and Immunity (2007) 8, 224–231 & 2007 Nature Publishing Group All rights reserved 1466-4879/07 $30.00 www.nature.com/gene

ORIGINAL ARTICLE CCL3L1 and CCL4L1: variable copy number in adolescents with and without human immunodeficiency virus type 1 (HIV-1) infection

W Shao1, J Tang2, W Song1, C Wang1,YLi2, CM Wilson2,3 and RA Kaslow1,2 1Department of Epidemiology, University of Alabama at Birmingham, Birmingham, AL, USA; 2Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA and 3Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL, USA

As members of the family, macrophage inflammatory 1 alpha (MIP-1a) and MIP-1b are unique in that they both consist of non-allelic isoforms encoded by different genes, namely chemokine (C-C motif) ligand 3 (CCL3), CCL4, CCL3- like 1 (CCL3L1) and CCL4L1. The products of these genes and of CCL5 (encoding RANTES, i.e., regulated on activation, normal T expressed and secreted) can block or interfere with human immunodeficiency virus type 1 (HIV-1) infection through competitive binding to chemokine (C-C motif) receptor 5 (CCR5). Our analyses of 411 adolescents confirmed that CCL3 and CCL4 genes occurred invariably as single copies (two per diploid genome), whereas the copy numbers of CCL3L1 and CCL4L1 varied extensively (0–11 and 1–6 copies, respectively). Neither CCL3L1 nor CCL4L1 gene copy number variation showed appreciable impact on susceptibility to or control of HIV-1 infection. Within individuals, linear correlation between CCL3L1 and CCL4L1 copy numbers was moderate regardless of ethnicity (Pearson correlation coefficients ¼ 0.63–0.65, Po0.0001), suggesting that the two loci are not always within the same segmental duplication unit. Persistently low serum MIP-1a and MIP-1b (in the pg/ml range) compared with high CCL5 concentration (ng/ml range) implied that multi-copy genes CCL3L1 and CCL4L1 conferred little advantage in the intensity of expression among uninfected or infected adolescents. Genes and Immunity (2007) 8, 224–231. doi:10.1038/sj.gene.6364378; published online 1 March 2007

Keywords: CCL3L1; CCL4L1; genetics; HIV-1

Introduction CCL3/CCL3L1 (MIP-1a, i.e., macrophage inflammatory protein 1 alpha), CCL4/CCL4L1 (MIP-1b) and CCL5 (chemotactic ) are small, secreted (RANTES, i.e., regulated on activation, normal T ex- molecules involved in immunological and inflammatory pressed and secreted), can inhibit or reduce viral entry responses. More than 50 human chemokine receptors into CD4 þ T-cells through competitive binding to and ligands have been identified and classified into four CCR5 or other indirect mechanisms.8–11 CCL3 and major groups based on their conserved C-X-C (a), C-C CCL4, located on the long arm of human (b), C (g), C-X3-C (d) motifs.1–3 The importance of 17, are both single-copy genes (two copies per diploid chemokines and their receptors in health and disease genome, Figure 1) regardless of population backgrounds, has been well documented, especially with the identifi- but the copy numbers of the neighboring CCL3- and cation of allelic variants that differentially influence CCL4-like genes CCL3L1 and CCL4L1 are highly variable disease susceptibility and severity.4–7 owing to a hot spot for segmental duplication,12–17 with In the context of human immunodeficiency virus type most individuals having 1–6 copies of CCL3L1 and 1 (HIV-1) infection, members of the C-C and C-X-C CCL4L1.14,17 chemokine subfamilies are of great interest (reviewed by CCL3L1 gene dosage (copy number variation, CNV) Berger et al.;4 Tang and Kaslow6 and Lederman et al.7). has been reported to influence HIV-1 infection and More specifically the, chemokine (C-C motif) receptor 5 pathogenesis.17 More specifically, CCL3L1 copy numbers (CCR5) serves as the major coreceptor for primary HIV-1 lower than the average for each ethnic group evaluated isolates. Natural CCR5 ligands (chemokines), mostly were associated with unfavorable outcomes, presumably due to lower expression of CCL3 (MIP-1a) and reduced capacity to block or interfere with HIV-1 binding to Correspondence: Professor RA Kaslow or Dr J Tang, Program in CCR5.17 In addition, CCL3L1 gene dose and CCR5 Epidemiology of Infection and Immunity, Schools of Medicine genotypes appeared to have synergistic or interactive and Public Health, University of Alabama at Birmingham, 1665 effects, being consistent with the notion that CCR5 University Boulevard, Birmingham, AL 35294-0022, USA. E-mail: [email protected] or [email protected] genotypes also influence HIV/AIDS, often in a popula- 6,18–20 Received 9 October 2006; revised 21 January 2007; accepted 22 tion-specific manner. These findings remain to be January 2007; published online 1 March 2007 confirmed in other populations and settings. Thus, we Variable CCL3L1 and CCL4L1 copy number W Shao et al 225 UTR ORF

