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Now showing items 1 - 16 of 35

  • Vaccines to prevent genital herpes

    Egan, Kevin   Hook, Lauren M.   LaTourette, Philip   Desmond, Angela   Awasthi, Sita   Friedman, Harvey M.  

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  • Zika Virus: Prospects for the Development of Vaccine and Antiviral Agents

    Awasthi, Sita  

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  • Zika Virus: Prospects for the Development of Vaccine and Antiviral Agents

    Awasthi, Sita  

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  • Successful application of prime and pull strategy for a therapeutic HSV vaccine

    Bernstein, David I.   Cardin, Rhonda D.   Bravo, Fernando J.   Awasthi, Sita   Lu, Peiwen   Pullum, Derek A.   Dixon, David A.   Iwasaki, Akiko   Friedman, Harvey M.  

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  • The Absence of DHHC3 Affects Primary and Latent Herpes Simplex Virus 1 Infection.

    Wang, Shaohui   Mott, Kevin R   Cilluffo, Marianne   Kilpatrick, Casey L   Murakami, Shoko   Ljubimov, Alexander V   Kousoulas, Konstantin G   Awasthi, Sita   Luscher, Bernhard   Ghiasi, Homayon  

    UL20, an essential herpes simplex virus 1 (HSV-1) protein, is involved in cytoplasmic envelopment of virions and virus egress. We reported recently that UL20 can bind to a host protein encoded by the zinc finger DHHC-type containing 3 (ZDHHC3) gene (also known as Golgi-specific DHHC zinc finger protein [GODZ]). Here, we show for the first time that HSV-1 replication is compromised in murine embryonic fibroblasts (MEFs) isolated from GODZ-/- mice. The absence of GODZ resulted in blocking palmitoylation of UL20 and altered localization and expression of UL20 and glycoprotein K (gK); the expression of gB and gC; and the localization and expression of tegument and capsid proteins within HSV-1-infected MEFs. Electron microscopy revealed that the absence of GODZ limited the maturation of virions at multiple steps and affected the localization of virus and endoplasmic reticulum morphology. Virus replication in the eyes of ocularly HSV-1-infected GODZ-/- mice was significantly lower than in HSV-1-infected wild-type (WT) mice. The levels of UL20, gK, and gB transcripts in the corneas of HSV-1-infected GODZ-/- mice on day 5 postinfection were markedly lower than in WT mice, whereas only UL20 transcripts were reduced in trigeminal ganglia (TG). In addition, HSV-1-infected GODZ-/- mice showed notably lower levels of corneal scarring, and HSV-1 latency reactivation was also reduced. Thus, normal HSV-1 infectivity and viral pathogenesis are critically dependent on GODZ-mediated palmitoylation of viral UL20.IMPORTANCE HSV-1 infection is widespread. Ocular infection can cause corneal blindness; however, approximately 70 to 90% of American adults exposed to the virus show no clinical symptoms. In this study, we show for the first time that the absence of a zinc finger protein called GODZ affects primary and latent infection, as well as reactivation, in ocularly infected mice. The reduced virus infectivity is due to the absence of the GODZ interaction with HSV-1 UL20. These results strongly suggest that binding of UL20 to GODZ promotes virus infectivity in vitro and viral pathogenesis in vivo. Copyright =C2=A9 2018 American Society for Microbiology.
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  • Taking aim at novel vaccines market

