Finding malaria’s Achilles’ heel

From the Pancera Group, Vaccine and Infectious Disease Division

Malaria is a parasitic disease spread by mosquitos that is endemic to many equatorial regions of the world, where infection can cause mortality, primarily in infants and young children. Malaria is caused by Plasmodium falciparum (Pf), which is introduced into humans through a mosquito bite and then travels through the blood to hepatocytes for further development in the liver. Current work on preventing malaria is hindered by the parasite’s many life cycle stages, which makes immune invasion easy. Pf is covered in circumsporozoite protein (PfCSP), which is important for the mosquito stage of malaria as well as entry into human hepatocytes. Since PfCSP is located on the surface of the parasite, it is a major target for neutralizing antibodies. The PfCSP protein is made up of a N-terminal domain that is comprised of a pentapeptide sequence (called region I), this is followed by a central repeat region made up of many NANP motifs, NVDP motifs and finally the C-terminal domain. At present, there has been some protective success with high-titer antibodies that target the immunodominant NANP repeats, however protection has typically waned over time. In a study published in Nature Medicine, carried out by researchers in the Vaccine and Infectious Disease Division at Fred Hutch and their collaborators, new monoclonal antibodies against malaria were isolated and screened for protective properties.

Human subjects were vaccinated using an attenuated, whole sporozoite vaccine which had previously shown protection against controlled human malaria infection. After immunization, blood and plasmablasts were screened for recognition of PfCSP. From this screen, the researchers found numerous antibodies that bound and inhibited sporozoite invasion of hepatocytes in vitro. Of these antibodies, the group selected the most promising to test in two malaria mouse models. One model uses a transgenic stain of Plasmodium berghei that has PfCSP as its surface protein, thus making it permissive to C57BL/6 mice. In this model, animals were infected intravenously with transgenic malaria followed by passive transfer of the antibody. This method showed a liver-stage burden reduction of 2-4 logs for antibody CIS43, 2 logs reduction for antibody CIS34, and 1-2 log reduction for antibody mAb10. Next, a more natural route of infection was tested; in this experiment, mice were subjected to mosquito bites by infected insects. All animals in the CIS43 group were free from parasites in the blood up to 12 days after infection. The other antibodies also had protective effects but to a lesser degree.

The second mouse model used to test protective capacity of the antibodies was a human liver-chimeric mouse. In this study, mice were infected with Pf and then the antibody was administered and parasite load was measured. Due to low animal availability, only CIS43 was tested in this model. The results showed an 80-90% reduction in Pf liver burden. Together, these in vivo experiments demonstrate that CIS43 is a potent neutralizing antibody against PfCSP in two mouse models.

Crystal structural of CIS42 (green/tan) and CIS43 (gray/tan) bound to peptide 21 (pink). Figure provided by Connor Weidle and Dr. Marie Pancera

To further characterize the antibodies, the N and C-termini and central repeat regions of PfCSP were tested for antibody binding in epitope mapping studies. These experiments confirmed binding of all antibodies to the central repeat region. To further narrow down the location of epitope binding, linear peptides overlapping the repeat region were screened for binding. A large portion of the antibodies bound to the NANP and NVDP repeats. Interestingly, the most potent in vivo antibody (CIS43) bound to a unique region at the junction of the N terminus and the central repeat, called the junction epitope.  Further identification of binding between CIS43 and the linear epitopes identified two sequential high-affinity binding events, suggesting an initial binding to the junctional epitope that may induce a conformational change of PfCSP. Co-crystallization of CIS43 and PfCSP was unsuccessful due to the flexibility of the proteins, however the group was able to crystalize the antibody binding fragment with the individual peptides. Data from these structures showed that the peptides are inserted into a hydrophobic groove found in the interface between heavy and light chains and that binding involved all complementarity- determining regions (CDRs). Interestingly, the four overlapping peptides that bound CIS43 adopted slightly varied conformations when bound to CIS43, which also showed movement between peptides. When compared to the crystal structures of CIS42 (a less effective antibody) bound to peptides, there was little variation in peptide conformation (see video for conformational differences between antibodies). This information suggests that CIS43 binds to a rare conformation of the junctional epitope and this region can be outcompeted or masked by the immunodominant NANP repeat region.

This video demonstrates the confirmation flexibility of peptide21. The video starts with a FAB(Antibody binding fragment) of 42 shown in green and tan bound to peptide21. It then zooms in on peptide21 and shows a linear morph of the confirmation seen with 42 to the confirmation seen with 43. It then zooms out demonstrating how 43 binds to peptide21. Lastly it overlays 42 and 43, it can been seen they have a different angle of approach. Video provided by Connor Weidle.

Through epitope mapping and co-crystallization, this study identified a new epitope that induces the best protective antibody to date against PfCSP. These data can be used to inform future vaccine development, since the current malaria vaccine (RTS/S) does not contain this epitope and thus cannot induce these specific potent antibodies. When asked what questions this paper raises, Dr. Pancera said, “Can we use the antibody for passive transfer? Can we make a better PfCSP-based vaccine?” As a follow up to this study, Dr. Pancera and her collaborators are currently working on a second generation CSP vaccine that takes into account this newly discovered site of weakness.