Spread of HIV-1 Infection

Formation of CladesRec0mbinant Strains

Differential rates of evolutionNovel strategies for development of

vaccines and therapies

HIV env in 8 men infected in the USA

Pt.7

Pt.9

HIV1U36148

HIV1U36015

HIV1U35980

HIV1U36073

HIV1U35926

HIVU95460

Pt.2

Patient #6 fromWolinsky et al.

Pt.5

Pt.3Pt.1Pt.8

Pt.6

10%

Subtype B (US)Subtype B (US)

1996

1992

10%

88

87

90

92

100

95

95

97

100

Diversity 1.5% higher in 1996 vs.

1992 (p< 0.001)

/A D

Central China

outbreak: B/C

Kaliningrad: B/A E

M Group

B

D

J

CHA

G

F

I

U

SIVcpz

SIVcpz

10%

O Group

N Group

Clades within the HIV-1 phylogenetic tree are becoming more obscure. Virus chimeras (recombinants) are becoming more evident and are significant in emerging epidemics

HIV-1 env Genes

Evolution from genomic chimerization (recombination)

One person is infected from two sources

Two virions from different sources infect the same cell

Chimera formation

The hybrid virion infects a new cell, reverse transcription occurs and RT switches between templates, creating a chimeric proviral genome

Each virion goes through reverse transcription, integration and produces viral RNA

One of each RNA is packaged in a single virion

HIVSC

HIVBa1aHIVSFAAA

HIVYU10XpNL43

0.01

635Days

PI

HIVSCHIVBa1a

HIVSFAAAHIVYU10X

pNL430.01

PATIENT BUNIQUE

PATIENT B

PATIENT A152/164Days PI

COMMON

887 Days

PI

HIVSCHIVBa1a

HIVSFAAAHIVYU10X

pNL43

0.01

Dual Infection and Virus Chimeras in UW PIC Pt. 14

Chimera

Evidence for Recombination

• Topological Incongruence along the Genome

• Bootscan Analysis

• Pairwise Distance Analysis

Topological incongruence of 3’genome sequences in MACS Pt. 6(3.0 yrs PI)

quartergenome

104109

18267855

3493

2830

4351

3260

62582

4954

57

0.00198%

100%

79%

72%

10443

10928

783026

1855

8260

325

9334

5462

5149

57

74%

0.01

gp41

10449

5109

6260

309343

51342818

2678

3255

8254

57

0.01

100%

78%

vif

1045754

18109

3043

2832

606255

3493

4951

5822678 0.01

94%71%

76%

nefU3

10454

5710918

822678

325

9334

5560

51622849

3043

0.01

97%

94%

5582

1045430

18109

2893

5734

492656078

3243

51

62

0.01vpu

What is bootscan analysis? A sliding window phylogenetic analysis that moves along

the genome and looks to see when (or if) the sequence being tested changes its clade association on the tree

e.g., The unknown grouping with clade 1 in the gag region and the unknown grouping with clade 2 in the env region

HIV-1-E

10

20

30

40

50

60

70

80

90

100

Boots

trap V

alu

e (

%)

P2

P1

90Rec

P2

P1

95

Rec

Boots

trap V

alu

e (

%) 100

50

0

Region of the Genome

Bootscan Example

500 bp500 bp

500 bp

Tree 1

Tree 3Tree 2

500 bp500 bp

500 bp

Tree 11

Tree 13Tree 12

Cautions for the interpretation of Bootscan Analysis The Bootscan analysis has several potential problems:

It normally uses neighbor joining to produce the tree topologies and often uses the evolutionary substitution models of F84 or Jukes and Cantor.

Neighbor joining has the potential to producing incorrect topologies and this potential can be enhanced by using substitution models that do not accurately describe the pattern of substitution in HIV

Bootscanning procedures look at a sliding window of sequences and produce a number describing the percent of time that the unknown groups with different clades.

The ability to decide if a recombination event has occurred is an arbitrary one. You have to decide if the line dips low enough and long enough to call it a recombination event. Using this technique is in no way a statistical measure of recombination events. There can be several random dips in the bootscan plot that are just random events.

