Sources, Consequences and Uses of Antigenic Diversity in Streptococcus pneumoniae
Marc Lipsitch
#ABPHM15
Antigenic diversity: a key concern for public health microbiologyMany current vaccines focus on diverse antigens
“Easy” vaccines already here: relatively conserved Ag, good natural immune response
Now the hard ones remainreverse vaccinologyserotype/strain
replacement
Malaria
Meningococcal disease
MMR, DPT
Pneumococcal disease
Serotype replacement in pneumococci: the quest to understand and predict
Serotype replacement: outgrowth of nontargeted serotypes (in carriage and disease) when a serotype-specific vaccine is deployed
Serotype replacement presupposes standing diversity. What maintains that diversity in the first place?
T Pilishvili et al. JID 2010Invasive disease, US, 65+
PC Wroe et al. PIDJ 2012, carriage in kids, MA
Evolutionary explanations for standing genetic diversity• Neutral variation: mutation and drift• Negative frequency-dependent selection:
common variants become disadvantageous- Acquired immunity often assumed responsible- Must outweigh directional selection
• Host-specific adaptation and linkage to other loci maintained by epistasis (S Gupta, M Maiden et al.)
Broader context: diversity at many antigenic loci, not just capsule
Mediated by:
Targeted on:
Antibody Antibody CD4+ T Cell
Capsule(polysaccharides)
Protein(Ab antigen)
sterilizing
Protein(T cell antigen)
duration-reducing
Host immunity
Talk outline
1. Causes and consequences of antigenic diversity: the capsule
2. Causes and consequences of antigenic diversity: proteins
3. Using antigenic (and other genetic) diversity for gene discovery
1. Diversity of capsules
Mediated by:
Targeted on:
Antibody Antibody CD4+ T Cell
Capsule(polysaccharides)
Protein(Ab antigen)
sterilizing
Protein(T cell antigen)
duration-reducing
Host immunity
Pneumococcal capsule and serotypes
• Sugar coating around the bacterial cell
• Encoded by polysaccharide biosynthetic enzymes of cps locus
• 92+ serotypes• Affects many properties of
the pneumococcus – host interactionJO Kim et al. I&I 1999
Standing diversity of pneumococcal serotypes
O Abdullahi et al. PLoS One 2012
Comparing pairs of carriage studies, ~7 of the top 10 serotypes in any study were in the top 10 found in any other study (91 pairwise comparisons) S Cobey & Lipsitch Science 2012
Certain serotypes have no “right to exist” – they are poor on all measured fitness components
Less encapsulated
Easily cleared by phagocytes, short duration
Low Prevalence
More encapsulated
Resistant to phagocytes,long duration
High Prevalence
Polysaccharide structure(few carbons/repeat)More negative charge
Less negative chargePolysaccharide structure
(high carbon/repeat)
Less polysaccharide/Less rigid capsule
More polysaccharide/More rigid capsule
Low acquisition rate
High acquisition rate
Poor competitors
Good competitors
Serotypes1,4,5 etc
Serotypes19A, 19F,23F, 6A etc
Daniel Weinberger (modified) reflecting work by himself, Krzysztof Trzcinski, Yuan Li, Claudette Thompson, Richard Malley, Derrick Cordy, Andrew Bessolo
Some examplesCompetition of isogenic capsule variants in mouse nose
In vitro survival from human neutrophil phagocytosis predicts prevalence in human population
K Trzczinski et al. in prep D Weinberger et al. PLoS Path 2009
Weak, partial, serotype-specific immunity reduces acquisition of previously-experienced serotypesTable 3. Effect of prior colonization with a particular serotype on new acquisition of the same or different serotype in toddlers in Israel. GEE analysis. Odds ratio with 95% confidence interval. Adjusted for prior exposure to other types, age, age at study entry, swab + at prior visit.
