Low immunogenicity of malaria pre‐erythrocytic stages can be overcome by vaccination
2021; Springer Nature; Volume: 13; Issue: 4 Linguagem: Inglês
10.15252/emmm.202013390
ISSN1757-4684
AutoresKatja Müller, Matthew P. Gibbins, Mark Roberts, Arturo Reyes‐Sandoval, Adrian V. S. Hill, Simon J. Draper, Kai Matuschewski, Olivier Silvie, Julius Clemence R. Hafalla,
Tópico(s)HIV Research and Treatment
ResumoArticle11 March 2021Open Access Transparent process Low immunogenicity of malaria pre-erythrocytic stages can be overcome by vaccination Katja Müller orcid.org/0000-0001-5100-0981 Parasitology Unit, Max Planck Institute for Infection Biology, Berlin, Germany Department of Molecular Parasitology, Institute of Biology, Humboldt University, Berlin, GermanyThese authors contributed equally to this work Search for more papers by this author Matthew P Gibbins orcid.org/0000-0002-7166-044X Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UKThese authors contributed equally to this work Search for more papers by this author Mark Roberts Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK Search for more papers by this author Arturo Reyes-Sandoval orcid.org/0000-0002-2648-1696 Jenner Institute, University of Oxford, Oxford, UK Search for more papers by this author Adrian V S Hill orcid.org/0000-0003-0900-9629 Jenner Institute, University of Oxford, Oxford, UK Search for more papers by this author Simon J Draper orcid.org/0000-0002-9415-1357 Jenner Institute, University of Oxford, Oxford, UK Search for more papers by this author Kai Matuschewski orcid.org/0000-0001-6147-8591 Parasitology Unit, Max Planck Institute for Infection Biology, Berlin, Germany Department of Molecular Parasitology, Institute of Biology, Humboldt University, Berlin, Germany Search for more papers by this author Olivier Silvie orcid.org/0000-0002-0525-6940 Sorbonne Université, INSERM, CNRS, Centre d’Immunologie et des Maladies Infectieuses, CIMI-Paris, Paris, France Search for more papers by this author Julius Clemence R Hafalla Corresponding Author [email protected] orcid.org/0000-0002-5452-9263 Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK Search for more papers by this author Katja Müller orcid.org/0000-0001-5100-0981 Parasitology Unit, Max Planck Institute for Infection Biology, Berlin, Germany Department of Molecular Parasitology, Institute of Biology, Humboldt University, Berlin, GermanyThese authors contributed equally to this work Search for more papers by this author Matthew P Gibbins orcid.org/0000-0002-7166-044X Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UKThese authors contributed equally to this work Search for more papers by this author Mark Roberts Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK Search for more papers by this author Arturo Reyes-Sandoval orcid.org/0000-0002-2648-1696 Jenner Institute, University of Oxford, Oxford, UK Search for more papers by this author Adrian V S Hill orcid.org/0000-0003-0900-9629 Jenner Institute, University of Oxford, Oxford, UK Search for more papers by this author Simon J Draper orcid.org/0000-0002-9415-1357 Jenner Institute, University of Oxford, Oxford, UK Search for more papers by this author Kai Matuschewski orcid.org/0000-0001-6147-8591 Parasitology Unit, Max Planck Institute for Infection Biology, Berlin, Germany Department of Molecular Parasitology, Institute of Biology, Humboldt University, Berlin, Germany Search for more papers by this author Olivier Silvie orcid.org/0000-0002-0525-6940 Sorbonne Université, INSERM, CNRS, Centre d’Immunologie et des Maladies Infectieuses, CIMI-Paris, Paris, France Search for more papers by this author Julius Clemence R Hafalla Corresponding Author [email protected] orcid.org/0000-0002-5452-9263 Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK Search for more papers by this author Author Information Katja Müller1,2, Matthew P Gibbins3,†, Mark Roberts3, Arturo Reyes-Sandoval4,†, Adrian V S Hill4, Simon J Draper4, Kai Matuschewski1,2, Olivier Silvie5 and Julius Clemence R Hafalla *,3 1Parasitology Unit, Max Planck Institute for Infection Biology, Berlin, Germany 2Department of Molecular Parasitology, Institute of