Introduction and Background of the Project

Introduction

Malaria remains a major global health problem, and a significant economic burden in disease-endemic countries. In 2020, there were approximately 229 million cases worldwide and 409,000 deaths, mostly among children under the age of five. Importantly, the number of deaths has increased as compared to 2019. Considering the loss of progress in the reduction of deaths, now, more than ever, the public health community is resolute in its commitment to the goal of malaria elimination and eradication. 

Malaria develops after the transmission of Plasmodium parasites from infected Anopheles mosquitoes (the vector for the disease) to humans as part of a complex life cycle that requires different developmental stages in the mosquito and humans. Most cases of malaria are caused by two out of five infectious Plasmodium species, P. falciparum, which is dominant in large parts of Africa, and P. vivax, which is predominant throughout Asia and the Americas. In recent years, a growing body of evidence suggests that P. vivax malaria has once again established in Africa. 

Although significant gains have been achieved with respect to malaria elimination in several countries, a growing population of insecticide resistant mosquitoes and antimalarial drug resistant parasites are challenging the current effort. Therefore, new tools are urgently needed to effectively block the transmission of the parasite to further reduce the number of new cases and overall disease prevalence with the aim of finally eradicating malaria entirely.

Project objective

A very promising approach to reduce malaria transmission is the development of so-called “Transmission Blocking Vaccines” or “TBVs” that could block the transmission of the parasite from humans to the mosquito. Targeted vaccination of individuals in high transmission areas promise an immediate and effective reduction in the number of malaria cases. Although a TBV would not directly prevent immunized individuals from developing the disease, it has a clear, delayed clinical benefit for the population. Moreover, a TBV could provide effective means to prevent the spread of antimalarial drug-resistant parasites, and parasites that break through the most advanced malaria vaccine (Mosquirix™) to date. Thus, the development of new TBVs is one of the research priorities for a cost-effective intervention that can directly support the malaria eradication effort. The development of TBVs has mostly focused on P. falciparum ookinete surface protein 25 (Pfs25) and its P. vivax homolog Pvs25, for which Phase 1 clinical trials have been initiated, as well as gametocyte proteins such as Pfs48/45 and Pfs230. However, successful suppression of malaria transmission in most parts of the world will require TBVs that effectively block transmission of both P. falciparum and P. vivax, as the most common causes of disease. A parasite-centric approach requires the development of multiple TBVs using protein immunogens from different species. Instead, the partners focused our studies on developing a vaccine based on a highly conserved mosquito protein that acts as a receptor for the parasite and has the potential to block malaria transmission regardless of the Plasmodium species, i.e., a so-called universal malaria TBV.

Project design

The anopheline mosquito midgut-specific alanyl aminopeptidase N (AnAPN1) is a luminal midgut surface protein involved in blood meal digestion. At present, AnAPN1 is the only TBV candidate, which blocks parasite transmission of P. falciparum and P. vivax in different Anopheles species. Working with a mosquito protein further reduces the risk that the parasite could develop resistance against the intervention, potentially allowing for a long-term use of the vaccine under elimination settings.

AnAPN1 has been studied extensively in transmission-blocking experiments, where the protein induced very high titers in immunized animals. A detailed analysis of the protein structure and consecutive mapping of epitope domains identified critical transmission-blocking epitopes of AnAPN1 that can elicit antibodies that completely reduced parasite development in the mosquito. Since any TBV will require very high antibody titers within vaccinated individuals to be effective, it is mandatory to develop an optimized antigen to ensure transmission-blocking activity. The partners have achieved this goal, by re-designing the AnAPN1 antigen and immuno-focusing the humoral response to the key epitopes. The optimized AnAPN1 immunogen, UF6b, has no purification tags, and when formulated with the GLA-LSQ adjuvant elicits potent transmission-blocking activity in mice and non-human primates against natural P. falciparum strains.

This project represents a joint effort by the University of Florida, CellFree Sciences, Ajinomoto Bio-Pharma Services, Center of Medical Research Lambaréné (CERMEL), and the University Hospital Tübingen. The partners envision that during the two-stage project they will complete preclinical manufacturing of UF6b and toxicology testing to obtain ethical approval for use of the vaccine and then enter directly into Phase IA/B clinical trials in Lambaréné, Gabon. As the end points of the clinical trial, safety and dose will be confirmed. Antibodies obtained from immunized individuals will be fully evaluated using a set of functional, immunological, and biological assays established at the University of Florida and CERMEL.

