Intradermal Immunization with Live Attenuated Sporozoites in a Mouse Model

Published: May 31, 2024

Abstract

Source: Mac-Daniel, L., et al. Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites. J. Vis. Exp. (2016).

This video demonstrates an assay for immunizing a mouse model with live attenuated sporozoites. The radiation-attenuated Plasmodium sporozoites are injected into the dorsal side of the mouse's ear. After injections at multiple locations on both ears, the mouse is allowed to recover.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Materials and Reagents

  1. Use female Anopheles stephensi mosquitoes (Sda500 strain) that feed on infected mice 3-5 days after emergence and rear as described previously.
  2. Use parasites Plasmodium berghei ANKA clone expressing the Green Fluorescent Protein (GFP) gene under the control of the constitutive Heat Shock Protein 70 (HSP70) promoter. This yields bright fluorescence throughout the parasite life cycle.
  3. Use female C57BL/6JRj mice (7 week-old).
    NOTE: All the reagents used in the described protocol are listed in the Table of Materials/Equipment.

2. Radiation-attenuated Sporozoite Isolation from Mosquito Salivary Glands

  1. Between 18-25 days after the infectious blood meal, collect and cold-anesthetize mosquitoes into a 15 ml tube on ice as described previously. Carefully transfer them onto a Petri dish by inverting the tube. Maintain the insects on ice and expose the mosquitoes to a 12 krad dose of γ- or x-irradiation.
  2. Isolate attenuated sporozoites from irradiated mosquito salivary glands as previously described. Collect infected salivary glands in a small volume (around 15 μl) of 1x Dulbecco's phosphate-buffered saline (DPBS) on ice and gently crush them to release sporozoites.
    NOTE: The injection of a highly concentrated parasite suspension in a small volume being critical, it is therefore essential to harvest a sufficient number of salivary glands from a high number of preselected well-infected mosquitoes, as evidenced by their robust expression of green-fluorescence.
  3. Filter the sporozoite suspension through a 35 µm cell strainer cap adapted to a low adhesion 1.5 ml microcentrifuge tube in order to eliminate clumps and mosquito debris.
  4. Count the total number of isolated sporozoites as previously described and adjust the concentration of parasite suspension with cold 1x DPBS to obtain 83,000 sporozoites/μl. Store the parasites on ice while continuing the next steps.
  5. Collect the equivalent number of salivary glands from uninfected mosquitoes that will be used as negative control and process the sample as described in steps 2.2 and 2.3. Dilute the sample as indicated above to obtain similar concentration of salivary gland extracts in the suspension.
    NOTE: Sporozoites can be stored on ice for a maximum of 2 hr. However, ideally they are injected within an hour after crushing the salivary glands.

3. Injection of Sporozoites into the Dermal Layer of the Ear

  1. Anesthetize animals by intraperitoneal injection of ketamine (50 mg/kg)/xylazine (5 mg/kg) mixture. Wait 5-8 min for the mouse to be in a state of unconsciousness and apply ophthalmic ointment to the eyes to prevent corneal drying. Evaluate the depth of anaesthesia by performing a gentle toe pinch on both rear feet.
    NOTE: The dose of anaesthesia lasts for less than 20 min, so the next steps must be done quickly.
  2. Stabilize the ear pinna of the mouse by fixing the ventral side with clear tape previously placed under a stereomicroscope (Figure 1A). Gently press the ear to avoid damage of the vasculature.
  3. Load a 10 μl syringe/35 gauge bevelled needle with 0.6 µl of resuspended parasites.
  4. Deliver the sporozoite suspension in 4 injection sites (0.15 µl per site) by carefully inserting the needle on the dorsal side of the ear (Figure 1A), beneath the epidermis, with the bevel up, taking care to avoid any blood vessels. Allow the needle to remain in the dermis for few sec to prevent backflow of the injectant before removing it. Observe a characteristic papule at each injection site at the end of the procedure (Figure 1B and 1C).
  5. After injection, carefully remove the ear from the tape.
  6. Inject the contralateral ear with the same dose of parasites by following the steps 3.2-3.4.
  7. Inject 2 additional mice with either 0.6 µl of 1x DPBS or 0.6 µl of 1x DPBS + salivary gland extract from uninfected mosquitoes in order to evaluate the inflammatory response mediated by the injection alone and the deposition of mosquito material in the dermis.
  8.  Monitor mouse recovery from anaesthesia before placing it back into the cage.
    NOTE: The proper sporozoite deposition in the skin can be monitored by fluorescence microscopy (Figure 2A and 2B), the mouse being prepared as described previously.

Representative Results

Figure 1
Figure 1: Intradermal Injection of Sporozoites into the Ear of Mouse. (A) Image showing intradermal injection of sporozoites in the ear pinna of an anesthetized mouse. The ventral side of the ear is stabilized with clear tape previously placed under a dissecting microscope. The needle is carefully inserted on the dorsal side of the ear, beneath the epidermis, with the bevel up. (BC) Pictures showing the dorsal side of the ear pinna prior (B) and after (C) intradermal injection of 0.1-0.2 μl of parasite suspension. A characteristic papule (black arrow) is observable at the injection site at the end of the operation.

Figure 2
Figure 2: Imaging of Sporozoites Deposited in the Ear Dermis of Mouse. (A) Fluorescence microscopy showing RAS migrating from the injection site (indicated by the dashed lines) in the mouse skin 15 min post-injection (Scale bar = 60 μm; 10X magnification). The path of sporozoites is represented by the maximum intensity projection of the fluorescent signal. Green and red fluorescence respectively show parasite position in the skin at the beginning and after 10 sec of acquisition. (B) Higher magnification of RAS (green) injected in the skin (Scale bar = 20 μm; 25X magnification).

Declarações

The authors have nothing to disclose.

Materials

Ketamine: Imalgene® 1000 Merial
Xylazine: Rompun® 2% Bayer
NanoFil syringe + 35 gauge needle World Precision Instruments
Omnican® 50 Insulin syringe 0,5 ml/50 I.U. B. Braun Medical 9151125
MultiwellTM 6 well tissue culture plate – Flat Bottom BD Falcon 353046
70 µm cell strainer BD Falcon 352350
2 ml syringe Terumo SS-02S
BLUE MAXTM 15ml Polypropylene conical tube BD Falcon 352097
BLUE MAXTM 50ml Polypropylene conical tube BD Falcon 352098
5ml Polystyrene Round-Bottom Tube with 35µm Cell-Strainer Cap BD Falcon 352235
DPBS 1X Cacl2– and MgCl2-free Life Technologies 14190-094
DMEM 1X + GlutaMAXTM Life Technologies 31966-021
Deoxyribonuclease I from bovine pancreas, type IV Sigma-Aldrich D5025 50 µg/ml
Female C57BL/6JRj mice (7-week-old) Janvier Laboratories

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Intradermal Immunization with Live Attenuated Sporozoites in a Mouse Model. J. Vis. Exp. (Pending Publication), e22279, doi: (2024).

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