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Search Results: 1 - 10 of 50828 matches for " Niranjan Y. Sardesai "
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Heterosubtypic Protection against Pathogenic Human and Avian Influenza Viruses via In Vivo Electroporation of Synthetic Consensus DNA Antigens
Dominick J. Laddy, Jian Yan, Michele Kutzler, Darwyn Kobasa, Gary P. Kobinger, Amir S. Khan, Jack Greenhouse, Niranjan Y. Sardesai, Ruxandra Draghia-Akli, David B. Weiner
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0002517
Abstract: Background The persistent evolution of highly pathogenic avian influenza (HPAI) highlights the need for novel vaccination techniques that can quickly and effectively respond to emerging viral threats. We evaluated the use of optimized consensus influenza antigens to provide broad protection against divergent strains of H5N1 influenza in three animal models of mice, ferrets, and non-human primates. We also evaluated the use of in vivo electroporation to deliver these vaccines to overcome the immunogenicity barrier encountered in larger animal models of vaccination. Methods and Findings Mice, ferrets and non-human primates were immunized with consensus plasmids expressing H5 hemagglutinin (pH5HA), N1 neuraminidase (pN1NA), and nucleoprotein antigen (pNP). Dramatic IFN-γ-based cellular immune responses to both H5 and NP, largely dependent upon CD8+ T cells were seen in mice. Hemaggutination inhibition titers classically associated with protection (>1:40) were seen in all species. Responses in both ferrets and macaques demonstrate the ability of synthetic consensus antigens to induce antibodies capable of inhibiting divergent strains of the H5N1 subtype, and studies in the mouse and ferret demonstrate the ability of synthetic consensus vaccines to induce protection even in the absence of such neutralizing antibodies. After challenge, protection from morbidity and mortality was seen in mice and ferrets, with significant reductions in viral shedding and disease progression seen in vaccinated animals. Conclusions By combining several consensus influenza antigens with in vivo electroporation, we demonstrate that these antigens induce both protective cellular and humoral immune responses in mice, ferrets and non-human primates. We also demonstrate the ability of these antigens to protect from both morbidity and mortality in a ferret model of HPAI, in both the presence and absence of neutralizing antibody, which will be critical in responding to the antigenic drift that will likely occur before these viruses cross the species barrier to humans.
Elucidating the Kinetics of Expression and Immune Cell Infiltration Resulting from Plasmid Gene Delivery Enhanced by Surface Dermal Electroporation
Janess M. Mendoza,Dinah H. Amante,Gleb Kichaev,Christine L. Knott,William B. Kiosses,Trevor R. F. Smith,Niranjan Y. Sardesai,Kate E. Broderick
Vaccines , 2013, DOI: 10.3390/vaccines1030384
Abstract: The skin is an attractive tissue for vaccination in a clinical setting due to the accessibility of the target, the ease of monitoring and most importantly the immune competent nature of the dermal tissue. While skin electroporation offers an exciting and novel future methodology for the delivery of DNA vaccines in the clinic, little is known about the actual mechanism of the approach and the elucidation of the resulting immune responses. To further understand the mechanism of this platform, the expression kinetics and localization of a reporter plasmid delivered via a surface dermal electroporation (SEP) device as well as the effect that this treatment would have on the resident immune cells in that tissue was investigated. Initially a time course (day 0 to day 21) of enhanced gene delivery with electroporation (EP) was performed to observe the localization of green fluorescent protein (GFP) expression and the kinetics of its appearance as well as clearance. Using gross imaging, GFP expression was not detected on the surface of the skin until 8 h post treatment. However, histological analysis by fluorescent microscopy revealed GFP positive cells as early as 1 h after plasmid delivery and electroporation. Peak GFP expression was observed at 24 h and the expression was maintained in skin for up to seven days. Using an antibody specific for a keratinocyte cell surface marker, reporter gene positive keratinocytes in the epidermis were identified. H&E staining of treated skin sections demonstrated an influx of monocytes and granulocytes at the EP site starting at 4 h and persisting up to day 14 post treatment. Immunological staining revealed a significant migration of lymphocytic cells to the EP site, congregating around cells expressing the delivered antigen. In conclusion, this study provides insights into the expression kinetics following EP enhanced DNA delivery targeting the dermal space. These findings may have implications in the future to design efficient DNA vaccination strategies for the clinic.
