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699. Induction of Protective Immunity Against Human Malaria Using Synthetic DNA in a Chimeric Sporozoite Challenge Model
VACCINES AND IMMUNOTHERAPY 698. Broadly Reactive Adenoviral-Vectored Multivalent Influenza Vaccine Eric A. Weaver.1 Medicine, Division of Infectious Diseases, Mayo Clinic, Rochester, MN.
1
There is a definite need to improve the current influenza vaccine technology. With the exception of the cold-adapted FluMist vaccines there have been no significant improvements. Here we report the use of Adenoviral-vectored anti-influenza vaccines that show protective immunity against all human-associated influenza subtypes. Centralized genes representing H1, H2, H3 and H5 hemagglutinin (HA) genes were computationally derived, codon-optimized and incorporated into replication-defective Adenovirus (Ad) types 4 and 5. Mice were immunized with various doses of Ad4 expressing all 4 HA proteins individually. The mice were boosted with the same doses at 4 weeks and test bleeds were obtained 2 weeks post-boost. The highest dose of vaccine, 1E10 vp/mouse, induced protective HA inhibition titers (HI) against 9 of 11 influenza viruses that represent a wide degree of divergence within the 4 HA subtypes. The lowest dose, 1E7 vp/mouse, induced protective levels of HI titers against 8 of 11 influenza isolates. The immunized mice were challenged 4 weeks post-boost with 100 MLD50 of H1N1, H3N1 and H5N1 mouseadapted influenza viruses. The lowest dose of 1E7 vp/mouse protected 100%, 100% and 40% of H1N1, H3N1, and H5N1 challenged mice, respectively. A dose of 5E7 vp/mouse protected 100% of mice against all three influenza challenges. The highest dose of 1E10 vp/mouse showed absolutely no signs of influenza infection and 100% survival against all influenza challenges. These data indicate that very low levels of Ad vaccines that express centralized HA genes are needed to induce broad levels of immunity against a wide divergence of influenza subtypes. These doses are easily translatable to human vaccine equivalents and may provide the foundation for complete and long-lasting anti-influenza immunity.
699. Induction of Protective Immunity Against Human Malaria Using Synthetic DNA in a Chimeric Sporozoite Challenge Model
Bernadette Ferraro,1 Diego A. Espinosa,2 Rebecca N. Lee,1 Matthew P. Morrow,3 Yan Jian,3 Niranjan Y. Sardesai,3 Fidel Zavala,2 David B. Weiner.1 1 Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelpha, PA; 2Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD; 3Inovio Pharmaceuticals, Blue Bell, PA.
The predominant P. falciparum (Pf) malaria sporozoite coat protein, circumsporozoite protein (CSP), is the target multiple vaccine approaches ,including the most clinically advanced vaccine candidate, RTS,S. Protective efficacy observed by vaccine approaches targeting CSP in animal models and humans is predominantly ascribed to antibody responses directed at the central repeat region. If immune responses to the N and C terminal regions of CSP contribute to vaccine efficacy remains unclear. Here, we report the preclinical assessment of a synthetic, highly optimized plasmid DNA Pf CSP consensus antigen (SynCon CSP), which was designed based on all available full-length Pf CSP sequences. Immune responses conferred by SynConCSP delivered intramuscularly with electroporation (EP) were assessed in Balb/c and C3H mice. In Balb/c mice (n=10), SynConCSP+EP induced IFNγ production as measured by ELISpot (1868 SFU) and CSP titers 15,918 (GMT OD=1). CSP responses were found in both the CD4+ (0.45%) and CD8+ (2.9%) T-cell compartments. CD8+ T cell responses were composed primary of IFNγ+ cells. In C3H mice (n=5), SynConCSP+EP elicited IFNγ production by ELISpot (4577 SFU) and high CSP titers 35,105 (GMT OD=1). CSP responses were found in both the CD4+ (0.31%) and CD8+ S270
