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Date Created: 10/23/15
MMBB 432532 Fall 2006 Topic 10 Vaccines and Antivirals Chapter 19 I General Considerations regarding the use of antivirals and vaccines A Use of vaccines and antivirals will impose a selection for mutants B There are inherent risks involved C Common sense and social measures are the BEST antiviral defense II Vaccines A Smallpox the big victory This was a serious threat to all humans and was the rst virus eradicated Polio and measles are on the hitlist Host restriction and effective vaccination are a key to success here B Question to ponder Should we destroy the stocks C Vaccine basics l Stimulation of immune memory 7 must stimulate T and B cell production 2 Active injecting a vaccine vs passive injectinginfusing Ab and CTL immunization 3 SAFETY FIRST There are many issues to consider 4 Other considerations tailoring of vaccine for best response generation of mutants costefficacy considerations D Type I Attenuated viruses see Fig 1 1 How to make them 7 grow virus in nonnormal hosts or under nonphysiological conditions 2 They work because they replicate but have reduced ability to spread 3 Problems associated with reversions other potential problems regarding immune responses and contaminants E Type II Killed vaccines 1 Generally whole virus which is inactivated by chemical or physical processing so that it is no longer infectious but IS antigenic 2 Derivation of the yearly In uenza vaccines 7 a complicated process that is NOT foolproof F Type III Subunit vaccines 7 only a subset of the viral proteins or even just small virally derived peptides are used to elicit an immune response 1 Generally structural proteins are used but problems come with incomplete induction of immune response 2 Success story Hepatitis B surface antigen vaccine G Type IV Viral vectors see Fig 2 Can utilize a NONPATHOGENIC virus like vaccinia to deliver a VERY antigenic protein from a PATHOGENIC virus into the host Our example here is using the HA protein of in uenza Delivery of the protein via the vaccinia vector is able to elicit a protective immune response in the mouse tested H Type V DNA vaccines 7 1 These are quotnakedquot DNA injections of very antigenic proteins into the muscle or subcutaneously 2 This works via MHC I presentation of the produced viral antigen Antivirals A A little bit of history starts around 1950s but to present there are only about 30 total antiviral drugs on the market B The path to drug discovery 7 importance of ef cacy delivery ease toxicity 7 SAFETY FIRST C Screens for drugs 1 Mechanism based screens see Fig 3 7 mostly 13911 w39tro based Looking for the ability of a given compound to INHIBIT a particular reaction of a viral protein 2 Cell based screens see Fig 4 7 These tests are in vivo based in tissue culture cells or bacteria or yeast generally A site for viral protein action is generally engineered and you look for the ability of a compound to inhibit the action of the protein Oftentimes the inhibition has consequence for the survival of the cells involved Biological relevance is much higher here 3 High throughput screening 7 we now have the ability to screen thousands of drugs a DAY through the use of combinatorial chemistry 4 Structure based drug design Xray crystallography based Many inhibitors particularly of proteases with active cleavage sites have been derived this way D Some examples of drugs on the market today see Fig 5 and 6 Many different classes of drugs most have very speci c targets The large majority target herpesviruses and HIV In uenza A targeting drugs include amantadine and rimantidine targets M2 protein 1 Many of the antivirals are chain terminators see Fig 5 2 Other targets for antivirals entry proteases nucleic acid synthesis proteinssee Fig 6 3 The problem of RESISTANCE MMBB 432532 Topic 11 Replication and mRNA expression strategies of strand RNA viruses I Some general considerations for ALL RNA Viruses A All use RNA dep RNA polymerase rst ID d in poliovirus B These enzymes are in general PRIMER independent some exceptions are found C Viral RNA synthesis is VERY EFFICIENT D There are accessory proteins to the polymerase they direct pol to correct site facilitate recognition of initiation site stimulate pol activity and act as helicases E There may be host proteins involved both in replication and transcription processes by the polymerase F RNA Viruses MUST keep the level of dsRNA within the cell at a minimum strand and strand Viruses have different methods of achieving this goal II The general schemes for replication and transcription of the different classes of RNA Viruses SEE FIGURE 1 A strand RNA Viruses 7 generally unimolecular in origin Have two different strategies for production of mRNA either they make a single polyprotein that is then cleaved to make different subunits eX picomaviruses or they make subgenomic mRNA fragments eX alpha Viruses like Sindbis Virus Note that protein production is the rst thing that happens here B 7 strand RNA Viruses 7 can be either 39 39 39 or A If 39 39 39 eX VSV they generally use subgenomic fragments for the production of multiple mRNAs If they are segmented eX in uenza each segment is transcribed separately to make a different mRNA Note they generally transcribe make strands of RNA rst before they can replicate C Ambisense and DSRNA genomes each have their own peculiarities which we will go into if we have time III Some speci cs about two particular strand RNA Viruses that illustrate several key points A Poliovirus 1 Poliovirus has a unique structure that has secondary structures in both the 5 and 3 UTR regions that are essential for replication and transcription of RNA SEE FIGURE 2 top This includes a protein called VpG that is attached to the 5 end of the Virus During translation this protein is cleaved from the end SEE FIGURE 2 bottom and the ribosomes utilize the internal ribosome entry sites 2 The polymerase shows template speci city by recognizing