Characterization of Subtype F HIV-1 Strains from Patients

with Initial Poorer Treatment Response Compared to

Individuals Infected with Subtype B HIV-1 Strains

E. Poveda 1, D. Winner 2, S. Joussef 3, B. Pernas 1, A. Aguilera 4, A. Castro-Iglesias 1, A.

Mena 1, P. Cid 1, and M.E. Quiñones-Mateu 3

1 Instituto de Investigación Biomédica de A Coruña-Complexo Hospitalario Universitario de A Coruña, Spain. 2 University Hospitals

Cleveland Medical Center, Cleveland, Ohio, USA 3 Case Western Reserve University, Cleveland, Ohio, USA. 4 Universidad de

Santiago de Compostela, Santiago de Compostela, Spain

CONFIDENTIAL

INTRODUCTION

o Prevalence of non-B HIV-1 subtypes has been on the rise in Spain during the last 15 years,

reaching double digits most likely to an increase in immigration from Africa and South America 1.

o In NW Spain the number of new cases increased in the last 5 years. Non-B variants are 34.4% of

patients, being subtype F (25.8%) the most common non-B sub type 3.

o Subtype F HIV-1 was introduced in Spain approximately 20 years ago, growing to epidemic

proportions in the last 5 years 2.

o Infection with subtype F HIV-1 strains in NW Spain has been mainly associated with MSM

transmission 3 and in Europe is found in the Balkans mainly in Romania (prevalence >70%) not

associated to any transmission route.

o We have observed significant delay achieving virological suppression among subtype F HIV-

infected individuals, showing evidence of impaired response to antiretroviral treatment 4,5

OBJECTIVEOBJECTIVEo To characterize subtype F HIV-1 strains circulating in NW Spain and its potential correlation with

initial poor response to antiretroviral treatment.

1 Monge et al 2012. Clin Microbiol Infect. 18:E485. 2 Paraskevis et al 2015. Infect Genet Evol. 30:96. 3 Pernas et al 2015. J Med Virol. 87:1319. 4 Pernas et al

2014. AIDS. 28:1837. 5 Cid-Silva et al 2017. AIDS. in press

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METHODS

o Clinical samples. Twenty plasma samples from individuals infected with subtype F (n=10) or B (n=10)

were obtained from two hospitals in Galicia (Spain) and sent to Cleveland, OH for further analysis.

Clinical, virological, and demographics data, including HIV-1 drug resistance results based on standard

Sanger sequencing, were obtained from patient care databases at the respective hospitals.

o HIV-1 genotyping. HIV-1 drug resistance and tropism was analyzed using a deep sequencing-based

HIV-1 genotyping assay (DEEPGEN™) as described 6

o Construction of recombinant viruses. Two sets of viruses were constructed using patient-derived PCR

products: p2-INT-recombinant viruses carrying gag-p2/NCp7/p1/p6/pol-PR/RT/IN fragments and env-

recombinant viruses harboring gp160 fragments. These viruses were used in all phenotypic tests

described below.

o HIV-1 phenotyping. Susceptibility of the p2-INT-recombinant viruses to 22 antiretroviral drugs (PI,

NRTI, NNRTI, and INSTI) was determined using our HIV-1 phenotypic assay (VIRALARTS™) as described7

6 Gibson et al 2014. Antimicrob Agents Chemother. 58:2167 7 Weber et al 2011. Antimicrob Agents Chemother. 55:3729. 8 Sarzotti-Kelsoe et al 2014. J Immunol Methods. 409:131.9 Quiñones-Mateu et al 2000. J Virol. 74:9222. 10 Weber et al 2017. AIDS Res Ther. 14:15. 11 Shao et al 2014. J Virol Methods. 203:73

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4

oHIV-1 neutralization assay. Susceptibility of the env-recombinant viruses to 2 broadly neutralizing

antibodies (VRC01 and 10E8) was determined using the TZM-bl assay as described 8

oHIV-1 replicative fitness assay. The ability of the p2-INT or env-recombinant viruses to replicate in vitro

in the absence of drug pressure or host immune response pressure was determined using viral growth

kinetics assays as described 9

oNear full-length HIV-1 genome sequencing. Six overlapping fragments covering almost the entire

