20150506 Applied Bioinformatics in Public Health Micro Keynote 2015 WTSI

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:



Protein(Ab antigen)

sterilizing


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:



Protein(Ab antigen)

sterilizing


duration-reducing

Host immunity

The whole Spn genome varies

N Croucher et al. Nat Genet 2013

… especially in surface proteins


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


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


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


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


Proper genetics confirmed role of SP_1645 SNP in changing surface killing survival and competitive ability of frozen stock


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


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

20150506 Applied Bioinformatics in Public Health Micro Keynote 2015 WTSI

Science

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