Quantification of Proviral Load of Human Immunodeficiency Virus Type 2 Subtypes A and B Using Real Time PCR

0095-1137/01/$04.00⫹0 DOI: 10.1128/JCM.39.12.4264–4268.2001

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Quantification of Proviral Load of Human Immunodeficiency

Virus Type 2 Subtypes A and B Using Real-Time PCR

FLORENCE DAMOND,

1,2

_{* DIANE DESCAMPS,}

1,2

_{ISABELLE FARFARA,}

1,2

_{JEAN NOE¨L TELLES,}

1,2,3

SOPHIE PUYEO,

4

_{PAULINE CAMPA,}

1,2

_{ANNIE LEPREˆTRE,}

1,2

_{SOPHIE MATHERON,}

1,2

FRANC¸OISE BRUN-VEZINET,

1,2_AND

_{FRANC¸OIS SIMON}

1,2,5

Laboratoire de Virologie

1

_{et Service des Maladies Infectieuses et Tropicales A,}

2

_{Hoˆpital Bichat-Claude Bernard, Paris,}

Biomerieux S.A., 69280 Marcy l’Etoile,

3

_{INSERM, Unite´ de Recherche U330, Universite´ Victor Segalen, Bordeaux,}

4

and Laboratoire de Virologie, CHU Charles Nicolle, Rouen,

5

_France

Received 14 March 2001/Returned for modification 14 September 2001/Accepted 20 September 2001

We have developed and evaluated a new method to quantify human immunodeficiency virus type 2 (HIV-2)

proviral DNA based on LightCycler real-time PCR. The assay has a detection limit of 5 copies/10

5

_peripheral

blood mononuclear cells (PBMC) and is insensitive to HIV-2 strain variability: HIV-2 subtypes A and B are

both recognized and quantified. The intra- and interassay coefficients of variation range from 16 to 40% for

high provirus concentrations (5

ⴛ

10

5

_{copies) and from 41 to 39% for low concentrations (5 copies). We used}

this method to compare the proviral DNA load and viral RNA load in plasma with clinical and immunological

status for 29 patients infected by HIV-2 (subtype A in 17 and subtype B in 12). The proviral load (median, 201

copies/10

5

_{PBMC) was similar to that reported for HIV-1 infection. The median proviral loads did not correlate}

with the CD4

ⴙ

_{cell count categories and were as follows for CD4}

ⴙ

_{cell counts of >400, 200 to 400, and <200}

cells/mm

3

_{, respectively: 121 copies/10}

5

_{PBMC (}

_n

_ⴝ

_{8; range, <5 to 712 copies/10}

5

_{PBMC); 114 copies/10}

5

PBMC (

n

ⴝ

9; range, <5 to 1,907 copies/10

5

_{PBMC); and 285 copies/10}

5

_{PBMC (}

_n

_ⴝ

_{12; range, 53 to 2,524}

copies/10

5

_{PBMC). Proviral load did not correlate with plasma HIV-2 RNA positivity. As HIV-2 is considered}

to replicate less efficiently than HIV-1, these high proviral loads might be explained by the proliferation of

infected cells.

Human immunodeficiency virus type 2 (HIV-2) was first

isolated in 1986 from peripheral blood mononuclear cells

(PBMC) from patients in the Cape Verde Islands and

Guinea-Bissau (7). Heterosexual and vertical HIV-2 transmission rates

are lower than those of HIV-1 (4, 21, 24). HIV-2-infected

patients exhibit a longer clinical latency period and progress

more slowly toward AIDS (17, 22). The origins of these viruses

have been clearly linked to cross-species transmission events

between mangabeys (

Cercocebus atys

, small West African

mon-keys) and humans (23). Six HIV-2 subtypes have so far been

described (6, 14). Only HIV-2 subtypes A and B are prevalent,

the others being considered self-limiting infections at the

epi-demiological level. HIV-2 infection is mostly confined to West

Africa (12).