Enhanced Delivery and Potency of Self-Amplifying mRNA Vaccines by Electroporation in Situ
Yen Cu,Kate E. Broderick,Kaustuv Banerjee,Julie Hickman,Gillis Otten,Susan Barnett,Gleb Kichaev,Niranjan Y. Sardesai,Jeffrey B. Ulmer,Andrew Geall
Vaccines , 2013, DOI: 10.3390/vaccines1030367
Abstract: Nucleic acid-based vaccines such as viral vectors, plasmid DNA (pDNA), and mRNA are being developed as a means to address limitations of both live-attenuated and subunit vaccines. DNA vaccines have been shown to be potent in a wide variety of animal species and several products are now licensed for commercial veterinary but not human use. Electroporation delivery technologies have been shown to improve the generation of T and B cell responses from synthetic DNA vaccines in many animal species and now in humans. However, parallel RNA approaches have lagged due to potential issues of potency and production. Many of the obstacles to mRNA vaccine development have recently been addressed, resulting in a revival in the use of non-amplifying and self-amplifying mRNA for vaccine and gene therapy applications. In this paper, we explore the utility of EP for the in vivo delivery of large, self-amplifying mRNA, as measured by reporter gene expression and immunogenicity of genes encoding HIV envelope protein. These studies demonstrated that EP delivery of self-amplifying mRNA elicited strong and broad immune responses in mice, which were comparable to those induced by EP delivery of pDNA.
Enhanced Efficacy of a Codon-Optimized DNA Vaccine Encoding the Glycoprotein Precursor Gene of Lassa Virus in a Guinea Pig Disease Model When Delivered by Dermal Electroporation
Kathleen A. Cashman,Kate E. Broderick,Eric R. Wilkinson,Carl I. Shaia,Todd M. Bell,Amy C. Shurtleff,Kristin W. Spik,Catherine V. Badger,Mary C. Guttieri,Niranjan Y. Sardesai,Connie S. Schmaljohn
Vaccines , 2013, DOI: 10.3390/vaccines1030262
Abstract: Lassa virus (LASV) causes a severe, often fatal, hemorrhagic fever endemic to West Africa. Presently, there are no FDA-licensed medical countermeasures for this disease. In a pilot study, we constructed a DNA vaccine (pLASV-GPC) that expressed the LASV glycoprotein precursor gene (GPC). This plasmid was used to vaccinate guinea pigs (GPs) using intramuscular electroporation as the delivery platform. Vaccinated GPs were protected from lethal infection (5/6) with LASV compared to the controls. However, vaccinated GPs experienced transient viremia after challenge, although lower than the mock-vaccinated controls. In a follow-on study, we developed a new device that allowed for both the vaccine and electroporation pulse to be delivered to the dermis. We also codon-optimized the GPC sequence of the vaccine to enhance expression in GPs. Together, these innovations resulted in enhanced efficacy of the vaccine. Unlike the pilot study where neutralizing titers were not detected until after virus challenge, modest neutralizing titers were detected in guinea pigs before challenge, with escalating titers detected after challenge. The vaccinated GPs were never ill and were not viremic at any timepoint. The combination of the codon-optimized vaccine and dermal electroporation delivery is a worthy candidate for further development.
Altered Response Hierarchy and Increased T-Cell Breadth upon HIV-1 Conserved Element DNA Vaccination in Macaques
Viraj Kulkarni, Antonio Valentin, Margherita Rosati, Candido Alicea, Ashish K. Singh, Rashmi Jalah, Kate E. Broderick, Niranjan Y. Sardesai, Sylvie Le Gall, Beatriz Mothe, Christian Brander, Morgane Rolland, James I. Mullins, George N. Pavlakis, Barbara K. Felber
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0086254
Abstract: HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24gag elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55gag increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist.
HIV-1 Env DNA Vaccine plus Protein Boost Delivered by EP Expands B- and T-Cell Responses and Neutralizing Phenotype In Vivo
Kar Muthumani, Megan C. Wise, Kate E. Broderick, Natalie Hutnick, Jonathan Goodman, Seleeke Flingai, Jian Yan, Chaoran B. Bian, Janess Mendoza, Colleen Tingey, Christine Wilson, Krzysztof Wojtak, Niranjan Y. Sardesai, David B. Weiner
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0084234
Abstract: An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs), and the elicitation of antibody-dependent cellular cytotoxicity (ADCC). Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP). However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted.
A DNA Vaccine against Chikungunya Virus Is Protective in Mice and Induces Neutralizing Antibodies in Mice and Nonhuman Primates
Karthik Mallilankaraman equal contributor,Devon J. Shedlock equal contributor,Huihui Bao,Omkar U. Kawalekar,Paolo Fagone,Aarthi A. Ramanathan,Bernadette Ferraro,Jennifer Stabenow,Paluru Vijayachari,Senthil G. Sundaram,Nagarajan Muruganandam,Gopalsamy Sarangan,Padma Srikanth,Amir S. Khan,Mark G. Lewis,J. Joseph Kim,Niranjan Y. Sardesai,Karuppiah Muthumani ,David B. Weiner
PLOS Neglected Tropical Diseases , 2011, DOI: 10.1371/journal.pntd.0000928
Abstract: Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus indigenous to tropical Africa and Asia. Acute illness is characterized by fever, arthralgias, conjunctivitis, rash, and sometimes arthritis. Relatively little is known about the antigenic targets for immunity, and no licensed vaccines or therapeutics are currently available for the pathogen. While the Aedes aegypti mosquito is its primary vector, recent evidence suggests that other carriers can transmit CHIKV thus raising concerns about its spread outside of natural endemic areas to new countries including the U.S. and Europe. Considering the potential for pandemic spread, understanding the development of immunity is paramount to the development of effective counter measures against CHIKV. In this study, we isolated a new CHIKV virus from an acutely infected human patient and developed a defined viral challenge stock in mice that allowed us to study viral pathogenesis and develop a viral neutralization assay. We then constructed a synthetic DNA vaccine delivered by in vivo electroporation (EP) that expresses a component of the CHIKV envelope glycoprotein and used this model to evaluate its efficacy. Vaccination induced robust antigen-specific cellular and humoral immune responses, which individually were capable of providing protection against CHIKV challenge in mice. Furthermore, vaccine studies in rhesus macaques demonstrated induction of nAb responses, which mimicked those induced in convalescent human patient sera. These data suggest a protective role for nAb against CHIKV disease and support further study of envelope-based CHIKV DNA vaccines.
DNA and Protein Co-Immunization Improves the Magnitude and Longevity of Humoral Immune Responses in Macaques
Rashmi Jalah, Viraj Kulkarni, Vainav Patel, Margherita Rosati, Candido Alicea, Jenifer Bear, Lei Yu, Yongjun Guan, Xiaoying Shen, Georgia D. Tomaras, Celia LaBranche, David C. Montefiori, Rajasekhar Prattipati, Abraham Pinter, Julian Bess, Jeffrey D. Lifson, Steven G. Reed, Niranjan Y. Sardesai, David J. Venzon, Antonio Valentin, George N. Pavlakis, Barbara K. Felber
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0091550
Abstract: We tested the concept of combining DNA with protein to improve anti-HIV Env systemic and mucosal humoral immune responses. Rhesus macaques were vaccinated with DNA, DNA&protein co-immunization or DNA prime followed by protein boost, and the magnitude and mucosal dissemination of the antibody responses were monitored in both plasma and mucosal secretions. We achieved induction of robust humoral responses by optimized DNA vaccination delivered by in vivo electroporation. These responses were greatly increased upon administration of a protein boost. Importantly, a co-immunization regimen of DNA&protein injected in the same muscle at the same time induced the highest systemic binding and neutralizing antibodies to homologous or heterologous Env as well as the highest Env-specific IgG in saliva. Inclusion of protein in the vaccine resulted in more immunized animals with Env-specific IgG in rectal fluids. Inclusion of DNA in the vaccine significantly increased the longevity of systemic humoral immune responses, whereas protein immunization, either as the only vaccine component or as boost after DNA prime, was followed by a great decline of humoral immune responses overtime. We conclude that DNA&protein co-delivery in a simple vaccine regimen combines the strength of each vaccine component, resulting in improved magnitude, extended longevity and increased mucosal dissemination of the induced antibodies in immunized rhesus macaques.