Bootscann procedures have used sliding windows of varied size. Small windows can add to the problem of identifying the incorrect topology

using neighbor joining

HIV-1-E

0

10

20

30

40

50

60

70

80

90

100

vprenvpol

vif nefgag

Boots

trap V

alu

e (

%)

Bootscan: HIV-1-E & Simulations

HIV-1-E

0

10

20

30

40

50

60

70

80

90

100

vprenvpol

vif nefgag

Boots

trap V

alu

e (

%)

Bootscan: HIV-1-E & Simulations

What the Kashino-Hasegawa (KH) does It asks: “Are these two trees significantly different from

one another The comparison is done on the likelihood scores of the two

topologies It tests the null hypothesis that there is no statistical difference in

the likelihoods of the two trees.)

Overall, we suggest that the bootscann analysis should be used in conjunction with the KH test to try and statistically prove that a recombination event has taken place.

Bootscanning analysis can locate potential recombination events and the KH test can test the hypothesis of a recombination event taking place.

Intrasubtype

0

Intersubtype

5

10

15

20

25

30

35

40

gag

vprenvpol

vif nefSequen

ce D

ista

nce

(%

Div

erg

ence

)Pairwise Distance Analysis

A/E

B/D

Simulated DataSimulated DataReal DataReal Data

0

5

10

15

20

25

30

35

40

gag

vprenvpol

vif nef

Sequen

ce D

ista

nce

(%

Div

erg

ence

)Simulated Pairwise Distance Analysis

Comparing Subtypes A and E

Statistical tests of reported recombinant HIV strains (1)

Assigned Reported Virus Gene region subtype Test Score Best p value Significance

ZAM184 pol 3173-3973 C C 6690.35 (best) A 6747.78 57.43 0.0032 YES

env-nef 6511-9463 A A 32524.71 (best) C 32619.66 94.940.0001 YES

92RW009 pol-vpr 2573-6071 C C 28378.06 (best) A 28806.18 428.12 <0.0001 YES

env 6109-8195 A A 22831.00 (best) C 22967.94 136.94 <0.0001 YES

MAL gag 789-1883 A A 9571.50 (best) D 9713.38 141.87 <0.0001 YES

vpr-env 5559-8751 D D 33543.66 (best) A 33725.95 182.29 <0.0001 YES

IBNG gag 789-2292 A A 13962.19 (best) G 14049.00 86.80 <0.0001 YES

pol 2293-3173 G G 6442.77 (best) A 6468.69 25.92 0.0096 YES

93BR029 pol 2085-5096 B B 23128.62 (best) F 23276.56 147.94 <0.0001 YES

env 6220-8795 F F 27885.77 (best) B 28273.97 388.19 <0.0001 YES

Assigned Reported Virus Gene region subtype Test Score Best p value Significance

BFP90 gag 789-1583 A A 7309.43 (best) J 7346.98 37.54 0.0261 YES G 7338.20 28.77 0.0133 YES env 6261-7312 G G 12646.18 (best) J 12675.35 29.17 0.0225 YES A 12749.43 103.24 <0.0001 YES nef 8424-9463 J J 12280.21 (best) A 12335.76 55.55 0.0003 YES G 12327.69 47.47 0.005 YES

94CY032 gag 789-1883 A A 9611.10 (best) G 9682.93 71.82 <0.0001 YES I 9635.65 24.55 0.012 YES pol 2189-4673 I I 20009.92 (best) A 20140.33 130.41 <0.0001 YES G 20036.07 26.14 0.0423 YES env 6311-6773 G G 4386.51 (best) A 4404.26 17.75 0.0605 NOI 4387.39 0.88 0.9251 NO

Statistical tests of reported recombinant HIV strains (2)

The weight of the evidence indicates that E and A are separate phylogenetic groups and not recombinants

Nearly all other reported recombinants reported to date satisfy our criteria

Variations in the rate of evolution along the HIV-1 genome can resemble a recombination event

Conclusions

Lineages observed through time

Time or Sequence Divergence

Semi logarithmic Transformationof the number of lineages

time ( )present

Lo

g N

1

10

100

00.020.040.060.080.10.12

Time ()

