plasmodium vivax should encompass the following two objectives, for use by the international public health community:
i) First, it should have a protective efficacy of at least 75 percent against clinical malaria and be suitable for administration to appropriate at-risk groups in malaria- endemic areas.
ii) Secondly, it should reduce transmission of the parasite and thereby substantially reduce the incidence of human malaria infection; enable elimination in multiple settings and be suitable for administration in mass campaigns.
Currently, the most promising candidate seems to be the whole malaria sporozoite approach, which is formed of cryopreserved, purified whole live-attenuated (either by irradiation or genetic attenuation) sporozoites. One of the novel tools used to analyze induced vaccine efficacy in sub-Saharan Africa experimentally vaccinated volunteers is controlled human malaria infection (CHMI). Many CHMIs using infectious mosquito bites or purified sporozoites have been successfully conducted in the USA and Europe over many years, but this approach had not been employed in sub-Saharan Africa until 2012. The aim of the second part of this thesis was to describe the potential of using CHMI as a tool to accelerate malaria vaccine development in sub-Saharan Africa and to dissect malaria- specific immunity induced by CHMI based on our trial conducted in 2012 in Bagamoyo.
Methods and findings
In the first part of this thesis (Chapter 4), the aim was to investigate safety, efficacy, cellular and humoral immunity in RTS,S/AS01 vaccinated Tanzanian paediatric populations. Adverse events were used to determine the safety of the RTS,S/AS01 vaccine in this age group (paper I), ELISA to measure the vaccine-induced CS-specific antibodies and Luminex to measure vaccine-induced cytokine responses (paper II and III). Furthermore, flow cytometry was used to investigate vaccine-induced cellular immune responses (paper III). We also looked into the implications and practicalities of immunological sampling in the African paediatric population. We did community sensitization and collected blood samples from 400 children for immunological study (paper IV). We showed that in 6-12 week old infants, vaccine efficacy against clinical malaria 14 months after first vaccination was 30.1% (95% CI, 23.6 to 36.1) in the intention-to-treat (ITT) and 31.3% (97.5% CI, 23.6 to 38.3) in the per-protocol (PP) population. Furthermore, the vaccine efficacy against severe malaria was 26.0% (95% CI, −7.4 to 48.6) and 36.6% (95% CI, 4.6 to 57.7) in the ITT and PP populations, respectively. The safety of the vaccine in terms of serious adverse events showed similar trends in both study groups. We identified two main RTS,S/AS01 vaccine induced cellular immune mechanisms:- (i) Th1-related responses such as CS-specific IFN-g, GM-CSF and IL-15 are associated with protection and (ii) Th2-related responses mediated by CS-specific IL5 and RANTES are associated with increased odds of malaria. Moreover, antibody avidity alone did not predict protective efficacy in the current study. The induction of RTS, S/AS01 protective Th1 and pro-inflammatory responses was lower in infants compared to children; a scenario that might explain the lower efficacy observed in the infant cohort. Furthermore, we also showed that immunology studies in the paediatric population can feasibly be conducted in African research institutions.
In the second part of this thesis (Chapter 5), we conducted in 2012 the first CHMI using cryo-preserved purified non-attenuated sporozoites in Tanzanian adult volunteers with previous malaria exposure (paper V). In this study, the humoral and cellular immune responses elicited following CHMI were evaluated (paper VI and VII). We used adverse events to determine the safety of the CHMI model in malaria pre-exposed volunteers. We also used blood slide microscopy to define sporozoite infectivity rates, Luminex assays to examine the sporozoite-induced antibodies, B-cell Elispot analysis, single cell RNA sequencing, flow cytometry and cell sorting followed by in vitro stimulation assays to investigate and define the affected innate and adaptive immune responses following CHMI (paper VIII). Our studies showed that: (i) CHMI is safe, tolerable and infective when used in malaria endemic regions, (ii) a single dose of intradermal sporozoite (PfSPZ) challenge elicited long-lived merozoite-opsonizing antibodies and long-lasting innate and innate-like lymphocyte populations, (iii) When we compared Dutch (malaria naïve) and Tanzanian (malaria exposed) subjects undergoing the same challenge study, Dutch subjects responded differently to PfSPZ challenge compared to Tanzanian (malaria pre-exposed) subjects.
