Figures
Abstract
The development of an efficacious vaccine against the Plasmodium parasite remains a top priority. Previous research has demonstrated the ability of a prime-boost virally vectored sub-unit vaccination regimen, delivering the liver-stage expressed malaria antigen TRAP, to produce high levels of antigen-specific T cells. The liver-stage of malaria is the main target of T cell-mediated immunity, yet a major challenge in assessing new T cell inducing vaccines has been the lack of a suitable pre-clinical assay. We have developed a flow-cytometry based in vitro T cell killing assay using a mouse hepatoma cell line, Hepa1-6, and Plasmodium berghei GFP expressing sporozoites. Using this assay, P. berghei TRAP-specific CD8+ T cell enriched splenocytes were shown to inhibit liver-stage parasites in an effector-to-target ratio dependent manner. Further development of this assay using human hepatocytes and P. falciparum would provide a new method to pre-clinically screen vaccine candidates and to elucidate mechanisms of protection in vitro.
Citation: Longley RJ, Bauza K, Ewer KJ, Hill AVS, Spencer AJ (2015) Development of an In Vitro Assay and Demonstration of Plasmodium berghei Liver-Stage Inhibition by TRAP-Specific CD8+ T Cells. PLoS ONE 10(3): e0119880. https://doi.org/10.1371/journal.pone.0119880
Academic Editor: Olivier Silvie, INSERM, FRANCE
Received: November 20, 2014; Accepted: February 3, 2015; Published: March 30, 2015
Copyright: © 2015 Longley et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper.
Funding: Funding was provided by the Wellcome Trust (http://www.wellcome.ac.uk/), grant number 095540/Z/11/Z. Additional funding was provided by the Rhodes Trust and a Nuffield Department of Medicine Studentship to support RJL. AVSH is a Jenner Institute Investigator; AJS is a James Martin Fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Malaria, caused by the parasite Plasmodium, remains a serious public health concern particularly in developing countries where it is a significant cause of morbidity and mortality [1], and promotes the cycle of poverty. Vaccines are considered one of the most cost-effective public health tools, yet a highly efficacious vaccine against malaria has yet to be achieved. One promising avenue has been the use of viral vectored vaccines to deliver the pre-erythrocytic antigen thrombospondin-related adhesion protein (TRAP) [2], also known as SSP2. This vaccination regimen provided 21% sterile efficacy and a higher rate of partial efficacy manifest as delay to patency in a controlled human malaria infection trial; protection was associated with interferon-gamma (IFN-γ) producing CD8+ T cells [3], suggesting TRAP might be the target of cell-mediated immunity at the liver-stage of infection. TRAP is a type 1 transmembrane protein essential for attachment and invasion of both the mosquito salivary gland and the liver, whilst also contributing to the sporozoite gliding motility [4–8]. TRAP has also been demonstrated to provide protection in a number of murine models, utilizing both the P. falciparum TRAP construct used in clinical trials [9, 10] and independently with the P. vivax ortholog [11].
Unfortunately, there is currently no standardized assay available to confirm the mechanism by which IFN-γ producing CD8+ T cells are associated with protection. P. falciparum cannot naturally infect small animals, and even if transgenic parasites or humanized mice are utilized to overcome this barrier, the murine immune system is still not equivocal to that of humans, particularly in terms of major histocompatibility complex (MHC) restriction and immunodominance. Hence, the assay of choice to assess the functionality of human T cells against P. falciparum liver-stages is an in vitro model. Whilst such an assay does not currently exist, similar methodologies have been used extensively to assess the effect of antibodies on liver-stage growth and development [12–18], and this assay has recently been standardized [19]. However, assays measuring cellular inhibition are more complicated given the additional need for MHC antigen matching between the hepatocytes and the effector T cells.
Whilst murine in vitro cellular assays have been reported in the past [20–24], they have not been used regularly to measure cellular inhibition. Hoffman et al. used splenocytes from mice vaccinated with irradiated sporozoites [20], whilst Weiss et al. used splenocytes from mice vaccinated with irradiated sporozoites that had been pre-cultured with the P. yoelii (Py) circumsporozoite protein (CSP) peptide and interleukin 2 (IL-2), to determine whether T cells directed against the PyCSP peptide could specifically inhibit liver-stage parasites [23]. Renia et al. utilized both cells from lymph nodes taken from mice vaccinated with a PyCSP synthetic peptide [22] and a CD4+ T cell clone generated from the same vaccination regimen [21]. Trimnell et al. more recently assessed the inhibitory activity of CD8+ enriched cells from both the spleens and livers of mice immunized with P. yoelii genetically attenuated parasites (PyUIS4−/−) [24]. All studies were able to detect inhibition of liver-stage parasites by fixation, staining and manual counting, and have contributed substantially to our understanding of the role of T cells during liver-stage malaria infection. Given our recent advances in the development of a liver-stage vaccine, it is timely to revisit and reassess the feasibility and utility of such an assay.
In this study we have aimed to further develop such an in vitro assay, focusing first on a murine model utilizing P. berghei (Pb), in order to simplify MHC matching between effector and target cells. The P. berghei ortholog of PfTRAP (PbTRAP) is also protective against homologous challenge in a C57BL/6 mouse model, with CD8+ T cells implicated in protection [25]. We have used our assay to demonstrate P. berghei TRAP-specific CD8+ T cell inhibition of liver-stage parasites in an effector-to-target (E:T) ratio dependent manner. As we have demonstrated the feasibility of this assay using a simplified murine model, we now plan to further develop this assay using human hepatocytes and P. falciparum. If successful, this will provide a new method to pre-clinically screen P. falciparum vaccine candidates and to elucidate mechanisms of protection in vitro, without reliance on murine models.
