Protocols for making and taking the vaccine

The RaDVaC intranasal vaccine is simple to both make and take for those with basic training in the biomedical sciences, chemistry, and related fields. You will find extensive documentation on all protocols, materials, and methods in our White Paper, but for easier access and convenience, key protocols are summarized below.

Preparation of chitosan nanoparticle vaccine

Given that the vaccine is being used in pandemic conditions, to ensure that the vaccine is free of infectious virus contaminants during administration, solutions should be sterilized prior to mixing. Small volumes of chitosan/peptide and of TPP can be rendered free of infectious virus by heating in capped conical tubes to 55° C for at least 10 minutes, allowed to cool to room temperature, then mixed in a small beaker while magnetic stirring rapidly (>500rpm) for a few minutes.

Stock solutions

Once prepared, heat stock solutions to 55° C (15 minutes for 50 ml conical tube; 10 minutes for 15 ml conical tube) to ensure that they are free of infectious virus. 5 M NaCl does not require heat sterilization but can be treated at the same time for an added safety precaution. Container sizes and amounts of each solution will depend on the number of doses of vaccine being produced. Multiple batches of 25 doses each can be made with the following stock volumes.

  • dH2O
  • 5M NaCl (in dH2O)
  • 3 mg/ml chitosan in 150 mM NaCl:
    • Add 48.5 ml dH2O to a 50ml conical tube
    • Add 1.5 ml 5M NaCl to the tube
    • Weigh 150 mg chitosan on a jeweler’s scale, and add to the tube
    • Cap the tube and shake well until the chitosan is dissolved
    • Dilute to desired working concentration. To make 10 ml working concentration of 1.2 mg/ml chitosan in 60 mM NaCl, add 4 ml stock solution to 6 ml dH2O
  • 1.0 to 1.5 mg/ml peptide solution(s). Mix peptides into dH2O 
  • 10 mg/ml Na5P3O10 (sodium triphosphate, a.k.a. tripolyphosphate, TPP) in 100 mM NaCl
  • OPTIONAL but recommended: dilute NaOH (e.g. 1M a.k.a. 1N), and acetic acid for adjusting pH of final vaccine solution.

Working solutions

  • 1.2 mg/ml chitosan in 60 mM NaCl
  • 1.0 to 1.5 mg/ml peptide solution(s)
  • 1 mg/ml TPP in 10 mM NaCl

Mixing the vaccine – sample protocol to make about 25 doses of vaccine (about 12 doses plus priming waste)

  1. Heat the working solutions to 50 to 60 deg C (15 minutes for 50 ml conical tube; 10 minutes for 15 ml conical tube) to ensure they are virus free.
  2. Sterilize the small beaker and stir bar by cleaning with soap and water, and then drying by wiping with an alcohol-soaked paper towel. 
  3. Place the beaker with the magnetic stir bar inside on a magnetic stir plate. 
  4. Add 7.2 ml chitosan working solution to the beaker.  Turn on the stir plate and slowly increase the stir speed to at least 500 rpm.
  5. Add peptide solutions up to 1.5 ml (total of 1500 to 2500 micrograms peptide). If adding less peptide solution, make up the volume in dH2O to bring the total to 1.5 ml.
  6. Slowly add 1.3 ml TPP solution one drop at a time, and after the last drop is added continue to stir for at least 5 minutes.
  7. Aliquot 800 microliters vaccine into sterile capped microfuge or 2 ml tubes. Each tube provides excess for priming the nasal sprayer, and about a 500 microliter dose.
  8. Optional but recommended: test the pH of the vaccine, and adjust to between 5.0 and 5.5. Nasal mucosal pH is about 5.5 to 6.5.

Administration of the vaccine

Two strategies have been used to achieve higher vaccine effectiveness (VE), especially in older people: high doses and multiple doses over time (a prime dose followed by booster doses). We have employed both of these strategies for increasing the likelihood of successful vaccination.

Dosage amount. Human studies have successfully used as little as 7.5 micrograms inactivated virus or other replication incompetent antigen. The intranasal inactivated influenza vaccine trial in healthy human adults conducted by Illum and colleagues used 7.5 micrograms and 15 micrograms per dose, with greater than double vaccine effectiveness at the higher dose. We have used 50 micrograms to 100 micrograms total peptide for the initial priming and boost doses. Typical doses have been in the range of 70 micrograms. As of mid July, 2020, over twenty self-experimenters have shown good tolerance for this dose.