Lin

eages (N

)

US B 1992

US Lineages through time

US B 1996

0

1

2

3

4

5

6

7

8

Year

Epidemic transform:

US’96

US ‘92

Thai EThai B

1976 1980 1984 1988 1992 1996

Time estimates are predicted from values assuming a substitution rate of 0.55%/year

m (T

ransformed N

umber of Lineages)

mt= ln n0-ntn0nt

US and Thai lineages through time

Epidemics in the US and Thailand are growing exponentially, with the Thai HIV-1B epidemic growing more rapidly

Coalescent dates for subtype B epidemics in the USA and Thailand are in accordance with epidemiologic data

Coalescent date for subtype E epidemic in Thailand is earlier than predicted from epidemiologic data. Potential reasons to account for this discrepancy:

Multiple introductions of HIV-1 (no evidence from phylogenetics) HIV-1 was undetected in Thailand for about 7 years Mutation rates for subtype E in the C2-C3 region are higher than

for subtype B

Conclusions

Novel approaches to HIV Prophylaxis

Ancestral Virus Reconstructions To minimize differences between vaccine

strain and infecting virus Epidemic-, subtype- and M group-specific Recovers antigenic recognition sites???

Antiretroviral Chemoprophylaxis Once-a-day pill provides protection??? Eventual induction of protective

immunity???

HIV-1-B env C2-V5 in the USA

HIV-1-B env C2-V5 in the USA

10%

88

87

90

92

100

95

95

97

100

~1992 (n=40)

~1996 (n=57)

The accumulation in the number of substitutions along lineages terminating in late timepoint sequences was 1.5% higher in 1996 vs. 1992 (p< 0.001)

Thus, temporally accurate strains are more divergent than an ancestor would be from any currently circulating strain

10 % Divergence

SIVCPZUS

SIVCPZGAB

Group O

Group NGroup M

A

B

C

D

E

F

AGAGI

J

G

H

Phylogenetic Classification of HIV-1

C- Clade Ancestor(s).

10%

ZF Z

RR FKKEZ

FZF

ZC

Z

FZ

ZFKZJ

F

F

H

E

G

J

F

H

K

N

H

A

Z

B

1

ZGG

b

Z

H

b

b

H G

b

H

G

b

bb

Ab

b

b

bbb

bb

bbb

b

bbb

bbb

bb

b

bb bb

bbb

bZ

G9Z Z

ZZZ ZZZ

Z

G

GR

9

Z

Z

Z

ZZ

ZG

b

H

Z Z

Z

Z

Z

b

Z

GG

G

GG

G

Z Z

M

G

GG

G

G

S

G

G

G

G

Z

M

GG

G

G

G

GG

Z

GG

GGG G

H

Z

M ZGGG

G

G H

Z

A

E

GZ Z

N GG G G

GG MZ F

ZGG

G Z H G

KK

GGS

A

b

C'

G

No C' aa340E350A429E

340E/350A350E/429E340E/429E340E/350A/429E

B BelgiumH BotswanaR BrazilF BurundiC Democratic Republic of the CongoJ DjiboutiE EthiopiaF France1 Gabonb IndiaA IsraelK KenyaM MalawiN New ZealandS SenegalS SomaliaE SwedenG RussiaG South AfricaZ TanzaniaH Uganda9 ZambiaZ Zimbabwe

Accuracy ofancestor predictions

MRCA estimate of 1000 bp region of gp120 97.4% identical to BK28 inoculum 98.2% when 5 convergent glycosylation sites are removed

5 changes

H915

H591

L35597U18033

MM316ZJMM132ZC

PHTBE5SIVT5

MNE170

186 H915M2191 H915M2190 H915M2

188 H915M2108 H915W6

111 H915W6114 H915W6

115 H915W6112 H915W6

118 H915W6109 H915W6

110 H915W6

43 H59155 H591

47 H591

56 H591

45 H59146 H591

48 H591

51 H59157 H59154 H59149 H591

50 H591

117 H915W6

(two macaques infected with the same clone-derived SIV inoculum)

