[Demelerlab] Discussion this afternoon

[Borries Demeler]

Hello everyone,
for this afternoon's discussion of the lipid nanoparticle samples I would
like for everyone to please review Bao Le's project request before the
meeting:
Thanks, -Borries
Project Description: Study the interaction of charged liposomes and antigen
protein- COVID manuscript.
Project GUID: f3e8913a-34fc-b774-01ae-2a0712ca8e85
Goals: We want to study the potential interaction between 3 types of
liposomes (neutral charged, negatively charged and positively charged) and
the Spike protein antigen (of SAR-CoV2).
In theory, liposome carriers can interact with protein via several
mechanism like electrostatic or van der Waals interactions, but
electrostatic interaction might be the main force. This would depend on the
charges of liposome and protein which in turns depends on the pH of the
buffer.
When testing vaccines made by mixing charged adjuvanted liposomes with
Spike antigen on mice, we observed higher antibody titers against Spike
protein with positively charged liposomes. Therefore, we want to know if
there is a correlation between antibody responses and the adsorption of
antigen protein on liposomes.
If there is interaction/adsorption of antigen onto liposome, we think that
the density of the 'complex' will increase and AUC technique might measure
that and give us an insight of how much antigen protein being adsorbed and
how much antigen exists in free form (non-adsorbed).
Molecules: There are 5 liposome samples:
- 1057-014-01
- 1057-014-02
- 1057-014-03
- 1057-014-04
- 1057-014-06
Liposomes were made from phospholipid (DOPC, DOPC, or EPC 18:1),
cholesterol (or DC-Cholesterol), and loaded with a lipophilic vaccine
adjuvant INI-2002. Total lipid content is 15-16 mM.

The Spike protein (Spike 2P- Florian Krammer) is approximately 140 kDa and
can from trimer. The protein solution was made in PBS buffer.
Purity: n/a
Expense: - Spike protein: 2 x 50 uL/vial, 2 mg/mL
- liposome samples: ~ 0.2 mL each, lipid content 15-16 mM
- The vaccine adjuvant INI-2002 loaded in liposomes can be detected at UV
210 nm but signal is low.
Buffer Components: Liposome buffer: 10 mM phosphate and 150 mM NaCl, pH 6.2
Spike protein buffer: PBS (10 mM phosphate, 140 mM Chloride, 4.5 mM
potassium, 147 mM sodium)
Sample Handling: - The Spike antigen protein should be kept at -80 C.
Non-infectious reagent.
- The liposomes should be kept at 2-8 C, do not freeze!
No toxic effect has been observed when ingesting or injecting these
formulations and protein on lab animals. As a precaution, DO NOT SWALLOW!
Wearing proper PPEs (gloves and googles) when handling these.
AUC Questions: Is there difference in interaction of differing charged
liposomes (neutral, +, -) with the Spike antigen?
We make 3 types of liposome (neutral, +, -), incubate them with Spike
antigen and analyze with AUC.
Experimental Design: - For in vivo study, ~30 uL liposome containing 10 ug
INI-2002 is mixed with 0.5 uL of the Spike antigen stock solution
(containing 1 ug antigen) to make the vaccine.
- Optimally, that would be nice if we can mimic this condition. However, to
make it easy for the measurement, we can adjust the ratio between liposome
and Spike protein to have the optimized condition for AUC measurement.
Notes:
Status: Submitted
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