Centromere Telomere

CCL5CCL3 CCL4 CCL4L1 CCL3L1 CCL4L2

224 kb Figure 1 Genomic organization of CCL3L1, CCL4L1 and related genes at 17q11–q12. Segmental duplication starts in a region telomeric to the CCL4 locus. The exons (boxes) as shown correspond to open-reading frames (ORF) and untranslated regions (UTR) at each locus (drawn to approximate scale). Dashed arrows indicate directions of transcription. Differing from other CCL genes (e.g., CCL5 gene in an 8.9-kb region), the CCL3L1 and CCL4L1 loci are rather small in size (1.8–1.9 kb), so are the CCL3 and CCL4 loci (based on NCBI Gene at http:// www.ncbi.nlm.nih.gov/entrez/query.fcgi?db ¼ gene&cmd ¼ search&term).

have examined the relationships of CCL3L1 and CCL4L1 CCL3L1 and CCL4L1 gene dose in relation to HIV infection, gene dose to gene expression and HIV-1-related out- viral load, CD4 þ T-cell counts and rates of disease progression comes in a racially diverse cohort of North American When the copy number of CCL3L1 and CCL4L1 was adolescents. each dichotomized at the population-specific median for comparison of patient groups, possession of either gene at a copy number lower than the median was not Results associated with HIV-1 seropositivity (P40.50) (Table 2). The copy number of CCL3L1 or CCL4L1, either as a Variable CCL3L1 and CCL4L1 gene copy number continuous or as a categorical variable, was not In quantitative 50 exonuclease (TaqMan) assays done associated with HIV-1 viral load or with CD4 þ T-cell for 184 HIV-1 seronegative (population control) and count. Likewise, neither CCL3L1 nor CCL4L1 copy 227 HIV-1 seropositive adolescents (Table 1), CCL3 number was differentially distributed across the three and CCL4 were detected constantly at two copies per distinct disease groups (controllers, intermediates and diploid genome, whereas CCL3L1 and CCL4L1 occurred noncontrollers)21 among the 227 HIV-1-seropositive with considerable variation in their copy number patients (P40.50). (Figure 2). The number of CCL3L1 copies ranged from zero (rarely) to 11 (also rare) per genome. African Americans Joint analyses of CCL3L1 and CCL4L1 gene CNV in relation had a greater number of CCL3L1 copies per genome: to HIV-1-related outcomes 55.8% of them had one to four and 44.2% had more than In further analyses of HIV-1 viral load and CD4 þ T-cell four. In the non-African American group (mainly counts, the 207 HIV-1-infected, AIDS-free patients were Hispanic and European Americans), 58.0% had one to divided into two subgroups. A subgroup (n ¼ 88) that three copies and 41.2% had more than three (Figure 2). displayed fewer copy numbers of both CCL3L1 and The median copy number for CCL3L1 was four CCL4L1 than their respective population means was for African Americans and three for those grouped as compared with the remainder as a reference group others. (n ¼ 119) for possible differences in repeated measure Similarly, the CCL4L1 copy number varied from one to analyses of HIV-1 viral load or CD4 þ T-cell counts. six per diploid genome (Figure 2). In African Americans, Neither of these analyses demonstrated associations 74.1% had one to three copies, whereas 25.9% had more (P-values adjusted for age, gender and ethnicity ¼ 0.77 than three. The respective proportions among others and 0.30, respectively). Likewise, no association was were 87.8 and 12.2% (Po0.01 between the two groups). observed in similar analyses restricted to African Amer- The median number of CCL4L1 copies was three for icans (n ¼ 54 in the group with low copy numbers and African Americans and two for those grouped as others. 94 in reference group; P-values ¼ 0.66 and 0.24). Another Regardless of ethnicity, CCL3L1 and CCL4L1 gene copies subgroup (n ¼ 103), redefined as having fewer copy showed a moderately strong overall linear correlation numbers of joint CCL3L1 and CCL4L1 than their (Pearson correlation coefficients r ¼ 0.63–0.65, Po0.0001). respective population means, was compared with the However, at the individual level, CCL3L1 gene duplica- reference group (all others, n ¼ 104). The adjusted P- tion was more frequent than CCL4L1, especially when values for viral load and CD4 þ T-cell count were 0.84 the number of CCL3L1 genes exceeded three copies per and 0.14, respectively; corresponding P-values in tests on genome. African Americans (54 subjects in subgroup and 94 in Testing of six chimpanzee (Pan troglodytes) samples reference group) were 0.98 and 0.08. For tests based on a with the same TaqMan assay detected CCL3 in none and single visit (e.g., visit 1) (Supplementary Table), the study CCL3L1 in all of them, with numbers of the latter gene population provided 80% power to detect differences (at ranging from 9 to 13. Similar findings were also reported a ¼ 0.05) that ranged from þ 0.37 to þ 0.46 log10 for HIV- by others.17 1 viral load and À89 to À111 cells for CD4 þ T-cell count.