Biology, Humboldt University, Berlin, Germany 3Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK 4Jenner Institute, University of Oxford, Oxford, UK 5Sorbonne Université, INSERM, CNRS, Centre d’Immunologie et des Maladies Infectieuses, CIMI-Paris, Paris, France †Present address: Wellcome Centre for Integrative Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, UK †Present address: Instituto Politécnico Nacional, IPN. Av. Luis Enrique Erro s/n, Unidad Adolfo López Mateos, Mexico City, Mexico *Corresponding author. Tel: +44 020 7958 8129; E-mail: [email protected] EMBO Mol Med (2021)13:e13390https://doi.org/10.15252/emmm.202013390 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Immunogenicity is considered one important criterion for progression of candidate vaccines to further clinical evaluation. We tested this assumption in an infection and vaccination model for malaria pre-erythrocytic stages. We engineered Plasmodium berghei parasites that harbour a well-characterised epitope for stimulation of CD8+ T cells, either as an antigen in the sporozoite surface-expressed circumsporozoite protein or the parasitophorous vacuole membrane associated protein upregulated in sporozoites 4 (UIS4) expressed in exo-erythrocytic forms (EEFs). We show that the antigen origin results in profound differences in immunogenicity with a sporozoite antigen eliciting robust, superior antigen-specific CD8+ T-cell responses, whilst an EEF antigen evokes poor responses. Despite their contrasting immunogenic properties, both sporozoite and EEF antigens gain access to antigen presentation pathways in hepatocytes, as recognition and targeting by vaccine-induced effector CD8+ T cells results in high levels of protection when targeting either antigen. Our study is the first demonstration that poorly immunogenic EEF antigens do not preclude their susceptibility to antigen-specific CD8+ T-cell killing, which has wide-ranging implications on antigen prioritisation for next-generation pre-erythrocytic malaria vaccines. Synopsis Key benchmarks for malaria vaccine design were investigated. Antigen immunogenicity and accessibility were studied with results indicating the proof-of-concept that a poorly immunogenic exo-erythrocytic form (EEF) antigen is comparably vulnerable as a strongly immunogenic sporozoite antigen to targeting by vaccine-induced effector CD8+ T cells. Contrasts in immunogenicities are brought about by antigen origin in malaria pre-erythrocytic stages, with a sporozoite surface antigen evoking strong and superior antigen-specific CD8+ T cell responses, whilst an EEF vacuolar antigen eliciting inferior responses. Antigen presentation pathways in hepatocytes are accessed by both sporozoite surface and EEF vacuolar antigens, notwithstanding their disparate immunogenicities. Recognition by vaccine-induced, antigen-specific effector CD8+ T cells leads to high levels of protection when targeting either antigen. EEF antigens were demonstrated to have marginal impacts on the level of CD8+ T cell responses to whole sporozoite immunisation. The paper explained Problem Antigen selection is critical for malaria vaccine discovery. To date, sporozoite antigens are prioritised according to their immunogenicity, accessibility and capacity to elicit inhibitory antibodies, and, to a limited extent, cellular responses. Very little is known about the vaccine potential of Plasmodium antigens expressed during liver infection. Results We have demonstrated in a mouse malaria model that despite having poor immunogenicity, antigens expressed during liver infection are excellent targets of vaccine-induced protection through cellular immunity. Impact Our data provide a rationale for systematic evaluation of previously unrecognised parasite-derived antigens for malaria vaccine discovery and pre-clinical evaluation in formulations aimed at eliciting strong cell-mediated immunity. Introduction Malaria caused by the apicomplexan parasite Plasmodium is responsible for more than 229 million clinical cases and over 409,000 deaths annually worldwide, with more than 97% of cases attributable to Plasmodium falciparum (WHO, 2020). Whilst current malaria control