Viral eye infections…. and antivirals to treat them

1. Viral eye infections…. and antivirals to treat them

2. Herpes simplex virus type 1 Human herpesvirus, infects 40-70% -Primary infection  neuronal latency and persistence for life  sproradic or induced reactivation  Recurrence disease Recurrent stromal keratitis -immune mediated- - collagen disorganization and scarring -blindness requires corneal transplant • defined by the genetics of virus and host • Many shed very frequently- no disease! epithelial keratitis Stromal keratitis Primary infection

3. Infection of neuronslatencyreactivation of HSV-1axonal transport

4. Regulating HSV Lytic/ latent switch -Latency in sensory neurons ( autonomic also?) -viral DNA is episomal and highly chromatin regulated -Only main expression is HSV Latency Associated Transcript -LAT abundantly made at latency -No protein miRNAs? -innate and adaptive cellular Immunity play a role in latency

5. Treating HSV-1 ocular disease Primary infection and Epithelial keratitis Antivirals– ACV or Valtrex- to remove and kill virus Keratitis and stromal disease -disease is immune mediated -goal is to reduce inflammation -STEROIDS -ALWAYS USE AN ANTIVIRAL COVER!!!

6. Varicella zoster virus (VZV) Human herpesvirus, related and like HSV-1 Chickenpoxneuronal latency (decades) zoster (Shingles), pain and PHN

7. Primary VZV infection  Chickenpox • Inhaled by aerosol • Infects immune T cells • tonsils/Waldemyers ring • Transfer to T cells in lymph nodes • Reaches skin by T cell viremia systemic spread • T cell Infiltrate skin –virus seeded in dermal skin layers- • 10-21 days- regulated by innate (IFNa/b) until adaptive immunity develops • VZV cleared by adaptive immunity • Problematic in adults- Pneumonia- Perry and Whyte 1998 Lumpkin E A et al. 2010

8. Establishment of the VZV latent state • VZV in skininfects sensory nerve axon endings retrograde axon transport • Also VZV in T cells directly infiltrate ganglia neuronal latency • VZV systemic spread - entire neuraxis over body autonomic and sensory ganglia

9. Vaccination Live Attenuated varicella vaccine- OKA • Now mandated in US - 5+ years of age • Live attenuated by passage in cuture • -1200 pfu cell sonicate given in arm x 2 • Virus Infects body, but impaired in skin growth long term protection from disease (not VZV infection) • 86-95% effective in children • Induces Herd Immunity- reduce spread in those not protected Shift the incidence of chicken pox to older ages (adults)? If no varicella-will this reduce boosting of immunity throughout life from subclinical exposure? • Lead to earlier zoster?

10. Herpes zoster -in 1/3 of adult lifetimes -one sided --1 in 20 get zoster on head Termed HZO if VZV reactivates from 5th cranial nerve Disease limited to a dermatome What’s different from HSV-1 reactivation? - frequency and age - ganglionic (zosteriform) spread - ganglionitis and damage - one neuron involved (HSV) versus many (VZV)

11. Zoster- Risk Factors Triggers of zoster? • Age – • 84% of cases occur after 60 • more in women • Cellular immune status • AIDS • Radiation Therapy • Cancer (esp. lymphoma) • medical immunosupression therapies • BMT & Transplants (30-55% in a year!) • CD4 appear more important than CD8 in preventing zoster • Is there subclinical reactivation during life? Harpaz R et al 2008

12. Examples of ocular zoster VZV Can infect every ocular tissue to cause:- • Punctate epithelial keratitis (PEK) • Dendritic keratitis • -w/o terminal bulbi • Stromal keratitis/inflammation • -Harder to treat than HSV-1 • Neurotrophic keratitis • -Total loss of corneal sensation • -iatrogenic ulceration • Rarer Findings • Uveitis, retinitis, • Acute retinal necrosis.

13. NeurotrophicKeratopathyThe “diabetic foot” of the eye • ~ 8% of HZO patients develop total loss of corneal sensation ~ 3% of HZO patients develop neurotrophic ulceration • Iatrogenic insults are the main reason that neurotrophic corneas get into trouble.

