173 the effects of the calreticulin on the immune response induced by DNA vaccine containing the classical swine fever virus e2 gene in mouse model

173 The Effects of the Calreticulin on the Immune Response Induced by DNA Vaccine Containing the Classical Swine Fever Virus E2 Gene in Mouse Model Molecular Therapy Volume 18, Supplement 1, May 2010[.]

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INFECTIOUS DISEASES AND VACCINES: VACCINE

RELATED GENE TRANSFER RESEARCH

measles, mumps and rubella and has an excellent safety record after

millions of doses administered in children On the other hand, MV is

an attractive vector platform for expression of foreign antigens and

vaccine development Here we present characterization and immune

response to MV strains encoding HP-NAP antigen

Results: HP-NAP from two different H pylori strains was cloned

into the MV Edmoston vector platform (NAP-26695 and

MV-NAP-43504) in position upstream of N gene To facilitate production

and extracellular secretion of the protein, the NAP gene was inserted

in the human lambda light immunoglobulin chain replacing a large

part of variable domain We generated MV strains expressing two

different secretory NAP forms: MV-lambda-NAP (encoding the full

length constant lambda domain) and MV-s-NAP (encoding only the

N-terminus of immunoglobulin molecule with the leader peptide)

Both vectors expressed high levels of the secretory NAP antigen (>

4µg/106 infected cells) Growth kinetics data showed that the NAP

insert did not interfere and did not inhibit viral replication Secretion

of biologically active NAP by MV-s-NAP infected cells triggered

signi cant Th1 cytokine and IL-8 production in THP-1 monocytic

cells A singe i.p injection of NAP-expressing strains induced a

robust humoral and cellular response against MV in Ifnarko-CD46Ge

transgenic mice There was no signi cant difference in MV-speci c

antibody response between mice immunized with NAP-expressing

MV strains and those injected with control MV-GFP (MV expressing

green  uorescent protein) The anti-measles immunity was

long-lasting and after 9 months the serum neutralizing titer was still

above level considered protective for humans (>1:120 PNT50) The

animals immunized with MV-lambda-NAP and MV-s-NAP developed

stronger anti-NAP response compared to MV-NAP injected groups

The NAP-speci c antibody titer reached the peak (>1:10,000 in

ELISA) 2-4 weeks post vaccination In addition, puri ed HP-NAP

antigen stimulated IFN-γ expression in lymphocytes from immune

animals indicating that NAP-encoding MV vectors induced also

NAP-speci c cell-mediated immunity

Conclusions: Our data demonstrated that MV is an excellent

platform for expression of bacterial antigens and development of

vaccines against H pylori Expression of HP-NAP does not affect

virus replication and induction of protective anti-measles immunity

In contrast to puri ed H pylori antigen formulated vaccines, MVs

encoding secretory NAP induced strong humoral and cellular immune

response * This work was supported by Atwater grant, P50CA116201

and Paul Leibson Memorial Fund

171 Generation of Chimeric GB Virus B Clones

Encoding the Hepatitis C Virus E1E2p7 and

coreE1E2p7 toward the Infected Marmoset Models

Tingting Li,1 Lifang Shuai,1 Zixuan Chen,1 Anqi Wang,1 Wenjing

Wang,1 Chengyao Li.1

1 School of Biotechnology, Southern Medical University,

Guangzhou, Guangdong, China.

The development of novel therapy and vaccination for hepatitis C

virus (HCV) has been hampered by a lack of a small primate model

GB virus B (GBV-B) is closely related to HCV that causes acute or

chronic hepatitis in marmosets, and thus, is an attractive surrogate

model for HCV Even more attractive, for mostly mimicking HCV

infection in marmoset models, is the idea of constructing chimeric

viruses by replacing GBV-B elements with its HCV counterpart of

interest Two infectious GBV-B chimeric genomes, HCV E1E2p7/

GBV-B and HCV coreE1E2p7/GBV-B, containing the coding region

of E1 E2 p7 proteins and core E1 E2 p7 proteins were constructed,

respectively Overlapping PCR and restriction-enzyme digestion

methods were used to generate the chimeric plasmids and then RNA

transcripts were synthesized in vitro The chimera may prove useful

for establishing HCV infected marmoset models and determining

HCV epitopes and immune responses in vivo

172 Characterization of Immune Response

in Mice Induced by Recombinant Vaccinia Virus (Tiantan) Based Multivalent H5N1 Avian In uenza Vaccines

Wen Wang, Yao Deng, Wenjie Tan, Hong Chen, Li Ruan, Yuelong Shu

National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.