strategies have led to marked reduction in incidence rate, cases and mortality for the past 16 years, a highly efficacious vaccine is likely essential to approach the ambitious World Health Organisation’s (WHO) vision of “a world free of malaria”. Targeting the malaria pre-erythrocytic stages, an obligatory and clinically silent phase of the parasite’s life cycle, is considered an ideal and attractive strategy for vaccination; inhibiting parasite infection of, and development in hepatocytes results in preclusion of both disease-causing blood stages and transmissible sexual stages. Yet, despite intensive research for over 35 years, a highly efficacious pre-erythrocytic stage vaccine remains elusive (Draper et al, 2018). An in-depth characterisation of how the complex biology of pre-erythrocytic stages influences the generation and protective efficacy of immune responses is warranted to inform the design of future malaria vaccines. CD8+ T cells are crucial mediators of protective immunity to malaria pre-erythrocytic stages (Doolan & Hoffman, 2000). Whilst often considered as a single phase of the parasite’s life cycle, the malaria pre-erythrocytic stage is comprised of two different parasite forms: (i) sporozoites, which are motile extracellular parasites that are delivered by infected mosquitoes to the mammalian host, and (ii) exo-erythrocytic forms (EEF; also known as liver stages), which are intracellular parasites resulting from the differentiation and growth of sporozoites inside a parasitophorous vacuole (PV) within hepatocytes (Hafalla et al, 2011). How these two spatially different parasite forms and the ensuing temporal expression of parasite-derived antigens impact the magnitudes, kinetics and phenotypes of CD8+ T-cell responses elicited following infection is poorly understood. Furthermore, the complexity within the pre-erythrocytic stages has fuelled a long-standing debate focussed on the contributions of distinct sporozoite and EEF antigens in parasite-induced responses, and whether sporozoite or EEF proteins are better targets of vaccines. Our current understanding of CD8+ T-cell responses to malaria pre-erythrocytic stages has been largely based on measuring responses to the H-2-Kd-restricted epitopes of rodent P. yoelii (Py) (Weiss et al, 1990) and P. berghei (Pb) (Romero et al, 1989) circumsporozoite proteins (CSP), the major surface antigen of sporozoites. Many of these fundamental studies have focussed on using infections with irradiated sporozoites, the benchmark vaccine model for malaria. Infection with Py sporozoites elicits an expected T-cell response typified by early activation and induction of effector CSP-specific CD8+ T cells followed by contraction and establishment of quantifiable memory populations (Sano et al, 2001). CSP-specific CD8+ T cells are primed by dendritic cells that cross-present sporozoite antigens via the endosome-to-cytosol pathway (Cockburn et al, 2011). Yet, CSP is a unique antigen because it is expressed in both sporozoites and EEFs (Hollingdale et al, 1983). Whilst the expression of CSP mRNA ceases after sporozoite invasion, the protein on the parasite surface is stable and endures in EEFs during development in hepatocytes (Silvie et al, 2014). In vitro data indicate that primary hepatocytes process and present PbCSP-derived peptides to CD8+ T cells in a proteasome-dependent manner, involving export of antigen to the cytosol (Cockburn et al, 2011). Taken together, these data imply that sporozoite antigens induce quantifiable CD8+ T-cell responses after infection. Antigens that have similar expression to the CSP, persisting to EEFs and with epitope determinants presented on hepatocytes, are excellent targets of CD8+ T cell-based vaccines. The paucity of EEF only-specific epitopes has hindered not only our ability to understand the immune responses that are evoked whilst the parasite is in the liver, but also their utility as targets of vaccination. Accordingly, the contribution of EEF-infected hepatocytes in the in vivo induction of CD8+ T-cell responses is poorly understood. The liver is an organ where the primary activation of CD8+ T cells is generally