HIV-1 genome were amplified and deep sequenced using a variation of the DEEPGEN™ assay as

described 10

oPhylogenetic and viral diversity analysis. Read mapping, alignment and phylogeny reconstruction was

performed as described 10. Variant calling (i.e., single nucleotide and amino acid polymorphisms,

including indels) and their frequencies in the virus population were quantified using a proprietary

pipeline (Alouani & Quiñones-Mateu). Intra-patient HIV-1 quasispecies diversity was determined based

on the p-distance model as described for deep sequencing 11

METHODS

RESULTS Total

(n=20) Subtype B

(n=10) Subtype F

(n=10) p

Sex (male) 18 (90%) 8 (80%) 10 (100%) 0.474

Age (years) a 34 ± 10 36 ± 12 33 ± 8 0.596

Route of Transmission

MSM

Heterosexual

16 (88.9%)

2 (11.1%)

6 (75%)

2 (25%)

10 (100%)

0 (0%)

0.183

Spanish origin b 18 (90%) 9 (90%) 9 (90%) 0.999

HCV antibodies c 1 (5%) 1 (10%) 0 (0%) 0.999

Late Diagnosis d 7 (35%) 3 (30%) 4 (40%) 0.999

CD4+ cell count at diagnosis

e 520 ± 355 647 ± 406 393 ± 256 0.112

Plasma HIV-1 RNA (log copies/ml) at diagnosis f 5.26 ± 0.84 4.89 ± 0.78 5.64 ± 0.76 0.013

ART g 19 (95%) 9 (90%) 10 (100%) 0.999

Time to ART initiation (months) 14.1 ± 16.2 16.2 ± 13.5 12.2 ± 18.8 0.252

CD4+ cell count at ART initiation 407 ± 279 486 ± 325 337 ± 224 0.327

ART Regimen h

PI

NRTI

NNRTI

INSTI

5 (26.3%)

20 (100%)

8 (42.1%)

6 (31.6%)

2 (22.2%)

10 (100%)

3 (33.3%)

4 (44.4%)

3 (30%)

10 (100%)

5 (50%)

2 (20%)

n/a

n/a

n/a

n/a

Time to plasma HIV-1 RNA

<50 copies/ml (weeks)

<20 copies/ml (weeks)

36 ± 31

43 ± 28

20 ± 8

31 ± 11

49 ± 36

53 ± 35

0.026

0.135

Polymorphisms i

PR L10I

L10V

G16E

K20R

M36I

D60E

I62V

L63P

A71T

V77I

L89M

I93L

RT V106I

1 (5%)

10 (50%)

8 (40%)

1 (5%)

9 (45%)

7 (35%)

2 (10%)

5 (25%)

1 (5%)

4 (20%)

8 (40%)

4 (20%)

10 (50%)

0 (0%)

1 (10%)

1 (10%)

0 (0%)

2 (20%)

0 (0%)

1 (10%)

5 (50%)

1 (10%)

4 (40%)

1 (10%)

4 (40%)

0 (0%)

1 (10%)

9 (90%)

7 (70%)

1 (10%)

7 (70%)

7 (70%)

1 (10%)

0 (0%)

0 (0%)

0 (0%)

7 (70%)

0 (0%)

10 (100%)

0.999

<0.001

0.020

0.999

0.070

0.003

0.999

0.033

0.999

0.087

0.020

0.087

<0.001

Plasma HIV-1 RNA (log copies/ml) at sampling j

5.28 + 0.83 4.91 + 0.77 5.65 + 0.76 0.013

� No major differences in

demographics and clinical

characteristics, with the exception

of plasma HIV-1 RNA load at

baseline and time to reach

undetectable viremia

5CONFIDENTIAL

Table 1. Clinical and virological parameters

of HIV-infected individuals

HIVdb Score - Subtype F vs B

PI-F

PI-B

NR

TI-F

NR

TI-B

NN

RTI-F

NN

RTI-B

INS

TI-F

INSTI-B

-50

0

50

100

150

200

250

300

350

HIV

db

Sc

ore

� Although DEEPGEN™ identified substitutions in the

PR-, RT-, and integrase-coding regions, drug resistance

interpretation using Stanford HIV Database classified

all 20 HIV-1 strains as fully susceptible to all

antiretroviral drugs tested.

