Proteomic Characterization of Malaria Sporozoites Reveals Potential Vaccine Targets

In mammals, the malaria infection starts with the transmission of Plasmodium sporozoites during an Anopheles mosquito bite. Sporozoites travel through the skin and infect the hepatocytes in the liver, where they transform ultimately into exo-erythrocytic merozoites. The merozoites invade red blood cells, causing the telltale clinical symptoms and disease of malaria. Vaccines that interfere with the infection at the earliest stages constitute one of the most important intervention strategies to prevent malaria and reduce its transmission. Despite intense research and development in this field over the last few decades, no licensed malaria vaccine is available. Achieving complete sterile protection against malaria infection with sporozoite and liver-stage-based subunit vaccines has so far proven elusive.¹

Over 20 subunit vaccines are currently being evaluated in clinical trials or are in advanced preclinical development. RTS,S/AS01 is the most advanced vaccine candidate to date against the most deadly form of human malaria, Plasmodium falciparum. Its immune response is attributed to a single recombinant fusion protein containing a repeat domain from the major surface-exposed sporozoite coat protein, fused to a highly immunogenic hepatitis B antigen. The Phase III trial data show partial efficacy.^2,3 However, RTS,S/AS01 confers no similar protection against P. vivax malaria. RTS,S could be a “first generation” malaria vaccine that is partially effective—in other words, capable of reducing the number of cases of malaria in vaccinated children but not preventing all episodes of the disease.⁴

The RTS,S/AS01 results highlight the need to discover additional antigen targets for the development of the next-generation, multivalent subunit vaccines, in an effort to protect people against infection. “Comprehensively elucidating the protein composition of sporozoites will be invaluable in identifying novel targets for blocking infection,” state Lindner, Swearingen and Harupa et al. (2013), reporting in Molecular and Cellular Proteomics.¹

Efforts until now to identify and purify the proteins—separating the parasite from its vector or host material—expressed in Plasmodium mosquito stages have been hampered by overwhelming protein contamination, rendering chromatographic column capacity and mass analyzer duty cycles ineffective. Researchers led by Stefan H. I. Kappe, PhD, Full Professor and Malaria Program Director at the Seattle Biomedical Research Institute and Affiliate Professor, Department of Global Health, University of Washington, therefore sought to analyze the proteome of highly purified salivary gland sporozoites using a combination of improved sample purification and high accuracy mass spectrometry (MS).

P. yoelii salivary gland sporozoites coated with bovine serum albumin (BSA) on ice were purified via passage through DEAE-cellulose. P. falciparum sporozoites were first purified over an Accudenz cushion and then coated with BSA on ice and passed through DEAE-cellulose. After processing for an indirect immunofluorescence assay and  biotinylation with 2 mM EZ-Link ® Sulfo-NHS-LC-Biotin (Thermo Scientific) and a western blot detection of putative sporozoite surface-exposed proteins, samples were subjected to one-dimensional SDS-PAGE and in-gel tryptic digestion. The surface-exposed protein gels were fractionated and analyzed via liquid chromatography and tandem MS (LC-MS/MS) in duplicate. Almost 10 ⁷ sporozoites of P. yoelii and P. falciparum were fractionated by means of one-dimensional SDS-PAGE and analyzed via nanoLC-MS/MS in triplicate.

Combining careful sample preparation and improved purification protocols with high-resolution mass spectrometric analysis using an LTQ Orbitrap Velos mass analyzer (Thermo Scientific) coupled to nanoLC, the researchers accomplished the most complete proteome coverage to date for a pre-erythrocytic stage of salivary gland sporozoites from the human-infective P. falciparum and the rodent-infective P. yoelii. They identified a total of 1,991 P. falciparum sporozoite proteins and 1,876 P. yoelii sporozoite proteins—over 86% of them using high sequence coverage. The findings showed a high degree of overlap of orthologous genes, with 71.9% of P. falciparum proteins overlapping with P. yoelii proteins, and 78.9% of P. yoelii proteins overlapping with P. falciparum proteins.

