Researchers suggest a genetically attenuated malaria parasite (GAP) that is engineered to arrest during the latter period of its residence in the human liver could represent a vaccine against malaria. They say that this technique is more effective than existing approaches based on radiation-attenuated sporozoites (RAS).
A team at the Seattle Biomedical Research Institute, the University of Iowa, and the University of Washington, Seattle has shown that immunizing mice with late liver stage-arresting GAP induces larger and broader CD8 T-cell responses than RAS or early-arresting GAP immunization and offers better protection against subsequent parasite challenge.
Vaccination using the late liver state-arresting GAP also resulted in high levels of protection across different species of the Plasmodium parasite. Additionally, unlike current RAS techniques, the GAP strategy protected animals from red blood cell-infected parasites.
The University of Iowa’s John T. Harty, Ph.D., and colleagues, suggest their findings support the potential utility of late-arresting GAP as a broadly protective live-attenuated malarial vaccination candidate. They also believe that it could aid in the search for a protein-based vaccine candidate that is effective against infection in the liver and the blood.
The team reports its findings in Cell Host & Microbe in a paper titled “Superior Antimalarial Immunity after Vaccination with Late Liver Stage-Arresting Genetically Attenuated Parasites.”
Although halting the infection during the clinically silent liver stage is an attractive vaccination proposition, in practice even the most promising pre-erythrocytic subunit vaccine candidates have proved only partly effective in clinical trials.
Instead, using the RAS approach has remained the main focus of vaccine development efforts. However, the technology has yet to be translated into a licensed vaccine, the researchers point out, partly because of the problems associated with the need to use a dose of radiation that is high enough to cause enough DNA damage that will attenuate the parasite before the red blood cell stage but at the same time maintain sporozoite infectivity and immunogenicity.
In contrast to RAS approaches, GAP generated by targeted gene deletions represent an attractive vaccine proposition because the parasites could be designed to arrest at specific points of liver-stage development, the authors note. Indeed, studies have already shown that GAPs engineered to arrest at the early stage of replication in hepatocytes do provide CD8 T cell-related immunity in vaccinated mice, and early liver stage-arresting GAPs have recently moved into clinical evaluation as vaccine candidates.
What isn’t yet known is whether late-stage liver-arresting GAPs would represent a better proposition for vaccination, the team admits. Neither have studies been carried out to directly compare the CD8 T-cell responses resulting from immunization with either RAS-, early-, or late-arresting GAP sporozoites.
In a series of studies in mice, the researchers tested the ability of late liver stage-arresting GAP to protect against sporozoite infection. They initially evaluated the vaccine in outbred mice that not only represent a model that mimics the immunogenetic complexity of humans but has proven difficult to protect against sporozoite challenge even after multiple RAS immunizations.
Groups of these animals were vaccinated using either RAS, an early liver stage-arresting GAP, or a previously developed late liver stage-arresting GAP. Sixty days after vaccination the animals were challenged with infectious Plasmodium sporozoites.
The results showed that there was little protection (measured as blood-stage parasitemia) from subsequent infection with liver-stage sporozoites in the mice vaccinated using RAS or early liver stage-arresting GAP. In contrast, a single vaccination with the late liver stage-arresting GAP provided significant levels of protection. Administering a booster vaccination after an initial priming immunization did increase protection in the RAS and early liver stage-attenuated GAP recipients, but the prime-boosted mice in the late stage-attenuated GAP group were again significantly better protected.
The researchers found that the late-stage liver-attenuated GAP vaccine triggered far larger short-term and longer-lasting CD8 T-cell responses than the other two vaccines, both after initial immunization and after homologous boosting. Interestingly, outbred mice immunized with early-arresting parasites exhibited at least twice the variability in the peak of both primary and secondary effector CD8 T-cell responses compared with late-arresting GAP-vaccinated mice.
This finding supports the notion that additional antigens expressed by late-arresting GAP parasites recruit a broader repertoire of CD8 T cells in outbred mice, leading to uniformly high CD8 T-cell responses, the researchers suggest.
Their next set of studies using the three vaccines was carried out in a particular strain of inbred mice known to be substantially more difficult to protect using RAS vaccination than other strains of inbred mice or the outbred mice. Little protection from subsequent parasite challenge was observed in any of the singly vaccinated mice or those receiving a prime-boost regimen of the RAS or early stage-attenuated GAP vaccine.
In contrast, animals receiving prime-boost vaccinations of the late stage-attenuated vaccine showed 100% protection against subsequent sporozoite challenge. The enhanced protection was completely CD8 T cell-dependent. Analysis of the T-cell responses suggested that increased protection in the late stage-attenuated GAP prime-boost-vaccinated mice was likely due to numerically larger secondary memory CD8 T-cell responses and not due to specific phenotypic or functional differences among memory CD8 T cells induced by each sporozoite vaccine.
Even in a mouse model that does demonstrate protection after vaccination with the RAS or early stage-attenuated GAP vaccine, administering the late stage-attenuated GAP vaccine provided longer-lasting (300 days) CD8 T-cell dependent immunity to subsequent high dose sporozoite challenge than the RAS or early stage GAP vaccine.
Not only did immunity in these animals protect against different species of Plasmodium, but encouragingly, 100% protection was afforded when the late stage-attenuated vaccine was administered by either intradermal or subcutaneous routes. Previous work had established that RAS vaccination only elicits complete (100%) sterilizing immunity when administered intravenously, the authors note.
Subsequent tests in the same strain of mice in addition showed that the late stage-attenuated GAP vaccine provided significant protection against parasite-infected red blood cells. The immunized animals were able to control and clear both a nonlethal and lethal challenge with parasite-infected red blood cells.
“Mechanistically, our data link both enhanced liver-stage protection and the induction of larger CD8 T-cell responses after late liver stage-arresting GAP vaccination to diversification of the antigenic targets of responding CD8 T cells,” the authors conclude. “In support of this, an analysis of early-mid-stage (24 hr) versus late (40–50 hr) liver stage parasites directly isolated from infected mice revealed differential expression of more than 770 transcripts.” In fact, the larger CD8 T-cell response induced by the late stage-attenuated GAP vaccine was directed against Plasmodium antigens not expressed by early-arresting RAS parasites.
“Collectively, our data indicate that in concert with improved identification of vaccine-induced T cells, late liver stage-arresting GAP constitute a powerful model for identifying late liver stage antigens that might provide cross-stage protection and underscore the potential utility of late-arresting GAP as broadly protective second generation live-attenuated malaria vaccine candidates.”