Viruses and immunology Flashcards
MHC class I
Uses endogenous antigens of 8-9 aa.
MHC class II
Uses exogenous antigens, which are taken up, and degraded to peptides of variable length.
Associates with invariant chain.
HLA-DM replaces CLIP with the peptide.
NK cell receptors
Inhibitory e.g. KIRs
Activating e.g. NCR
ISGs
2'5 (A) synthetase PKR Mx proteins dsRNA-specific deaminase Inhibitory microRNAs
Mx proteins
dynamin-like domains, GTP binding/GTPases
Interact with GTP-microtubular/vesiclar pathway components, inhibiting viral protein movement in the cell.
IFNy activity
Some ISGs
iNOS
Immunomodulatory activity.
IFNy immunomodulatory activity
Increase expression
- MHC
- proteasome and TAP
- adhesion molecule expression.
IFNa/B pathway
Binds receptor. Cytoplasmic side binds JAK1 and TYK2.
These phosphorylate STAT1 and STAT2, which activate ISGF3 for activity on ISRE.
IFNy receptor pathway
Binds IFNGR.
Binds JAK2 and JAK1.
Phosphorylates STAT1.
Stimulates GAS and some ISREs.
IL-1 receptor pathway
Same as IFNa/B
PAMPs - dsRNA and viral rep in cytoplasm
RLRs - RIG1 binds. Downstream IRF-3 and NFkB are activated.
PAMPs - dsDNA in cytoplasm.
DNA dep activater of IFN regulatory factors binds dsDNA in cytosol, activating IRF3.
NOD-like receptors
Stimulate inflammasomes.
TLRs
Sense endosomal nucleic acids.
Production of cytokines in response to viruses.
Due to activation of PAMPs like TLR7 and TLR9, via NFkB, producing IL-6, TNF and IL-12.
Viral immune evasion - general strategies.
Hiding Antigenic variation Molecular mimicry. Inhibition of effector arm. Immunomodulation.
Viral immune evasion - hiding
Integration vs episomal
Immunoprivileged sites
Reactivation.
Viral immune evasion - hiding, integration vs episomal
Silencing transcription
Passing to next cell (latency program vs integrated vs type of cell)
Passing to offspring?
Viral immune evasion - hiding, immunoprivileged sites.
Neurons, liver. Both have low MHC class I expression and so are poor targets for CTL.
Viral immune evasion - antigenic variation.
o RNA viruses due to high error rate of RNA polymerases
o Segmented viruses
o Recombination.
Inhibiting the effector arm.
Innate, adaptive.
Inhibiting the effector arm - innate.
Intracellular, complement, NK cells.
Inhibiting the effector arm - innate, intracellular.
Inhibit apoptosis
Inhibit interferon pathways
Inhibit cytokine production.
Restriction factors.
Inhibiting the effector arm - innate, complement
Express or capture membrane bound regulators.
Express soluble regulators.
Inhibiting the effector arm - adaptive
Antibodies and CTL mediated killing.
Inhibiting the effector arm - innate, NK cells.
Express homologues of MHC class I. regulate MHC class I expression levels. Inhibit activating receptor function. Express ligands for inhibitory receptors.
Inhibiting the effector arm - adaptive, antibodies.
Do not leave cell.
Bind Fc receptors
Mask antibody binding site.
Inhibiting the effector arm - adaptive, CTL mediated killing.
Prevent generation of viral peptides Inhibit peptide transport into ER Destabilies MHC class I in ER Inhibit MHC class I transport Remove MHC class I from surface.
Immunomodulation,
Inhibit cytokine maturation
Synthesise steroids.
Influence cell to cell interactions.
HIV immune evasion: inhibition of effector arm depends on…
Nef, vif, vpu, vpr and vpx.
Tend to act as adaptors, linking cellular proteins. Resist restriction factors.
HIV immune evasion, innate, intracellular, interferon. INDUCTION OF INTERFERON.
Viral RNA present as ssRNA briefly, RNA:DNA hybrids, ssDNA and dsDNA before latter is transported to nucleus.
Capsid structure also recognised by TRIM5a.
Pathways promote on IRF3 which translocates to nucleus and stimulates transcription of IFN-B, also promotes apoptosis.
HIV immune evasion, innate, intracellular, interferon. INHIBITION OF INTERFERON.
Vpu especially important in targeting IRF3 and preventing IRF3-driven gene induction. Degradation of IRF3 probably via lysosome mediated destruction and blocking of IRF3 activation.
HIV immune evasion, innate, intracellular, RESTRICTION FACTORS.