Previous Colonization with type:
6A 6B 14 15 19A 19F 23A 23F
New acquisition of type:
6A 0.48*(0.27-0.84)
0.55(0.28-1.08)
1.02(0.59-1.75)
1.27(0.78-2.06)
0.63(0.31-1.28)
0.62(0.37-1.02)
0.87(0.45-1.68)
0.75(0.47-1.21)
6B 0.76(0.31-1.90)
0.87(0.26-2.87)
0.79(0.24-2.62)
2.16(0.88-5.31)
2.09(0.69-6.36)
1.38(0.58-3.28)
1.46(0.50-4.28)
1.95(0.44-2.60)
14 0.96(0.46-2.03)
1.02(0.39-2.67)
0.08*(0.01-0.66)
0.37(0.43-1.93)
0.72(0.24-2.17)
1.01(0.49-2.08)
0.69(0.23-2.10)
0.82(0.40-1.68)
15 1.14(0.71-1.84)
1.24(0.70-2.20)
1.15(0.67-1.99)
1.07(0.64-1.79)
1.42(0.80-2.50)
1.09(0.68-1.73)
2.07*(1.24-3.46)
1.16(0.72-1.85)
19A 1.32(0.58-3.03)
1.95(0.74-5.15)
2.24(0.99-5.11)
0.53(0.22-1.26)
0.58(0.15-2.21)
1.32(0.60-2.93)
1.28(0.49-3.37)
1.15(0.52-2.56)
19F 1.43(0.85-2.41)
0.87(0.44-1.73)
0.65(0.34-1.27)
1.02(0.60-1.72)
0.80(0.37-1.74)
0.90(0.50-1.61)
1.16(0.60-2.26)
0.86(0.52-1.42)
23A 1.37(0.64-2.95)
1.57(0.69-3.56)
1.22(0.54-2.75)
0.95(0.45-2.02)
1.73(0.74-4.03)
1.65(0.81-3.36)
0.51(0.14-1.84)
1.38(0.67-2.86)
23F 0.63(0.36-1.09)
1.57(0.83-2.95)
1.41(0.78-2.54)
1.00(0.56-1.79)
1.17(0.60-2.30)
0.90(0.52-1.56)
0.45(0.17-1.17)
0.47*(0.26-0.86)
NOTE: * p<0.05. D. Weinberger et al. 2008 JID
Mouse experiments: Acquired immunity that transcends serotype is duration-reducing, not sterilizing
Y Lu, et al. PLoS Pathogens 2008
This immunity is antibody-independent, dependent on CD4+ Th17 cells
In humans: duration of carriage declines with age (~immunity?)
M Lipsitch et al. Epidemiology 2012
Immunity: summary
• Weak serotype-specific immunity- Provides advantage of rare types, balancing fitness
differences
• Acquired serotype-nonspecific immunity- Reduces duration not acquisition- CD4+ Th17 cells and neutrophils
- Greatest reduction in duration for fittest serotypes, reducing fitness differences
Together, these two forms of immunity permit realistic levels of serotype coexistence
Cobey & Lipsitch 2012 Science
Other patterns reproduced• Increase in serotype diversity
with age*• Stability of rank order*• Decrease in carriage duration
with age• Frequency of co-colonizations*• Epidemics of rarer serotypes*• Serotype replacement after
vaccination*
* Obtained from public health surveillance data
Cobey & Lipsitch 2012 Science
Adapting the model to full fit of carriage prevalence in MA before and after PCV7
T Fussell, D Weinberger, S Cobey unpublished
Vaccine-related serotypes systematically exceeded predicted prevalence postvaccine: suggests natural immunity between serotypes within a serogroup
19A
6C23A
23B
Genomic perspective: serotype switching more common within serogroup than between
We excluded several non-immunologic hypotheses to explain this phenomenon. By exclusion, a leading candidate for conservation of serogroup in switches is cross-immunity within-serogroup, coupled with strain structure maintained by immune-mediated epistasis (S. Gupta et al.)
2. Diversity of protein antigens
Mediated by:
Targeted on:
Antibody Antibody CD4+ T Cell
Capsule(polysaccharides)
Protein(Ab antigen)
sterilizing
Protein(T cell antigen)
duration-reducing
Host immunity
The whole Spn genome varies
N Croucher et al. Nat Genet 2013
… especially in surface proteins
N Croucher et al. Nat Genet 2013
Hypothesis: diversification of proteins (esp surface) represents selection to escape immunity while maintaining function
Proteins targeted by Ab, but not T cells, show evidence of diversifying selection
Y Li et al. PLoS Pathog 2012
Diversifying selection strongest for epitope regions of Ab-targeted proteins
Y Li et al. PLoS Pathog 2012
Hypothesis: Escaping from a T cell response provides little in vivo advantage• Immunize transgenic mouse whose CD4 cells
see only one peptide (OVA), with this peptide• Challenge mouse with a mix of SpnOVA+ (OVA)
and SpnOVA- (AVO)• Advantage of escaping immune response:
increase AVO/OVA ratio in immunized mice
Escaping from a T cell response provides little in vivo advantage
Y Li et al. PLoS Pathog 2012
Diversifying selection strongest on Ab epitopes
Mainly purifying
seln
Mainly purifying
seln
Little advantage to escape
Little advantage to escape
Selectn for escape
Selectn for escape
?Other host adaptation??Other host adaptation?
Diversifying selection on gene content?
• If a gene performing a useful but nonessential function is an immune target, it should be preferentially present in naïve hosts
• Tradeoff between function and vulnerability
• Proxy for naïve: young age
N Croucher et al. Nat Genet 2013
Most S. pneumoniae genes are not present in all isolates
Declining prevalence of some genes/alleles w host age
N Croucher et al. Nat Gen 2013
Category Diversifying selection Purifying selection
Resolution
Capsule Anti-CPS Ab Antiphagocytic effects
Nonspecific immunity compresses fitness differences enough for DS>PS
Surface proteins: Ab
antigens
dN/dS suggests antiprotein Ab create diversifying selection. But ??evidence antiprotein Ab protective??
Function? Age-structuring in presence/absenceIs there age-structure in diversity?
T cell Ag maybe v weak: in trans effects
Function No evidence of diversifying selection
Protein immunity: Back to public health
• New vaccines almost all involve proteins, some involve cellular immunity
• Replacement phenomena may differ• Predicting vaccine effects requires understanding the prevaccine
status quo
3. Using diversity
Nightmare on Huntington Avenue
After multiple confirmatory repeats of this experiment including multiple different experimental conditions, one day 19F completely disappeared in competition, a result that itself was repeatable with the same stock. This was probably the largest experimental story our lab has ever put together.
K Trzczinski et al. in prep
This stock (YL101) was different from the old stock (TIGR4:19F), suggesting mutations during passage/storage
Competition in mouse Survival from surface phagocytosis
WGS to the rescue
SNP separating the two stocks
Strategic laziness: narrowing the choices
Y Li et al. in press BMC Genomics
low-cost proxy phenotype
low-cost proxy phenotype
Costly stepCostly step
Obtained serotype- and phylogenetically- matched pairs with differences at candidate loci from our collection
Only one pair differed in surface killing
Y Li et al. in press BMC Genomics
Proper genetics confirmed role of SP_1645 SNP in changing surface killing survival and competitive ability of frozen stock
Y Li et al. in press BMC Genomics
SP_1645 is in stringent response pathway
SP_1645
What about other stringent response genes?• Modify the “creative laziness” approach:- Find pathway of interest- Find and perform low-cost phenotyping natural isolate
pairs with • same serotype• low overall genetic distance• high genetic distance at a candidate locus
- If pair differs, switch sequence at candidate locus. Then check low- and high-cost phenotype of isolates with switched sequence to properly confirm causal role of variation at that locus
SP_1097, the other GTP pyrophosphokinase in SR pathway, affects surface killing and growth
Y Li et al. in press BMC Genomics
Interplay of surveillance, mechanistic biology, transmission modeling, population genomics
• Exploit surveillance: many conclusions depend on representative strain collections and on associated host data (eg age)
• Extend surveillance: shows value of phenotyping and sequencing surveillance collections
• Enhance surveillance: mechanistic models suggest what we should be measuring and watching for, and improves our predictions of vaccine effects
CollaborationsHarvard TH Chan SPHSarah CobeyClaudette ThompsonKrzysztof TrzcinskiDan WeinbergerDebby BogaertGili Regev-YochayAlex D’AmourEric Tchetgen TchetgenYuan LiThomas FussellNick CroucherBill HanageLisa Kagedan
FundingNIH/NIAID, NIH/NIGMSPATH (Malley lab)Pfizer
KEMRI/LSHTMOsman AbdullahiAnthony Scott
Children’s Hospital BostonRichard MalleyPorter AndersonYingjie Lu
SangerSte BentleyJulian Parkhill
Imperial College LondonChristophe FraserCaroline Colijn
SPARCJonathan FinkelsteinGrace LeeSusan HuangSteve PeltonSPARC team