adaptive immunity: understanding cross-reactive responses lorena e. brown the university of...
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Adaptive Immunity:Understanding cross-reactive responses
Lorena E. BrownThe University of Melbourne
Australia
Is vaccination of everyone from birth onwards with current inactivated vaccines desirable?
Would a population devoid of infection-induced cross-protective immunity be worse off in situations of:
• Vaccine mismatch/shortage• Emergence of a new subtype• Elderly experiencing OAS or waning B cell immunity• Lack of vaccine efficacy in >30% population
Modelling of the evolution of influenza virus suggests short-lived crossreactive immunity is essential to explain the linear nature of antigenic drift.
• Without this would we start to see more strain diversity and corresponding difficulty in infection control?
What adaptive immune mechanisms are cross-subtype reactive?
• CD8+ cytotoxic T cells (CTL)– kill cells infected with all type A viruses– can recognise peptides from internal proteins– thought to speed recovery – if present in high numbers at site of infection as
memory cells may lead to subclinical outcome
• Non-neutralising antibodies that bind to viral antigens on infected cells– Lyse infected cells by Ab +C’ or ADCC– Best studied is M2 ectodomain
What adaptive immune mechanisms are cross subtype reactive?
Stem domain:– Mabs from human B cells (Throsby et
al. PLoS ONE 3(12): e3942; Ekiert et al. Science. 2009 324:246; Corti et al. J. Clin
Invest 2010 120:1663)– Induced by DNA/ split vaccine
prime boost (Wei et al., Science 2010 329, 1060)
Site B epitope– Yoshida et al. PLoS Pathog. 2009 5(3):
e1000350
10.1126/science.1195116
• Antibodies to conserved regions of HA
Site B
Investigating cross-protective mechanisms against H5N1 in the ferret model
Days
3-5 mth old male/femaleseronegative
Challenge 106 EID50 wt H5N1
A/Vietnam/1203/2004
VaccinergA/Vietnam/1194/2004 or
other split virus formulations i.m.
• serology (HI, micro virus neut.)• weight• temperature (rectal, transponder)• clinical symptoms • activity score• post mortem tissues for histology
• virus isolation- rectal swab- nasal wash- oral swab- organs post mortem
Days 3, 5 and 7 and 14 or at
humane endpoint
(systemic and pulmonary disease)
0 3 wk 7 wk
H5N1 inactivated split vaccines are fully protective when formulated with adjuvant
Alert and playfulAlert, play only when inducedAlert but not playfulNeither alert nor playfulCulled at humane endpoint
*IMX = ISCOMATRIXTM
Middleton et al. J. Virol 2009 83:7770
Activity scoreD
ay
po
st
ch
all
en
ge
165 166 167 168 177 178 179 180161 162 163 164
1
3
567
14
4
2
PBSFluvaxTM + AlPO4
FluvaxTM + IMX*
FluvaxTM
157 158 159 160
* IMX = ISCOMATRIXTM adjuvant
Seasonal influenza vaccine can protect against H5N1
173 174 175 176 169 170 171
H3N2 + IMX
H1N1 + IMX
The H1N1 component is inducing the cross-protection
Despite any crossreactive immunity measured by HI or VN assays
Vaccine Pre-challenge HIGMT fraction respond
Pre-challenge VNGMT fraction respond
H5N1 + AlPO4 45 4/4 45 4/4
H5N1 + IMX 45 4/4 128 4/4
Fluvax + AlPO4 <4 0/4 <4 0/4
Fluvax + IMX <4 0/4 <4 0/4
* IMX = ISCOMATRIXTM adjuvant
Crossreactive responses potentially against N1 BUTreassortant H3N1 vaccine shows no protection against H5N1
0
0.5
1
1.5
2
2.5
1 2 4 8 16 32 64 128
Dilution
NA
acti
vit
y
H3N1H1N1H3N2rNA
However, to maintain the HA/NA activity balance the H3N1 virus: • has 8-fold less enzyme activity (fetuin cleavage assay)• contains 8-fold less NA (Western blot)
Da
y p
os
t c
ha
lle
ng
e
1
3
567
14
4
255 256 257 258
H1N1 + IMX
251 252 253 254
H3N1 + IMX
259 260 261 262
H3N2+ IMX
PBS
271 272 273 274
Activity score
Ferret number
There is an imperfect correlation between neuraminidase inhibition activity of ferret serum and protection
PBS H1N1 H3N1 H3N2 rNA rNP
Vaccines formulated with ISCOMATRIXTM adjuvant
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
= survivors
NI
acti
vity
of
pre
-ch
alle
ng
e fe
rret
ser
a a
gai
nst
H5N
1
Conclusions from ferret data:
• Seasonal influenza vaccine can provide some protection against H5N1, particularly when formulated with an adjuvant.
