Treating viruses Flashcards
1
Q
How does pathogenesis affect ease of vaccination?
A
Acute diseases with no animal reservoir are the easiest
Antigenic variation makes it v difficult
Latent infection - immune response isn’t enough to eradicate so difficult
Type of immunity needed (antibody or cell mediated) effects vaccine strategy
2
Q
What are the different types of vaccine?
A
Killed vaccines
Live attenuated vaccines (through multiple passages or related species)
Subunits of virus (purified components e.g. surface antigen or VLPs)
Therapeutic vaccines
3
Q
What are the advantages and disadvantages of killed vaccines?
A
Safe, may be better in immunocompromised patients, no need to cold store.
Boosters and adjuvants required as not immunogenic, need to be completely inactivated
4
Q
What are the advantages and disadvantages of live attenuated vaccines?
A
Broad range of immune responses induced, don’t need boosters (life long immunity), rapid immunity induced
Could revert to virulent phenotype, often have to be stored at -70, often grown in tissue culture so could end up with contamination (e.g. polio with SV40 which could transform cells), could spread from vaccines (increasing immunity? issue with consent), may be problem in immunocompromised
5
Q
What are the scenarios in which therapeutic vaccines may be administered?
A
Post virus exposure (e.g. rabies)
Treatment of established disease
Prevention of recurrence
6
Q
What qualities of smallpox made eradication possible?
A
No animal reservoir
Acute infection
DNA genome (less mutation from vaccine and virus)
Life long immunity from vaccine
Only 1 serotype
Subclinical cases rare so infections can be isolated
7
Q
Describe the 2 polio vaccines?
A
Inactivated vaccine was made first: Salk
Then oral live attenuated vaccine made: Sabin
8
Q
How is the live attenuated vaccine for polio made?
A
Many passages and plaque purifications. Has mutations in the 5’ UTR: 3 mutations in loop 5
9
Q
Why is polio difficult to eradicate?
A
Failure to vaccinate effectively: has to be between 2/3yrs of age or doesn’t take as well and immunological state is poor
Failure of vaccine uptake
Epidemiology of polio - high density populations, less seasonality (affects timing of vaccine) in tropical climates, poor sanitation
Emergence of revertant viruses from vaccine
10
Q
Describe the measles vaccine
A
Protects against measles infection (acute and occasionally persistent in the CNS - serious)
Live attenuated vaccine within the MMR cocktail. Gives life long immunity. Can target vaccine as measles is seasonal - give before peak.
11
Q
What are the issues with the measles vaccine?
A
People being idiots - resurgence of measles whenever herd immunity drops because people believe vaccine side effect propaganda (e.g. USA 1989-91 and Swansea 2013).
12
Q
Describe the hepatitis B vaccine
A
Subunits vaccine - purified surface antigen from envelope (as can’t culture Hep B in vitro so can’t do inactivated or live attenuated). At first had plasma derived vaccines - purify plasma from asymptomatic individuals and heat inactivate.
13
Q
What factors effect the response to the hepatitis B vaccine?
A
Immunological factors (dosage, immunisation schedules, site and rout of vaccination, adjuvant) Host factors (age, weight, immune status (e.g. immunosuppression), gender, genetics)
14
Q
Describe the Rubella vaccine
A
Risk of congenital infection (transmission to foetus). Originally targeted adolescent females, then moved to everyone to induce herd immunity. Attenuated live vaccine in MMR
15
Q
Describe vaccination against rabies
A
Animal reservoir makes eradication v difficult
Can give a post exposure vaccine
Only need to vaccinate those at risk e.g. vets, animal handlers, some lab workers, people who work with bats/other rapid species, travelling to risk areas
16
Q
Describe yellow fever vaccination
A
A flavivirus with an insect vector (so only need to vaccinate in areas where vector is found). Live attenuated vaccine made in hens eggs (was passaged through mice and chickens in the past). Vaccine gives life long protection against all serotypes; neutralising antibody forms. Vaccine is very safe with very rare adverse effects (more in the elderly)
17
Q
Describe the HPV vaccination
A
The first vaccine to be genetically engineered - 2 capsid proteins (L1 and L2) that self assemble if grown in yeast/baculovirus - empty shell
18
Q
Describe the development of the HPV vaccine
A
GSK trial: virus like particles from HPV16 + 18 made in baculovirus and given to HPV seronegative women (placebo and vaccine). Did 3 doses and a 27 month follow up. Led to a fast track licence
Merck vaccine was also developed in a different expression system. Included more HPV strains (6 and 11)
19
Q
Why is the HPV vaccine so effective?
A
In an HPV infection, there are very few virions. In the vaccine there are many empty shells so a stronger immune response occurs
20
Q
What are the issues surrounding HPV vaccination policy?
A
At the moment only vaccinate teenage women, but vaccinating men as well would protect them from male HPV cancers and induce herd immunity. Also worth considering vaccinating older women
Have to consider which vaccine to use and what strains it includes
21
Q
Describe the VZV vaccine
A
Developed as infection in immunosuppressed patients lead to a severe primary infection. Live attenuated vaccine made by passage through human and guinea pig cells
22
Q
Describe the policy of VZV vaccination
A
Policy varies between countries - is compulsory in USA but in UK and Germany use a targeted program as chicken pox isn’t a super bad disease in most people. However, there is a vaccine against shingles available to people over the age of 70 in the UK
23
Q
Describe the flu vaccine
A
Is a killed vaccine - HA and NA, sometimes given with adjuvants. The HA is from different virus strains - H3N2, H1N1, Influenza B HA. Vaccine strains are isolated in hens. Vaccine is updated regularly and tested in ferrets.
24
Q
Describe zoonotic vaccine development in flu
A
Can clone the HA from H5N1 and apply site directed mutagenesis to the HA cleavage site to attenuate. This can be tested for in ferrets and poultry.
