Generating Genetically Modified Plasmodium berghei Sporozoites

Despite advances in drug development and research into malaria prevention and treatment, the global disease burden of malaria remains high. Over half a million people die of malaria each year, with the highest levels of mortality seen among children living in malaria-endemic regions, such as sub-Saharan Africa¹. Malaria is caused by the parasite Plasmodium, which is transmitted to humans through the bite of female Anopheles mosquitoes bearing the parasite in their salivary glands. The infectious stage of Plasmodium-the sporozoites-are deposited in the skin of the vertebrate hosts during a blood meal and travel through the bloodstream to infect liver cells, where they undergo mandatory development (constituting pre-erythrocytic malaria) prior to infecting the erythrocytes. The infection of the erythrocytes initiates the blood-stage of malaria and is responsible for the entirety of the morbidity and mortality associated with the disease^2,3.

The obligate nature of the pre-erythrocytic development of Plasmodium has made it an attractive target for prophylactic vaccine and drug development efforts⁴. A prerequisite for studying the biology of pre-erythrocytic malaria, as well as the development of vaccines or drugs targeting the liver stage, is access to Plasmodium sporozoites. Furthermore, our ability to generate genetically modified Plasmodium sporozoites has been instrumental in the success of such research endeavors^5,6,7,8,9. Transgenic Plasmodium lines expressing fluorescent or luminescent reporter proteins have allowed us to track their development in vivo and in vitro^10,11. Genetically attenuated parasites (GAPs), generated through the deletion of multiple genes in Plasmodium, are also some of the most promising vaccine candidates^12,13.

Rodent and non-human primate malaria models have helped us understand the mechanisms of host-parasite interactions in human malaria due to the similarities in biology and life cycle among Plasmodium species¹⁴. The use of Plasmodium species that infect rodents, but not humans (e.g., P. berghei) allows the maintenance of the complete parasite life cycle and the generation of infectious sporozoites for studying liver-stage malaria in a controlled, biosafety level 1 setting. A variety of separate protocols already exist for the generation of transgenic blood-stage Plasmodium parasites¹⁵, infection of mosquitoes¹⁶, and isolation of sporozoites¹⁷. Here, we outline a comprehensive protocol combining these methodologies in order to generate and isolate transgenic P. berghei sporozoites, utilizing the novel transgenic strain PbGFP₁₁ as an example. PbGFP₁₁ traffics the 11th β-strand of super-folder green fluorescent protein (GFP), GFP₁₁, into the parasitophorous vacuole (PV) generated in the host hepatocytes. PbGFP₁₁ is used in conjunction with transgenic hepatocytes (Hepa1-6 background) expressing residues that constitute the GFP 1-10 fragment (GFP_1-10) in the cytoplasm (Hepa GFP_1-10 cells). PbGFP₁₁ reports PV lysis in the host hepatocytes through self-complementation and the reformation of functional GFP and the green fluorescence signal¹⁸. Of note, GFP₁₁ is encoded as a series of seven tandem sequences in PbGFP₁₁ to enhance the resulting fluorescence signal. Upon staining PbGFP₁₁ sporozoites with the cytoplasmic dye CellTrace Violet (CTV), we can track the parasites. The lysis of such CTV-stained intracellular parasites itself results in leakage of CTV into the host cell cytoplasm and staining of the host cell. In addition to visualizing and distinguishing the lysis of Plasmodium PV and/or the parasite in host hepatocytes, this system can be reliably used to study the immune pathways responsible for either of these processes, through the genetic or therapeutic perturbation of the molecular components of such pathways.

All research involving vertebrate animals in our laboratory was performed in compliance with the University of Georgia animal use guidelines and protocols.

