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

Introduction 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

Understanding the role of serological and clinical data on assessing the dynamic of malaria transmission: a case study of Bagamoyo district, Tanzania

A research article is submitted in Research | Volume 43, Article 60, 07 Oct 2022Introduction: naturally acquired blood-stage malaria antibodies and malaria clinical data have been reported to be useful in monitoring malaria change over time and as a marker of malaria exposure. This study assessed the totalimmunoglobulin G (IgG) levels to Plasmodium falciparum schizont among infants (5-17 months), estimated malaria incidence using routine health Facility-based surveillance data and predicted trend relation between anti-schizont antibodies and malaria incidence in Bagamoyo. Methods: 252 serum samples were used for assessment of total IgG by enzyme-linked immunosorbent assay and results were expressed in arbitrary units (AU).147/252 samples were collected in 2021 during a blood-stage malaria vaccine trial [ClinicalTrials.gov NCT04318002], and 105/252 were archived samples of malaria vaccine trial conducted in 2012 [ClinicalTrials.gov NCT00866619]. Malaria incidence was calculated from outpatient clinic data of malaria rapid test or blood smear positive results retrieved from District-Health-Information- Software-2 (DHIS2) between 2013 and 2020. Cross-sectional data from both studies were analyzed using STATA version 14. Results: this study demonstrated a decline in total anti-schizont IgG levels from 490.21AU in 2012 to 97.07AU in 2021 which was related to a fall in incidence from 58.25 cases/1000 person-year in 2013 to 14.28 cases/1000 person-year in 2020. We also observed a significant difference in incidence when comparing high and low malaria transmission areas and by gender. However, we did not observe differences when comparing total anti-schizont antibodies by gender and study year. Conclusion: total anti-schizont antibody levels appear to be an important serological marker of exposure for assessing the dynamic of malaria transmission in infants living in malaria-endemic regions

Whole blood transcriptome changes following controlled human malaria infection in malaria pre-exposed volunteers correlate with parasite prepatent period

Malaria continues to be one of mankind's most devastating diseases despite the many and varied efforts to combat it. Indispensable for malaria elimination and eventual eradication is the development of effective vaccines. Controlled human malaria infection (CHMI) is an invaluable tool for vaccine efficacy assessment and investigation of early immunological and molecular responses against Plasmodium falciparum infection. Here, we investigated gene expression changes following CHMI using RNA-Seq. Peripheral blood samples were collected in Bagamoyo, Tanzania, from ten adults who were injected intradermally (ID) with 2.5x104 aseptic, purified, cryopreserved P. falciparum sporozoites (Sanaria® PfSPZ Challenge). A total of 2,758 genes were identified as differentially expressed following CHMI. Transcriptional changes were most pronounced on day 5 after inoculation, during the clinically silent liver phase. A secondary analysis, grouping the volunteers according to their prepatent period duration, identified 265 genes whose expression levels were linked to time of blood stage parasitemia detection. Gene modules associated with these 265 genes were linked to regulation of transcription, cell cycle, phosphatidylinositol signaling and erythrocyte development. Our study showed that in malaria pre-exposed volunteers, parasite prepatent period in each individual is linked to magnitude and timing of early gene expression changes after ID CHMI

Molecular malaria surveillance using a novel protocol for extraction and analysis of nucleic acids retained on used rapid diagnostic tests

The use of malaria rapid diagnostic tests (RDTs) as a source for nucleic acids that can be analyzed via nucleic acid amplification techniques has several advantages, including minimal amounts of blood, sample collection, simplified storage and shipping conditions at room temperature. We have systematically developed and extensively evaluated a procedure to extract total nucleic acids from used malaria RDTs. The co-extraction of DNA and RNA molecules from small volumes of dried blood retained on the RDTs allows detection and quantification of P. falciparum parasites from asymptomatic patients with parasite densities as low as 1 Pf/µL blood using reverse transcription quantitative PCR. Based on the extraction protocol we have developed the ENAR (Extraction of Nucleic Acids from RDTs) approach; a complete workflow for large-scale molecular malaria surveillance. Using RDTs collected during a malaria indicator survey we demonstrated that ENAR provides a powerful tool to analyze nucleic acids from thousands of RDTs in a standardized and high-throughput manner. We found several, known and new, non-synonymous single nucleotide polymorphisms in the propeller region of the kelch 13 gene among isolates circulating on Bioko Island, Equatorial Guinea

drLumi: An open-source package to manage data, calibrate, and conduct quality control of multiplex bead-based immunoassays data analysis