To develop an effective and broad immune protective H5N1 vaccine, We  rst developed two recombinant vaccinia(Tiantan strain) virus(rTTV) based H5N1 vaccines, which consisted of bicistron expressing the hemagglutinin(HA) and matrix protein 2(M2),or bicistron expressing the neuraminidase(NA) and matrix protein 1(M1) The expression of H5N1 protein in rTTVs was con rmed

We immunized the Balb/C mice twice with two kind of dose (104 pfu,

107 pfu) using different combination Subsequently, we assessed the humoral and cellular immune response in vaccinated mice Our data showed that rTTV-based H5N1 vaccine induced rapidly robust HA- and NA-speci c antibody level and IFNγ secreting form cell(SFC) with either single dose of 107 pfu or twice dose of 104 pfu or 107 pfu

We also detected signi cant neutralizing antibody and matrix-speci c immune response In addition, we found that immunization with two kind of rTTV-based H5N1 vaccines induced much high level

of matrix 2-speci c antibody than that with single of rTTV-based H5N1 vaccine In conclusion, rTTV-based H5N1 vaccines elicit board array of immunity and our study offers a promising alternative H5N1 prevaccine candidates with favorable potential to prevent various H5N1 pandemic

173 The Effects of the Calreticulin on the Immune Response Induced by DNA Vaccine Containing the Classical Swine Fever Virus E2 Gene in Mouse Model

Ming Kun Hsieh, Chia Chin You, Chienjin Huang

Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan.

The structural glycoprotein E2 of the Classical swine fever virus (CSFV) can stimulate the production of neutralizing antibody which can protect pigs against CSF Previous reports showed that DNA vaccine containing the E2 gene of CSFV was able to provide protective immunity Calreticulin (CRT) has demonstrated the ability

to enhance MHC class I presentation and elicit antigen-speci c CD8+

T cell immune responses The purpose of this study was to determine whether the mouse CRT gene can enhance the immune response

of DNA vaccine by fusing with the E2 gene of CSFV in mouse model Protein expression of plasmids containing CSFV E2 gene (pcDNA4/E2), mouse CRT gene (pcDNA4/CRT) or both (pcDNA4/

CRT-E2) were con rmed by immuno uorescent assay (IFA) In the trial one, six-week-old BALB/c mice were intramuscularly injected four times with various doses of pcDNA4/E2 or plasmid vector at two-week intervals Mice receiving 100 mg of pcDNA4/E2 showed signi cantly higher (P<0.1) anti-E2 ELISA titers and signi cantly higher (P<0.05) stimulation index (SI) of splenocytes proliferative response with stimulations of Con A or 20 mg of CSFV E2 protein than those of mice receiving plasmid vector only The results of trial one showed that DNA vaccine containing CSFV E2 gene was able

to induce humoral and cellular immune responses in mouse model

In the trial two, mice were vaccinated with vector, pCDNA4/CRT, pcDNA4/E2 or pcDNA4/CRT-E2 three times at two-week intervals

by intramuscularly injection or gene gun Mice receiving pcDNA4/E2 and pcDNA4/CRT-E2 showed signi cantly higher (P<0.05) anti-E2 ELISA titers and signi cantly higher (P<0.05) SI of splenocytes proliferative response with stimulation of 20 mg of CSFV E2 protein than those of mice receiving plasmid vector or pcDNA4/CRT only

CANCER - IMMUNOTHERAPY I

The result of trial two indicated that both DNA vaccine pcDNA4/E2 and pcDNA4/CRT-E2 were also able to induce humoral and cellular immune responses in mouse model However, the mouse CRT fusing

to E2 gene did not enhance the immune responses when compare

to those of mouse receiving pcDNA4/E2, no matter which deliver method, intramuscular injection or gene gun was used

174 Evaluation of the Multi-Electrode Array for

Use in DNA Vaccination Against Bacillus anthracis

Amy L Donate,1 Richard Heller.2

1 Molecular Medicine, University of South Florida, Tampa, FL;

2 Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA.

The Multi-electrode array (MEA) has been developed by our lab to enhance gene expression and decrease discomfort from Electroporation (EP) Our group has demonstrated that as compared

to other non-penetrating skin electrodes the MEA enhances reporter gene expression and decreases muscle twitching The MEA has also been previously evaluated for use in DNA vaccination Our lab has

previously used Hepatitis B as a model We have shown that EP with

the MEA signi cantly increased antibody levels in serum as compared

to injection alone Here we evaluate the MEA for use in DNA

vaccination against a potential bioterrorist agent, Bacillus anthracis

The current available FDA approved vaccine for B anthracis,

Biothrax®, requires several initial intramuscular injections (0, 4, 6, 12, and 18 months) and annual boosters are recommended This method of injecting Biothrax® has been shown to decrease the side effects from vaccination as compared to the original s.c vaccine However, new methods will still need to be developed to alleviate production burdens and make vaccination possible for the general public We propose that electrically mediated DNA vaccination with the MEA will be an

effective mediator of immunity against B anthracis.To determine the

effectiveness of the MEA in DNA vaccination, a plasmid expressing

a protective antigen (PA), a component of the B anthracis toxins,

was injected i.d and immediately EP’ed Various EP conditions were evaluated including  eld strength, number of injections/boosts, and injection volume Total serum antibodies were measured by indirect ELISA at several time-points over 12 weeks Toxin neutralizing