1. Generation of P. berghei -infected mice

1. Initiate blood-stage infection in male or female, 6-8-week-old C57BL/6 (B6) mice using wild-type P. berghei parasites. To do this, transfer cryopreserved P. berghei-infected blood (2 x 10⁵ infected RBCs), reconstituted in 400 µL of phosphate-buffered saline (PBS), into one or two donor mice intraperitoneally (i.p.).
2. Transfer fresh blood collected through retro-orbital bleeding from the donor mice at 5 days post-infection (d.p.i.) into two naïve B6 mice. To do this, collect 200 µL of blood, reconstitute with 300 µL of PBS, and inject 200 µL i.p. into the recipient mice. Ensure that the parasitemia in the donors at this time point is 2%-5%^19,20.
3. Monitor parasitemia as previously described^19,20, starting from 2 d.p.i. in the recipients. When parasitemia ranges from 3%-5% (around 5 d.p.i.), euthanize the recipient mice and then collect blood through cardiac puncture or retro-orbital bleeding. Euthanize the mice using carbon dioxide inhalation followed by cervical dislocation, or according to institutional guidelines.
   ​NOTE: Once parasitemia exceeds 5%, the transfection efficiency may be reduced due to the increased prevalence of multiply infected RBCs.

2. Generation of schizonts in culture

1. Collect blood from the infected mice in step 1.3 (approximately 2 mL in total from two mice) into 5 mL of Alsever's solution (see Table of Materials) in a sterile environment. Perform steps 2.1 to 3.12 under sterile conditions.
   NOTE: Sterile conditions include wearing gloves, wiping gloves and surfaces with 70% ethanol, employing sterile consumables and materials, and working within a biosafety cabinet.
2. Centrifuge the blood for 8 min at 450 x g at room temperature (RT). Discard the supernatant and resuspend the cell pellet in 10 mL of complete RPMI media (RPMI 1640 with 20% fetal bovine serum [FBS] and 1% penicillin-streptomycin, v/v).
3. Centrifuge for 8 min at 450 x g at RT. Resuspend the pellet in 25 mL of complete RPMI media and transfer to a T75 culture flask with a filter cap.
4. Place in an incubator at 36.5 °C and 5% CO₂, while gently shaking to keep the cells in suspension for 16 h.
5. At the 16 h time point, check for schizont development. To do this, collect 500 µL of the sample, centrifuge at 450 x g for 8 min, resuspend the pellet in 10 µL of complete RPMI media, and prepare a thin blood smear. Stain the blood smear with Giemsa stain¹⁹ (see Table of Materials) and determine the frequency of schizonts (Figure 1).
6. For optimal transfection efficiency, 60%-70% of the parasites should be in the schizont stage. If fewer than this threshold is determined, continue culturing as in step 2.4 and check every hour to ensure the above threshold is crossed before proceeding.

3. Transfection of Plasmodium schizonts

1. Prepare a gradient centrifugation buffer by reconstituting 13 mL of density gradient stock medium (see Table of Materials) with 1.44 mL of 1.5 M NaCl and 5.6 mL of 0.15 M NaCl in a 50 mL centrifugation tube.
2. Transfer 25 mL of blood culture to a fresh 50 mL centrifugation tube. Carefully layer 20 mL of gradient centrifugation buffer to the bottom of the centrifugation tube using a narrow glass pipette to create a gradient column. Take care not to disturb the liquid layers and interfaces and ensure a clear division between the buffer and the media.
3. Centrifuge for 20 min at 450 x g with no brake at RT. Using a Pasteur pipette, carefully remove the schizont layer¹⁵ located at the interface of the culture media and the gradient centrifugation buffer, and place it in a new 50 mL centrifugation tube.
4. Resuspend the isolated schizonts in complete RPMI media and bring up the total volume to 40 mL. Centrifuge at 450 x g for 8 min at RT and discard the supernatant.
5. Resuspend the schizonts in 10 mL of complete RPMI media. Then, transfer 1 mL of this solution into a 1.5 mL microcentrifuge tube for each transfection, and centrifuge at 16,000 x g for 5 s to pellet the infected RBCs. The infected RBCs derived from two mice in the above manner can be used for up to 10 separate transfections.
6. Combine 100 µL of the electroporation buffer (nucleofector solution from the electroporation kit; see Table of Materials) at RT with 5 µg of circular or linearized target plasmid DNA (in up to a maximum of 10 µL volume), as applicable for each transfection in separate 1.5 mL microcentrifuge tubes. A "no plasmid" sample must be included as a control for transfection and antibiotic selection.
7. Remove the supernatant after centrifugation from step 3.5 and carefully resuspend the infected RBCs in the prepared nucleofector solution + plasmid DNA (from step 3.6).
8. Transfer the suspension (nucleofector solution + plasmid + schizonts; 110 µL maximum) into electroporation cuvettes. Transfect using a suitable nucleofector program (U-033, see Table of Materials).
   NOTE: Ensure that steps 3.8 to 3.10 are performed at RT.
9. Add 100 µL of complete RPMI media to the cuvette and transfer the entire solution (now 200 µL total volume) into a 1.5 mL microcentrifuge tube and keep at RT.
10. Inject the 200 µL solution of transfected parasites into mice intravenously (i.v.), working to minimize the time between transfection and the final injection into mice.
    ​NOTE: Transfected schizonts are injected into mice less than 5 min after electroporation to maximize the efficiency of the protocol.

