Human leukocyte antigen genotype and risk of HIV disease progression before and after initiation of antiretroviral therapy.

Abstract

While the human leukocyte antigen (HLA) genotype has been associated with the rate of HIV disease progression in untreated patients, little is known regarding these relationships in patients using highly active antiretroviral therapy (HAART). The limited data reported to date identified few HLA-HIV disease associations in patients using HAART and even occasional associations that were opposite of those found in untreated patients. We conducted high-resolution HLA class I and II genotyping in a random sample (n = 860) of HIV-seropositive women enrolled in a long-term cohort initiated in 1994. HLA-HIV disease associations before and after initiation of HAART were examined using multivariate analyses. In untreated HIV-seropositive patients, we observed many of the predicted associations, consistent with prior studies. For example, HLA-B*57 (β = -0.7; 95% confidence interval [CI] = -0.9 to -0.5; P = 5 × 10⁻¹¹) and Bw4 (β = -0.2; 95% CI = -0.4 to -0.1; P = 0.009) were inversely associated with baseline HIV viral load, and B*57 was associated with a low risk of rapid CD4+ decline (odds ratio [OR] = 0.2; 95% CI = 0.1 to 0.6; P = 0.002). Conversely, in treated patients, the odds of a virological response to HAART were lower for B*57:01 (OR = 0.2; 95% CI = 0.0 to 0.9; P = 0.03), and Bw4 (OR = 0.4; 95% CI = 0.1 to 1.0; P = 0.04) was associated with low odds of an immunological response. The associations of HLA genotype with HIV disease are different and sometimes even opposite in treated and untreated patients.

Full text

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University of Nebraska - Lincoln
DigitalCommons@University of Nebraska - Lincoln
Public Health ResourcesPublic Health Resources
1-1-2011
Human Leukocyte Antigen Genotype and Risk of
HIV Disease Progression before and after Initiation
of Antiretroviral Therapy
Mark H. Kuniholm
Albert Einstein College of Medicine, mark.kuniholm@einstein.yu.edu
Xiaojiang Gao
SAIC-Frederick, Inc.
Andrea Kovacs
University of Southern California
Kathryn Anastos
Montefiore Medical Center
Darlene Marti
Harvard University
See next page for additional authors
Follow this and additional works at:http://digitalcommons.unl.edu/publichealthresources
Part of thePublic Health Commons
This Article is brought to you for free and open access by the Public Health Resources at DigitalCommons@University of Nebraska - Lincoln. It has
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Kuniholm, Mark H.; Gao, Xiaojiang; Kovacs, Andrea; Anastos, Kathryn; Marti, Darlene; Greenblatt, Ruth M.; Cohen, Mardge H.;
Minkoff, Howard; Gange, Stephen J.; Fazzari, Melissa; Young, Mary A.; Strickler, Howard D.; and Carrington, Mary, "Human
Leukocyte Antigen Genotype and Risk of HIV Disease Progression before and after Initiation of Antiretroviral Therapy" (2011).
Public Health Resources.Paper 130.
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Authors
Mark H. Kuniholm, Xiaojiang Gao, Andrea Kovacs, Kathryn Anastos, Darlene Marti, Ruth M. Greenblatt,
Mardge H. Cohen, Howard Minkoff, Stephen J. Gange, Melissa Fazzari, Mary A. Young, Howard D. Strickler,
and Mary Carrington
This article is available at DigitalCommons@University of Nebraska - Lincoln:http://digitalcommons.unl.edu/
publichealthresources/130