How can your partnership (project) address global health challenges?

AnAPN1 is highly conserved in different Anopheles mosquito species. Antibodies raised against the AnAPN1 protein in animals greatly reduced parasite development in the mosquito and thus can subsequently block the cascade of secondary infection of other individuals. Along with its ability to block transmission of P. falciparum and P. vivax, an AnAPN1-derived TBV has the potential to be globally applied for the control of malaria transmission regardless of the parasite and mosquito species combination in local settings. Working with a mosquito protein further reduces the risk that the parasite could develop resistance against the intervention, potentially allowing for a long-term use of the vaccine in malaria elimination settings. Thus, the impact of the AnAPN1-based TBV on global health and malaria elimination could be profound. In the short-term, if delivered in tandem with Mosquirix™ or another malaria vaccine, it can provide synergistic reduction in the total number of new cases (disease incidence) of malaria. Importantly, TBVs in the near term can extend the efficacy of artemisinin-based combination therapies, by reducing the spread of drug-resistant parasites that have been described recently in Southeast Asia and Africa. In the long term, once malaria is controlled in a specific region, and there is a predominance of asymptomatic carriage that can lead to epidemic malaria, the TBV can be used in a top-down approach to wipe out the last vestiges of parasite reservoirs in the human population. Importantly, TBVs offer immediate benefits to the population by reducing infection rates in households and communities during and beyond the malaria transmission season. Protection can be further extended by combining TBV delivery with another malaria vaccine or administration of artemisinin-combined therapy to wipe out low-level parasitemia.

What sort of innovation are you bringing in your project?

Shifting the focus of TBV development from the use of parasite proteins towards the mosquito-derived AnAPN1 protein offers several principal advantages for blocking the transmission of multiple Plasmodium species on a global scale by a single TBV. AnAPN1-derived antibodies have already been demonstrated to reduce, if not completely block the transmission of P. falciparum and P. vivax. We previously applied a structure-guided vaccine design strategy to produce the optimized AnAPN1 vaccine antigen UF6 and performed laboratory and field infection studies in mosquitoes to quantify the impact of this TBV antigen on P. falciparum transmission. We then developed a process for large-scale expression of a low-endotoxin, purification tag-free UF6b antigen and showed that UF6b formulated with the adjuvant GLA-LSQ produced a durable humoral response in non-human primates. Finally, considering that many previous malaria vaccines that performed well in Phase IA studies in the United States failed when tested in Sub-Saharan Africa, the proposed Phase IA/B study for UF6b:GLA-LSQ will be conducted immediately in Gabon having a population in a malaria endemic region.

Role and Responsibility of Each Partner

(1) University of Florida (PRIME): Overall project management and proposal design, oversight of out-sourced toxicology studies, immunological assays qualification, membrane feeding assays. UF will provide the adjuvant, which will be purchased from the Infectious Diseases Research Institute (IDRI) as part of the co-funding support.

(2) Ajinomoto Bio-Pharma Services (SUB): cGMP clinical manufacturing of AnAPN1 antigen.

(3) CERMEL (SUB): Clinical study sponsor, study design, research site, clinical research team and acquisition of ethical approval for the study.

(4) University Hospital Tübingen (SUB): Discussions with regulatory bodies in Europe, study design, statistics, and clinical trial platform.

(5) CFS (Collaborating institute): Project coordination in close collaboration with the University of Florida.

Others (including references if necessary)

WHO World Malaria Report November 30, 2020

Bender NG, Khare P, Martinez J, Tweedell RE, Nyasembe VO, López-Gutiérrez B, Tripathi A, Miller D, Hamerly T, Vela EM, Davis RR, Howard RF, Nsango S, Cobb RR, Harbers M, Dinglasan RR. Immunofocusing humoral immunity potentiates the functional efficacy of the AnAPN1 malaria transmission-blocking vaccine antigen. NPJ Vaccines. 2021. In press.

