Introduction and Background of the Project

Introduction

Malaria continues to be a major global health problem, and economic burden in disease-endemic countries. In 2015 alone, the WHO reported 214 million cases worldwide and approximately 438,000 deaths mostly among children under the age of five. With almost half of the world’s population at risk, the public health community has once again rallied around 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. Large efforts have been undertaken to reduce the global malaria burdens, where between 2000 and 2015 the rate of new cases fell globally by 37%; this led to an even greater reduction in the malaria death rate by an impressive 60% globally over all age groups. However, insecticide resistant mosquitoes and antimalarial drug resistant parasites are challenging the success of malaria elimination efforts. Therefore, new tools are 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, even though a TBV would not directly prevent immunized individuals from developing the disease. 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 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, we 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.

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 blocked 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 eradication settings. AnAPN1 has extensively been studied in transmission blocking experiments, where the protein induced very high titers in immunized animals. Antibodies elicited against specific epitopes of AnAPN1 completely reduced parasite development in the mosquito. Detailed analysis of the protein structure and consecutive mapping of epitope domains identified different antigenic regions within the full-length protein, out of which only one domain is required for blocking transmission. Since any TBV will require very high antibody titers within the vaccinated individuals to be effective, it is mandatory to develop well-defined antigens comprising only the domain required for transmission blocking activity, while avoiding the triggering of an immune response against regions that do not contribute to transmission blocking activity. In a joint effort by the University of Florida and CellFree Sciences Co., Ltd., the project aims to accelerate AnAPN1 vaccine development by selecting the most promising antigen domain within AnAPN1. Different epitopes on AnAPN1 will be expressed along with control proteins in a wheat germ cell-free protein expression system, a proven strategy for the rapid production of soluble proteins to screen for multiple antigens. The antigens will be tested in mice in combination with different FDA-approved adjuvants to reach durable and very high antibody titers against the critical TBV epitopes on AnAPN1. Antibodies obtained from these mice will be fully evaluated using a set of functional immunological and biological in vivo assays established at the University of Florida along with additional studies to validate epitope conformation. Hopefully, these experiments will potentiate the induction of functional antibodies in the animals through an optimal antigen/adjuvant combination. By comparing the new AnAPN1 immunogens to the well-characterized AnAPN1 control proteins used in previous studies, we anticipate proceeding to preclinical evaluation of up to two superior AnAPN1 constructs to support the clinical development of an AnAPN1 derived TBV.

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

AnAPN1 has been extensively well studied in transmission blocking experiments, where the protein induced very high titers in immunized animals. Antibodies raised against AnAPN1 in those animals greatly reduced parasite development in the mosquito and thus subsequently blocked the cascade of secondary infection of other individuals. The locus encoding the AnAPN1 protein is highly conserved in the genome of different Anopheles mosquito species suggesting that an AnAPN1-derived TBV could be effectively used in different areas regardless of the local vectors. Along with its ability to block transmission of P. falciparum and P. vivax, an AnAPN1-derived TBV therefore 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 under eradication settings. We hope that the project will allow us to rapidly move an AnAPN1-derived antigen to vaccine development with final goal to make a significant contribution to the control of malaria transmission and in particular blocking the further spread of antimalarial drug resistant parasites.

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 a number of principle 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, and the solution of the AnAPN1 protein structure and epitope mapping studies have allowed for highly targeted epitope designs not yet achieved for other TBV candidates. Applying the wheat germ cell-free protein expression system for rapid antigen preparation in combination with a comprehensive battery of assays in a medium-throughput screen at the University of Florida will identify the most suitable AnAPN1 TBV construct to carry forward for further development.

CFS will support UF on the design of the AnAPN1 target protein candidates. For each candidate protein, CFS will prepare the expression template using gene synthesis methods, and perform the protein expression experiments using its wheat germ cell-free protein expression system. Each candidate will be characterized prior to shipment to UF.

UF will formulate the candidate antigens with adjuvants and perform the competitive profiling immunization study for each antigen-adjuvant formulation. UF will implement a battery of assays to evaluate the immune response of immunized mice as well as standard membrane feeding assays to examine the transmission-blocking activity of immune sera following immunization with antigen-adjuvant combinations against Plasmodium sp. (human malaria parasite) in Anopheles gambiae mosquitoes.