Genes and Immunity Variable CCL3L1 and CCL4L1 copy number W Shao et al 226 Table 1 Oligonucleotide primers and fluorescent reporters used in real-time 50 exonuclease (TaqMan) assays that define CCL3L1 and CCL4L1 copy numbers

Primer/reporter specificity Annealing sites 50–30 Sequence

CCL3L1 30156-30175a Forward primer 118199-118218a TTC TGG ACC CAC TCC TCA CT 131730-131749a CCL3L1 30205’302226a Reverse primer 118248’118269a CTT CTC CAC AGC TTC CTA ACC A 131779’131800a CCL3L1 30191’30204a Reporter 118234’118247a FAM-CTG CCG GCC TCT CT 131765’131778a CCL3 Forward primer 26502-26523b CTT CTC CAC AGC TTC CTA ACC A CCL3 Reverse primer 26553’26573b TTC TGG ACC CAC TCC TCA CT CCL3 Reporter 26524’26539b FAM-CCT GCC GGC TTC GCT CCL4L1 Forward primer 46016-46036a CGT GCT AGT AGC TGC CTT CTG 147582-147602a CCL4L1 Reverse primer 46064’46088a CGA AAT AGC AGC TGC AAA AGT AGA C 147630’147654a CCL4L1 Reporter 46041’46059a FAM-TTG GTG CTG AGA GTG CTA G 147607’147625a CCL4 Forward primer 11269’11292b CTC ATG CTA GTA GCT GCC TTC TG CCL4 Reverse primer 11218-11242b CGA AAT AGC AGC TGC AAA AGT AGA C CCL4 Reporter 11253’11270b FAM-CTC TCC AGC GCT CTC AG HBBc Forward primer 70936’70955d AGC CAG GCC ATC ACT AAA GG HBBc Reverse primer 70896-70917d AAC CCT AAG GTG AAG GCT CAT G HBBc Reporter 70919’70934d FAM-ACC GAG CAC TTT CTT G

aBased on GenBank sequence GI49487494. bBased on GenBank sequence GI20377057. cHBB, hemoglobin, beta gene (as an internal reference). dBased on GenBank sequence GI28380636.

