Vaccine materials overview
The RaDVaC intranasal vaccine is very simple and consists of a small number of simple ingredients. 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 mildly acidic solution of chitosan is mixed with mildly basic and 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 the following ingredients: peptide(s), chitosan, sodium triphosphate, sodium chloride, dilute acetic acid (vinegar), 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).
- White vinegar or 5% acetic acid
- OPTIONAL: Sodium bicarbonate (NaHCO3, baking soda) 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 2.0 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
Given the growing number of epitope peptides in recent RaDVaC vaccine designs and questions about a preferred selection of “core” peptides, the Gen 9 design is the first to provide a selective list of core peptides and a separate list of optional peptides. This more compact design reduces the initial cost of peptide synthesis, simplifies production, better balances the epitope ratios (B-cell/CD4/CD8), and increases the relative concentration of each peptide in the vaccine. Several peptides in Gen 8 and previously have been retired for the Gen 9 design (for more detail, see the white paper section entitled SUMMARY OF THE PROTECTIVE STRATEGY OF Gen 7+ VACCINE DESIGNS). Two new additions are broad-coverage HLA class I/CD8 T-cell epitope peptides, which were selected based on data from Adaptive Biotechnologies,. The selection of peptides has changed to emphasize our shift in focus to membrane fusion neutralizing B-cell epitopes, rather than ACE2 interactions with RBD, and to further emphasize the critical importance of MHC class I/CD8 T-cell epitopes. We are using optional peptides in our current vaccine compositions because they likely provide potentially beneficial features, but we think data presently available suggest that the core group will provide most of these in a smaller number of peptides. 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. Retired peptides might similarly be considered for inclusion in a vaccine.
In the lists below, primary functions of each peptide are listed next to each peptide name. 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. Two of the optional HLA class I (CD8) peptides provide the additional HLA alleles B15 and B58. Bold and underlined portions show CD8 epitopes. Number signs (#) indicate peptides for disulfide formation. A letter T at the end of the sequence name indicates a T-cell 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; B07, Cw07)
- Nuc 321-345, GMEVTPSGTWLTYTGAIKLDDKDPN (combo CD4/CD8; B40, C15, C17)
- Nuc 359T, AYKTFPPTEPK (combo CD4/CD8; A03, A11)
- Orf1 1636T, HTTDPSFLGRY (CD8; A01, A11, A26, A33)
- Orf8 117T, DFLEYHDVRVVL (CD8; A02, A24, B39, B40, C05)
- Orf1 5471T, KLSYGIATVR (CD8; A02, A03, A33)
Gen 9.1 Optional peptides
- Nuc 263T, ATKAYNVTQAFGRRG (combo CD4/CD8; B15, B58, C02, C03, C12, C14, C16, C17)
- Spike 21, RTQLPPAYTNSFTRGVYYPDK (combo B-cell/CD4/CD8; C02, C03, C12, C16)
- Nsp7 21T, RVESSSKLWAQCVQLH (combo CD4/CD8; A02, B58)
- Spike 442-460, DSKVGGNYNYLYRLFRKSN (combo B-cell/CD4/CD8; A24)
If you have all materials and are ready to take the next step, click here to access Protocols for making and taking the vaccine