The investigators used the proteomic data to confirm the presence of components critical for sporozoite infection. They include the sporozoite motility and invasion apparatus (glideosome), sporozoite signalling pathways, and the contents of the apical secretory organelles. Several well-characterized sporozoite proteins identified include those involved in gliding motility (e.g., TRAP, myosin A, GAP50, inner membrane complex (IMC) proteins), cell traversal (e.g., SPECT, SPECT2, CelTOS) and hepatocyte invasion (e.g., CSP, TRAP, SIAP-1, P52, P36). The coat protein CSP, which is strongly shed during gliding motility, was among the most abundant Plasmodium proteins the investigators identified in both P. falciparum and P. yoelii. Aldolase 1 and Actin 1 play a role in gliding motility in blood stage parasites. Aldolase 2 was significantly more abundant than Aldolase 1 (4-fold in P. falciparum and 9-fold in P. yoelii). G-protein-coupled receptors, adenylyl and guanylyl cyclases, and a carbonic anhydrase allow the sporozoite to detect external stimuli and trigger calcium-dependent signalling cascades. The researchers detected apical organelle proteins involved in the  invasion process, including those that localize to the micronemes (20 proteins), the rhoptry neck (6 proteins), the rhoptry body (20 proteins), and the dense granules (4 proteins): they are usually released in this sequence before, during and after host cell invasion. The sporozoite signalling response to external stimuli (such as highly sulfated heparan sulfate proteoglycans, albumin or bicarbonate) results in a sequential secretion of proteins involved in gliding, movement and migration across the skin and blood vessels, until the sporozoite reaches the liver, where additional proteins help in endothelial invasion and infection of hepatocytes.

The researchers have identified several known surface and secreted proteins: CSP, TRAP, thrombospondin-related sporozoite protein, apical membrane antigen-1, and a hexose transporter. They have also identified proteins not previously identified in Plasmodium sporozoites (e.g., SUB2, Pf34, RON6, RAP1, CLAG3.2, RhopH2, RhopH3, RAMA, RALP1 and ASP/RON1) that require further characterization. Several secreted proteins, including UIS3, were present in the analyses of P. yoelii sporozoites but absent in those of P. falciparum.

“Taken together, the data constitute the most comprehensive analysis to date of the protein expression of salivary gland sporozoites and reveal novel potential surface-exposed proteins that might be valuable targets for antibody blockage of infection,” the researchers write. Indeed, if any of the newly characterized surface-exposed proteins prove to be clinically effective as antigens, they could be combined with the major surface-exposed sporozoite coat protein of the RTS,S/AS01 vaccine to significantly improve the antibody-based vaccine efficacy by producing a multivalent malaria subunit vaccine, for a broader immune response to Plasmodium parasites and improved protection against malaria infection.

References

1. Lindner, S.E., Swearingen, K.E., Harupa, A., et al. (2013, May) “Total and putative surface proteomics of malaria parasite salivary gland sporozoites,” Mol. Cell Proteomics, 12(5) (pp. 1127–1143), doi: 10.1074/mcp.M112.024505, available at http://www.mcponline.org/content/12/5/1127.long

2. Agnandji, S.T., Lell, B., Soulanoudjingar, S.S., et al. (2011) “First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children,” N. Engl. J. Med., 365 (pp. 1863–1875), doi: 10.1056/NEJMoa1102287.

3. RTS,S Clinical Trials Partnership, Agnandji, S.T., Lell, B., Fernandes, J.F., et al. (2012) “A Phase 3 trial of RTS,S/AS01 malaria vaccine in African infants,” N. Engl. J. Med., 367 (pp. 2284–2295), doi: 10.1056/NEJMoa1208394.

4. World Health Organization (2012) “Questions and Answers on Malaria Vaccines,” available at  http://www.who.int/vaccine_research/development/malaria_vaccine_q_and_a.pdf