biased towards the induction of tolerance (Thomson & Knolle, 2010; Bertolino & Bowen, 2015). Yet, studies in other model systems have demonstrated antigen-specific primary activation within the liver (Bertolino et al, 2001). Another confounding issue with EEFs is their development in PVs with constrained access to the hepatocyte’s cytosol (Hafalla et al, 2011). Nonetheless, if CD8+ T cells specific for EEF antigens are primed, do they expand and contract with distinct kinetics? Moreover, are EEF-specific epitopes efficiently generated for recognition and targeting by vaccine-induced CD8+ T cells? Answers to these questions will be key for antigen selection and design of future malaria vaccines. In this study, we compared the initiation and development of CD8+ T-cell responses—elicited following parasite infection—to CSP, a sporozoite antigen, and to upregulated in infective sporozoites gene 4 (UIS4), an EEF-specific vacuolar protein (Mueller et al, 2005). UIS4, a member of the early transcribed membrane protein (ETRAMP) family, is abundantly expressed in EEFs and associates with the PVM (Mueller et al, 2005). Whilst UIS4 mRNA expression is present in sporozoites, translation is repressed until when EEFs develop (Silvie et al, 2014). To control for epitope specificity, we generated Pb transgenic parasites that incorporate the MHC class I H-2-Kb epitope SIINFEKL, from ovalbumin, in either the CSP or UIS4 protein. The resulting transgenic parasites develop normally as wild-type (WT) Pb in the mosquito vector and mammalian host. However, SIINFEKL would be expressed at the same time and space as its respective Plasmodium protein, enabling the CD8+ T-cell response against these proteins to be tracked in an epitope-specific physiological manner. In line with previous studies (Cockburn et al, 2011; Montagna et al, 2014), to augment low numbers of CD8+ T cells in the naïve response, cells from OT-I mice, which express SIINFEKL-specific T-cell receptors (TCRs) on their CD8+ T cells, were initially adoptively transferred to mice prior to them receiving sporozoite immunisations. Furthermore, we evaluated the capacity of vaccine-induced CD8+ T cells to target these parasites in a mouse challenge model. Our data show disparate immunogenic properties between a sporozoite and an EEF vacuolar membrane antigen but equivalent susceptibility to vaccine-induced CD8+ T cells. Results Transgenic CSPSIINFKEL and UIS4SIINFEKL parasites display normal sporozoite motility and liver invasion We generated, by double homologous recombination, transgenic Pb parasites expressing the immunodominant H-2-Kb-restricted CD8+ T-cell epitope of ovalbumin (SIINFEKL) in the context of the sporozoite surface antigen CSP or the EEF vacuolar membrane antigen UIS4 (Figs 1A and EV1A and B). Constructs included the TgDHFR/TS-positive selection cassette and incorporated SIINFEKL in the context of the gene open reading frame. For CSPSIINFEKL, SIINFEKL replaced SYIPSAEKI, the immunodominant H-2-Kd-restricted CD8+ T-cell epitope of CSP, which allowed for recognition in H-2-Kb-carrying C57BL/6 mice. For UIS4SIINFEKL, the SIINFEKL epitope was added to the immediate C terminus of the UIS4 protein. Appending the C terminus was chosen because it had been shown in Toxoplasma gondii that the potency of the immunodominant epitope of GRA6 was associated with its C-terminal location, which may have enhanced the presentation by parasite-infected cells (Feliu et al, 2013). Whilst undefined for UIS4 itself, it has been shown for several other ETRAMPs that the C terminus faces the host cell cytoplasm (Spielmann et al, 2003), which is likely to facilitate exposure to the MHC I complex. Figure 1. Generation and characterisation of recombinant CSPSIINFEKL and UIS4SIINFEKL P. berghei parasites Plasmodium berghei parasites expressing the CD8+ T-cell epitope of ovalbumin, SIINFEKL, in the context of CSP or UIS4 were generated using double homologous recombination. Schematic overview of transgenic parasite lines. To generate CSPSIINFEKL, SIINFEKL replaced amino acids SYIPSAEK in CSP. To generate UIS4SIINFEKL, SIINFEKL was adjoined to the carboxyl-terminus of the UIS4 protein. Sporozoite immunofluorescent antibody staining of WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites after gliding on BSA-coated glass slides. Shown are microscopic images of the respective sporozoites that were stained with anti-CSP (green), anti-UIS4 (red) and nuclear stain Hoechst (blue). Scale bars, 10 μm. The numbers show mean percentage (± SD) of sporozoites with trails assessed from ≥ 220 sporozoites. Fluorescent-microscopic images of EEF-infected Huh7 hepatoma cells. 24 and 48 h after infection with WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites, the cells were fixed and stained with anti-UIS4 (red), anti-HSP70 (green) and the nuclear stain Hoechst (blue). Scale bars, 10 µm. The numbers show mean numbers (± SD) of intracellular parasites counted per well of 8-well Labtek slides. Data information: The data shown are representative from one of two independent experiments. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Generation of transgenic CSPSIINFEKL and UIS4SIINFEKL P. berghei lines Plasmodium berghei parasites expressing the CD8+ T-cell epitope of ovalbumin, SIINFEKL, in the context of CSP or UIS4 were generated using double homologous recombination, combining drug-resistance selection (through incorporation of the dhfr/ts gene from Toxoplasma gondii) and cloning by limiting dilution to select for correctly recombined parasites. A, B. Diagrams illustrate the reverse genetics strategy. (A) In CSPSIINFEKL, SIINFEKL replaces the immunodominant CD8+ T-cell epitope SYIPSAEK(I) of CSP. (B) In UIS4SIINFEKL SIINFEKL is adjoined to the carboxyl-terminus of the UIS4 protein. Purified schizonts of WT P. berghei ANKA were transfected with linearised plasmid by electroporation as described (Janse et al, 2006), and immediately injected intravenously in the tail vein of a mouse. The day after transfection, pyrimethamine (70 mg/l) was orally administered in the drinking water for selection of transgenic parasites. Transgenic clones were generated in mice by in vivo cloning by limiting dilution. Correct integration of the constructs and purity of the transgenic lines were verified by diagnostic PCR using primer combinations specific for the unmodified CSP or UIS4 locus, and for the 5’ and 3’ recombination events as indicated by lines, arrows and expected fragment sizes. C. Oocyst midgut infectivity of mosquitoes infected with WT, CSPSIINFEKL or UIS4SIINFEKL. The mean percentage (± SD) of infected midguts was enumerated 10–14 days after infection (14 biological replicates). D. Salivary glands were isolated from WT-, CSPSIINFEKL- or UIS4SIINFEKL-infected mosquitoes and mean sporozoite numbers (± SD) were enumerated between 18–30 days after infection (CSPSIINFEKL, 21 biological replicates; UIS4SIINFEKL, 18 biological replicates; WT, 18 biological replicates). E. Numbers of intracellular parasites of hepatoma cells 24, 48 and 72 h after infection with WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites. The cells were fixed, stained and then were enumerated from two independent experiments. Values are percent of intracellular parasites compared with the mean of WT intracellular parasites ± SD (*P < 0.05; one-way ANOVA with Tukey’s multiple comparison test). See Appendix Table S4 for exact P-values. F. Kaplan–Meier curve showing the prepatency of WT, CSPSIINFEKL or UIS4SIINFEKL sporozoite infections after intravenous injection of 800 sporozoites (n = 4 per group). Download figure Download PowerPoint The resulting parasites showed a phenotype comparable to WT parasites, with comparable mosquito infectivity and number of salivary gland sporozoites (Fig EV1C and D), functional sporozoite motility (Fig 1B) and normal invasive capacity and development inside hepatocytes (Figs 1C and EV1E). Thus, the introduced mutations to generate CSPSIINFEKL and UIS4SIINFEKL parasites did not interfere with the completion of the life cycle, in either mosquito vector or mouse. All C57BL/6 mice that received 800 sporozoites of either CSPSIINFKEL or UIS4SIINFEKL intravenously developed a detectable (patent) blood stage infection by day 4, comparable to infection with WT sporozoites (Fig EV1F). Peripheral blood CD8+ T-cell responses are superior if elicited by a sporozoite surface protein in contrast to a vacuolar membrane protein in the infected liver We first wanted to determine whether the generated transgenic parasites allow antigen-specific responses to be tracked using SIINFEKL as a surrogate CD8+ T-cell epitope for sporozoite surface and EEF vacuolar membrane antigens. To this end, we assessed the kinetics of the CD8+ T-cell response following intravenous immunisation with CSPSIINFEKL or UIS4SIINFEKL sporozoites. To augment the CD8+ T-cell response, mice were adoptively transferred with 2 × 106 OT-I cells expressing a SIINFEKL-specific TCR (Cockburn et al, 2011), prior to receiving 10,000 γ-radiation attenuated WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites. Prior work showed that γ-radiation attenuation of P. berghei sporozoites does not impact host cell invasion and UIS4 expression (Mac-Daniel et al, 2014). Peripheral blood was taken at days 4, 7, 14, 21, 42 and 88 after immunisation and CD8+ T-cell responses were analysed after staining with H-2-Kb-SIINFEKL pentamers and for CD11a, a marker for antigen-experienced T cells (Rai et al, 2009; Schmidt et al, 2010) (Fig 2A and B). Additional gating strategies for the characterisation of Kb-SIINFEKL+ CD8+ T cells are shown in Appendix Fig S1. A substantial proportion of Kb-SIINFEKL+ CD11ahi CD8+ T cells were observed in mice immunised with CSPSIINFEKL; the response was highest on day 4, reaching 5% of all antigen-experienced CD8+ T cells, and declined steadily until day 21, when the response stabilised and remained unchanged for several weeks (Fig 2C). In marked contrast, UIS4SIINFEKL immunisation induced a poor CD8+ T-cell response; the proportion of Kb-SIINFEKL+ CD11ahi CD8+ T cells was only higher than the control groups at day 4 after immunisation, and the response remained within background levels for the duration of the experiment. Control groups included mice receiving OT-I cells only or in addition to γ-radiation attenuated WT sporozoites, which lack SIINFEKL sequences. Figure 2. Sporozoite antigen exposure elicits superior peripheral blood CD8+ T-cell responses A. Schematic overview of experimental design. C57BL/6 mice received 2 × 106 OT-I cells alone or were additionally immunised with 10,000 γ-radiation attenuated parasites intravenously. B. Flow cytometry plots show the gating strategy for identifying Kb-SIINFEKL+ CD11ahi CD8+ T cells. C. Temporal analysis of antigen-specific CD8+ T-cell responses. Peripheral blood was obtained on days 4, 7, 14, 21, 42 and 88 post-immunisation with WT (triangles; n = 3–9), CSPSIINFEKL (orange squares; n = 4–8) or UIS4SIINFEKL (blue circles; n = 4–10) sporozoites, or no parasites (diamonds; n = 2–5) and stained for Kb-SIINFEKL+ CD11ahi CD8+ T cells. Line graph shows mean values (± SEM) from representative experiments. (*P < 0.05; **P < 0.01; ***P < 0.001; Welch’s t-test comparing CSPSIINFEKL and UIS4SIINFEKL). See Appendix Table S1 for the number of mice used per timepoint and per group. See Appendix Table S4 for exact P-values. D–H. C57BL/6 mice (n = 4 per group), which received 2 × 106 CFSE-labelled OT-I splenocytes, were immunised with 10,000 γ-radiation attenuated WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites intravenously. 5 days later, mice were sacrificed, spleens harvested and splenocytes assessed for (D) CFSE dilution of CD8+ T cells, (E) CFSE dilution of antigen-experienced Kb-SIINFEKL+ CD11ahi CD8+ T cells and stained ex vivo (F-H) for effector CD8+ T-cell surface markers. Shown are flow cytometry plots of Kb-SIINFEKL co-staining with markers of effector phenotypes: (F) CD11ahi, (G) CD62Llo and (H) CD49dhi. Download figure Download PowerPoint The poor CD8+ T-cell response induced by UIS4SIINFEKL sporozoites, as compared to CSPSIINFEKL, led us to characterise the early events in the proliferation and differentiation of these cells. Mice were adoptively transferred with CFSE-labelled OT-I cells and immunised with γ-radiation attenuated WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites. As shown by gating on CD8+ T cells (Fig 2D and E), after 5 days, immunisation with