Page 3

JOURNAL OF VIROLOGY, Oct. 2011, p. 10826–10833
0022-538X/11/$12.00 doi:10.1128/JVI.00804-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 20
Human Leukocyte Antigen Genotype and Risk of HIV
Disease Progression before and after Initiation
of Antiretroviral Therapy?‡
Mark H. Kuniholm,1* Xiaojiang Gao,2,3Xiaonan Xue,1Andrea Kovacs,4Kathryn Anastos,1,5
Darlene Marti,2,3Ruth M. Greenblatt,6Mardge H. Cohen,7Howard Minkoff,8
Stephen J. Gange,9Melissa Fazzari,1Mary A. Young,10
Howard D. Strickler,1† and Mary Carrington2,3†
Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, New York1; Cancer and
Inflammation Program, Laboratory of Experimental Immunology, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland2;
Ragon Institute of MGH, MIT and Harvard, Charlestown, Massachusetts3; Department of Pediatrics, University of
Southern California, Los Angeles, California4; Department of Medicine, Montefiore Medical Center, Bronx,
New York5; Department of Clinical Pharmacy, University of California, San Francisco, California6;
CORE Center, Cook County Bureau of Health Services, Chicago, Illinois7; Department of
Obstetrics and Gynecology, Maimonides Medical Center, Brooklyn, New York8;
Department of Epidemiology, Johns Hopkins Bloomberg School of
Public Health, Baltimore, Maryland9; and Department of Medicine,
Georgetown University Medical Center, Washington, DC10
Received 20 April 2011/Accepted 5 August 2011
While the human leukocyte antigen (HLA) genotype has been associated with the rate of HIV disease
progression in untreated patients, little is known regarding these relationships in patients using highly active
antiretroviral therapy (HAART). The limited data reported to date identified few HLA-HIV disease associa-
tions in patients using HAART and even occasional associations that were opposite of those found in untreated
patients. We conducted high-resolution HLA class I and II genotyping in a random sample (n ? 860) of
HIV-seropositive women enrolled in a long-term cohort initiated in 1994. HLA-HIV disease associations before
and after initiation of HAART were examined using multivariate analyses. In untreated HIV-seropositive
patients, we observed many of the predicted associations, consistent with prior studies. For example, HLA-
B*57 (? ? ?0.7; 95% confidence interval [CI] ? ?0.9 to ?0.5; P ? 5 ? 10?11) and Bw4 (? ? ?0.2; 95% CI ?
?0.4 to ?0.1; P ? 0.009) were inversely associated with baseline HIV viral load, and B*57 was associated with
a low risk of rapid CD4?decline (odds ratio [OR] ? 0.2; 95% CI ? 0.1 to 0.6; P ? 0.002). Conversely, in treated
patients, the odds of a virological response to HAART were lower for B*57:01 (OR ? 0.2; 95% CI ? 0.0 to 0.9;
P ? 0.03), and Bw4 (OR ? 0.4; 95% CI ? 0.1 to 1.0; P ? 0.04) was associated with low odds of an immunological
response. The associations of HLA genotype with HIV disease are different and sometimes even opposite in
treated and untreated patients.
Human leukocyte antigen (HLA) molecules play a central
role in the immune response to HIV by presenting viral anti-
gens to T cells. Specifically, HLA class I molecules are ex-
pressed on the cell surfaces of most nucleated cells, where they
present intracellularly produced antigens to CD8?cytotoxic T
lymphocytes (CTLs), the major effector cells of the adaptive
immune system. HLA class II molecules are expressed by spe-
cialized antigen-presenting cells and present antigens from ex-
tracellular sources to CD4?T cells, major regulators of the
adaptive immune response.
HLA genes are highly polymorphic, and these variations can
result in differences in the antigen binding characteristics of
HLA molecules. Prior studies have reported that specific HLA
class I alleles are associated with the rate of HIV disease
progression. In particular, a series of well-conducted prospec-
tive investigations have shown that HLA-B*57, B*27, the Bw4
allele group, and heterozygosity at HLA class I loci are each
strongly associated with slower HIV disease progression (re-
viewed in references 9, 11, and 18). Conversely, the B*35(Px)
group has been associated with rapid HIV disease progression
(10, 15).
These prior studies, though, focused on specimens and data
obtained prior to the use of highly active antiretroviral therapy
(HAART). Few studies have examined the relationship of
HLA polymorphism and HIV disease progression in individu-
als currently using HAART (2, 7, 35). Nonetheless, there is
substantial interindividual heterogeneity in disease progression
among patients using HAART, suggesting that genetic or
other host factors may continue to influence HIV disease pro-
gression following HAART initiation. For example, 15% to
30% of individuals receiving virologically suppressive HAART
* Corresponding author. Mailing address: Department of Epidemi-
ology & Population Health, Albert Einstein College of Medicine,
Belfer Building, Room 1308, 1300 Morris Park Ave., Bronx, NY 10461.
Phone: (718) 430-4129. Fax: (718) 430-8780. E-mail: mark.kuniholm
@einstein.yu.edu.
‡ Supplemental material for this article may be found at http://jvi
.asm.org/.
† Both senior authors contributed equally to this study.
?Published ahead of print on 17 August 2011.
10826
This article is a U.S. government work, and is not subject to copyright in the United States.