The CD4

⫹

_{cell count correlates negatively with clinical}

sta-tus in both HIV-2 and HIV-1 infections (3, 33). In contrast,

HIV-2 is more difficult to detect in plasma than HIV-1 (3, 28).

This fact has led us and others to consider HIV-2 poorly

replicative, at least early in the infection (3, 12, 28, 33). This

factor could explain the epidemiological and clinical

differ-ences between HIV-2 and HIV-1 infections. The reasons for

this poorer fitness of HIV-2 in humans is unclear. The animal

counterpart of HIV-2, SIVsm (infecting mangabeys), is highly

replicative in its natural host, with high cellular and plasma

viral loads, but is nonpathogenic (30). The reason for this lack

of replicative robustness remains unclear.

For HIV-1 infection, it has been demonstrated that proviral

load correlates with disease progression (32). Previous

quan-titative assessments of HIV-2 proviral load have proposed, as

for HIV-1, an inverse correlation with the CD4

⫹

_{cell count and}

clinical outcome (2, 27, 31). However, recent reports have

suggested that HIV-2 proviral load does not correlate with the

CD4

⫹

_{cell count (15, 29).}

The development of new quantitative methods using

real-time PCR technology has led us to evaluate those methods for

proviral HIV-2 DNA quantitation in 29 HIV-2-infected

pa-tients living in France. We also tested plasma RNA detection

and compared the findings with immunological and clinical

status.

MATERIALS AND METHODS

Study population and sample collection. The study group consisted of 29 patients (17 males and 12 females) who had been enrolled since 1991 in the French National HIV-2 Cohort. These patients originated from various countries in West Africa, North Africa, and Europe. The infecting strains had been pre-viously sequenced in theenvregion: 17 were subtype A, and 12 were subtype B (11). The epidemiological and clinical characteristics of the patients are summa-rized in Table 1.

According to Centers for Disease Control and Prevention criteria (5), 23 patients were in stage A and 6 were in stage C. Eight patients had CD4⫹_cell counts above 400⫻106_{/liter (median, 511; range, 413 to 832), 9 had counts}

between 200⫻106_{and 400⫻10}6_{/liter (333; 210 to 391), and 12 had counts below}

200⫻106_{/liter (68; 13 to 199). Fourteen patients were receiving antiretroviral}

treatment at enrollment in this study (one drug for 9, two drugs for 3, and three drugs for 2).

Sample preparation and DNA extraction. Whole blood was collected in EDTA-containing Vacutainer tubes, and PBMC were isolated by

Ficoll-* Corresponding author. Mailing address: Laboratoire de Virologie,

Hoˆpital Bichat-Claude Bernard, 46 Rue Henri Huchard, 75877 Paris

Cedex 18, France. Phone: 00 33 1 40 25 88 96. Fax: 00 33 1 40 25 67 69.

E-mail: [email protected].

4264

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Hypaque density gradient centrifugation. Plasma was clarified and stored at

⫺80°C until viral RNA assays were done. DNA was extracted using a QIAamp DNA mini kit (QiAgen) and quantified spectrophotometrically. Cellular and plasma viral culturing was performed as previously described (33).

Preparation of HIV-2 DNA and RNA assay standards.DNA templates were derived from a PCR-amplified region of thegaggene of HIV-2 strain ROD. HIV-2 ROD DNA in fresh PBMC was amplified by nested PCR with a first round of long PCR. The LTR1-5⬘–LTR2-3⬘primer pair was used for the first amplification step as previously described (10). Nested PCR was done using the following primers containing enzyme restriction sequences at their 5⬘extremities: QC1Bam, 5⬘-GATTGCGGATCCGTGGGAGAATGGGCGCGAGA-3⬘, and GAG OG AS1 Hind, 5⬘-CTGCATAAGCTTGCCTTCTGAGAGTGCCTGAA ATCC-3⬘(Genset, Paris, France). The resulting PCR product was cloned into vector pGEM3Z (Promega, Madison, Wis.). Plasmids were purified on columns with a Qiagen kit and quantified byA260measurements. To control the validity of our primers, the amplified product was sequenced.

