Background
Immune responses to pre-erythrocytic stages of
Plasmodium induced after immunization with attenuated sporozoites or vaccine candidates confer protective immunity by inhibiting sporozoite infection and the development of liver stages [
1,
2]. Work in rodent and non-human primate models of malaria have enabled significant advances in the characterization of immune mechanisms involved in protection against infection by
Plasmodium sporozoites. The primary target of these immune mechanisms is the circumsporozoite protein (CSP).
Circumsporozoite protein in Plasmodium berghei and other Plasmodium species that infect animals have significant functional similarity to Plasmodium falciparum CSP. However, they have major differences in amino acid sequences. Therefore, studies on the immunogenicity and efficacy of vaccine candidates using non-human plasmodial antigens have limited relevance for the design of human vaccine candidates. Furthermore, because of the strict species-specificity of malaria-causing parasites, the efficacy of vaccines designed for humans cannot be accurately evaluated using animal models; this results in an over-reliance on complex and costly human vaccine trials for initial proof-of-concept data.
The development of transgenic rodent parasites in which the murine CSP is replaced by the
P. falciparum orthologue helps to overcome some of these limitations. This strategy has previously been used to evaluate the immunogenicity and efficacy of vaccine constructs against
P. falciparum [
3,
4], and
Plasmodium vivax CSP [
5‐
7], but a detailed analysis of the key features of such an assay has not been reported. Here, in vivo challenge assays are described that use
P. berghei parasites expressing the
P. falciparum CSP (3D7) in place of the
P. berghei CSP; the infectivity of these transgenic parasites is evaluated and quantitative methods are used to assess liver stage development. Critical parameters that affect parasite infection and development in mice are defined, including route of infection, viability and maturity. In addition, host factors that affect the development of liver stages, such as age, sex and genetic background of mice used in these assays are evaluated. Systematic studies using qPCR or other quantitative assays have not addressed all these variables. It is also demonstrated that this in vivo model can be used effectively to determine and compare the protective efficacy of antibodies specific for
P. falciparum CSP. The aim of this study is to standardize a method that can quantitatively assess sporozoite infectivity and liver stage development under different experimental and physiological conditions. An important objective is also to develop a high throughput assay that may allow the evaluation of multiple immune or pharmacological interventions, permitting a quantitative comparison of reagents that may affect sporozoite infectivity and/or development of the liver stage.
Methods
Mice
Six to seven weeks old female mice were used in all experiments, unless otherwise stated for experiments described in Fig.
4c. They were purchased from Charles River Laboratories and maintained at the animal facility of Bloomberg School of Public Health, Johns Hopkins University. All procedures performed in this study were approved by the IACUC Committee under the Protocol MO17H369.
Parasites
Plasmodium berghei used for the transfection express GFP and luciferase and was obtained through BEI Resources, NIAID, NIH:
P. berghei, strain ANKA 676m1c11, MRA-868, contributed by Chris J Janse and Andrew P Waters [
8]. To generate
P. berghei expressing full-length
P. falciparum CSP and GFP-luciferase, the same transfection strategy described in detail by Espinosa et al. was followed [
4]. Sporozoites were generated by infecting
Anopheles stephensi mosquitoes by allowing 5-days old mosquitoes to feed on parasite-infected mice at approximately 1–3% parasitaemia. Prior to feeding mosquitoes, the blood of each infected mouse was examined for the presence of gametocyte exflagellation to ensure mosquito infection. This is done by microscopic examination of blood in ookinete medium [
7]. After infection, mosquitoes were maintained in an incubator at 19–20 °C. On top of the cages, mosquitoes were supplied with a sterile cotton pad soaked in 10% sucrose, which was changed every 48 h.
Sporozoites were generally harvested at days 20–22 post infection, unless otherwise described. In experiments to evaluate the effect of sporozoite age on infectivity, sporozoites were harvested between 18 and 28 days.