25
Describe the past rotavirus vaccine
Mixing segments from human and monkey reassortment virus - including a mixture of VP7 (surface protein). Had side effects of bowel disease so was withdrawn
26
Describe the Merck vaccine against rotavirus
Segmented live viruses, mix of human and bovine. Cocktail of up to 5 strains with various surface proteins (VP4 and VP7)
27
Describe the GSK vaccine against rotavirus
A live attenuated strain from severe child infection with many passages
28
Why are vaccines difficult to make?
```
Antigenic variation
Latency
Congenital disease
Antibody enhancement
Lack of growth in vitro
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29
What strategies can be used to rationally attenuate a virus for a vaccine?
Deletion/mutation of a gene
Modify replication fidelity of RNA polymerases
Codon de-optimise
Add miRNAs to the virus genome (can use to target vaccine to only certain areas of the body)
Zn finger nuclease control of virus production
30
What are the goals of an HSV vaccine?
Herpes simplex virus. A prophylactic vaccine to prevent acute clinical disease, infection (viral replication in genital tract), and reduce establishment of latency. A therapeutic vaccine to reduce clinical recurrence and viral shedding and transmission
31
What is DISC technology?
Disabled infectious singly cycle technology.
32
How has DISC technology been used in trying to creating an HSV vaccine?
Delete the gH gene in the virus that it requires for entry. Grow virus on a complementing cell line that expresses gH in trans. When virus moved to a non-complementing cell line, will have gH so can enter but won't pick it up as it exits the cell so the virus is no longer infectious. Virus can still induce an immune response, so is good for a vaccine
33
What methods have been used in an attempt to create an HSV vaccine?
Live deletion mutants (replication limited)d
Pox virus and adenovirus vectors for glycoproteins
Inactivated whole virus
Inactivated infected cell extracts
Subunit glycoproteins
DNA plasmids
Peptides
34
What makes creation of a vaccine against Dengue virus difficult?
Antibody enhancement: if have antibody against one serotype, if another serotype infects the antibody binds the virus but doesn't neutralise it. The antibody-virus complex then binds to monocytes enabling entry and makes the disease much worse
35
How has a Dengue vaccine attempted to be created?
Make a chimeric virus that expresses all structural genes. Can delete 30 residues at the 3' end to attenuate the virus (combine forms of deletion)
36
What are the issues with RSV vaccination?
Respiratory syncytial virus - need to immunise new borns. Issues with immunological immaturity, maternal antibodies inhibiting an effective response, mucosal immunity to respiratory viruses is short lived, 2 antigenic subgroups, animal models not fully permissive, vaccine for older children is dangerous in infants.
37
What are the prospects for RSV vaccine development?
Rational attenuation with reverse genetics
Subunit vaccine - have one for older children using F and G proteins
DNA vaccines - plasmid expressing F gene is protective in rodents
38
What is the rationale behind modifying replication fidelity of RNA viruses?
If can make the polymerase less error prone, the virus is less likely to revert to a virulent phenotype. Has been attempted with polio - mutate the active site to a less error prone residue
39
What has been the result of looking at codon-deoptimisation in regards to vaccine development?
If can make the virus less fit, might be a safer vaccine - in picornavirus found that codon-deoptimisation of some and all of the virus resulted in less and smaller plaques. Were still some present which is good for a vaccine (need some replication to occur)
40
How could miRNAs be used in polio vaccine development?
Polio is normally a GI infection and not severe - only bad in the CNS. If could engineer a CNS miRNA into the vaccine, the miRNA would result in cleavage of viral RNA in the CNS with no virus produced. Would be good for immunosuppressed individuals
41
How would attenuation of a virus for a vaccine using Zn finger nucleases work?
The nuclease would cleave DNA - operates in pairs by recognising 2 lots of a 9 nucleotide sequence. Engineer the viral genome so it had a Zn finger recognition site and the nuclease. The nuclease could be expressed under temporal control so there was still immunogenic proteins expressed.
42
What is MVA?
A highly attenuated version of VACV - many passages through chick cells, 31kB lost from the genome including immune evasion genes. Is a poxvirus so can take up foreign DNA so is a candidate for a live delivery vector
43
What is the advantages and disadvantages of using MVA as a live delivery vector?
Is a poxvirus so can take up a lot of DNA, induces interferon, has lost many genes including those for immune evasion e.g. soluble interferon receptor.
Doesn't replicate well in certain cell types
44
What has been the result of using MVA as a live delivery vector for a flu vaccine?
MVA could be used to deliver internal proteins of flu virus that would be under less selection. M1 and NP are more stable so wouldn't need to update vaccine every year. In a human trial saw CD8 and CD4 T cell responses to the vaccine.
45
What are the advantages of a DNA vaccine?
Plasmids are safe, stable and can be transported long distances. Can give a cocktail of plasmids with multiple antigens. Unlikely to generate an adverse immune response as there is no live vector.
46
What methods have been used for development of an Ebola vaccine?
DNA vaccine development - good as there is a significant safety risk with growing the vaccine in vitro. DNA plasmids can produce an antibody response
47
What are the issues with developing an Ebola vaccine?
```
No implémentation of safety trials - want to develop quickly
No small rodent model - only primates
Cost and stability
Strain variation
Host genetic variation
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48
What are the different mechanisms of vaccine trial in the field?
```
Double blind (2 different vaccines given, and not vaccinated)
Stepped wedge (gradually roll out vaccine)
Ring method (vaccinate close contacts of infected)
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49
What has been the result of trying to developing an HCMV vaccine?
Congenital infection. Looking at glycoprotein as a vaccine. Have to use seronegative people in trial. No significant changes in chances of becoming infected; no significant changes in pregnancy outcomes
50
What are the examples of potential adjuvants?
