Transcript TAT-mediated gp96 transduction to APCs enhances gp96

Enhanced Heat Shock Protein
gp96-induced antiviral and
antitumor T cell responses
By: Brandon Davis
Role of Vaccine Adjuvants
 Immunological adjuvant are any substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used in
combination with specific vaccine antigens by augmenting the activities of dendritic cells (DCs), lymphocytes, and macrophages by
mimicking a natural infection
 Adjuvants are primarily pathogen-associated molecular pattern (PAMP) constructs, which include:





Liposomes
Lipopolysaccharide (LPS)
Molecular cages for antigen
Components of bacterial cell walls
Nucleic acids



double-stranded RNA (dsRNA)
single-stranded DNA (ssDNA)
unmethylated CpG dinucleotide-containing DNA
 Adjuvant may also take the form of damage-associated molecular patterns (DAMPs)

Heat shock proteins
 Innate and adaptive immunity have evolved to recognize these antigenic moieties through pattern recognition receptor (PRR)



Toll-like receptors (TLRs)
C-type Lectin receptors (CLRs)
NOD-like receptors (NLRs)
 The efficient binding of ligand - either in the form of adjuvant used in vaccinations or in the form of invasive moieties during times of
natural infection - to the TLR triggers the key molecular events that ultimately lead to innate immune responses and the development of
antigen-specific acquired immunity
Heat Shock Protein 96 (HSP gp96)