    Awasthi, Sita  

    The World Vaccine Congress Washington 2009 was held in Chantilly, VA USA April 20-23(rd). The Vaccine congress attracted over 400 participants from across the world, including leading vaccine manufacturers, biotech companies, governmental agencies, NGOs, research and academic institutes, venture capital and legal firms, and contract service and equipment manufacturers. The speakers covered a wide range of topics, including the role of government and regulatory agencies, funding availability, research and development, manufacturing, packaging and post-vaccine evaluations. Past vaccine development efforts have historically focused on infectious diseases. With advancements in the field of immunology, molecular biology and vaccinology, the vaccine field has begun moving in new directions. The "Taking aim at novel vaccines market" session chaired by Dr. Una Ryan, Chief Executive Officer of Waltham Technologies, focused on traditional approaches to novel targets (nosocomial infections), novel approaches to traditional targets (flu and rabies), novel approaches to novel targets (Type 1 diabetes, multiple sclerosis and smoking) and vaccines for developing markets (TB, malaria, rabies). The importance of collaboration among academic institutions, industries and philanthropic foundations for developing markets was also emphasized.
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  • HERPES SIMPLEX VIRUS COMBINED SUBUNIT VACCINES AND METHODS OF USE THEREOF

    This invention provides vaccines comprising two or more recombinant Herpes Simplex Virus (HSV) proteins selected from a gD protein, a gC protein and a gE protein; and methods of vaccinating a subject against HSV and treating, impeding, inhibiting, reducing the incidence of, or suppressing an HSV infection or a symptom or manifestation thereof, comprising administration of a vaccine of the present invention.
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  • Next generation of human vaccines: What does the future hold?

    Awasthi, Sita  

    The World Vaccine Congress was held in Arlington, VA April 21st-24th, 2008. Tevi Troy, the deputy secretary of the US Department of Health and Human Services, set the tone of the meeting during his keynote address. He discussed the government's plan to deliver a strategic outlook and follow a road map for vaccine development. He also emphasized the importance of ongoing cooperation between industry and the government's many departments. In an electrifying keynote address Gregory Poland, Professor of Medicine and Infectious Diseases at the Mayo Clinic in Rochester, MN discussed the role of recent advancements in the fields of Immunology, Genetics, Molecular Biology, Bioinformatics and the completion of the Human Genome Project. Poland described the recent emergence of the field of Vaccinomics and laid out his vision for an era of personalized medicine. Next-generation vaccine approaches targeting cervical cancer, meningitis, childhood diarrhea and renal cell carcinoma were presented by leaders in the field. Preclinical and early-stage clinical successes of vaccines against Malaria, TB and Ebola were discussed along with a road map for HIV, TB and Malaria vaccine development. The importance of collaborations among government departments, academic institutions, industries and philanthropic foundations was a common theme stressed throughout the conference.
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  • An HSV-1 gD mutant virus as an entry-impaired live virus vaccine

    Awasthi, Sita   Lubinski, John M.   Eisenberg, Roselyn J.   Cohen, Gary H.   Friedman, Harvey M.  

    HSV-1 glycoprotein D (gD) interacts with HVEM and nectin-1 cell receptors to initiate virus entry. We prepared an HSV-1 strain with mutations in the gD gene at amino acid residues 3 and 38 by changing alanine to cysteine and tyrosine to cysteine, respectively (A3C/Y38C). These mutations were constructed with the intent of evaluating infection in vivo when virus enters by HVEM but not nectin-1 receptors and were based on prior reports demonstrating that purified gDA3C/Y38C protein binds to HVEM but not to nectin-1. While preparing a high-titered purified virus pool, the cysteine mutation at position 38 reverted to tyrosine, which occurred on two separate occasions. The resultant HSV-1 strain, KOS-gDA3C, had a single amino acid mutation at residue 3 and exhibited reduced entry into both HVEM and nectin-1 expressing cells. When tested in the murine flank model, the mutant virus was markedly attenuated for virulence and caused only mild disease, while the parental and rescued viruses produced much more severe disease. Thirty days after KOS-gDA3C infection, mice were challenged with a lethal dose of HSV-1 and were highly resistant to disease. The KOS-gDA3C mutation was stable during 30 passages in vitro and was present in each of 3 isolates obtained from infected mice. Therefore, this gD mutant virus impaired in entry may represent a novel candidate for an attenuated live HSV-1 vaccine. (C) 2008 Elsevier Ltd. All rights reserved.
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  • Smart cup:A minimally-instrumented,smartphone-based point-of-care molecular diagnostic device

    Liao, Shih-Chuan   Peng, Jing   Mauk, Michael G.   Awasthi, Sita   Song, Jinzhao   Friedman, Harvey   Bau, Haim H.   Liu, Changchun  

    Nucleic acid amplification-based diagnostics offer rapid, sensitive, and specific means for detecting and monitoring the progression of infectious diseases. However, this method typically requires extensive sample preparation, expensive instruments, and trained personnel. All of which hinder its use in resource limited settings, where many infectious diseases are endemic. Here, we report on a simple, inexpensive, minimally-instrumented, smart cup platform for rapid, quantitative molecular diagnostics of pathogens at the point of care. Our smart cup takes advantage of water-triggered, exothermic chemical reaction to supply heat for the nucleic acid-based, isothermal amplification. The amplification temperature is regulated with a phase-change material (PCM). The PCM maintains the amplification reactor at a constant temperature, typically, 60-65 degrees C, when ambient temperatures range from 12 to 35 degrees C. To eliminate the need for an optical detector and minimize cost, we use the smartphone's flashlight to excite the fluorescent dye and the phone camera to record real-time fluorescence emission during the amplification process. The smartphone can concurrently monitor multiple amplification reactors and analyze the recorded data. Our smart cup's utility was demonstrated by amplifying and quantifying herpes simplex virus type 2 (HSV-2) with LAMP assay in our custom-made microfluidic diagnostic chip. We have consistently detected as few as 100 copies of HSV-2 viral DNA per sample. Our system does not require any lab facilities and is suitable for use at home, in the field, and in the clinic, as well as in resource-poor settings, where access to sophisticated laboratories is impractical, unaffordable, or nonexistent. (C) 2016 Elsevier B.V. All rights reserved.
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  • Molecular association of herpes simplex virus type 1 glycoprotein E with membrane protein Us9

    Awasthi, Sita   Friedman, Harvey M.  

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  • Trivalent glycoprotein subunit vaccine prevents neonatal herpes simplex virus mortality and morbidity.

    Patel, Chaya D.   Taylor, Sean A.   Mehrbach, Jesse   Awasthi, Sita   Friedman, Harvey M.   Leib, David A.  

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  • An HSV-2 Trivalent Vaccine Is Immunogenic in Rhesus Macaques and Highly Efficacious in Guinea Pigs

    Awasthi, Sita   Hook, Lauren M.   Shaw, Carolyn E.   Pahar, Bapi   Stagray, Jacob A.   Liu, David   Veazey, Ronald S.   Friedman, Harvey M.  

    A genital herpes vaccine is urgently needed to prevent pain and suffering, reduce the incidence of neonatal herpes, and decrease the risk of HIV acquisition and transmission that accompanies genital infection. We evaluated a trivalent HSV-2 subunit antigen vaccine administered with CpG and alum in rhesus macaques and guinea pigs. The vaccine contains glycoproteins C, D and E (gC2, gD2, gE2) to block virus entry by gD2 and immune evasion by gC2 and gE2. In rhesus macaques, the trivalent vaccine induced plasma and mucosa neutralizing antibodies, antibodies that block gC2 and gE2 immune evasion activities, and stimulated CD4 T cell responses. After intravaginal challenge, a self-limited vaginal infection of brief duration was detected by histopathology and immunohistochemistry in naive, but not in trivalent immunized macaques. Vaccine efficacy was evaluated in female guinea pigs. Animals were mock immunized, or immunized with gD2, the trivalent vaccine or the trivalent vaccine followed by a booster dose of gD2 (trivalent + gD2). The trivalent and trivalent + gD2 groups were 97% and 99% efficacious, respectively in preventing genital lesions and both outperformed gD2 alone. As a marker of transmission risk, vaginal swabs were evaluated daily for HSV-2 DNA and replication competent virus between five and seven weeks after challenge. HSV-2 DNA shedding was reduced in all groups compared with mock. Shedding of replication competent virus occurred on fewer days in the trivalent than gD2 immunized animals while the trivalent + gD2 group had no shedding of replication competent virus. Overall, the trivalent group had genital lesions on < 1% days and shedding of replication competent virus on 0.2% days. The vaccine has outstanding potential for prevention of genital herpes in humans.
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  • Protein oxidation biomarkers in hyperoxic lung injury in rats: effects of U-74389

    Awasthi, Sita   et. al.  