Immunostimulants
Vehicles for optimal antigen presentation
Rational adjuvant design by stimulating TLRs
51
What are some examples of immunostimulants used as adjuvants?
Attenuated version of LPS (MLP), Cholera toxin B subunit (engineered) as a mucosal adjuvant for orally delivered vaccines
52
What are some examples of vehicles used as adjuvants?
Oil emulsions, mineral salts, liposomes, virosomes, biodegradable polymer microspheres
53
What are the theories behind rational adjuvant design?
Want to stimulate TLRs. dsRNA binds TLR3 - use poly I:C as an analogue and see if get increased immunogenicity (get more interferon induction). Flagellin binds TLR5 - use to enhance immunogenicity of HA head group (fusion protein)
54
What are the issues with developing an HCV vaccine?
Limited tropism in vitro and in vivo
Cell culture systems poor - have now identified human entry receptors so may be able to use this knowledge to add to rodent cells
55
What is a 'humanised mouse' used for hepatitis virus vaccine testing?
Immune deficient mouse with a liver toxin expressed. Kill mouse liver cells and implant human hepatocytes. Is now susceptible to HBV and HCV but mice are infertile, expensive and limited in numbers. Human cell quality is also important
56
What are some vaccines in trial for HCV?
```
Peptide vaccine (core, NS3, NS4)
MVA (NS3, NS4, NS5B)
Purified gpE1/E2 protein
DNA (NS3/4A)
DNA and protein (E1/E2 plasmid then core protein)
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57
How can viruses be stabilised by cryopreservation?
Important for vaccination in the 3rd world. Trehalose-sucrose dried on a glass slide. Can put virus particles in this and store for a long time - has shown to be promising with adenoviruses: get significant interferon production after thawing.
58
Why is an HIV vaccine challenging?
Many subtypes with geographical variation
Many routes of transmission
Need serum antibody and mucosal immunity
59
Describe the Merck vaccine trial for HIV
Was bad - saw increased risk of HIV infection in the vaccinated group compared to the placebo. Was an adenovirus expressing gag, pol and nef
60
Describe the Thailand HIV vaccine trial
Was reported to be successful (31% protection). Used a prim/boost protocol in which a pox vector was the prime and DNA was the boost. Used strains found in the region. However, no real significant difference in infection risk.
61
What are the types of antiviral treatments?
Antiviral chemotherapy
Vaccines
Newer/other (e.g. Zmapp - mix of 3 monoclonal antibodies against Ebola surface glycoprotein)
62
How does basic virology focus on discovering drugs?
By identifying key events in the virus life cycle, solving chemical/crystal structure of targets, identifying target cells, identifying off-target toxicity (human analogues), developing reliable models, identifying mechanisms of resistance latency and reservoirs.
63
What are the stages of human drug development?
Synthesis of a drug candidate
In vitro studies
Animal studies
Phase I studies (human in vivo safety)
Phase II studies (small efficacy trial)
Phase III studies (large pre-licensing efficacy trial)
Phase IV studies (post licensing safety reporting)
64
What characteristics does an ideal antiviral posses?
Potent, specific and non-toxic
Reliable models (in vitro, in vivo and in silico) and reliable to produce
Easy to store and administer
High barrier to resistance
65
What are the challenges in developing antivirals?
Virus factors: if the virus is difficult/dangerous to culture in vitro, no animal model, heterogeneity of the target, resistance develops
Host-virus interaction factors: off target toxicity, compartmentalisation of infection, delayed access to testing and treating
Viral replication uses host metabolism - need to inhibit only the virus. May have antiviral activity in vitro but not in vivo, only works against replicating viruses (not latent)
66
What are the goals of antiviral chemotherapy?
Want to treat (cure/control) AND prevent infection/disease
67
What are category 1 antivirals?
Antivirals that are direct inhibitors of the virus, usually at a cellular level. Could block virus entry/egress, inhibit nucleic acid synthesis, inhibit protease, inhibit integrase
68
What drugs block virus entry?
Maraviroc (HIV) is a CCR5 cellular chemokine receptor antagonist
Enfuvirtide (HIV) inhibits gp41 mediated fusion
Monoclonal antibody palivizumab (RSV)
VZV antibody drugs
Docosano (HSV)
69
What drugs inhibit nucleic acid synthesis?
Nucleoside analogues such as Acyclovir - requires thymidine kinase which isn't in uninfected cell)
Nucleotide analogues (as above but with phosphate) such as cidofovir and foscarnet (pyrophosphate, blocks binding site of viral DNA pol
Inhibition of the helicase-primase complex (in clinical trials)
Inhibition of reverse transcriptase for HIV
70
What drugs inhibit proteases?
Many for HIV to prevent cleavage of polyprotein
| Have some for Hep C
71
How do integrase inhibitors work?
Inhibits strand transfer, the final step of provirus integration
72
What are category 2 antivirals?
Drugs that modulate the host immune response to up regulate/modify
73
What are some approaches that can be used to modulate the host immune response?
Reduce immunosuppression
Antiviral prophylaxis and pre-emptive therapy
Interferons to induce antiviral state
Innate immunity stimulators
74
What is the definition of antiviral resistance?
Lack of clinical and virologic response to treatment. Clinical response - drug sensitive virus in an immunocompromised host fails due to lack of immune response
75
What are the factors involved in the emergence of resistant virus strains?
Mutations in viral genomes (e.g. SNPs that give amino acid substitutions that result in resistance)
Selective pressure (the drug)
High replicative load
High intrinsic viral mutation rate
76
What are the consequences of antiviral resistance?
If the resistant strain is as fit as the parent get prolonged and severe disease. If not, there is less impact
77
How can antiviral resistance be found and how can this knowledge be used in treatment?