Materials and Methods
Experimental animals and parasites
Female C57BL/6 mice (H-2b) of at least six weeks of age (Harlan, UK) were used in all experiments. P. berghei blood-stage parasites expressing the green fluorescent protein (GFP) under control of the elongation factor 1α promoter were provided by Prof. Robert Sinden at Imperial College, London [26]. Sporozoites were obtained by dissection and homogenization of salivary glands from infected Anopheles stephensi mosquitoes.
Ethics statement
All animal work was conducted in accordance with the UK Animals (Scientific Procedures) Act 1986 and approved by the University of Oxford Animal Care and Ethical Review Committee for use under Project License PPL 30/2414 or 30/2889. Animals were group housed in individually ventilated cages under specific pathogen free conditions, with constant temperature, humidity and with a 12:12 light-dark cycle (8am to 8pm). For induction of short-term anesthesia, animals were either injected intramuscularly (i.m.) with rompun and Domitor or anaesthetized using vaporized IsoFlo. All animals were humanely sacrificed at the end of each experiment by an approved Schedule 1 method (cervical dislocation). All efforts were made to minimize suffering.
Labeling and infection of the Hepa1-6 cell line
The murine Hepa1-6 cell line (C57L hepatoma, H-2b) (European Collection of Cell Cultures) [27–29] was propagated in supplemented DMEM (2mM L-glutamine, 100U penicillin, 100μg streptomycin, 50μm 2-mercaptoethanol and 10% fetal calf serum (FCS)). In order to discriminate between hepatocytes and added splenocytes, Hepa1-6 cells were first labeled with the membrane dye Vybrant DiD (Life Technologies) according to the manufacturer’s instructions. 5x104 labeled cells were added per well of a 96-well flat bottom plate and left to form a monolayer overnight, prior to infection with 40 000 P. berghei GFP sporozoites (resulting in the most reliable and consistent levels of infection from the recommended range [30]). Plates were centrifuged at 500xg for five minutes and incubated for three to six hours prior to addition of cells, to allow time for the sporozoites to invade [31]. Experimental wells were performed at least in duplicate, and in triplicate where possible. If no cells were added, the medium was changed at three hours post-infection to reduce potential contamination.
Assessment of infectivity via flow cytometry
After 24 hours at 37°C, cells were incubated with trypsin for four minutes prior to collection into 10% FCS in phosphate buffered saline, centrifugation and resuspension in 2% FCS in phosphate buffered saline, as previously described [32]. Immediately prior to acquisition on a LSR II flow cytometer (BD Biosciences), 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, final concentration 1μg/ml, Sigma Aldrich) was added to stain dead cells. Cells were gated on size and doublet negativity, followed by exclusion of dead cells. The hepatocytes were selected by DiD positivity and GFP positive hepatocytes were considered infected. Data were analysed using FlowJo (Tree Star Inc.). The background GFP fluorescence was always subtracted based on measurements from at least six uninfected wells.
P. berghei TRAP vaccine
P. berghei TRAP (PbTRAP) (NCBI AAB63302.1) was expressed in the viral vectors chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) following previously described methods [33, 34]. The vaccines were administered in a prime-boost regimen with 1x108 infectious units ChAd63 administered i.m. followed at least eight weeks later by 1x106 pfu MVA (always one week prior to splenocyte harvest).
Enrichment of CD8+ splenocytes
One-week post-MVA boost, spleens were harvested and single cell suspensions were prepared by passing splenocytes through a 70μm cell strainer followed by incubation with ammonium-chloride-potassium lysate buffer to remove red blood cells. Splenocytes were enriched for CD8+ cells by negative depletion using MACS Anti-Biotin MicroBeads and a biotin antibody cocktail, according to the manufacturer’s instructions. Briefly, splenocytes were first labeled with a biotin-antibody cocktail containing monoclonal antibodies against CD4 (clone GK1.5), CD11b (clone M1/70), CD11c (clone N418), CD19 (clone M1319-1), CD45R (B220) (clone RA3-6B2), CD49b (clone DX5) and MHC Class II (clone M5/114.15.2) (all from BioLegend), followed by addition of Anti-Biotin MicroBeads before separation using a MACS Separator and LD Column. Purity of the fractions was determined by staining with anti-CD8α-FITC (clone 53–6.7, eBiosciences) and compared with unfractionated splenocytes.
Intracellular cytokine staining (ICS)
To determine the frequency of TRAP specific cells, an aliquot of pre-enrichment splenocytes were stimulated for six hours with 5μg/ml of a single pool of PbTRAP peptides (60 20mers overlapping by 10aa), 1μg/ml GolgiPlug (BD Biosciences), and CD107a-PE (clone 1D4B). The PbTRAP pool includes the previously mapped CD8+ epitope PbTRAP130 [25]. CD107a is a glycoprotein present in the membrane of cytotoxic granules and can be detected on the cell surface following degranulation; it is therefore an indirect marker of cytotoxic activity and identifies cells with this capability. Degranulation is an obligatory process for subsequent cytotoxic killing mediated by release of perforin or granzymes, and CD107a expression has been shown to represent cells capable of cytotoxicity in an antigen-specific manner [35].