CCL3L1 and CCL4L1 gene dose in combination with found the joint analyses of CCR2–CCR5 genotypes in CCR2–CCR5 haplotypes in relation to HIV-1-related outcomes combination with CCL3L1 and CCL4L1 copy numbers. In an earlier analytic scheme,17 individuals with a favorable ligand gene dose (i.e., copy number higher Quantification of serum MIP-1a (CCL3 þ CCL3L1) and than the population-specific median) combined with a MIP-1b (CCL4 þ CCL4L1) favorable receptor genotype showed greater protection For 32 HIV-1-seronegative and 32 HIV-1-seropositive from HIV-1 and its consequences than those with a subjects (two visits per subject) with representative deleterious ligand gene dose (i.e., lower than the median CCL3L1 and CCL4L1 gene copy numbers (Table 2), serum copy number) combined with a deleterious receptor MIP-1a and MIP-1b concentrations did not differ by genotype. Here, CCL3L1 and CCL4L1 copy numbers gender, ethnicity or HIV-1 infection status (Table 3). could also be analyzed in conjunction with CCR2–CCR5 Although gene copy number was greater for CCL3L1 haplotypes. For improved statistical power, mixed than CCL4L1 (Table 2), median ratios of serum MIP-1a to models were applied to all subjects with data from MIP-1b were consistently less than 1.0 (Table 3). Within eligible (treatment-free) visits. In the 207 chronically the HIV-1-seropositive group, serum MIP-1a and MIP-1b infected patients (CD4 þ T cell 4200/ml of blood), neither concentrations did not correlate with HIV-1 viral load or þ the extremes of viral load nor the extremes of CD4 þ T- CD4 T-cell counts (P40.50). cell count showed any appreciable overall association The third ligand of CCR5, RANTES (CCL5), was with particular receptor/ligand gene combinations. In present in serum at much higher concentrations (in the alternative analyses (t-tests), slight trends in the pre- ng/ml range) when compared with serum MIP-1a and dicted direction were noted at some but not all MIP-1b (both in the pg/ml range) (Table 3). Serum CCL5 treatment-free visits (PX0.17). Further comparison of concentration was higher in HIV-1 seropositives than three patient groups defined as controllers, intermediates seronegatives (P ¼ 0.043 and 0.0001 by Mann–Whitney and noncontrollers21 also led to negative findings U-test, for visit 1 and visit 2, respectively), although (PX0.14). correlation between serum CCL5 and HIV-1 viral load Of note, the CCR2–CCR5 haplotype HHD found was weak (Spearman ro0.30, P40.05 for measures primarily in populations of African ancestry20,22,23 was averaged for the two visits) (data not shown). overrepresented (Pp0.002) in subjects with CCL3L1 and CCL4L1 copy numbers equal to or greater than the population-specific median numbers. However, HHD Discussion was not associated with HIV-1-related outcomes in this Host genetic variations are increasingly recognized study population,24 and the relationship did not con- as partly accounting for individual and population

Genes and Immunity Variable CCL3L1 and CCL4L1 copy number W Shao et al 227 differences in the initial acquisition of HIV-1 infection (a non-allelic variant of CCL3) is probably the most and in the course of disease following natural and potent ligand for CCR5 and that production of CCL3L1 treated HIV-1 infection.25–30 Recent findings implicate transcripts and protein (MIP-1a) correlates positively variations in CCL3L1 gene copy number17 as well as with CCL3L1 gene copy number.14,17 nucleotide sequences in other related genes31 whose Unlike many other CNVs found in about 12% of the products are known to block or interfere with HIV-1 ,36 CNVs for CCL3L1 and CCL4L1 can be binding to CCR5 (the major coreceptor for primary viral readily assessed for their relation to protein expression. isolates).32–35 Overall, these studies indicate that CCL3L1 However, apart from confirming the variability and comparable median numbers of CCL3L1 and CCL4L1 copies in the two ethnic groups represented, our work 35 did not detect any relationship of CCL3L1 and CCL4L1 CCL3L1 CNVs to quantifiable differences in the systemic circula- 30 African Americans tion of MIP-1a and MIP-1b as measured by serum Others 25 concentration. The enzyme-linked immunosorbent assay (ELISA) detected overall MIP-1a without distinguishing 20 CCL3L1 from CCL3 (the two mature products differ by three amino-acid residues), but the correlation analysis 15 was not compromised as CCL3 occurs as a single-copy 10 gene uniformly in all subjects. Therefore, differences in serum MIP-1a levels are more likely attributable to the 5 variability in CCL3L1 copy number than to expression of Frequency distribution (%) distribution Frequency 14,17 0 the single-copy CCL3 gene. The same inference 012345678911 would apply to MIP-1b, which corresponds to CCL4 60 plus CCL4L1 (their mature peptides are identical in CCL4L1 amino-acid sequence). 50 The HIV-1 serostatus and key outcome measures of African Americans Others HIV-1 disease control were not associated with CCL3L1 40 or CCL4L1 copy numbers, either alone (as continuous or categorical variables) or in combination with the CCR2– 30 CCR5 haplotypes. These findings were somewhat consistent with that of an earlier study based on 150 20 subjects.14 Additional convincing evidence against a prominent association of these two chemokine gene 10

