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Journal Club Fall 2012

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Amruta Mhashilkar

on 18 October 2012

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Transcript of Journal Club Fall 2012

Protective immune responses to biolistic DNA vaccination of Brugia malayi abundant larval transcript-2 photo credit Nasa / Goddard Space Flight Center / Reto Stöckli Lymphatic filariasis is an infectious parasitic disease affecting over 120 million people around the world leading to severe impairment, loss of productivity and social stigma
Chemotherapy and vector control are the methods employed for control of this disease
These control methods have limited utility because of high drug and pesticide resistance, high cost of the chemoprophylactic programs and inaccessibility to endemic areas Why the choice of DNA vaccine? Bm-ALT-2 plasmid was constructed by inserting Bm-ALT-2 gene into pVR1020 vector and was PCR amplified
Green Florescent Protein was constructed at the EcoR1 and XhoI site of the BmALT-2pVAX plasmid
Empty pVAX vector served as controls
The plasmids were purified using endotoxin free plasmid extraction kit. The endotoxin level were undetectable and determined by ToxinSensor Chromogenic LAL Endotoxin Assay kit Twist 1 S.K. Joseph et al. Amruta Mhashilkar Overview A vaccine can prove a better strategy to eliminate filariasis
There is no prophylactic or therapeutic vaccine currently available to control the disease
Previous studies showed that vaccination with recombinant paramyosin could offer partial protection to larval challenge
Vaccine studies using BmVAL-1, heat shock protein 70, myosin, and 1-type IV collagen have been under research as potential vaccine target
Of the recombinant vaccine candidates, ALT (Abundant Larval Transcript) family of proteins appeared to elicit significantly high protective responses against B. malayi The Bm-ALT-2 protein is synthesized in the infective stages of the parasite and it is believed to have vital role in transmission and infectivity of the parasite
The sera from endemic normal population without the disease or asymptomatic cases, have antibodies against Bm-ALT-2 protein
The ALT protein and the gene has no homologues with any other non-filarial organism
This makes it an ideal candidate for vaccine development against lymphatic filariasis Why Bm-ALT-2 as a candidate? DNA vaccines are relatively simple to formulate and inexpensive.
The protein response can be expressed in the skin
The drawback of the DNA vaccine is that the immune response generated does not increase with the increase in dosage
The route of administration largely influences the immune response The intradermal injections is most common route to administer DNA vaccines
In this paper the authors have used a gene gun to administer DNA vaccine and evaluate the protective immune responses generated Methods Plasmids T7 expression vector pRSET B was used to express recombinant Bm-ALT-2 as histidine tagged protein. Plasmid was transformed to Escherichia coli for expression.
Endotoxin levels were below 1EU/mg
Ten Balb/c mice were injected subcutaneously with 4 doses of 15 ug of rBmALT-2 in Imject® alum and at 2 weeks interval and serum was collected for antibodies. Recombinant BmALT-2 and anti BmALT-2 antibodies Gene Gun cartridges A Helios Gene Gun was used for bolistic vaccination sonicated in water bath for 20sec BmALT-2pVAX or BmALT-2GFPpVAX or empty pVAX were added
co-precipitated by addition of 100ul of 1M CaCl2 100ul of 0.05 M spermidine added to 1um gold microcarriers The precipitate was resuspended in Poly Vinyl Pyrrolidine (PVP) in ethanol and loaded onto pre-dried tefzel tubing
Optimization showed that 0.1mg/ml PVP allowed maximum binding of gold carrying DNA 0.5 inch cartridge were prepared with a DNA loading ratio (DLR) of 2 and a microcarrier loading quantity (MLQ) of 0.5 mg, yielding a cartridge having 1 ug plasmid in 0.5 mg gold carrier Bm ALT-2GFP expression in COS-1 cells and in the skin of mice COS-1 cells bombarded with BmALT-2GFPpVAX DNA using the Gene Gun at 50, 100 and 150 psi helium pressures
After 48 hours, GFP is visualized under florescent microscope. Lipofectamine transfected cells were used as control
5ug BmALT-2GFPpVAX plasmid was bombarded into shaved abdominal skin of mouse using gene gun at 200, 300 and 400 psi helium pressures
The skin was excised and serial sections were prepared in a cryotome. GFP was observed under florescent microscope
BmALT-2 protein expression was evaluated in the skin homogenates by immunoblot using polyclonal mouse antiBmALT-2 antibodies Dose standardization studies using BmALT-2 DNA in mice 5 mice each were vaccinated using gene gun as above with 2.5, 5, 7.5 and 15 ug (2×7.5 ug) BmALT-2pVAX plasmid.
All animals were boosted same DNA after 28 days and blood was collected by retro-orbital bleed
Presence of antibodies was detected using indirect ELISA Biolistic gene gun vaccination protocol in mice 5 groups of 5 mice each were made
Group1: 5ug of pVAX via gene gun
Group 2: 5ug BmALT-2pVAX via gene gun
Group 3: 5ug of BmALT-2pVAX intradermally
Group 4: 100 ug of BmALT-2pVAX intradermally
Group 5: 100 ug pVAX vector intradermally Parasite challenge studies using micropore chambers 20 infective L3s suspended in RPMI1640 medium supplemented with 15% heat inactivated fetal calf serum (FCS), were inoculated into the chambers and implanted into the peritoneum of the mice