Phenotypic assays in cell culture - difficult to do and standardise
Specific genotypic assays - look for changes (need to know what they are and update primers/probes frequently)
For HSV and VZV, only look for resistance if treatment fails as they rarely develop it de novo - could be clinical (immune system failure) or a higher viral load than expected
Resistance is common in HIV, so test for with a baseline assay and select treatment appropriately
78
What is Ribavirin?
A nucleoside analogue, mode of action uncertain. Inhibits synthesis of viral genome. Works on a large range of DNA and RNA viruses including RSV and HCV (not used as frequently any more). Causes anaemia
79
How can herpes viruses be treated?
Acyclovir, Cidofovir, Foscarnet
80
What is acyclovir?
A drug used against HSV, Herpes B and VZV (not as effective against VZV; an attenuated dose can be used for VZV prophylaxis but unlicensed). The viral thymidine kinase phosphorylates acyclovir (so benign in non-infected cells). The product is an analogue of dGTP and is a chain terminator which is more specific to the viral polymerase.
81
Describe resistance to acyclovir
More common in immunocompromised patients. Resistance can develop in the thymidine kinase (reduced phosphorylation/absent), phosphorylation of acyclovir can be decreased or the viral DNA polymerase can have a decreased affinity for acyclovir triphosphate.
82
What is ganciclovir/valganciclovir?
Ganciclovir is used in HCMV (valganciclovir is an oral prodrug converted to ganciclovir). Similar to acyclovir but the initial phosphorylation is done by a viral protein kinase (no thymidine kinase present)
83
What are the mechanisms of resistance to Ganciclovir?
Mainly in immune compromised patients due to increased exposure. Mutations in UL97 (viral protein kinase) - point or deletion. Mutation in UL54 (DNA polymerase) - gives high resistance to ganciclovir and cross-resistance to cidofovir.
84
What is foscarnet?
Used to treat HCMV and HSV that are resistant to classical nucleoside analogues. Is also effective against HIV but has many side effects and there are other options so not used. Blocks the pyrophosphate binding site on the viral DNA polymerase/reverse transcriptase. Has high toxicity
85
What are the mechanisms of resistance to foscarnet?
Doesn't require phosphorylation - resistance can only develop by point mutations in viral DNA polymerase (or HIV reverse transcriptase)
86
What is cidofovir?
A monophosphorylated nucleotide analogue - cellular kinases triphosphorylates. Activity against thymidine kinase negative HSV and UL97 phosphotransferase negative HCMV. Has dose related nephrotoxicity
87
What are the mechanisms of resistance to cidofovir?
Mutations in viral DNA polymerase gene
88
What is brincidofovir?
Similar to cidofovir. Has a lipid tail to aid entry which is gotten rid of once inside and stops viral replication. Due to ease of entry is less toxic to kidneys than cidofovir. Hasn't been licensed yet.
89
What are the drugs used against HIV?
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Maraviroc
Enfuvirtide
Abacavir
Non-nucleoside RT inhibitors
Integrase inhibitors
Protease inhibitors
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90
What is Maraviroc?
A drug against HIV that blocks entry. Is a CCR5 antagonist. Resistance can develop if there are substitutions in gp120 or if virus uses CXCR4 instead (can do an assay to find this out)
91
What is Enfuvirtide?
A drug that prevents HIV fusing with the cell - HIV-2 is intrinsically resistant. Has to be administered via an injection (oral would be better). Not many side effects
92
What is Abacavir?
A nucleoside reverse transcriptase inhibitor used to treat HIV. Has adverse side effects due to hypersensitivity with some HLA-B alleles.
93
What are some examples of nucleoside reverse transcriptase inhibitors used to treat HIV?
Abacavir
Emtricitabine (also has activity against HBV)
Lamivudine (HBV too)
Tenofovir (HBV too)
94
What are some examples of non-nucleoside reverse transcriptase inhibitors used to treat HIV?
Delavirdine
Efavirenz
Etravirine
Target HIV-1: HIV-2 is resistant. Is often the first line of treatment. Side effects include CNS toxicity and elevation of liver function enzymes
95
What are some examples of protease inhibitors used to treat HIV?
Atazanavir
Darunavir
Ritonavir
Limited distribution to the CNS, adverse effects include GI and metabolic (e.g. insulin resistance)
96
What are some examples of intergrase inhibitors used to treat HIV?
Raltegravir
Dolutegravir
Elvitegravir
Side effects include nausea, diarrhoea, hepatitis, hypersensitivity
97
Describe resistance in HIV
Is common - test before starting treatment and if treatment starts to fail/patient stops taking drugs
Mechanisms contributing include compartmentalised infection (e.g. CNS), transmitted drug resistance mutations, latency, recombination, high mutation rate
98
How was HCV treated in the past?
Pegylated interferon and ribavirin (a nucleoside analogue). Only 40-50% clearance
99
How is HCV treated today?
4 classes of new drugs - NS3/4A protease inhibitors, NS5A protein inhibitors, NS5B polymerase inhibitors (including nucleoside analogues and non-nucleoside methods)
100
What are the guidelines for treatment of HCV?
Depends on genotype of virus and presence/absence of cirrhosis. Each country has national guidelines specific to their population and subpopulations (e.g. injecting drug users). Have high clearance (90%) over 12 weeks or less of treatment.
101
What are some examples of immunomodulators?
Podofilox - antimititic inhibitor used against HPV
| Fomiversen
102
Describe the use of interferon as a drug
Was used for HBV and HCV - now have others. Infrequently used as has many side effects and is expensive and has to be injected.
103
What do neuraminidase inhibitors do?
Prevent influenza virus shedding, but don't prevent already shed virus infecting more cells and initiating more replication and further innate immunity stimulation or stop production of viral proteins in already infected cells which may reduce innate immunity stimulation
104
What were the first drugs used against influenza?