Multiplex bead-based immunoassays are used to measure concentrations of several analytes simultaneously. These assays include control standard curves (SC) to reduce between-plate variability and normalize quantitation of analytes of biological samples. Suboptimal calibration might result in large random error and decreased number of samples with analyte concentrations within the limits of quantification. Suboptimal calibration may be a consequence of poor fitness of the functions used for the SC, the treatment of the background noise and the method used to estimate the limits of quantification. Currently assessment of fitness of curves is largely dependent on operator and that may add additional error. Moreover, there is no software to automate data managing and quality control. In this article we present a R package, drLumi, with functions for managing data, calibrating assays and performing quality control. To optimize the assay the package implements: i) three dose-response functions, ii) four approaches for treating background noise and iii) three methods for estimating limits of quantifications. Other implemented functions are focused on the quality control of the fitted standard curve: detection of outliers, estimation of the confidence or prediction interval, and estimation of summary statistics. With demonstration purpose, we apply the software to 30 cytokines, chemokines and growth factors measured in a multiplex bead-based immunoassay in a study aiming to measure correlates of risk or protection from malaria of the RTS,S malaria vaccine nested in the Phase 3 randomized controlled trial of this vaccine

Experience and Challenges from Clinical Trials with Malaria Vaccines in Africa.

Malaria vaccines are considered amongst the most important modalities for potential elimination of malaria disease and transmission. Research and development in this field has been an area of intense effort by many groups over the last few decades. Despite this, there is currently no licensed malaria vaccine. Researchers, clinical trialists and vaccine developers have been working on many approached to make malaria vaccine available.African research institutions have developed and demonstrated a great capacity to undertake clinical trials in accordance to the International Conference on Harmonization-Good Clinical Practice (ICH-GCP) standards in the last decade; particularly in the field of malaria vaccines and anti-malarial drugs. This capacity is a result of networking among African scientists in collaboration with other partners; this has traversed both clinical trials and malaria control programmes as part of the Global Malaria Action Plan (GMAP). GMAP outlined and support global strategies toward the elimination and eradication of malaria in many areas, translating in reduction in public health burden, especially for African children. In the sub-Saharan region the capacity to undertake more clinical trials remains small in comparison to the actual need.However, sustainability of the already developed capacity is essential and crucial for the evaluation of different interventions and diagnostic tools/strategies for other diseases like TB, HIV, neglected tropical diseases and non-communicable diseases. There is urgent need for innovative mechanisms for the sustainability and expansion of the capacity in clinical trials in sub-Saharan Africa as the catalyst for health improvement and maintained

Virosome-Formulated Plasmodium falciparum AMA-1 & CSP Derived Peptides as Malaria Vaccine: Randomized Phase 1b Trial in Semi-Immune Adults & Children

BACKGROUND\ud \ud This trial was conducted to evaluate the safety and immunogenicity of two virosome formulated malaria peptidomimetics derived from Plasmodium falciparum AMA-1 and CSP in malaria semi-immune adults and children.\ud \ud METHODS\ud \ud The design was a prospective randomized, double-blind, controlled, age-deescalating study with two immunizations. 10 adults and 40 children (aged 5-9 years) living in a malaria endemic area were immunized with PEV3B or virosomal influenza vaccine Inflexal®V on day 0 and 90.\ud \ud RESULTS\ud \ud No serious or severe adverse events (AEs) related to the vaccines were observed. The only local solicited AE reported was pain at injection site, which affected more children in the Inflexal®V group compared to the PEV3B group (p = 0.014). In the PEV3B group, IgG ELISA endpoint titers specific for the AMA-1 and CSP peptide antigens were significantly higher for most time points compared to the Inflexal®V control group. Across all time points after first immunization the average ratio of endpoint titers to baseline values in PEV3B subjects ranged from 4 to 15 in adults and from 4 to 66 in children. As an exploratory outcome, we found that the incidence rate of clinical malaria episodes in children vaccinees was half the rate of the control children between study days 30 and 365 (0.0035 episodes per day at risk for PEV3B vs. 0.0069 for Inflexal®V; RR  = 0.50 [95%-CI: 0.29-0.88], p = 0.02).\ud \ud CONCLUSION\ud \ud These findings provide a strong basis for the further development of multivalent virosomal malaria peptide vaccines.\ud \ud TRIAL REGISTRATION\ud \ud ClinicalTrials.gov NCT00513669

Mixed Th1 and Th2 Mycobacterium tuberculosis-specific CD4 T cell responses in patients with active pulmonary tuberculosis from Tanzania.

