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Muthumani K.,University of Pennsylvania | Lambert V.M.,University of South Florida | Kawalekar O.,University of Pennsylvania | Heller R.,Old Dominion University | And 3 more authors.
Vaccine | Year: 2010

By virtue of its ability to induce cell cycle arrest and apoptosis, the HIV accessory protein Vpr (viral protein R) has been evaluated by us and others as an anti-proliferative/anti-cancer agent. We have demonstrated that Vpr, when delivered to established experimental B16.F10 melanoma tumors in mice as a DNA expression plasmid through in vivo electroporation, can result in complete regression of the established tumors. We have also demonstrated that Vpr peptides from the carboxy region of the protein can inhibit in vitro growth of both B16.F10 melanoma as well as human HeLa cervical carcinoma tumor cells. These findings, summarized in this report, underscore the potential of Vpr as an anti-cancer agent and warrants, we believe, further experimental as well as clinical evaluation. © 2009 Elsevier Ltd. All rights reserved.


Patel V.,U.S. National Cancer Institute | Valentin A.,U.S. National Cancer Institute | Kulkarni V.,U.S. National Cancer Institute | Rosati M.,U.S. National Cancer Institute | And 12 more authors.
Vaccine | Year: 2010

Naïve Indian rhesus macaques were immunized with a mixture of optimized plasmid DNAs expressing several SIV antigens using in vivo electroporation via the intramuscular route. The animals were monitored for the development of SIV-specific systemic (blood) and mucosal (bronchoalveolar lavage) cellular and humoral immune responses. The immune responses were of great magnitude, broad (Gag, Pol, Nef, Tat and Vif), long-lasting (up to 90 weeks post third vaccination) and were boosted with each subsequent immunization, even after an extended 90-week rest period. The SIV-specific cellular immune responses were consistently more abundant in bronchoalveolar lavage (BAL) than in blood, and were characterized as predominantly effector memory CD4+ and CD8+ T cells in BAL and as both central and effector memory T cells in blood. SIV-specific T cells containing Granzyme B were readily detected in both blood and BAL, suggesting the presence of effector cells with cytolytic potential. DNA vaccination also elicited long-lasting systemic and mucosal humoral immune responses, including the induction of Gag-specific IgA. The combination of optimized DNA vectors and improved intramuscular delivery by in vivo electroporation has the potential to elicit both cellular and humoral responses and dissemination to the periphery, and thus to improve DNA immunization efficacy. © 2010.


Brenndorfer E.D.,Karolinska University Hospital | Brass A.,Karolinska University Hospital | Soderholm J.,Inovio Biomedical Corporation | Soderholm J.,Gothenburg University | And 4 more authors.
Gut | Year: 2012

Background: The non-structural (NS) 3/4A protease/ helicase of the hepatitis C virus is known to modulate signalling pathways in the infected hepatocyte by cleaving CARD adaptor inducing IFNb (Cardif), T-cell protein tyrosine phosphatase (TC-PTP) and TIR domain-containing adaptor inducing IFNb (TRIF), but the effects of NS3/4A in vivo still remain unclear. Aim: To investigate the influence of NS3/4A on intracellular and intercellular signalling in vivo by analysing the intrahepatic inflammatory response of naïve, lipopolysaccharide (LPS)/D-galactosamine (D-galN) or tumour necrosis factor-α (TNFα)/D-galN-treated NS3/4A-transgenic (Tg) mice. Methods: The intrahepatic immunity of nä?ve and LPS/D-galN- or TNFα/D-galN-treated NS3/4A-Tg mice was determined using western blot, ELISA, real-time PCR, flow cytometry and survival monitoring. The injection of cytokines or antibodies against signalling components was performed to analyse the relevance of the respective pathways for the investigated issues. A Tg mouse lineage expressing an inactivated NS3/4A protease (NS3/4A Ile1073Ala- Tgs) was generated to examine if protective effects were NS3/4A protease dependent. Results: The activation of hepatic signal transducer and activator of transcription 1 and 2 was impaired in NS3/4A-Tg mice after treatment with LPS/D-galN or TNFα/D-galN. This was paralleled by a reduction in hepatic interferon-γ (IFNγ). Reconstitution of IFNγ reverted the resistance to LPS/TNFα in NS3/4A-Tg mice. Subsequently, blocking IFNγ in vivo rendered wild-type mice resistant against treatment with LPS/TNFα. A new Tg mouse expressing an inactivated NS3/4A protease had the same phenotype as wild-type mice with respect to hepatic IFNγ levels and sensitivity to LPS/D-galN. Finally, the chemokine profile was altered in the NS3/4A-Tg mice towards an anti-inflammatory state, which helps to explain the altered immune cell subsets and reduction in hepatic IFNγ production. Conclusions: Our data demonstrate that the NS3/4A protease reduces the intrahepatic production of IFNγ and alters TNFα-mediated effects, thereby impairing the hepatic inflammatory response. This may contribute to viral persistence.


