My slides from talk at Sanger ABPHM 2015 on S pneumoniae

1. Sources, Consequences and Uses of Antigenic
Diversity in Streptococcus pneumoniae
Marc Lipsitch
#ABPHM15

2. Antigenic diversity: a key concern
for public health microbiology
Many current vaccines focus
on diverse antigens
“Easy” vaccines already here:
relatively conserved Ag, good
natural immune response
Now the hard ones remain
reverse vaccinology
serotype/strain
replacement
Malaria
Meningococcal disease
MMR,
DPT
Pneumococcal disease

3. 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 2010
Invasive disease, US, 65+
0%
5%
10%
15%
20%
25%
30%
2001 2003-4 2006-7 2008-9
non-PCV7
PCV7-related
PCV7 type
PC Wroe et al. PIDJ 2012, carriage in kids, MA

4. 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.)

5. 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

6. 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

7. 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

8. 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

9. Standing diversity of
pneumococcal serotypes
0%
2%
4%
6%
8%
10%
12%
19F*
6B*
6A
23F*
23B
14*
9V*
11A
13
15A
16F
35B
15C
19A
4*
10A
15F
15B
17F
20
29
34
3
7C
10B
18C*
19B
23A
37
non-typable
other†
Percentcolonized
SerotypeO 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

10. 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 charge
Polysaccharide structure
(high carbon/repeat)
Less polysaccharide/
Less rigid capsule
More polysaccharide/
More rigid capsule
Low acquisition rate
High acquisition rate
Poor competitors
Good competitors
Serotypes
1,4,5 etc
Serotypes
19A, 19F,
23F, 6A etc
Daniel Weinberger (modified) reflecting work by himself, Krzysztof Trzcinski, Yuan Li,
Claudette Thompson, Richard Malley, Derrick Cordy, Andrew Bessolo

11. Some examples
19A 19F 23F 6B 14 35B
10-5
10-4
10-3
10-2
10-1
100
serotype of clinical strain
fractionofrecoveredpopulation
B
DAY 6
Competition 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

12. Weak, partial, serotype-specific
immunity reduces acquisition of
previously-experienced serotypes
Table 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

13. 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

14. In humans: duration of carriage
declines with age (~immunity?)
M Lipsitch et al. Epidemiology 2012

15. 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

16. Together, these two forms of immunity
permit realistic levels of serotype
coexistence
Cobey & Lipsitch 2012 Science

17. 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

18. Adapting the model to full fit of carriage
prevalence in MA before and after PCV7
T Fussell,
D Weinberger,
S Cobey
unpublished0
0.01
0.02
0.03
0.04
0.05
0.06
6A
23F
19F
6B
11A
15B/C
19A
35A/B
14
22F
9A
18C
NT
10
6C
9N
23A
35F
23B
3
34
4
31
15A
38
15F
29
25A
7F
16F
33F
PoolI
17F
21
37
9V
7C
33A
13
18F
36
20
24F
Prevalence
Serotype
2007
Observed
Expected
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
6A
23F
19F
6B
11A
15B/C
19A
35A/B
14
22F
9A
18C
NT
10
6C
9N
23A
35F
23B
3
34
4
31
15A
38
15F
29
25A
7F
16F
33F
PoolI
17F
21
37
9V
7C
33A
13
18F
36
20
24F
Prevalence
Serotype
2001 - "prevaccine"
Observed
0% Cross-Immunity
Vaccine-related
serotypes systematically
exceeded predicted
prevalence postvaccine:
suggests natural
immunity between
serotypes within a
serogroup
19A
6C
23A
23B

19. 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.)

20. 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

21. The whole Spn genome varies
N Croucher et al. Nat Genet 2013

22. … especially in surface proteins
N Croucher et al. Nat Genet 2013

23. Hypothesis: diversification of proteins
(esp surface) represents selection to
escape immunity while maintaining
function

24. Proteins targeted by Ab, but not T cells,
show evidence of diversifying selection
Y Li et al. PLoS Pathog 2012

25. Diversifying selection strongest for
epitope regions of Ab-targeted proteins
Y Li et al. PLoS Pathog 2012

26. 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

27. Escaping from a T cell response
provides little in vivo advantage
Y Li et al. PLoS Pathog 2012

28. 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?

29. 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
ageN Croucher et al. Nat Genet 2013
Most S. pneumoniae genes are
not present in all isolates

30. Declining prevalence of some
genes/alleles w host age
N Croucher
et al. Nat
Gen 2013

31. 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/absence
Is there age-
structure in
diversity?
T cell Ag maybe v weak: in trans
effects
Function No evidence of
diversifying
selection

32. 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

33. 3. Using diversity

34. 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.19A 19F 23F 6B 14 35B
10-5
10-4
10-3
10-2
10-1
100
serotype of clinical strain
fractionofrecoveredpopulation
B
DAY 6
K Trzczinski et al. in prep

35. This stock (YL101) was different from
the old stock (TIGR4:19F), suggesting
mutations during passage/storage
Competition in mouse Survival from surface phagocytosis

36. WGS to the rescue
Positiona
Old
(TIGR4:19F)
New
(YL101)
CDS Codonb
SNP Namec
150259 A C SP_0152 N SP_0152:C380A
626569 C T SP_0655 S SP_0655:C906T
1052463 A G SP_1119 N SP_1119:G652A
1542955 G A SP_1645 N SP_1645:C1019T
1543998 G A - I -
SNP separating the two stocks

37. Strategic laziness: narrowing the
choices
Y Li et al. in press
BMC Genomics
low-cost proxy
phenotype
low-cost proxy
phenotype
Costly stepCostly step

38. Obtained serotype- and phylogenetically-
matched pairs with differences at candidate
loci from our collection

39. Only one pair differed in surface
killing
Y Li et al. in press
BMC Genomics

40. 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

41. SP_1645 is in stringent response
pathway
SP_1645

42. 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

43. SP_1097, the other GTP
pyrophosphokinase in SR pathway,
affects surface killing and growth
Y Li et al. in press
BMC Genomics

44. 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

45. Collaborations
Harvard TH Chan SPH
Sarah Cobey
Claudette Thompson
Krzysztof Trzcinski
Dan Weinberger
Debby Bogaert
Gili Regev-Yochay
Alex D’Amour
Eric Tchetgen Tchetgen
Yuan Li
Thomas Fussell
Nick Croucher
Bill Hanage
Lisa Kagedan
Funding
NIH/NIAID, NIH/NIGMS
PATH (Malley lab)
Pfizer
KEMRI/LSHTM
Osman Abdullahi
Anthony Scott
Children’s Hospital Boston
Richard Malley
Porter Anderson
Yingjie Lu
Sanger
Ste Bentley
Julian Parkhill
Imperial College London
Christophe Fraser
Caroline Colijn
SPARC
Jonathan Finkelstein
Grace Lee
Susan Huang
Steve Pelton
SPARC team