Itzstein 2007: adamantane based M2 ion channel protein inhibitors. Only in flu A (has M2), rapid rise in resistant strains, side effects in CNS
105
How were neuraminidase inhibitors developed?
Used rational drug design from knowledge of neuraminidase structure (incl. active site with adjoining pockets for binding) and enzymatic function (including intermediates formed). Structural derivatives of the neuraminidase substrate were designed to bind the active site and be metabolically stable and not cleared quickly. These were confirmed and refined with structural studies of the neuraminidase-inhibitor complex.
106
What was the first and second neuraminidase inhibitor developed?
Zanamivir, was inhaled (couldn't be taken orally)
Oseltamivir could be taken orally, was refined from a cyclohexene model so it could be taken orally, though there is now resistance against it.
107
What drugs are being developed against influenza?
Replication inhibitors e.g. cap binding inhibitors, endonuclease inhibitors
Monoclonal antibodies
108
What were the outcomes of a cap endonuclease inhibitor trial to treat flu?
Results from Oct 2017. Found that a small molecule inhibitor for the cap endonuclease activity of the viral polymerase reduced viral load in 24hrs compared to 72hrs when treated with oseltamivir.
109
How is HBV treated?
```
Pegylated interferon (immunomodulatory but interferon related side effects; ow HBsAg clearance)
Nucleus(t)ide analogues (effectively suppress HBV DNA, lifelong therapy required, low HBsAg clearance)
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110
What is the goal of HBV treatment?
Want a functional cure (complete sterilising cure is unlikely) - finite treatment length with sustained HBsAg loss and/or control by host mechanisms. This will reduce the risk of progression to fibrosis and cancer
111
How is HBV drug development being targeted?
Virus approches: entry inhibitors, capsid/core protein modulators, viral expression inhibitors, cccDNA destabilisers, HBx inhibitors
Immune-Stimulatory approaches: TLR7 or TLR8 agonists, RIG-I agonists, therapeutic vaccines, checkpoint inhibitors
112
How has RNA treatment been used to develop a drug against the Ebola virus?
Kole 2012: PMO-based compounds used RNA-blockign oligomers to inhibit viral translation and greatly improve survival, as well as surviving monkeys having immune resistance to reinfection
113
How can RNA oligonucleotides be used to treat HBV?
Can be targeted to hepatocytes through binding to GalNAc (a trivalent carbohydrate cluster) which has high affinity for ASGPR, a receptor on hepatocytes. Get high rate of uptake of oligonucleotide and recycling of GalNAc. The oligonucleotides delivered can then inhibit HBV through RNAi methods, targeting all 4 transcripts
114
How are we prepared for epidemics?
WHO has a list of emerging diseases likely to cause major epidemics
Coalition for epidemic preparedness innovations supports vaccine development
115
What kind of vectors are used in gene therapy trials?
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Adenoviruses
Retroviruses
Naked/Plasmid DNA
Adeno-associated viruses
Vaccinia virus
Lentivirus, lipofection, poxvirus, HSV
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116
What diseases are addressed by gene therapy trials?
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Cancer
Monogenic diseases
Infectious diseases
Cardiovascular diseases
Neurological diseases, ocular diseases etc
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117
What is gene therapy?
The transfer of a gene into a cell with the purpose of correcting a gene defect
118
What are the requirements for transduction mechanisms for gene therapy?
The critical first step in gene therapy, can be in vivo or ex vivo (on organisms tissue but outside the body)
Must be safe and result in stable/regulatable gene expression for therapeutic benefit
Viral and non viral methods of delivery are under development
119
What are the ideal properties of a viral vector for gene delivery?
Large transgene capacity for single/multiple gene delivery
Gene of interest are expressed appropriately or under the control of an inducible promoter
Transduction efficiency must be high and targeted to specific cell types
Must avoid promoter inactivation, loss of vector sequences, cytotoxicity, immune response to vector or transgene, insertional mutagenesis
120
How is cGAS involved in DNA sensing?
L Sun 2013: cGAS binds DNA in the cytoplasm, inducing its cyclase activity. It produces cGAMP (from ATP and GTP) which goes on to bind STING. This binding activates the type 1 interferon response. cGAS is a sensor of retroviral DNA as shown by D Gao 2013.
121
How does STING activate the type 1 interferon response?
Signals to IRF3 and NFkB to activate production of type 1 interferon
122
How can non-enveloped viruses be restricted?
Staring 2017: By autophagy. Virus entry into the cytoplasm is aided by PLA2G16, a cellular factor potentially by altering/maintaining pore size or inducing membrane curvature of the membrane the capsid is bound to, triggering genome release. When PLA2G16 is deleted, translation of the viral genome is lowered; this can be recovered by deletions in autophagy proteins, suggesting a restriction mechanism.
123
How do retroviruses (R), HSV, adenoviruses (AV), adeno-associated viruses (AAV) and lentiviruses (L) compare as virus vectors?
All will infect non-dividing cells apart from R
HSV and AV have the highest efficiency of infection (R only in dividing cells)
R, AAV and L induce long term expression (HSV is short, AV is moderate)
HSV can have large inserts
AV require high titres, AAV and LV low, R and HSV moderate
HSV and AV have cytotoxicity and immunological problems (others are limited)
124
What are the advantages and disadvantages of HSV vectors?
Can establish latency in neurones - potential as a neuronal gene delivery vector
Advantages: Can be grown to high titres, infect many different cell types, can accommodate large DNA inserts
Disadvantages: toxicity and difficulty of achieving long term gene expression, large genome with many genes, at least half are needed for replication
125
Describe the first generation of retrovirus vectors
Mutate the packaging signal in wild type viruses, then transfect a cell line with the packaging signal followed by the transgene. Transduce packaging signal-mutated virus, get viruses containing the wild type packaging signal followed by the transgene (some wt virus due to recombination). Use this as a delivery vector
126
Describe the second generation of retrovirus vectors
Similar to the first generation but the packaging cell line was designed to minimise generation of wild type virus by recombination - packaging functions were split
127
Describe the third generation of retrovirus vectors
Packaging cell lines were designed to minimise recombination to form wt replication competent viruses by splitting packaging functions. Also, used VZV G protein in place of Env to widen the cell tropism and produce a more stable virion
128
What are the problems with third generation retroviruses?