Conclusion
Substantial investment in research and development is needed to develop a highly efficacious malaria vaccine. To date, the recombinant subunit vaccines are yet to give the desired levels of protection for malaria elimination but seem to prevent malaria disease in high transmission settings. Large scale manufacturing, storage and distribution of live whole malaria sporozoite-based vaccines for mass administration need further development. So far, data generated from the PfSPZ vaccine trials conducted in the USA, Europe and in African research institutions imply that malaria naive individuals respond better to malaria vaccines than malaria pre-exposed individuals. The question remains to be, “what exactly constitutes the reason for lack of durable protection against malaria infection in endemic areas?” The most important factor in accelerating future vaccine development is a better understanding of the biology and nature of acquired immunity, which will lead to improved vaccine design. We have established the foundation for using CHMI to assess efficacy of new interventions against malaria and to study the mechanisms of the lack of protection conferred by different malaria vaccines in endemic settings. This study has opened new doors in the field of malaria intervention, whereby malaria vaccine and drug efficacy can be easily tested using CHMI in the target population
Molecular epidemiology of Mansonella perstans on Bioko Island: identification of risk factors, co-infection with malaria and Loa loa and impact in pregnant women
No full textImplementation of an effective and targeted mass drug administration program against filarial worms needs an accurate identification and mapping of the distribution of these parasites. Molecular diagnostic methods like polymerase chain reaction (PCR) based techniques have shown high sensitivity to detect and distinguish parasites at low infection levels and even in people that are amicrofilaremic by microscopy. However, larger scale implementation over time remains a challenge for low-income countries because of logistical complexity and costs, including collection, transportation and preservation of biological samples and running of the analytical tests.
In this study, we aimed to develop and implement a novel molecular based diagnostic approach for monitoring highly neglected filariasis causing parasites in Equatorial Guinea, West Africa. The ENAR protocol is an approach based on the use of nucleic acids (NA) extracted from dried blood retained on used malaria rapid diagnostic test (mRDT). Malaria RDTs used in this work were stored at room temperature for around 18 months before used for molecular analysis and served as reliable source for NA based detection of blood-dwellings pathogens beyond malaria. Our approach of repurposing used mRDTs for filarial parasites detection provides a versatile and cost-effective approach to monitor prevalence, genotype, infection intensity and co-infections of filarial nematodes and other blood borne infectious diseases
Heterogeneity in Plasmodium falciparum whole sporozoite vaccine induced humoral immune responses and protection in African volunteers: the role of age, human pegivirus and human immunodeficiency virus co-infections.
No full textMalaria, a vector borne disease caused by Plasmodium species remains a major public health problem especially in sub-Saharan Africa. In 2018 alone, there were an estimated 228 million clinical cases and 405,000 deaths attributed to malaria. New tools such as efficacious vaccines, better drugs and diagnostics are needed to supplement the current malaria control tools that rest mainly on vector control measures. Clinical trials in malaria naïve volunteers have demonstrated high level of sterile vaccine induced protection in healthy individuals immunized with live, metabolically active, irradiation attenuated purified sporozoites (PfSPZ Vaccine) or live non-attenuated purified sporozoites given under chloroquine chemoprophylaxis (PfSPZ-CVac). Immunogenicity and protective efficacy against malaria induced by these whole sporozoite based vaccines varies widely between European/US versus African volunteers. Interestingly, variations in protection and immunogenicity of these malaria vaccines have been observed among African volunteers residing in different malaria endemic regions of East and West Africa. These different outcomes could be linked to levels of malaria pre-exposure and co-infections at the time of vaccination. Until recently safety, tolerability, immunogenicity and protective efficacy of PfSPZ Vaccine in immunocompromised populations including individuals infected with the Human Immunodeficiency Virus (HIV) was unknown. Given the high geographical overlap of HIV and malaria, an effective malaria vaccine deployed in this population could possibly support the long-term objective of regional elimination of malaria by using mass vaccination. Furthermore, the role of asymptomatic, under researched, highly prevalent viruses like Human Pegivirus (HPgV-1) circulating in Sub-Saharan Africa on malaria pathogenesis and vaccination outcomes remains elusive.
