Vaccine materials overview
The RaDVaC intranasal vaccine is very simple and consists of five ingredients: epitope peptide(s), chitosan, sodium triphosphate, sodium chloride, and water.
The vaccine delivery vehicle is chitosan. Chitosan is a deacetylated form of chitin, which is found in mushrooms and the shells of crustaceans such as shrimp and crabs (seafood allergies are not allergies to chitin). Deacetylation produces free amino groups, and a high density of positive charges on chitosan. A solution of chitosan is mixed with negatively charged triphosphate (sodium triphosphate, STP a.k.a. tripolyphosphate, TPP) under rapid stirring or vortexing. Ionically bonded chitosan gel nanoparticles form spontaneously upon mixing with TPP, including when mixed together with peptide. The method we use is adapted from published methods for creating peptide-loaded nanoparticles. You will find extensive documentation on all materials and methods in our White Paper, but for easier access and convenience, key materials and equipment are listed below.
Acquiring vaccine ingredients
All materials and ingredients are commercially available. Again, the vaccine is very simple and consists of five ingredients: peptide(s), chitosan, sodium triphosphate, sodium chloride, and water.
- Epitope/antigen peptides. Multiple vendor options are available for synthetic peptides. These peptides are small synthetically produced portions of viral sequences.
- Chitosan. Multiple molecular weights and suppliers have been tested and published. Acceptable deacetylation range = 75% to 90%.
- Sodium triphosphate, Na5P3O10. Multiple suppliers.
- Sodium chloride (NaCl). 5M stock solution.
- dH2O, deionized water (widely available).
- pH strips ranging at least from 2.0 to 9.0; NaOH and acetic acid to adjust pH.
Preferred equipment and materials
Certain equipment is required for efficient vaccine production and administration. Vaccine can be produced without specialized laboratory equipment but the process is more laborious and the results are likely to be more variable.
- Pipettes: 1000 microliter, 200 microliter
- Sterile filtered pipette tips: 1000 microliter, 200 microliter
- Scale: gram scale accurate to 0.1 grams, or jeweler’s scale for very small amounts
- Clean spatula for dry reagents
- Small beakers for mixing and stirring. 10 ml to 25 ml beaker for stirring
- Small bottles for stock solutions; 100 ml to 250 ml
- 15 ml conical tubes
- 50 ml conical tubes
- 1.5 to 2 ml microcentrifuge tubes
- Stirring apparatus: magnetic stir plate and small stir bar to fit in beaker
- OPTIONAL but recommended: pH strips; range at least 4.0 to 9.0, but ideally from 0 to 10.0
- Nasal spray apparatus. These are available through multiple vendors. We selected small nasal spray bottles (about 5 milliliter) with a pump-top apparatus. A tube stem about 4 cm long and approximately 1 mm inner diameter extends from the bottom of the apparatus, which can be placed into the bottom of a 2-ml tube containing vaccine. Our spray apparatus delivers about 100 microliters per pump.
Peptides for Gen 9 vaccine; About 4 to 6 micrograms each peptide per dose of vaccine. The list below features virus protein ID, starting amino acid or range , and amino acid sequences for each peptide. Substitutions are shown in lower case. The purpose of most substitutions is increased solubility, amino acid coupling efficiency for synthesis, or disulfide circularization for conformational stability and resistance to proteolysis. Listed optional peptides have been used in prior vaccine generations, and might be included to increase certain properties of a vaccine, such as additional CD4 immunogenicity or HLA allelic coverage.
Complete epitope peptide sequences are shown; a number sign (#) at the end of a sequence means a peptide has been circularized by disulfide bond formation. A letter T at the end of the sequence name indicates a T-cell epitope. Most peptides are combination HLA class II (CD4) and B-cell epitopes, or HLA class II (CD4) and HLA class I (CD8) epitopes. HLA allele restrictions are shown for each HLA class I (CD8) epitope.
- Spike 802-823cir, FSQcLPDPSKPSKRSFcEDLLF# (combo CD4/B-cell)
- Spike 660, YECDIPIGAGICASYQTQTNSPRRA# (combo CD4/B-cell)
- Spike 1145, LDSFKEELDKYFKNHTSP (combo CD4/B-cell)
- Nuc 100-120, KMKDLSPRWYFYYLGTGPEAG (combo CD4/CD8; B*07, Cw*07)
- Nuc 321-345, GMEVTPSGTWLTYTGAIKLDDKDPN (combo CD4/CD8; B*40)
- Nuc 359T, AYKTFPPTEPK (combo CD4/CD8; A*03, A*11)
- Orf1 1636T, HTTDPSFLGRY (CD8; A*01, A*11, A*26, A*33)
- Orf8 117T, DFLEYHDVRVVL (CD8; A*02, A*24, B*39, B*40)
- Orf1 5470T, KLSYGIATVR (CD8; A*02, A*03, A*33)
Gen 9 Optional peptides
- Spike 21, RTQLPPAYTNSFTRGVYYPDK (combo CD4/B-cell)
- Nuc 263T, ATKAYNVTQAFGRRG (combo CD4/CD8; B*15, B*58)
- Nsp7 21T, RVESSSKLWAQCVQLH (combo CD4/CD8; A*02, B*58)
- Spike 442-460, DSKVGGNYNYLYRLFRKSN (combo CD4/B-cell)
- Mem 172T, TSRTLSYYKLGASQRVA (Immunodominant HLA class II/CD4)
If you have all materials and are ready to take the next step, click here to access Protocols for making and taking the vaccine