Cells were subsequently surface stained with CD16/32 (clone 93), CD4-eFluor 450 (clone RM4-5) and CD8α-PerCPCy5.5 (clone 53–6.7) followed by fixation with 10% neutral buffered formalin (containing 4% paraformaldehyde) (Sigma Aldrich). Staining of intracellular cytokines was achieved using tumour necrosis factor-alpha (TNF-α)-FITC (clone MP6-XT22), IL-2-PeCy7 (clone JES6-5H4) and IFN-γ-APC (clone XMG1.2) diluted in Perm/Wash buffer (BD Biosciences). All antibodies were purchased from either eBiosciences or BD Biosciences. Data were acquired on a LSRII flow cytometer and analysed using FlowJo. Cells were gated on size (FSC vs SSC) and exclusion of doublet cells (FSC-A vs FSC-H), followed by gating on CD4 or CD8 positive cells and identification of cytokine or CD107a positivity. Background responses were measured from unstimulated samples (without peptide) and these were subtracted from the overall response in stimulated samples.
In vitro T cell killing assay
DiD labeled Hepa1-6 cells were infected with P. berghei GFP sporozoites. Three to six hours later CD8+ enriched splenocytes from PbTRAP vaccinated mice were added to the assay at various ratios, typically 2x106 total splenocytes. Control wells contained CD8+ enriched splenocytes from naïve or vector control vaccinated mice (ChAd63-MVA luciferase), as stated. Cells were incubated for 24 hours, prior to analysis by flow cytometry. The E:T ratio was back calculated using the purity of the CD8+ enrichment and the percentage of antigen-specific cells measured via ICS. The percentage inhibition was calculated using the following formula [20]:
Statistical analysis
Prism version 5 (Graphpad, USA) was used for all analyses. Non-parametric data are shown as the median ± the interquartile range, or with individual data points plotted. The Mann Whitney test was used to compare the medians of two groups of non-parametric data, and correlations tested using Spearman’s rank correlation. The significance threshold was 0.05.
Results
Infectivity and measurement of liver-stage P. berghei cultures
As various methodologies, cell lines and parasites had been previously used [20–23] we first needed to establish a reliable and standard method for infecting and measuring infection in our laboratory. Isolation and culture of primary murine hepatocytes requires technical expertise and highly specialized reagents and protocols, therefore we chose to use a cell line, Hepa1-6, instead. Using a cell line reduces the variability in the quality of cells, but limits the assay to H-2 backgrounds on which such cell lines are available. We chose to use the Hepa1-6 cell line given the relatively high infectivity of P. berghei sporozoites previously achieved [36], and given our target vaccine PbTRAP induces a good response in C57BL/6 mice of the same H-2 background (see ‘Characterization of splenocytes’ below). We defined our gating strategy based on selection of viable hepatocytes, with positive GFP expression representing P. berghei infected cells (Fig. 1); this method of measuring infectivity has previously been shown to correlate with the number of infected cells as per traditional microscopy or PCR [32].
Cells were gated by size, followed by exclusion of doublets. Viable cells were selected based on the absence of DAPI staining. Hepatoma cells were labeled with the Vybrant DiD membrane dye prior to seeding, thus enabling identification with DiD fluorescence. Finally, the hepatoma cells that were GFP positive were selected. Uninfected hepatoma cells were run as a control to guide gating of the population of GFP+ cells.
Centrifugation of the plates immediately after addition of sporozoites was shown to increase infectivity from a median of 0.56% to 2.76% (Mann Whitney test, p = 0.0001) (Fig. 2), consistent with recent reports [19, 30] and with other infectious agents [37, 38]. We also confirmed that dead sporozoites no longer express GFP by the addition of heat-killed sporozoites into the assay (Fig. 2).
40 000 P. berghei GFP sporozoites were added per well with (n = 69) or without (n = 6) centrifugation. To determine whether dead or aborted sporozoites maintained expression of GFP, sporozoites were heat-killed for 20 minutes at 95°C prior to infection of Hepa1-6 cells (n = 6). 24 hours post-infection cells were harvested and run on a flow cytometer. (A) Representative example of the flow cytometry plots. (B) Data from multiple experiments was pooled and results are expressed as the percentage of GFP positive viable Hepa1-6 cells. The effect of centrifugation on infectivity was assessed using the Mann Whitney test, *** p = 0.0001.
Characterization of splenocytes and their addition to the in vitro assay
After establishment of the liver-stage P. berghei cultures, we developed a cellular inhibition assay (Fig. 3) based on those previously described. Given TRAP is an antigen of keen interest for a pre-erythrocytic malaria vaccine [3] we wanted to determine the ability of TRAP-specific cells to inhibit liver-stage parasites in vitro. Thirteen experiments were conducted, using splenocytes from twelve sets of independently vaccinated mice (Table 1). After vaccination of these mice in a prime-boost regimen, the median PbTRAP-specific CD8+ IFN-γ+ response in the spleen was 20.22% of total CD8+ cells, CD8+ TNF-α+ 15.43% and CD8+ CD107a+ 20.10%, compared to no PbTRAP-specific response from naïve or vector control vaccinated mice (ChAd63-MVA luciferase) (Fig. 4A).
C57BL/6 mice were vaccinated with a ChAd63-MVA prime-boost regimen using vaccines expressing P. berghei TRAP. Six days post-MVA boost, Hepa1-6 cells were labeled with the membrane dye Vybrant DiD and seeded into a 96-well flat bottom plate. The following day salivary glands were dissected from P. berghei GFP infected mosquitoes, sporozoites isolated and 40 000 added per well to the Hepa1-6 cell cultures, followed by centrifugation at 500xg for five minutes. Subsequently, spleens from vaccinated mice were enriched for CD8+ cells via negative depletion prior to addition to the sporozoite infected Hepa1-6 cell cultures approximately three hours post-infection. Splenocytes were also harvested from vector control vaccinated mice and treated in the same way, to determine the effect of non-specific killing. Plates were then incubated for approximately 24 hours prior to trypsin treatment to harvest cells for acquisition on the flow cytometer.