antibodies were also measured as an in vitro model for protection

Our results indicate that speci c antibodies can be generated and are signi cantly increased over injection only Antibodies levels achieved from the MEA are similar to those generated from s.c injection of recombinant PA Toxin neutralizing antibodies can also be generated with Electroporation conditions of 150-175 V/cm Future work will compare immunity to the newer vaccine modality of i.m injection of recombinant PA This work demonstrates the potential for the MEA

to be an effective method of enhancing DNA vaccination

Cancer – Immunotherapy I

175 Enhancing the Priming of Tumor Antigen Speci c T Cell Responses Using Oncolytic Viruses

Phonphimon Wongthida,1 Rosa Diaz,1 Feorillo Galivo,1 Karen Kaluza,1 Jill Thompson,1 Timothy Kottke,1 Richard Vile.1

1 Department of Molecular Medicine, Mayo Clinic, Rochester, MN.

We have reported that direct intratumoral injection of VSV leads

to the generation of low levels of CD8+ T cells speci c for tumor associated antigens (TAA), consistent with the hypothesis that viral-mediated oncolysis of established tumors will release tumor associated antigens in the context of a pro-in ammatory immune

activatory signal (Cancer Res 2007 15:2840) However, the

predominant T cell responses primed in vivo are a) speci c for viral associated antigens and b) non-antigen speci c (Hum.Gene Ther

2009, in press) In contrast, when we engineered the virus to encode

a model TAA, the frequency of TAA-speci c T cells generated by intratumoral oncolytic virotherapy was signi cantly enhanced in the tumor draining lymph node, which also correlated with improved anti tumor therapy These data are consistent with our previous reports that oncolytic viruses, which can both access and induce release

of TAA in the tumor draining lymph nodes, are more effective in

priming therapeutic anti tumor immune responses (Nat Med 2008

14: 37; Clin.Can.Res 2009 15: 4374) These data suggest that the

pathways of antigen presentation associated with delivery by viral gene infection/gene expression leads to improved T cell priming and activation compared to that induced by antigen presentation of the same TAA expressed solely within the tumor Moreover, these data also indicate that increasing the frequency of TAA-speci c T cells

in vivo can enhance immune-mediated tumor clearance Therefore,

to further increase the frequency of circulating TAA-speci c T cells,

we combined oncolytic virotherapy with a TAA encoded by the virus with adoptive transfer of T cells with speci city for the viral encoded TAA Direct intratumoral injection of VSV-ova, combined with adoptive transfer of naive OT-I T cells, led to signi cantly enhanced traf cking of TAA-speci c T cells to tumors, higher levels

of activated TAA-speci c T cells in the lymph nodes and better anti tumor therapy compared to the use of virus or adoptive T cell therapy alone Taken together, our results suggest that it is possible to improve the therapeutic effects of oncolytic virotherapy by manipulating the pathways by which TAA are presented to the immune system In particular, this can be optimized by engineering the virus to present TAA, by enhancing access of these TAA-encoding viruses to the lymph nodes, as opposed to the tumor itself, and by combining these TAA-priming viruses with adoptive transfer of T cells with speci city for the one or more TAA

176 AdCD40L Immunogene Therapy for Bladder Carcinoma – A Phase I/IIa Trial

Per-Uno Malmstrom,1 Angelica S I Loskog,2 Camilla A

Lindqvist,2 Sara M Mangsbo,2 Moa Fransson,2 Alkwin Wanders,3

Truls Gardmark,1 Thomas H Totterman.2

1 Division of Urology, Uppsala University & University Hospital, Uppsala, Sweden; 2 Division of Clinical Immunology, Uppsala University, Uppsala, Sweden; 3 Division of Pathology, Uppsala University & University Hospital, Uppsala, Sweden.

Immunotherapy such as bacillus Calmette-Guerin (BCG) instillations is recommended for high-risk non-muscle invasive bladder cancer but is not effective in more advanced tumors Immunostimulating gene therapy with adenoviral vectors (AdCD40L) expressing CD40L has shown ef cacy in experimental tumor models CD40L is a potent stimulator of systemic immunity and may be effective in both local and invasive disease Patients with invasive bladder cancer scheduled for cystectomy or patients with high-risk super cial tumors were enrolled in a Phase I/IIa trial Patients were treated with three cycles of Clorpactin WCS-90 bladder wash followed by AdCD40L instillation seven days apart Safety, gene transfer, and anti-tumor responses were monitored Eight recruited patients were treated and therapy was well tolerated The main adverse effect was transient local pain during Clorpactin wash No adverse events were considered due to vector therapy Gene transfer was detected in biopsies and the bladders were heavily in ltrated with T-cells The effector marker IFNg increased in the biopsies while levels of systemic T regulatory cells were reduced Histological evaluation indicated that AdCD40L therapy reduced the load of malignant cells In conclusion, local AdCD40L gene therapy was safe, increased immune cell in ltration into the bladder, and should

be further evaluated as single or adjuvant therapy for urothelial malignancies

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