Mycobacterium tuberculosis (Mtb) and helminth infections elicit antagonistic immune effector functions and are co-endemic in several regions of the world. We therefore hypothesized that helminth infection may influence Mtb-specific T-cell immune responses. We evaluated the cytokine profile of Mtb-specific T cells in 72 individuals with pulmonary TB disease recruited from two Sub-Saharan regions with high and moderate helminth burden i.e. 55 from Tanzania (TZ) and 17 from South Africa (SA), respectively. We showed that Mtb-specific CD4 T-cell functional profile of TB patients from Tanzania are primarily composed of polyfunctional Th1 and Th2 cells, associated with increased expression of Gata-3 and reduced expression of T-bet in memory CD4 T cells. In contrast, the cytokine profile of Mtb-specific CD4 T cells of TB patients from SA was dominated by single IFN-γ and dual IFN-γ/TNF-α and associated with TB-induced systemic inflammation and elevated serum levels of type I IFNs. Of note, the proportion of patients with Mtb-specific CD8 T cells was significantly reduced in Mtb/helminth co-infected patients from TZ. It is likely that the underlying helminth infection and possibly genetic and other unknown environmental factors may have caused the induction of mixed Th1/Th2 Mtb-specific CD4 T cell responses in patients from TZ. Taken together, these results indicate that the generation of Mtb-specific CD4 and CD8 T cell responses may be substantially influenced by environmental factors in vivo. These observations may have major impact in the identification of immune biomarkers of disease status and correlates of protection

Maturation and mip-1β production of cytomegalovirus-specific T cell responses in Tanzanian children, adolescents and adults : impact by HIV and Mycobacterium tuberculosis co-infections

It is well accepted that aging and HIV infection are associated with quantitative and functional changes of CMV-specific T cell responses. We studied here the expression of Mip-1β and the T cell maturation marker CD27 within CMVpp65-specific CD4+ and CD8+ T cells in relation to age, HIV and active Tuberculosis (TB) co-infection in a cohort of Tanzanian volunteers (≤16 years of age, n = 108 and ≥18 years, n = 79). Independent of HIV co-infection, IFNγ+ CMVpp65-specific CD4+ T cell frequencies increased with age. In adults, HIV co-infection further increased the frequencies of these cells. A high capacity for Mip-1β production together with a CD27low phenotype was characteristic for these cells in children and adults. Interestingly, in addition to HIV co-infection active TB disease was linked to further down regulation of CD27 and increased capacity of Mip-1β production in CMVpp65-specific CD4+ T cells. These phenotypic and functional changes of CMVpp65-specific CD4 T cells observed during HIV infection and active TB could be associated with increased CMV reactivation rates

Understanding the role of serological and clinical data on assessing the dynamic of malaria transmission: a case study of Bagamoyo district, Tanzania

This research article was published in Pan African Medical Journal, Volume 43, 2022.Introduction: naturally acquired blood-stage malaria antibodies and malaria clinical data have been reported to be useful in monitoring malaria change over time and as a marker of malaria exposure. This study assessed the total immunoglobulin G(IgG) levels to Plasmodium falciparum schizont among infants (5-17 months), estimated malaria incidence using routine health facility-based surveillance data and predicted trend relation between anti-schizont antibodies and malaria incidence in Bagamoyo. Methods: 252 serum samples were used for assessment of total IgG by enzyme-linked immunosorbent assay and results were expressed in arbitrary units (AU). 147/252 samples were collected in 2021 during a blood-stage malaria vaccine trial [ClinicalTrials.gov NCT04318002], and 105/252 were archived samples of malaria vaccine trial conducted in 2012 [ClinicalTrials.gov NCT00866619]. Malaria incidence was calculated from outpatient clinic data of malaria rapid test or blood smear positive results retrieved from District-Health-Information Software-2 (DHIS2) between 2013 and 2020. Cross sectional data from both studies were analysed using STATA version 14. Results: this study demonstrated a decline in total anti-schizont IgG levels from 490.21AU in 2012 to 97.07AU in 2021 which was related to a fall in incidence from 58.25 cases/1000 person-year in 2013 to 14.28 cases/1000 person-year in 2020. We also observed a significant difference in incidence when comparing high and low malaria transmission areas and by gender. However, we did not observe differences when comparing total anti-schizont antibodies by gender and study year. Conclusion: total anti-schizont antibody levels appear to be an important serological marker of exposure for assessing the dynamic of malaria transmission in infants living in malaria-endemic regions