(A) Cellular immunogenicity of ChAd63-MVA P. berghei TRAP in C57BL/6 mice. Each data point represents splenocytes from two mice pooled together, with twelve pairs in total that were used in thirteen assays (one pair provided enough cells for two experiments). Cellular immunogenicity was assessed by ICS, after six hours stimulation with a pool of P. berghei TRAP peptides. Vector control mice were vaccinated with ChAd63-MVA luciferase and treated identically to the experimental mice. Results are expressed as the percentage of CD8+ cells, with median and individual data points shown. (B) Prior to addition of the splenocytes into the in vitro assays, samples were enriched for CD8+ cells. Results are expressed as the percentage of CD8+ cells out of total splenocytes, with both median and individual data points shown. (C) In each in vitro assay conducted, CD8+ enriched splenocytes from vector control vaccinated mice were included in the assay, along with wells containing sporozoites only, to act as controls. Results are expressed as the percentage infectivity, with the median shown for each experiment and error bars representing the interquartile range. (D) These controls allowed calculation of the background level of non-specific inhibition. Results are expressed as the percentage inhibition of splenocytes from vector control vaccinated mice compared to sporozoite only wells (no splenocytes), with median and individual data points shown for each experiment. In Exp1 and Exp2 sporozoite only wells were not included and hence the background inhibition could not be calculated.
In order to limit the effect of non-specific killing, these splenocytes were then enriched for CD8+ T cells prior to addition into the in vitro assay (Fig. 4B). Given a previous report of such non-specific killing from naïve (unvaccinated mice) (22), in each experiment conducted CD8+ enriched splenocytes from vector control vaccinated mice were used as a control. The background inhibition from the vector control vaccinated mice did vary substantially between the independent experiments, from 0–70%, when compared to wells containing only sporozoites and no splenocytes (Fig. 4C and 4D). To account for the variation in background inhibition, the effect of addition of PbTRAP-specific CD8+ T cell enriched splenocytes was compared to the vector control in each experiment.
Ability of P. berghei TRAP-specific CD8+ T cell enriched splenocytes to inhibit liver-stage parasites
Overall, the addition of PbTRAP-specific CD8+ T cell enriched splenocytes was shown to significantly reduce the number of liver-stage parasites compared with vector control wells, p = 0.0479 (Wilcoxon matched-pairs signed rank test) (Fig. 5A and B). This effect was seen in eleven out of thirteen experiments (Fig. 5B). However, the percentage inhibition achieved did vary between experiments (Fig. 5C), despite addition of the same number of splenocytes in the majority of experiments (Table 1). To account for changes in the frequency of PbTRAP-specific cells and purity of CD8+ enrichment between experiments, the E:T ratio was further defined as the number of antigen-specific CD8+ T cells (determined by ICS) to the number of infected hepatocytes (calculated from sporozoite only wells).
(A) Representative example of the flow cytometry plots, from ‘Experiment 3’ (see Table 1). (B) Results are expressed as the percentage infectivity, with the median shown for each experiment and error bars representing the interquartile range. A statistically significant difference was evident overall between wells containing splenocytes from PbTRAP compared to vector control vaccinated mice, p = 0.0479 (Wilcoxon matched-pairs signed rank test). (C) Results are expressed as the percentage inhibition compared to the wells containing cells from vector control vaccinated mice (equal number of CD8+ enriched splenocytes from ChAd63-MVA luciferase vaccinated mice), with median and individual data points shown for each experiment. If the percent inhibition was negative, it was considered to be zero.
Addition of PbTRAP-specific CD8+ T cell enriched splenocytes was shown to inhibit liver-stage parasites in an E:T ratio dependent manner (Fig. 6A), however there was no statistically significant correlation observed (Spearman r = 0.35, p = 0.24). Of the thirteen independent experiments performed, four experiments did not fit the pattern observed (i.e. a greater number of effector cells were required to achieve similar levels of inhibition) and corresponded to significantly lower levels of infectivity (Mann Whitney test, p<0.0001) (Fig. 6B). When ‘outlier’ experiments were excluded, a statistically significant correlation between the percentage inhibition and the E:T ratio was observed (Spearman r = 0.82, p = 0.011) (Fig. 6C).
(A) Results are expressed as the percent inhibition compared to mock-vaccinated control wells (equal number of CD8+ enriched splenocytes from ChAd63-MVA luciferase vaccinated mice). Across all thirteen experiments there was no significant correlation between E:T ratio and inhibition (Spearman r = 0.35, p = 0.24), although a trend can be observed. Full circles represent those experiments that fit this trend, whilst empty circles represent those that did not. Experiments were then divided into data that fitted the E:T pattern (full circles) and those that did not (empty circles) and a graph (B) of the infectivity measured in wells containing only Hepa1-6 cells and sporozoites are shown. Statistical difference was assessed using the Mann Whitney test, **** p<0.0001. (C) Correlation of the E:T ratio with the percentage inhibition (n = 9 experiments), excluding the outliers. Spearman r = 0.82, p = 0.011.
Discussion
Currently, there is no standardized in vitro cellular assay to test the effectiveness of P. falciparum vaccine candidates in the pre-clinical phase of development or to assess immunological correlates of protection clinically. Here we have described the development of an improved murine in vitro assay and used it to demonstrate the ability of PbTRAP-specific CD8+ T cell enriched splenocytes to inhibit liver-stage parasites. The correlation between the number of PbTRAP-specific CD8+ T cells with the percentage inhibition, whilst not excluding the possibility that CD4+ T cells could have an effect, does suggests that CD8+ cells were the important effectors in our CD8+ enriched splenocyte suspension. This finding demonstrates the ability of PbTRAP to be processed and presented on the hepatocyte cell surface in association with MHC Class I molecules, in order to be recognized by CD8+ T cells. The only other antigen for which this has been demonstrated is CSP [21–23], and this could be how protection is mediated in the clinical TRAP vaccine [3].