The aims of this PhD thesis include: 1) Evaluate the safety, tolerability, efficacy, and immunogenicity of the PfSPZ Vaccine in different populations and age groups residing in malaria endemic countries. 2) Compare the safety, tolerability, efficacy, and immunogenicity of two different whole sporozoite based vaccination approaches, PfSPZ Vaccine and PfSPZ-CVac in Equatorial Guinean adults. 3) The unbiased assessment of PfSPZ Vaccine induced humoral immunity using protein microarrays probed with serum samples of HIV positive and HIV negative volunteers to understand immune status before vaccination, immuno-dominance of vaccine induced antibody targets, and distinct antibody profiles that might be associated with vaccine-induced protection. 4) Investigate the interaction of chronic HPgV-1 co-infection on PfSPZ Vaccine induced humoral immunity and protection against homologous CHMI. These aims are structured around 6 manuscripts presented in this PhD thesis.
Manuscript 1-3: Safety, immunogenicity, and efficacy of radiation attenuated whole sporozoite vaccine (PfSPZ Vaccine) in African populations of different ages
In this chapter, we include the outcomes of clinical trials conducted in Bagamoyo, Tanzania. These trials for the first time i) evaluated the safety, immunogenicity and efficacy against homologous CHMI of irradiation attenuated purified Plasmodium falciparum sporozoites (PfSPZ Vaccine) in adult volunteers; ii) tested increasing dosages of PfSPZ Vaccine in different age groups including adults, adolescents, children and infants. We found PfSPZ vaccine to be safe and well tolerated and that vaccine inoculation by intravenous inoculation is well accepted even in younger age groups. Protective efficacy varied in the different trials leading to the identification of a vaccine regimen of 9x105PfSPZ per dose as suitable for further development. PfSPZ Vaccine induce immune responses, both cellular and humoral, were age dependent with infants mounting no measurable malaria specific cellular immunity in peripheral blood. Surprisingly, compared to other age groups, older children and adolescents mounted higher cellular and humoral immune responses. These findings are relevant for further optimization of PfSPZ vaccine regimen that might need to be adapted to different age groups to optimize vaccine induced protection. As an extension of these trials, we have compared PfSPZ Vaccine safety, immunogenicity and efficacy in HIV positive versus HIV negative volunteers. We observed marked differences in PfSPZ Vaccine induced efficacy between HIV positive (0%) and HIV negative individuals (80%) undergoing homologous CHMI(Manuscript in preparation).
Manuscript 4: Immunogenicity and protective efficacy of radiation-attenuated and chemo-attenuated PfSPZ vaccines in Equatoguinean adults
This work describes the outcome of a first time side-by-side comparison of two whole sporozoite based vaccine approaches (PfSPZ Vaccine and PfSPZCVac) in malaria pre-exposed individuals of Equatorial Guinea. We evaluated PfSPZ Vaccine dosages (2.7 X106) given three times at 8-week interval and PfSPZ CVac dose (1X105) given three times at 4 weeks interval. Homologous CHMI was employed for assessment of vaccine efficacy. Both approaches were safe and well tolerated in malaria pre-exposed individuals but the immunogenicity and protective efficacy differed. Vaccine efficacy was lower in the PfSPZ Vaccine group (27%) compared to the PfSPZ CVac group (55%), despite induction of about 2.9 times higher antibody titres against the circumsporozoite protein in the PfSPZ Vaccine group prior to CHMI. These results highlight the potential involvement of different protective immune mechanisms induced by each of the two whole sporozoite vaccines approaches and the effect of malaria pre-exposure on pfSPZ CVac vaccine induced efficacy in comparison to malaria naïve volunteers. We show that induction of high antibody titres against the circumsporozoite protein does not correlate with protection since no difference was observed between CHMI protected and non-protected volunteers.
Manuscript 5: HIV-1 positive and HIV-1 negative Tanzanian adults undergoing whole irradiation attenuated Plasmodium falciparum sporozoite vaccination mount antibody responses targeting the circumsporozoite protein and merozoite surface protein 5
In this manuscript, we investigated antibody profiles binding to 262 pre-selected antigens of Pf before and after vaccination as well as after homologus CHMI. We aimed to identify antibody profiles that might explain the observed poor vaccine induced protection in HIV positive individuals. We found a lower - albeit not statistically significant - antigen breadth in HIV positive volunteers at baseline before first vaccine inoculation. Immunization with PfSPZ Vaccine induced IgG and IgM isotypes specific for the Merozoite surface protein 5 (PfMSP 5) and the circumsporozoite protein (PfCSP) regardless of HIV infection status. Interestingly, volunteers displayed a highly personalized IgG and IgM immune profiles targeting Pf antigens before vaccination and these remained unchanged after PfSPZ vaccination confirming our previous results of antigenic imprinting in malaria.