� Despite natural variation in the phenotypic

susceptibility to the 22 antiretroviral drugs tested with

VIRALARTS™, no significant difference was observed

between 3’Gag/PR/RT/INT-recombinant viruses

constructed from patients infected with subtype F

and B HIV-1 strains

6CONFIDENTIAL

Figure 1. HIV-1 genotyping based on deep sequencing (DEEPGEN™)

Figure 2. HIV-1 phenotyping (VIRALARTS™)

� (A) With the exception of a few env-recombinant viruses,

which had slightly higher EC50 values against VRC01 or

10E8 than the rest, not significant differences were

observed in the capacity of these two broadly

neutralizing antibodies to block the replication of

subtype F and B viruses.

� (B) Natural polymorphisms observed in the subtype F

and B viruses at VRC01 (gp120) and 10E8 (gp41)

epitopes.CONFIDENTIAL

Figure 3. HIV-1 neutralization

Full-length (minus LTRs)

0.0

0.5

1.0

1.5

2.0

2.5

p-d

ista

nc

e

Subtype F

Subtype B

� Similar replicative fitness (viral growth

kinetics) were observed among

3’Gag/PR/RT/INT-recombinant

viruses.

� Although no significant, subtype F env-

recombinant viruses showed slightly

higher replication rates compared to

subtype B viruses (median 0.036 vs.

0.015, p=0.119).

� Viral diversity was slightly higher in

individuals infected with subtype F vs.

patients with subtype B viruses (1.08

vs. 0.89, p=0.37).

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Figure 4. HIV-1 replicative fitness

Figure 5. Intra-patient HIV-1 diversity

� Three gag sequences (14-750, 14-751 and

14-755) and one env sequence (14-755)

clustered with subtype B strains rather than

subtype F viruses, suggesting potential

intersubtype HIV-1 recombination

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Figure 1. Phylogenetic and HIV-1 subtyping

analysis

� Deep sequencing of the full HIV-1 genomes identified 37 polymorphisms only present in subtype F10

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Table 2. Frequency of polymorphisms identified by deep sequencing of near full-length HIV-1 genomes

CONCLUSIONS

o HIV-1 phenotypic (VIRALARTS™) and genotypic (DEEPGEN™) analysis showed that subtype F

and B HIV-1 strains were susceptible to all antiretroviral drugs tested

o All subtype F and B env-recombinant viruses were equally neutralized by the bNABs VRC01

(gp120-CD4) and 10E8 (gp41-MPER)

o Although not significant, a slightly higher replication capacity was observed in subtype F env-

recombinant viruses compared to subtype B viruses

o Deep sequencing analysis of the near full-length HIV-1 genomes identified 37 polymorphisms

in subtype F that were absent in all subtype B viruses analyzed

o Ongoing growth competition experiments, as well as further characterization of the identified

polymorphisms, will allow us to verify the potentially higher replicative fitness of subtype F

HIV-1 strains, which could potentially be responsible for the delay in virological suppression in

patients infected with these viruses in NW SpainCONFIDENTIAL

ACKNOWLEDGEMENTS

Biobank of A Coruña (SERGAS)

CWRU/UH CFAR Catalytic Award2015/17

Fondo de Investigación SanitariaCPII14/00014, PI10/02166, PI13/02266, CM13/00328,

CM15/00233, PI16/02159, MV16/00054

amfAR109348-59-RGRL

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Fundación Profesor Novoa Santos, A Coruña