Malaria, caused by Plasmodium parasites and transmitted by mosquito bite, remains a major global public health challenge. Plasmodium falciparum is the deadliest species affecting humans and is responsible for the majority of the 600,000 malaria-related fatalities annually. Significant progress has been made in malaria prevention but we still need a highly efficacious vaccine. One promising approach is the use of genetically attenuated parasite (GAP) based vaccines, which arrest in the liver promoting a protective immune response from infection. This study identifies a novel gene, LINUP that when deleted, creates a GAP that arrests late in liver stage development.

 What are GAPs, and Why Are They Important?

GAPs are whole parasite-based vaccines that are engineered to develop normally until they arrest during liver stage development. In contrast to other whole parasite vaccine modalities, GAPs are intrinsically attenuated and need no further manipulation. GAP vaccines stimulate the immune system and can provide protection from infection. Previous research shows that GAPs that arrest late in liver stage development elicit stronger immunity against infection compared to those that arrest early. This is because the late-arresting GAPs are far larger and contain a larger but also more diverse antigen repertoire leading to a superior immune response. The challenge lies in developing a GAP that replicates fully in the liver but arrests before reaching the pathogenic blood stage. Late-arresting GAPs therefore, represent an aspirational goal in malaria vaccine development.

The Discovery of LINUP and Its Role in Malaria Parasite Development

This study used the late liver stage transcriptome from another human malaria parasite species, Plasmodium vivax, generated from infected human-liver chimeric FRG huHep mice, to identify novel orthologous genes crucial only during Plasmodium liver stage development ¹. The study identified the gene we named LINUP, which has syntenic orthologues, with high amino acid similarity in Plasmodium vivax, Plasmodium falciparum and the rodent malaria parasite, Plasmodium yoelii.

To determine the spatial-temporal expression of LINUP, a transgenic parasite line was created with an mCherry tag fused to the C-terminus of the endogenous P. yoelii LINUP gene. LINUP was only expressed during liver stage development and localized to the nucleus of replicating liver stage parasites, known as schizonts (see the image below). This nuclear localization led to the naming of the gene liver stage nuclear protein (LINUP). Its nuclear localization suggests that LINUP may regulate gene expression during late liver stage development.

The image shows the nuclei of a late liver stage Plasmodium yoelii schizont. LINUP (red) localizes to the liver stage nuclei (blue).

Effects of LINUP Deletion on Parasite Development 

Deletion of LINUP in both rodent Plasmodium yoelii and human Plasmodium falciparum parasites, resulted in parasites that grew normally in blood and mosquito stages, but experienced severe defects in growth and differentiation in the liver. These included defective organelle segregation and poor formation of infectious merozoites, resulting in a compromised transition from the liver stage to the blood stage.

The impact of LINUP gene deletion was more pronounced in P. falciparum compared to P. yoelii. In P. yoelii, the administration of 50,000 P. yoelii linup^- sporozoites led to blood stage infection in 45% of highly susceptible mice albeit with a delay of 4 to 9 days in the time to blood stage patency. In contrast, P. falciparum linup^- parasites failed to transition to the blood stage in human liver-chimeric mice, even after four weeks of in vitro culture of transitioned blood from infected mice.

Immunogenicity of LINUP as a GAP vaccine

A prime-boost immunization with 10,000 P. yoelii linup^- sporozoites conferred complete protection against a challenge with 10,000 infectious sporozoites. This demonstrates that the deletion of LINUP does not impair the parasite’s ability to elicit a protective immune response in mice, reinforcing its potential as an effective GAP vaccine.

Developing the Next-Generation Malaria Vaccine: LINUP and Beyond

The discovery of LINUP is part of a broader effort to identify genes that are essential only for liver stage development. Deletion of these genes could ultimately create effective LARC GAP vaccine. Although LINUP deletion in P. falciparum showed complete liver stage attenuation in preclinical models, it is possible that high doses of Plasmodium falciparum linup^- infection in clinical trials could lead to breakthrough blood stage parasitemia. To mitigate this risk, rather than a standalone vaccine, LINUP gene deletion can be combined with further gene knockouts to create synthetic lethal phenotype. A promising development is the recent creation of a GAP vaccine, which combines LINUP gene deletion with a second gene deletion, Mei2 ². This vaccine arrests very late in liver stage development and is scheduled to enter dose-escalation safety trials in malaria-naive adults, followed by trials in malaria-endemic regions.

Conclusion: A New Era in Malaria Vaccines

The discovery of LINUP and its role in liver stage development marks a significant milestone in malaria research. Late liver stage-arresting GAPs offer a promising path toward to creating effective malaria vaccines by preventing the transition to the blood stage while triggering a strong immune response.

As clinical trials move forward, the success of GAP vaccines could transform malaria prevention and bring us closer to eliminating this deadly disease. The journey from laboratory discovery to clinical application highlights both the complexity and promise of malaria vaccine development. With each new finding, the goal of a highly effective malaria vaccine moves closer to reality, offering hope to millions at risk from this disease.

References:

1.      Goswami, D. et al. A conserved Plasmodium protein that localizes to liver stage nuclei is critical for late liver stage development. Commun. Biol. 7, (2024).

2.      Goswami, D. et al. A replication competent Plasmodium falciparum parasite completely attenuated by dual gene deletion. EMBO Mol. Med. 16, 723–754 (2024).