Synergy in anti-malarial pre-erythrocytic and transmission-blocking antibodies is achieved by reducing parasite density

Anti-malarial pre-erythrocytic vaccines (PEV) target transmission by inhibiting human infection but are currently partially protective. It has been posited, but never demonstrated, that co-administering transmission-blocking vaccines (TBV) would enhance malaria control. We hypothesized a mechanism that TBV could reduce parasite density in the mosquito salivary glands, thereby enhancing PEV efficacy. This was tested using a multigenerational population assay, passaging Plasmodium berghei to Anopheles stephensi mosquitoes. A combined efficacy of 90.8% (86.7–94.2%) was observed in the PEV +TBV antibody group, higher than the estimated efficacy of 83.3% (95% CrI 79.1–87.0%) if the two antibodies acted independently. Higher PEV efficacy at lower mosquito parasite loads was observed, comprising the first direct evidence that co-administering anti-sporozoite and anti-transmission interventions act synergistically, enhancing PEV efficacy across a range of TBV doses and transmission intensities. Combining partially effective vaccines of differing anti-parasitic classes is a pragmatic, powerful way to accelerate malaria elimination efforts

Table 1 - Fueling Open Innovation for Malaria Transmission-Blocking Drugs: Hundreds of Molecules Targeting Early Parasite Mosquito Stages

Despite recent successes at controlling malaria, progress has stalled with an estimated 219 million cases and 435,000 deaths in 2017 alone. Combined with emerging resistance to front line antimalarial therapies in Southeast Asia, there is an urgent need for new treatment options and novel approaches to halt the spread of malaria. Plasmodium, the parasite responsible for malaria propagates through mosquito transmission. This imposes an acute bottleneck on the parasite population and transmission-blocking interventions exploiting this vulnerability are recognized as vital for malaria elimination

SPCLIP1 is required for triggering the melanization cascade.

<p>(<b>A</b>) Reducing western blot analysis of CLIPA8 in hemolymph collected from control <i>LacZ</i> dsRNA-injected (top) and <i>SPCLIP1</i> kd mosquitoes (bottom) after injection with <i>E. coli</i> bioparticles. Full-length and cleaved CLIPA8 are labeled CLIPA8-F and CLIPA8-C, respectively. Images are representative of two independent biological replicates. (<b>B</b>) PO activity measured in hemolymph samples collected from ds<i>LacZ</i>, ds<i>SPCLIP1</i> and ds<i>CLIPA8</i> treated mosquitoes 6 h after injection with bacteria. Data are representative of two independent biological replicates. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003623#ppat.1003623.s002" target="_blank">Figure S2</a>. (<b>C</b>) GFP-expressing <i>P. berghei</i> oocysts (green circles) and melanized ookinetes (gray squares) in ds<i>LacZ</i>, ds<i>SPCLIP1</i>, ds<i>CTL4</i> and ds<i>CTL4</i>/ds<i>SPCLIP1</i> injected mosquitoes 7 days post infection were counted. Lines indicate median infection intensity values. Data were combined from three independent biological replicates. For statistical analysis, ds<i>CTL4</i> and ds<i>SPCLIP1</i> injected mosquitoes were compared to ds<i>LacZ</i> while ds<i>CTL4</i>/ds<i>SPCLIP1</i> injected mosquitoes were compared to ds<i>CTL4</i>. Asterisks indicate Kruskal-Wallis P-values<0.01.</p

SPCLIP1 and TEP1 interact after challenge with <i>E.</i><i>coli</i> bioparticles.

<p>IP beads containing SPCLIP1 antibody or control beads were used to capture proteins from hemolymph 15 min after injection with <i>E. coli</i> bioparticles (+) or PBS (−). The beads were separated and samples of the unbound and bound fractions were analyzed by western blot under reducing and non-reducing conditions for TEP1 and SPCLIP1, respectively. Images are representative of two independent biological replicates.</p

SPCLIP1 is a component of the mosquito complement cascade.

