Understanding drivers of experimental malaria sub-unit vaccine induced immunity in Tanzania volunteers

Despite 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

Epitope mapping and fine specificity of human T and B cell responses for novel candidate blood-stage malaria vaccine P27A

P27A is a novel synthetic malaria vaccine candidate derived from the blood stage Plasmodium falciparum protein Trophozoite Exported Protein 1 (TEX1/PFF0165c). In phase 1a/1b clinical trials in malaria unexposed adults in Switzerland and in malaria pre-exposed adults in Tanzania, P27A formulated with Alhydrogel and GLA-SE adjuvants induced antigen-specific antibodies and T-cell activity. The GLA-SE adjuvant induced significantly stronger humoral responses than the Alhydrogel adjuvant. Groups of pre-exposed and unexposed subjects received identical vaccine formulations, which supported the comparison of the cellular and humoral response to P27A in terms of fine specificity and affinity for populations and adjuvants. Globally, fine specificity of the T and B cell responses exhibited preferred recognized sequences and did not highlight major differences between adjuvants or populations. Affinity of anti-P27A antibodies was around 10−8 M in all groups. Pre-exposed volunteers presented anti-P27A with higher affinity than unexposed volunteers. Increasing the dose of GLA-SE from 2.5 to 5 μg in pre-exposed volunteers improved anti-P27A affinity and decreased the number of recognized epitopes. These results indicate a higher maturation of the humoral response in pre-exposed volunteers, particularly when immunized with P27A formulated with 5 μg GLA-SE

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

Developing the OpenFlexure Microscope towards medical use: technical and social challenges of developing globally accessible hardware for healthcare

The OpenFlexure Microscope is an accessible, three-dimensional-printed robotic microscope, with sufficient image quality to resolve diagnostic features including parasites and cancerous cells. As access to lab-grade microscopes is a major challenge in global healthcare, the OpenFlexure Microscope has been developed to be manufactured, maintained and used in remote environments, supporting point-of-care diagnosis. The steps taken in transforming the hardware and software from an academic prototype towards an accepted medical device include addressing technical and social challenges, and are key for any innovation targeting improved effectiveness in low-resource healthcare. This article is part of the Theo Murphy meeting issue 'Open, reproducible hardware for microscopy'

Safety and tolerance of lymph node biopsies from chronic HIV-1 volunteers in rural Tanzania

HIV-1 rapidly establishes a persistent infection that can be contained under life-long antiretroviral therapy (ART) but not cured. One major viral reservoir is the peripheral lymph node (LN) follicles. Studying the impact of novel HIV-1 treatment and vaccination approaches on cells residing in germinal centers is essential for rapid progress towards HIV-1 prevention and cure.; We enrolled 9 asymptomatic adult volunteers with a newly diagnosed HIV-1 infection and CD4 T cell counts ≥ 350/ml. The patients underwent venous blood collection and inguinal lymph node excision surgery in parallel. Mononuclear cells were extracted from blood and tissues simultaneously. Participants were followed up regularly for 2 weeks until complete healing of the surgical wounds. All participants completed the lymph node excision surgery without clinical complications. Among the 9 volunteers, one elite controller was identified. The number of mononuclear cells recovered from lymph nodes ranged from 68 to 206 million and correlated positively with lymph node size. This is the first study to show that lymph node biopsy is a safe procedure and can be undertaken with local experts in rural settings. It provides a foundation for detailed immune response investigations during future clinical trials

The adjuvant GLA-SE promotes human Tfh cell expansion and emergence of public TCRβ clonotypes

The generation of protective humoral immunity after vaccination relies on the productive interaction between antigen-specific B cells and T follicular helper (Tfh) cells. Despite the central role of Tfh cells in vaccine responses, there is currently no validated way to enhance their differentiation in humans. From paired human lymph node and blood samples, we identify a population of circulating Tfh cells that are transcriptionally and clonally similar to germinal center Tfh cells. In a clinical trial of vaccine formulations, circulating Tfh cells were expanded in Tanzanian volunteers when an experimental malaria vaccine was adjuvanted in GLA-SE but not when formulated in Alum. The GLA-SE–formulated peptide was associated with an increase in the extrafollicular antibody response, long-lived antibody production, and the emergence of public TCRβ clonotypes in circulating Tfh cells. We demonstrate that altering vaccine adjuvants is a rational approach for enhancing Tfh cells in humans, thereby supporting the long-lived humoral immunity that is required for effective vaccines.</jats:p

