A T-cell response to a liver-stage Plasmodium antigen is not boosted by repeated sporozoite immunizations Sean C. Murphya, Arnold Kasb, Brad C. Stoneb, and Michael J. Bevanc,d,1 aDepartment of Laboratory Medicine, University of Washington Medical Center, Seattle, WA 98195; bBenaroya Research Institute, Seattle, WA 98101; and cDepartment of Immunology, and dThe Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195 Contributed by Michael J. Bevan, March 1, 2013 (sent for review February 2, 2013) + Development of an antimalarial subunit vaccine inducing protective instead associated with antibodies and CD4 T-cell responses (21). cytotoxic T lymphocyte (CTL)-mediated immunity could pave the way Thus, CSP is an important but currently insufficient target for use for malaria eradication. Experimental immunization with sporozoites as a single-antigen vaccine. Because responses to non-CSP anti- induces this type of protective response, but the extremely large gens can protect in the absence of CSP-specific immunity in mice number of proteins expressed by Plasmodium parasites has so far (11, 22–25), we and others are working to discover novel protective prohibited the identification of sufficient discrete T-cell antigens to antigens to broaden the immune repertoire. develop subunit vaccines that produce sterile immunity. Here, using Single immunization using CPS or late-arresting GAP ap- mice singly immunized with Plasmodium yoelii sporozoites and high- proaches usually provides greater protection than an equivalent throughput screening, we identified a unique CTL response against number of RAS parasites (11, 26). RAS parasites arrest shortly after the parasite ribosomal L3 protein. Unlike CTL responses to the circum- hepatocyte infection, whereas GAPs progress further depending on sporozoite protein (CSP), the population of L3-specific CTLs was not the specific genetic lesion. CPS completes liver-stage development expanded by multiple sporozoite immunizations. CSP is abundant in and progresses to a brief RBC infection halted by drug treatment. the sporozoite itself, whereas L3 expression does not increase until GAP and CPS approaches express a wider array of proteins than the liver stage. The response induced by a single immunization with RAS. Accordingly, late-arresting GAPs were shown to induce more sporozoites reduces the parasite load in the liver so greatly during diverse CTL responses than RAS vaccination in mice (27). Thus, subsequent immunizations that L3-specific responses are only gen- proteins expressed de novo in the liver [e.g., by CPS and some GAPs erated during the primary exposure. Functional L3-specificCTLscan, like 3-oxoacyl-ACP synthase I/II; PY04452 (FabB/F)-deficient IMMUNOLOGY however, be expanded by heterologous prime-boost regimens. Thus, parasites] and in the red blood cell (RBC) stages (e.g., by CPS) have although repeat sporozoite immunization expands responses to pre- received attention as candidate antigens. However, despite efforts formed antigens like CSP that are present in the sporozoite itself, this by several groups, few non-CSP antigens remain confirmed as CTL immunization strategy may not expand CTLs targeting parasite pro- targets and none are reported to be individually protective (28). teins that are synthesized later. Heterologous strategies may be It remains unclear why there is a paucity of protective antigens needed to increase CTL responses across the entire spectrum of Plas- after sporozoite immunization. The lack of targets may reflect modium liver-stage proteins. technical limitations in antigen discovery, antigenic bias due to homologous sporozoite vaccination, lack of late-stage antigen ex- vaccination | epitope pression, and/or CSP immunodominance. Traditionally, pooled synthetic peptides are used in IFN-γ (IFNγ) enzyme-linked im- alaria parasites cause tremendous morbidity and mortality munosorbent spot (ELISPOT) assays to identify T-cell targets (29), Mworldwide. The only vaccine approach known to induce but even with pooling strategies (30), these approaches require sterile protection against challenge relies on whole organism spo- complex, expensive peptide libraries. In lieu of peptides, plasmid- rozoite-stage parasites. This vaccination is experimentally achieved encoded parasite genes can be transfected into antigen-presenting fi in mice and humans by using radiation-attenuated sporozoites cells (APCs) (31), but this approach is low throughput and dif cult (RAS) (1, 2), genetically attenuated parasites (GAPs) (3), or wild- because of the A/T-richness of Plasmodial genes. Instead here, we type (WT) sporozoites with chloroquine prophylaxis (CPS) (4, 5). used a minigene-based high-throughput screen (HTS) for CTLs (32) to study a library of Plasmodium yoelii-derived antigens in These approaches induce protective antibodies and T cells, with fi cytotoxic T lymphocytes (CTLs) particularly important for pro- sporozoite-vaccinated mice. Our approach identi edauniqueCTL tection against experimental sporozoite challenge (6). target antigen that provided surprising insights about CTL Because sporozoites are difficult to manufacture, a multicom- responses in multiply-immunized mice. ponent subunit vaccine that induces protective CTLs is desirable; Results however, protective antigens must be identified first. Because Plasmodia have >5,000 genes, there are many candidates yet few HTS Reveals Greater CTL Diversity Following Different Sporozoite Immunizations. To identify novel malaria vaccine candidates, we bona fide antigens have been described since the identification of developed a HTS approach based on massively parallel synthesis the circumsporozoite protein (CSP) as a critical target more than of thousands of oligonucleotides that encode candidate peptides 24 y ago (7, 8). CSP coats the sporozoite surface, is shed from the (32) (Fig. S1). Liver-stage expressed genes (33) were subjected to motile sporozoite (9), and is transported to the hepatocyte cyto- H2d class I MHC prediction to produce a sequence library plasm (10). CSP is a target of protective class I-dependent CTL (Dataset S1) encoding 1,988 distinct 17-aa peptides (centered on responses (10–12). Depending on the mouse model, CSP can also + a predicted 9-mer peptide) in 199 pools of 10 minigenes each. elicit important CD4 T-cell responses (13, 14) and MHC class II- dependent IgG responses (15). In some systems, large numbers of adoptively transferred CSP-specific T-cell clones or endogenously Author contributions: S.C.M., B.C.S., and M.J.B. designed research; S.C.M., A.K., and B.C.S. generated CTLs can protect mice against sporozoite challenge (8, performed research; S.C.M., A.K., and B.C.S. contributed new reagents/analytic tools; 12, 14, 16, 17). However, it remains unclear whether CSP-specific S.C.M., B.C.S., and M.J.B. analyzed data; and S.C.M., B.C.S., and M.J.B. wrote the paper. CTLs in naïve subjects can be primed to achieve protection with The authors declare no conflict of interest. existing vaccination approaches. In addition, the CSP-based RTS, 1To whom correspondence should be addressed. E-mail: [email protected]. S vaccine in phase 3 human trials does not trigger strong CTL This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. responses (18, 19). The partial protection (20) from RTS,S is 1073/pnas.1303834110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1303834110 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 Pools were transfected into H2d P815 cells and used for ELISPOT and a single peptide from the PY05881 ribosomal protein L3 assays of mouse splenocytes 6 d after immunization. (GYKSGMSHI) predicted to bind H2-Kd generated ELISPOT In general, low frequency T-cell responses were observed for all responses (Fig. 2C). primary immunization regimens. The background in CPS-immu- The P. yoelii L3 ribosomal protein is expressed in liver and nized mice was significantly higher [22.9 ± 20.2 to 25.5 spot-forming RBC stages (33) and is conserved in Plasmodium berghei ANKA units (SFUs), 95% confidence interval (CI)] than for GAP- (4.7 ± (PBANKA_051190), Plasmodium falciparum (PF10_0272), and 4.7 to 9.6 SFU, 95% CI) or RAS-immunized mice (5.6 ± 4.5 to 6.7 Plasmodium vivax (PVX_111330). By ELISPOT, L3-specific SFU, 95% CI) (P < 0.0001, unpaired Student’s t test, 16 wells for cells responded to low nanomolar peptide concentrations, sim- CPS and RAS, 15 wells for GAP), possibly due to natural killer cell ilar to the behavior of CSP-specificcells(Fig.3A). The L3 re- + activation by infected RBCs (iRBCs) (34). CPS spleens were larger sponse was mediated by CD8 T cells because anti-CD8 but not and contained more pigment than normal-appearing spleens of anti-CD4 antibodies blocked the response (Fig. 3B). By H2-Kd GAP- and RAS-immunized mice, likely due to uptake of drug- binding studies using RMA/S lymphoma cells, both L3 and CSP killed iRBCs. After normalization, CPS and GAP triggered the peptides demonstrated low nanomolar-strength MHC-specific d most positive results (each with 27 incompletely overlapping pools) binding to H2-K (L3 Kd = 0.7 nM compared with CSP Kd 1.6 followed by RAS immunization (17 pools) at P < 0.05 (Fig. 1A and nM) (Fig. 3C). The L3-specific ELISPOT response could also be Fig. S2). Most positive responses produced fewer IFNγ spots than blocked by using anti–H2-Kd antibodies (Fig. S3). the CSP control well. Only two pools were positive in all screens in CSP- and L3-specific responses could be detected in liver lym- CPS, GAP, and RAS immunized mice (pools 79 and 171). All phocytes from CPS- and RAS-immunized mice by ELISPOT 6 d immunizations induced strong ELISPOT responses to the column- after immunization (Fig. S4). Because L3 lacks a canonical signal synthesized CSP-encoding control minigene (Fig. 1A and Fig. S2). sequence and may not traffic to the parasitophorous vacuole or Pool 102 also contained the microarray-synthesized minigene hepatocyte cytosol, we tested responses to cross-presented L3 as encoding the SYVPSAEQI peptide of CSP and was positive in well.