1. Dynamics
of
adap.ve
immunity
during
LASV
infec.on
and
proposed
mechanism
for
immune
impairment
Jessica
N.
Hartne>
Graduate
Program
in
Biomedical
Sciences
July
1,
2015
2. Overview
•Lassa Virus
•The Immune System
•Lassa Fever Lab
•Evaluation of the Host Immune Response to LASV Infection
•Antibodies
•Cytokines
•LASV Immune Impairment
•NP Exonuclease Activity
•Impact on TLR3 Pathway
•Conclusions
•Future Directions
3. Arenaviridae
• The first Arenavirus was identified
in 1933 during the St. Louis
encephalitis epidemic (LCMV)
• Other viruses with similar
morphology, serology, and
biochemical features were
discovered by the 1960s and
were classified under the
Arenaviridae family
• Arenaviridae describes the sandy
appearance of the virions
• LASV first reported in Nigeria in
1969 after three missionary-
nurses contracted the illness T. Geisbert et al., 2005
4. Lassa Virus
• Fatal in 5% - 20% of cases
• Deafness most common
complication
• Should be suspected in any
febrile patient in endemic areas
• Pharyngitis, retrosternal pain,
gastrointestinal complaints, and
proteinuria
• Often misdiagnosed or
diagnosed when treatment is no
longer effective
• Estimated number
300,000-500,000 per year
5. Reservoir
• LASV rodent host Mastomys
• Prolific breeders
• Often found in homes in
endemic areas
• Capable of horizontal (and
vertical - shown in LCMV)
transmission
• Viral transmission thought to be
through contact with the feces
or urine of MastomysCourtesy of D. Bausch
6. Prevention and Treatment
• Prevention is important
• Rodent control
• Containment of the sick
• Proper barrier nursing
techniques
• Ribavirin is currently the only
treatment
• Nucleotide analogue antiviral
drug
• Effective only if administered
early in infection
• Other drugs under development
7. Virion Structure
• 50-300nm structure
• Pleomorphic
• Enveloped
• Bisegmented genome
• Single stranded RNA
• Ambisense coding
• Viral proteins include L polymerase, NP, GP1, GP2, and Z
• Ratio in LAS virion of L:NP:GP1:GP2:Z = 1:160:60:60:20
• As reported in Strecker et al. (2003)
Y
Y
Y
Y
Y
YYYY
Y
Y
Y
YY
YYYY
YYYY
vv
Y
Zn
Y
Y Y
Y
YYYY
Y
YYY
Y
YYY
YY
YYY
Y
YY
Y
Y
Y
Y
Y
YYYY
Y
Y
Y
YY
YYYY
YYYY
vv
Y
Zn
Y
YY
Y
YY
YY
Y
Y
YY
YY
YYYY
YYYY
v
v
Y
Y
Glycoprotein 1:
GP1, 42 kDa
Stable Signal Peptide:
SSP, 7 kDa
Glycoprotein 2:
GP2, 38 kDa
Large RNA (L):
7279 b.p.
Small RNA (L):
3417 b.p.
Large protein
(L): RDRP,
290 kDa
Z matrix protein:
Z, 12 kDa
Nucleoprotein:
NP, 60 kDa
host cell
ribosome
envelope
60
60
20?
20
160 1
#?
L RNA < S RNA (multiple copies/ virion)
Subgenomic RNA species
Cellular RNA (28S, 18S, 4-6S, ribosomes)
tRNA?
Glycoprotein
Complex:
GPC, 87 kDa
Courtesy of L. Branco
8. Virion Structure
• 50-300nm structure
• Pleomorphic
• Enveloped
• Bisegmented genome
• Single stranded RNA
• Ambisense coding
• Viral proteins include L polymerase, NP, GP1, GP2, and Z
• Ratio in LAS virion of L:NP:GP1:GP2:Z = 1:160:60:60:20
• As reported in Strecker et al. (2003)
K. Hastie et al., 2011
9. Cellular Tropism
• Virion binds to surface receptor
alpha-dystroglycan (α-DG)
• Endothelial cells, monocytes,
macrophages, dendritic cells
• 99% of α-DG was found on
dendritic cells in the spleen
• LASV targets DC early in infection
in NHP
• Pathogens targeting DC shown to
impair immune arms
• Virion is internalized by uncoated
vesicles
• pH-dependent membrane fusion with
the endosome releases viral contents
Y. Hara et al., 2011
16. ReLASV IgM/IgG-Capture
ELISA Diagnostic
• ELISA plates coated with recombinant NP and
GPC produced in bacteria and mammalian
cells respectively
• captures LASV epitope-specific antibodies
from serum of suspected patients
• data used for serosurveys and as predictor
of outcome
• Suspected patients confirmed by reLASV
antigen capture ELISA and/ or LASV PCR
• Data presented here grouped by:
1. Antigen status (positive/negative for
LASV NP antigen)
2. Antibody class status (positive/
negative for any LASV antigen)
3. Antibody epitope specificity (GPC or
NP)
4. Change in antibody status over time
(increase/positive/decrease/negative)
23. Antibody Profiles
US N = US Normal
MOY and BOM NHS = non-febrile
Sierra Leone donors with no reported
history of LASV infection
LF FU = LF convalescent
LF NF = acute, survived
LF F = acute, succumbed
• IgM present at varying levels
during acute illness
• IgM persists into conva-
lescence
• Sierra Leonian population has
been exposed to LASV
• IgG takes longer to respond
• IgG doesn’t have an opportu-
nity to appear in fatal cases
Adapted from Branco, Grove et al., 2011
24. Antibody Profiles
US N = US Normal
MOY and BOM NHS = non-febrile
Sierra Leone donors with no reported
history of LASV infection
LF FU = LF convalescent
