Enhanced Heat Shock Protein gp96-induced antiviral andantitumor 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

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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 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

http://www.aryagastro.com/images/hep-c.jpg

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 pigment-producing 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 andantitumor T cell responses

Hypothesis: Efficient transduction of gp96 adjuvant complexes, via TAT (trans-activator 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 gp96-peptide 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.