Half of the global population is at risk for malaria, which caused over 400,000 deaths worldwide in 2015 alone. Due to its global prevalence, many steps have been taken to curb infection rates. Even with the use of bed nets, insecticides, and increased drug treatment malaria is still a major global health threat in need of a preventative vaccine. One of the major hurdles of malaria vaccine design is the fact that the malaria parasite (Plasmodium falciparum, PF) has multiple life stages (figure 1). This means that protection against a single life stage needs to be wholly sterilizing (not allowing escape) or that protection needs to be aimed at multiple life stages to circumvent escape effects. This being said, there have been many attempts at an anti-malaria vaccine. The two main formats have been: 1) a subunit vaccine against the major surface protein on the sporozoite and 2) a whole parasite formulation that has either been inactivated or is given alongside anti-malarial drugs. Current vaccine candidates using these formulations have exhibited some success but have caveats including safety, ease of production, and adherence to drug regimen.
In work featured on the cover of Science Translational Medicine Dr. James Kublin (Vaccine and Infectious Disease Division) and colleges from University of Washington and Seattle Biomedical Research Institutes used genetic engineering to create a new genetically attenuated parasite (GAP) vaccine candidate. Dr. Kublin commented, “Attenuating an entire organisms, whether it be a virus or a cell, has traditionally been the first approach to vaccine development and one of the most effective ways of protecting against infection. Vaccination with the weakened form of the malaria parasite is the most promising approach to date that confers sterilizing immunity against infection”. To create an attenuated Pf GAP, Dr Kublin’s group created three genetic deletions (called Pf GAP3KO) causing the parasite to arrest at various developmental stages preventing the parasite from progressing to the blood-stage infection. Attenuation of the triple knockout was verified in a mouse malaria model using Plasmodium yoelii, Py. Mice were challenged with high doses of the Py GAP3KO parasite and their ability to reach blood-stage infection was measured. No mice developed blood-stage parasites, verifying the triple knockout as completely attenuated during liver infection. To further test the knockout, mice were vaccinated twice with Py GAP3KO then challenged either 7, 30, or 180 days after last immunization with non-attenuated Py. Results from this study showed complete protection from blood-stage infection with vaccination, regardless of challenge day. These data provided the foundation for a phase I clinical trial, in which vaccine safety and antibody response was evaluated in ten human volunteers.
In order to test the effects of Pf GAP3KO in human volunteers laboratory mosquitoes were infected with the modified parasite using membrane feedings, resulting in 91% salivary glad parasite prevalence with greater than 40,000 sporozoites per mosquito. Infected mosquitoes (150-200) were allowed to feed on volunteer forearms for twn minutes allowing transmission of the sporozoites. Over the 28-day study no subject exhibited malaria symptoms, all had negative blood-stage parasitemia by blood smear, and no Pf 18S ribosomal RNA was detected at anytime by qualitative reverse transcriptase PCR (qRT-PCR) and there were no reported laboratory toxicities. This result demonstrated that in humans the Pf GAP3KO was attenuated and lead to no blood-stage infection. Overall, most adverse reactions to the trial (all grade 1 or 2) were related to the large number of mosquito bites. Sera from the volunteers were also collected on days 0, 7, 13, and 28 after immunization to test for anti-malaria antibody (a-CSP) titers by IgG ELISA. On day seven, eight of ten subjects had values over baseline and by day 13 and 28 all were positive with a peak being achieved at day 13. Serum neutralization to block sporozoite infection was tested using an in vitro assay: inhibition of sporozoite traversal and invasion (ISTI). Results from the ISTI assay showed around 50% inhibition (in 9 out of 10) by day 7 and over 50% inhibition at both day 13 and 28 in all subjects. Despite the drop in sera titer over time the inhibition assays stayed constant suggesting that a-CSP does not correlate with the ISTI results, as seen in figure 2.
To test if Pf GAP3KO could protect in vivo, researchers used mice modified to have human immune components. Human IgG antibodies were purified from 5 participants (day 0 and 13) with high levels of inhibition and varied a-CSP levels and passively transferred into the humanized mice. Once loaded with anti-malaria antibodies, researchers measured the amount of non-attenuated Pf that persisted in the liver of these mice 13 days later. Day 0 (pre-immune) antibodies were compared to day 13 and three out of the five mice showed over 69% inhibition. Interesting the sera that showed inhibition had low a-CSP titers. To further characterize the antibodies produced by human vaccination, sporozoites and liver parasites were immunofluorescently stained with pooled sera. The staining revealed both an a-CSP pattern and intra-parasite staining, suggesting that antibodies produced by vaccination recognize both sporozoite and liver-stage parasites. Taken together this paper demonstrates that a GAP vaccine can elicit safe and possible protective responses to malaria and that further work should be completed to develop this malaria vaccine candidate. When asked about the future of the Pf GAP3KO, Dr. Kublin said, “The next step will be to infect volunteers with wild type malaria after they have received 3-5 administrations of the genetically attenuated parasite to assess the protective efficacy of this novel attenuation approach”.
This work was supported by an award from the U.S. Army Medical Research and Material Command and the Telemedicine and Advanced Technology Research Center.
Kublin JG,Mikolajczak SA,Sack BK,Fishbaugher ME,Seilie A,Shelton L,VonGoedert T,Firat M,Magee S,Fritzen E,Betz W,Kain HS,Dankwa DA,Steel RW,Vaughan AM,Noah Sather D,Murphy SC,Kappe SH. 2017. Complete attenuation of genetically engineered Plasmodium falciparum sporozoites in human subjects. Sci Transl Med, 9(371).