Booster schedule. The vaccine should be used 3 times, minimum (as shown by animal studies and human clinical trial data): a priming dose, and administration of 2 booster doses. Booster doses have been taken as soon as a few days after the prior dose, or as long as a few weeks. Ideally, doses are spaced by about 2 weeks. As of mid-October, 2020, members of the Boston RaDVaC group have taken as few as 3 and as many as 12 doses of progressive generations of vaccine. Groups in other locations have taken between 3 and 4 doses each. Given that this is rapidly evolving research, these numbers are subject to frequent change.

Pre-administration. To assess immunity, it is ideal to obtain saliva, nasal wash or swab, and blood draws prior to vaccination. Of these, blood draw is most difficult, and possibly least important, although it is easier to compare results to known standards.

Administration. Vaccination is achieved by nasal administration with the use of a small (5ml to 20ml) commercial nasal sprayer. Depression of the sprayer top of our bottle delivers approximately 100ul of fine mist. This should be tested empirically by spraying into a small tube or beaker, and measuring by pipette. Dose is adjusted accordingly. Spray should be directed into each nostril. The sprayers we obtained come as a top sprayer unit, and either opaque white plastic or clear bottle. The sprayer unit stem stretches to the bottom of the bottle. The delivered total volumes of vaccine should be in the range of 200 to 500 microliters, which is too small to be delivered reliably using the bottle. Therefore, we do not use the bottle for vaccination, and instead place the sprayer stem directly into the smaller vaccine vial. The stem of our sprayer unit is slightly longer than a 2 ml microcentrifuge tube. We pipette an aliquot of vaccine into the tube, then place the stem of the sprayer into the vaccine for spray administration. 

Protocol for vaccine administration

To administer vaccine, it is ideal to wear gloves and have a spray bottle of 70% alcohol (ethanol or isopropanol) to sterilize your gloved hands, vials, and equipment. Select a well lit work surface that can be sterilized with alcohol.

Required materials:

  • Clean and well lit work surface
  • Gloves (nitrile, latex, etc)
  • 70% alcohol (ethanol or isopropanol), preferably in a spray bottle
  • Small (5ml to 20ml) commercial nasal sprayer bottle
  • Vial of vaccine containing 1 dose plus extra for priming the sprayer (total,  800 microliter)
  • Clean and preferably sterile paper towels or tissues
  • OPTIONAL: saline wash solution and collection tube(s)
  • OPTIONAL: rack or holder for vaccine vial
  1. Sterilize the work surface with alcohol. Place a paper towel or tissue on the clean work surface, and spray with alcohol. You can place items on the surface and use the towel or tissue to dab or wipe items (e.g. the sprayer stem) while maintaining sterility.
  2. Wipe the vaccine vial with an alcohol wipe. Loosen the cap so that it can be removed by lifting it off, but leave the cap in place, and set the vial upright on the work surface. If you choose to use a tube holder or rack for the vial, make sure it is sterilized.
  3. Blow your nose thoroughly with a tissue or paper towel to clear your nasal passages. Use an alcohol soaked wipe or tissue to clean the outer area of the nose and just inside each nostril. This will help prevent inadvertent contamination of the sprayer tip with infectious virus that might be present on or just inside the nose. We do not recommend cleaning deep into the nasal passage; this is likely to do more harm than good.
  4. Sterilize your spray bottle by placing 2 to 4 ml of 70% alcohol into the spray bottle, screw on the cap, and deploy the sprayer until a fine mist is sprayed.
  5. Remove the spray top, and spray the residual alcohol. While you are doing this DO NOT TOUCH THE SPRAYER TIP OR STEM to maintain sterility. After sterilization or the following optional nasal wash, do not return the spray top to the bottle prior to using the vaccine.
  6. OPTIONAL. You can use the sterilized spray top apparatus at this point to spray 100 mM NaCl into nostrils for collection of nasal wash samples. Spray saline into nostrils, inhale slightly, and then evacuate nasal wash into a collection tube. Collect at least 500 microliters.
  7. While holding the spray top in one hand, uncap the vaccine vial with the other hand, and place the clean and sterile stem of the sprayer directly into the vaccine vial so that the stem touches the bottom of the vial. 
  8. Grasp the vaccine vial with one hand, and the spray top with the other hand so that your index finger and middle finger are on the side handles of the sprayer. 
  9. Deploy the sprayer until you see a fine mist. Spray once more to clear residual alcohol and/or saline from the sprayer.
  10. Insert the spray tip into a nostril. While inhaling through the nose, depress the sprayer (ideally, a total of about 100 to 150 microliters per spray). Sniff in the vaccine. Repeat the dosing, for a total of 200 to 300 microliters into each nostril. The entire procedure can take seconds or minutes but don’t let the vaccine drip out of the nose, and don’t blow your nose for at least an hour.