Manuscript 6: Role of Pegivirus infections in whole Plasmodium falciparum sporozoite vaccine induced humoral immunity and controlled human malaria infections in African volunteers
In this study, we wanted to understand the role of human pegivirus infections in East and Western African adult volunteers and its impact on PfSPZ Vaccine induced humoral immune responses and homologous CHMI. We found HPgV-1 to be highly prevalent in our volunteers (29.2%) with circulating genotypes 1, 2 and 5 as described in other African settings. HPgV-1 infection did not alter PfSPZ vaccine induced antibody responses and parasite multiplication rates during CHMI. However, a higher proportion of individuals were protected against homologous CHMI that had ongoing, active human pegivirus infections. Significantly higher serum concentrations of IL-2 and IL-17A were measured in HPgV-1 positive volunteers likely indicating chronic activation of the immune system. CHMI was safe and well tolerated in HPgV-1 positive individuals since the viremia did not change upon acute asexual blood stage parasitemia. These results highlight the potential impact of chronic, asymptomatic viral infections on PfSPZ vaccine efficacy that needs confirmation in larger cohorts and in field studies of naturally occurring malaria infections
Improved treatment options for the control of soil-transmitted helminthiasis : from repurposed drugs to reinfection patterns
No full textBackground: Soil-transmitted helminths (STHs) are a group of intestinal dwelling parasitic nematode worms that disproportionally affect socio-economically deprived populations in warm and tropical environments living with inadequate sanitation, poor hygiene and unsatisfactory educational coverage. Intestinal helminths are transmitted through contamination of soil with human feces and by subsequent accidental ingestion of soil with parasite eggs (Ascaris lumbricoides, Trichuris trichiura and occasionally Ancylostoma duodenale) or by penetration of the worm larvae (hookworm) into the skin and body of the human host. Soil-transmitted helminths are responsible for the largest burden of neglected tropical diseases, with about 1.5 billion infected people worldwide. Although most infections are asymptomatic and of light intensity, heavier intensity infections can cause severe morbidity. Chronic high-intensity manifestations caused by STH infections can lead to physical and intellectual growth retardation, perpetuating a vicious cycle of poor health and poverty. The World Health Organization (WHO) has advocated targeted preventive chemotherapy (PC), the periodic mass drug administration (MDA) of single dose benzimidazoles (i.e., albendazole and mebendazole) to at-risk population groups, without prior diagnosis. The main goal of this strategy is to reduce morbidity by decreasing infection intensities and to ultimately eliminate STH infections as a public health problem. This is defined as the decrease of prevalence of moderate and heavy infection intensities to below 2% as assessed in preschool- and school-aged children by 2030. However, several factors might jeopardize the success of PC, including the low efficacy of the currently used benzimidazoles, its inability to prevent reinfections and the potential emergence of anthelmintic resistance due to mounting drug pressure. On Pemba Island, Tanzania, STH infections were recognized as a major public health problem in the early 1990s. Since then, PC has been widely implemented (coverage rate >80%), but STH prevalence remains high to date. Hence, development of new and safe broad-spectrum drugs, repurposing of available drugs or the use of drug combinations to expand the armamentarium of treatment options is of paramount importance to help control and eliminate STH infections.
Goal and specific objectives: The first objective of my PhD was to test the efficacy and safety of ascending doses of moxidectin alone or combined with albendazole (400 mg) against trichuriasis. The second objective was to evaluate the short-term and long-term outcomes 14-21 days, six and 12 months post-treatment of ivermectin-albendazole and albendazole alone in an expanded study population (6-60 years) aiming to inform and update STH control guidelines and programs. The third objective was to compare the performance of the microscopic Kato-Katz method to the molecular polymerase chain reaction (qPCR) and its impact on drug efficacy and day-to-day variation. The fourth objective was to test fecal calprotectin (FC) and fecal occult blood (FOB) as potential surrogate markers for STH attributable morbidity. Insights gained from the ivermectin-albendazole trial on trial methodology, trial procedures and mitigation strategies to overcome challenges faced during clinical research taking place in resource-limited environments is presented as fifth objective.
Methods: This PhD work consisted of two clinical trials. The first was a phase II, randomized, placebo-controlled, dose-finding study on moxidectin in adolescents aged between 16 and 18 years on Pemba Island in 2018. Screened individuals were asked to provide two stool samples at baseline to assess STH ova by the Kato-Katz method. Eligible adolescents were physically examined and questioned for clinical symptoms by a trial physician prior to treatment administration. Trichuris trichiura-infected adolescents were randomly assigned to seven treatment arms: 8, 16, or 24 mg of moxidectin monotherapy; 8, 16, or 24 mg of moxidectin plus 400 mg of albendazole combination therapy; or placebo. The primary outcome was cure rate (CR) against T. trichiura, analyzed 13 to 20 days post-treatment. Adverse events were assessed 3h, 24h, 48h, 72h and 13-20 days after treatment, graded on severity, relatedness and expectedness as specified in the trial protocol. The second study was a Phase III, multi-country, randomized, standard of care-controlled, blinded, parallel group, single dose, superiority trial on ivermectin-albendazole in Côte d’Ivoire, Lao People’s Democratic Republic (Lao PDR) and on Pemba Island, Tanzania between 2018 and 2020. The study was conducted in communities aged 6-60 years. Screened individuals provided two stool samples at baseline, 14-21 days, six and 12 months post-treatment. Similar to the first study, the Kato-Katz method was employed for STH diagnosis and, in addition, an aliquot of stool (~1 g) was mixed with 80% ethanol and preserved at 4°C and later shipped at room temperature to Swiss Tropical and Public Health Institute in Basel, Switzerland for subsequent qPCR analyses. Furthermore, fecal rapid tests (FC and FOB), were used as potential proxy markers for STH attributable morbidity. Hence, a semi-quantitative chromatographic immunoassay (Actim® Fecal Calprotectin test/Actim® Fecal Blood test, Medix Biochemica, Finland) was applied for FC and FOB detection from participants diagnosed positive for T. trichiura and concomitant STH infections and identified STH negative participants as controls. Before treatment administration, all participants underwent a physical examination and a rapid diagnostic test for hemoglobin levels, pregnancy in all female participants (≥12 years), malaria (Côte d’Ivoire and Lao PDR) and lymphatic filariasis (Côte d’Ivoire and on Pemba Island, Tanzania) was applied. Drug efficacies (in terms of egg reduction rates (ERRs) and CRs), reinfections and new infections were assessed 14-21 days, six and 12 months post-treatment. Adverse events were captured 3h, 24h and 14-21 days after treatment, graded on severity, relatedness and expectedness as specified in the trial protocol.
Results: We found that 8 mg of moxidectin (the lowest tested dose) performed as well as 16 mg and 24 mg, and that the combination of moxidectin and albendazole was significantly more efficacious against T. trichiura than albendazole alone. Likewise, we revealed superiority of the ivermectin-albendazole combination therapy compared to albendazole alone against T. trichiura infections in Lao PDR and on Pemba Island. Similarly, the ivermectin-albendazole combination therapy led to a larger reduction of moderate and heavy T. trichiura infections and successfully reduced the prevalence of these infections to below 1.5% within 12 months. However, ivermectin-albendazole was not found to be superior to albendazole alone in Côte d’Ivoire. Moreover, we observed a higher sensitivity of qPCR compared to quadruple Kato-Katz, revealing significantly lower CRs for ivermectin-albendazole, when two qPCR samples were assessed pre- and post-treatment. In addition, we did not find an association between the presence of intestinal inflammation or mucosal bleeding, assessed with FC and FOB as respective proxy markers, and STH infection status or infection intensity.
Conclusion: Promising efficacies and safety for moxidectin-albendazole and ivermectin-albendazole against T. trichiura were found. Hence, both drug combinations might be valuable alternatives in PC programs. The combination of 8 mg moxidectin and 400 mg albendazole should be further investigated in younger age groups, with longer follow-up periods and in different settings to help guiding recommendations for future STH control. Prior to MDA implementation with ivermectin-albendazole, careful decisions on the frequency of deworming adapted to the epidemiological parasite profile in each setting have to be made, while variations in treatment responses should be considered. In addition to that, standardized and accurate molecular diagnostic tools, which are applicable in peripheral field settings for the assessment of drug efficacy and for future monitoring within STH control and/or elimination programs, should be developed. Further studies are needed to identify suitable, standardized, low-cost proxy markers of STH attributable morbidity to monitor the clinical impact of STH control interventions. A strategic plan adapted to each setting with a distinct focus on community engagement and workforce is crucial for successful preparation, screening and implementation of randomized controlled trials. Gained knowledge on improvements of trial methodology, trial procedures and mitigation strategies to overcome challenges faced during clinical research in resource-constrained healthcare environments are valuable information that should be made available to the related research network. Moreover, potential drug donors and preferably local anthelmintic drug production facilities will need to be identified to meet the demand for STH control programs
Bacteriological and immunological studies towards effective control of "Mycobacterium ulcerans" disease (Buruli ulcer)
No full textAfter tuberculosis and leprosy, Buruli ulcer (BU) which is caused by Mycobacterium
ulcerans, is the most common mycobacterial infection in immuno-competent humans.