CSPSIINFEKL sporozoites led to greater expansion of Kb-SIINFEKL+ CD8+ T cells, as compared to that observed with UIS4SIINFEKL sporozoites (Fig EV2A), in good agreement with the peripheral blood data described above (Fig 2C). Consistent with the activation of these cells, the proliferation of antigen-specific CD8+ T cells by both parasites was associated with the development of effector and effector memory phenotypes as evidenced by upregulation of CD11a and CD49d, and downregulation of CD62L, respectively (Figs 2F–H and EV2B–D). Click here to expand this figure. Figure EV2. Sporozoite surface antigen leads to greater expansion of antigen-specific CD8+ T cells than EEF vacuolar membrane antigen A–D. C57BL/6 mice (n = 4 per group), which received 2 × 106 CFSE-labelled OT-I splenocytes, were immunised with 10,000 γ-radiation attenuated WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites intravenously. 5 days later, mice were sacrificed, spleens harvested and splenocytes assessed for (A) CFSE dilution of antigen-experienced Kb-SIINFEKL+ CD8+ T cells and stained ex vivo for effector CD8+ T-cell surface markers (B) CD11ahi, (C) CD62Llo and (D) CD49dhi. Bar charts show mean values (± SEM) from representative experiments (***P < 0.001; one-way ANOVA with Tukey’s multiple comparison test). See Appendix Table S4 for exact P-values. Download figure Download PowerPoint Figure 3. Sporozoite surface antigen induces a higher CD8+ T-cell response than EEF vacuolar membrane antigen in the spleen and liver A. Schematic overview of experimental design. C57BL/6 mice received 2 × 106 OT-I cells alone (n = 2–4) or were additionally immunised with 10,000 γ-radiation attenuated WT (n = 3–6), CSPSIINFEKL (n = 4) or UIS4SIINFEKL (n = 4) sporozoites intravenously. See Appendix Table S2 and S3 for the number of mice used for pentamer staining and peptide restimulation. Spleens and livers were harvested either at day 14 or day 42. B–G. Proportions and numbers of (B-D) Kb-SIINFEKL+ CD8+ T cells were enumerated, or (E-G) IFN-γ-secreting CD8+ T cells following restimulation ex vivo with SIINFEKL peptide were quantified. Flow cytometry plots show representative percentages of CD8+ T cells co-stained with CD11a and (B) Kb-SIINFEKL or (E) IFN-γ. The upper panel of bar charts (C, F) show the percentage of co-stained CD8+ T cells and the lower panel (D, G) the absolute cell counts. Bar charts show mean values (± SEM) from representative experiments (*P < 0.05; **P < 0.01; ***P < 0.001; one-way ANOVA with Tukey’s multiple comparison test). See Appendix Table S4 for exact P-values. Download figure Download PowerPoint Figure 4. Higher magnitude of endogenous SIINFEKL-specific CD8+ T-cell responses induced by a sporozoite antigen A. Schematic overview of experimental design. B–G. C57BL/6 mice received 10,000 γ-radiation attenuated WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites, either (B-D) intravenously (n = 6–8 mice per group) or (E-G) intradermally (n = 4 mice per group). Additional control mice did not receive sporozoites (n = 6–8 intravenously, n = 4 intradermally). Spleens and livers were harvested either at day 14 (n = 5 livers and n = 6 spleens for intravenous immunisation) or day 42 (n = 8 for intravenous immunisation), and IFN-γ-secreting lymphocytes following restimulation ex vivo with SIINFEKL peptide were quantified. Flow cytometry plots show representative percentages of CD8+ T cells co-stained with IFN-γ and CD11a (B, E). The upper panel of bar charts (C, F) show the percentage of CD11ahi IFN-γ+ CD8+ T cells and the lower panel (D, G) the absolute cell counts. Bar charts show mean values (± SEM) from representative experiments (*P < 0.05; **P < 0.01; ***P < 0.001; one-way ANOVA with Tukey’s multiple comparison test). See Appendix Table S4 for exact P-values. Download figure Download PowerPoint Figure 5. Increasing antigen dose does not improve antigen-specific CD8+ T-cell responses to an EEF vacuolar membrane protein Schematic overview of experimental design. C57BL/6 mice (n = 4 per group) received an intravenous dose of 8,000 γ-radiation attenuated CSPSIINFEK
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