1. Viral Persistence

2. Viral persistence in vivo: Some Examples
Viral Titer (Log10 scale)
Sites of persistence are usually terminally differentiated cells

3. Viral persistence in vivo: Some Examples and Rules
RNA and DNA viruses can persist
Strategies for persistence can range from “high-titer” replication to “latency” to “smoldering” infections
Corresponding strategies for evasion of host immune responses
High titer replication:
Noncytocidal vs rapid replenishment of target cells
Ineffective immune clearance due to tolerance, immune complex formation, viral variation etc.
Latency:
Viral genome is maintained in non-replicative mode
“Hidden” from immune surveillance
Smoldering infections:
Continuous replication at low levels
Effective immune clerance is prevented by antigenic variation, infectious immune complex, transmission via intracellular bridges etc.

4. Cell culture models of viral persistence

5. Determinants of viral persistence can be mapped
Reovirus: Respiratory Enteric Orphan Virus (dsRNA genome)
Reovirus is a lytic virus but can be induced to cause persistent infections in cell culture by co-infecting cultures with a lytic wild type virus (Type 2 wt) and a temperature sensitive variant (Type 3 ts).
The virus isolated late after persistent infection is a reassortant carrying the genes of the T2 wt virus except for the S4 and S1 genes of the T3 ts variant virus, and it appears that these two gene segments are responsible for the persistent phenotype
S4(s3: major outer capsid protein) and S1(s1 viral attachment protein) from Type 3ts reduce the efficiency of viral entry, thus reducing likelihood of overwhelming lytic infection

6. Evidence for immune clearance of viral infection: example
West Nile Virus: IC injection of
suckling mouse LD50 into adult rats
If immune system
can clear virus,
what accounts for
virus persistence
Log10 titer per gm
No difference in titer in first week,
implies difference is due to subsequent immune response
(which is abrogated by Cytoxan treatment)

7. Mechanisms of persistence and escape from immune surveillance
High titer persistence
Not acutely cytocidal or
Target cells are replenished at high rate, and
Tolerance (absence of virus-specific immunity)
Deletion of naive T-cell clones
Exhaustion of peripheral virus-specific T-cell clones
Absence of specific Ab response
E.g. HBV (fig. 6.4), LCMV (fig. 7.4) and HIV

8. Mechanisms of High Titer Persistence: LCMV & Immune tolerance
(Fig. 7.4)
Mouse infected at high dose---> viral persistence
Viremia
CTL
Log10 titer per gm
Log10 titer per gm
Exhaustion
of LCMV-specific
CTL

9. Mechanisms of persistence and escape from immune surveillance
High titer persistence
Not acutely cytocidal or
Target cells are replenished at high rate, and
Tolerance (absence of virus-specific immunity)
Deletion of naive T-cell clones
Exhaustion of peripheral virus-specific T-cell clones
Absence of specific Ab response
E.g. HBV (fig. 6.4), LCMV (fig. 7.4) and HIV
Nonlytic viruses
a-LCMV Abs circulate as immune complexes
LCMV persistence can be terminated by adoptive transfer of virus specific CTL
Similar for HBV ( see fig. 5.11)
“Lytic” viruses
SIV/HIV
Constant replenishment of target cell pool

10. Mechanisms of persistence and escape from immune surveillance
Latency (e.g HSV, VZV, EBV, CMV)
Virus enters and replicates in permissive cells at portal of entry, after immune induction, virus appears cleared but actually becomes latent in another cell type
Genome may be maintained chromosomally (integrated) or episomally
If genome is in terminally differentiated cells (e.g. neurons for HSV), no need to replicate genome, but signals required for re-activation (e.g. fever, sunburn, trigeminal nerve insult)
Axoplasmic spread towards periphery, conducts virus to skin-->”cold sores”

11. Herpes Simplex Virus
Retrograde transport of virions from exposure site
to dorsal root ganglion
Remains latent
Activation results in anterograde transport to
epithelial surfaces via peripheral sensory nerves,
replication in epithelium results in vesicles

12. Mechanisms of persistence and escape from immune surveillance
Latency (e.g HSV, VZV, EBV, CMV)
Virus enters and replicates in permissive cells at portal of entry, after immune induction, virus appears cleared but actually becomeslatent in another cell type
Genome may be maintained chromosomally (integrated) or episomally
If genome is in terminally differentiated cells (e.g. neurons for HSV), no need to replicate genome, but signals required for re-activation (e.g. fever, sunburn, trigeminal nerve insult)
Latently infected cells express little if any viral proteins, permitting escape from immune surveillance

13. Mechanisms of persistence and escape from immune surveillance
Smoldering Infections
Infectious virus is produced, but at minimal levels
Virus continues to spread, may produce progressive chronic disease
Detectable immune response, sometimes immune response may even be hypernormal (due to chronic viral antigenic challenge)
Paradox: why does virus continue to spread in the presence of a potentially effective immune response