Page 4

do not attain substantial increases in CD4?T cell levels (16).
However, the host factors associated with the risk of immuno-
logical nonresponse are not well known (16).
Among the three studies of which we are aware that
examined the relationship of HLA genotype with outcomes
in HAART users, only a single finding in one study was con-
sistent with those in untreated patients, a positive association
between class I heterozygosity and subsequent increases in
CD4?count (7). In fact, B*57 (in two studies) and Bw4 (in one
study) were associated with a smaller increase in CD4?count
following HAART initiation (2, 35), inconsistent with the pro-
tective effects of these alleles found in numerous studies of
patients prior to HAART.
The reasons for these counterintuitive results are unclear.
However, no studies to our knowledge have examined the
relation of HLA genotype with long-term risk of AIDS events
in HAART users, nor have any studies examined HLA alleles
other than those reported to be significant prior to HAART,
i.e., to identify HLA associations with HIV disease progression
that might only be significant in patients using HAART. In the
current investigation, therefore, we studied the relation of the
HLA genotype with HIV disease progression both before and
after HAART initiation in a large, long-term prospective co-
hort of HIV-seropositive women.
MATERIALS AND METHODS
Study population. The Women’s Interagency HIV Study (WIHS) is a prospec-
tive, multicenter cohort study of 2,793 HIV-seropositive and 975 at-risk HIV-
seronegative women enrolled through similar sources at six clinical sites. The
initial enrollment was conducted between October 1994 and November 1995,
and a second recruitment occurred during 2001 and 2002. WIHS women are
followed semiannually with physical exams, specimen collection including blood,
and detailed questionnaires regarding health and behavior (5). The WIHS pro-
tocol was approved by each local institutional review board, and all participants
signed informed consent. The current analyses used data and specimens col-
lected through February 2008.
Most HIV-seropositive WIHS women provided consent for genetic testing
(n ? 2,556). Among these women, we selected a stratified random sample (n ?
899) for HLA testing. These strata were defined by CD4?count at enrollment
(?500 [n ? 256], 200 to 500 [n ? 125], and ?200 [n ? 125] cells/?l) and also
included all women who reported injection drug use (IDUs) at enrollment if not
already included through sampling by CD4?count (n ? 393). IDUs were over-
sampled in this fashion to ensure sufficient prevalence of hepatitis C virus (HCV)
for studies of HLA and HCV viremia. We then excluded women who had
received HAART prior to study enrollment (n ? 29) and women with unknown
HCV serostatus (n ? 10).
Clinical AIDS was defined as self-report of any of the 23 individual AIDS-
defining conditions specified in the 1993 CDC classification system for AIDS
surveillance (4) and excluded “immunological AIDS” (i.e., a CD4?count of
?200 cells/?l or a CD4?cell percentage of less than 14%). HAART was defined
according to the recommendations of the DHHS Panel on Antiretroviral Guide-
lines for Adults and Adolescents (DHHS 2008), as follows: the use of three or
more antiretroviral medications, one of which has to be a protease inhibitor, a
nonnucleoside reverse transcriptase inhibitor, one of the nucleoside reverse
transcriptase inhibitors abacavir or tenofovir, an integrase inhibitor (e.g., ralte-
gravir), or an entry inhibitor (e.g., maraviroc or enfuvirtide).
Trends in HAART and non-HAART use in the WIHS cohort have been
previously reported in detail (13, 37). Briefly, monotherapy and non-HAART
combination regimens were relatively common in the 1990s but declined over
time such that these regimens have made up ?5% of all regimens during the last
10 years. As measured by person visits (because the same woman could be on
different regimens at different times), 80% of all reported regimens have been
HAART, while 7% were monotherapy and 13% were non-HAART combination
therapy.
Clinical laboratory testing. T cell subsets (no. of cells/?l) were determined by
flow cytometry in laboratories participating in the AIDS Clinical Trials Quality
Assurance Program (8). Plasma HIV RNA levels were measured through visit 6
with a nucleic acid sequence-based amplification method that had 4,000
copies/ml as its lower threshold of detection (Organon Teknika Corp., Durham,
NC). Similar methods with greater sensitivity were used thereafter as they be-
came clinically available (i.e., the lower threshold of detection was 400 copies/ml
during visits 7 to 9 and 80 copies/ml thereafter). Phylogenetic analysis to deter-
mine HIV-1 subtype has not been conducted for all WIHS women, but all 18
subjects who have been typed were subtype B (23, 34). HCV serostatus was
determined at baseline using a commercial second- or third-generation enzyme