4. Selection of the transfected parasites

1. Check the parasitemia levels in the mice inoculated with the transfected schizonts daily from 2 d.p.i. to verify infection.
2. Once the infection is verified, start drug selection using oral administration of the drug in drinking water, offered ad libitum.
   NOTE: Pyrimethamine was employed as the selectable drug marker for the plasmid. In this case, 17.5 mg of pyrimethamine was added to 250 mL of clean water (final concentration of 70 mg/L). In addition, 10 g of sugar was added to this water to encourage adequate consumption of the pyrimethamine-containing water by mice.
3. Monitor parasitemia every two days using blood smears made with 5 µl of blood, starting from 6 days after the initiation of pyrimethamine treatment. The lack of detectable parasites after 15 days indicates a failed transfection; in this case, reinitiate the process from step 1.1 for a fresh attempt.
4. When parasitemia reaches at least 1%, transfer the parasites to naïve B6 mice for the creation of blood-stage parasite stocks. Continue selection in the newly infected mice until parasitemia reaches 2%-5%, at which point generate stocks and cryopreserve them²¹.

5. Infection of mosquitoes with the transgenic lines

1. Infect B6 mice with 200 µL of blood containing the selected parasites (2 x 10⁵ infected RBCs/mouse, i.v.). Determine parasitemia in these mice on the day of mosquito feeding and infection. Parasitemia between 2%-5% is optimal for the infection of mosquitoes. Once verified, immediately transfer the infection to the female Anopheles stephensi mosquitoes according to steps 5.2-5.4).
   NOTE: Incubators containing mosquitoes for infection should be maintained at 20.5 °C, at 80% humidity, and on a 12 h light/dark cycle (lights on between 07:00 a.m. to 07:00 p.m.).
2. The day before mosquito infection, separate the female mosquitoes by placing a heat source on top of the cage containing both male and female mosquitoes, such as a thin plastic bladder or glove filled with water heated to 37 °C. Remove the female mosquitoes, which are attracted to the heat source, using an insect aspirator and transfer these to a separate, smaller cage for infection.
   NOTE: Male mosquitoes can be distinguished from female mosquitoes by their slightly reduced body size and plumose antennae.
3. Inject the infected mice with a general anesthetic (e.g., 2% tribromoethanol/avertin). Ensure complete anesthesia by firmly pinching the foot pad of each mouse. If they do not react, they are sufficiently sedated and ready to transfer the infection into mosquitoes.
4. Place the anesthetized mice on top of the caged female mosquitoes (from step 5.2) under sternal recumbency. Manually spread the front and back legs to provide the greatest surface area for mosquito feeding. Leave the infected mice over each cage for a total of 15 min, checking every 5 min to ensure the mice are not awake. Once feeding is complete, euthanize the mice using cervical dislocation, or as per the institutional animal use protocol, and dispose of them safely.