RNA transcripts were obtained from the linearized templates by using a Riboprobe in vitro transcription system according to the manufacturer’s instruc-tions (Promega). RNA was quantified spectrophotometrically at 260 nm. Tran-scripts were diluted to 106_{copies/␮l, divided into aliquots, and stored at⫺80°C.}

Subsequent dilutions were made in a solution containing carrier tRNA (from

Escherichia coli; 30␮g/ml; Sigma, St. Louis, Mo.).

Plasma RNA detection.Viral RNA was extracted from 1 ml of plasma and ultracentrifuged for 1 h at 4°C using an HCV specimen preparation kit (Roche Diagnostics Systems). RNA was amplified by a one-step procedure using a Titan one-tube RT-PCR kit (Roche) with primers located in thegagregion (GAG OG S1 and GAG OG AS1). Amplified products were subjected to nested PCR with primers GAG OG S2 and GAG OG AS2 as previously described (16, 19). The efficiency and sensitivity of qualitative reverse transcription-PCR for detection of viral RNA isolated from plasma were determined by using a transcript standard diluted in 200␮l of RPMI medium to obtain 5,000, 1,000, 500, and 250 copies (seven replicates each). The standard was extracted and processed as described above. The sensitivities of reverse transcription-PCR were 100% at 5,000, 1,000, and 500 copies/ml and 66% at 250 copies/ml.

PCR primers and labeled hybridization probe.In order to avoid major mis-matches due to HIV-2 variability, the primers and probe were designed using specific software from TibmolBiol (Berlin, Germany), on the basis of all HIV-2 subtype A and B sequences so far published, according to the Los Alamos National Laboratory database (26). Primers used for amplification were located in the highly conservedgagregion: U3, 5⬘-GGGAGATGGGCGCGAGA-3⬘, and L140, 5⬘-TCCAACAGGCTCTCTGCTAATCC-3⬘. Probe S65GAG2 had the se-quence 5⬘-R-TAGGTTACGGCCCGGCGGAAAGA-Q-3⬘, where reporter R indicates a 6-carboxyfluorescein group and quencher Q indicates a 6-carboxytet-ramethylrhodamine group conjugated through a linker arm nucleotide as de-scribed previously (35). The specificity of the primers was evaluated with refer-ence samples (11).

Proviral load quantitation.Real-time quantitative PCR was performed using an LC Fast Start DNA master mix hybridization probe kit (Roche Molecular Diagnostics). Each PCR mixture (20-␮l total volume) contained the follow-ing: 2␮l of DNA master mix, 2.4␮l of 25 mM MgCl2, primers U3 and L140 (0.8␮M each), TaqMan probe S65 (100 nM), and 20␮l of H2O qsp. Cycling

parameters were as follows: denaturation for 8 min at 95°C, followed by 45 cycles of 10 at 95°C and 40 at 60°C. For each run, a standard curve was generated from purified HIV-2gagplasmid ranging from 5⫻105_{to 5 copies.}

Fresh dilutions of the control were prepared with salmon sperm DNA (100 ng/ml) before each experiment from a stock stored at⫺20°C. Five hundred nanograms of DNA from each patient was analyzed. Results were expressed in copies per 105_PBMC.

[image:2.587.44.541.83.377.2]

Reproducibility of DNA quantitation with the LightCycler system.To assess the inter- and intra-assay reproducibilities of the LightCycler system, we first analyzed two- and fivefold differences in the starting copy number using 25,000 to 50,000 and 5,000 to 25,000 copies per capillary (12 replicates each). Calcula-tions were determined with the cycle threshold (CT). We also tested 10-fold differences in copy number with a broader concentration range: from 5 to 50 copies (10 replicates each), 500 to 5,000 copies (6 replicates), and 50,000 to 500,000 copies (4 replicates). The last experiment was used to assess the inter-assay reproducibility (10 runs at each point from 5 to 5⫻105_copies).

