week 12 Flashcards
- Types of virus vectors and applications
- protein expression
- functional rna expression
- gene therapy
- vaccine
- Vector design features
- must remove virulence factors so they dont cause disease
- expression can be chronic (gene therpy) or transient (for example vaccines, when you want to body to have a rapid response) and depends on which viral vector is chosen
- different gene sizes can be inserted in different viruses. herpesvirus cn carry the biggest genes. many rna viruses cant carry long genes.
- also needs to consider if the body produces antibodies against the vecot. produces a lot for adenoviruses because about 40% of the population has neutralizing antibodies from previous infection
- only lentivirus can infect non-dividing cells and is used for terminally differentiated cells
- genes required for replication needs to be in vector. for example LTR in retroviruses. might need a packaging signal
SIN vector
self inhibiting vector. in a retrovirus vector you can replace the U3 so when it undergoes reverse transcription it has no promoter. instead you insert a homologous promoter from ex CMV which stops the uncontrolled integration and expression of the gene as well as the downstream effects that the retropromoter induce on genes that can drive oncogenesis.
issues with retroviral gene transfer vectors
- if the recipient has a retrovirus in its genome that the vector recombines with or if the patient gets infected with HIV, the vector can recombinate and we get an active virus. to evade this, the best way is to split up all the genes from HIV onto different plasmid which makes it more difficult to recombine to yield functioning viruss.
- since integration is random it can promote cancer
- limited tropism. can be solved by using a different envelope protein like the VSV-G protein.
- some of the transcription elements can be unsafe. therefore its good to use the self inhibiting promoter and remove the tat transctiptional trans activator whihc can also have other consequences for the cell.
applications of retroviral/ lentiviral vectors
- gene correction therapy
- cancer therapy: can direct the vector to cancer cells to kill them
- vaccines: transient high level expression, causes strong activation of both arms of the immune system
adenovirus application as vector, main features and limitations
- efficient transduction of mammalian cells, almost 100% becomes transfected
- grows to high titers in suspension cells meaning its easy to manufacture
- can be used for gene correction therapy
- vaccine and cancer therapy
- limitations: preexisting immunity causing reduced capacity to deliver to cells and also stronf t cell response to vector proteins which clears transfected cells and causes only a short term expression.
- wide tissue tropism
- a new version using the cre loxP system that is safer and can insert bigger genes.
generation and features of adeno-accosiated virus vectors (AAV)
- does not require replicatinf cells for gene transfer, so both dividing and non dividing cells
- co transfect with plasmif expressing rep and capsid proteins into the 293 cell line where packaging occurs with the assistence of adenoviral helper proteins into replication defective AAV particles.
- may integrate into host genomic dna which doesnt seem to promote cancer. has a preferred insertion site on chromosome 19, may be used for gene therapy to cause persistent expression
- size limitation
- low titrer if virus and low gene expression
rna replicons
self-replicating RNAs derived from RNA viruses. lack the ability to form complete virus particles and cause infection. repliactino occurs in the cytoplasm and they are capable of high gene expression. can be delivered as naked RNA, naked DNA or virus-like particles. can be generated by deleting the structural proteins from the rna genome.
west nile virus strain kunjin replicons
causes a high level of expression, no integration and no replication. VLPs has a small cloning capacity and there can be some preexisting immunity. replicons can be introduced by making them as cDNA that will generate mRNA in the cell which will start to replicate bc of all the replicative genes int ge genome, forming heterologous gene. can also be introuced through virus like particles that have replication inside of them or through naked RNA usinf the T7 or sp6 promoter from polio which cna be in vitro transcripbed and introduce the rna to cytoplasm.
what is a vaccine?
preparation derived from a pathogen that when administred to the host does not cause disease but indices protective immunity against the pathogen. aims to prime the adaptive immune response to the antigens of the pathogen so that first infection induces a seondary immune response. should be effective, safe, stable and low cost.
maternal IgG
in our early months, up to 6 months of age we are protected by our mothers passively transferred antibodues. this is kind of the same as vaccine
different approaches to vaccine design, what methods are there and from which viruses?
- inactivated virus vaccine: the virus have been neutralized using chemicals so it cannot replicayte but it can present to the immune system. used for hepatitis A, influenza, polio and rabies
- attenuation: still capable of replication but dont induce disease because the virulence genes have been removed. used for measels, mumps, runella, polio, chicjen pox, yellow fever etc. most common method
- “subviral”: virus like particles. for example injecting the structural proteins or make the structural proteins recombinantely: hepatitis B and papilloma
- nucleic acid based, mRNA
how was small pox eradicated?
vaccine was generated in 1790s. there is no secondary host meaning there is no reservoir, the disease is only infectous after inubation period and its easily diagnosed. there is only one serotype and the vaccine is bery effective and cheap.
control of polio
polio is not eradicated on a global stale and remains in endemic only in afganistan and pakistan. there si also sporadic outbreaks in africa, probably form the middle east. there are 2 different types of vaccines which caused an enormous decrease in cases.
poliovirus 2 vaccines
- inactivated poliovirs vaccine, IPV: virus treated with formalin to inactivate it. works in the blood.
- oral poliovirus vaccine, OPV: 3 different strains of poliovirus was attenuated in neurovirulence via passage in monkey cells. works at the mucosal surfaces which is the viruses natural site of infection. is then carried to the lymoth nodes.
the main difference between these 2 is its ability to cause produciton of IgA. since poliovirus route of entry is through the mucosa of GI or rectal, much of the uímmunity of due to iga in the mucosa whihc is provided by the oral vaccine. most of the cases of polio today is its ability to revery back to virulent form in very low frequency.
