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Transcript of Thesis
GENE GUN AS A TOOL
FOR DNA VACCINATION AIM III
OPTIMIZATION OF DNA DOSAGE
FOR VACCINATION USING
IMMUNE RESPONSES OF
GENE GUN VS INTRADERMAL VACCINATION Need for Vaccine Best Route of Administration Aim I
Construction of pVAX BmALT-2
DNA vaccine Tropical infectious disease
Parasitic disease caused by thread like nematode worms – Brugia malayi, Brugia timori and Wucheraria bancrofti.
Filarial worms lodge in lymphatic system and causes obstruction of lymph fluid that results in the enlargement of entire leg, arm, genitals &breasts. Third larval stage(L3) Second larval stage(L2) Microfilariae (L1) MOSQUITO Pathology of the disease HUMAN Fourth larval stage(L4) Adult worms Elephantiasis Lymphedema Tropical pulmonary
Eosinophilia Hydrocele Elephantiasis Manifestations + + Diethyl Carbamazine (DEC) - Anti-parasitic drug that kills larval form of the parasite. However, infected individuals can still develop lymphoedema.
Ivermectin – Antiparasitic drug
Albendazole – Antihelminthic drug – kills the parasite by diminishing its energy production.
Side effects – dizziness, headache, fever, nausea, vomiting,Temporary hair loss Need to develop an effective vaccine that will provide sustained protection. Lymphatic filariasis is a mosquito borne parasitic infection that is endemic in 83 countries
Most Disabling and Disfiguring disease
#2 infectious disease in the World ( WHO)
Drugs and Treatments - completely not effective. Nanograms
No Cold chain
Both cell mediated & humoral responses
RESPONSE DNA vaccine Recombinant protein Killed Live attenuated + Keratinocytes secreted cytokines upregulate the expression of LC Bone marrow derived LC Activation of APC and presentation of the antigen in lymph nodes Myocytes & low frequency of residential DC Intradermal Intramuscular Deliver many drugs,
Infection Targeting LC,
Limited clinical history Low quantities of DNA,
Cost Traditional method
Risk of infection Microneedles Electroporation Gene gun Injection “Gene gun-based DNA vaccine delivery induces higher level of protective antibody responses in mice compared to intradermal injection of the same plasmid DNA” Tangled bits of dead
worms plug up
the lymphatic system STATEMENT OF PROBLEM Bm ALT 2
(Wuchereria bancrofti Abundant
Larval Transcript -2) L-3 stage L-2 stage Codon optimization
of Wb ALT 2 Codon optimization : Adaptation of codon usage of the transcript gene to the typical codon usage of the host G+C content = 35% to 57% Cloning codon optimized WbALT 2 into pVAX vector BmALT 2 L-3 stage L-2 stage Codon optimization
of BmALT 2 LYMPHATIC FILARIASIS ENDEMIC COUNTRIES AND TERRITORIES DRUGS AND TREATMENT ROUTE OF ADMINISTRATION pVAXBmALT-2 VACCINE CODON OPTIMIZED Vs NON CODON OPTIMIZED CONSTRUCTION OF pVAXBmALT-2 VACCINE SEQUENCING RESULTS ADVANTAGES
Immune responses can be induced with very small quantities of DNA
Highly efficient and simple to use
Overcome physical barriers (eg. Stratum corneum of epidermis)
No pain Delivery into the epidermal layer Gold particles
Coated with DNA - Biolistic particle delivery system
- Method uses microscopic gold particles to deliver the genes
into skin Size of the gold particle PVP MLQ & DLR Helium pressure Gene gun immunization Cartridges for gene gun Coating of Gold-DNA onto Tefzel tube DNA-coated gold pVax WbAlt-2 Production of
antibodies Tolerance Fails to provoke immunity Antigen dose determines the class and strength of immune response Western blot analysis Reverse transcriptase PCR Mouse was sacrificed, abdominal skin section and lymph nodes were collected After 72 hours 2µg of pVAXWbALT-2 was immunized in mice using gene gun Varying DNA doses – 2µg, 4µg, 7µg, 14µg 373 bp 24 kDa Lane 1: cDNA extracted from skin cells of mice bombarded with pVAX control.
Lane 2: cDNA extracted from skin cells of mice bombarded with 2µg of pVAXWbALT-2.
Lane 3: cDNA from lymph node of mice bombarded with 2µg of pVAXWbALT-2 Lane 1: Proteins extracted from skin cells mice bombarded with pVAX vector.