To our knowledge, this is the first report of an in vitro cellular assay utilizing flow cytometry to measure infection based on GFP positivity. An added difficulty compared to antibody based assays is the need to distinguish between hepatocytes and effector T cells in order to reliably determine the percentage of GFP positive hepatocytes; we successfully utilized a membrane dye in order to stain and gate on hepatocytes only. A surprisingly high level of non-specific background inhibition was observed from vector control vaccinated mice; this is likely due to the presence of other cell types such as natural killer cells, and due to the large number of cells included in the assay. Calculating inhibition of PbTRAP vaccinated mice in comparison to infectivity of wells containing splenocytes from vector control vaccinated mice enabled us to normalize for this high level of background inhibition. The results suggested that a greater E:T ratio was required when a lower level of infectivity of hepatoma cells was achieved; this is likely an effect of the culture model and highlights that a standard level of infectivity will be required to compare results across experiments and between groups.
For the majority of our independent experiments we added 2x106 total splenocytes, based on the total number of cells required to achieve the highest levels of cellular inhibition (60–80%) in in vitro assays previously reported [20, 22]. Our inhibition ranged from 0–60% and was associated not only with the effector-to-target ratio but also the level of infectivity of the hepatocytes. Comparatively, other groups have previously used a higher number of sporozoites to hepatocytes (varying from 1:1 to 5:1, compared to our 0.8:1) [21–24], and hence conceivably could have achieved higher levels of infection and hence inhibition, based on our current findings. Of those studies that achieved greater levels of inhibition with fewer total immune cells (Weiss et al. with 1x106 immune cells [23], Renia et al. with 0.3x106 immune cells [21], and Trimnel et al. with 0.05x106 immune cells [24]), they were also using vaccination models that result in 100% protection (irradiated sporozoites or genetically attenuated parasites) or clonal cells, and hence it is not surprising that they outperformed the inhibition achieved with a sub-unit vaccine. Our current results are therefore physiologically relevant and highlight the value of this assay.
Apart from these aforementioned in vitro studies that have demonstrated the ability of T cells to inhibit liver-stage malaria parasites, two other experimental findings in murine models highlight the importance of CD8+ T cells: (i) adoptive transfer of CD8+ T cell clones specific for TRAP or CSP can induce protection against P. berghei and P. yoelii in naive mice [39–42] and (ii) depletion of CD8+ T cells completely abolishes the protection seen in radiation and genetically attenuated sporozoite models [24, 43–46]. Hence, there is a strong rationale for the development of a T cell-inducing malaria vaccine, and a need to be able to assess such vaccines based on P. falciparum antigens in vitro.
Whilst pre-clinical testing of vaccine candidates using murine models is widely accepted, there are a number of inherent limitations, most critically the limited MHC repertoire of inbred mice and the generation of single immunodominant epitopes. Hence, future work will aim to transfer the described in vitro assay from a murine-based system to an assay using P. falciparum sporozoites, primary human hepatocytes or cell lines and human T cells. Such an assay faces more difficulties: human leukocyte antigen (HLA) matching of human samples, the limited HLA type of available cell lines [47, 48] and the lower level of infectivity in human hepatocytes [48, 49], as well as the increased difficulty in obtaining P. falciparum sporozoites. However, these obstacles are not insurmountable.
Recently, cryopreserved primary human hepatocytes have become commercially available, enabling a new source of hepatocytes with a greater range of HLA types than cell lines. Furthermore, methods of culturing primary human hepatocytes have also improved [50], and such improvements are also expected to improve the subsequent infectivity. In addition, P. falciparum parasites expressing GFP are now available [51, 52], suggesting that direct transfer of our flow-cytometry based assay should be possible. The ability to cryopreserve P. falciparum sporozoites [53] will enable a reduction in the batch-batch variation in infectivity levels seen in the current study. The major difficulty in moving to a human assay will be inclusion of appropriate controls to account for the likely high non-specific background inhibition (based on our current findings). We suggest use of T cells stimulated with irrelevant antigens (such as influenza peptides), or the use of pre-vaccination T cells if evaluating the mechanisms of protection in clinical trials. Given many of the components of the human based assay have already been optimized and developed individually, and the remaining challenges can be addressed, the feasibility and utility of this assay is very promising.
Acknowledgments
The authors wish to thank R. Sinden for providing P. berghei GFP parasites; V. Clark and H. Gray for their animal husbandry; A. Williams, S. Zakutansky, S. Biswas and V. Unwin for the production of mosquitoes; M. Dicks for provision of the vector control (ChAd63-MVA luciferase); A. Worth for assistance with flow cytometry; and J. Furze for general laboratory assistance.
Author Contributions
Conceived and designed the experiments: RJL KJE AVSH AJS. Performed the experiments: RJL AJS. Analyzed the data: RJL. Contributed reagents/materials/analysis tools: KB. Wrote the paper: RJL.
References
- 1.
World Malaria Report. Geneva, Switzerland: World Health Organisation, 2012.
- 2. O'Hara GA, Duncan CJ, Ewer KJ, Collins KA, Elias SC, Halstead FD, et al. Clinical assessment of a recombinant simian adenovirus ChAd63: a potent new vaccine vector. The Journal of infectious diseases. 2012;205(5):772–81. Epub 2012/01/26. pmid:22275401; PubMed Central PMCID: PMCPMC3274376.
- 3. Ewer KJ, O'Hara GA, Duncan CJ, Collins KA, Sheehy SH, Reyes-Sandoval A, et al. Protective CD8+ T-cell immunity to human malaria induced by chimpanzee adenovirus-MVA immunisation. Nature communications. 2013;4:2836. Epub 2013/11/29. pmid:24284865; PubMed Central PMCID: PMCPMC3868203.
- 4. Ghosh AK, Devenport M, Jethwaney D, Kalume DE, Pandey A, Anderson VE, et al. Malaria parasite invasion of the mosquito salivary gland requires interaction between the Plasmodium TRAP and the Anopheles saglin proteins. PLoS pathogens. 2009;5(1):e1000265. Epub 2009/01/17. pmid:19148273; PubMed Central PMCID: PMCPMC2613030.
- 5. Kappe S, Bruderer T, Gantt S, Fujioka H, Nussenzweig V, Menard R. Conservation of a gliding motility and cell invasion machinery in Apicomplexan parasites. The Journal of cell biology. 1999;147(5):937–44. Epub 1999/12/01. pmid:10579715; PubMed Central PMCID: PMCPMC2169348.
- 6. Muller HM, Scarselli E, Crisanti A. Thrombospondin related anonymous protein (TRAP) of Plasmodium falciparum in parasite-host cell interactions. Parassitologia. 1993;35 Suppl:69–72. Epub 1993/07/01. pmid:8233617.
- 7. Pradel G, Garapaty S, Frevert U. Proteoglycans mediate malaria sporozoite targeting to the liver. Molecular microbiology. 2002;45(3):637–51. Epub 2002/07/26. pmid:12139612.
- 8. Sultan AA, Thathy V, Frevert U, Robson KJ, Crisanti A, Nussenzweig V, et al. TRAP is necessary for gliding motility and infectivity of plasmodium sporozoites. Cell. 1997;90(3):511–22. Epub 1997/08/08. pmid:9267031.
- 9. Reyes-Sandoval A, Rollier CS, Milicic A, Bauza K, Cottingham MG, Tang CK, et al. Mixed vector immunization with recombinant adenovirus and MVA can improve vaccine efficacy while decreasing antivector immunity. Molecular therapy: the journal of the American Society of Gene Therapy. 2012;20(8):1633–47. Epub 2012/02/23. pmid:22354374; PubMed Central PMCID: PMCPMC3412496.
- 10. Reyes-Sandoval A, Sridhar S, Berthoud T, Moore AC, Harty JT, Gilbert SC, et al. Single-dose immunogenicity and protective efficacy of simian adenoviral vectors against Plasmodium berghei. European journal of immunology. 2008;38(3):732–41. Epub 2008/02/13. pmid:18266272.
- 11. Bauza K, Malinauskas T, Pfander C, Anar B, Jones EY, Billker O, et al. Efficacy of a Plasmodium vivax malaria vaccine using ChAd63 and modified vaccinia Ankara expressing thrombospondin-related anonymous protein as assessed with transgenic Plasmodium berghei parasites. Infection and immunity. 2014;82(3):1277–86. Epub 2014/01/01. pmid:24379295; PubMed Central PMCID: PMCPMC3957994.
- 12. Brahimi K, Badell E, Sauzet JP, BenMohamed L, Daubersies P, Guerin-Marchand C, et al. Human antibodies against Plasmodium falciparum liver-stage antigen 3 cross-react with Plasmodium yoelii preerythrocytic-stage epitopes and inhibit sporozoite invasion in vitro and in vivo. Infection and immunity. 2001;69(6):3845–52. Epub 2001/05/12. pmid:11349050; PubMed Central PMCID: PMCPMC98406.
- 13. Hollingdale MR, Appiah A, Leland P, do Rosario VE, Mazier D, Pied S, et al. Activity of human volunteer sera to candidate Plasmodium falciparum circumsporozoite protein vaccines in the inhibition of sporozoite invasion assay of human hepatoma cells and hepatocytes. Transactions of the Royal Society of Tropical Medicine and Hygiene. 1990;84(3):325–9. Epub 1990/05/01. pmid:2175464.
- 14. Hollingdale MR, Ballou WR, Aley SB, Young JF, Pancake S, Miller LH, et al. Plasmodium falciparum: elicitation by peptides and recombinant circumsporozoite proteins of circulating mouse antibodies inhibiting sporozoite invasion of hepatoma cells. Experimental parasitology. 1987;63(3):345–51. Epub 1987/06/01. pmid:2438152.
- 15. Hollingdale MR, Nardin EH, Tharavanij S, Schwartz AL, Nussenzweig RS. Inhibition of entry of Plasmodium falciparum and P. vivax sporozoites into cultured cells; an in vitro assay of protective antibodies. Journal of immunology (Baltimore, Md: 1950). 1984;132(2):909–13. Epub 1984/02/01. pmid:6317752.
- 16. Mazier D, Mellouk S, Beaudoin RL, Texier B, Druilhe P, Hockmeyer W, et al. Effect of antibodies to recombinant and synthetic peptides on P. falciparum sporozoites in vitro. Science (New York, NY). 1986;231(4734):156–9. Epub 1986/01/10. pmid:3510455.
- 17. Mellouk S, Berbiguier N, Druilhe P, Sedegah M, Galey B, Yuan L, et al. Evaluation of an in vitro assay aimed at measuring protective antibodies against sporozoites. Bulletin of the World Health Organization. 1990;68 Suppl:52–9. Epub 1990/01/01. pmid:2094592; PubMed Central PMCID: PMCPMC2393043.
- 18. Finney OC, Keitany GJ, Smithers H, Kaushansky A, Kappe S, Wang R. Immunization with genetically attenuated P. falciparum parasites induces long-lived antibodies that efficiently block hepatocyte invasion by sporozoites. Vaccine. 2014;32(19):2135–8. Epub 2014/03/04. pmid:24582635.
- 19. Zou X, House BL, Zyzak MD, Richie TL, Gerbasi VR. Towards an optimized inhibition of liver stage development assay (ILSDA) for Plasmodium falciparum. Malaria journal. 2013;12:394. Epub 2013/11/07. pmid:24191920; PubMed Central PMCID: PMCPMC3831258.
- 20. Hoffman SL, Isenbarger D, Long GW, Sedegah M, Szarfman A, Waters L, et al. Sporozoite vaccine induces genetically restricted T cell elimination of malaria from hepatocytes. Science (New York, NY). 1989;244(4908):1078–81. Epub 1989/06/02. pmid:2524877.
- 21. 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. Journal of immunology (Baltimore, Md: 1950). 1993;150(4):1471–8. Epub 1993/02/15. pmid:7679428.
- 22. 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. Proceedings of the National Academy of Sciences of the United States of America. 1991;88(18):7963–7. Epub 1991/09/15. pmid:1680235; PubMed Central PMCID: PMCPMC52425.
- 23. Weiss WR, Mellouk S, Houghten RA, Sedegah M, Kumar S, Good MF, et al. Cytotoxic T cells recognize a peptide from the circumsporozoite protein on malaria-infected hepatocytes. The Journal of experimental medicine. 1990;171(3):763–73. Epub 1990/03/01. pmid:1689762; PubMed Central PMCID: PMCPMC2187765.
- 24. Trimnell A, Takagi A, Gupta M, Richie TL, Kappe SH, Wang R. Genetically attenuated parasite vaccines induce contact-dependent CD8+ T cell killing of Plasmodium yoelii liver stage-infected hepatocytes. Journal of immunology (Baltimore, Md: 1950). 2009;183(9):5870–8. Epub 2009/10/09. pmid:19812194.
- 25. 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 pathogens. 2013;9(5):e1003303. Epub 2013/05/16. pmid:23675294; PubMed Central PMCID: PMCPMC3649980.
- 26. Franke-Fayard B, Trueman H, Ramesar J, Mendoza J, van der Keur M, van der Linden R, et al. A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle. Molecular and biochemical parasitology. 2004;137(1):23–33. Epub 2004/07/29. pmid:15279948.
- 27. Grimm CF, Ortmann D, Mohr L, Michalak S, Krohne TU, Meckel S, et al. Mouse alpha-fetoprotein-specific DNA-based immunotherapy of hepatocellular carcinoma leads to tumor regression in mice. Gastroenterology. 2000;119(4):1104–12. Epub 2000/10/21. pmid:11040197.
- 28. Harimoto H, Shimizu M, Nakagawa Y, Nakatsuka K, Wakabayashi A, Sakamoto C, et al. Inactivation of tumor-specific CD8(+) CTLs by tumor-infiltrating tolerogenic dendritic cells. Immunology and cell biology. 2013;91(9):545–55. Epub 2013/09/11. pmid:24018532; PubMed Central PMCID: PMCPMC3806489.
- 29. Zimmermann M, Flechsig C, La Monica N, Tripodi M, Adler G, Dikopoulos N. Hepatitis C virus core protein impairs in vitro priming of specific T cell responses by dendritic cells and hepatocytes. Journal of hepatology. 2008;48(1):51–60. Epub 2007/11/14. pmid:17998148.
- 30. Sinnis P, De La Vega P, Coppi A, Krzych U, Mota MM. Quantification of sporozoite invasion, migration, and development by microscopy and flow cytometry. Methods in molecular biology (Clifton, NJ). 2013;923:385–400. Epub 2012/09/20. pmid:22990793; PubMed Central PMCID: PMCPMC3951895.
- 31. Mota MM, Pradel G, Vanderberg JP, Hafalla JC, Frevert U, Nussenzweig RS, et al. Migration of Plasmodium sporozoites through cells before infection. Science (New York, NY). 2001;291(5501):141–4. Epub 2001/01/06. pmid:11141568.
- 32. Prudencio M, Rodrigues CD, Ataide R, Mota MM. Dissecting in vitro host cell infection by Plasmodium sporozoites using flow cytometry. Cellular microbiology. 2008;10(1):218–24. Epub 2007/08/19. pmid:17697130.
- 33. Cottingham MG, Carroll F, Morris SJ, Turner AV, Vaughan AM, Kapulu MC, et al. Preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vectors. Biotechnology and bioengineering. 2012;109(3):719–28. Epub 2012/01/19. pmid:22252512.
- 34. Draper SJ, Moore AC, Goodman AL, Long CA, Holder AA, Gilbert SC, et al. Effective induction of high-titer antibodies by viral vector vaccines. Nature medicine. 2008;14(8):819–21. Epub 2008/07/29. pmid:18660818.
- 35. Betts MR, Brenchley JM, Price DA, De Rosa SC, Douek DC, Roederer M, et al. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. Journal of immunological methods. 2003;281(1–2):65–78. Epub 2003/10/29. pmid:14580882.
- 36. Silvie O, Franetich JF, Boucheix C, Rubinstein E, Mazier D. Alternative invasion pathways for Plasmodium berghei sporozoites. International journal for parasitology. 2007;37(2):173–82. Epub 2006/11/23. pmid:17112526.