TABLE 1. Epidemiological, clinical, and virological characteristics of the 29 HIV-2-infected patients

Patient Age (yr) Sexa Geographic

origin Subtype Stage Treatmentb CD4

⫹_{cell count}

(106_cells/liter) Cell culture_resultc _detectionRNA c DNA viral load (copies)/₁₀5_PBMC

1

45

M

Mali

A

C

AZT

54

⫹

⫹

199

2

41

M

Mali

A

C

AZT-3TC

24

⫹

⫹

2,524

3

46

F

Mali

A

C

DDC

25

⫹

⫹

1,287

4

43

M

France

A

C

AZT

31

⫹

⫹

369

5

42

M

Burkina Faso

A

A

AZT-3TC

37

⫹

⫹

2,342

6

45

M

Ivory Coast

B

A

D4T-3TC-IDV

83

⫹

⫹

69

7

47

M

Senegal

B

C

AZT

120

⫹

⫹

161

8

63

M

France

B

A

AZT-3TC-IDV

13

⫺

⫺

53

9

39

M

Ivory Coast

B

A

AZT-DDI

153

⫹

⫹

80

10

32

F

Martinique

B

A

AZT

178

⫹

⫹

791

11

57

F

France

A

A

AZT

189

⫹

⫹

457

12

31

M

Guinea-Bissau

A

A

DDC

199

⫹

⫹

201

13

43

F

Senegal

A

A

NT

210

⫹

⫺

1,907

14

25

F

Ivory Coast

A

A

NT

240

⫹

⫺

1,730

15

36

F

Cape Verde Islands

A

A

NT

333

⫺

⫺

⬍5

16

45

M

Mali

B

C

AZT

283

⫹

⫺

92

17

57

M

France

B

A

NT

328

⫺

⫺

⬍5

18

64

F

Cape Verde Islands

A

A

NT

339

⫹

⫹

354

19

27

F

Ivory Coast

B

A

NT

347

⫹

⫺

5

20

35

M

Guinea-Bissau

A

A

NT

375

⫹

⫹

709

21

39

M

Morocco

A

A

NT

391

⫹

⫺

114

22

37

M

Mali

A

A

NT

540

⫺

⫹

⬍5

23

34

F

Ivory Coast

A

A

NT

644

⫺

⫺

223

24

41

M

France

A

A

AZT

474

⫺

⫺

⬍5

25

33

F

Ivory Coast

B

A

NT

799

⫺

⫺

712

26

33

F

Senegal

A

A

NT

832

⫹

⫹

334

27

62

F

France

B

A

NT

418

⫹

⫹

390

28

27

M

Ivory Coast

B

A

NT

413

⫹

⫹

19

29

55

M

Congo

B

A

NT

483

ND

⫺

⬍5

a_{M, male; F, female.}

b_{AZT, zidovudine; 3TC, lamivudine; DDC, dideoxycytidine; IDV, indinavir; NT, no treatment; DDI, didanosine; D4T, stavudine.} c_{⫹, positive;⫺, negative; ND, not done.}

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RESULTS

Reproducibility of the LightCycler system for HIV-2 DNA

quantitation.

DNA concentrations of 5,000, 25,000, and 50,000

copies were distinguished with high confidence. The

coeffi-cients of variation (CVs) ranged between 0.31 and 0.56% for

the CT and between 7.5 and 12.9% for the copy number (Table

2). At a high virus concentration (mean, 534,340 copies per 10

5

PBMC), the intra-assay CV was 16%. At a mean copy number

of 586,692 per 10

5

_{PBMC, the interassay CV was 40%. At the}

lowest concentration (five copies), the intra-assay CV was 41%

and the interassay CV was 39%. Intra- and interassay CVs are

summarized in Table 3.

Sensitivity of the LightCycler system for HIV-2 DNA

quan-titation.

The sensitivities of the assay on the basis of repeated

testing (eight replicates) of the lower concentrations of our

plasmid standard were 100% at 5 copies, 62% at 2.5 copies,

and 75% at 1 copy. Consequently, we set the detection limit of

the assay at five copies.

HIV-2 proviral DNA detection according to subtype.