Frequency distribution (%) products (MIP-1a and MIP-1b) with HIV-1 infection or disease outcomes in our study population came from the 0 123456 quantification of all three principal chemokine ligands of Range of gene copy number per individual CCR5. Serum concentrations of MIP-1a and MIP-1b showed inter-individual differences, but they were too Figure 2 Distribution of CCL3L1 and CCL4L1 gene copy numbers far lower than that of CCL5 (RANTES) to be biologically in the study population (n ¼ 411) stratified by ethnicity (about 70% dominant. Even if CCL3L1 (detected as MIP-1a in ELISA are African Americans). Overall, variability is less in CCL4L1 than CCL3L1 gene copy numbers. Median copy number is greater in assays) is considered the most potent ligand for CCR5, African Americans (four CCL3L1 and three CCL4L1 per individual) with 10 times the affinity of other CCR5 ligands (e.g., than in those loosely defined others (three CCL3L1 and two CCL4L1 CCL5),14 the amounts of serum MIP-1a (in the pg/ml per individual). range) relative to amounts of CCL5 (in the ng/ml range)

Table 2 CCL3L1 and CCL4L1 gene copy numbers in the study population (n ¼ 411) and a subset (n ¼ 64) selected for quantification of serum MIP-1a, MIP-1b and RANTES (CCL5)

Group and HIV-1 status Sample size (n) CCL3L1a CCL4L1a

Mean Median (IQR) Mean Median (IQR)

All subjects 411 4.2 4 (2–6) 2.7 3 (2–3) HIV-1 seropositivesb 227 4.3 4 (3–6) 2.7 3 (2–3) HIV-1 seronegatives 184 4.1 4 (2–6) 2.7 3 (2–3)

Selected subjects 64 3.8 3 (2–6) 2.6 2 (2–3) HIV-1 seropositives 32 3.8 4 (2–5) 2.6 3 (2–3) HIV-1 seronegatives 32 3.9 3 (2–6) 2.7 2 (2–4) aAs quantified in real-time 50 exonuclease (TaqMan) assays normalized against the hemoglobin, beta gene (HBB) in each subject (see text and Table 1). Data stratified by race are shown in Figure 2. IQR: interquartile range (the 25–75th percentile). bTwenty of these patients had advanced infection (CD4+ T-cells o200/ml of blood) at treatment-free visits; they were not used for analyses of HIV-1 viral load or CD4+ T-cell counts.

Genes and Immunity Variable CCL3L1 and CCL4L1 copy number W Shao et al 228 Table 3 Serum concentrations of three chemokine (C-C motif) ligands in adolescents with (HIV-1+) and without (HIV-1À) human immunodeficiency virus type 1 (HIV-1) infection

Analytesa Visit 1: median (IQR) Visit 2: median (IQR)

HIV-1+ (n ¼ 32) HIV-1À (n ¼ 32) HIV-1+ (n ¼ 32) HIV-1À (n ¼ 32)

MIP-1a 62.8 (56.4–72.1) 53.9 (48.7–60.3) 61.6 (56.5–66.2) 54.7 (48.5–60.9) MIP-1b 78.8 (60.8–131.4) 68.8 (49.7–101.0) 79.5 (62.6- 136.8) 77.3 (48.1–107.2) CCL5 (RANTES) Â 10À3 83.7 (55.0–134.3)b 65.8 (33.4–85.0)b 91.1 (74.2–206.9)c 61.9 (40.4–77.7)c MIP-1a/MIP-1b ratio 0.7 (0.5–1.0) 0.8 (0.6–1.1) 0.7 (0.5–1.0) 0.7 (0.6–1.0) CCL5/MIP-1a ratio 1273 (661–2021) 1244 (517–1996) 1697 (1143–3591)d 1197 (441–1478)d CCL5/MIP-1b ratio 790 (516–1613) 807 (336–1392) 1505 (779–2244)e 771 (427–1260)e

aAs quantified by ELISA-based assays and expressed in pg/ml of serum. Note that MIP-1a ¼ CCL3+CCL3L1 and MIP-1b ¼ CCL4+CCL4L1. IQR: interquartile range (the 25–75th percentile). bP ¼ 0.043 between HIV-1À and HIV-1+ groups, by Mann–Whitney U-test. cP ¼ 0.0001 by Mann–Whitney U-test. dP ¼ 0.003 by Mann–Whitney U-test. eP ¼ 0.010 by Mann–Whitney U-test.