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  • Protein oxidation biomarkers in hyperoxic lung injury in rats: Effects of U-74389

    Awasthi, Sita   Gyurasics, Agnes   Knight, Scott A.   Welty, Stephen E.   Smith, Charles V.  

    Hyperoxia, used therapeutically in the treatment of respiratory insufficiencies, can cause lung injury, probably through the actions of reactive oxygen species. The present studies were designed to test the hypothesis that oxidation of specific proteins would provide useful biomarkers of the onset of tissue injury, and thereby provide clues as to the mechanisms responsible. We exposed adult male Sprague-Dawley rats to room air or to greater than 95% O2 for 60 h and examined proteins in pleural effusion and broncho-alveolar lavage (BAL) fluids, and in lung tissue homogenates and subfractions. Oxidation of protein thiols was assessed by derivatization with monobromobimane, separation by electrophoresis, and visualization of the fluorescent thioether derivatives. Derivatization with 2,4-dinitrophenylhydrazine (DNPH), electrophoresis, and western analysis was employed to assess a different class of oxidative modifications, frequently termed 'protein carbonyls'. In addition, we investigated the effects of the 21-aminosteroid U-74389G, 10 mg/kg, given intraperitoneally every 12 h, on biomarkers of protein oxidation and on manifestations of lung injury. Hyperoxia caused lung injury evidenced by pleural effusions, increases in BAL protein concentrations, and pulmonary edema; U-74389G attenuated the first two indices of lung injury, but did not alter edema. Protein thiol status of the fractions studied were not affected notably by hyperoxia, or by the aminosteroid. The formation of DNPH-reactive sites on a limited number of proteins by hyperoxia was observed, and some of these effects were attenuated in the animals given U-74389G. Histological examination of lung tissues showed accumulation of intra-alveolar protein exudates in hyperoxic rats, and a significant attenuation of this effect was observed in the animals treated with U-74389G. In conclusion, studies of shifts in protein thiol status that may be caused by hyperoxia will require increasingly specific methods of analysis, and characterization of the specific DNPH-reactive proteins formed in hyperoxia may provide critical insights into the mechanisms of lung injury. Administration of U-74389G offers some degree of protection against hyperoxia and attenuation of these biomarkers of oxidation, but the precise mechanisms by which this protection is effected will require additional study.
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  • Anterograde Spread of Herpes Simplex Virus Type 1 Requires Glycoprotein E and Glycoprotein I but Not Us9

    McGraw, Helen M.   Awasthi, Sita   Wojcechowskyj, Jason A.   Friedman, Harvey M.  

    Anterograde neuronal spread (i.e., spread from the neuron cell body toward the axon terminus) is a critical component of the alphaherpesvirus life cycle. Three viral proteins, gE, gI, and Us9, have been implicated in alphaherpesvirus anterograde spread in several animal models and neuron culture systems. We sought to better define the roles of gE, gI, and Us9 in herpes simplex virus type 1 (HSV-1) anterograde spread using a compartmentalized primary neuron culture system. We found that no anterograde spread occurred in the absence of gE or gI, indicating that these proteins are essential for HSV-1 anterograde spread. However, we did detect anterograde spread in the absence of Us9 using two independent Us9-deleted viruses. We confirmed the Us9 finding in different murine models of neuronal spread. We examined viral transport into the optic nerve and spread to the brain after retinal infection; the production of zosteriform disease after flank inoculation; and viral spread to the spinal cord after flank inoculation. In all models, anterograde spread occurred in the absence of Us9, although in some cases at reduced levels. This finding contrasts with gE- and gI-deleted viruses, which displayed no anterograde spread in any animal model. Thus, gE and gI are essential for HSV-1 anterograde spread, while Us9 is dispensable.
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