Table 2.

Challenges facing vaccine discovery against eukaryotic parasites.

Challenge	Impact
Complex multiple life cycle stages yet to be fully understood and/or studied in detail
Expression of protein antigens can be different at each life-cycle stage and under altered environmental conditions, such as interactions with a host during infection	Choice of candidate can be specific to a life cycle stage
Antigens (in terms of protein sequences and/or 3D structure) can vary over time e.g. ancient and ongoing interactions between protozoans and the immune system have influenced their coevolution	Efficacy of candidate can change over time
Parasites are always mutating. Mutations can change parasites in ways that allow better resistance to immune defences and opportunities for multiple mechanisms of immune evasion e.g. mutations introduce variability in vaccine targets	Efficacy of candidate can change over time
In the endeavour to greatly improve safety, advances in vaccine candidates such as subunit components tend to be less immunogenic or efficacious than traditional live, whole pathogen vaccines	Requires potent adjuvants and appropriate delivery vectors
In a laboratory discovery approach, the expression of proteins is different in vitro than those proteins expressed during infection in vivo. Furthermore, abundantly expressed proteins are more easily identified in the laboratory	Potential vaccine candidates are missed
Unknown contributing factors that may be detrimental to vaccine discovery e.g. contributions from definitive and intermediate hosts, and transmission vectors (all complex biological systems)
Limited knowledge of precise interactions between parasites and host immune system, but more specifically, the interactions between antigens and immune cells	Difficult to assess the contribution of candidate to overall efficacy
Types of immune responses needed for protection is not completely understood for many protozoans
Different strains of parasites have different levels of virulence	Protective immunity may be better in some strains than others
No standardized testing protocol	A quantitative comparison of claimed protection levels between studies is difficult
Coinfection with other pathogens can influence the host response to vaccination
For any protozoal disease, methods must be developed to cultivate the pathogen	Some pathogens are too difficult and/or dangerous to cultivate in the laboratory
Many protozoan diseases are exacerbated by poverty and challenging environmental conditions in developing tropical and subtropical countries
A protozoan disease needs to attract the attention of vaccine manufacturers e.g. some diseases maybe considered commercially less profitable than others	Influences research and development for vaccines
Geopolitics can play a role in which diseases obtain the necessary funding	Influences research and development for vaccines
Regulatory limitations and safety concerns can impede vaccine licensing, especially for human recipients
The number of protozoan diseases in need of a vaccine is not static due to changing factors such as the rapid population growth in areas with weak health systems, climate change, antimicrobial resistance, and the changing nature of pathogen transmission between human and animal populations
For targeted human diseases, inability to test vaccine candidates on humans during vaccine discovery	Reliance on natural animal disease models
Species barriers to clinical translation e.g. antigens identified in mice may not protect humans or food-producing animals	Efficacy tested on animal model may not translate to target host
Limited research funding, although more funding is available for human diseases