14. Mechanisms of persistence and escape from immune surveillance: Smoldering Infections
Immunological privileged sites
Brain
Blood brain barrier limits trafficking of lymphocytes thru the brain
Neurons express little or no MHC Class I (resulting in little presentation of viral antigens and ineffective CTL response)
Experimental evidence: allogeneic/xenogeneic grafts survive better in the brain than in the skin or other sites
Kidney
LCMV is cleared more slowly from kidney than any other tissues
??inability of lymphocytes to cross subendothelial basement membrane

15. Mechanisms of persistence and escape from immune surveillance: Smoldering Infections
Immunological privileged sites
Intracelluar Bridges
Cell to cell spread of virus without exposure to immune effector mechanisms (e.g. Abs)
Measles in SSPE: neuron to neuron spread in the presence of high titers of neutralizing antibodies
About 1:100,000 primary measles infection results in SSPE
Virus implicated in SSPE is maturation defective--either mutations in matrix protein or envelope glycoprotein, thus, selects for efficient cell to cell spread

16. Mechanisms of persistence and escape from immune surveillance: Smoldering Infections
Immunological privileged sites
Intracelluar Bridges
Suppression of MHC Class I Expression
Virus infected cells rendered less sensitive to CTL attack
Adenovirus E1A
SIV/HIV nef
Viremia

17. Mechanisms of persistence and escape from immune surveillance: Smoldering Infections
Immunological privileged sites
Intracelluar Bridges
Suppression of MHC Class I Expression
Infectious Immune Complexes
Ab-coated virus remains infectious
Ab-virus complex may be internalized by Fc receptors on macrophages-- Ab dissociates from virus in vacuoles, permitting infection of macrophages
LCMV, Aleutian Disease Virus can form infectious immune complexes and macrophages are major host cell
Addition of anti-mouse IgG
remove infectivity
Virus
a-LCMV Y Y Y Y
a-mouse IgG

18. Mechanisms of persistence and escape from immune surveillance: Smoldering Infections
Immunological privileged sites
Intracelluar Bridges
Suppression of MHC Class I Expression
Infectious Immune Complexes
Impaired CTL function
Ag specific CTL may be deficient in effector molecules
HIV specific CTL identified by tetramer staining are deficient in perforin content
What would be your controls?
E (Effector Cell)
CTL
MHC Class I
HIV antigen
T (Target Cell) Y

Staining using
a-perforin Abs
HIV-specific
CTL
SAV

19. Mechanisms of persistence and escape from immune surveillance: Smoldering Infections
Immunological privileged sites
Intracelluar Bridges
Suppression of MHC Class I Expression
Infectious Immune Complexes
Impaired CTL function
Antigenic variation
Selection for neutralization resistance; allows for viral persistence in the presence of Ab response
11 X 105
pfu/ml

20. Mechanisms of persistence and escape from immune surveillance: Smoldering Infections
Immunological privileged sites
Intracelluar Bridges
Suppression of MHC Class I Expression
Infectious Immune Complexes
Impaired CTL function
Antigenic variation
Selection for neutralization resistance; allows for viral persistence in the presence of Ab response
In vivo selection for neutralization escape variants
Ab escape
EIAV
--Immune sera neutralizes virus
from earlier time points but not
concurrent virus or virus thereafter
--evidence of antigenic drift, and
explains viral persistence even in the
face of Ab response

21. Mechanisms of persistence and escape from immune surveillance: Smoldering Infections
Indicates humoral immunity
plays a role in viral clearance
Immunological privileged sites
Intracelluar Bridges
Suppression of MHC Class I Expression
Infectious Immune Complexes
Impaired CTL function
Antigenic variation
Selection for neutralization resistance; allows for viral persistence in the presence of Ab response
In vivo selection for neutralization escape variants
Ab escape
EIAV
LCMV
Log10 titer/ml
Viremia
Neutralizing Ab titer

22. Mechanisms of persistence and escape from immune surveillance: Smoldering Infections
Immunological privileged sites
Intracelluar Bridges
Suppression of MHC Class I Expression
Infectious Immune Complexes
Impaired CTL function
Antigenic variation
Selection for neutralization resistance; allows for viral persistence in the presence of Ab response
In vivo selection for neutralization escape variants
Ab escape
EIAV
LCMV
HIV
Viral Env from Day 16
138
212
381
772
X.Wei. et. al. (2003) Antibody neutralization and
escape by HIV. Nature 422:307

23. Mechanisms of persistence and escape from immune surveillance: Smoldering Infections
Immunological privileged sites
Intracelluar Bridges
Suppression of MHC Class I Expression
Infectious Immune Complexes
Impaired CTL function
Antigenic variation
Selection for neutralization resistance; allows for viral persistence in the presence of Ab response
In vivo selection for neutralization escape variants
Ab escape
EIAV
LCMV
HIV
CTL escape
HIV, LCMV

24. DNA vaccinated rhesus macaques challenged with
pathogenic SHIV 89.6P ?
20 weeks

25. single amino acid change
CTL escape via
single amino acid change
in immunodominant
CTL epitope
Viral Load
CD4
T-cell
count
p11C
Tetramer
(Immunodominant)
p41A
tetramer
p68A
tetramer
weeks

26. Diseases associated with persistant viral infections: selected examples