Background

            Malaria is a disease caused by the Apicomplexan parasite Plasmodium, which currently claims around 400,000 lives yearly, striking hardest pregnant women and children under 5 years of age. Five Plasmodium species are responsible for various degrees of morbidity and mortality amongst humans, each with particular challenges that hinder their eradication. The most lethal of those species is P. falciparum, and as such, it has been the species receiving greatest attention in terms of anti-malarial drug development, vaccine research, and other malaria control strategies (1). Vaccine development has been very elusive, yet it continues to be necessary in the fight against malaria, particularly in view of the fast increase in anti-malarial drug resistance worldwide.

The most successful vaccine so far, is called RTS,S/AS01, which was developed by GlaxoSmithKline, and licensed in 2015. In 2019, pilot implementation in Ghana, Malawi and Kenya began. This vaccine consists of a recombinant protein containing a region of the P. falciparum circumsporozoite antigen (CSP) fused to a hepatitis B surface protein, and reconstituted with adjuvant AS01. CSP is a surface protein in sporozoites, which are the stages transmitted by the mosquito to the human host, and which initiate parasite infection in the liver. CSP has three domains: a conserved N-terminal region, a central region which contains the immunodominant B cell epitope, and a polymorphic C-terminal region that elicits a T cell response (for further insight, readers are referred to (2)).

During phase III clinical trials, the vaccine achieved reduction of clinical malaria in children by no more than 50%. It has been recognized that Pf CSP harbours great genetic diversity, with great inter- and intra-regional geographic variation, together with the challenge of multi-clonal infections. Altogether, it has been suggested that allele-specific immunity is important for the response elicited by the vaccine. While responses elicited to the C-terminus of PfCSP have been well studied, the N-terminal region has less diversity, yet remains understudied as a vaccine possibility. Consistent with intense research efforts worldwide towards designing a more efficacious and durable anti-CSP vaccine, Thai et al (3) investigate the molecular recognition and functional efficacy of a N-CSP-specific monoclonal antibody (Figure 1).

Key findings and developments

Baseline

• To date, no human monoclonal antibodies (mAbs) specific for N-CSP have been reported, and only a handful of murine mAbs from immunization studies with recombinant Pf N-CSP have been carried out. These mAbs recognized N-CSP epitopes adjacent to Region I (RI), a site with high conservation across Plasmodium species, suggesting that RI may be a good target for cross-species vaccine development. Moreover, proteolytic cleavage of RI was linked to efficient sporozoite invasion of host hepatocytes.
• Based on these observations, it has been proposed that adding N-CSP, including the RI motif, into a Pf CSP subunit vaccine may improve protective efficacy, compared to the current RTS,S/AS01 vaccine, which lacks this domain.
• However, passive transfer of the most potent RI-targeting mAb, called 5D5 showed inconclusive results in P. berghei models expressing a chimeric PbCSP with the Pf N-CSP domain, while impact on P. falciparum is unknown. Therefore, an important question that remained unaddressed, and which is the focus of the work by Thai et al is how mAb 5D5 binds and inhibits Pf sporozoites.

Structural findings

• The authors solved the crystal structure of the 5D5 Fab in complex with a peptide derived from N-CSP and conducted binding and functional experiments with Pf sporozoites.
• The crystal structure at 1.85 A resolution showed that 5D5 binds an alpha-helical epitope in N-CSP_83-91 with high affinity through extensive shape and charge complementarity, and the unusual utilization of an N-linked glycan.
• The first N-linked GlcNAc moiety contacts the aliphatic portion of the lysine residue K90 of PfCDP_81-98, contributing to mAb 5D5 occlusion. K90, K85, K88 and K92 are lysine residues directly upstream of RI, and have been previously proposed to be important for binding heparan sulfate proteoglycans on the surface of hepatocytes to initialize liver invasion.
• The molecular description of PfCSP recognition by mAb 5D5 demonstrates optimal antibody-antigen characteristics associated with high affinity binding to a putative functional site on Pf sporozoites. The authors highlight the importance of the H.Asn₉₈-linked GlcNAc moiety for high affinity mAb 5D5 binding to recombinant PfCSP,
• Altogether, shape and charge complementarity were determined to be important for high affinity binding, as mutations of side chain length and electrostatic properties resulted in significant binding affinity variations.