In 2000, third generation retroviruses were used to transduce a normal copy of the gamma-c signalling chain receptor gene ex vivo into children with SCID XI. Was initially successful, but 4/9 children developed leukaemia due to point of retrovirus insertional and activation of an oncogene - now determine insertion sites by deep sequencing
129
What are SIN vectors?
Self inactivating vectors. Contain a deletion in the U3 region off the 3' viral LTR so the viral enhancer promoter elements are inactivated following reverse transcription.
130
How was adenosine deaminase deficiency treated with viral therapy?
Causes SCID, requires bone marrow transplantation for enzyme replacement (expensive). Found that a mouse knock out can be given a lentivirus vector to deliver the ADA gene to correct the enzyme deficiency. Is now attempted in patients with success.
131
What are the advantages and disadvantages of adenovirus vectors?
Advantages: efficiently infect non-dividing cells, replicate to high titres and have large cargo capacity
Disadvantages: non-integrative so stable transduction is difficult and can get potent immune responses. Virus is complex and packaging cel lines haven't been developed - rely on replication defective virus lacking E1 expressed on complementing cell line
132
What are the advantages and disadvantages of adeno-associated virus vectors?
Is naturally defective - requires HSV or adenovirus to replicate. Has a high transduction efficiency of many cell types including non-dividing cells; in the absence of helper virus it integrates
Can only accommodate small DNA inserts (less than 5kb) and is difficult to produce in high titre
133
What has been the focus of developing adeno-associated virus vectors?
Improving the yield - originally replaced REP and CAP in wd AAV with promoter and transgene, infected wt (or similar) and transgene into a cell along with a helper virus. Got low yields. Now do a mini adenovirus genome along with AAV vectors and AAV helper genes to get an improved yield
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What is the future of gene therapy?
Using CRISPR-cas9 to edit DNA in our bodies
135
How has small pox affected the british royal family?
House of Stuart - line of succession ended when all of Queen Anne's siblings and children died of small pox. Throne was passed to Sophia of Hanover in the act of settlement 1701 to establish the hanoverian dynasty (our current royal family)
136
How was small pox first controlled?
Edward Jenner: vaccination using cow/horsepox. Was spread around the world by arm to arm transfer, but this was then banned as other diseases could also have been transmitted (measles, syphilis)
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What was the important step in eradication of small pox that occurred in 1950?
Freeze dried vaccine was developed so it could be shipped around the world without refrigeration
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How was small pox eradicated?
Intensive vaccination by ring vaccination protocol. Massive effort from WHO to have last natural case in 1977 (last actual case in 1978 when escaped from a lab)
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Why was eradication of small pox possible?
No animal reservoir
No latent or persistent infection
Easily recognisable (can quarantine and vaccinate contacts)
High fidelity DNA polymerase so vaccine worked against all strains
Vaccine was efficient, potent, low cost, abundant, heat stable and easily administered
WHO determination to erradicate
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Why was the small pox vaccine successful?
Efficient, potent, low cost, abundant, heat stable and easily administered
Antigens of vaccine were shown to be highly conserved (after eradication!)
141
What is the evolutionary history of Variola Virus? (VARV)
Complete virus sequence has been obtained from a child who died in the 1650s - was basal to all genome sequences from the 20th century and both clades of the virus (major and minor). Suggested that most of the variation has arisen recently, potentially triggered by the start of vaccination.
142
What are the issues post small pox eradication?
Vaccination has stopped - more people are immunologically naive
Virus still exists, has been catalogued and centralised
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What are the arguments for and against destroying the remaining variola virus stocks?
Destroy: logical next step after eradication, removes risk of virus escaping, removes need and cost of storing virus safely and securely
Retain: virus needed for testing of safer vaccines, anti-viral drugs and diagnostic tests (need live virus to authenticate), increase understanding of why it was so virulent and could overcome innate immunity, don't want to destroy biological diversity
144
What are the arguments for retaining variola virus stocks?
Need the live virus to authenticate testing for safer vaccines, anti-viral drugs and diagnostic tests
Study virus to understand why it was so virulent and could overcome the innate immune system - host pathogen interactions and design new strategies for treatment
Don't destroy biological diversity - may have useful genes e.g. soluble proteins that bind TNFalpha
145
Why has the delay of variola virus destruction been authorised?
Research for public health benefits.
Diagnostic tests
Antiviral drugs
Safer vaccine
146
What is the benefit of developing diagnostic tests for small pox?
PCR and protein based tests
| Distinguish from herpes and other orthopox virsues
147
What is the benefit of developing antiviral drugs against small pox?
Was eradicated with no drugs...
USA won't allow destruction until have 2 drugs that act at different stages in case of use as a weapon. Difficulties in licensing drugs as can't show drug works against VARV in its natural host (animal models and surrogate viruses)
148
What is the benefit of developing a safer small pox vaccine?
The old vaccine had safety concerns - was withheld from pregnant mothers, people with eczema and potentially people with HIV
149
How does WHO regulate work with small pox?
Work happens at 2 collaborating centres, any proposals have to be submitted to the WHO committee.
No genetic engineering of the virus is allowed
No expression of VARV genes in other orthopox viruses (no make super virulent)
No lab can have more than 20% of the genome
No lab can have VARV DNA and another infectious orthodox virus (no accidental hybrid viruses)
There is an advisory committee on variola virus research which oversees proposals and approves/disapproves. Debates at the world health assembly
150
What has been the progress towards developing drugs against small pox?
