1. MITD/sec: MHC class I signal peptide fragment / secretion signal
(sec)
Development of a novel modified RNA based vaccine
: a proof of concept
Sonia Mohinta1, Michael Goldberg1.
1 Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Harvard Medical School, 450, Brookline Avenue, Boston MA 02215.
CONCLUSION
Fig.1 BMDCs were generated with GMCSF and IL-4 and tranfected on day 6 of culture with
different combinations of modified mRNA as indicated and and SIINFEKL expressing DCs were
evaluated with H2kb and CD11c antibodies by flow cytometry .
1. Showed sustained antigen presentation by
dendritic cells transfected with SIINFEKL
encoded mRNA.
2. SIINFEKL expressing DCs were able to present
antigen to OTI CD8+T cells, leading to clonal
proliferation and effector cytokine secretion in
vitro.
3. SIINFEKL encoded mRNA induce antigen-specific
CD8+T cell proliferation, activation and expansion
in vivo.
ACKNOWLEDGEMENTS
I would like to thank Ellese Carmona and Dr Jianmei Leavenworth of
Department of Cancer Immunology and Virology at Dana-Farber Cancer
Institute for designing the SIIFEKL mRNA and helping with in vivo
injections respectively.
ABSTRACT
Nucleic acid-based vaccines (NAVs) were conceptualized over two
decades ago and since then there have been considerable
improvements in its development and have emerged as a novel and
promising candidate for oncology, autoimmune and infectious
diseases. DNA-based vaccines, though more stable, harbor the risk
of genomic integration and induction anti-DNA autoantibodies. On
the other hand mRNA based vaccines are an attractive alternative as
it contains information relating to both specificity (encoded antigen)
and function (danger signal, TLR7/8 activation), albeit less stable.
We hypothesize that modification of mRNA can increase its stability
and thereby enable prolonged expression of the desired target
epitopes, resulting in improved efficacy of vaccination. Our ultimate
goal is to develop a vaccine that generates both primary and
boosting responses following a single administration. Using
ovalbumin as a model antigen we show that the same antigen-
presenting cell can confer both antigen specificity and adjuvancity to
elicit an immune response circumventing immunological tolerance,
one of the biggest hurdles in vaccine development. We show that
iDCs transfected with mRNA encoding SIINFEKL (dominant CD8+ T
cell epitope in ovalbumin protein), were able to present antigen. iDCs
transfected with linear and circular mRNA significantly enhanced
antigen presentation compared iDCs transfected with linear or
circular mRNA alone. Furthermore, these transfected iDCs were able
to promote clonal expansion and effector function of CD8+ T cells
both in vitro and in vivo. These results are extremely valuable as we
aim is to develop prophylatic and therapeutic mRNA based vaccines
for potentiation of adaptive CD8+ T cell responses in the context of
cancer.
Construction of Linear and Circular RNA encoding the
SIINFEKL peptide
Linear
PolyA
MITD/sec
MITD/sec SIINFEKL
Circular
Removal of 5’ and 3’ exonuclease
targets
Anti-H2Kb IgG1-isotype FMO control
Linear (100%)
Circular (100%)
Linear +Circular
(50%)
H2Kb
CD11c
Combination of linear and circular mRNA enhances presentation of
SIINFEKL
Inclusion of circular mRNA sustains presentation of SIINFEKL
past day 2
Fig.2 BMDCs were generated with GMCSF and IL-4 and tranfected on day 6 of culture with
different combinations of modified mRNA as indicated and and SIINFEKL expressing DCs were
evaluated with H2kb and CD11c antibodies for 24, 48 and 72 hrs by flow cytometry .
Fig.3 BMDCs were generated with GMCSF and IL-4 and tranfected on day 6 of culture with
different combinations of modified mRNA as indicated and co-cultured with OTI CD8+T cells for 3
days. A) Effector cytokine and B) proliferation was measured by indicated antibodies by
intracellular staining and flow cytometry.
IFNγ
CD8
Circular(100%) Linear(100%) Circular (50%)+Linear(50%)
Increased IFNγ produced by
CD8+TCRβ+ T cells that were co-
cultured with DCs transfected
with combined Circular+Linear
mRNA
Gated on CD8+TCRβ+ T cells
IFNγ
KI67
Increased Ki67 expression in
CD8+TCRβ+ T cells co-cultured
with DCs transfected with
combined Circular+Linear mRNA
Gated on CD8+TCR β+ T cells
Circular(100%) Linear(100%) Circular(50%)+Linear(50%)
RESULTS
SIINFEKL encoded mRNA induce antigen-specific CD8+T
cell proliferation, activation and expansion in vivo
Central memory
Effector memory
Fig4. OTI mice were injected i.v. with SIINFEKL encoded mRNA compleaxed with
RNAiMAX and following 3days post injection spleen was harvested to check for
CD8+T cell proliferation A), central memory and effector memory (B,C,D) expansion
with indicated antibodies by flow cytometry .
PolyAMITD/sec SIINFEKL
+
Primary Boost
Ki67
CD8
Control
Linear
RNA- 20µg
CD44
CD62L
Control Linear RNA-
20µg
A) B)
C)
D)
A)
B)