SELF-ADJUVANTING
GENETICALLY ATTENUATED
RODENT MALARIA PARASITES
Ahmad Syibli Othman1,2, Blandine M Franke-Fayard1,
Séverine Chevalley-Maurel1, Catherin Marin-Mogollon1,
Antonio M. Mendes3, Helena Nunes-Cabaço3,
Hans Kroeze1, Jai Ramesar1, Miguel Prudêncio3,
Chris J. Janse1 and Shahid M. Khan1*
1 Leiden Malaria Research Group, Parasitology, Leiden University Medical
Center (LUMC), Leiden, The Netherlands
2 Faculty of Health Sciences, Universiti Sultan Zainal Abidin,
Terengganu, Malaysia
3 Instituto de Medicina Molecular João Lobo Antunes, Faculdade
de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal
* The corresponding author
Generation and pr
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ABSTRACT
In this study we created genetically attenuated rodent malaria parasites (GAPs) that express putative immunomodulatory proteins to increase GAP immunogenicity. Four different proteins were selected with known adjuvant activity: nontoxic cholera toxin B (CTB) subunit, mouse heat shock protein Gp96, Mycobacterium heat shock protein X (HspX) and
Salmonella flagellin (FliC). These proteins were C- terminally tagged to UIS4, a protein expressed in liver stages where it is located on the parasitophorous vacuole membrane (PVM). The genes encoding the fusion proteins, were introduced into the genome of a Plasmodium yoelii GAP and were expressed under control of the P. yoeliiuis4 promoter. To create the adjuvant GAPs, we first developed a P. yoelii GAP GIMO parent line (GIMOPyGAP-fabb/f) for rapid introduction of the adjuvant fusion-transgenes into the genome without retention of a drug selectable marker (SM). Specifically, in this GIMO parent line the hdhfr::yfcu positive::negative SM is introduced into the fabb/f gene, creating a GAP that arrests during late liver-stage development. The four adjuvant-expression cassettes were introduced into the fabb/f locus of GIMOPyGAP-fabb/f by performing GIMO transfection and negative selection. Adjuvant GAP-immunogenicity was determined by analysis of protective immunity induced by sporozoite immunization of BALB/c mice. When compared to immunization performed with non-adjuvanted P. yoelii fabb/f GAP, we were unable to observe a significant enhancement in protection (>10×) against wild type P. yoelii sporozoite challenge after immunization with the four adjuvant GAPs.
Generation and pr
otective ef
ficacy of ‘self-adjuvanting’ GAP
4
INTRODUCTION
Complete protection against a malaria infection can be obtained after immunization with live attenuated sporozoites, both in rodent models of malaria and in humans [1-3]. Sterile protection against a malaria infection has been achieved in humans after immunization with Plasmodium falciparum sporozoites, that have either been attenuated by radiation or administered under chemoprophylaxis [4-6]. A prerequisite for induction of protective immunity by sporozoite-based vaccines is that sporozoites retain their capacity to invade liver cells after their administration. The most advanced live-attenuated vaccine is based on radiation-attenuated sporozoites (PfSPZ-Vaccine), which is currently being evaluated both in the clinic and in field trials [2, 7]. In rodent models of malaria, immunization with sporozoites of genetically-attenuated parasites (GAP) can induce similar, or even better, levels of protective immunity compared to irradiated sporozoites (Irr-Spz) [1, 8-10]. These rodent GAP studies have been critical in the creation of two P. falciparum GAP vaccines, which are currently undergoing clinical evaluation [11-13].
A number of studies from both the clinic and the field have shown that Irr-Spz can generate strong protective immunity in humans [7, 14, 15]. However, in order to achieve high levels of protection, multiple immunizations with high doses of attenuated sporozoites are required [4, 7]. Immunization with high sporozoite doses increases the costs of sporozoite- based vaccines and complicates their production, compromising mass administration in malaria-endemic countries. A major challenge is to produce a highly immunogenic live-attenuated vaccine, which requires the fewest attenuated sporozoites per dose and the fewest doses to induce sustained sterile protection against a malaria infection.
While the precise mechanisms of protection mediated by immunization with attenuated sporozoites remain unknown, T cells, in particular CD8+ T cells, appear to be critical for
protective immunity as they are thought to play a major role in eliminating infected hepatocytes [16, 17]. Recent mechanistic investigations into immune responses induced by sporozoite-based immunization have shown that protective immune responses encompass diverse and robust immune responses that include not only CD8+ but also CD4+ T cells,
and a significant contribution from antibodies [17, 18].