(2.0%) T-cell compartments. CD8+ T cell responses were composed primary of IFNγ+ cells. Plasmodium host specificity does not allow for Pf challenges in mouse models. Chimeric Pf-P. berghei (Pb) CSP sporozoite (spz) challenge models, Pb spz that contain the N-terminus (Pf-Pb NT) or C-terminus (Pf-Pb CT) of Pf CSP, were used to evaluate if immune responses induced by SynConCSP+EP could decrease liver-stage parasite burden. MHC I class-restricted epitopes were previously identified in the N- and C- terminus of Pf CSP, but it has not yet been determined if responses directed at these epitopes could confer efficacy in malaria vaccine approaches. SynConCSP+EP immunized Balb/c mice were challenged intravenously with Pf-Pb NT spz (n=10) and C3H mice with Pf-Pb CT spz (n=10) 10 days after the last immunization. Liver parasitemia was determined 40 hours post-challenge by QPCR quantifying Pb spz 18s rRNA. Pb spz levels were significantly lower in mice receiving SynConCSP+EP (4.7 fold) (p=0.007) compared to naïve mice (n=5). CD8+ T cell responses to the N-terminal CD8 epitope (3.52%) were observed by ICS 40 hours post-challenge. Lower parasitemia levels correlated with higher CD8+ T cell responses to the N-terminal epitope (r=-0.674) (p£0.01). In C3H mice challenged with Pf-Pb CT spz, Pb spz levels in the liver were significantly lower in mice immunized with SynConCSP+EP (5.0 fold) (p=0.001) compared to naïve mice (n=5). Robust CD8+ T cell responses to the C-terminal CD8 epitope (0.72%) were observed by ICS 40 hours post-challenge. Lower parasitemia levels correlated with higher CD8+ T cell responses to the C-terminal epitope (r=0.789) (p£0.001).
700. Direct Plasmid Based Generation of In Vivo Protective Monoclonal Antibodies Against Chikungunya Virus
Colleen Tingey,1 Seleeke Flingai,1 Emma Reuschel,1 Gopalsamy Sarangan,2 Padma Srikanth,2 Nagarajan Muruganantham,3 Paluru Vijayachari,3 Kenneth E. Ugen,4 Niranjan Y. Sardesai,5 Joseph J. Kim,5 David B. Weiner,1 Kar Muthumani.1 1 Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA; 2Dept of Microbiology, Sri Ramachandra Medical College & Research Institute, Chennai, Tamil Nadu, India; 3Regional Medical Research Centers, Indian Council of Medical Research, Port Blair, Andaman & Nicobar Islands, India; 4Department of Molecular Medicine, University of South Florida Morsani College of Medicine, Tampa, FL; 5Inovio Pharmaceuticals Inc, Blue Bell, PA. Chikungunya virus (CHIKV) has re-emerged as a serious mosquitoborne alphavirus responsible for several recent epidemics in tropical Africa and Asia. Recent evidence suggests that CHIKV, which is primarily transmitted to human by the Aedes species of mosquitoes, can also be transmitted by other mosquito carriers, raising concern for continued pathogenic spread. Despite the recent resurgence of CHIKV, no vaccine or therapeutic against this virus is currently available. Considering the current potential for a global CHIKV pandemic, a novel development of a CHIKV vaccine or therapy which could generate potent and protective immune responses, is of paramount importance. Previously, it has been shown that monoclonal antibody (mAb) therapy can effectively control CHIKV infection and disease both in vivo and in vitro. We therefore developed a novel DNA plasmid encoding an highly engineered immunoglobulin encoding a CHIKVmAb to directly generate in vivo production of a anti-CHIKV mAb following electroporation (EP) in mice. We demonstrate that the serum of transfected animals exhibited the specific ability to bind to CHIKV envelopes and this serum possessed CHIKV-neutralizing activity. This activity appeared within a few hours and lasted for several weeks. This method is distinct from an active antigen based vaccine or passive immunotherapeutic strategy. Importantly, this direct plasmidbased method generated the mAbs in vivo at a concentration and Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