glycoprotein of 96 kDa
localized to the endoplasmic reticulum, but can be present at the cell surface
GP96 acts as a molecular chaperone for protein maturation
Expressed constitutively in virtually all cell types, but is upregulated by
endoplasmic reticulum stress
 GP96 is known as a potent viral adjuvant as well as a stress-inducible tumor
rejection antigen GP96
 GP96 activates dendritic cells via the Toll-like receptor-2 and Toll-like receptor-4
 GP96 is expressed in a variety of tumors and its glycosylation pattern differs
considerably from that observed in normal tissues
Gp96 Tumor Rejection
http://www.nature.com/nri/journal/v3/n5/images/nri1089-f3.jpg
Hepatitis C Virus (HCV)
 RNA virus family: Flaviviridae
 Positive-sense, single-stranded RNA
 RNA genome of approximately 9,500 nucleotides. The genome
contains highly conserved untranslated regions
 Approximately 50nm in diameter
 Virus envelope derived from virally-encoded glycoproteins (E1 and E2)
inserted into host membrane enclosed in a nucleocapsid
 Structural proteins: Core, E1 and E2, are located in the N-terminal
with the non- structural (NS) proteins (NS2,NS3, NS4A, NS4B, NS5A
and NS5B)
https://gi.jhsps.org/Upload/200710261000_13974_000.jpg
Hepatitis C (Disease)
 Hepatitis is an infectious disease primarily characterized by its ability to cause liver damage
 Cirrhosis
 Hepatocellular carcinoma
 Acute vs Chronic
 80% of those infected with the acute form of the virus develop chronic disease
 Mode of transmission:
 Primarily Blood borne contact
 Blood transfusions
 Poorly sterilized instruments
 Sexual transmission
 150-200 million people infected world wide
http://www.aryagastro.com/images/hep-c.jpg
 There is currently no established vaccine for hepatitis C, however research in this area is ongoing
 Obstacles:
 Polyclonal and multispecific immune response is required to recognize multiple epitopes
 Low efficacy of HCV antigens to stimulate a significant immune response
 Hypothesis: Polytope (PT) DNA vaccine encoding both structural and
non-structural antigens of HCV with multiple HLAs will induce a
greater immune response over conventional vaccines when fused
with the N-terminal domain of gp96 (NT(gp96)).
HCV Vaccine Development Strategy
 Generating a polytope (PT) DNA vaccine that contains B and T cell
epitopes on the N-terminus domain of heat shock protein gp96
(NT(gp96)), which acts as an adjuvant
 PT DNA Encoding:
 4 HCV immunodominant CD8+ cytotoxic T lymphocyte epitopes
 HD-2Dd restricted antigens: Core132-142 and E2405-414
 HLA-A2 restricted antigens: NS31073-1081 and NS5B2727-2735
 1 HCV T Helper CD4+ Epitope
 NS31248-1262
 1 B cell epitope
 E2412-426
 NT(gp96) adjuvant was fused with either the C- or N-Terminal of PT
DNA epitopes to generate the PT-NT(gp96) vaccine particle
Experimental Mouse Model
 CB6F1 mouse line used for in vivo vaccine evaluation
 Cellular and Humoral immune responses against expressed peptides
 Murine cell culture
 HeLa cell monolayer
 Splenocytes
 hepatocytes
Fluorescent microscopy
Cytotoxicity analysis
Conclusions
 The use of multi-epitope DNA vaccines is a promising approach for inducing a safe and
effective immunity against highly variable pathogens like HCV, but their efficacy should
be improved.
 Fusion of HBV-specific antigens to the N-terminal domain of gp96 gene could improve
the potency of HBV DNA vaccines
 PT-NT(gp96) DNA vaccine significantly decreased the percentage of regulatory CD4+ Tb
cells. This effect could also contribute to further enhancement of PT-specific immune
responses
 Increase in the frequency of IFN-γ/TNF-α double producers in mice vaccinated with two
fusion DNAs compared to the PT-immunized group
 PT-NT(gp96) DNA was able to produce a significantly higher amount of IgG2a antibody
compared to PT DNA, preferentially priming Th1-type immune responses.
 N-terminal fusion of NT(gp96) to PT results in a conformational change or steric
hindrance, which reduces the efficacy of the PT DNA vaccine. The position of the fusion
may affect the pattern of ubiquitination of the fusion constructs
Future Studies in PT DNA Vaccine
 Further studies are needed to determine whether differences in
conformation due to differences in the position of the adjuvant in the
polypeptide interactions affect immune response.
 Preclinical studies are required to analyze the CTL immune responses
against HLA-A2-restricted epitopes incorporated in PT in HLA-A2
transgenic mice.
Hepatitis B (Disease)
 Hepatitis is an infectious disease primarily characterized by its ability to cause
liver damage
 Cirrhosis
 Hepatocellular carcinoma
 Acute vs Chronic
 Mode of transmission:
 Primarily Blood borne contact
 Blood transfusions
 Poorly sterilized instruments
 Sexual transmission
 Mother to child vertical transmission
 350 million people world wide are infected with chronic Hbv
 A vaccine is available for Hbv, however, for those who develop a chronic infection
there is no medical cure
Melanoma
 Melanoma is primarily characterized
by cancerous growth in skin
epithelium
 Instigated by intense ultraviolet light
exposure to the skin
 Cancerous growths are triggered by
mutation of skin epithelial cells to rapidly
multiply and form malignant tumors
 These tumors originate in the pigmentproducing melanocytes in the basal layer of
the epidermis
 Malignant Melanoma is the most
dangerous form of skin cancer killing
an estimated 9,000 people in the US
annually
http://www.aimatmelanoma.org/wp-content/uploads/benign-or-malignant.png
TAT-mediated gp96 induced antiviral and
antitumor T cell responses
 Hypothesis: Efficient transduction of gp96 adjuvant complexes, via TAT (transactivator of transcription), into APCs can significantly enhance the outcome and
efficiency of gp96-based immunotherapy
 Experimental design: Generate vaccines potent enough to elicit significant T Cell
responses to both HBV and B16 Melanoma antigen presentation in rodent
models
 Mode of action: Direct antigen internalization into APCs utilizing gp96
recombinant fusion with the cell-penetrating peptide TAT
Gp96 Immunity Initiation (Innate)
 Rodent models and clinical trials have shown that gp96 can act as an
adjuvant to stimulate both T cell and antibody immunity against
viruses and tumors
 Gp96 interaction with the immune system relies on cell surface
receptors of antigen presenting cells (APCs) and lymphocytes via TLRs
 TLR-2, TLR-4 and TLR-9
 APC recognition stimulates the secretion of pro-inflammatory
cytokines activating the innate immune system
 TNF-α, IL-1β, and IL-12
Gp96 Immunity Initiation (Adaptive)
 Gp96 effectively bind antigenic peptides derived from tumor, viral and
intracellular bacterial sources to enhance APC recognition
 Interaction of gp96 with cell surface receptor CD91 or scavenger
receptor-A (SRA) on APCs promotes internalization of the gp96peptide complexes
 Cross-presentation of gp96-antigen complex peptides to MHC class I
and II activate peptide-specific cytotoxic T lymphocyte (CTL)-mediated
adaptive immune responses
 Additionally gp96–CD91 binding phosphorylates CD91 triggering a
signaling cascades that activates NFkB which in turn upregulates
cytokine production
Gp96 Immunity Initiation Challenges
 Receptor associated protein (RAP) is able to bind to CD91 with high
affinity, competitively blocking its association with gp96
 Specific tumor cells types have the ability to constitutively produce
RAP and render gp96 ineffectual as a vaccine adjuvant
Potential Solutions
 HIV-1 derived trans-activator of transcription (TAT) protein has shown
itself to be able to mediate protein transduction in both mice and
cultured cells
 TAT fusion proteins have been generated to deliver a wide variety of
size-independent molecules into cells, including peptides, proteins,
antisense oligonucleotides, large iron beads and liposomes
 TAT is able to directly translocate gp96-peptide complexes directly
into APC regardless of RAP cell surface interference
Experimental Constructs
 The various gp96–peptide complexes were prepared by incubating
 native mouse gp96 (mgp96)
 recombinant TAT-gp96 (rTAT-gp96)
 Recombinant gp96 (rgp96) protein
 Three Hbv Kd-restricted epitopes:




HBc87–95 (SYVNTNMGL)
HBs362–371 (WYWGPSLYSI)
HBV pol140–148 (HYFQTRHYL)
One control peptide HBc18–27 (FLPSDFFPSV)
 RAW264.7 cells
 P815 cells
 B16.F10 melanoma cells
HBV Mouse Vaccination Model
 Female HBV transgenic BALB/c and C57BL/6 mice (6–8 weeks old)
 The mice (+/-) for rgp96, rTAT-gp96 or mgp96 as adjuvant were
immunized with HBc87–95, HBs362–371 and HBV pol140–148
peptides at weeks 1, 2 and 4, respectively
 Native mouse gp96 (mgp96) and recombinant TAT-fused albumin
(TAT-BSA) expressed in E. coli were used as positive and negative
controls
 Mice were sacrificed at week 8, and splenocytes were isolated and
collected
T cell activation marker CD69
IFN-γ-secreting CD8+
IFN-γ-secreting CD4+
Conclusions
 TAT PTD dramatically increases the uptake of gp96
 Recombinant TAT-fused gp96 protein has the ability to induce antigen-specific T cell responses,
making rTAT-gp96 a powerful adjuvant for augmenting T cell responses
 Therapeutic effect against HBV by immunization with rTAT-gp96 adjuvant vaccine can significantly
inhibit HBV replication
 rTAT-gp96 induces anti-tumor T cell response - immunization with rTAT-gp96 effectively induced
CTL with high tumor specificity and high cytotoxicity against B16 tumors . This result suggests that
TAT dramatically increases the antitumor T cell activation function of gp96
 Fusion of gp96 with TAT may be able to subjugate the tumor-induced suppression of gp96
internalization, and thus enhance gp96-induced antitumor T cell immune responses and
overcome RAP-mediated blockage of binding of CD91
 These results provide further evidence that the increased internalization of gp96 into APCs is a
powerful strategy to enhance gp96-mediated T cell responses by overcoming the bioavailability
barrier, and could help to optimize the efficiency of gp96-based vaccines against cancer and
infectious diseases
Specific Aim
 Design a immunotherapeutic vaccine utilizing nanoparticles to carry
multiple melanoma tumor epitopes fused to a gp96/Tat construct to
elicit an cytotoxic immune response potent enough to counter tumor
growth and induce regression of malignant melanoma in preclinical
trial transgenic mice.