2 compounds: CMX-001 and ST-246. CMX-001 has been used to treat Herpes viruses (has bad side effects and has been altered), targets the viral DNA polymerase to block DNA replication. ST-246 acts on F13 and stops phospholipase activity to prevent IMV wrapping at the Golgi. May be licensed soon
151
What has been the progress towards developing new diagnostic tests against small pox?
PCR based diagnostic test has been licensed in the USA
152
What has been the progress towards developing safer vaccines for small pox?
Modified VACV Ankara (MVA) has been licensed in Europe and bought by the USA
153
What are the current views on the progress towards VARV destruction?
Don't require the virus for making vaccines or further diagnostic tests
May require the virus for drug discovery for a bit longer.
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What is the significance of the work done in mouse pox published in 2000 to the small pox debate?
Found that adding IL-4 (a Th2 cytokine) to the virus induced mouse pox in resistant mice, and mice that were recently immunised. Was concerning, as could mean that engineering small pox with Th2 cytokines would render the vaccine useless.
155
What are the fears to do with small pox and bioterrorism?
Population is immunologically naive
Vaccine is old and in limited supply
Takes 7-19 days for infection to develop - release in a public place would cause widespread dissemination before it could be identified
Highly infectious
156
What is the UK small pox policy?
Purchase a stockpile of vaccine (Lister strain)
Immunise 350 health care workers, maintain vaccination
Don't vaccinate whole population
Ve vigilant
Train infectious disease physicians to recognise small pox and distinguish from chicken pox
Monitor immune response in vaccinees
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How can monitoring the immune response in small pox vaccinees aid preparation for potential outbreak?
Measure antibody response against vaccinia antigens (compare to unvaccinated teenagers) - many antigens on IMV surface, only B5 (neutralising antibody) on EEV
Found that people with multiple vaccinations had higher antibody levels
Use to analyse residual immunity and immunogenicity of new candidate vaccines (compare to old vaccines)
158
How can pox viruses be constructed from nothing?
Make 20kb DNA chunks with short overlapping fragments and terminal hairpins
Put into cells and co-infect with another pox virus from a different genus (necessary viral proteins to replicate)
Transcription and recombination and replication occurs, get a pox virus
Take progeny, grow on a permissive cell line (but not permissive for helper virus)
Can be done with cloned and synthetic DNA
159
What are the phases of polio in history?
Phase 1: 1400BCE-mid 19th century - no poliomyelitis
Phase 2: epidemics in developed countries 1900-1960
Phase 3: developing countries begin to control, eradication is target 1985-present
Phase 4: getting rid of vaccine
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Why did polio epidemics arise in the early 1900s?
Theorised to be due to better hygiene standards meaning children exposed later in life when don't have mother's antibodies for protection
161
What have been the issues with polio eradication?
Some polio is caused by the vaccine
| Vaccines don't work as well in tropical countries or those with poor infrastructure
162
What is the polio vaccination strategy?
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Routine immunisation at 3 months (in developed countries, focus is on individual protection not large population. Works in a temperate climate as polio is only around in the summer. Goal is to reduce pool of susceptibles until there are no more)
Mass vaccination (swamp the country all at once, targeting babies under 5)
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163
What was the status of polio eradication in 2002/2003?
Vaccination by WHO was focussed on in Nigeria, however some groups thought it was a US plot and stopped vaccination, allowing polio to spread across central africa - then spread to yemen as people took religious journeys.
164
What was the status of polio eradication in 2016?
India has eliminated polio (2011) after a heroic effort
Still a few cases in Pakistan and Afghanistan. Book Haram won't allow polio vaccination, and the population is large enough to sustain the virus; don't know what is going on there.
cVDPV viruses have evolved from the vaccine which can be paralytic and infectious
165
What are the issues with eradicating polio?
Most infections are silent unless get poliomyelitis (only 1/200 cases), can't tell if someone is infected/infectious
Can't tell if someone is vaccinated without extensive tests
Have to eradicate the agent, not just the disease
166
Describe the course of the disease caused by polio
Infection is asymptomatic
Minor disease 3-4 days post infection (malaise, fever, sore throat)
Major disease 8-30 days post infection only in 1% of cases
167
Describe the major disease caused by polio
8-30 days post infection, only in 1% of cases. Different presentations including Aseptic meningitis (non-paralytic poliomyelitis), spinal (legs)/bulbar (breathing, potentially lethal) poliomyelitis (paralysing), encephalitis (inflammation of the brain)
168
How is polio transmitted from host to host?
Ingested through contaminated water/similar. Virus goes to intestine mucosa and grows there (cell unknown). Virus leaks into lymphoid tissue, some virus is found in the blood (if the host has antibodies, can't spread in blood and stays in the gut). Can then seed into other tissues e.g. extra neural tissue, regional nerve ganglia, CNS and tonsils
169
What is the structure of the polio virus?
Non-enveloped positive sense RNA virus. A picornavirus (group C enterovirus). 60 unit icosahedral capsid with 4 different proteins VP1-4.
170
What is the structure of the polio virus genome?
5' non coding region containing an IRES, VPg linked. Structural VP proteins are first, then P2 and P3 regions (non-structural) and a small 3' non-coding region
171
What are the 2 types of polio vaccine?
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Inactivated polio vaccine - Salk
Oral poliovaccine (OPV) - Sabin (is live attenuated)
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172
What is the difference between the live virus and the live attenuated vaccine?
Only 10 nucleotides different. The ones that matter are a mutation in VP3 and changes in the 5' non-coding region which is highly structured
173
How does the vaccine change after it has been administered?