LF NF = acute, survived
LF F = acute, succumbed
• IgM present at varying levels
during acute illness
• IgM persists into conva-
lescence
• Sierra Leonian population has
been exposed to LASV
• IgG takes longer to respond
• IgG doesn’t have an opportu-
nity to appear in fatal cases
Adapted from Branco, Grove et al., 2011
25. Antibody Profiles
US N = US Normal
MOY and BOM NHS = non-febrile
Sierra Leone donors with no reported
history of LASV infection
LF FU = LF convalescent
LF NF = acute, survived
LF F = acute, succumbed
• IgM present at varying levels
during acute illness
• IgM persists into conva-
lescence
• Sierra Leonian population has
been exposed to LASV
• IgG takes longer to respond
• IgG doesn’t have an opportu-
nity to appear in fatal cases
Adapted from Branco, Grove et al., 2011
26. Antibody Profiles
US N = US Normal
MOY and BOM NHS = non-febrile
Sierra Leone donors with no reported
history of LASV infection
LF FU = LF convalescent
LF NF = acute, survived
LF F = acute, succumbed
• IgM present at varying levels
during acute illness
• IgM persists into conva-
lescence
• Sierra Leonian population has
been exposed to LASV
• IgG takes longer to respond
• IgG doesn’t have an opportu-
nity to appear in fatal cases
Adapted from Branco, Grove et al., 2011
27. Antibody Profiles
US N = US Normal
MOY and BOM NHS = non-febrile
Sierra Leone donors with no reported
history of LASV infection
LF FU = LF convalescent
LF NF = acute, survived
LF F = acute, succumbed
• IgM present at varying levels
during acute illness
• IgM persists into conva-
lescence
• Sierra Leonian population has
been exposed to LASV
• IgG takes longer to respond
• IgG doesn’t have an opportu-
nity to appear in fatal cases
Adapted from Branco, Grove et al., 2011
28. Antibody Status
does not correlate with time in convalescence
Adapted from Branco, Grove et al., 2011
42. Conclusions about the humoral
response to LASV infection
Courtesy of R. Garry and L. Branco
43. Conclusions about the humoral
response to LASV infection
1. NP-IgM response tends to be stronger than GPC in acute illness
Courtesy of R. Garry and L. Branco
1
44. Conclusions about the humoral
response to LASV infection
1. NP-IgM response tends to be stronger than GPC in acute illness
2. NP-IgG generated in late-phase of acute illness more likely than GPC-IgG
Courtesy of R. Garry and L. Branco
1 2
45. Conclusions about the humoral
response to LASV infection
1. NP-IgM response tends to be stronger than GPC in acute illness
2. NP-IgG generated in late-phase of acute illness more likely than GPC-IgG
3. IgM lasts into convalescence and is most likely specific to NP because
GPC-IgM tends to decrease in convalescence
Courtesy of R. Garry and L. Branco
1 2
3
61. Conclusions about the cytokine
response to LASV infection
1. Dysregulation of cytokines early in acute illness with
quiescence once humoral response was established
62. Conclusions about the cytokine
response to LASV infection
1. Dysregulation of cytokines early in acute illness with
quiescence once humoral response was established
2. LF F cohorts tended to have elevated levels of
cytokines
63. Conclusions about the cytokine
response to LASV infection
1. Dysregulation of cytokines early in acute illness with
quiescence once humoral response was established
2. LF F cohorts tended to have elevated levels of
cytokines
3. Pro-inflammatory cytokines tended to be higher in
individuals with decreasing or negative antibody levels
64. Conclusions about the cytokine
response to LASV infection
1. Dysregulation of cytokines early in acute illness with
quiescence once humoral response was established
2. LF F cohorts tended to have elevated levels of
cytokines
3. Pro-inflammatory cytokines tended to be higher in
individuals with decreasing or negative antibody levels
4. Memory T cells were not abundant in convalescent
individuals
77. Conclusions
• Antibodies:
• NP is immunodominant antigen
• IgM lasts into convalescence
• Cytokines:
• Dysregulation during early infection
• Elevated levels associated with mortality
• Elevated levels associated with decreasing or negative antibody response
• NP Exonuclease activity
• Both N-terminus and C-terminus WT and mutants possess exonuclease
activity
• Capable of interfering with immune activation via the TLR3 pathway by
degrading dsRNA
78. Future Directions
• Perform radioassays with full length rNP and mutants
• Determine if N-terminus NP has cap-snatching
capabilities
• Perform experiments HEK-Blue™ hTLR3 cells using live
virus
• Transfect human dendritic cells with rNP and mutants
and stimulate with PiC, and infect human dendritic cells
with live virus
• Use TLR3 k/o line as control