speci c structural units within the genomic RNA SEE FIGURE 3 top The polymerase must discriminate between the Viral and cellular RNAs in the cytoplasm as to which is the template 7 polio uses the 5 cloverleaf and the 3 pseudoknot structures Several cellular proteins are also involved 3 Initiation and elongation Via the polymerase DOES require a primer in the case of polio most don t It is the VpG protein SEE FIGURE 3 bottom The VpG protein is added to both the and strands during synthesis 4 mRNAs of MOST RNA Viruses are polyadenylated For RNA Viruses the genome is equivalent to an mRNA so the polyA is encoded at the 3 end for polio it is a string of 62 As For 7 RNA Viruses the 539 end ofthe genome has a string of Us which is copied 5 In poliovirus there is only one mRNA which equals the strand genome so there is not much regulation required for the switch between transcription and replication The switch is primarily regulated by the concentration of the capsid proteins These proteins are made by proteolytic cleavage of the large single precursor protein SEE FIGURE 4 B Sindbis virus 1 This is a unimolecular genome that uses subgenomic mRNA transcription to get different proteins expressed see FIGURE 5 2 Regulation of initiation at the different sites on the genome and for replication vs transcription comes from the speci city of the polymerase see FIGURE 6 Recognition is probably induced by conformational changes due to cleavage reactions MMBB 432532 Virology Handouts for Topics 2 and 3 Topic 2 Methods of Detection A Virus cultivation 7 the advent of tissue culture see FIGURE 1 1 Most tissue culture is performed in either epithelial or endothelial cell monolayers There are primary tissue cells and then there are cell lines which are immortal The cells can be synchronized in different cell cycle phases to see the effects on both the virus and the host N B The evidence for viral growth in culture cytopathic effects see FIGURE 2 7 the virus often causes very distinct phenotypic changes that can easily be viewed under the microscope C The need for animal work still exists for many viruses lack of good model pathogenesis studies important D Detection of viruses 7 there are many methods used to detect virus depending upon how it interacts with the host cell 1 Measurement of infectious units 7 All of these methods involve SERIAL DILUTION of a viral stock and then reinfection onto a new monolayer of cells a Plaque Assays see FIGURE 3 7 Here we are looking for PFU plaque forming units Virus will lyse the original cell and will spread to surrounding cells in the monolayer forming a clearly visible plaque The spread of the virus is inhibited by an agarose overlay b There are variations on this theme depending upon what the virus does to the cell If it does not LYSE the cell we can perform infectious center assays must stain for a viral antigen and if the virus transforms the cells we can use focus forming assays c There is an important concept here known as PARTICLE T0 PFU RATIO ie ofthe stuff that s coming out ofan infected cell how much is INFECTIOUS 2 Measurement of viral particles and components 7 this is not so much to specifically quantitate how much virus is in your stock but to get a feel for relative concentrations ie 7 to compare different samples to one another a Hemagglutination assays 7 see FIGURE 4 7 these are used to get relative concentrations of viral particles in different samples Twofold serial dilutions are performed on stocks which are then mixed with RBCs We look for lattice formation at the highest dilution We can detect speci c viral protein components using speci c antibodies that have been raised against these proteins These antibodies can then be used to track and quantitate proteins via Fquot i immuno uorescence see FIGURE 5 for localization of particular antigens There are now also ways to metabolically label the viral genome or speci c proteins using labeled nucleotide analogs or by engineering virus to express a GFP tagged protein so that we can follow the progress of the virus into the cell see FIGURE 6 Western blot analysis for the analysis of the steady state levels of the proteins see FIGURE 7 c The genome can also be quantitated using either Southern or Northern hybridization with virus speci c probes 3 Measurements of effects on the cells a New techniques like microarray analysis see FIGURE 8 can also now be used to assess the effects ofthe virus on the host cell on a global scale Mock versus infected cells are compared for mRNA expression of host genes Remember though that working with RNA can often be very problematic Therefore to do these microarray analyses we rst convert the mRNAs single stranded RNAs into cDNA copyDNA double stranded DNA copies of those messages through the action of a reverse transcriptase see FIGURE 9 The cDNA copies are much more stable and can then be hybridized to our arrays We can also assess how well the virus will infect and successfully reproduce in a given cell type by performing single step growth curves We infect at a high multiplicity and assay what comes out the other side of the cells at given times post infection Fquot Topic 3 Virus Classi cations A The classical strati cation phylum class order family genus and species For viruses there are 3 orders 56 families see tables at the back of handouts 233 genus and 1550 known species What de nes these subtypes In the classical methodology viruses are generally classi ed according to nature of the genome symmetry of the capsid whether there is an envelope and the dimensions of the particle see FIGURE 10 The Baltimore classi cation system see FIGURE 11 This scheme is based upon the fact that all viruses must direct the synthesis of mRNA which is then translated by the cellular ribosomes to produce viral proteins The scheme therefore is a classi cation according to how the genome of the virus relates to the all importan mRNA moiety
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