Bioluminescence measurement
Twenty-four hours after sporozoite injection, mouse abdominal hair was removed using Nair cream. Forty-two hours after injection, the challenged mice were intraperitoneally injected with 100 µL of
d-luciferin (30 mg/mL), and then anesthetized in an isoflurane chamber. After mice were immobilized, they were then placed in an IVIS Spectrum imager to evaluate bioluminescence by measuring the radiance for 5 min from the abdomen. For comparison to RT-qPCR quantitation, mouse livers were harvested, and ribosomal RNA was quantified as described previously [
9].
Freezing and thawing parasites
Sporozoites were harvested as described above, counted, re-suspended in FCS, 7% DMSO in FCS or HBSS, and then placed in a pre-cooled NALGENE Cryo 1 °C Freezing Container (Cat. No. 5100-0001). The container was placed in a − 80 °C freezer and samples were left to freeze overnight. Parasites were then thawed on ice prior to challenge.
Route of infection
Sporozoites were obtained as described above, and 2000 sporozoites suspended in HBSS-2% FCS and injected through indicated routes. A total volume of 50 µL was injected for intramuscular injections in the leg, subcutaneous injection in the tail base, or subcutaneous injections close to the head. For IV and IP injections, sporozoites were administered in a final volume of 200 µL.
Mosquito bite challenge
Mice were exposed to the bites of 1–5 infected mosquitoes obtained from a population of mosquitoes that was 70–80% infected as determined by microscopic examination of salivary glands. Mice were anesthetized with 2% Avertin and then placed on top of cages containing mosquitoes for 10 min. Individual mosquitoes used for infection were examined to determine if the mosquitoes successfully fed on mice by assessing the presence of blood in the mosquito midguts. Four days after mosquito bite infection, Giemsa-stained blood smears were examined by light microscopy to look for blood stage infection.
Protection studies
For both intravenous sporozoite and mosquito bite challenge experiments, mice were first injected in the tail vein with 200 µL of the desired concentration of mAbs, 2 h prior to challenging mice. For intravenous sporozoite challenge experiments, sporozoites were harvested and diluted to 10,000 sporozoites/mL in Hanks balanced salt solution (HBSS) supplemented with 2% fetal calf serum (HBSS-2% FCS), and mice were injected with 2000 sporozoites (200 µL). Bioluminescence data were collected 42 h later. For mosquito bite challenge experiments, mice were anesthetized with 2% Avertin and placed on a cup containing 5 infected mosquitoes for 10 min. Blood stage infection was determined by light microscopy of Giemsa-stained blood films from days 4 to 10.
To test the effect of mAb treatment after infection, mAbs were injected in the tail vein 2 h after sporozoite infection, and bioluminescence data were collected 42 h later.
Statistical analysis
The data were analysed by using the non-parametric test, the Mann–Whitney test, considering the sample size and lack of evidence for normal distribution.
Discussion
The rapid and accurate assessment of sporozoite infectivity of the liver is critical for the development of new immunological or pharmacological interventions aimed at blocking this stage of the parasite life cycle. The mouse challenge model reported here allows the comparative screening of antibodies administered through passive or active immunization in mice. A transgenic
P. berghei strain that expresses
P. falciparum CSP was generated. As determined by immunofluorescence and ELISA assays, the transgenic sporozoites express nearly identical amounts of CSP compared to
P. falciparum sporozoites, and have identical infectivity to wild type
P. berghei ANKA parasites (Additional file
1: Figure S1C). Critical parameters were investigating regarding the method of detection of liver infection, sporozoite preparation, and host factors and each demonstrated a dose response range for inhibition of infection.
