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What limit can be put to this power (natural
selection), acting during long ages and rigidly
scrutinising the whole constitution, structure,
and habits of each creature,-favouring the good
and rejecting the bad.

• Charles Darwin Origin of the Species

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Natural Selection
All species produce more offspring than can
possibly survive and reproduce. Organisms
differ in their ability to survive and
reproduce, in part due to differences in
genotype. In every generation, those genotypes
that promote survival in the current environment
are present in excess at the reproductive age and
therefore contribute disproportionately to the
offspring of the next generation. For natural
selection to operate there must be heritable
variation for traits that are correlated with
reproductive success.
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Average Rates of Nucleotide Substitution in
Different Organisms

Substitution

Rate
Organism/Genome
(per site, per year)
-9
Plant
chloroplast DNA
1 x 10
-9
Mammalian nuclear DNA
3.5 x 10
-9
Plant nuclear DNA
5 x 10
-9
E.
coli
and
Salmonella
enterica
bacteria
5 x 10
-8
Drosophila
nuclear DNA
1.5 x 10
-8
Mammalian mitochondrial DNA
5.7 x 10
-3
HIV-1
6.6 x 10
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The Degeneracy of the Genetic Code
Proline (4 synonymous codons) CCT CCC CCA CCG His
tidine (2 synonymous codons) CAT CAC CAA -
Gln CAG - Gln
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Measuring Selection Pressures in Genes
Selection pressures can be measured by
comparing the relative rates (ratio) of
synonymous (silent) (dS) and nonsynonymous
(amino acid changing) (dN) substitutions Ser M
et Leu Gly Gly Seq 1 TCA ATG TTA GGG GGA
Seq
2 TCG ATA CTA GGT ATA Ser Ile Leu Gly Ile
Synonymous substitution Nonsynonymous
substitution dN/dS lt 1.0 negative selection
(functional constraint - most genes) dN/dS 1.0
neutral evolution (pseudogenes) dN/dS gt 1.0
positive selection
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Genetic Variability of HIV

• HIV exhibits extremely high levels of genetic
  variation the virus evolves about 1 million
  times faster than human DNA (HIV-1 10-3
  subs/site/year, human DNA 10-9 subs/site/year)
  .
• This is mainly due to
• Extremely high mutation rate, particularly
  because the replication enzyme reverse
  transcriptase that converts RNA to DNA is very
  error prone
• High turnover of virus within infected individual
  1010 viruses produced in each patient each day
• Rapid generation time (2.6 days)

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Phylogeny of global HIV-1 isolates 197
strains from the Congo
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Genetic diversity at transmission

• Early analyses concentrated on the env gene and
  revealed a low level of diversity in
  seroconverters
• Ho D., Science, Leigh-Brown A., J. Virol
• So the infection was thought to be established by
  a small founder set of clones
• But that work did produce evidence of greater
  diversity in gag, nef and pol than env

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Four Sexual Transmitters
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UNRELATED SEROCONVERTER
Maximum Likelihood phylogenetic tree
DONOR
RECIPIENT
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Evading Cytotoxic T-Lymphocyte Recognition May
Enhance Viral Fitness
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Evading Cytotoxic T-Lymphocyte Recognition May
Enhance Viral Fitness
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Selection of escape mutants
replication
A
A1-A101
SELECTION PRESSURE
e.g. A6
e.g. A12
Some lead to increased fitness - survive
Some mutations lead to reduced fitness - die off
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Continued selection of escape mutants
replication
A6
A6.1-A6.101
SELECTION PRESSURE
e.g.A6.4
e.g. A6.9
Some mutations lead to reduced fitness -
extinction
Some lead to increased fitness - survival
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Extinction has only separated groups it has by
no means made them for if every form which has
ever been were suddenly to reappearall would
blend together by steps as fine as those between
the finest existing varieties

• Charles Darwin Origin of the Species

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Do CTL Select for Escape Variants During Acute
Infection?
CTL Response
Viral Load
Weeks
Years
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5 CTL Epitopes Accumulate Variation at Different
Rates During Acute SIV Infection
Consensus
GDYKLVEI
QGQYMNTPW
ARRHRILDIYL
IRFPKTFGW
KRQQELLRL
Inoculum
........
.........
...........
.........
.........
........
.........
...........
T.Y......
.........
........
...H.....
...........
T.Y......
.........
........
.........
E..........
T.Y......
.........
........
......N..
E.......T..
T.Y..I...
.........
........
......N..
E.......M.F
T.Y..I...
.........
........
......N..
E.......K..
T.Y..I...
.........
.....I.V
....I.N..
E.......K..
..Y..I...
...H.....
.....I.V
......N..
E.......K..
T.Y..I...
...H.....
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Epitope Variants Reduce CTL Recognition
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SIV Nef variation (yellow blocks) clusters within
epitopes defined by MHC Class I (green blocks)
C
D
A
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Summary