Analyses of human vaccine-specific circulating and bone marrow-resident B cell populations reveal benefit of delayed vaccine booster dosing with blood-stage malaria antigens

We have previously reported primary endpoints of a clinical trial testing two vaccine platforms for the delivery of Plasmodium vivax malaria DBPRII: viral vectors (ChAd63, MVA), and protein/adjuvant (PvDBPII with 50µg Matrix-M™ adjuvant). Delayed boosting was necessitated due to trial halts during the pandemic and provides an opportunity to investigate the impact of dosing regimens. Here, using flow cytometry – including agnostic definition of B cell populations with the clustering tool CITRUS – we report enhanced induction of DBPRII-specific plasma cell and memory B cell responses in protein/adjuvant versus viral vector vaccinees. Within protein/adjuvant groups, delayed boosting further improved B cell immunogenicity compared to a monthly boosting regimen. Consistent with this, delayed boosting also drove more durable anti-DBPRII serum IgG. In an independent vaccine clinical trial with the P. falciparum malaria RH5.1 protein/adjuvant (50µg Matrix-M™) vaccine candidate, we similarly observed enhanced circulating B cell responses in vaccinees receiving a delayed final booster. Notably, a higher frequency of vaccine-specific (putatively long-lived) plasma cells was detected in the bone marrow of these delayed boosting vaccinees by ELISPOT and correlated strongly with serum IgG. Finally, following controlled human malaria infection with P. vivax parasites in the DBPRII trial, in vivo growth inhibition was observed to correlate with DBPRII-specific B cell and serum IgG responses. In contrast, the CD4+ and CD8+ T cell responses were impacted by vaccine platform but not dosing regimen and did not correlate with in vivo growth inhibition in a challenge model. Taken together, our DBPRII and RH5 data suggest an opportunity for protein/adjuvant dosing regimen optimisation in the context of rational vaccine development against pathogens where protection is antibody-mediated

autohaem: 3D printed devices for automated preparation of blood smears.

The process of making blood smears is common in both research and clinical settings for investigating the health of blood cells and the presence of blood-borne parasites. It is very often carried out manually. We focus here on smears for malaria diagnosis and research, which are frequently analyzed by optical microscopy and require a high quality. Automating the smear preparation promises to increase throughput and to improve the quality and consistency of the smears. We present here two devices (manual and motorized) designed to aid in the making of blood smears. These are fully documented, open-source hardware, and an important principle was to make them easily fabricated locally anywhere. Designs and assembly instructions are freely available under an open license. We also describe an image analysis pipeline for characterizing the quality of smears and use it to optimize the settings and tunable parameters in the two devices. The devices perform as well as expert human operators while not requiring a trained operator and offering potential advantages in reproducibility and standardization across facilities

Transitioning from academic innovation to viable humanitarian technology:The next steps for the OpenFlexure project

Academic interest in designing medical technology appropriate for Africa continues to grow, with funding available for innovations that answer complex questions. However, there is significant engineering work required to realise the promised impact of an innovation, even when it is shared as an Open Source design for others to build on. With academic innovation more highly prized by journals, funding bodies and academic institutions, this results in split priorities, and can lead to a difficult balance between the humanitarian aims of the project and pursuit of novel research. We present the OpenFlexure Microscope project as an example of an innovative academic project pushing the limits of 3D printed instrumentation. The microscope is already undergoing trials for malaria diagnosis, but significant product development is still necessary to transition the project from a prototype to a certified in-vitro diagnostic device. In this paper, we consider the engineering work that is needed to move from prototype to product, and how best to structure this work to support distributed manufacturing across Africa. We highlight the need to focus not just on the necessary engineering, but also on documenting this work so it can be understood and reproduced by any potential manufacturer.</p

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