Rodent models of malaria have been used to explore different approaches to enhance immunogenicity of vaccines consisting of attenuated sporozoites [9, 10, 19]. For example, it has been shown that immunization of mice with GAP that arrest growth late during liver stage development induce higher levels of protective immunity than GAP that arrest early after invasion of hepatocytes [8]. In a limited number of studies, adjuvants have been co-administered with attenuated sporozoites to enhance protective immune responses after immunization. It has been shown that the co-administration of the glycolipid
α
-galactosylceramide (α
-GalCer) and its analog 7DW8-5 with sporozoites can enhance the recruitment and activation/maturation of dendritic cells in draining lymph nodes at the site of sporozoite administration, thereby enhancing parasite- specific T cell immunogenicity [20, 21]. We recently demonstrated that immunizationGeneration and pr
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ficacy of ‘self-adjuvanting’ GAP
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with attenuated sporozoites in combination with treatment with agonistic antibodies, targeting the costimulatory receptor on activated T–cells, OX40, can enhance protective immunity [22].
Although these preclinical studies provide useful information about the mechanisms underlying protective immunity, the use of adjuvants in human vaccination studies may be hampered by cost, applicability or side-effects. Further, induction of protective immune responses by GAP immunization is dependent on sporozoites migrating to the liver and invading hepatocytes. The administration of adjuvants at the site of GAP injection will result in systemic distribution of the adjuvant which will therefore be considerably diluted at the sites where parasite antigens are taken up by antigen presenting cells (APCs), i.e. the liver, spleen or proximal lymph nodes [23]. In order to maximize the adjuvant effect (i.e. increase antigen uptake by APCs and/or provide stimulatory signals to enhance APC function) it is important to maximize the adjuvant effect at the point of antigen uptake and processing [23, 24].
Due to the limitations of co-injecting adjuvants with attenuated sporozoites, we explored the possibility of creating GAPs that express immunomodulatory proteins in sporozoites and/or liver stages, so called adjuvant GAPs [25-28] . Self-adjuvanting vaccines, in which the antigenic and adjuvanting moieties of the vaccines are present in the same molecule, have been developed for subunit vaccines targeting cancer cells, viruses [29, 30], nematodes [31] and bacteria [32, 33], for example by conjugation of lipopeptide-based Toll-like receptor (TLR) agonists to the target protein [28]. In vaccine development against malaria, the vaccine candidate antigen CSP has been fused to bacterial flagellin [34], a protein which is a potent TLR5 agonist [35]. However, to the best of our knowledge, no sporozoite-based vaccine has been reported that expresses additional immunomodulatory/adjuvant molecules [9, 10, 19].
We selected four TLR agonists that can increase adaptive immune responses and have the ability to improve cross-presentation of antigens, as has been demonstrated in other animal and/or human studies. The selected adjuvant proteins are: (i) nontoxic cholera toxin B subunit from Vibrio cholerae (CTB)[36, 37]; (ii) heat shock protein Gp96 of mice (Gp96)[38-40]; (iii) heat shock protein X from Mycobacterium tuberculosis (HspX) [41, 42]; and (iv) the TLR5 binding region of Salmonella typhimurium flagellin (amino acids 89–96; FliC) [35, 43, 44]. The genes encoding these proteins were fused to a Plasmodium protein expressed in liver stages, UIS4 (PY17X_0502200), which is located at the parasitophorous vacuole membrane (PVM) in infected hepatocytes. We fused these proteins to a PVM protein as it has been shown that ovalbumin (OVA) fused to proteins located in the PV/ PVM induce stronger T cell responses than ovalbumin expressed in the cytoplasm of transgenic parasites [45, 46]. The fusion genes were introduced by GIMO transfection [47, 48] into a novel GIMO GAP parasite line (GIMOPyGAP-fabb/f) whose growth is arrested late during liver stage development. The four adjuvant GAP were analyzed using the P. yoelli-BALB/c screening model for assessing protective immunity after GAP immunization [49]. We describe the immunogenicity studies and compared the protective immunity
Generation and pr
otective ef
ficacy of ‘self-adjuvanting’ GAP