HIV-1 p24gag Derived Conserved Element DNA Vaccine Increases the Breadth of Immune Response in Mice
Viraj Kulkarni, Margherita Rosati, Antonio Valentin, Brunda Ganneru, Ashish K. Singh, Jian Yan, Morgane Rolland, Candido Alicea, Rachel Kelly Beach, Gen-Mu Zhang, Sylvie Le Gall, Kate E. Broderick, Niranjan Y. Sardesai, David Heckerman, Beatriz Mothe, Christian Brander, David B. Weiner, James I. Mullins, George N. Pavlakis, Barbara K. Felber
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0060245
Abstract: Viral diversity is considered a major impediment to the development of an effective HIV-1 vaccine. Despite this diversity, certain protein segments are nearly invariant across the known HIV-1 Group M sequences. We developed immunogens based on the highly conserved elements from the p24gag region according to two principles: the immunogen must (i) include strictly conserved elements of the virus that cannot mutate readily, and (ii) exclude both HIV regions capable of mutating without limiting virus viability, and also immunodominant epitopes located in variable regions. We engineered two HIV-1 p24gag DNA immunogens that express 7 highly Conserved Elements (CE) of 12–24 amino acids in length and differ by only 1 amino acid in each CE (‘toggle site’), together covering >99% of the HIV-1 Group M sequences. Altering intracellular trafficking of the immunogens changed protein localization, stability, and also the nature of elicited immune responses. Immunization of C57BL/6 mice with p55gag DNA induced poor, CD4+ mediated cellular responses, to only 2 of the 7 CE; in contrast, vaccination with p24CE DNA induced cross-clade reactive, robust T cell responses to 4 of the 7 CE. The responses were multifunctional and composed of both CD4+ and CD8+ T cells with mature cytotoxic phenotype. These findings provide a method to increase immune response to universally conserved Gag epitopes, using the p24CE immunogen. p24CE DNA vaccination induced humoral immune responses similar in magnitude to those induced by p55gag, which recognize the virus encoded p24gag protein. The inclusion of DNA immunogens composed of conserved elements is a promising vaccine strategy to induce broader immunity by CD4+ and CD8+ T cells to additional regions of Gag compared to vaccination with p55gag DNA, achieving maximal cross-clade reactive cellular and humoral responses.
AN EFFICIENT IP TRACEBACK THROUGH PACKET MARKING ALGORITHM
Y.Bhavani,P.Niranjan Reddy
International Journal of Network Security & Its Applications , 2010,
Abstract: Denial-of-service (DoS) attacks pose an increasing threat to today’s Internet. One major difficulty todefend against Distributed Denial-of-service attack is that attackers often use fake, or spoofed IPaddresses as the IP source address. Probabilistic packet marking algorithm (PPM), allows the victim totrace back the appropriate origin of spoofed IP source address to disguise the true origin. In this paperwe propose a technique that efficiently encodes the packets than the Savage probabilistic packet markingalgorithm and reconstruction of the attack graph. This enhances the reliability of the probabilistic packetmarking algorithm.
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