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Defining rules of CD8+ T cell expansion against pre-erythrocytic Plasmodium antigens in sporozoite-immunized mice
© Billman et al. 2016
Received: 6 January 2016
Accepted: 14 April 2016
Published: 26 April 2016
Whole Plasmodium sporozoites serve as both experimental tools and potentially as deployable vaccines in the fight against malaria infection. Live sporozoites infect hepatocytes and induce a diverse repertoire of CD8+ T cell responses, some of which are capable of killing Plasmodium-infected hepatocytes. Previous studies in Plasmodium yoelii-immunized BALB/c mice showed that some CD8+ T cell responses expanded with repeated parasite exposure, whereas other responses did not.
Here, similar outcomes were observed using known Plasmodium berghei epitopes in C57BL/6 mice. With the exception of the response to PbTRAP, IFNγ-producing T cell responses to most studied antigens, such as PbGAP50, failed to re-expand in mice immunized with two doses of irradiated P. berghei sporozoites. In an effort to boost secondary CD8+ T cell responses, heterologous cross-species immunizations were performed. Alignment of P. yoelii 17XNL and P. berghei ANKA proteins revealed that >60 % of the amino acids in syntenic orthologous proteins are continuously homologous in fragments ≥8-amino acids long, suggesting that cross-species immunization could potentially trigger responses to a large number of common Class I epitopes. Heterologous immunization resulted in a larger liver burden than homologous immunization. Amongst seven tested antigen-specific responses, only CSP- and TRAP-specific CD8+ T cell responses were expanded by secondary homologous sporozoite immunization and only those to the L3 ribosomal protein and S20 could be re-expanded by heterologous immunization. In general, heterologous late-arresting, genetically attenuated sporozoites were better at secondarily expanding L3-specific responses than were irradiated sporozoites. GAP50 and several other antigens shared between P. berghei and P. yoelii induced a large number of IFNγ-positive T cells during primary immunization, yet these responses could not be re-expanded by either homologous or heterologous secondary immunization.
These studies highlight how responses to different sporozoite antigens can markedly differ in recall following repeated sporozoite vaccinations. Cross-species immunization broadens the secondary response to sporozoites and may represent a novel strategy for candidate antigen discovery.
Pre-clinical and clinical vaccine studies have demonstrated that whole Plasmodium sporozoites can induce sterile protection against infectious challenge [1–5]. Sporozoite formulations include radiation-attenuated sporozoites (RAS) [3, 6], genetically attenuated parasites (GAP)  or wild-type (WT) sporozoites administered under anti-malarial drug prophylaxis [2, 8]. Such approaches induce protective antibodies and T cells with IFNγ-producing cytotoxic T lymphocytes (CTL), which are particularly important for protection during the liver stage . Plasmodium are complex eukaryotic pathogens that express thousands of different proteins throughout their lifecycle . Until recently, this enormous array of proteins made it difficult to study antigen-specific immune responses on a large scale. Consequently, relatively little is known about the repertoire of T cell responses and to some extent antibody responses that target pre-erythrocytic stage antigens.
The most well-studied antigen is the circumsporozoite protein (CSP) [11, 12]. CSP encounters antigen-processing cells after being shed from the sporozoite surface  and alternatively after transport into the hepatocyte cytoplasm in sporozoite-invaded hepatocyte . CSP induces protective Class I-dependent CTL responses [14–16] and can also induce CD4+ T cell responses [17, 18] and major histocompatibility complex (MHC) Class II-dependent IgG responses . While CSP-specific cells can induce protection when present at extremely high frequencies through experimental manipulations [12, 16, 18, 20, 21], such frequencies are not commonly achieved following sporozoite exposures. The CSP-based RTS,S vaccine in humans does not trigger strong CTL responses [22, 23] and instead seems to rely on antibodies and CD4+ T cell responses  to achieve partial protection . Moreover, non-CSP antigens are increasingly appreciated as potential vaccine candidates since mice can be protected against challenge even in the absence of CSP-specific immunity [15, 26–29]. In addition to CSP, thrombospondin-related adhesive protein (TRAP, also called sporozoite surface protein 2 or SSP2) can induce CD8 + T cells  and TRAP-specific CD8 T cells can kill infected liver cells . Like CSP, TRAP is also shed from the sporozoite surface, a process required for gliding motility and sporozoite infectivity . These two proteins have been the focus of most pre-clinical and clinical studies of pre-erythrocytic antigens. While a handful of new antigens have been recently identified , the remaining antigens targeted by humoral and cellular immune responses are not well understood.
Minigene library screening was recently employed in an effort to identify novel pre-erythrocytic antigens  and identified the L3 ribosomal protein as a target of the CD8 T cell response. Whereas the response to CSP increased with repeated sporozoite exposures in BALB/c mice, the response to L3 was not strongly recalled by subsequent sporozoite exposures. The L3-specific T cells were functionally cytotoxic and could be re-expanded by a non-sporozoite booster in the form of Listeria monocytogenes recombinantly expressing the L3 epitope . Although single dose immunizations with attenuated sporozoites do not usually lead to sterile protection in mice , a single immunization does achieve a significant level of partial protection, as measured by liver burden assessments . Subsequent immunizations further increase this protective effect, indicative of gradual acquisition of sterile protective immunity. At the vaccination stage, gradually acquired protection leading to significant reductions in liver burden also correlated with significant reductions in the L3 antigen load . Since L3 was mostly expressed in the liver and later erythrocyte stages but not the sporozoite stage, the acquisition of immunity against the sporozoites used for vaccination were reducing the immunogenicity of the vaccine for antigens that needed to be expressed de novo in the hepatocyte . Here, a series of known Plasmodium berghei antigens were examined in C57BL/6 mice multiply immunized with sporozoites to determine if the distinction between boostable versus non-boostable responses was generalizable beyond BALB/c mice. In addition, heterologous cross-species/strain immunization was tested to determine if this modified the immunogenicity of shared sporozoite antigens.