immunoassay, and HCV viremia was determined in HCV-seropositive women
using either the COBAS Amplicor Monitor 2.0, as previously described (3), or
the COBAS TaqMan assay (both from Roche Diagnostics, Branchburg, NJ).
HLA genotyping. Genomic DNA was prepared from subjects’ lymphoblastoid
B cell lines or from peripheral blood lymphocytes. Protocols for HLA genotyping
have been standardized through the International Histocompatibility Working
Group (http://www.ihwg.org). Briefly, HLA class I genes (HLA-A, -B, and -C)
were amplified using locus-specific PCR primers flanking exons 2 and 3, the
polymorphic segments of the class I genes. The 1-kb PCR products were blotted
on nylon membranes and hybridized with a panel of sequence-specific oligonu-
cleotide (SSO) probes. The HLA alleles were assigned by the reaction patterns
of the SSO probes, according to known HLA sequences. Any ambiguous SSO
probing was resolved by sequencing analysis, as previously described (15). HLA
class II typing was conducted using high-resolution SSO typing for HLA-DQA,
-DQB, and -DRB1 loci, using the polymorphic exon 2. DRB genotyping involved
a two-step procedure. First, the broad serological DR types were determined
using a pair of DRB generic primers and a panel of SSO probes. Allele-level
DRB typing was then achieved by using group-specific primers to amplify the
DRB alleles determined in the generic typing, followed by SSO hybridization.
For DQA and DQB, locus-specific PCR was performed, followed by SSO hy-
bridization. The number of women with complete allele information varied by
HLA locus, as follows: 850 for HLA-A, 827 for HLA-B, 810 for HLA-C, 763 for
HLA-DRB1, 470 for HLA-DQA1, and 471 for HLA-DQB1.
Statistical methods. (i) HIV disease prior to HAART initiation. Initial analyses
examined the relation of HLA genotype with HIV viral load and CD4?T cell
count at enrollment in HAART-naïve women. HIV viral load and CD4?count
were log10and square root transformed, respectively, to normalize these values
and were analyzed using multivariable linear regression. These models were
adjusted for self-reported race/ethnicity (i.e., non-Hispanic white, non-Hispanic
black, Hispanic, and other) and HCV infection, as parameterized using three
HCV dummy variables (i.e., HCV serostatus, HCV viremia, and unknown
viremia). This addressed the fact that 18% of HCV-seropositive patients were
not HCV viremic and that 46 HCV-seropositive patients lacked HCV RNA data
(for these individuals, HCV serostatus was the only data available). As men-
tioned, 10 patients lacking both HCV serologic and RNA data were excluded.
The number of covariates in these models was carefully limited in part because
recent statistical studies have demonstrated that genetic association models are
generally unaffected by control for multiple covariates and have recommended
restricting the number of adjustment variables (27). Therefore, factors such as
smoking and other behavioral covariates—factors not thought to be influenced
by the HLA genes under study—were not included in our models. HCV was
included in these models because HCV infection may influence HIV disease
progression and is also associated with HLA (12, 26). Similarly, race/ethnicity is
strongly related to HLA genotype and may be related to other genetic factors
related to HIV disease progression.
We then examined HLA associations with incident clinical AIDS (among
women free of clinical AIDS at baseline) using continuous-time (i.e., exact-
calendar-date) Cox models that controlled for race/ethnicity, HCV infection,
baseline CD4?count (?500, 200 to 500, and ?200 cells/?l), and baseline HIV
RNA level (?100,000 versus ?100,000 copies/ml). Inclusion of baseline CD4?
count, HIV RNA level, and HCV infection in these models controlled for both
initial disease status and the probability of selection by our stratified random
sampling design; i.e., we essentially addressed the following question: among
women who were otherwise similar in relation to starting immune status and
HCV infection, did HLA genotype help explain the heterogeneity in HIV disease
progression? These analyses were limited to the period prior to the widespread
use of HAART to minimize the possibility of “confounding by indication” (i.e.,
selection bias related to early initiation of HAART [1]). Specifically, we censored
all data after September 1996 when the prevalence of HAART use first exceeded
5% of WIHS subjects. Women who died, were lost to follow-up, or missed more
than a single visit (two or more consecutive visits) were censored at their last
appropriate study visit.
We additionally examined the relation of HLA genotype with CD4?decline.
The short pre-HAART follow-up time, though, limited our ability to accurately
estimate slopes, because these levels are known to have substantial intraindi-
VOL. 85, 2011 HLA AND HIV DISEASE PROGRESSION BEFORE AND AFTER HAART10827