1
There is a definite need to improve the current influenza vaccine technology. With the exception of the cold-adapted FluMist vaccines there have been no significant improvements. Here we report the use of Adenoviral-vectored anti-influenza vaccines that show protective immunity against all human-associated influenza subtypes. Centralized genes representing H1, H2, H3 and H5 hemagglutinin (HA) genes were computationally derived, codon-optimized and incorporated into replication-defective Adenovirus (Ad) types 4 and 5. Mice were immunized with various doses of Ad4 expressing all 4 HA proteins individually. The mice were boosted with the same doses at 4 weeks and test bleeds were obtained 2 weeks post-boost. The highest dose of vaccine, 1E10 vp/mouse, induced protective HA inhibition titers (HI) against 9 of 11 influenza viruses that represent a wide degree of divergence within the 4 HA subtypes. The lowest dose, 1E7 vp/mouse, induced protective levels of HI titers against 8 of 11 influenza isolates. The immunized mice were challenged 4 weeks post-boost with 100 MLD50 of H1N1, H3N1 and H5N1 mouseadapted influenza viruses. The lowest dose of 1E7 vp/mouse protected 100%, 100% and 40% of H1N1, H3N1, and H5N1 challenged mice, respectively. A dose of 5E7 vp/mouse protected 100% of mice against all three influenza challenges. The highest dose of 1E10 vp/mouse showed absolutely no signs of influenza infection and 100% survival against all influenza challenges. These data indicate that very low levels of Ad vaccines that express centralized HA genes are needed to induce broad levels of immunity against a wide divergence of influenza subtypes. These doses are easily translatable to human vaccine equivalents and may provide the foundation for complete and long-lasting anti-influenza immunity.
699. Induction of Protective Immunity Against Human Malaria Using Synthetic DNA in a Chimeric Sporozoite Challenge Model
Bernadette Ferraro,1 Diego A. Espinosa,2 Rebecca N. Lee,1 Matthew P. Morrow,3 Yan Jian,3 Niranjan Y. Sardesai,3 Fidel Zavala,2 David B. Weiner.1 1 Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelpha, PA; 2Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD; 3Inovio Pharmaceuticals, Blue Bell, PA.
The predominant P. falciparum (Pf) malaria sporozoite coat protein, circumsporozoite protein (CSP), is the target multiple vaccine approaches ,including the most clinically advanced vaccine candidate, RTS,S. Protective efficacy observed by vaccine approaches targeting CSP in animal models and humans is predominantly ascribed to antibody responses directed at the central repeat region. If immune responses to the N and C terminal regions of CSP contribute to vaccine efficacy remains unclear. Here, we report the preclinical assessment of a synthetic, highly optimized plasmid DNA Pf CSP consensus antigen (SynCon CSP), which was designed based on all available full-length Pf CSP sequences. Immune responses conferred by SynConCSP delivered intramuscularly with electroporation (EP) were assessed in Balb/c and C3H mice. In Balb/c mice (n=10), SynConCSP+EP induced IFNγ production as measured by ELISpot (1868 SFU) and CSP titers 15,918 (GMT OD=1). CSP responses were found in both the CD4+ (0.45%) and CD8+ (2.9%) T-cell compartments. CD8+ T cell responses were composed primary of IFNγ+ cells. In C3H mice (n=5), SynConCSP+EP elicited IFNγ production by ELISpot (4577 SFU) and high CSP titers 35,105 (GMT OD=1). CSP responses were found in both the CD4+ (0.31%) and CD8+ S270