All animal studies were approved by the University of Washington Institutional Animal Care and Use Committee (IACUC protocol 4317-01). Female BALB/cj and C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). A breeding pair of C57BL/6-derived μMT mice (B6.129S2-Ighmtm1Cgn/J) were also obtained from Jackson Laboratories and were bred at the University of Washington. All mice were housed in standard IACUC-approved small animal facilities and used in compliance with IACUC-approved protocols.
Sporozoite vaccination and challenge
WT Plasmodium yoelii 17XNL, WT P. berghei ANKA and P. yoelii fabb/f −/− (GAP) sporozoites were obtained by salivary gland dissection from Anopheles stephensi mosquitoes reared at the Research Insectary at the Center for Infectious Disease Research (formerly Seattle Biomed, Seattle, WA, USA). Where indicated, RAS were generated by exposure to 10,000 rads using an X-ray irradiator (Rad-Source, Suwanee, GA, USA). Unless stated otherwise, RAS, GAP and WT sporozoites were administered by intravenous tail vein injection in a volume of 150 μL. Multiply immunized mice were vaccinated at three-week intervals. Where indicated, sporozoites were purified using the Accudenz gradient method . For challenge, mice were intravenously challenged with 1000 or 10,000 WT sporozoites.
Ex vivo IFNγ enzyme-linked immunosorbent spot (ELISPOT) assays
For ELISPOTs, peptides corresponding to known CD8+ T cell epitopes (1 µg/mL final) were combined with 1 × 106 murine splenocytes by murine interferon-γ (IFNγ) ELISPOT (Affymetrix, Santa Clara, CA, USA) and cultured in antibody-coated ELISPOT plates 18 h at 37 °C as previously reported . Plasmodium berghei peptides used to assess responses included those for PbTRAP130–138 (H2-Db-restricted SALLNVDNL from PBANKA_1349800 TRAP ), PbS20318–326 (H2-Kb-restricted VNYSFLYLF from PBANKA_1429200 sporozoite-specific gene 20 (S20) ), PbGAP5041–48 (SQLLNAKYL from PBANKA_0819000 glideosome-associated protein 50 (GAP50) [38, 39]), PbF4 (EIYIFTNI from PBANKA_0416600 replication protein A1 ), PbNCY (NCYDFNNI from PBANKA_0714500 ) and PbCSP245–253 (H2-Kd-restricted SYIPSAEKI from PBANKA_0403200 circumsporozoite protein (CSP). Plasmodium yoelii peptides used to assess responses included those for PyCSP280–288 (H2-Kd-restricted SYVPSAEQI from PY03168) and PyL3 (H2-Kd-restricted GYKSGMSHI from PY05881 ). All gene identifiers refer to PlasmoDB names . ELISPOT plates were developed using a colorimetric substrate as reported . All ELISPOT wells were tested in two to three wells per mouse per antigen and cumulative ELISPOT data were evaluated using the mean spot forming units (SFU) per million splenocytes for each animal.
Bioinformatics analysis of class I peptide-sized homology
FASTA files of all protein sequences were downloaded from PlasmoDB  for Plasmodium species (P. falciparum 3D7, P. vivax Sal-1, P. berghei ANKA, P. yoelii 17XNL). Comparisons were made between proteins of P. falciparum 3D7 and P. vivax Sal1 and between the proteins of P. berghei ANKA and P. yoelii 17XNL. For each pair of species, an exhaustive search was carried out on the full protein sets of the two species to find all common 8-amino acid sequences. The search strategy was to loop through the first protein set using 8-amino acid by 8-amino acid comparisons. For the second protein set, proteins were concatenated end-to-end 100 at a time, with a separator symbol ($) between successive proteins. The concatenated string was searched using a sorted suffix array (reviewed in ). Matching 8-amino acid regions were then extended to find the longest exact matching peptide of length ≥8 amino acids. This strategy was implemented in a Python program. Data consisting of the common peptide sequence, species 1 protein identifier and species 2 protein identifier were filtered to include syntenic orthologues only and further categorized by available expression data (e.g., sporozoite or liver stage). In addition, highly repetitive peptides such as pure runs or nearly pure runs of a single amino acid were removed from the database—this last criterion was especially useful for P. falciparum where asparagine repeats are extremely common . Expression data consisted of mass spectrometry data from several large published datasets [10, 45–47] transformed by syntenic orthology to generate lists of proteins where the protein of interest or its syntenic orthologue was identified as a sporozoite (spz) and/or liver stage protein. Proteins were included if mass spectrometry data demonstrated at least one peptide and one spectra minimum. Stage-specific datasets included P. berghei and P. yoelii salivary gland sporozoites , P. falciparum salivary gland sporozoites [10, 45, 46] and P. yoelii liver stage proteins . The lengths of the homologous peptides were recorded and percent homologous sequence compared to the total encoded protein sequence for each species.
B cell depletion experiments
Anti-CD20 antibody (clone 5D2 IgG2a) was provided by Genentech. BALB/cj mice were injected with 250 μg of anti-CD20 one day prior to primary immunization with 1 × 104 purified P. yoelii RAS. Four weeks later, mice were injected again with anti-CD20 and then administered a second homologous dose of 1 × 104 purified P. yoelii RAS 2 days later. B cell depletion was confirmed by evaluating peripheral blood for the presence of B220+ cells in the single cell lymphocyte gate on a Canto flow cytometer (BD, Franklin Lakes, NJ, USA).
Liver stage Plasmodium 18S rRNA assay
At the indicated time post-challenge, mice were sacrificed, and half of the total liver was excised and pulverized by bead beating in 5 mL NucliSENS lysis buffer (bioMérieux, Durham, NC, USA). Total RNA was extracted by processing 100 μL of the NucliSENS buffer-treated sample diluted 1:10 in NucliSENS lysis buffer on the EasyMag system (bioMérieux) as described for whole blood . In some experiments, livers were pulverized in 5 mL Trizol (Life Technologies/Invitrogen, Carlsbad, CA, USA) and total RNA was Trizol extracted as described . RNA was subjected to RT-PCR using the One Step AgPath RT-PCR kit (Invitrogen) using a predesigned HEX-labelled mouse GAPDH RT-PCR assay (IDT Inc, Coralville, IA, USA) multiplexed with a Pan-Plasmodium 18S rRNA assay. The Pan-Plasmodium 18S rRNA reagents consisted of a CalFluor Orange560-labelled Pan-Plasmodium probe (5′[CAL Fluor Orange 560]-ACCGTCGTAATCTTAACCATAAACTA[T(BHQ1)]GCCGACTAG-3′; Biosearch Technologies, Navato, CA, USA) and adjacent primers (forward 5′-AAAGTTAAGGGAGTGAAGA-3′; reverse 5′-AAGACTTTGATTTCTCATAAGG-3′) under the following conditions (45 °C for 20 min, 95 °C for 15 min and 45 cycles of 95 °C for 20 s, 50 °C for 30 s, 60 °C for 30 s) on a CFX96/1000C real-time PCR machine (Biorad, Hercules, CA, USA). Data were normalized to mouse GAPDH and transformed to relative log10 values to compare log10 copy number reduction in Plasmodium 18S rRNA versus the control group.
Most malaria-specific CD8+ T cell responses contract with repeated whole sporozoite immunization except for those targeting protective TRAP and CSP antigens
Heterologous cross-species immunization has the potential to generate a more diverse T cell repertoire
Plasmodium species share considerable amino acid homology. Despite this, most immunization-challenge models have consisted almost exclusively of homologous immunization followed by homologous challenge. Recently, some groups have begun to evaluate homologous P. falciparum (single strain) immunizations followed by heterologous challenge with a different P. falciparum strain in CHMI studies .
Since heterologous challenge circumvents some antigen-specific immune responses , it was possible that a heterologous (cross-species) immunization regimen could expand CD8+ T cell responses against shared epitopes more so than a homologous regimen. To gauge the potential breadth of shared Class I epitopes, bio-informatic analyses were performed to look for interspecies homology of syntenic orthologues using a minimum homology length of 8 aa, the shortest length of a typical Class I MHC-binding peptide. First, coding sequences for all predicted proteins [P. yoelii 17XNL 7724 (3.3 × 106 total aa), P. berghei ANKA 4952 (3.4 × 106 total aa), P. falciparum 5398 (4.1 × 106 total aa), P. vivax Sal-1 5530 (3.9 × 106 total aa)] were filtered to include only syntenic orthologous pairs. For P. yoelii 17XNL/P. berghei ANKA, there were 4191 pairs comprising 4166 P. yoelii 17XNL proteins of 2.4 × 106 total aa and 4075 P. berghei ANKA proteins of 2.9 × 106 total aa. For P. falciparum/P. vivax Sal1, there were 4777 pairs comprised of 4638 unique P. falciparum proteins of 3.6 × 106 total aa and 4584 P. vivax Sal1 proteins of 3.4 × 106 total aa) (Additional file 2).
Shared 8-mer linear peptidome of rodent and human Plasmodium parasites
Homologous windows (#≥8 aa)
Mean length (aa)
Max length (aa)
Total aa conserved in ≥8 aa windows
% of syntenic proteomea (%)
1.72 × 106
70.5 % Py/59.3 % Pb
6.49 × 105
18.1 % Pf/18.9 % Pv
1.12 × 106
All: 46.5 % Py/38.4 % Pb
Spz: 76.2 % Py/61.6 % Pb
4.73 × 105
All: 13.2 % Pf/13.8 % Pv
Spz: 20.8 % Pf/13.8 % Pv
2.67 × 105
All: 10.9 % Py/9.2 % Pb
LS: 79.5 % Py/77.9 % Pb
1.51 × 105
All: 4.2 % Pf/4.4 % Pv
LS: 38.6 % Pf/39.3 % Pv
Heterologous cross-species immunizations achieve larger secondary liver infections than homologous immunizations
Previous work on cross-species protection in homologously immunized C57BL/6 mice suggested that such protection was T cell dependent since adoptively transferred antisera generated by homologous immunization could not confer protection against cross-species challenge . To determine if heterologous immunization resulted in larger liver burdens by circumventing the antibody responses stimulated by the priming immunization, several experiments were conducted on the C57BL/6 and BALB/c backgrounds. The following B-cell deficient mouse experiments were measured by liver-stage RT-PCR at 24 h post-immunization in order to separate effects of cell-mediated killing and antibody-mediated blockade of invasion. First, WT C57BL/6 mice and C57BL/6-derived μMT mice were compared in homologous P. berghei RAS immunization experiments. μMT mice lack mature B cells and do not make antibody responses. WT C57BL/6 and μMT mice were immunized with P. berghei RAS once or twice and the parasite liver burden was measured at 24 h after the last immunization as a measure of initial liver-stage infection. At 24 h post-challenge, homologously immunized B6 mice showed a reduction in liver burden compared to singly exposed animals (Fig. 2b). In contrast, there was no such reduction in B-cell deficient μMT mice regardless of one or two exposures (Fig. 2b). This finding suggests that a single exposure to sporozoites elicits antibody-mediated ‘debulking’ of subsequent immunizations in immunocompetent C57BL/6 mice. μMT mice could not be used to assess whether the resulting increase in liver burden increased overall immunogenicity because repeated immunizations led to the development of small brittle spleens that demonstrated unacceptably high lymphocyte mortality (>80 %) upon splenocyte harvest. To test the role of antibodies in BALB/c mice, anti-CD20 antibodies (kind gift of Genentech) were used to eliminate B cells before each of two P. yoelii RAS immunizations in BALB/cj mice. Anti-CD20 antibody-treated mice showed no demonstrable B cells by anti-B220 staining compared to control mice (Additional file 6: Figure S3). Antibody-treated, B cell-deficient BALB/c mice showed a more than twofold increase in parasite liver burden (+0.45 log10 copies Plasmodium 18S rRNA) at 24 h post-challenge compared to untreated mice (Fig. 2c). Changes in liver burden observed in B-cell deficient mice (through genetic- and antibody-mediated approaches) show that antibodies reduce the liver burden in homologously immunized mice. These findings support the idea that heterologous immunization circumvents homologous antibody-dependent protection that otherwise would debulk the intended liver infection.
Heterologous immunization recalls some but not all CD8+ T cell responses that normally contract following homologous immunizations
Although repeated homologous sporozoite immunization protects humans from WT P. falciparum challenge, the regimen that achieves protection requires four to five intravenous doses of 1.35 × 105 irradiated, cryopreserved sporozoites at each dose . Less attenuated sporozoites such as late-arresting GAPs  or WT sporozoites administered under anti-malarial drug coverage  may have a dose-sparing effect but GAP vaccines have not yet been manufactured in a vialed format. Vialed, cryopreserved WT sporozoites under a concurrent drug treatment-vaccination approach known as CVac  are being developed but may require optimization. At this time, it is difficult to make a head-to-head comparison about the immunogenicity of PfSPZ, GAP and/or CVac-type approaches because optimal vaccination schedules have not been finalized for any of these approaches. Modifications that reduce dosing frequency and/or dosage while increasing immunogenicity (and ultimately efficacy) may improve delivery of whole sporozoite vaccine product. This study was undertaken in the mouse model to evaluate sporozoites vaccination modifications that could generally enhance the immunogenicity of sporozoite-based vaccines.
In previous work in the BALB/cj model , two model P. yoelii-derived antigens were found to induce very different CD8+T cell responses upon repeated sporozoite immunizations. The well-studied CSP antigen-induced cytotoxic CD8+ T cells that increased in frequency with multiple doses of 1 × 104 sporozoites (RAS or GAP). In contrast, the L3 ribosomal protein induced a comparable number of cytotoxic CD8+ T cells as CSP upon primary immunization but these responses did not increase and generally contracted with multiple immunizations. Here, the same pattern of secondary recall or contraction was observed for T cell responses to a panel of known P. berghei epitopes in C57BL/6 mice. PbTRAP130-138 (SALLNVDNL) is an H2-Db-restricted cytotoxic immunodominant CD8+ T cell epitope in C57BL/6 mice derived from PBANKA_1349800 TRAP that contributes to protection against liver-stage infection . PbS20318–326 (VNYSFLYLF) is an H2-Kb-restricted but non-cytotoxic, non-protective CD8+ T cell epitope derived from PBANKA_1429200 sporozoite-specific gene 20 (S20) . PbGAP5041–48 (SQLLNAKYL) is derived from PBANKA_0819000 glideosome-associated protein 50 known as GAP50 and targeted by cytotoxic CD8+ T cells [38, 39] but is not protective . PbGAP5041–48-specific cells have amongst the highest precursor frequency identified for any T cell with ~2200 PbGAP5041–48-specific cells per animal . PbF4 (EIYIFTNI) is derived from PBANKA_0416600 replication protein A1 and is a blood stage CD8+ epitope . Recently, T cells responding to Pb1, F4 and another epitope from PBANKA_113700/bergheilysin (IITDFENL) were shown to damage the blood–brain barrier in a murine model of P. berghei-induced cerebral malaria . PbNCY (NCYDFNNI) is derived from PBANKA_0714500 and is the target of so-called ‘PbT1′ T cells that have been shown to respond to P. berghei sporozoites and infected erythrocytes . This antigen is encoded on sporozoites and PbT1 cells are able to protect mice from liver-stage challenge infection following a spleen-centered CD8α+ dendritic cell-dependent immune response, indicating that the antigen may be partly cross-presented .
Comparison of immunogenicity data to available mass spectrometry-based expression data
Conserved in Pb/Py?
No. spectra in Py/Pf spzb (Py rank/Pf rank)
No. spectra from Py/Pf spz surface?b
(Py rank/Pf rank)
Δ Transcription (24/40 h LS vs sgSPZ)c
To address whether a simple ‘debulking’ model explained the contraction of responses to S20, GAP50, F4, and NCY, heterologous immunization was tested to determine if this approach could secondarily expand these specific T cell populations. Bio-informatic analysis demonstrated that there is a considerable amount of protein sequence homology that constitutes peptides of sufficient length to be Class I MHC targets. To pursue this approach experimentally, BALB/cj mice immunized with P. berghei RAS then P. yoelii RAS achieved larger liver burdens upon secondary immunization as compared to mice immunized twice with P. yoelii RAS. Although a major goal of vaccine developers is to produce a malaria vaccine that achieves cross-species protection, the imperfect cross-species immunity achieved after a single immunization with just one species means that the vaccine ‘take’ upon secondary heterologous immunization is improved. This debulking is at least partially due to antibodies directed against homologous parasites since circumventing such antibodies can increase liver burden upon secondary vaccination. The T cell repertoire is potentially extremely large and could benefit from secondary boosting of responses to shared epitopes. Although L3 and PbS20 are both non-protective, these antigens could be recalled by heterologous but not homologous immunization. The assumption is that these antigens are emblematic of a class of antigens that do not re-expand due to debulking of the secondary immunization by the immunity achieved by primary immunization. If this class of antigens contains as yet undiscovered protective targets, heterologous immunization could accelerate their discovery. L3 responses were more vigorously recalled by secondary heterologous immunization with a late-arresting GAP compared to the early-arresting RAS approach. Thus, a more ‘fit’ parasite may also improve recall of liver-stage antigens. It seems unlikely that the degree of attenuation would profoundly affect responses to antigens that are primarily pre-formed in the sporozoite. This may explain why abundant pre-formed CSP and TRAP antigens expand responses while less abundant pre-formed antigens like PbS20 and PbGAP50 do not. The strong primary responses to PbS20 and PbGAP50 and lack of secondary homologous responses also suggest that these responses probably depend on sufficient hepatocyte infection. Heat-killed sporozoites can induce responses to CSP but at considerably lower frequencies than with live sporozoites [34, 55], which also implies that responses to CSP also depend on liver infection. Responses to the CSP and TRAP epitopes tested here did not benefit from heterologous immunization because there are amino acid differences between the species that preclude cross-reactive responses by species-specific T cells. Heterologous RAS immunization increased the secondary response to the conserved PbS20 antigen. For some conserved antigens however (PbGAP50, F4, NCY), even heterologous RAS immunization did not lead to secondary recall, suggesting that these antigens may not be of sufficient quantity even when the liver burden is modestly increased by heterologous exposure.
How much of the Pb20 and GAP50 antigens come from sporozoite versus intrahepatic liver expression is unknown. Mass spectrometry data shows that peptides from P. falciparum S20 can also be found on the surface of sporozoites  and it is possible that secondary recall is enhanced by antigen surface exposure in addition to heterologous RAS exposure. Little is known about the role of GAP50 in the sporozoite. Although GAP50 peptides are found in sporozoites , GAP50 is principally expressed in blood and gametocyte stages. In the blood, GAP50 is a constituent protein of the glideosome, an actin-myosin motor complex critical for erythrocyte invasion [56–58]. GAP50 expression continues into gametocytes , where its interaction with factor H  is being targeted as a candidate transmission blocking strategy . At this time, it remains unknown whether a late-arresting/less-attenuated sporozoite would secondarily improve recall responses for GAP50 and other antigens tested in C57BL/6 mice.
The type of bio-informatic analysis conducted here has not been previously reported. Previous work has shown that rodent Plasmodia (P. yoelii/P. berghei) are evolutionarily much closer to one another than P. falciparum/P. vivax [61–65]. P. yoelii/P. berghei both arise from a rodent Plasmodium clade whereas P. falciparum is in the hominid clade and P. vivax in the monkey clade [66, 67]. Not surprisingly, there was greater amino acid homology of sufficient lengths to constitute potential class I MHC peptides (≥8 amino acids) for P. yoelii/P. berghei compared to P. falciparum/P. vivax. Nonetheless, the listings of shared syntenic peptides may help to identify protein targets that are in the right time and place to be T cell targets in heterologously immunized mice. These peptides also constitute one strategy for developing pan-Plasmodium T cell vaccines. Given the large evolutionary distance between P. falciparum and P. vivax, more closely related P. falciparum strains should also be evaluated. A number of these P. falciparum strains are now available for CHMI studies (e.g., P. falciparum 7G8 and P. falciparum NF135.C10) and will certainly share many more homologous syntenic peptides with P. falciparum than for the P. falciparum/P. vivax pair. Continued bio-informatics and experimental analysis of common peptides between species and strains may reveal novel strategies for developing malaria vaccines against one or more of the human-infecting strains.
One potential limitation of this study is that the infectivity of P. yoelii and P. berghei for mice differs. P. berghei infectivity is generally greater than P. yoelii in C57BL/6 mice whereas P. yoelii is more infectious in BALB/c mice (reviewed in ). However, the same number of parasites was administered regardless of species since there is no clear method for titrating the dose of one species against the other. The less infective species was used as the priming dose (P. yoelii in C57BL/6 mice or P. berghei in BALB/c mice). In addition, the same species was always used as the final dose, thereby minimizing the infectivity difference at the time of measurement. Nonetheless, infectivity differences may need to be accounted for in efforts to design optimally protective vaccination regimens. For instance, it may be desirable to use the most infectious species first to prime a larger number of responses or it may prove better to use the more infectious sporozoites secondarily to induce a larger recall response.
In summary, these data categorize CD8+ T cell responses induced by Plasmodium sporozoites into (a) those primed but never recalled by sporozoite re-exposure; (b) primed and recalled by homologous sporozoites; or, (c) primed and recalled by heterologous but not homologous re-sporozoites. The next phase of this work is to determine how heterologous immunizations affect antibody responses and to determine if two-dose heterologous or combination dose immunizations can be designed to achieve sterile protection. A possible strategy is to prime mice or humans with sporozoites of a single species or strain and then boost mice with that same strain plus sporozoites from an additional species or a different strain. In addition, if low abundance and/or late liver stage antigens are found to be important through sporozoite- or subunit-based experimentation, sporozoite-based vaccines could potentially be empowered to expand such responses if recombinant parasites were engineered to express such live stage antigens earlier as pre-formed proteins in salivary gland sporozoites.
ZPB performed the research and analysed the data. BCS designed and performed the research, contributed new reagents, analysed the data and wrote the paper. AK performed the bio-informatics work. SCM designed and performed the research, contributed new reagents, analysed the data and wrote the paper. All authors read and approved the final manuscript.
The authors wish to thank Annette Seilie for the liver-stage 18S rRNA assay, Kevin Zhou for bio-informatics assistance and Emma Fritzen, Matt Fishbaugher, Heather Kain, Will Betz and Stefan Kappe of CID Research for sporozoite production. This work was partially funded by the NIH (Grant 1K08AI097238 to SCM). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
The authors declared that they have no competing interests.
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- Seder RA, Chang LJ, Enama ME, Zephir KL, Sarwar UN, Gordon IJ, et al. Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine. Science. 2013;341:1359–65.View ArticlePubMedGoogle Scholar
- Roestenberg M, McCall M, Hopman J, Wiersma J, Luty AJ, van Gemert GJ, et al. Protection against a malaria challenge by sporozoite inoculation. N Engl J Med. 2009;361:468–77.View ArticlePubMedGoogle Scholar
- Nussenzweig RS, Vanderberg J, Most H, Orton C. Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. Nature. 1967;216:160–2.View ArticlePubMedGoogle Scholar
- Butler NS, Schmidt NW, Vaughan AM, Aly AS, Kappe SH, Harty JT. Superior antimalarial immunity after vaccination with late liver stage-arresting genetically attenuated parasites. Cell Host Microbe. 2011;9:451–62.View ArticlePubMedPubMed CentralGoogle Scholar
- Butler NS, Vaughan AM, Harty JT, Kappe SH. Whole parasite vaccination approaches for prevention of malaria infection. Trends Immunol. 2012;33:247–54.View ArticlePubMedGoogle Scholar
- Clyde DF, Most H, McCarthy VC, Vanderberg JP. Immunization of man against sporozite-induced falciparum malaria. Am J Med Sci. 1973;266:169–77.View ArticlePubMedGoogle Scholar
- Mueller AK, Labaied M, Kappe SH, Matuschewski K. Genetically modified Plasmodium parasites as a protective experimental malaria vaccine. Nature. 2005;433:164–7.View ArticlePubMedGoogle Scholar
- Putrianti ED, Silvie O, Kordes M, Borrmann S, Matuschewski K. Vaccine-like immunity against malaria by repeated causal-prophylactic treatment of liver-stage Plasmodium parasites. J Infect Dis. 2009;199:899–903.View ArticlePubMedGoogle Scholar
- Doolan DL, Hoffman SL. The complexity of protective immunity against liver-stage malaria. J Immunol. 2000;165:1453–62.View ArticlePubMedGoogle Scholar
- Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD, et al. A proteomic view of the Plasmodium falciparum life cycle. Nature. 2002;419:520–6.View ArticlePubMedGoogle Scholar
- Kumar S, Miller LH, Quakyi IA, Keister DB, Houghten RA, Maloy WL, et al. Cytotoxic T cells specific for the circumsporozoite protein of Plasmodium falciparum. Nature. 1988;334:258–60.View ArticlePubMedGoogle Scholar
- Romero P, Maryanski JL, Corradin G, Nussenzweig RS, Nussenzweig V, Zavala F. Cloned cytotoxic T cells recognize an epitope in the circumsporozoite protein and protect against malaria. Nature. 1989;341:323–6.View ArticlePubMedGoogle Scholar
- Stewart MJ, Vanderberg JP. Malaria sporozoites leave behind trails of circumsporozoite protein during gliding motility. J Protozool. 1988;35:389–93.View ArticlePubMedGoogle Scholar
- Bongfen SE, Torgler R, Romero JF, Renia L, Corradin G. Plasmodium berghei-infected primary hepatocytes process and present the circumsporozoite protein to specific CD8+T cells in vitro. J Immunol. 2007;178:7054–63.View ArticlePubMedGoogle Scholar
- Kumar KA, Baxter P, Tarun AS, Kappe SH, Nussenzweig V. Conserved protective mechanisms in radiation and genetically attenuated uis3(-) and uis4(-) Plasmodium sporozoites. PLoS One. 2009;4:e4480.View ArticlePubMedPubMed CentralGoogle Scholar
- Balam S, Romero JF, Bongfen SE, Guillaume P, Corradin G. CSP-A model for in vivo presentation of Plasmodium berghei sporozoite antigens by hepatocytes. PLoS One. 2012;7:e51875.View ArticlePubMedPubMed CentralGoogle Scholar
- Renia L, Marussig MS, Grillot D, Pied S, Corradin G, Miltgen F, et al. In vitro activity of CD4+ and CD8+T lymphocytes from mice immunized with a synthetic malaria peptide. Proc Natl Acad Sci USA. 1991;88:7963–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Renia L, Grillot D, Marussig M, Corradin G, Miltgen F, Lambert PH, et al. Effector functions of circumsporozoite peptide-primed CD4+T cell clones against Plasmodium yoelii liver stages. J Immunol. 1993;150:1471–8.PubMedGoogle Scholar
- Molano A, Park SH, Chiu YH, Nosseir S, Bendelac A, Tsuji M. Cutting edge: the IgG response to the circumsporozoite protein is MHC class II-dependent and CD1d-independent: exploring the role of GPIs in NK T cell activation and antimalarial responses. J Immunol. 2000;164:5005–9.View ArticlePubMedGoogle Scholar
- Rodrigues MM, Cordey AS, Arreaza G, Corradin G, Romero P, Maryanski JL, et al. CD8 + cytolytic T cell clones derived against the Plasmodium yoelii circumsporozoite protein protect against malaria. Int Immunol. 1991;3:579–85.View ArticlePubMedGoogle Scholar
- Schmidt NW, Podyminogin RL, Butler NS, Badovinac VP, Tucker BJ, Bahjat KS, et al. Memory CD8 T cell responses exceeding a large but definable threshold provide long-term immunity to malaria. Proc Natl Acad Sci USA. 2008;105:14017–22.View ArticlePubMedPubMed CentralGoogle Scholar
- Barbosa A, Naniche D, Aponte JJ, Manaca MN, Mandomando I, Aide P, et al. Plasmodium falciparum-specific cellular immune responses after immunization with the RTS, S/AS02D candidate malaria vaccine in infants living in an area of high endemicity in Mozambique. Infect Immun. 2009;77:4502–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Pichyangkul S, Kum-Arb U, Yongvanitchit K, Limsalakpetch A, Gettayacamin M, Lanar DE, et al. Preclinical evaluation of the safety and immunogenicity of a vaccine consisting of Plasmodium falciparum liver-stage antigen 1 with adjuvant AS01B administered alone or concurrently with the RTS, S/AS01B vaccine in rhesus primates. Infect Immun. 2008;76:229–38.View ArticlePubMedPubMed CentralGoogle Scholar
- Ndungu FM, Mwacharo J, Kimani D, Kai O, Moris P, Jongert E, et al. A statistical interaction between circumsporozoite protein-specific T cell and antibody responses and risk of clinical malaria episodes following vaccination with RTS, S/AS01E. PLoS ONE. 2012;7:e52870.View ArticlePubMedPubMed CentralGoogle Scholar
- Kester KE, Cummings JF, Ofori-Anyinam O, Ockenhouse CF, Krzych U, Moris P, et al. Randomized, double-blind, phase 2a trial of falciparum malaria vaccines RTS, S/AS01B and RTS, S/AS02A in malaria-naive adults: safety, efficacy, and immunologic associates of protection. J Infect Dis. 2009;200:337–46.View ArticlePubMedGoogle Scholar
- Kumar KA, Sano G, Boscardin S, Nussenzweig RS, Nussenzweig MC, Zavala F, et al. The circumsporozoite protein is an immunodominant protective antigen in irradiated sporozoites. Nature. 2006;444:937–40.View ArticlePubMedGoogle Scholar
- Gruner AC, Mauduit M, Tewari R, Romero JF, Depinay N, Kayibanda M, et al. Sterile protection against malaria is independent of immune responses to the circumsporozoite protein. PLoS ONE. 2007;2:e1371.View ArticlePubMedPubMed CentralGoogle Scholar
- Mauduit M, Tewari R, Depinay N, Kayibanda M, Lallemand E, Chavatte JM, et al. Minimal role for the circumsporozoite protein in the induction of sterile immunity by vaccination with live rodent malaria sporozoites. Infect Immun. 2010;78:2182–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Mauduit M, Gruner AC, Tewari R, Depinay N, Kayibanda M, Chavatte JM, et al. A role for immune responses against non-CS components in the cross-species protection induced by immunization with irradiated malaria sporozoites. PLoS One. 2009;4:e7717.View ArticlePubMedPubMed CentralGoogle Scholar
- Pearson FE, O’Mahony C, Moore AC, Hill AV. Induction of CD8(+) T cell responses and protective efficacy following microneedle-mediated delivery of a live adenovirus-vectored malaria vaccine. Vaccine. 2015;33:3248–55.View ArticlePubMedGoogle Scholar
- Longley RJ, Bauza K, Ewer KJ, Hill AV, Spencer AJ. Development of an in vitro assay and demonstration of Plasmodium berghei liver-stage inhibition by TRAP-specific CD8+T cells. PLoS One. 2015;10:e0119880.View ArticlePubMedPubMed CentralGoogle Scholar
- Ejigiri I, Ragheb DR, Pino P, Coppi A, Bennett BL, Soldati-Favre D, et al. Shedding of TRAP by a rhomboid protease from the malaria sporozoite surface is essential for gliding motility and sporozoite infectivity. PLoS Pathog. 2012;8:e1002725.View ArticlePubMedPubMed CentralGoogle Scholar
- Longley RJ, Salman AM, Cottingham MG, Ewer K, Janse CJ, Khan SM, et al. Comparative assessment of vaccine vectors encoding ten malaria antigens identifies two protective liver-stage candidates. Sci Rep. 2015;5:11820.View ArticlePubMedPubMed CentralGoogle Scholar
- Murphy SC, Kas A, Stone BC, Bevan MJ. A T-cell response to a liver-stage Plasmodium antigen is not boosted by repeated sporozoite immunizations. Proc Natl Acad Sci USA. 2013;110:6055–60.View ArticlePubMedPubMed CentralGoogle Scholar
- Schmidt NW, Butler NS, Badovinac VP, Harty JT. Extreme CD8 T cell requirements for anti-malarial liver-stage immunity following immunization with radiation attenuated sporozoites. PLoS Pathog. 2010;6:e1000998.View ArticlePubMedPubMed CentralGoogle Scholar
- Kennedy M, Fishbaugher ME, Vaughan AM, Patrapuvich R, Boonhok R, Yimamnuaychok N, et al. A rapid and scalable density gradient purification method for Plasmodium sporozoites. Malar J. 2012;11:421.View ArticlePubMedPubMed CentralGoogle Scholar
- Hafalla JC, Bauza K, Friesen J, Gonzalez-Aseguinolaza G, Hill AV, Matuschewski K. Identification of targets of CD8(+) T cell responses to malaria liver stages by genome-wide epitope profiling. PLoS Pathog. 2013;9:e1003303.View ArticlePubMedPubMed CentralGoogle Scholar
- Howland SW, Poh CM, Gun SY, Claser C, Malleret B, Shastri N, et al. Brain microvessel cross-presentation is a hallmark of experimental cerebral malaria. EMBO Mol Med. 2013;5:916–31.View ArticlePubMedPubMed CentralGoogle Scholar
- Poh CM, Howland SW, Grotenbreg GM, Renia L. Damage to the blood-brain barrier during experimental cerebral malaria results from synergistic effects of CD8+T cells with different specificities. Infect Immun. 2014;82:4854–64.View ArticlePubMedPubMed CentralGoogle Scholar
- Lau LS, Fernandez Ruiz D, Davey GM, de Koning-Ward TF, Papenfuss AT, Carbone FR, et al. Blood-stage Plasmodium berghei infection generates a potent, specific CD8+T-cell response despite residence largely in cells lacking MHC I processing machinery. J Infect Dis. 2011;204:1989–96.View ArticlePubMedGoogle Scholar
- Lau LS, Fernandez-Ruiz D, Mollard V, Sturm A, Neller MA, Cozijnsen A, et al. CD8+T cells from a novel T cell receptor transgenic mouse induce liver-stage immunity that can be boosted by blood-stage infection in rodent malaria. PLoS Pathog. 2014;10:e1004135.View ArticlePubMedPubMed CentralGoogle Scholar
- PlasmoDB website. http://plasmodb.org (Accessed 2014–2016).
