SPEC TOP PATHOB SCI
SPEC TOP PATHOB SCI PBS 7003
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This 18 page Class Notes was uploaded by Ms. Josiane Braun on Tuesday October 13, 2015. The Class Notes belongs to PBS 7003 at Louisiana State University taught by Staff in Fall. Since its upload, it has received 28 views. For similar materials see /class/222503/pbs-7003-louisiana-state-university in Pathology at Louisiana State University.
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Date Created: 10/13/15
Rational for use of viral vectors Desirable ro erties of viral vectors Host response to virus infections Examples of viral vectors Edward Jenner s experiment 1776 to com bat smallpox with live cowpox virus Vaccinia virus closely related to cowpox virus mu later used Crossprotection with nonpathogenic human viruses Jenner accurately predicted that annhilation of small pox must be the final result of vaccination The Cuw Pux The Wandeme E efts at New Innmlatinn Nonpathogenic Elicit appropriate protective immune response IoIerate foreign gene Insertions Stable genome Limited host range Relatively simple and cost effective to produce Intracellular expression processing and correct presentation to the immune system Vaccines contain only the necessary antigens in a heterologous background Potential for incorporating multiple antigens in a single vector Hinges m V Potential for recombination to generate virulent henotype Instability of the inserted sequences Need to have sufficient information about the correct antigen as well as the appropriate vector Presence of prior immunity to the viral vector Antigen WV Innate Immune System Acquired Immune System Immunological Memory The Inn mum The innate defense system comprises of IFNa p 7 TNFa IL6 IL12 etc APCs produce large amounts of ctyokines and also trigger adaptive Tcell mediated immunity perform cytolysis opsonization activate of inflammation solubilization of immune complexes recognize missing self or altered self and kill virusinfected cells B cells bone marrowderived lymphocytes make antibodies that bind to foreign molecules Cellm ec Immature T cells fferentiate to Thelper Th and cytotoxic T lymphocytes CTL that are responsible for elimination ofinfected cells Came I113 05 a Activation of intracellular sensors dsRNA produced by DNA and RNA viruses Activation of APCs Increased processing and presentation of both virus epitopes and heterologous antigens Activation of adaptive immune response leading to immunological memory Binding and adsorption 2 Penetration and uncoating 3 Macromolecular synthesis Transcription Translation Replication 4 Virus assembly 5 Budding and release Goals should be to Reduce virulence Reliance on functional immune system for V restri n Restrict spread to other hosts Genetic sta y of WV lmmunogenicity of the encoded antigen Poxvirus vectors Adenovirus vectors AdenoAssociated virus vectors Herpesvirus vectors PositiveStrand Viruses NegativeStrand Viruses Viruses contain linear doublestranded DNA of approximately 200000 bp Replication occurs exclusively in the cytoplasm of infected cells Poxviruses are thermostable suitable for storage Can accommodate at least 25 kbp of foreign DNA Restricted host range makes these viruses ideal forVW F Replicationdeficient Poxviruses in humans Modlf39ed Vaccinia Wrus Ankara MVA Canarypoxvirus Fowlpoxviruses and attenuated vaccine strains Replicationcompetent viruses in avian cells t ave incomplete viral life cycle in human or primate cells Early example of success of poxvirus vector Recombinant vaccinia virus Copenhagen strain with rabies virus glycoprotein Replicationdeficient poxviruses are safe in humans Several recombinants encoding SN and HIV1 glycoproteins have been generated SN and HlV1specific immune response in nonhuman primates HIV1 specific immune response in humans have been disappointin Moderate neutralizing antibodies Moderate CTL response Synthesis of proteins in cell culture Protection against experimental infections Tumor immunization minute Veterinary vacclne trials Human vaccine trials Determination of targets of humoral and cell medlated immune response E mm A pimmmilyom A w Ger reunion mm anviruslransler H 39quot quot9 39 S Recomhlnatlon veclnr TKVL alumni Thy Adenuvirus Adenovirus as m name Slum nun 6mm Enhyllrnuqh can rec lnr and mum mum VTX Adiquot evilIs 39 actors unmemelnuen nmses Genuine Is linear Ilnuhlerslramletl 35733 km nun Wmely used m exnenmmlal anathele Remmaummmuaan veclnrs HIIJII callac y muncaum cmlllmlenl veclnrs smuwem Ad qmm ml Earl Generation Adenoviral Vector replication defective Latest Generation Adenoviral Vector Gutless Helperdependent Minimal Ad Replicationcompetent recombinant Ad expressing HIV1 gp160 Nonreplicating Ad virus expressing HIV1 gp160 Immunological Impediments Host Immune Response potent and consms nf bath x uglng 39neunlnr response and h mubodyrpmduung humnml msnons ynnn ymphncy39 the h cellular respnnse mulls m 51ng nfvnnuy mfecled cells by c A a GIL al esp the gmmnrm nfanllbudles to am my subsequem mfeccozls plesms a major nu n m nslng ademwlml mm m human gene xmpy smce must fth lnnnnn nnnnlnnnn nu hm annlznnns m adennvimses lnnnm nun Insults n zdenovml pmenn and will pm 939 Adeuovilal Vectors szmble futures can bnnmmrncum safety lad nrassncmnnn mu nn gem v n n a and a y lnrlllplllnlrl smlnlny and hlgll um uflecnmlllunnf vclnrs abuny m lnfcd n broad range clme Iypes induzlmg 1min lng cells Clancy of cellular uptake large ulscn capanlly up m 37 kbp Lmle nsk of madam chm mnsomal lntcglatlon Helperdependent Adenovinlses gnntmkd by epiaclng all arpans afthe nan genes ElA andElE u mnnmmmz cclor Cunullllllg m E null an In pl holl fecm e nl rrphcatmn drpmds nn helper fllnnluns ptovldcd m an Eldclclc sdcna lml wch can bc pmpagalcll and ampk cd to Ingh lers using pl39essed cell llnn an lnfen calls in viva and express the mgcnc a lug c pmssinn lasts rm only 510 days dun m an mnnnnn response Herpes viruses Alphaherpes viruses HSV VZV Betaherpes viruses CMV Gammaherpesviruses EBV Genomes linear dsDNA range in size from 130 kbp to 3oo kbp Replicationcom petent and replicationdefective amplicons vectors Recombinants generated through homologous recombination Macaques immunized with the recombinants Challenged with SleacZSB 27 of the vaccinated ani als were protected 57 showed a clear reduction of viral load Major impediments for HSV based vectors HSV reactivation from latency may boost specific 39 mune response Preexisting immunity against HSV in human population may limit its use in gene therapy Enveloped singlestranded positivestrand RNA viruses Advantages Two types of vectors ll Alphavirus IMDNEStr ctnraall ml Alphavlrus mb39rLTS39Ii39Tl39 t E ll ml Alphavirus mt m c39tll Attenuated VEE str nhased vaccine vector expressing gag region of HIV1 VEE repliconhased vectors expressing SIV gag an env SFV replicons expressing HIV1 and SIN gp160 VSV as V rccine Nectar L3 J M EE Mononegavirales Rhahdovirus family Genome 11 Kb enomic RNA SU of N and smaller uanti es ofL and P M imal infectious unit 9 so Consists of 23 nucleotides u 339AUACU polyadenylation and transcription termination El SACA intergenic dinucleotide u UUGUCNNUAGS39 start complimentryto beginning of each mRNA Sufficient to express GOI at VSV noncoding 3 end Singlestranded negativestrand RNA genomes of 1115 kb Prototypes Replication in the cytoplasm No recombination and no integration Modular arrangement of genes V Rhabdovu us SV Vectors Reverse genetics allows recovery of infectious viruses with altered genomes Modular organization of the viral genome can be conviniently manipulated to insert foreign genes 3 VSV can be pseudotyped with a variety of viral and cellular glycoproteins l4 f mbdom us ISV Vectors Foreign genes are stably expressed over multiple assag s High levels of expression lntranasal immunization is very ef caceous Immunity to VSV G protein lowers ef ciency of booster inoculatio Very cytolytic No evidence oflongterm persistence Potential safety concerns in immunocompromised 39 NSquot Vectors VSV expressing a variety of viral glycoproteins including HIV1 gp160 and Sleac239gag have shown signi cant promIse r Protection against disease but not against infection Indiana El NewJelsey c ChandipuraG developed an AIDS vacclne based on auenuated enes e M vsv vectors are promising cam man AIDS vaccine mals because they prop high 1 ers and can he delivered without Injection Altering the gene order in SV genome results in marked attenuation of VSV virulence Stable expression of foreign genes Replicates in cytoplasm Unable to undergo natural recombinationre assortment and integration Can be easily attenuated AGchange order of enes Strong cellular and antibody responses Protection in presence of maternal antibodies Multiple foreign inserts up to 42 Kb Can incorporate foreign membrane proteins up to 30 of G protein Noninvasive vaccination l6 J m exam 11ng umw Libya Ezwswtem 527 21 j as quotJim M2 2233391393 nun rEnresenlinu vsv uennme nvsvrxuz Scrategy for recovery or recomblnant vsv mmmm I mm axweungi mu m Knew Wm Knew Med Knew nnshda mm mummy 1 m w DIS N 5w DIS F m DIS l vsvr XNZ mumm nuslrutl Reverse genetics has allowed the production of recombinant viruses that can efficiently express foreign genes Fundamental knowledge of viral genetics coupled with virushost interactions and host immunological responses are needed to design an effective VWstrategy 18
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