The RT-qPCR assay is based on the measurement of plasmodial 18s rRNA in liver, while bioluminescence imaging measures sporozoite-induced gene expression in the liver as luciferase activity. It was found that both assays can efficiently detect liver infections generated by as few as 25 sporozoites. An important factor favouring the bioluminescence approach is the complexity of the RT-qPCR methodology that involves a number of different steps, such as RNA purification from livers, generation of cDNA, and PCR amplification, all of which are likely to accumulate experimental errors and increase variability. In contrast, the assessment of liver burden by bioluminescence is a procedure made in live mice, without manipulation of the parasites, is sensitive, reproducible and much easier to perform, allowing increased throughput without significant loss of specificity or sensitivity (Additional file
1: Figure S1A, B; Table S1).
The bioluminescence-imaging assay enabled definition of basic assay features that influence detection of sporozoite liver infection. Using the bioluminescence assay, it was possible to discern differences in sporozoite infectivity due to route of infection, sporozoite age and treatment, as well as host factors, including mouse age, strain, and sex.
Importantly, it was determined that mice could be reproducibly infected by exposure to bites of 4–5 infected mosquitoes, resulting in clearly measurable levels of parasite liver load. In contrast, the exposure of mice to the bites of 1–3 mosquitoes does not reliably result in blood stage infection, even though it was confirmed that those mosquitoes were infected by the presence of sporozoites in their salivary glands. These results are consistent with prior studies, which indicate that the bites from single infected mosquitoes have a 30–70% probability to induce blood stage parasitaemia and that this may depend on sporozoite load in salivary glands [
15,
16].
This assay was used to evaluate the protective capacity of antibodies specific for
P. falciparum CSP and found a clear dose response in conferring inhibition of liver stage development. Since this inhibition is strictly dependent on the injection of antibodies before parasite challenge, we conclude that the protective activity of antibody AB317 results from neutralization of sporozoites before invading hepatocytes. Importantly, this antibody does not inhibit the infectivity of wild type
P. berghei sporozoites (Additional file
1: Figure S1D). The lack of effect of AB1245 is expected, as this is an irrelevant antibody that does not recognize sporozoites. It was determined that this in vivo model could also be used to assess the capacity of these of antibodies to confer sterile immunity after challenge of mice through the bites of infected mosquitoes. When challenging mice by exposure to the bites of 5 infected mosquitoes, a dose-dependent protection was found, with 100% protection conferred with mice receiving 300 µg of AB317.
Conclusions
A bioluminescence assay that provides data similar to RT-qPCR assessment of
Plasmodium liver-stage infection in mice, which is more rapid than RT-qPCR and can use fewer mice in order to assess pre-erythrocytic interventions, was developed. This in vivo model should greatly help to evaluate the efficacy of multiple CSP-based vaccine constructs and immunization regimens as a screen to select the most effective and efficient vaccine candidates prior to evaluation in controlled human malaria infection trials. It should also be helpful to define the efficacy of passively transferred mAbs, including those obtained from immunized, challenged and protected (or non-protected) individuals. These mAbs can provide valuable information on the precise conformation of epitopes recognized by protective antibodies, thus eliminating unnecessary or inhibitory epitopes and guiding vaccine design. In addition, this method offers the advantage of measuring both liver infection and blood-stage infection in the same mouse, reducing the number of mice required for measuring both liver load and the pre-patency period, allowing studies to more precisely link the two phenomena. Finally, this tool can be modified to evaluate other human plasmodial antigens of interest. Recent surface proteome work in
P. falciparum [
17] and
P. vivax [
18] has identified other possible vaccine candidates. Transgenic
P. berghei parasites expressing other plasmodial antigens could be generated and this model could interrogate whether or not these surface proteins may have potential as vaccine candidates.
Acknowledgements
The authors thank the Insectary and Parasitology Core Facilities of Johns Hopkins Malaria Research institute. We also thank the Bloomberg Philanthropies for continued support. The following reagent was obtained through BEI Resources, NIAID, NIH: Plasmodium berghei, Strain (ANKA) 676m1c11, MRA-868, contributed by Chris J. Janse and Andrew P Waters.
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