Trim5a, APOBEC1, Tetherin, SAMHD
HIV immune evasion, innate, intracellular, restriction factors - resisting APOBEC.
o Vif probably links APOBEC to E3 ubiquitin ligases to lead to mislocalisation and degradation. Otherwise this is packaged into virion, and is a deaminase, so hypermutates the virus until it is incompetent otherwise.
HIV immune evasion, innate, intracellular, restriction factors - resisting tetherin
Inhibits release of virions. Vpu acts as an adaptor between proteins in the ubiquitination pathway and tetherin, leading to its degradation.
HIV immune evasion, innate, complement
Express or capture membrane bound regulators.
Importance of CTL mediated killing in HIV.
Important in control of HIV infection. – up to 19% of CD8+ T cells can target HIV. Gag specific response appears especially important at the start. But chronic antigenic stimulation can lead to an exhausted phenotype, e.g. with overexpression of PD-1
HIV: counteracting CTL mediated killing.
Impaired T cell help. Stronger CD4+ responses seem to lead to slower progression to AIDS. MHC class I degradation (Nef) Remove MHC class I from surface (Nef)
HIV: counteracting CTL mediated killing. Nef.
MHC class I degradation. Remove MHC class I from surface.
HIV: counteracting CTL mediated killing. Nef. MHC class I degradation.
Links MHC class I molecules to clathrin adaptor protein 1, leading to misdirection into endosomal/lysosomal pathway and degradation there.
This technique used for other proteins to, e.g. CD4, which prevents retention after budding.
Probably occurs later in infection.
HIV: counteracting CTL mediated killing. Nef. Remove MHC class I from surface.
- Nef mediates the endocytosis of MHC class I by binding membrane trafficking proteins. Activity is dependent on binding of src family kinases. I.e. it mediates the assembly of a kinase complex necessary for endocytosis. Like MHC class I from ER, these are then linked to clathrin adaptor protein 1 and targeted to endosomes.
- Nef also increases endocytosis of other cytokine receptors and CD4, but latter is clathrin dependent and uses AP2.
- Probalby occurs early in infection.
HCMV immune evasion
Hiding - latency, immunoprivilenged site, molecular mimicry.
Counteract effector arm - interferon induction and results, CTL mediated killing.
HCMV immune evasion, molecular mimicry
Prevents recognition of proteins.
HCMV, counteracting effector arm, interferon pathway.
Induction, production and reception.
Also, resultant antiviral state.
HCMV, counteracting effector arm, interferon pathway; INDUCTION
PRRs such as TLRs, NLRs. Probably a central role for dsDNAs in stimulating this, although there are many receptors that signal in response to HMCV infection.
HCMV, counteracting effector arm, interferon pathway; PRODUCTION
o IRF3 and NFkB pathways
UL123-encoded IE2 blocked transcription of NFkB
cmvIL-10 decreases IFNa production.
HCMV, counteracting effector arm, interferon pathway; RECEPTION.
o Unidentified IE or E protein prevents phosphorylation of Jak1, Tyk2 and STAT molecules, apparently by activating phosphatase SHP2, and promotes degradation of Jak1.
o IE1 also appears to relocalise STAT molecules.
HCMV, counteracting effector arm, interferon pathway; ANTIVIRAL STATE
Protein kinase R. TRS1 and IRS1 block this.
o Some genes induction is allowed, and gene-products are then ‘repurposed’ to allow pro-viral outcomes.
IFN-y inducible protein 16 is meant to detect dsDNA, but in HCMV bind the major immediate-early promoter, stimulating viral transcription.
BST-2, tetherin, has been hypothesized to facilitate viral capture to the plasma membrane and hence infection.
HCMV, counteracting effector arm, interferon pathway; CTL mediated killing.
All stages of antigen presentation targeted by members of the herpesviridae. HCMV inhibits expression, loading and presentation of MHC class I, as well as mimicking cellular proteins and inhibits MHC function on the surface with an inhibitory cytokine analogue.
HCMV, counteracting effector arm, interferon pathway; CTL mediated killing. DOWNREGULATION OF MHC class 1.
UL82 and UL83.
HCMV, counteracting effector arm, interferon pathway; CTL mediated killing. PREVENT GENERATION OF VIRAL PEPTIDES.
UL83
HCMV, counteracting effector arm, interferon pathway; CTL mediated killing. RETENTION OF MHC class 1.
US3 (binds tapasin, preventing loading leading to retention) , US10
HCMV, counteracting effector arm, interferon pathway; CTL mediated killing. DEGRADATION OF MHC class 1.
US11, US2. Use ER quality control mechanism meant to degrade misfolded proteins to degrade MHC class I molecules.
HCMV, counteracting effector arm, interferon pathway; CTL mediated killing. INHIBIT MHC class 1
UL82.
EBV: counteracting effect of IFNa/B.
LMP1 binds Tyk and prevents phosphorylation.