19 H824 2

110 H915W6 2

118 H915W6 2

112 H915W6 2

115 H915W6 2

114 H915W

6 2

111 H915W6 2108

H91

5W6

2

186

H915M

29 2

190

H91

5M29

2

191

H91

5M29

2

188

H915M

29 2

109

H91

5W6

2

11 H

824

2

10 H

824

2

14 H

824

2

18 H

824

2

16 H824 2

21 H824 215 H824 220 H824 212 H824 2

9 H824 213 H824 2

41 H500 2

123

H615M

4 239 H

500 2

63 I580 2

134 H042M4 240 H500 237 H500 2

180 H615LN 269 I580 2

135 H042M4 2

129 H615M4 235 H500 265 I580 2

50 H

591

2

45 H

591

257

H59

1 2

51 H

591

2

56 H591 2

48 H591 249 H591 2

54 H591 2

46 H591 255 H591 2

140 H042M4 2

117 H915W6 2

58 I580 247 H

591 2

33 H500 2

138 H042M

4 2

136 H042M

4 2

133 H042M

4 2

137 H042M

4 2143 H

042M4 2

60 I580 2139 H

042M4 2

67 I580 259 I580 2

68 I580 2

61 I5

80 2

32 H500 2

43 H

500

212

1 H

615M

4 2

120

H61

5M4

2

119 H615M

4 2

128

H61

5M4

2

176

H61

5LN

217

7 H

615L

N 2

179

H61

5LN

218

3 H

615L

N 2

182

H61

5LN

217

2 H

042L

N 2

173

H04

2LN

2

174

H04

2LN

2

171

H042L

N 2

170

H042L

N 2

MNE170

SIV

T5

U18033

MM132ZC

MM

316ZJ

PH

TB

E5L35597

84 H914 2

88 H914 2

83 H914 293 H914 292 H914 2

85 H914 2

86 H914 2

91 H914 2

89 H914 2

87 H914 2

126 H615M4 2

125 H615M4 2

127 H615M4 2

124 H615M4 2

43 H591 2

34 H500 2

70 I580 262 I580 2

64 I580 2

66 I580 2

130 H615M4 2

42 H500 238 H500 236 H500 2

0.001 changes

MRCA estimate is 98.5% identical

to 1- F965M4

Accuracy of ancestor predictions(macaques infected with in vivo passaged SIV-BK28 inoculum [F965 strain])

% of consensus epitopes missingwithinHIV-1-B gp160 proteins

HIVELICGHIVNDKHIVZ2Z6

B_ancestor

HIVLAICG87USSG3X88USWR27

89SP061AUC18

AUC18MBCAUMBC200AUMBC925

AUMBCC18BAUMBCC54AUMBCC98AUMBCD36CNRL42CG

D31HIVBH102HIVCAM1

HIVF12CGHIVHAN2HIVJC16

HIVJRCSFHIVJRFL

HIVMCK1HIVMN

HIVNL43HIVNY5CG

HIVOYIHIVPV22

HIVRFHIVSF2CG

HIVWEAU160HIVYU10X

HIVYU2XMANC

NLACH32OANLACH320B

US89.6USDH123

USAD8

0 5 10 15 20

COS-7 mixed with GHOST-R5

COS-7 mixed with GHOST-X4

COS-7

COS-7 mixed with GHOST-R5

COS-7 mixed withGHOST-X4

Expression of HIV-89.6 Env:

Expression of ANC1 Env:

Antiretroviral ChemoProphylaxis

1000

2000

3000

0.1

1

10

100

1000

0 10 20 30 40

vag SIV

PMPA

vag HIV-2

Controls

PMPACD4

IUPM

Novel Therapeutic Approaches

Inhibitor plus Mutagen

P24 (ng/ml)750

500

250

0

Con

trol

AZT 3.2nm5-FdU25nM

5-FdU 25nmAZT 3.2nM 5-FdU

50nM

5-FdU 50nMAZT 3.2nM

HIV alone

-6 -5 -4 0 36 60 84 108

5-FdU AZT HIV1 WASH 5-FdU 5-FdU 5-FdU Harvest 5-FdU+AZT AZT AZT AZT