Atkinson SC, Armistead JS, Mathias DK, Sandeu MM, Tao D, Borhani-Dizaji N, Tarimo BB, Morlais I, Dinglasan RR, Borg NA. The Anopheles midgut APN1 structure reveals a new malaria transmission-blocking vaccine epitope. Nat Struct Mol Biol. 2015 Jul;22(7):532-9. doi: 10.1038/nsmb.3048. Epub 2015 Jun 15.

Armistead JS, Morlais I, Mathias DK, Jardim JG, Joy J, Fridman A, Finnefrock AC, Bagchi A, Plebanski M, Scorpio DG, Churcher TS, Borg NA, Sattabongkot J, Dinglasan RR. Antibodies to a single, conserved epitope in Anopheles APN1 inhibit universal transmission of Plasmodium falciparum and Plasmodium vivax malaria. Infect Immun. 2014 Feb;82(2):818-29. doi: 10.1128/IAI.01222-13. Epub 2013 Dec 9.

Dinglasan RR, Kalume DE, Kanzok SM, Ghosh AK, Muratova O, Pandey A, Jacobs-Lorena M. Disruption of Plasmodium falciparum development by antibodies against a conserved mosquito midgut antigen. Proc Natl Acad Sci U S A. 2007 Aug 14;104(33):13461-6. Epub 2007 Aug 2.

Final Report

1. Project objective

This is a two-stage First-In-Human (FIH) study to test the hypotheses that the recombinant AnAPN1 candidate malaria TBV antigen formulated with GLA-LSQ [synthetic Glucopyranosyl Lipid A (GLA) in liposome Quillaja saponaria 21 formulation (LSQ)] adjuvant will be safe and induce a robust and effective humoral immune response in a dose-dependent manner compared to non-adjuvanted AnAPN1-immunized controls.

Stage 1 Objective: E. coli Master Cell Bank expression of the UF6b recombinant AnAPN1 (G2017-211) by Aji Bio Pharma for cGMP manufacture.

Stage 2 Objective: The primary clinical study objective is to assess the safety, tolerability, and reactogenicity of the AnAPN1 vaccine [UF6b(AnAPN1):GLA-LSQ].

2. Project design

A randomized, double-blind, controlled, dose-escalation Phase 1a/b clinical trial was conducted in Lambaréné, Gabon among thirty-three healthy adult volunteers between 18-45 years old. The vaccine is administered intramuscularly following a 0, 28, and 56-day schedule to assess safety. Close participant monitoring for any adverse effects occurring during up to 392 days after the initial vaccination was performed to evaluate safety. The secondary objective is to assess immunogenicity via ELISA, intracellular cytokine staining, and flow cytometry. A non-clinical objective assesses the transmission-reducing activity (TRA) of immune sera via direct or standard membrane feeding assay.

3. Results, lessons learned

Between October and December 2023, of the fifty-five participants that were screened, thirty-three eligible participants were recruited and assigned to a vaccine dosing cohort, with three sequential cohorts of eleven individuals each. The median age was 29 years (range: 18-43 years old). Participants in cohort 1 (n=10), cohort 2 (n=10), and cohort 3 (n=11) received the complete 3-dose vaccination schedule. The most frequently solicited local adverse events were mild to moderate injection site pain, representing 92% (44/48) of local events, and mild headache 36% (29/81), and nausea 19% (15/81) at a systemic level. No vaccine related serious adverse events were reported. The vaccine was found to be consistently immunogenic across all three cohorts. We observed a vaccine dose-dependent increases for the humoral responses but the differences between the 20µg and 50µg vaccine dose cohorts were modest. The 50µg dose (cohort 2) induced the strongest humoral responses, with 100% of the subjects responding to the vaccine.  For both the 20µg (cohort 1) and 100µg (cohort 3), only 70-75% of the subjects produced a robust antibody response. While we observed statistically significant TRA for each cohort, the TRA level varied within and across the dosing cohorts. Initial cellular immune response profiling and antibody isotyping data could not explain the variability in individual responses and functional activity of antibodies by cohort. Overall, these results suggest that the administration of the AnAPN1 malaria candidate vaccine is immunogenic, safe, and well tolerated in healthy Gabonese adults. The observed consistency in the magnitude of the humoral response and the inconsistency in the functionality of the elicited antibodies among adults may not be reflected in a future clinical study in a pediatric population. Furthermore, the data also suggest that a more potent adjuvant may be useful in improving the overall immune response, especially if dose sparing measures are considered.