Since the 1980s BU has gained significant public health importance in the tropics
especially in West Africa, including Ghana. The establishment of control measures is
hampered as a result of the scarcity of understanding of many features of the disease.
Priority areas for research defined by WHO include: understanding the mode of
transmission, development of simpler methods for early diagnosis, development of
effective antibiotic treatment, and the understanding of protective immune responses to
support vaccine development.
The availability of M. ulcerans isolates from endemic areas is necessary for detailed
transmission studies and the analysis of efficacy of antibiotics for the treatment of BU.
However, cultivation of M. ulcerans from clinical specimens is burdensome; reported
recovery rates are as low as 20%. We evaluated four different decontamination methods
and one non-decontamination procedure in combination with four egg-based media for
the primary isolation of M. ulcerans from tissue specimens excised from BU lesions.
Oxalic acid decontamination and culture on LJ medium supplemented with glycerol was
the most efficient procedure and achieved a recovery rate of 75.6%. The success of
cultivation depended also on a good sampling procedure. The use of the optimised
cultivation method has allowed the production of a large isolate collection.
For efficient case management and confirmation of epidemiological data, it is necessary
to reconfirm clinical diagnosis by laboratory procedures. We used culture together with
PCR and direct AFB staining to establish a system of reconfirming cases clinically
diagnosed at the Amasaman Health Centre, Ghana. All three methods showed a
comparable sensitivity and the laboratory analysis demonstrated a high accuracy of
clinical judgment by an experienced clinician.
Current recommendation by the WHO requires that BU patients be treated with a
combination of rifampicin and streptomycin for 8 weeks before surgical excision. In
many infectious diseases, the development of drug resistance has a serious impact on
patient management. It is therefore essential to monitor the drug susceptibility of M.
ulcerans. We analysed the susceptibility of 28 isolates to rifampicin, streptomycin
isoniazid and ethambutol and identified both streptomycin and rifampicin resistant strains
in Ghana. Findings from this study call for reconsideration of the current treatment
guidelines.
Currently, micro-epidemiological studies aiming to reveal transmission chains cannot be
done in BU. This is due to the low degree of genetic polymorphism in M. ulcerans
revealed by routinely used genetic fingerprinting procedures. We used VNTR typing
based on a newly identified polymorphic locus designated ST1 and the previously
described locus MIRU 1 to detect genetic diversity in isolates from Ghana. Analysis
revealed three different genotypes in isolates from Ghana, demonstrating for the first time
genetic diversity among M. ulcerans isolates in an African country.
Ex vivo ELISpot analysis of IFN-γ secreting cells was carried out by stimulating PBMCs
from BU patients with PPD, IPP and IRIV. Data from the study demonstrated for the first
time that M. ulcerans infection-associated systemic reduction in IFN-γ responses is not
confined to stimulation with live or dead mycobacteria and their products but extends to
other antigens. We also showed that the immune suppression reversed after surgical
treatment and that the suppression is not related to reduction in IL-12 secretions. This
indicates that the observed systemic immunosuppression was not the consequence of a
genetic defect in T cell function predisposing for BU but is rather related to the presence
of M. ulcerans bacteria.
In a longitudinal study, we compared recovery of immediate effector function of Vγ2Vδ2
T cells in surgically treated BU patients to that of TB patients under chemotherapy. At
the time of diagnosis, systemic production of IFN-γ after IPP stimulation was suppressed
in both disease states but reverses after treatment. Restoration of Vγ2Vδ2 reactivity was
slow such that an optimum response was not yet achieved by two months in both
populations. Our result demonstrates that immunosuppression in BU may not be caused
by the terpenoid toxin of M. ulcerans (mycolactone) alone
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