• Antiretrovirus T lymphocytes recognise peptide
  antigens dictated by, and bound to MHC Class I
  molecules
• Genetic variants of the virus can alter or even
  abolish immune recognition of infected target
  cells
• Viruses bearing these sequences have a survival
  advantage
• Escape mutants grow out (at different rates) and
  are positively selected

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26 year old man admitted with aseptic meningitis

• Had unprotected anal sex on 20 Sept.1995
• HIV Elisa negative on 10 October 1995
• P24 gag antigen positive
• Viral load gt10 millions copies per ml
• CTL assay 19 October 1995
• Single nef response

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HLA B8NEF EPITOPEFLKEKGGL
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Intra-Host Evolution of HIV-1
SIHIGPGRAFYTTGE SIPIGPGRAFYTTGQ SIHIGPGGAFYTTGQ SI
HIGPGRAFYTTGD SIPIGPGRAFYTTGD GIHIGPGSAFYATGD SIHI
GPGRAFYTTGG SIHIGPGRAVYTTGQ GIHIGPGSAFYATGG GIHIGP
GRAVYTTEQ RIHIGPGRAVYTTEQ GIHIGPGSAFYATGR RIYIGPGR
AVYTTEQ GIHIGPGSAVYATGG RIYIGPGSAVYTTEQ GIHIGPGSAF
YATGG RIGIGPGRSVYTAEQ GIHIGPGSAVYATGD GIHIGPGRAFYA
TGD GIHIGPGRAVYTTGD RIYIGPGRAVYTTDQ
Tip of the V3 loop (part of the envelope protein
of HIV-1) - diversity in a single patient
The HIV-1 envelope protein is under very strong
positive selection to help the virus escape from
the human immune response (the V3 loop contains
epitopes for neutralising antibodies and
cytotoxic T-lymphocytes (CTLs). V3 loop dN/dS
13.182 (Nielsen Yang. Genetics 148, 929.
1998).
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Does transmission of CTL escape viruses impact on
the global epidemic?
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Questions

• Child can inherit a HLA Class I molecule which
  has dictated a response in the mother
• The maternal immune response can select escape
  mutants
• Are these immune escape viruses detectable in the
  mother?
• Can the mother transmit these viruses?
• Do these escape viruses propagate infection in
  the child?

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Father
Mother
Child
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Father
Mother
B27
B27
Child
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Father
Mother
B27
B27
Child
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The HLA B27 Gag peptide is not recognized in
children of HLA B27-positive mothers WHY?
1925
2000
1600
IFN-g SFC/ million PBMC
1200
800
400
0
0
0
0
043-C
002-C
048-C
049-C
B27-ve mother
B27ve mothers
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The HLA B27 bearing MOTHERS acquired an escape
virus and transmitted it to the CHILDREN
No P2 anchor mutation
2000
P2 anchor mutation shared with mother
1600
IFN-g SFC/ million PBMC
1200
800
400
0
0
0
0
043-C
002-C
048-C
049-C
B27-ve mother
B27ve mothers
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Father
Mother
B27
B27
B27
KRWIILGLNK - K- - -M- - - -
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HIV IN PERTH, AUSTRALIAHypotheses

• If CTL-driven selection operates on HIV-1 in
  populations then selected mutations should
• be within or close to CTL epitopes
• because of genetic restriction of immune
  responses, mutations should be linked to
  particular HLA variants
• viral escape mutations would be evident as
  HLA-associated polymorphisms.

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HLA alleles significantly associated with HIV-RT
polymorphisms
Moore et al (2002) Science 296, 1439
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Natural selection by AIDSWho wins the race?

• HIV-1 10-3 subs/site/year
• Human genome (eg HLA) 10-9 subs/site/year

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Nothing in biology makes sense, except in the
light of evolution

• Theodosius Dobzhansky 1973

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Acknowledgments

• Eddie Holmes
• Paul Klenerman
• David Watkins
• Angela McLean
• Simon Mallal
• David OConnor
• David Price
• Annette Oxenius
• Andrew McMichael
• Charles Bangham

• Philippa Easterbrook
• Jonathan Weber
• Sarah Fidler
• George Scullard
• Anele Waters
• Anne Edwards
• Philip Goulder
• The patients of The Chelsea and Westminster
  Hospital St Marys Hospital KwaZulu Natal