Role and Responsibility of Each Partner

CFS will support UF on the design of the AnAPN1 target protein candidates. For each candidate protein, CFS will prepare the expression template using gene synthesis methods, and perform the protein expression experiments using its wheat germ cell-free protein expression system. Each candidate will be characterized prior to shipment to UF. UF will formulate the candidate antigens with adjuvants and perform the competitive profiling immunization study for each antigen-adjuvant formulation. UF will implement a battery of assays to evaluate the immune response of immunized mice as well as standard membrane feeding assays to examine the transmission-blocking activity of immune sera following immunization with antigen-adjuvant combinations against Plasmodium sp. (human malaria parasite) in Anopheles gambiae mosquitoes.

Others (including references if necessary)

On malaria: WHO Malaria Fact sheet, updated January 2016 World Malaria Report 2015 by the WHO

On AnAPN1: The Anopheles-midgut APN1 structure reveals a new malaria transmission-blocking vaccine epitope. Atkinson SC, Armistead JS, Mathias DK, Sandeu MM, Tao D, Borhani-Dizaji N, Tarimo BB, Morlais I, Dinglasan RR, Borg NA. Nat Struct Mol Biol. 2015 Jul;22(7):532-9. doi: 10.1038/nsmb.3048. Epub 2015 Jun 15.

Antibodies to a single, conserved epitope in Anopheles APN1 inhibit universal transmission of Plasmodium falciparum and Plasmodium vivax malaria. 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. Infect Immun. 2014 Feb;82(2):818-29. doi: 10.1128/IAI.01222-13. Epub 2013 Dec 9.

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

Final Report

1. Project objective

Regardless of determined efforts by the international community, about half of the world population is still at risk of suffering from malaria. For 2015, the World Health Organization (WHO) estimated 212 million malaria cases leading to 429,000 deaths, most of them children below 5 years of age. One of the most promising approaches to reduce malaria burden is blocking the transmission cycle of the parasites through mosquitoes; substantially reducing the number of malaria cases and the spread of disease. Thus, transmission-blocking interventions are an essential part to all malaria elimination strategies.

2. Project design

Malaria is a vector-borne disease that depends on human-mosquito-human transmission. Therefore, blocking the transmission of parasites from an infected human to a mosquito by a Transmission-Blocking Vaccine (TBV) is a critical tool for malaria elimination. Previous studies showed that the mosquito midgut protein AnAPN1 is a very potent TBV candidate that could prevent the transmission of P. falciparum and P. vivax – the most common malaria parasites. Based on structural information on the AnAPN1 protein, we tested new AnAPN1-derived antigens for vaccine optimization. These new antigens in combination with potent adjuvants would permit the generation of very high antibody titers with potent transmission-blocking activity.

3. Results, lessons learned

Previous studies on the transmission-blocking activity of antibodies to AnAPN1 in combination with data on the protein structure indicated that there are multiple epitopes on the AnAPN1 protein, but only some of those contributed to the transmission blocking activity. Hence, we used this information to design new antigens to specifically present only the relevant transmission-blocking regions and thus focusing the immune responses. Using a wheat germ cell-free protein expression system, we prepared as a reference the originally used AnAPN1 antigen along with four new antigen candidates. All these antigens were tested in immunization experiments in mice in combination with three different adjuvants (Alhydrogel, GLA-LSQ, and Stable Emulsion adjuvant). In these experiments, two of the four new antigen candidates showed high immunogenicity that clearly exceeded the immunogenicity of the original antigen. Serum samples from the immunized mice were used to further determine the transmission-blocking activity of the different antigens in mosquito feeding assays. These feeding assays were conducted on laboratory strains as well as wild type strains working together with partners in Cameroon. Including the wild type strains helped us to further demonstrate the very high transmission-blocking potential of the two new antigen candidates that had also shown the highest endpoint titers in the immunization studies. However, the experiments also indicated that there is no clear correlation between the antibody titer and the transmission-blocking activity. Instead, we observed different effects depending on the antigen/adjuvant combination that must be considered for the final immunogen formulation that would move forward to preclinical development. Overall, our experiments allowed us to successfully optimize our mosquito-based AnAPN1 candidate for further development as a potent malaria TBV. Our studies further demonstrated the benefits of using structural information in antigen optimization as a crucial step in the development of a TBV candidate. The two new antigens identified in this study meet the benchmark for a successful TBV, which requires high, neutralizing (transmission-blocking) and target-specific antibody titers.