Replacement Polymorphism of DENV E Asn-67 with Lys-67
GCURS Presentation
1. GCURS 2016
“The effect of modified PEG and GP100
on AuNV induced IL-12 production”
Ángel A. Garcés, Emily Reiser Evans, Rebekah Drezek
Drezek Lab
October 22, 2016
2. What is cancer immunotherapy?
• Immune system recognizes foreign entities as harmful
• After recognition, cytotoxic T-cells attack the entity and destroy it
• Cancer cells difficult to recognize as foreign in the body
• Retrain body to recognize cancer tumors and destroy them
• Components of a successful vaccine: antigen and adjuvant
3. Project Introduction
• Developing gold nanoparticle (AuNP) vaccines (AuNV) for cancer
immunotherapy applications in vitro in BMDCs
• Specific antigen overexpressed in mouse melanoma tumors
conjugated to AuNP induces immune response
• Optimize the design of AuNVs for maximum effectiveness in vitro
4. Specific project goals
• To modify the GP100 peptide by replacing the terminal leucine
residue with an aromatic one to induce a strong immune response
• To quantify the immune response via IL-12 p70 ELISA
5. Previous literature
• Yang et. Al (2011): Aromatic amino acids conjugated to the end of
AuNPs found to increase AuNV cellular uptake in vitro
• Xia et. Al (2012): IL-12 crucial to maturation of CD8+ T-cells
• Almeida et. Al (2015): AuNVs developed elicit successful immune
responses in vivo via proof of concept antigen
6. AuNV
CD4+
AuNV activates
APC
CD8+
CD4+ recruits CD8+
cytotoxic T-cells
Melanoma
Tumor
APC
IL-12 cytokine
secretion
IL-12
Figure 1: Overview of AuNV immunology
Antigen presenting cells (APCs), which represent either J774.A macrophages or BMDCs, produce
cytokines to communicate with T-cells to coordinate an immune response. The more cytokines
produced correlates to a stronger immune response.
7. Modifying the GP100 peptide
• Preliminary results: AuNP-PEG-GP100 showed highest induced IL-12 cytokine
production in J774.A macrophages
• GP100 had stronger immune response than other melanoma associated antigens
• Unmodified GP100: KVPRNQDWL
• Replaced end amino acid (“L”) with an aromatic amino acid
Figure 2: Unmodified GP100 peptide structure
The terminal Leucine has been highlighted for emphasis.
10. Results of preliminary ELISA testing
-100
0
100
200
300
400
500
600
700
PBS AuNP-PEG (COOH) AuNP-PEG-GP100
(normal)
AuNP-PEG-GP100
(W)
AuNP-PEG-GP100
(Y)
AuNP-PEG-GP100
(F)
AuNP-PEG-GP100
(-L)
LPS
IL-12concentration(pg/mL)
Preliminary AuNV induced IL-12 production observed in BMDCs in vitro
11. Results of secondary ELISA testing
0
50
100
150
200
250
300
IL-12concentration(pg/mL)
Secondary AuNV induced IL-12 production in vitro in BMDCs
12. ELISA data analysis
• Preliminary/secondary ELISA shows a significant immune response for
PEGylated and GP100 variants
• No discernable advantage to conjugating AuNVs with terminal
aromatic residues; no discernable effect on IL-12 production
• AuNP-PEG (COOH) induced higher IL-12 production than AuNP-mPEG
• Terminal aromatic residues had no discernable effect on IL-12
production in BMDCs
14. Possible advantages to AuNP-PEG (COOH)
• COO- group (at pH 7.4) is more hydrophilic than methyl group
• PEG (COOH) has a net negative charge at physiological pH
• Possibility for nucleophilic substitution to make covalent bonds arises
• Kumar (2012): Covalent coupling to biological molecules
• PEG (COOH) has a COO- similar to a C-terminus on proteins
15. Conclusions
• Terminal aromatic residues on AuNVs do not increase IL-12
production in BMDCs
• Ability of AuNP-PEG (COOH) to covalently interact with cell receptors
increases its applications for treating cancer
16. Future directions
• Further ELISA testing further confirm presence of PEG effects
• Flow cytometry with anti-CD40, anti-CD86, and anti-CD80 cell surface
markers to more accurately quantify immune response (paper?)
Adjuvants stimulate the immune response to respond to the antigen of interest, but don’t provide immunity themselves (Wikipedia). One of adjuvant’s functions is to help present the antigen to the cells of interest, expose the body to the antigen over a long period of time. Basically helps your actual stuff “antigen” get to where it needs to go Aluminum hydroxide is an example of an adjuvant for vaccines.
Antigen: A molecule capable of inducing an immune response on a part of a host organism (Wikipedia boi). It causes the body to produce antibodies against something foregin inside of it. Antibodies match antigens!
Introduce what I do very clearly. I make gold nanovaccines for use in vitro to retrain the natural immune response to recognize cancer tumors (the specific peptide on the thing) and destroy them.
Use mainly as a transition slide. Objectives should be readily obvious but just make sure to emphasize it hard.
Note in the Xia paper that CD8+ T cells are a type of th1 cell, which are the t cells that “attack” stuff in the body!
Don’t forget to mention that we characterized the AuNVs with DLS and UV-Vis spec normally!
BMDCs: Bone marrow derived cells (from femur of mice; dendritic cells)
Mention the 3 day protocol for synthesizing gold nanovaccines very quickly
Note: Mice are B57BL6/J mice (naïve of course)
Media is RPMI-1640; Growth factor is GCM
ELISA is Quantikine IL-12 p70 ELISA
This was the 06192016 ELISA run. Talk about how we initially saw that AuNP-Peg (cooh) was pretty huge. Decided from here to compare to AuNP-mPEG
This is the 2nd “good” ELISA that we did back in September. Provides further proof that carboxylated peg is better than methylated peg for inducing the immune response Exciting stuff
Talk in detail about the differences between AuNP-PEG (COOH) and AuNP-mPEG
No one GP100 variant was significantly better than the others. They are all over the place in terms of trends go
Talk about how that our results on aromaticity that uptake does not necessarily correlate to activation of dendritic cells for the Th1 immune pathway
Main difference between these two forms of PEG is CHARGE DIFFERENCES. Carboxylated PEG is negatively charged while methylated PEG is not charged at all. They both have the same chain length, chemical composition otherwise
Make sure to note that traditionally, PEG is in the mPEG form!
Note that PEG in general helps to “hide” the nanoparticles from getting expelled by the RES (reticuloendothelial system), resists protein absorption
Uptake mechanism isn’t too well known but Liu et al (2012) possibly thinks endocytosis mech bc they center around cytoplasm
So…
According to Kumar (2012); “the free carboxyl group… strongly participates in covalent coupling with various biological molecules by cross-linking to reactive amino acids.”
COO- is similar to proteins which means that protein uptake mechanisms more likely to see it like that?