6. Collection of sporozoites

1. Check mosquito midguts for oocysts at approximately 10 d.p.i. to verify successful infection and the development of Plasmodium within the mosquitoes. Isolate mosquito midguts from the abdomen by removing the terminal abdominal segment using forceps. Visualize the midguts under polarized light for the presence of oocysts²².
2. Wild-type sporozoites are expected to be present in the salivary glands of mosquitoes 18-21 days after feeding on infected mice. At day 18, dissect 5-10 mosquitoes to assess sporozoite numbers.
   NOTE: Mosquitos are washed and killed in ethanol. Subsequently, dead mosquitos are washed twice with PBS to remove debris prior to dissection.
3. To isolate and count the sporozoites, carefully remove the head of the mosquito while applying pressure to the mosquito thorax using forceps or a fine dissecting needle. The salivary glands remain attached to the removed head (Figure 2).
4. Remove any additional mosquito debris from the salivary glands and place in a microcentrifuge tube containing 400 µL of mosquito dissection media (Dulbecco's modified Ealge medium [DMEM] with 1% mouse serum, 100 U/mL penicillin, and 100 µg/mL streptomycin; see Table of Materials).
5. Disrupt the isolated salivary glands by passing through a 30 G needled syringe 15-20 times. Place 10 µL of the disrupted salivary glands in mosquito dissection media into a hemocytometer and count. Resuspend in the desired concentration of mosquito dissection media or PBS for injection into mice, inoculation into cell cultures, or long-term cryopreservation^23,24.

Determining the frequency and development of schizonts is critical for assuring that enough viable parasites are in the optimal stage for transfection. Immature schizonts can be differentiated from fully mature schizonts by the presence of fewer merozoites that do not fill the entire intracellular space of the RBC (Figure 1B). It is important to note that when making blood smears from cultured blood, infected RBCs may break open, resulting in the observation of free, extracellular merozoites in the blood smear (Figure 1). Such merozoites do not count toward the assessment of the frequency of schizonts in step 2.5.

The removal of salivary glands from mosquitoes can be challenging if the user is unfamiliar with mosquito physiology or small-scale dissections. During the isolation of salivary glands from mosquitoes, note that the translucence of the glands can be used to differentiate them from the opaque mosquito debris (Figure 2). The counting of sporozoites following disruption of the glands is most efficient at 400x magnification, and the use of phase-contrast allows for easier identification of the sporozoites within the counting chamber.

We have shown that over 90% of the Plasmodium in hepatocytes are possibly eliminated through cell-intrinsic immune mechanisms²³. Therefore, we expect parasites in a considerable number of hepatocytes to undergo lysis. As a tool to determine the lysis of Plasmodium or its PV in the host hepatocytes, we generated transgenic hepatocytes expressing the GFP_1-10 subunit (Hepa-GFP_1-10) and transgenic P. berghei expressing and trafficking the GFP₁₁ subunit to its PV (Pb-GFP₁₁). The GFP_1-10 fragment, which contains the residues that constitute the GFP chromophore, is nonfluorescent by itself and fluoresces only upon associating with GFP₁₁, potentially through self-complementation. In addition to functionally validating transgenesis in Plasmodium, the generation of green fluorescence signal in the Pb-GFP₁₁ infected Hepa-GFP_1-10 host cells indicated the lysis of the PV (Figure 3). Wild-type P. berghei-infected Hepa-GFP_1-10 cells or Pb-GFP₁₁-infected wild-type Hepa_1-6 cells failed to generate any green fluorescence signal (data not shown). We consider the lysis of the PV to be a key preceding step in the destruction of liver stage Plasmodium. The Pb-GFP₁₁ sporozoites were also stained with CTV to visualize the parasites in the hepatocytes. CTV is expected to leak into the host cell only upon lysis of the parasite itself (Figure 3). The dispersed CTV signal observed in the host hepatocyte likely indicates the lysis of the parasite within the hepatocyte. The close overlap between the CTV and GFP signals is expected with the concomitant lysis of both the PV and the parasite. This system allows us to query the distinct contributions of the individual host molecules in innate and cell-intrinsic immune pathways in modulating the lysis of Plasmodium or its PV.

Figure 1: Plasmodium berghei schizonts. Representative light microscopy images depicting P. berghei schizonts in parasitized mouse blood culture (16 h) stained with Giemsa stain. Arrows indicate fully matured (A) or immature (B) schizonts. Scale bars: 5 µm . Please click here to view a larger version of this figure.