EBV: Inhibiting peptide transport into the ER
Conflicting data, but may downregulate TAP and MHC class I.
Vaccinia: counteracting the effector arm.
Intracellular, complement, NK cells.
Vaccinia: counteracting the innate immune system, intracellular.
Inhibit apoptosis
Inhibit interferon pathways
Inhibit cytokine production
Vaccinia: counteracting the innate immune system, inhibiting complement
VCP is a cofactor for factor I, inducing cleavage of C3b and C4b. Can be localised to cell or EEV surfaces by binding the A56 protein.
Acquire host protiens CD55, CD59.
Vaccinia, counteracting NK cells.
Regulate MHC class I expression levels – modestly downregulate. Inhibit activating receptor function. ¬– A56 modulates.
Vaccinia, inhibiting apoptosis.
Pathway: Pro-apoptotic Bad and Bid bind effector proteins Bax and Bak, which oligomerise in outer mitochondrial membrane.
Inhibition: Protein F1 Bcl-2-like fold. Binds and inhibits Bak. N1 inhibits Bad and Bid.
Also, caspases. Inhibited by B13.
Vaccinia inhibiting interferon pathways. INDUCTION.
Minimizes production of PAMPs. Although transcription is from either strand, normally only one strand at a time, to prevent production of dsRNA.
Vaccinia inhibiting interferon pathways. PRODUCTION.
o Blocks induction of signaling pathways.
o Inhibits host protein synthesis, and increase degradation of host mRNAs (cap cleavage by D9 and D10)
o Many, many proteins target downstream of PRR pathway, preventing NFkB transcription. E.g. A49, a Bcl2 like protein which mimics phosphorylated IkB, outcompeting it for degradation by B-TrCP.
o Many inhibit IRF3 activation as well.
o Many proteins are multifunctional and redundant.
Vaccinia inhibiting interferon pathways. RECEPTION.
secretes proteins to capture in solution or on the surface.
o B18 binds IFNa best in solution, but IFN-B defence more intracellular.
o B8 acts as a decoy receptor for IFN-y.
VH1 dephosphorylates STAT proteins inside cell. Probable that there are other inhibitors of JAK/STAT pathway too.
Vaccinia inhibiting cytokine production.
Inhibit formation
Produce soluble receptors
TNF-a
Vaccinia inhibiting cytokine production - FORMATION.
o Some cytokines induced by NFkB – inhibition of this inhibits their production. E.g. IL-1. Can be induced by other pathways, also inhibited by NFkB.
o IL-18 depends on caspase-1. B13 inhibits that.
Vaccinia inhibiting cytokine production - RECEPTORS.
Produce soluble receptors e.g. B15 mimics IL-1R.
Chemokine binding proteins like vCKBP.
Production of all these soluble receptors means interest in using against inflammatory diseases such as rheumatoid arthritis.
Vaccinia inhibiting cytokine formation - TNF-a.
TNF-a has pleiotropic effects including anti-viral effects. Inhibit production via effects on NFkB, inhibit reception by producing soluble decoy receptors, inhibit effects by interfering with downstream signaling.
Key themes of viral immune evasion
Speed, mutability, camouflage and sabotage.
Example of NK cell inhibition; production of homologue of MHC class 1.
MCMV m144
Example of NK cell inhibition - regulating expression of MHC class Is.
Normally HLA-A and HLA-B present peptides for CTL recognition, while HLA-C and HLA-E are more important for repressing NK mediated cytotoxicity.
HIV-1 only significantly downregulates HLA-A, -B.
HCMV upregulates HLA-E.
Preventing production of peptides for loading onto MHC class I
EBV EBNA 1 protein has gly-ala repeat region that indigestible by proteosome
Preventing transport through TAP
HCMV US6 glycoprotein inhibits ATP binding to TAP1, HSV ICP-47 binds TAP inducing conformational change that prevents peptide binding.
Preventing MHC trafficking to surface.
HIV Nef
E gene products of MCMV virus retain in ER, or redirect it for degradation
Targetting MHC class II by EBV
EBV encodes protein BZLF2 to retain MHCII in ER.
Preventing activation of caspases
Vaccinia B13, NLRP-1.
Also inhibit apoptosis by producing mimics of human Golgi anti-apoptotic proteins, GAAPs.
Vaccinia inhibition of IRF3
target TANK kinases.
Altering cytokine production
Make your own - EBV vIL-10.
Inhibit host production
Upregulate production of some; MCMV upregulates IL-10
Diversity in HIV-1 clades - and antibodies
6 clades which can differ by 30% in Env sequence and 14% in Gag.
Even when antibodies are made, most of the epitopes involve recognising glycan moieties (of shield) which are easily changed.