seem trivial from the functional perspective. To date, concentration in human serum suggested that additional neither MIP-1a nor MIP-1b serum concentration has ever copies do not seem to provide any clear advantage been correlated with HIV-1-related outcomes,37 whereas for systemic production. Close similarity (over 95% circulating CCL5 (RANTES), especially as measured in sequence identity at both the genomic and the mRNA plasma depleted of CCL5-rich platelets,38,39 may be a levels) between CCL3 and CCL3L116 reinforces the useful biomarker in HIV-1 patients.37,40,41 The elevated redundancy of these loci. The regulatory elements for concentrations of serum CCL5 in our HIV-1-seropositive CCL3 and CCL3L1 do not seem to operate in a similar patients were quite consistent with all earlier observa- way; rather, CCL3 mRNA transcripts accumulate much tions.37,40,41 Recent array-based work has also corrobo- faster than does CCL3L1 mRNA in mitogen-stimulated rated the upregulation of CCL5 (RANTES) transcription peripheral blood mononuclear cells derived from healthy by HIV-1 infection,42 suggesting that CCL5 is likely to be subjects (mean CCL3L1 copy number ¼ 2).44 Overall, a dominant chemokine factor in HIV-1 infection. absence of CCL3L1 in some healthy (HIV-1-seronegative) Apart from sample size (statistical power), the mostly individuals here and elsewhere (in Australians)43 sug- female (B70%) and African American (B70%) adoles- gests that this locus may be expendable under certain cents from the REACH (Reaching for Excellence in conditions. On the other hand, because CNVs may be Adolescent Care and Health) project differ in age, more important in some cell lineages than others, racial/ethnic background, sex ratio, and follow-up CCL3L1 gene copy and function in humans merit intervals from other populations with defined CCL3L1 additional study in the setting of HIV-1 infection. They CNVs.14,17,43 Accordingly, findings from this cohort may should prove even more informative if monoclonal not always be generalizable. Nonetheless, our study was antibodies can be produced to distinguish CCL3 from expected to have adequate (80%) statistical power to CCL3L1 and their full-length products from those detect modest differences in HIV-1 viral load and CD4 þ slightly truncated at the N-terminus by CD26 and T-cell counts, before and after stratification by race quiescent cell proline dipeptidase.45,46 (Supplementary Table). Smaller differences would have been more difficult to detect, but trends toward the predicted relationships of the reported magnitude17 should have been observable. Overall, CCL3L1 and Subjects and methods CCL4L1 CNVs or their combination failed to exert major Study population and outcome measurements impact on early outcomes after HIV-1 infection in this All human subjects came from the REACH Study of the adolescent population. Adolescent Medicine and HIV/AIDS Research Network, Multiple CCL3L1 and CCL4L1 copies may have which recruited HIV-1-infected or high-risk uninfected resulted from segmental gene duplication, most likely adolescents (mostly females and African Americans) at through unequal crossing over. In this duplication 15 locations in 13 US cities. The characteristics of this process, CCL3L1 and CCL4L1 are often preserved in a cohort have been described previously.47,48 Between 1996 single unit.14–16 However, in our study population, the and 2000, data were collected at quarterly intervals for as copy numbers of CCL3L1 and CCL4L1 showed only a many as 550 subjects, of whom 530 had a sample suitable modest linear correlation, suggesting that these two for DNA extraction. Subjects selected for this study genes are not always duplicated simultaneously (i.e., at included 184 HIV-1 seronegatives (population controls) a 1:1 ratio). Such discordance in CCL3L1 and CCL4L1 and 227 HIV-1 seropositives (161 African Americans and gene copy numbers supports the existence of a second 66 others) with known history of exposure to antiretro- duplication unit.14 Our analyses of chimpanzee DNA viral therapy (ART) and up to four measurements of further imply that humans may have preferentially lost HIV-1 viral load (RNA copies per ml) in plasma and the extra copies of CCL3L1, as measurement of MIP-1a corresponding CD4 þ T-cell counts during ART-free