14. VZV and Pain • 1 million people a year in the US • 90% will seek prescribed medication for pain • 30% will develop debilitating chronic pain Post Herpetic Neuralgia (PHN) • Very difficult to treat (uses • Many PHN patients get no benefits from any form of treatment Why is there pain? Ganglionitis Ganglionic necrosis Neuronal connective plasticity and reorganization Demyelyination Cell-Neuron Fusion-cytoplasmic mixing

15. Other ocular/ neurological complications of VZV • Most are rare and mis-diagnosed • Persistent VZV in CNS vasculature or CNS/brain • leads to …. • Cognitive deficits • Stroke • Headaches • Migraines? • seizures

16. Vaccination to prevent zoster • Zostervax- same as varicella vaccine- 14X more virus • VZV immune people get it. • Recommended > 50 yrs, may eventually need two doses • Is the only human herpesvirus vaccine so far • Partial efficacy - not everyone is protected • 51% drop in zoster incidence • 68% fall in “burden of illness” (including PHN)

18. Adenovirus follicular conjunctivitisvs others-differential diagnosis

19. Adenoviral Infections • non-enveloped virus, • 34Kbp DS-DNA, many viral proteins • At least 57+ identified Serotypes • Three major ocular diseases • Epidemic Keratoconjunctivitis (8, 19 and 37+ several new serotypes and interrecombinant strains) • Pharyngoconjunctival fever (3,4, & 7)

20. Epidemic Kerato-conjunctivitis • transferred by hands, instruments, solutions. • Adenoviruses survive >35 days on dry surface • Epidemics arise from ophthalmologists offices. • Patients remain infectious for 14 days after onset Usually One eye, then other (milder) Lasts 7 days -2 weeks Source of new isolates- - Japan/Asia • Japan-EKC is a reportable disease • Hawaii west coast -mixing ground

21. Clinical Symptoms • Foreign Body Sensation • Tearing • Photophobia • Sore Throat • Breathing Problems • Conjuntivitis NO ANTIVIRAL – YET • -Correct timely diagnosis an issue After virus…... -Subepithelial infiltrates (immune mediated) -last weeks to months - treat with steroids - requires slow withdrawal

22. Adenovirus EKC management

23. Ebola Virus

24. EBV in Ocular Fluid during convalescence Varkey, JB et al NEJM, 2014

25. Zika virus • Human RNA alphavirus • transmitted by mosquitos • Also transmitted in utero and by sexual contact • 80% have no symptoms • Fever rash, arthritis and conjunctivitis are common • No known antiviral or vaccine – yet • Of most concern is ocular development in newborns with microcephaly

26. Ocular complicationsin newborns • Retinal bleeding (Hemorrhagic retinopathy) • Abnormal blood vessel development (missing vessels) • Torpedo maculopathy (topedo shaped lessions in macula

27. A model of Zika and eye disease? Miner et al., 2016, Cell Reports 16, 3208–3218 September 20, 2016 Highlights a. ZIKV infects several different regions of the eye, including the Retina. ZIKV infected the iris, cornea, retina, and optic nerve and caused conjunctivitis, panuveitis, and neuroretinitis in mice. b ZIKV RNA can be detected in tear fluid c Eye and brain infection in adult mice by ZIKV occurs independently of the AXL receptor d ZIKV infection results in apoptosis of neurons of the visual processing pathway

28. CMV and retinitis • 80% are seropositive • Virus is endemic- • early childhood • Usually asymptomatic • Retinitis Rare outside • of HIV/AIDS • -A major factor in “will to live” • Of AIDS patients Signs Photophobia Eye Pain/redness Floaters Vision loss Usually initiates monocular

29. Other Viruses causing conjunctivitis or Eye Disease • CM and EBV herpesviruses affecting most people • may cause conjuntivitis and ,rarely, corneal keratitis • Entero/coxsaccivirus- Hemorrhagic conjunctivitis -begins as eye pain, then red, watery eyes with swelling, - light sensitivity, and blurred vision. • HIV (and everything resulting from it) • Newcastle disease virus • VacciniaMollocsumcontageosum (lid lesions) • Papilloma (lid lesions) • Measles (Conujuntivitis)

30. Important Ophthalmic antivirals Triflorothymidine (viroptic) HSV-1>> VZV Acyclovir and valacyclovir HSV-1 and VZV Ganciclovir and valganciclovir CMV retinitis, Adeno, Foscarnet (phosphonoformate) CMV (GCVr)> HSV,VZV Cidofovir CMV (GCVr) HAART HIV/AIDs

31. Trifluridine (viroptic) Analog of deoxyuridine nucleoside Incorporated into DNA CF3 blocks base pairing in DNA Has higher affinity for viral DNA pol over cell pol Used topically only- toxic systemically

32. Acyclovir, gancyclovir and derivatives

33. Acyclovir (FDA licensed in 1981) • Use Herpes simplex virus 0.1-3 ug/ml Varicella Zoster virus 5-20 human cytomegalovirus 60-200 (not deemed “clinically effective”) • Safe in Long term Prophylaxis treatments • Prodrug mechanism- • only active in virus infected cells • Virtually non toxic in uninfected cell • 100 x more active in HSV—1 infected cell. • Once activated, has higher affinity (50x) for HSV DNA polymerase over cellular DNA polymerase.