<p>(<b>A</b>) Western analysis of mosquito hemolymph collected 4 days after injection with <i>LacZ</i> or <i>SPCLIP1</i> dsRNA. The blot was initially probed with a polyclonal antibody raised against recombinant SPCLIP1 (top panel) and re-probed with an APL1C antibody (bottom panel) to confirm equal loading. (<b>B</b>)(<b>C</b>) Mosquito hemolymph collected 4 days after injection of <i>LacZ</i> dsRNA or silencing <i>SPCLIP1</i>, <i>LRIM1</i> or <i>TEP1</i> (or combinations of those) was analyzed by western blot using SPCLIP1, APL1C and TEP1 antibodies. Blots were re-probed with SRPN3 and PPO6 antibodies to confirm equal loading. The labels on the right indicate protein or complex detected. Images are representative of three independent biological replicates.</p

SPCLIP1 is required for the utilization of TEP1-F.

<p>(<b>A</b>) Western blot analysis of hemolymph collected from mosquitoes after injection with PBS (left) or <i>E. coli</i> bioparticles (right) using a panel of different antibodies. Full-length and processed TEP1 are indicated as TEP1-F and TEP1_cut, respectively. Re-probing with SRPN3 was used to confirm equal loading. (<b>B</b>) Western blot analysis of hemolymph collected from control <i>LacZ</i> dsRNA-injected (left) and <i>SPCLIP1</i> kd (right) mosquitoes after injection with <i>E. coli</i> bioparticles. Re-probing with PPO6 was used to confirm equal loading. Images are representative of three independent biological replicates.</p

Model of TEP1 convertase formation.

<p>In steady state hemolymph a pool of TEP1-F is processed by an unknown protease to generate TEP1_cut, which interacts and circulates with the LRIM1/APL1C complex. Recognition of microbial surfaces leads to deposition of LRIM1/APL1C and TEP1_cut and subsequent recruitment of SPCLIP1. An unknown catalytically active protease is then recruited generating the mature TEP1 convertase, which processes TEP1-F causing it to rapidly interact with nearby surfaces. Steady state processing of TEP1-F and that performed by the TEP1 convertase are distinct, as only the latter requires SPCLIP1. Formation of the TEP1 convertase is required for phagocytosis, lysis, or CLIPA8 cleavage by an unknown protease and subsequent activation of the melanization cascade.</p

TEP1 and SPCLIP1 localization on dead parasites is mutually dependent.

<p>(<b>A</b>) TEP1 immunolocalization on the surface of GFP-expressing <i>P. berghei</i> parasites invading the mosquito midgut 26 h after infection. TEP1 positive parasites (arrows) do not express GFP and appear fragmented indicating that they are killed, while TEP1 negative parasites express GFP and are considered live. There is a dramatic reduction in TEP1 signal in mosquitoes treated with ds<i>SPCLIP1</i>. Lack of TEP1 signal in ds<i>TEP1</i> treated mosquitoes confirms the specificity of the antibody. A rare TEP1, GFP double positive parasite is visible in the upper left panel of the ds<i>LacZ</i> control. (<b>B</b>) SPCLIP1 immunolocalization on the surface of GFP-expressing <i>P. berghei</i> parasites invading the mosquito midgut epithelium 26 h after infection. SPCLIP1 positive parasites (arrows) are fragmented and lack GFP signal indicating they are dead. There is a dramatic reduction in SPCLIP1 signal in mosquitoes treated with ds<i>TEP1</i>. Lack of SPCLIP1 signal in the ds<i>SPCLIP1</i> treated mosquitoes confirms the specificity of the antibody. The background staining observed in all panels is non-specific antibody trapping by the trachea and muscle fibers present on the basolateral surface of the mosquito midgut. For both TEP1 and SPCLIP1 immunolocalization assays two independent biological replicates were performed with 5–10 midguts for each dsRNA. Panels are representative confocal projections of an approximately 20 µm thick section of the midgut basolateral surface. The scale bar is 10 µm.</p