(2.0%) T-cell compartments. CD8+ T cell responses were composed primary of IFNγ+ cells. Plasmodium host specificity does not allow for Pf challenges in mouse models. Chimeric Pf-P. berghei (Pb) CSP sporozoite (spz) challenge models, Pb spz that contain the N-terminus (Pf-Pb NT) or C-terminus (Pf-Pb CT) of Pf CSP, were used to evaluate if immune responses induced by SynConCSP+EP could decrease liver-stage parasite burden. MHC I class-restricted epitopes were previously identified in the N- and C- terminus of Pf CSP, but it has not yet been determined if responses directed at these epitopes could confer efficacy in malaria vaccine approaches. SynConCSP+EP immunized Balb/c mice were challenged intravenously with Pf-Pb NT spz (n=10) and C3H mice with Pf-Pb CT spz (n=10) 10 days after the last immunization. Liver parasitemia was determined 40 hours post-challenge by QPCR quantifying Pb spz 18s rRNA. Pb spz levels were significantly lower in mice receiving SynConCSP+EP (4.7 fold) (p=0.007) compared to naïve mice (n=5). CD8+ T cell responses to the N-terminal CD8 epitope (3.52%) were observed by ICS 40 hours post-challenge. Lower parasitemia levels correlated with higher CD8+ T cell responses to the N-terminal epitope (r=-0.674) (p£0.01). In C3H mice challenged with Pf-Pb CT spz, Pb spz levels in the liver were significantly lower in mice immunized with SynConCSP+EP (5.0 fold) (p=0.001) compared to naïve mice (n=5). Robust CD8+ T cell responses to the C-terminal CD8 epitope (0.72%) were observed by ICS 40 hours post-challenge. Lower parasitemia levels correlated with higher CD8+ T cell responses to the C-terminal epitope (r=0.789) (p£0.001).
700. Direct Plasmid Based Generation of In Vivo Protective Monoclonal Antibodies Against Chikungunya Virus
Colleen Tingey,1 Seleeke Flingai,1 Emma Reuschel,1 Gopalsamy Sarangan,2 Padma Srikanth,2 Nagarajan Muruganantham,3 Paluru Vijayachari,3 Kenneth E. Ugen,4 Niranjan Y. Sardesai,5 Joseph J. Kim,5 David B. Weiner,1 Kar Muthumani.1 1 Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA; 2Dept of Microbiology, Sri Ramachandra Medical College & Research Institute, Chennai, Tamil Nadu, India; 3Regional Medical Research Centers, Indian Council of Medical Research, Port Blair, Andaman & Nicobar Islands, India; 4Department of Molecular Medicine, University of South Florida Morsani College of Medicine, Tampa, FL; 5Inovio Pharmaceuticals Inc, Blue Bell, PA. Chikungunya virus (CHIKV) has re-emerged as a serious mosquitoborne alphavirus responsible for several recent epidemics in tropical Africa and Asia. Recent evidence suggests that CHIKV, which is primarily transmitted to human by the Aedes species of mosquitoes, can also be transmitted by other mosquito carriers, raising concern for continued pathogenic spread. Despite the recent resurgence of CHIKV, no vaccine or therapeutic against this virus is currently available. Considering the current potential for a global CHIKV pandemic, a novel development of a CHIKV vaccine or therapy which could generate potent and protective immune responses, is of paramount importance. Previously, it has been shown that monoclonal antibody (mAb) therapy can effectively control CHIKV infection and disease both in vivo and in vitro. We therefore developed a novel DNA plasmid encoding an highly engineered immunoglobulin encoding a CHIKVmAb to directly generate in vivo production of a anti-CHIKV mAb following electroporation (EP) in mice. We demonstrate that the serum of transfected animals exhibited the specific ability to bind to CHIKV envelopes and this serum possessed CHIKV-neutralizing activity. This activity appeared within a few hours and lasted for several weeks. This method is distinct from an active antigen based vaccine or passive immunotherapeutic strategy. Importantly, this direct plasmidbased method generated the mAbs in vivo at a concentration and Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy