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

Stock solutions only need to be prepared if you are planning to make large quantities of vaccine. If you are planning on making small-scale batches, concentrated stock solutions are probably unnecessary, and you can simply make working solutions. A vaccine recipe for about 10 ml (about 12 to 15 doses) is provided in a section below, and the working solution recipes below allow you to make several of these batches. If you require much more than this, consider making 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 as an added safety precaution.

  • dH2O
  • 5M NaCl (in dH2O)
  • 5 mg/ml chitosan in 100 mM NaCl and 1% acetic acid; 50 ml chitosan stock solution recipe: 
    1. Add 39 ml dH2O to a 50ml conical tube
    2. Add 10 ml distilled white vinegar (or 5% acetic acid).
    3. Add 1 ml 5M NaCl to the tube
    4. Weigh 250 mg chitosan on a jeweler’s scale, and add to the tube.
    5. Cap the tube and shake or vortex occasionally until the chitosan is dissolved. This probably will take at least 30 minutes. Heating helps speed the process.
    6. Dilute to desired working concentration. To make 50 ml working concentration of 1 mg/ml chitosan in 20 mM NaCl, add 10 ml stock solution to 40 ml dH2O
  • 1.5 mg/ml peptide solution(s). Mix peptides into dH2O. See protocol below for peptides that require disulfide circularization.
  • 5 mg/ml Na5P3O10 (sodium triphosphate, a.k.a. tripolyphosphate, TPP) in dH2O.
  • OPTIONAL: 1M solution of sodium bicarbonate (NaHCO3, baking soda) for adjusting pH of final vaccine solution. 
  • OPTIONAL: solutions containing additional adjuvants

Working solutions

Working solutions of chitosan and TPP can be created from 5x stock solutions by adding 1 part stock solution to 4 parts water. For example, for 50 ml TPP working solution, add 10 ml TPP stock solution to 40 ml dH2O. Individual working solution recipes are also provided in detail below.

  • 1 mg/ml chitosan in 20 mM NaCl and 0.2% acetic acid; 50 ml recipe:
    1. To a 50 ml conical tube, add 2 ml white vinegar (or 5% acetic acid)
    2. Add NaCl to 20 mM by one of the following methods: Pipette 200 microliters 5 M NaCl into the tube; or, weigh 59 mg NaCl and add to the tube
    3. Weigh 50 mg chitosan and add to the tube
    4. Add dH2O to 50 ml (about 49 ml dH2O )
    5. Cap and shake the tube vigorously for about a minute, and then occasionally until all of the chitosan is fully dissolved. This might take up to 30 minutes for larger clumps of chitosan to fully dissolve.
  • 1.5 mg/ml peptide solution(s). Mix peptides into dH2O. See protocol below for peptides that require disulfide circularization.
    1. Add sufficient water to a conical tube (leaving headspace for mixing)
    2. Add peptide powder. 
    3. Cap and invert or shake the tube until all of the peptide is dissolved.
  • 1 mg/ml TPP: recipe for 40 ml:
    1. Add 40 ml dH2O to a 50 ml conical tube
    2. Add 40 mg TPP to the tube
    3. Cap and shake the tube until all TPP is dissolved.
  • OPTIONAL: 1M solution of sodium bicarbonate (NaHCO3, baking soda); recipe for 10 ml:
    1. To a 15 ml conical tube, add 10 ml dH2
    2. Add 840 mg NaHCO3 to the tube
    3. Cap and shake the tube until all NaHCO3 is dissolved.

Charcoal-assisted circularization of peptides

For peptides that require disulfide circularization. After circularization, charcoal can be filtered out although we find that freezing peptide solutions with charcoal does not appear to cause problems, and the charcoal remains intact in the bottom of the tube upon thawing.

  1. Resuspend lyophilized peptide powder in dH2O at a concentration of 1.5 mg/ml in a tube with headspace and a tight fitting cap. 
  2. Estimate the amount of peptide in the tube and weigh approximately 1 to 2 equal weights of small (average 0.5 to 1 mm diameter) granular charcoal (e.g. for 10 mg peptide, use 10 to 20 mg charcoal). If the charcoal granules are larger, they can be broken up to increase surface area. Add the charcoal to the peptide solution and tightly cap the tube. 
  3. Place the tube on agitator (rocker, shaker, rotator, etc) Incubate at room temperature 3 to 5 hours, with mild agitation. Make sure that the charcoal is mixing through the peptide solution, and not simply sitting in the bottom of the tube. This can be done by placing the tube on its side and shaking, or by rocking or inverting the tube on a rotator.

Mixing the vaccine – sample protocol to make 10 ml of vaccine (about 15 to 20 doses, or about 12 to 15 doses plus nasal sprayer 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 magnetic stir bar inside the small beaker, and place the beaker onto a magnetic stir plate. 
  4. Add 8 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 1.0 ml peptide solution (total of 1500 micrograms peptide).
  6. OPTIONAL adjuvant solutions should be added at this time.
  7. Slowly add 1.0 ml TPP solution one drop at a time, and after the last drop is added continue to stir for at least 5 minutes.
  8. Test the pH of the vaccine. The pH should be in the range of 3.5 to 4.0. This is a mildly acidic pH, equivalent to very dilute vinegar, and can be used as is. If desired, adjust to between 4.5 and 5.0 with NaHCO3 solution. Only a very small amount (100 to 150 microliters) of NaHCO3 should be required.
  9. Aliquot 800 microliters vaccine into sterile capped microfuge or 2 ml tubes. Only about 500 to 600 microliters of this will be used as vaccine; some will be lost due to sprayer priming and other waste.

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.