DNA

viral load, expressed as copies per 10

5

_{PBMC, was determined}

for the 29 HIV-2-infected patients. DNA was detected in 24

(83%) of the 29 patients and was consistently below five copies

in the other 5 patients. The lack of HIV-2 provirus

amplifica-tion was not linked to the subtype, three subtype A and two

subtype B samples being below the cutoff. This result indicates

that the selected primers and probes cover a wide range of

HIV-2 diversity.

HIV-2 proviral load is high whatever the CD4

ⴙ

_{cell count.}

[image:3.587.300.544.85.189.2]

Levels of proviral DNA ranged from 5 to 2,524 copies per 10

5

FIG. 1. HIV-2 proviral load according to CD4

⫹

_{cell count category. Horizontal lines represent median values in each category. Samples below}

[image:3.587.43.283.93.157.2]

the limit of detection were given a value of four copies/10

5

_{PBMC. The number of samples in each category is shown. There is no correlation among}

the three groups (␹

2

_⫽

_3.071;

_P

_⬍

_{0.3; Kruskal-Wallis test).}

TABLE 2. CVs for quantitation of standards of 5,000, 25,000,

and 50,000 copies

Copies % CV for:

CT Copy no.

5,000

0.5

11.8

25,000

0.31

7.5

50,000

0.56

12.9

TABLE 3. Intra- and interassay precision of the LightCycler system

Copies

Reproducibility

Intra-assay Interassay

% CV Meana _{% CV Mean}a

5

41

11

39

9

50

31

82

35

44

500

34

727

39

430

5,000

11.8

2,363

32

3,951

50,000

12.9

39,880

30

44,559

5,000,000

16.3

534,340

40

586,692

a_{Values are in copy numbers per milliliter.}

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[image:3.587.51.531.391.675.2]

PBMC, with a median of 201 copies/10

5

_{PBMC. These values}

are similar to those previously reported for both HIV-2 and

HIV-1 (1, 2, 8, 15, 27, 29, 31). DNA viral load in these 29

patients was then examined according to the following CD4

⫹

cell count categories:

⬍

200/mm

3

_{(all these patients being on}

antiretroviral therapy), 200 to 400/mm

3

_{, and}

_⬎

_400/mm

3

_{. The}

respective median proviral loads per 10

5

_{cells were 285 (12; 53}

to 2,524 [

n

, range]), 114 (9;

⬍

5 to 1,907), and 121 (8;

⬍

5 to

712). This trend toward increasing proviral load according to

CD4

⫹

_{cell count was not significant. All the samples with}

negative provirus amplification were from patients with CD4

⫹

cell counts above 328

⫻

10

6

_{cells/liter. Viral load did not differ}

significantly among these three categories of CD4

⫹

_{cell count}

(

␹

2

_⫽

_3.071;

_P

_⬍

_{0.3; Kruskal-Wallis test) (Fig. 1).}

HIV-2 proviral load does not influence plasma RNA

detec-tion.

There was no significant relationship between the level of

HIV-2 proviral load and the rate of plasma RNA detection

(

U

⫽

61;

P

⬍

0.07, Mann-Whitney test). All but one of the

patients with a CD4

⫹

_{cell count below 200}

_⫻

₁₀

6

_{/liter were}

positive for plasma RNA; the remaining patient was receiving

antiretroviral tritherapy (Table 4). A correlation was found

between positive cell culture and viral DNA load (

U

⫽

26;

P

⬍

0.02; Mann-Whitney test).

DISCUSSION

We evaluated a real-time PCR assay with the LightCycler

system for HIV-2 proviral DNA quantitation in clinical

spec-imens. The LightCycler system is simple and rapid and offers a

standardized approach to HIV-2 DNA quantitation. It also

avoids false-positive results due to PCR contamination.