Genes and Immunity Variable CCL3L1 and CCL4L1 copy number W Shao et al 229 intervals within the first 3 years of follow-up. Except for MIP-1a (CCL3 þ CCL3L1), MIP-1b (CCL4 þ CCL4L1) and 20 patients with CD4 þ T-cell counts less than 200/mlof CCL5 (RANTES) concentrations in serum blood and two patients who acquired HIV-1 infection Serum MIP-1a and MIP-1b concentrations were analyzed during follow-up, HIV-1-seropositive patients here were by a multiplex SearchLight assay (Pierce Biotechnology in the early chronic phase, as judged by the time Inc., Rockford, IL, USA), which is similar to sandwich intervals from first sexual encounter or drug use to the ELISA, with improved sensitivity and reduced intra- and first positive test for HIV-1 infection. Combinations of inter-assay variability. The lower detection limit for HIV-1 viral load and CD4 þ T-cell counts at multiple SearchLight assay was 3.0 and 0.8 pg/ml for MIP-1a ART-free intervals allowed the classification of three and MIP-1b, respectively. CCL5 ELISA was done using a distinct disease groups (37 controllers, 134 intermediates commercial sandwich ELISA kit (R&D Systems, Minnea- and 56 noncontrollers) among HIV-1 seropositives.24 polis, MN, USA), as described elsewhere.54 The lower Several immunogenetic effects established in other detection limit for CCL5 was 2.0 pg/ml. studies of HIV-1 infection have been corroborated in this cohort.21,24 Statistical analyses Standard procedures in the SAS (Statistical Analysis Quantification of CCL3L1 and CCL4L1 gene copy number Software) version 9.1 (SAS Institute Inc., Cary, NC, USA) Several 50 nuclease (TaqMan) assays were designed to were used for descriptive and comparative statistics. quantify the copy numbers of CCL3L1, CCL3, CCL4L1 Briefly, the relationship between CCL3L1 and CCL4L1 and CCL4, when the single-copy gene hemoglobin, beta copy numbers was determined using the Pearson (HBB), served as the internal control. TaqMan assay for correlation coefficient. Linear regression models were CCL3L1 was similar to that used earlier,17 with minor used to test associations of genetic factors (CCL3L1 and modifications. This assay determined human CCL3L1 CCL4L1 gene dose and their combination with CCR2– gene dose by distinguishing CCL3L1 from CCL3 (Table 1). CCR5 genotypes) with cell-free HIV-1 viral load (RNA TaqMan assays for CCL3, CCL4 and CCL4L1 were new copies per ml of plasma) and CD4 þ T-cell counts (for from Applied Biosystems (Assay-by-Design) (Applied visits without ART). Differences in CCL3L1 and CCL4L1 Biosystems, Foster City, CA, USA), and specificity was gene copy numbers between HIV-1 seronegative and confirmed by bioinformatics. Real-time PCR was done seropositive were assessed by w2-tests or logistic regres- using the AB 7500 Fast System (Applied Biosystems Inc.), sion, with adjustment for other host factors like age, with the following universal cycling conditions: 2 min at gender, ethnicity, sexual behavior and other sexually 501C, 10 min at 951C, and 40 cycles of 15 s at 921C and transmitted infections (chlamydia, gonorrhea and hu- 1 min at 601C. All the samples were tested in duplicate. man herpes virus type 2).55,56 Moreover, analytic routines For each of the four target loci, gene copy number in each (mixed models) were used to stabilize the repeated individual sample was determined by the relative measure analysis of outcomes (viral load and CD4 þ T- quantification program (Applied Biosystems). Calcula- cell counts). tions were based on the estimates of template quantities, after comparing the threshold cycle numbers at which positive (over background) fluorescence was de- tected.49,50 The housekeeping gene HBB, which is present Acknowledgements at two copies per diploid genome, served to standardize This work was supported in part by Grants AI41951 (to the chemokine gene copy number counts: the copy RAK) and AI51173 (to JT) from National Institute of number of CCL3L1, CCL3, CCL4L1 and CCL4 each was Allergy and Infectious Diseases. The parental study taken as two times the ratio of its respective starting (REACH) was supported by the National Institute of template quantity to that of HBB. To gain additional Child Health and Human Development (U01 HD32830), evolutionary perspective, CCL3 and CCL3L1 TaqMan with supplemental funding from the National Institutes assays were further applied to DNA from six chimpan- on Drug Abuse, Allergy and Infectious Diseases, and zees (Pan troglodytes) (courtesy of Dr Patricia N Fultz, Mental Health. Investigators and staff of the Adolescent Department of Microbiology, University of Alabama at Medicine HIV/AIDS Research Network (1994–2001) are Birmingham). listed in J Adolesc Health 2001; 29 (suppl): 5–6. We are grateful to all participants in the REACH cohort for their valuable contributions. gene (CCR2 and CCR5) variants Nine major haplotypes at the CCR2 and CCR5 loci had been classified in the study population based on eight common SNPs and the 32-bp deletion in CCR5 genes.21,24 References This new classification scheme also captured other variants (e.g., the 59029G SNP and the P1 haplotype) 1 Baggiolini M. 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