Immunodominant antibodies are non-neutralising
Some antibodies stimulated are auto-reactive and so deleted by tolerance mechanisms
Autoreactive HIV antibodies example
Verkoczy et al noted that B cells producing antibodies to 2F5 and 4E10 were deleted by tolerance mechanisms. Only 5% survived as anergic B cells, this was substantiated by Yang et al 2013 who identified the autoantigens that these antibodies targeted as KYNU and SF3B3.
Antibodies as treatment to HIV
Many are non-neutralising, some autoreactive. Event the best may only act on 10-50% of HIV-1 isolates with an 80% inhibition concentration of 5 ug/ml so too high to be practical (Walker 2011).
Examples of passive immunisation
hCMV immunoglobulin for post-heart-lung transplantation. Given with anti-viral drug.
Zmapp (convalescent serum) given to Ebola patients.
Ideal antiviral
Potent, specific, non-toxic.
Reliable, reliable to produce.
Easy to store and administer.
High barrier to resistance.
Challenge to developing anti-virals - viral factors
Difficult or dangerous to culture in vitro.
No animal model.
Target heterogeneity (e.g. HIV)
Rapid resistance.
Challenges to developing anti-virals
Viral factors
Virus-host interactions.
Challenges to developing antivirals - host/virus interaction factors.
Off target toxicity
In vitro activity vs in vivo activity.
Compartmentalisation of infection.
Delayed access to testing and treatment.
Ways to discover antivirals
Serendipity
Screening
Antiviral drug design.
Ways to discover anti-virals, serendipity.
Sulphonamide antibiotic derivatives had activity against poxviruses.
Ways to discover anti-virals - screening.
Screen known compounds and derivatives.
E.g. adamantane derivatites.
Disadvantages: low success rate by blind screening. Only understand mechanisms retrospectively.
Anti-viral drug desing
Consider anti-virals of veterinary importance.
In silico screening - predict active site screening.
High throughput antiviral screening, automated with microtitre plates.
Human drug development stages
Synthesis of drug candidate In vitro studies Animal studies (sometimes humanised) Phase I - human safety Phase II - small efficacy Phase III - large, prelicensing efficacy Phase IV - post-licensing safety reporting.
Extra info about licensing
Can take 10+ years, costs millions, and licence may be withdrawn.
Compassionate use of unlicensed anti-virals or antivirals for unlicensed indications, based on available studies, by specialist clinicians.
DNA viruses with licensed antivirals
VZV
HSV
HCMV
HBV
RNA viruses with licensed anti-virals
HIV-1 and -2.
HCV
Influenza
RSV
Therapeutic classes of antivirals
Nucleoside analogues Nucleotide analogues Non-nucleoside inhibitors of polymerase or reverse transcriptase. Protease inhibitors Other drugs Immunomodulaters
Antiviral drugs that do not inhibit replication or proteases.
Integrase inhibitor Neuraminidase inhibitors NS5A inhibitor (HCV) Entry inhibitors (HIV) M2 proton channel inhibitors Terminase inhibitors Maturation inhibitors
Mechanisms of actions of antivirals
Inhibit... Replication of genome Assembly of new virions Virion release Maturation of virions Virion entry.
Nucleoside analogues
Aciclovir, guanosine analogue. Ganciclovir = ditto.
Ribavirin = guanosine analogue.
Nucleoside reverse transcriptase analogue
- cytidine analogues (lamivudine)
- thymidine analogues (zidovidine)
- guanosine analogues (abacavir).
Lamivudine
Licensed for HIV and HBV, useful in co-infection, must be used with caution to prevent resistance.
Guanosine
Guanine with ribose ring.
Aciclovir
Versatile administration.
Guanosine analogue; guanine with deoxyribose ring replaced.
Viral thymidine kinase phosphorylates to aciclovir monophosphate, which is a nucleotide analogue. Incorporated into DNA molecule by viral DNA pol. Lack of deoxyribose disrupts extension of DNA molecule by viral DNA polymerase.
Nucleotide analogues
Cidofovir, a DNA pol inhibitor for hCMV.
Nucleotide reverse transcritpase inhibitors for HIV-1 and HBV (tenofovir).
RNA pol inhibitor, sofosbuvir.
A49
Vaccinia protein: a Bcl2 like protein which mimics phosphorylated IkB, outcompeting it for degradation by B-TrCP.
UL82 and UL83.
Degradation of MHC class 1, HCMV
UL83
HCMV prevent generation of viral peptides for MHC loading.
UL82
Inhibit MHC class 1. HCMV.
Us3, Us10
Retention of MHC class 1, HCMV.
Us11, Us2
Use ER quality control mechanism meant to degrade misfolded proteins to degrade MHC class I molecules.