In our experiments, the reproducibility of the system was

similar to that of commercial viral load kits (9, 25). The high

sensitivity of our assay (detection limit, 5 DNA copies/10

5

cells) must be emphasized, as it provides ample opportunities

for further clinical studies. Given the importance of HIV-2

genetic diversity, we first tried to select conserved and efficient

segments of the viral genome to use as primers. During a

previous evaluation (data not shown) with different

gag

primers

(U5, 5

⬘

-GAGAATGGGCGCGAGAAACT-3

⬘

, and L130, 5

⬘

-AATTCATTCGCTGCCCACAC-3

⬘

), we successfully

ampli-fied all the A subtypes but failed to amplify any B subtypes.

This result convinced us of the underlying importance of

primer and probe design in genome-based detection assays.

Previous studies of HIV-2 quantitation were performed in

West Africa, where only subtype A circulates (1, 2, 31). In

contrast, subtype B is frequent in HIV-2-infected patients

liv-ing in France (11) and probably in other Western countries as

well (18), creating the need to assess a large panel of

repre-sentative HIV-2 strains.

The levels of HIV-2 proviral DNA found in the 29 patients

tested here were high and similar to those observed in studies

of HIV-2 subtype A infections (1, 2, 15). We found no

signif-icant differences in the amount of proviral DNA according to

subtype. Likewise, we found no correlation between proviral

DNA level and CD4

⫹

_{cell count or viral RNA positivity. These}

results for DNA quantitation are similar to those recently

reported by Popper et al. (29) and Gomes et al. (15), although

other studies have shown a correlation between these

param-eters (2, 27, 31). Popper et al. also reported a lack of

correla-tion between levels of viral RNA and proviral DNA (29).

Because of the low pathogenicity of HIV-2 compared to that

of HIV-1, we were intrigued in find low HIV-2 proviral loads

but relatively large amounts of HIV-2 DNA in our patients. A

selection bias can be ruled out, as the majority of the patients

were symptom free and the CD4

⫹

_{cell count distribution was}

normal. In a community-based study, Ariyoshi et al. found

similar high levels of HIV-2 DNA, regardless of the percentage

of CD4

⫹

_{cells (1). Although not statistically significant, we}

tended to find higher levels of HIV-2 DNA and a trend toward

lower CD4

⫹

_{cell counts with more advanced stages of}

infec-tion. The effect of antiretroviral treatment in patients with

CD4

⫹

_{cell counts of}

_⬍

_200/

_␮

_{l could explain the lack of}

corre-lation between proviral loads and CD4

⫹

_{cell counts.}

Alterna-tively, our results could be due to a high rate of defective

integrated virus. Low cellular pathogenicity of HIV-2 in the

early stages of infection, together with the long duration of the

symptom-free period, could explain the high proviral loads. As

with human T-cell leukemia virus type 1 infection, where high

proviral loads have been reported (13, 20, 34), normal

prolif-eration of infected cells could explain the large number of

PBMC harboring proviral DNA. This discordance between

high proviral loads and inefficient replication of HIV-2 might

help to explain the roles of viral replication and immune

re-sponses in HIV-2 disease.

ACKNOWLEDGMENT

This study was supported by French National Agency on AIDS

Research (A.N.R.S.) grant 98015.

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[image:4.587.43.543.85.156.2]

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TABLE 4. Relationships among the CD4

⫹

_{cell count, HIV-2 proviral load, cell culture positivity, and plasma RNA positivity}

CD4⫹_{cell count}

(no./mm3_{) patients tested}No. of

Median (range) No. positive/no. tested (%) for: CD4⫹_{cell count,}

106_{/liter copies/10}Viral DNA load,5_PBMC Cell culture Plasma RNA

⬍200

12

a

_{68 (13–199)}

_{285 (53–2,524)}

_{11/12 (92)}

_{11/12 (92)}

200–400

9

b

_{333 (210–391)}

_{114 (⬍5–1,907)}

_{2/9 (22)}

_{7/9 (78)}

⬎400

8

b

_{511 (413–832)}

_{121 (⬍5–712)}

_{3/7 (43)}

_{4/8 (50)}

a_{Eleven patients were treated.} b_{One patient was treated.}

on May 15, 2020 by guest

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