Functional findings

• Overall, the authors tested 5D5 inhibitory potency against Pf sporozoites through in vitro traversal assays and in vivo passive mAb transfer experiments in mosquitoes.
• The authors quantified reactivity of mAb 5D5 to single live Pf sporozoites isolated from the midgut and salivary glands of mosquitoes. In contrast to previous reports, they found that mAbs 5D5 did not bind the majority of sporozoites. In spite of high affinity interaction with the N-CSP epitope in recombinant PfCSP, the overall low levels of mAb 5D5 binding to live Pf sporozoites preclude efficient inhibition of parasite traversal.
• mAb 5D5 also exhibited low binding efficacy to immature sporozoites. As for salivary gland sporozoites, paratope glycosylation increased the proportion of mAb 5D5-bound midgut sporozoites.
• In vivo experiments in mosquitoes supported the above findings, concluding that mAb 5D5 fails to efficiently recognize its epitope on the surface of Pf parasites, and lacks inhibitory potency against sporozoites in both the vector and the host.

Overall

• The preprint by Thai et al provides a detailed molecular and functional understanding of mAb 5D5 recognition of its epitope, and highlights poor recognition of this N-CSP epitope on the Pf sporozoite surface. mAb 5D5 is unable to block Pf sporozoite development in the mosquito or inhibit sporozoite traversal of hepatocytes. Altogether, the findings do not support the inclusion of the 5D5 epitope into the next generation of CSP-based vaccines.

 

What I like about this paper

            Successful vaccine development remains relatively elusive, with concerns over the efficacy and protection rate of RTS,S/AS01. While CSP has been the focus of research for a long time, I like that the authors investigated a topic that has remained understudied for a long time. I also like the thorough scientific approach with which they answered the research questions, and the diverse methodology they used, joining the expertise of various leading groups. I think that an out-of-the-box approach is needed in the search for a malaria vaccine, and this work is in line with this idea.

 

Open questions

1. Why, in your opinion, was the functional relevance of N-CSP mAbs so understudied until now?
2. What led you to try and investigate its relevance now?
3. You show in your work poor recognition of 5D5 of its N-CSP epitope on the Pf sporozoite surface. However, from a structural point of view, it seemed a very promising candidate. This is biologically, very interesting. From an immunology and structural point of view, what should the field be looking for in a candidate?
4. In more general terms, can you discuss what, in your opinion (and based in your work and other’s tackling CSP), makes the malaria vaccine elusive?
5. Altogether, you discuss the relevance of RI, and you mention the importance of further studies to resolve the residues comprising RI, and the overall structure of Pf N-CSP. Can you expand further on why further understanding of this region is important?
6. There is discussion in the field of the need for multi-stage protective vaccines. CSP continues to be the candidate with most momentum. In your work, you took advantage of both classical and modern tools of research. How, in your opinion, can the search for malaria vaccine candidates change and progress, with the availability of novel research tools, not available in the past?
7. You discuss in your work, your findings in vivo in mosquitoes, and your findings on hepatocyte traversal. There has been work discussing the importance of skin inoculation-dependent protection (4). Did you perform skin experiments, and do you expect any relevant finding using this approach?

 

References

1. World Health Organization. 2019 World Malaria Report.
2. Pance A., How elusive can a malaria vaccine be?, Genome watch News and Analysis, 2019, doi:10.1038/s41579-018-0148-3.
3. Thai E, Costa G, Weyrich A, Murugan R, Oyen D, Prieto K, Bosch A, Valleriani A, Wu NC, Pholcharee T, Scally SW, Wilson IA, Wrdemann H, Julien JP, Levshina EA. A high-affinity antibody against the CSP N-terminal domain lacks Plasmodium falciparum inhibitory activity, bioRxiv, 2020, doi:10.1101/2020.01.13.904425.
4. Aliprandini E, Tavares J, Panatieri RH, Thiberse S, Yamamoto MM, Slivie O, Ishino T, Yuda M, Dartevelle S, Traincard F, Boscardin SB, Amino R, Cytotoxic anti-circumsporozoite antibodies target malaria sporozoites in the host skin, Nature Microbiology, 2018, 3(11).

Acknowledgements

I am very grateful to Elena Levashina and Mate Palfy for their feedback on this preLights highlight.

 

doi: https://doi.org/10.1242/prelights.17743

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