Nystrom J.,Karolinska University Hospital | Chen A.,Karolinska University Hospital | Frelin L.,Karolinska University Hospital | Ahlen G.,Karolinska University Hospital | And 8 more authors.
Journal of Infectious Diseases | Year: 2010

Hepatitis B virus core antigen (HBcAg) is thought to be a major target for specific cytotoxic T cells (CTLs) in hepatitis B virus infections. A single dose of hepatitis C virus nonstructural 3/4A DNA (<5 μg) effectively primes functional specific CTLs, independently of CD4+ T helper cells and by different routes of immunization. In contrast, HBcAg-specific CTL priming was T helper cell dependent and highly sensitive to the dose and route of delivery. Although CTL priming was improved 10-fold by codon optimization and in vivo electroporation, low levels of DNA still failed to prime CTLs effectively. Only high doses (≥5μg) of codon-optimized HBcAg delivered by in vivo electroporation primed in vivo lytic and polyfunctional CTLs. The ability of endogenous HBcAg to prime CTLs is surprisingly inefficient and differs from that of nonstructural 3/4A. This has important implications for the design of HBcAg-based therapeutic vaccines in humans. © 2010 by the Infectious Diseases Society of America. All rights reserved.


Morrow M.P.,University of Pennsylvania | Yan J.,University of Pennsylvania | Pankhong P.,University of Pennsylvania | Ferraro B.,University of Pennsylvania | And 4 more authors.
Clinical and Vaccine Immunology | Year: 2010

Adjuvant compounds are usually included in vaccinations in order to bolster total vaccine-specific responses or to tailor an immune response toward a desired endpoint, such as the production of gamma interferon or an increase in antibody titers. While most adjuvants are studied in regard to their impact on vaccine-specific responses during and just after the vaccination period, a detailed analysis of how adjuvants skew the Th1/Th2 axis at more distant time points is not often undertaken. In the current study, we present data that suggests that adjuvants differ in their relative abilities to bolster and skew immune responses in the short term compared with more distant time points. To that end, we have employed interleukin-12 (IL-12) and IL-28B as adjuvants for DNA vaccination of rhesus macaques. While both adjuvants were able to bolster Th1-biased responses, our analysis shows that this skewing was achieved through different mechanisms. Moreover, analysis 3 months after the final immunization revealed the activity of the IL-12 adjuvant to be short lived, while the IL-28B adjuvant continued to exert its influence on the immune system. Taken together, these data suggest that the scientific and medical communities would benefit from a more detailed analysis of adjuvant function, including the determination of long-term influences of administered adjuvants. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Bodles-Brakhop A.M.,Inovio Biomedical Corporation
Methods in molecular biology (Clifton, N.J.) | Year: 2011

For plasmid-mediated gene therapy applications, a major limitation to scale up from rodents to large animals is the low expression level of injected plasmid DNA. The electroporation technique, which results in the passage of foreign material through the cell membrane, is one method that has been shown to be effective at improving local plasmid uptake and consequently, expression levels. Previous studies have determined that optimized electroporation parameters (such as electric field intensity, number of pulses, lag time between plasmid injections and electroporations, and optimal plasmid formulation conditions) are dependent on the target muscle type and individual species. Here, we provide a detailed protocol to optimize conditions for the successful intramuscular electroporation of plasmid DNA to swine, a large animal model. Our results suggest that the technique is safe and effective for veterinary applications. Furthermore, these results provide evidence for the feasibility of upcoming human applications.


Morrow M.P.,University of Pennsylvania | Yan J.,University of Pennsylvania | Pankhong P.,University of Pennsylvania | Shedlock D.J.,University of Pennsylvania | And 6 more authors.
Molecular Therapy | Year: 2010

Type III/ interferons (IFNs) were discovered less than a decade ago and are still in the process of being characterized. Although previous studies have focused on the function of IFNλ-3 (also known as interleukin (IL)-28B) in a small animal model, it is unknown whether these functions would translate to a larger, more relevant model. Thus in the present study, we have used DNA vaccination as a method of studying the influence of IFNλ-3 on adaptive immune responses in rhesus macaques. Results of our study show for the first time that IFNλ-3 has significant influence on antigen-specific CD 8 T-cell function, especially in regards to cytotoxicity. Peripheral CD8 T cells from animals that were administered IFNλ-3 showed substantially increased cytotoxic responses as gauged by CD107a and granzyme B coexpression as well as perforin release. Moreover, CD8 T cells isolated from the mesenteric lymph nodes (MLN) of animals receiving IFNλ-3 loaded significant amounts of granzyme B upon extended antigenic stimulation and induced significantly more granzyme B-mediated cell death of peptide pulsed targets. These data suggest that IFN-λ3 is a potent effector of the immune system with special emphasis on CD8 T-cell killing functions which warrants further study as a possible immunoadjuvant. © The American Society of Gene & Cell Therapy.


PubMed | Inovio Biomedical Corporation
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2011

For plasmid-mediated gene therapy applications, a major limitation to scale up from rodents to large animals is the low expression level of injected plasmid DNA. The electroporation technique, which results in the passage of foreign material through the cell membrane, is one method that has been shown to be effective at improving local plasmid uptake and consequently, expression levels. Previous studies have determined that optimized electroporation parameters (such as electric field intensity, number of pulses, lag time between plasmid injections and electroporations, and optimal plasmid formulation conditions) are dependent on the target muscle type and individual species. Here, we provide a detailed protocol to optimize conditions for the successful intramuscular electroporation of plasmid DNA to swine, a large animal model. Our results suggest that the technique is safe and effective for veterinary applications. Furthermore, these results provide evidence for the feasibility of upcoming human applications.

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