Lane 2: Proteins extracted from skin cells mice bombarded with 2µg pVAXWbALT-2
Lane 3: Proteins from lymph node of mice bombarded with 2µg pVAXWbALT-2
3 2 1 1 2 3 3 Enzyme Linked ImmunoSorbent Assay Blood collection via retro-orbital bleeding from each group 14µg of pVAXWbALT-2 7µg of pVAXWbALT-2 4µg of pVAXWbALT-2 2µg of pVAXWbALT-2 DAY 28 DAY 14 DAY 0 14µg of pVAXWbALT-2 7µg of pVAXWbALT-2 4µg of pVAXWbALT-2 2µg of pVAXWbALT-2 Coated with rBmALT-2 Mice immunized with 4 µg of pVAX WbALT-2 elicited good immune response when compared to the other DNA doses Block Sera Goat antimouse
IgG HRP Absorbance at 405 nm 4µg pVAX 4µg pVAX Intradermal,
Gene gun Group B, C
pVAX control Intradermal 100µg pVAXWbALT-2 4µgpVAX WbALT-2 4µg pVAX WbALT-2 DAY 28 DAY 0 4µgpVAX WbALT-2 4µg pVAX WbALT-2 100µg pVAXWbALT-2 Group D
- Intradermal Group E
- Gene Gun Group A
control Checked for protection using ADCC Checked for antibody titre
using ELISA Sera obtained from all control and test groups Serum Blood collection via retro-orbital bleeding from control and test groups Mice immunized with 4µg of
pVAXALT-2 elicited immune response similar to positive control Coated with rBmALT-2 OPD Goat antimouse
IgG HRP Sera Block Absorbance at 405 nm Live larvae Dead larvae After 48 hours of incubation 10 L3 larvae Sera from immunized mice 2x10^5 peritoneal cell/well from normal mouse Effector cells within the Peritoneal population binds the antibodies through their Fc receptors and are then activated by respective antigens released or present on the L3 larvae. Antibody Dependent Cell mediated Cytotoxicity ADCC RESULTS In conclusion… Immune responses: Gene gun elicits better immune response for 4µg when compared to that of intradermal injection for the same dose in mice
Intradermal Injection – 7.4%
Gene gun – 27.18% Optimization of DNA dosage
In vitro – 4 and 8 µg
In vivo - 4 µg Optimization of gene gun parameters
PVP = 0.1 mg/ml
MLQ,DLR = 1, up to 4
Size = 1µm
Helim pressure – in vitro - 100 psi
- in vivo - 400 psi Intradermal
injection Immune responses and Protection : Intradermal Injection Vs Gene gun Gene gun based DNA vaccination is an excellent mode of delivery and can be developed for Lymphatic filariasis
Further optimization of gene gun parameters and booster doses with multiple vaccine candidates will make this approach a viable vaccination tool
Helps to alleviate the suffering, disability and social stigma undergone by the infected individuals Thesis Advisor: Ramaswamy Kalyanasundaram, Ph.D. This project is supported by the grant NIH RO1 AI064745 awarded to Dr.Ramaswamy.
Committee Members : Guoxing Zheng Ph.D , Rohit Kolhatkar Ph.D, Khalifah Sidik Ph.D.
Thomas Sutliff Ph.D, Penny Billman Ph.D, Gnanasekar Munirathinam Ph.D, Aoushang Chen Ph.D, Neelu Puri Ph.D, John Javaherian
Sujith Kurian Joseph (Post doctoral Associate), Gajalakshmi Dakshinamoorthy Ph.D
Jyotsna Sundar Production of
antibodies Tolerance Fails to provoke immunity Antigen dose determines the class and strength of immune response 3 Optimum pressure needed to lodge the DNA in the epidermal layers of skin Low pressure~ 100 psi DNA-coated gold particles DNA-coated gold particles High pressure~ 800 psi Strips the DNA-coated gold particles from the inner surface of the cartridge and propels
them onto the sample ALT not expressed ALT
expressed pVAX GFPWbALT-2 pVAX 1® WbALT-2 GFP pVAX GFPWbALT-2 For optimization purpose, pVAX 1®
3.0 kb WbALT-2 GFP PCMV Processed into
8µm sections using cryostat Frozen in OCT compound Frozen in OCT compound Frozen in OCT compound After 72 hours After 72 hours After 72 hours 200 psi 400 psi 300 psi Optimum helium pressure for in-vivo studies was determined to be 400 psi 400psi 300psi 200psi Normal GENE GUN CARTRIDGE PREPARATION OPTIMIZATION OF SIZE OF GOLD PARTICLES OPTIMIZATION OF HELIUM PRESSURE IN VIVO SKIN SECTIONS SHOWING GFP Why should we optimize DNA dosage? OPTIMIZATION OF DNA DOSE IN-VIVO Reverse transcriptase PCR 373 bp Expression of ALT protein 24 kDa To analyze the dose response relationship........ OPD Vaccination trials Antibody titre Antibody Dependent Cell Cytotoxicity