Very quick reversion of mutation in 5' end (472) - very strong deletion pressure
After 10 days, one of the temperature sensitive mutations revert and the strains may undergo recombination
After 28-40 days get another recombination to become completely non-temperature sensitive
Antigenic drift throughout
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What mutations are found in the Sabin vaccine? How do they revert?
Phenylalanine is substituted for serine in the Sabin strain - this destabilises promoter interactions in the capsid. Other mutations make the strain temperature sensitive. Don't necessarily get direct back mutation of the amino acids, may get compensatory mutations
Mutations are mainly at capsid interfaces (one in the centre, unsure of cause) either blocking assembly of the pentamers or assembly of the whole virus
175
How was the molecular clock of polio evolution established?
Look in someone who produced no serum antibodies to polio so virus production was for a long time (greater than 30 days). Found 2.78% changes per year
176
When can vaccination with OPV be stopped?
When wild type polio is eradicated IF the vaccine either causes no disease or vaccine related disease can't spread. Neither is true, however, it only spreads a bit and risk of paralysis is small, so may be sufficient.
177
Describe the outbreak of polio in Haiti in 2000?
Virus was related to the Sabin strain, had reverted to a poliomyelitis causing virus. Was a recombinant strain with another C type enterovirus - virus only has to regain transmission, then can reestablish virulence
178
What is the significance of chronic secretors of polio?
They secrete a highly virulent virus which has highly drifted - there is no way to cure, can only stop spread from gut by giving immunoglobulin
Arise from people who have been given the vaccine
Need to be able to detect these people, probably immunodeficiency related. Have found drifted vaccine based viruses in sewage across the world
179
How can OPV stop being used?
To tackle cases of vaccine associated paralysis poliomyelitis - stop using OPV
To tackle circulating vaccine derived poliovirus (cVDPV), stop using OPV, get rid of cVDPVs by use of monovalent OPV
To tackle immunocompromised vaccine derived polio virus (iVDPV), stop using OPV and develop and antiviral
For ambiguous VDPVs, stop using OPV, rest is tricky
Countries are moving to IPV
180
How has type 2 OPV been withdrawn?
No wild type case since 1999
Most vaccine associated polio cases are caused by type 2 - most cVDPV and iVDPV are type 2 as well (out competes, is well adapted to the gut)
Have to withdraw type 2 OPV all at once, replace with IPV given at birth
Monitor for cVDPV carefully and have a response reddy
Contain all type 2 virus in labs
181
What is the problem with IPV vaccine supply?
Current manufacture is mostly european
Supply is insufficient and is expensive to produce
Uses wild type polio strains that could cause paralysis - a problem in countries who have never made IPV and have poor infrastructure
Could make it with Sabin strains, but quality issues are very very poor (and are differently immunogenic and there is a question over how much safer this would be)
182
Who does the GAPIII for polio apply to?
WHO global action plan 3
Production units
Virology diagnostic labs
Virology research labs
Any lab which may have a polio specimen (e.g. GI, RI parasitology)
Any lab that may have a full length polio genome (is infectious)
Any molecular biology lab (can synthesis the genome)
183
What is the next stage in eradication of polio?
New modes of production: make new viruses that are safer than wild type and Sabin (for vaccine and serological assays). Want strains that don't revert or transmit, strains that only grow in culture and development of empty capsids
184
What are the determinants of attenuation of polio?
In wild type have CG, in Sabin have UG. Make a strain which is UA (needs more than 1 mutation to revert). This has been carried out - S15 strain is the same as sabin 3 with many UAs. S19 can only be grown at 33 degrees - could be a good option for IPV production
Make thermostable empty capsids - use what we know from temperature sensitive mutants that revert in babies to inform mutations that could make a capsid more stable
185
Describe the Ebola virus
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Filoviridae, negative sense RNA virus
Enveloped
19Kb with 7 ORFs
Filamentous
5 types of ebolavirus
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186
What is the history of Ebola?
Ebola virus disease (EVD) first appeared in 1976 in Sudan and Congo near the Ebola river
187
How does Ebola transmit between hosts?
Natural host is bat, can then spread to monkeys and bush meat
Once in humans, traditional funeral practice, unprotected healthcare workers and unprotected contact with blood and bodily fluids cause the virus to spread
188
What was identified during the first deployment in december 2014 of the Ebola outbreak?
A need for real time in country genetic sequencing. Had to ensure that repeated bottlenecks weren't compromising the ability to detect viral RNA and over come bottlenecks in EBOV sequencing. This needed to be provided to the scientific community on an open access basis allowing identification of epidemiological links in a valuable time frame
189
How can sequencing help in the ebola epidemic?
When the virus replicates, it picks up mutations over time. Sequencing allows an unbiased way to track the virus through the first, second and following waves of infection
190
What did sequencing of Ebola tell us?
There was cross-border movement of infected individuals despite controls
Geographical movement of infected individuals despite monitoring
Sexual transmission/persistance showed evidence of slow rates of evolution
Potential evidence for transmission via breast milk from an asymptomatic individual
191
What was the use in tracing sporadic cases in the Ebola outbreak?
Sequencing of an outbreak in a region free of Ebola for the previous 130 days showed that it was due to patient travelling into the area, not importation from Guinea or a new zoonosis
192
What did viral genomes tell us of the ebola virus disease?
The factors that spread and sustained the epidemic. A case in Seirra Leone arose after 42 Ebola-free days - was very closely related to initial outbreak, suggesting from persistence in a patient.
193
What went wrong in the Ebola response?
Geographical location wasn't ideal
Cultural practises allowed spread of virus e.g. burial practices including touching and kissing of the body
Lack of preparedness including not enough doctors and nurses (like, hardly any at all)
Slow response
Inadequate resources
194
Why were there no vaccines in Ebola outbreak?
Some candidate vaccines were developed, but none made it to human trials. Testing the vaccine was challenging and there was a lack of incentive to develop vaccines for pathogens that don't have a market value