- 37. O'Doherty U, Swiggard WJ, Malim MH. Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding. Journal of virology. 2000;74(21):10074–80. Epub 2000/10/12. pmid:11024136; PubMed Central PMCID: PMCPMC102046.
- 38. Scanlan PM, Tiwari V, Bommireddy S, Shukla D. Spinoculation of heparan sulfate deficient cells enhances HSV-1 entry, but does not abolish the need for essential glycoproteins in viral fusion. Journal of virological methods. 2005;128(1–2):104–12. Epub 2005/05/24. pmid:15908019.
- 39. Khusmith S, Sedegah M, Hoffman SL. Complete protection against Plasmodium yoelii by adoptive transfer of a CD8+ cytotoxic T-cell clone recognizing sporozoite surface protein 2. Infection and immunity. 1994;62(7):2979–83. Epub 1994/07/01. pmid:8005684; PubMed Central PMCID: PMCPMC302907.
- 40. 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. International immunology. 1991;3(6):579–85. Epub 1991/06/01. pmid:1716146.
- 41. 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(6240):323–6. Epub 1989/09/28. pmid:2477703.
- 42. Weiss WR, Berzofsky JA, Houghten RA, Sedegah M, Hollindale M, Hoffman SL. A T cell clone directed at the circumsporozoite protein which protects mice against both Plasmodium yoelii and Plasmodium berghei. Journal of immunology (Baltimore, Md: 1950). 1992;149(6):2103–9. Epub 1992/09/15. pmid:1517574.
- 43. Doolan DL, Hoffman SL. IL-12 and NK cells are required for antigen-specific adaptive immunity against malaria initiated by CD8+ T cells in the Plasmodium yoelii model. Journal of immunology (Baltimore, Md: 1950). 1999;163(2):884–92. Epub 1999/07/08. pmid:10395683.
- 44. Seguin MC, Klotz FW, Schneider I, Weir JP, Goodbary M, Slayter M, et al. Induction of nitric oxide synthase protects against malaria in mice exposed to irradiated Plasmodium berghei infected mosquitoes: involvement of interferon gamma and CD8+ T cells. The Journal of experimental medicine. 1994;180(1):353–8. Epub 1994/07/01. pmid:7516412; PubMed Central PMCID: PMCPMC2191552.
- 45. Tarun AS, Dumpit RF, Camargo N, Labaied M, Liu P, Takagi A, et al. Protracted sterile protection with Plasmodium yoelii pre-erythrocytic genetically attenuated parasite malaria vaccines is independent of significant liver-stage persistence and is mediated by CD8+ T cells. The Journal of infectious diseases. 2007;196(4):608–16. Epub 2007/07/13. pmid:17624848.
- 46. Weiss WR, Sedegah M, Beaudoin RL, Miller LH, Good MF. CD8+ T cells (cytotoxic/suppressors) are required for protection in mice immunized with malaria sporozoites. Proceedings of the National Academy of Sciences of the United States of America. 1988;85(2):573–6. Epub 1988/01/01. pmid:2963334; PubMed Central PMCID: PMCPMC279593.
- 47. Karnasuta C, Pavanand K, Chantakulkij S, Luttiwongsakorn N, Rassamesoraj M, Laohathai K, et al. Complete development of the liver stage of Plasmodium falciparum in a human hepatoma cell line. The American journal of tropical medicine and hygiene. 1995;53(6):607–11. Epub 1995/12/01. pmid:8561262.
- 48. Sattabongkot J, Yimamnuaychoke N, Leelaudomlipi S, Rasameesoraj M, Jenwithisuk R, Coleman RE, et al. Establishment of a human hepatocyte line that supports in vitro development of the exo-erythrocytic stages of the malaria parasites Plasmodium falciparum and P. vivax. The American journal of tropical medicine and hygiene. 2006;74(5):708–15. Epub 2006/05/12. pmid:16687667.
- 49. Mazier D, Beaudoin RL, Mellouk S, Druilhe P, Texier B, Trosper J, et al. Complete development of hepatic stages of Plasmodium falciparum in vitro. Science (New York, NY). 1985;227(4685):440–2. Epub 1985/01/25. pmid:3880923.
- 50. March S, Ng S, Velmurugan S, Galstian A, Shan J, Logan DJ, et al. A microscale human liver platform that supports the hepatic stages of Plasmodium falciparum and vivax. Cell host & microbe. 2013;14(1):104–15. Epub 2013/07/23. pmid:23870318; PubMed Central PMCID: PMCPMC3780791.
- 51. Talman AM, Blagborough AM, Sinden RE. A Plasmodium falciparum strain expressing GFP throughout the parasite's life-cycle. PloS one. 2010;5(2):e9156. Epub 2010/02/18. pmid:20161781; PubMed Central PMCID: PMCPMC2819258.
- 52. Vaughan AM, Mikolajczak SA, Camargo N, Lakshmanan V, Kennedy M, Lindner SE, et al. A transgenic Plasmodium falciparum NF54 strain that expresses GFP-luciferase throughout the parasite life cycle. Molecular and biochemical parasitology. 2012;186(2):143–7. Epub 2012/10/31. pmid:23107927.
- 53. Sheehy SH, Spencer AJ, Douglas AD, Sim BK, Longley RJ, Edwards NJ, et al. Optimising Controlled Human Malaria Infection Studies Using Cryopreserved Parasites Administered by Needle and Syringe. PloS one. 2013;8(6):e65960. Epub 2013/07/05. pmid:23823332; PubMed Central PMCID: PMCPMC3688861.