34. ACV Mechanism of Action • HSV VZV Thymidine (nucleoside) Kinase activates it • ACV TP binds Viral DNA polymerase >>>>> cell pol • Incorporated into DNA - acts as DNA chain terminator

35. ACV - Resistance • Readily arises in culture • Defect /loss of viral TK • Mutation of DNA polymerase- alters ACV affinity • rarely occurs in vivo- why? • Latency in neurons • TK needed for HSV reactivation • TK- viruses don’t reactivate • ACVR arise In AIDS patients with long treatments • Have Pol mutations: or • Low persistent viral replication: or • Have minimal TK levels • sufficient to enable reactivation from latency • Not sufficient to activate/ phoshorylate ACV

36. Oral forms of Acyclovir Liver Acyclovir Valacyclovir “Valtrex” • ACV alone is degraded by the stomach • Valine Ester derivative has high oral- bioavailability • e.g. 63-72% absorption vs 15% for ACV • Is de-esterified by liver on first pass  ACV • Allows reduced dosing and taking by mouth

37. N N N N N Ph O GMP Gancyclovir O O N N N N N O O To 5’ end O Gancyclovir and Valgancyclovir - drugs to combat HCMV disease O to 3’ end

38. Ganciclovir (Cytovene) • used for hCMV also used for stubborn VZV and HSV • works much better than ACV for CMV disease • retinitis and systemic disease in transplant patients BUT GCV Requires IV dosing but val-GCV (ester form) by mouth • GCV is More toxic than ACV- Why? • GCV-PPP also inhibits host cell polymerase- • much less selective • GCV Does not cross retinal/brain barriers well • Often use ocular implants for retinitis • GCV increases AZT toxicity- bad for HIV patients

39. GCV- Mechanism of action • -a DNA chain terminator • CMV has no TK gene • CMV uses a protein kinase (UL97) to phosphorylate GCV (and ACV) GCV O N N N N N GCV Resistance • Arises due to long treatment for CMV diseases (upto 10% In Retinitis and organ transplants) • Rare- mutations in UL97 protein kinase • ( UL97 is needed for hCMV to efficiently assemble) O O O • Mutations in DNA polymerase that alter affinity • High GCV-resistant CMV have both genes altered

40. OH O O OH OH P P OH OH P CH OH O O OH Foscarnet (phosphonoformate, PFA) P-P • Mechanism of action: • All polymerases need P-P as cofactor • PFA analog of pyrophosphate (P-P) • binds to DNA polymerase • PFA blocks P-P binding • resistance – altered DNA polymerase • Efficacy/toxicity • active on nucleoside resistant viruses • Acts at different site to GCV/ACV • Toxic in bone, kidney, neuronal deposits • Uses: • CMV retinitis and GCVr CMV in transplants • rare use on HSV and VZV ARN • Rare use on systemic HSV and VZV PFA

41. Nucleoside phosphonates • Cidofovir • Licensed for CMV retinitis • Analogs of dNMPs – no initial P step needed • CDV has long intracellular half life • Has activity to many viral DNA polymerases • Works against : many adenoviruses, poxviruses, -(used if smallpox resurrects?) herpesviruses , polyomaviruses, HBV? Could be the universal antiviral drug in not so toxic

42. Lipid Conjugate Technology Exploits Natural Phospholipid Pathways Lysolecithin Polar head Non-polar tail Lipid conjugation enhances absorption and distribution CMX001 Cidofovir

43. Broadly active against dsDNA viruses Approved for treatment of CMV retinitis in patients with AIDS Requires intravenous infusion Black box warning for renal impairment and neutropenia Orders of magnitude more potent than CDV; broadly active Initially being developed for adenovirus, CMV and smallpox Orally available No evidence of nephrotoxicity or myelotoxicity Cidofovir CMX001