- Shrestha AM, Frith MC, Horton P. A bioinformatician’s guide to the forefront of suffix array construction algorithms. Brief Bioinform. 2014;15:138–54.View ArticlePubMedPubMed CentralGoogle Scholar
- Muralidharan V, Goldberg DE. Asparagine repeats in Plasmodium falciparum proteins: good for nothing? PLoS Pathog. 2013;9:e1003488.View ArticlePubMedPubMed CentralGoogle Scholar
- Lindner SE, Swearingen KE, Harupa A, Vaughan AM, Sinnis P, Moritz RL, et al. Total and putative surface proteomics of malaria parasite salivary gland sporozoites. Mol Cell Proteomics. 2013;12:1127–43.View ArticlePubMedPubMed CentralGoogle Scholar
- Lasonder E, Janse CJ, van Gemert GJ, Mair GR, Vermunt AM, Douradinha BG, et al. Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity. PLoS Pathog. 2008;4:e1000195.View ArticlePubMedPubMed CentralGoogle Scholar
- Tarun AS, Peng X, Dumpit RF, Ogata Y, Silva-Rivera H, Camargo N, et al. A combined transcriptome and proteome survey of malaria parasite liver stages. Proc Natl Acad Sci USA. 2008;105:305–10.View ArticlePubMedPubMed CentralGoogle Scholar
- Murphy SC, Prentice JL, Williamson K, Wallis CK, Fang FC, Fried M, et al. Real-time quantitative reverse transcription PCR for monitoring of blood-stage Plasmodium falciparum infections in malaria human challenge trials. Am J Trop Med Hyg. 2012;86:383–94.View ArticlePubMedPubMed CentralGoogle Scholar
- Schats R, Bijker EM, van Gemert GJ, Graumans W, van de Vegte-Bolmer M, van Lieshout L, et al. Heterologous Protection against malaria after immunization with Plasmodium falciparum sporozoites. PLoS ONE. 2015;10:e0124243.View ArticlePubMedPubMed CentralGoogle Scholar
- Sedegah M, Weiss WW, Hoffman SL. Cross-protection between attenuated Plasmodium berghei and P. yoelii sporozoites. Parasite Immunol. 2007;29:559–65.View ArticlePubMedPubMed CentralGoogle Scholar
- Vaughan AM, O’Neill MT, Tarun AS, Camargo N, Phuong TM, Aly AS, et al. Type II fatty acid synthesis is essential only for malaria parasite late liver stage development. Cell Microbiol. 2009;11:506–20.View ArticlePubMedPubMed CentralGoogle Scholar
- Richie TL, Billingsley PF, Sim BK, Epstein JE, Lyke KE, Mordmuller B, et al. Progress with Plasmodium falciparum sporozoite (PfSPZ)-based malaria vaccines. Vaccine. 2015;33:7452–61.View ArticlePubMedGoogle Scholar
- Doll K, Pewe L, Harty J. Protective capacity of CD8 T cells targeting a spectrum of Plasmodium-specific epitopes (MPF6P.735). J Immunol. 2014;192(Suppl 1):195.4.Google Scholar
- Van Braeckel-Budimir N, Harty J. Highly focused TCR Vβ repertoire is associated with a large number of naive precursors and robust CD8 T cell responses specific for a Plasmodium antigen (IRM14P.450). J Immunol. 2015;194(198):10.Google Scholar
- Hafalla JC, Rai U, Morrot A, Bernal-Rubio D, Zavala F, Rodriguez A. Priming of CD8 + T cell responses following immunization with heat-killed Plasmodium sporozoites. Eur J Immunol. 2006;36:1179–86.View ArticlePubMedGoogle Scholar
- Ridzuan MA, Moon RW, Knuepfer E, Black S, Holder AA, Green JL. Subcellular location, phosphorylation and assembly into the motor complex of GAP45 during Plasmodium falciparum schizont development. PLoS ONE. 2012;7:e33845.View ArticlePubMedPubMed CentralGoogle Scholar
- Frenal K, Polonais V, Marq JB, Stratmann R, Limenitakis J, Soldati-Favre D. Functional dissection of the apicomplexan glideosome molecular architecture. Cell Host Microbe. 2010;8:343–57.View ArticlePubMedGoogle Scholar
- Baum J, Richard D, Healer J, Rug M, Krnajski Z, Gilberger TW, et al. A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites. J Biol Chem. 2006;281:5197–208.View ArticlePubMedGoogle Scholar
- Simon N, Lasonder E, Scheuermayer M, Kuehn A, Tews S, Fischer R, et al. Malaria parasites co-opt human factor H to prevent complement-mediated lysis in the mosquito midgut. Cell Host Microbe. 2013;13:29–41.View ArticlePubMedGoogle Scholar
- Beiss V, Spiegel H, Boes A, Scheuermayer M, Reimann A, Schillberg S, et al. Plant expression and characterization of the transmission-blocking vaccine candidate PfGAP50. BMC Biotechnol. 2015;15:108.View ArticlePubMedPubMed CentralGoogle Scholar
- Ayala FJ, Escalante AA, Rich SM. Evolution of Plasmodium and the recent origin of the world populations of Plasmodium falciparum. Parassitologia. 1999;41:55–68.PubMedGoogle Scholar
- Rich SM, Ayala FJ. Population structure and recent evolution of Plasmodium falciparum. Proc Natl Acad Sci USA. 2000;97:6994–7001.View ArticlePubMedPubMed CentralGoogle Scholar
- Escalante AA, Barrio E, Ayala FJ. Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene. Mol Biol Evol. 1995;12:616–26.PubMedGoogle Scholar
- Ayala FJ, Rich SM. Genetic variation and the recent worldwide expansion of Plasmodium falciparum. Gene. 2000;261:161–70.View ArticlePubMedGoogle Scholar
- Schaer J, Perkins SL, Decher J, Leendertz FH, Fahr J, Weber N, et al. High diversity of West African bat malaria parasites and a tight link with rodent Plasmodium taxa. Proc Natl Acad Sci USA. 2013;110:17415–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Duval L, Fourment M, Nerrienet E, Rousset D, Sadeuh SA, Goodman SM, et al. African apes as reservoirs of Plasmodium falciparum and the origin and diversification of the Laverania subgenus. Proc Natl Acad Sci USA. 2010;107:10561–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Hall N. Genomic insights into the other malaria. Nat Genet. 2012;44:962–3.View ArticlePubMedGoogle Scholar
- Nganou-Makamdop K, Sauerwein RW. Liver or blood-stage arrest during malaria sporozoite immunization: the later the better? Trends Parasitol. 2013;29:304–10.View ArticlePubMedGoogle Scholar