Lecture 4 influenza

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H5 and H7 are highly pathogenic to both birds and

  • The species barrier between avian and humans is mostly keeping highly pathogenic avian H5N1 out of human population
    - H5 has a greater specificity for alpha 2-3 SA than 2-6 SA
    - It is rare for a human to be infected by avian
    - It has never happened between humans 
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How could H-H spread of H5 be achieved?

  • A serious threat of humanity is that highly pathogenic H5N1 may develop capacity for human to human transmission 
  • 3 requirements
    1. Ability to enter cells of the human URT
    2. The ability to replicate to high numbers in URT 
    3. Ability to exit cells of the URT
  • The current H5N1 avian influenza cannot do these things very well
  • Likely to change - Requirement 1 is very simple 
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Binding to different target cells

  • Binding specificity is conferred by as few as 1 or 2 residues on HA
  • Thus SA binding specificity can easily change through mutation 

H1--> Residues 190 and 225
H2--> Residues 226 and 228 
H3--> Residues 226 and 228

To change alpha 2-3 to 2-6 specificity 

For this reason, H5 virus host range can almost certainly change too. For H5N1, change in HA= Q222L, G224S

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Ferret model

Closest to human 
- Receptor distribution
- Disease outcome 
- Transmission 
Requires high level of replication in human URT cells

  • Replication rate of avian H5N1 is reduced in humans
  • Mainly due to different temperature of 37 degrees in humans and 41 in avians. 
  • This reduces virus production in airways
  • This factor all prevents human to human transmission 

Mutation E627K in PB2 is known to affect peak replication temperature
- Avian H5N1 virus was engineered with all 3 220/224/627 mutations and given to ferrets
This caused entry into URT and increased URT replication but no aerosol transmission

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Experiment

The experiment was to select an aerosol transmissible virus
- Designed to identify what other AA changes were necessary 

1. Ferrets infected with H5N1 (220,224,627) 
2. Sneeze samples taken after 4 days (selection)
3. Used to infect next ferret
4. Wait another 4 days and repeat
5. Process is repeated 10 times 
- The virus released went up each passage so showed the virus was adapting 
6. The virus from p10 was isolated and used to infect multiple ferrets in isolation cages
- There was airflow but no direct contact
- Transmission occured and resulting virus was sequenced
- H103Y
- T156A
Additional changes to HA

The likelihood of this occuring in nature is approaching 100%

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Virus vaccine production

  • Dominant influenza strain is different each year
  • New influenza vaccine must reflect current circulating viruses
  • GISRS- Global Influenza Surveillance Response System 
    - Membersin 105 countries 
    - 175,000 sequences sampled 
    - GISRS predicts 3 likely strains in March 
    A/ Texas/ 361/ 2011 (H3N2)
    A/ California/7/ 2009 (H1N1)
    B/ Massachusettes/2/2012
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Traditional method of influenza vaccine production

  • Each of the 3 chosen strains must be weakened to reduce their ability to cause disease
  • Achieved by allowing segment exchange with the weak PR8 strain
  • This is called REASSORTMENT 

1. PR8 and wildtype viruses coinfect a single cell
2. Viruses replicate
3. Segments can mix to produce 256 possible new genotypes 
4. Reference strain is used to infect (seed)

Drawbacks 

1. Generation and selection of 3 reassortments is slow = 3 months 
From selection to production is a minimum of 9-10 months 
2. Dependence on egg production=170 million eggs
3. Some H5N1 viruses grow poorly in eggs

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New vaccine

A new vaccine manufacturing process is being used to remove all 3 problems 

Generation of reassortment flu viruses from cDNA's

  • 8 cDNA's encoding RNA genome sequence added (PB1, PB2, PA, NP, M, NS= PR8) (HA, NS = X) 
  • 4 cDNAs encoding influenza virus proteins required to form the 8 RNP's (PB1, PB2, PA, N) added
  • 8 functional RNPs 
  • Transcription of viral proteins 
  • RNP replication 
  • Assembly 

Allows rapid response to new virus, seed in 2 weeks
Should allow a response to most rapidly emerging pandemics
No issues with egg production
No issue with poor virus growth 
No issue with egg allergy

Has now been approved by FDA for Flucelvax, produced by Novartis 

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Anti-virals

Current targets:
- Endosome escape of RNA
- Release of virus 

Adamantes - Amantadine and rimantadine 
- Amantadine was 1st antiviral compounds approved for humans ( 1966)
- At low concentration less than 5mM, it is specific for influenza virus A
- At high concentration of 100mM it has some general antiviral activities

Rimantadine 
Has an additional methyl group
FDA approved in 1984, improved efficacy and fewer side effects
PROS=  Cheap, effective
CONS= Must be administered during the early stages of infection (before 2 days) 

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How they work

  • Prevent M2 ion channel activity
  • Binding either blocks or closes the pore
  • Prevents ion channel activity, Matrix layer intact-virus cant exit endosome 
  • Problems- Most humans flu's now resistant. This began in 2003

2003: Significant local strain resistance
2006: More than 50% of human isolates of H3N2 influenza virus were resistant 
2008: Global resistance

The resistance is confered by virus mutation within M2 channel itself. Those prevent amantadine binding ad blocking the channel.
Adamantes are no longer FDA approved for flu 

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Neuraminidase (NA)

Essential for virus release
Virus release depends on NA binding SA and cleaving it
SA is substrate for NA
If you block this interaction, you block infectivity 

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Strategy: Design molecule that could bind NA bette

1. An early example of structure based drug design
2. High resolution structure of NA determined in 1983
3. 1986- Structure of co-crystal of NA and SA was determined
4. Showed contact between 2 molecules (5 sites in a pocket) 

The aim then became to make a molecule that can better bind the pocket
Block pocket so prevent NA function 

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Site 2

Room for improvement

  • Interaction between C4 OH and Glutamic acid 119 was not optimal - could be altered
  • The addition of a positive charged Guanido group at C4 to interact with a negatively charged region of the pocket, Asp 151, Glu 227

This molecule has 100x higher affinity for NA than sialic acid 
- Thus SA is outcompeted

This led to development of Relenza ( Zanamir) an aerosol and Oseltamivir (tamiflu) a tablet

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Do they work?

Compared finding of 83 trials 
It must be given early on in infection
- Take within 48 hours- symptoms reduced by 1 day 
- But this may be enough to slow the pandemic spread
- Stockpiles of both tamiflu and relenza now exist - £500 million 

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NA resistance

  • Tamiflu and relenza should not encounter resistance
  • NA must maintain ability to bind SA for virus viability 
  • SA and tamiflu/relenza are structurally very similar 
  • A virus than no longer binds Tamiflu/Relenza will not bind SA
  • However 1% of H3N2 (seasona l) strains are resistant. 
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