Comparison of genetic sequences from donor and recipient viruses from linked

subtype A transmission pairs

Samantha McInally

Eric Hunter’s Laboratory

Emory University

Atlanta, GA

Global Distribution of HIV-1 subtypes

Adapted from McCutchan FE. J Med Virol 2006 and Arien, K.K. et al. 2007

Previous subtype A studies

• Female sex workers infected with nonsubtype A viruses were 8 times more likely to develop AIDS over the study period vs. those infected with subtype A (Kanki et al. 1999 Journal of Infectious Diseases)

• HIV-1 subtype A viruses replicate at a lower level compared to subtype D viruses (Baalwaet al. 2013 Virology)

• HIV-1 subtype A viruses were more virulent compared to subtype C viruses (Venner et al. 2016 EBioMedicine)

Differences between subtype A and C in IAVI Protocol C Acute Infection cohort

• 60% faster disease progression with subtype C vs. subtype A infections (Amornkul et al. 2013 AIDS)

• Higher post-counseling HIV-1 incidence in Zambia vs. Rwanda (Kamali et al. 2015 PLOS One)

• Higher proportion of non-progressors infected with subtype A vs subtype C (Price et al.

2017 PLOS Medicine submitted)

Comparison of subtype A and C viral loads

Prentice et al. 2014 Virology

Characteristics of Rwandan subtype A cohort and 10 transmission pairs

Rwandan subtype A cohort characteristics

Percentage of single and multi-variant infections in Rwandan cohort

25% Multi-variant transmission

75% Single-variant transmission

Percentage of subtypes and recombinants in Rwandan cohort

21 % AC recombinant 4 % CD recombinant

8% Subtype C 67% Subtype A

Ling Yue, Rui Xu, Giselle Umviligihozo, Erick Muok

Sample collection from transmission pairs

Epidemiologically linkedtransmission pair

Experimental parameters

• 10 subtype A infected transmission pairs

• Normalized for gender

• All single variant infections

• Exclude acutely infected donors

Donor Coded ID EDI

# of amplicons generated

(Y/N) Included in analysis

R880M 21 11 Y

R463M 21 11 Y

R53M 27 8 Y

R49F 26 12 Y

R65F 51 15 Y

R1135F 18 15 Y

R977M 17 17 Y

R269F 23 10 Y

R885M 32 7 Y

R3103F 13 11 Y

R3671M 27 16 N

Viral Kinetics of 10 linked recipients from the transmission pairs

0 3 6 9 12 15 18 21 24 27 300

2

4

6

8

TIme after Infection (months)

HIV

-1 V

iral L

oad

(L

og

10)

Viral Load 2 year kinetics

Subtype A

Subtype C

0 3 6 9 12 15 18 21 24 27 300

2

4

6

8

TIme after Infection (months)V

iral L

oad

(L

og

10)

Viral load 2 year individual kinetics

Subtype A

Subtype C

cDNA synthesis and limiting dilution genome-length PCR

Plasma sample near time of transmission

Plasma virus

Affinity purification of total RNA

Lyse cells, and spin to remove large particulates/cell debris

Apply lysate (containing nucleic acids and cellular contaminants)

to column with glass membrane

Wash with alcohol to remove contaminants; nucleic acids

stick to glass membrane while contaminants wash

through. Treat with DNase enzyme to remove

contaminating DNA.

Apply water to the column;

purified RNA washes off the

glass and is collected

RNA Extraction

Agarose Gel

DNA Sequencing9kb

Near-full length genome amplification

• Sequencing using Pacific Biosciences platform and MDseq (Dilernia et al. NAR 2015)

Bias for consensus-like selection in subtype C infections

Deymier, Ende et al 2015 PLOS Pathogens

Phylogenetic analysis of subtype A transmission pairs

Yue et al. PLOS Pathogens. 2015

Nucleotide sequence of TF is not closer to A1 consensus

R88

0

R26

9R53

R46

3R65

R49

R11

35

R97

7

R88

5

R31

03

Z331

Z3576

Z3618

Z3678

Z4248

Z4473

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

Pair

wis

e d

ista

nce t

o D

NA

co

nsen

su

s

(Patr

isti

c D

ista

nce)

Individual protein sequences are not closer to consensus

R88

0

R26

9R53

R46

3R65

R49

R11

35

R97

7

R88

5

R31

03

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Pair

wis

e D

ista

nce t

o A

A c

on

sen

su

s

(Patr

isti

c D

ista

nce)

Comparison of subtype A

gag sequences to A1 consensus

R88

0

R26

9R53

R46

3R65

R49

R11

35

R97

7

R88

5

R31

03

0.00

0.05

0.10

0.15

0.20

0.25

Pair

wis

e D

ista

nce t

o A

A c

on

sen

su

s

(Patr

isti

c D

ista

nce)

Comparison of subtype A

pol sequences to A1 consensus

R88

0

R26

9R53

R46

3R65

R49

R11

35

R97

7

R88

5

R31

03

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Pair

wis

e D

ista

nce t

o A

A c

on

sen

su

s

(Patr

isti

c D

ista

nce)

Comparison of subtype A

env sequences to A1 consensus

R88

0

R26

9R53

R46

3R65

R49

R11

35

R97

7

R88

5

R31

03

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Pair

wis

e D

ista

nce t

o A

A c

on

sen

su

s

(Patr

isti

c D

ista

nce)

Comparison of subtype A

nef sequences to A1 consensus

Summary• Amplified near full-length genomes for 10 subtype A

transmission pairs where the donors show significant evolution• Initial phylogenetic analyses suggests that subtype A TF viruses do not exhibit

a bias for consensus-like residues. However, this may reflect the small number of samples analyzed in the current study.

Future Directions• More detailed analysis of selection bias (Jonathan Carlson)

• Compare replicative capacity of subtype C and A IMCs

• Compare replicative capacity of subtype A TF and NT IMCs

AcknowledgementsEric Hunter, PhD

• Zach Ende, PhD

• Kelsie Brooks

• Sarah Connolly

• Elina El-Badry

• Ling Yue, MD

• Erick Muok, PhD

• Giselle Umviligihozo

• Dario Dilernia, PhD

• Daniela Monaco, PhD

• Evonne Woodson, PhD

• Danna Cunningham

• Daniel Wilkins

• Rui Xu

• Qianhong Qin

• Jon Allen

• Paul Farmer, PhD

• Effie Bartley

Rwanda-Zambia HIV

Research Group

(RZHRG)

• Study Participants

• Susan Allen, MD, MPH,

DTM&H

Funding

• R01 AI 51231

• R01 AI 64060

• IAVI