After 48 hours, the chambers were removed and the examined microscopically for cell adherence and death of parasites In vitro antibody-dependent cellular cytotoxicity (ADCC) assay ADCC assay was performed to verify whether the antiBmALT-2 antibodies from vaccinated animals can participate in the killing of B. malayi L3s
Larval viablility was noted Immune correlation of protection Indirect ELISA was performed on the sera to analyze Anti-BmALT-2 IgG antibody response
Levels of cytokines from spleenocytes were measured using BD Cytometric Bead Array Mouse Th1/Th2/Th17 Cytokine kit Results BmALT-2 is expressed in COS-1 cells and the mouse skin after gene gun delivery GFP protein was present in COS-1 cells after 48 hours after gene gun delivery with 150psi and 100psi in Fig B & C
No florescence was seen in empty pVAX transfection in Fig D
Positive control is lipofectamine transfection in Fig A
Increase in GFP was observed in skin of mice with increasing helium pressure -Fig F-H
No GFP was observed in control mice -Fig E 384 bp BmALT-2 mRNA transcripts were present in the skin site and skin draining lymph nodes of mice vaccinated with BmALT-2pVAX using gene gun Fig2A
Immunoblot analysis showed a 25kDa band comparable to rBmALT-2 was seen in skin homogenates Fig 2B
Lane 1 and 2 represent skin and node from BmALT-2pVAX vaccinated micewhereas 3 and 4 represent control empty pVAX vaccinated mice Gene gun vaccination induced antibody production IgG levels remained significantly high (P < 0.01) at all dilutions in mice vaccinated with 5, 7.5 and 15 μg of DNA vaccine against the control given 5 μg of pVAX vaccination using gene gun.
5ug of DNA was sufficient to produce significant titres, it was used as a standard in the in-vivo studies Gene gun vaccination offered protection against infective larvae About 25% of the L3 were dead in the micropore chamber implanted in the peritoneal cavity with 5ug of BmALT-2pVAX using gene gun
Intradermal vaccination with the same dose killed only 3% L3. 100ug of i.d gave a comparable result with 5ug of gene gun
Controls had all the larvae alive and no cell adherence
ADCC assay perform to show anti-ALT-2 antibodies generated following vaccination by gene gun and i.d.
It showed 34% protection from 5ug gene gun vaccinated mice which was comparable to 38% prtection using 100ug i.d vaccine Gene gun vaccination induced high IgG1 responses Gene gun delivery method induced IgG1 and IgG2a anti-BmALT-2 antibody response
The intradermal method induced Ig2a and Ig2b anti-BmALT-2 antibody response
IgG3 antibody were not significantly elevated in any group Gene gun vaccination promoted Th2 cytokine responses in splenocytes IL-4 were produced in spleen cells from vaccinated mice with 5ug ALT-2pVAX gene gun vaccine where as 100mg i.d. vaccine secreted IFN-gamma, IL-12 and TNF-alpha
IL-6, IL-10 and IL-17A remained unchanged in the groups
Th2 response was predominated following a gene gun vaccination whereas Th1 response in intradermal route Discussion The study compared protective responses elicited by conventional intradermal route vs the gene gun method
The results show that the gene gun method was more efficient than the intradermal method and require 20-fold less antigen to evoke the immune response
Neutralization of Bm-ALT-2 of the larva by the antibody mediated immune response leads to death of the larva making the BmALT-2 vaccine an ideal candidate for the study Early experiments in the COS-1 cells and mouse skin confirmed that the BmALT-2 protein can be expressed in mammalian cells.
Gene gun delivery system is believed to transfect the antigen directly into the Antigen Presenting Cells (APC) and transport the antigen to the draining lymph nodes
BmALT-2 mRNA transcripts and protein were demonstrated from the lymphnodes after 48 hours of vaccination via the gene gun The anti-BmALT-2 IgG antibodes generated by vaccination through gene gun were predominantly IgG1 isotype whereas intradermal method generated IgG2a isotype
B cell differentiation and IgG isotype regulation in mice is under the control of T cell products IL-4 and IL12 which directs IgG subclass switch of IgG1 and IgG2a respectively
This was shown from the mice spleen cells that secreted IL-4 in response to rBmALT-2 promoting IgG1 class switch following a gene gun delivery
Intradermal vaccinated mice spleen cells secreted IFN-gamma, IL-12 and TNF-alpha that promoted IgG2a class switch
Th2 cytokine response were generated following gene gun delivery method
The vaccine is equally efficient as compared to Th1 response generated by intradermal delivery of BmALT-2 DNA 25% protection was observed using 5ug of BmALT-2 DNA using gene gun approach where around 26% protection was observed in 100ug of BmALT-2 DNA using intradermal method
Provided only about 26% protection with the DNA vaccination whereas other DNA recombinant vaccines provide around 30-37% protection
But, when combined with DNA prime protein boost vaccination regime intradermal method, the response increased to 64% Summary The authors compared gene gun delivery method with intradermal method and found that the gene gun method provides significant protection against BmALT-2 specific to Brugia malayi
It requires 20-fold less DNA
It is minimally non-invasive, needleless approach and easy to administer
Can be of a potential use for control of lymphatic filariasis in the field Thank you!!!
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