Figure 2: Mosquito salivary glands and sporozoites. Light microscopy images depicting salivary glands isolated from Pb-GFP₁₁-infected female Anopheles mosquitoes. (A) Image of a mosquito head with the salivary glands (arrow) still intact during dissection, prior to its removal and further processing (scale bar: 1 mm). (B,C) Representative images of salivary glands at lower (B) and higher (C) magnifications (scale bars: 0.1 mm and 0.04 mm, respectively). Sporozoites can be seen both inside and outside of the gland (arrow). Please click here to view a larger version of this figure.

Figure 3: Detecting the lysis of Plasmodium and its parasitophorous vacuole in host hepatocytes. Differential interference contrast (DIC) and pseudo-colored immunofluorescence images with overlay depicting Hepa-GFP_1-10 hepatocytes infected with Pb-GFP₁₁ sporozoites. Sporozoites were stained with CTV prior to the infection (scale bar: 10 µm). A total of 1 x 10⁶ Hepa-GFP_1-10 hepatocytes were plated in a 35/10 mm glass-bottom culture dish and inoculated with 3 x 10⁵ PbGFP₁₁ sporozoites 4 h after plating. Images were taken with an inverted fluorescent microscope at 600x magnification and at 16 h post-infection. Abbreviations: DIC = differential interference contrast. Please click here to view a larger version of this figure.

We have used the above protocol in our laboratory to create several lines of transgenic P. berghei parasites. Though optimized for P. berghei, we have also successfully used this protocol to generate transgenic P. yoelii sporozoites. After injecting the transfected schizonts into mice, parasites are detectable typically no later than 3 d.p.i. in all groups, including the no plasmid control. Selection is started only once parasitemia has been detected to ensure the viability of parasites following electroporation. Additionally, when preparing for drug selection, acidification of the water with hydrochloric acid, bringing the pH down to 4, may be necessary for pyrimethamine to fully dissolve. We expect complete clearance of the parasites from the no plasmid control group, while the mice inoculated with the transfectants remain infected. Clearance in the control mice typically occurs 5-8 days after the initiation of pyrimethamine treatment. Of note, B6 mice infected with P. berghei may show signs of experimental cerebral malaria and succumb to death (follow humane endpoints according to institutional animal use guidelines), typically in the interval of 6-12 d.p.i. This can be avoided by performing drug selection of the transfectants in TCRaKO mice²⁵, or by transferring the parasites undergoing selection at 5 d.p.i. to new B6 recipients and continuing selection in the latter. Transgenesis in Plasmodium can be verified using a variety of approaches, such as genetic screens, phenotypic assessment, or gain or loss of specific functions.

It is important to note that the selection of a suitable plasmid is crucial for the successful transfection and retention of the introduced DNA in the parasite. Plasmids may also show limited efficacy in achieving transgenesis across different Plasmodium species. For example, the pSKspGFP11 plasmid (based on PL0017; BEI resources) we utilized to generate PbGFP₁₁ can be used to efficiently generate transgenic P. yoelli as well, but not P. chabaudi. To generate pSKspGFP11, we replaced the GFP mutant3 sequence in the parent pL0017 plasmid (BEI resources) with seven tandem sequences of the 11th β-strand of super-folder GFP, GFP₁₁ (GFP₁₁-7X)¹⁸. Although linearization of the plasmids before transfection allows better genomic integration, both linear and circular plasmids show similar transfection efficiencies using our protocol. The Plasmodium lines generated by transfection with circular plasmids also appear to maintain their transgenic properties over the course of the generation of sporozoites in the mosquitoes.

Notably, only female mosquitoes can harbor and transmit Plasmodium. Following the isolation of mosquitoes, it is recommended to not provide the isolated mosquitoes with a source of sugar water at this stage. Starving them in this manner would increase the chance of any male mosquitoes that may have been inadvertently transferred dying, and the females feeding the blood more efficiently from the infected mice. We typically return the sugar water 1 day after transferring the infection from mice to mosquitoes. It is noteworthy that the timeframe for the development and maturation of the sporozoite in mosquitoes may vary among transgenic Plasmodium lines. Therefore, it is important to assess sporozoite numbers in salivary glands at multiple time points after infection before a protocol specific for each transgenic line is established.