• The H1N1 component provides the cross protective immunity
• This is not reflected by HI or VN assays but imperfectly by NI assays, so the N1s share epitopes for Ab and we should not discount their role in crossprotection.
• An H3N1 virus was non-protective, possibly due to low N1 levels, nor was H3N2 virus so these crossreactive responses are NOT HETEROSUBTYPIC
• To induce true heterosubtypic responses we can prime CD8+ T cells (CTL)
• These are not induced efficiently by inactivated split virus vaccines
• A different type of vaccine that delivers antigen to the cytoplasm of dendritic cells is required
• Can’t adequately study these in ferrets
Testing benefits of adding CTL-inducing component to split virus vaccine for
seasonal influenza
Suboptimal dose of seasonal split vaccine• mimics people responding poorly to vaccine• mimics situation of vaccine mismatch
CD4+ T cell epitope
CD8+ T cell epitopeK
Pam2Cys
Suboptimal dose of lipopeptide• will induce influenza-specific CD4+ and CD8+ T cells• will not prevent infection but aid clearance
TLR2
immature DC mature DC
lipopeptide
costimulatory molecule
• lipopeptide binds to TLR2 on DC surface via Pam2Cys
• lipopeptide enters cells by TLR2-mediated endocytosis so Ag can enter the class II processing pathway
• Ag can also escape the endosome and so can enter the class I processing pathway
• DC maturation is induced by TLR signalling; costim. and MHC II upregulated
Help for:•Antibody production•priming of long lived memory CD8+ T cells
T
MHC molecules
CD8+T cells
The lipopeptide used here do not have any B cell epitopes
Lipopeptides
CD4+T cells
T
Kill virus-infected cells
10ug HA intranasally
0
1
2
3
4
5
0
1
2
3
4
5
6
10ug HA subcutaneously
0
1
2
3
4
5
0
1
2
3
4
5
6
Mixtures of suboptimal split vaccine and lipopeptide induce improved viral clearance
Mice given a single dose of vaccine
3 weeks later mice are bled then challenged with A/Memphis/1.71 virus
5 days later lung virus titres are determined
10ug HA intranasally
0
1
2
3
4
5
0
1
2
3
4
5
6
10ug HA subcutaneously
0
1
2
3
4
5
0
1
2
3
4
5
6
Mixtures of suboptimal split vaccine and lipopeptide induce improved viral clearance
Slight enhancement of Ab levels; sub cut combination vac. is approaching live virus levels
0.3ug HA intranasally
0
1
2
3
4
5
0
1
2
3
4
5
6
0.3ug HA subcutaneously
0
1
2
3
4
5
0
1
2
3
4
5
6
With even lower doses of split vaccine the improvement is best seen when vaccine is given by the i.n. route
Though slightly enhanced, Ab levels are still low – other mechanisms may need to come into play
0
5000
10000
15000
20000intranasal subcutaneous
Large numbers of activated CD8+ T cells are present in the lungs of intranasally-primed mice 5 days after
challenge with influenza
influenza-specific IFN-producing CD8+ T cells in the lungs measured by intracellular cytokine staining 5 days after infection
CTLs expanded by infected cells as not enough antibody to remove virusthese are as numerous as when induced by prior virus infection
note that 5 days is before any T cells are present as a result of the challenge virus
Conclusions from lipopeptide/split virus data:• Suboptimal doses (10µg and 0.3µg) of split virus leads to poor antibody
production and viral clearance
• Viral clearance can be improved by the addition of a suboptimal dose of lipopeptide that induces T cell responses
• Extra “help” from CD4+ T cells plus increased DC activation during priming may provide a greater quantity or quality of Ab to enhance the 10 µg dose of split virus.
• When antibody is insufficient to control infection rapidly, as with the 0.3 µg dose of split virus, CTL-memory cells in the lungs can be activated and expanded to aid clearance
• Triggering of CTL memory cells already present in the lung (i.n. delivery) may be more efficient than relying on trafficking of memory cells from other organs (sub. cut. delivery)
Different arms of the adaptive immune response are acting in synergy in these responses to the combination vaccines
Conclusion• Co-induction of T cell and antibody responses by influenza
vaccines may provide better protection against disease when seroconvertion has not been adequate due to: – vaccine mismatch, – waning B cell responsiveness, – original antigenic sin, – individuals not recently vaccinated – for emergence of a new subtype.
• Only through a greater understanding of the mechanisms of cross-protection and their induction can we hope to create vaccines that provide us with the much needed “safety net” of heterosubtypic immunity.
University of Melbourne• David Jackson• Joanna Cobbin • Weiguang Zeng
WHO Collaborating Centre for Influenza Reference and Research
• Ian Barr
Main contributors
CSL Limited• Steve Rockman• Martin Pearse
CSIRO Australian Animal Health Laboratory
• Deborah Middleton
Urgent Research into a Potential Avian Influenza-Induced Pandemic Grant Scheme