Page 5

vidual variability (28, 36). To address this issue, we used previously published
methods and compared cases who had a rapid and persistent CD4?T cell count
decline to those who maintained their CD4?count—two clearly defined groups
(38). Specifically, the cases were women with ?50 CD4?cells/mm3at baseline
who met the following definition: compared with baseline, the CD4?T cell
counts at both the second and third visit either (i) fell into a lower CD4?T cell
stratum (based on 4 strata, namely, ?500, 200 to 500, 50 to 200, and ?50 CD4?
T cells/mm3) or (ii) had a ?50% reduction in count (within the same stratum).
Control patients were women whose CD4?count remained at ?95% of the
baseline value through the first three visits. These analyses were conducted using
logistic regression and controlled for race/ethnicity, HCV infection, and baseline
CD4?count.
We focused our analysis on the alleles and allele groups with an a priori high
prior probability of association in HAART-naïve women, based on a recent
comprehensive review of the literature (18) and other large studies (10, 14, 30,
40). No adjustment for multiple comparisons was conducted for these a priori
hypothesized associations. However, we also conducted exploratory analyses of
alleles without a high prior probability of association. These exploratory analyses
adjusted for multiple comparisons by using Bonferroni corrections. Our study
included 114 alleles and allele groups with ?3% prevalence (a cutoff used
previously [25]), and statistical significance was defined as P values of ?0.05/
(114 ? 0.0004) for all exploratory analyses. While there are other less con-
servative methods for addressing multiple comparisons which are commonly
employed, none of these methods to our knowledge can readily account for
important covariates.
(ii) HIV disease after HAART initiation. The time of HAART initiation was
set as the visit an HIV-seropositive WIHS woman first reported using medica-
tions meeting the definition of HAART. Initial analyses examined the associa-
tion of HLA genotype with the short-term virological and immunological re-
sponse to HAART. Consistent with an earlier study (31), a virological response
was defined as a reduction in the HIV RNA level by ?90%, or to undetectable
levels, for at least two consecutive visits within 12 months of HAART initiation
and was limited to women who had detectable plasma HIV RNA levels prior to
HAART initiation. In additional sensitivity analyses, we more restrictively de-
fined a virological response as only undetectable HIV RNA to determine if this
affected our findings. The short-term immunological response was evaluated
among the subset of women who met the definition of a virological responder
and was defined as in prior studies (19, 32) as a CD4?T cell count increase of
?50 cells/?l following at least two sequential consecutive visits (6 months)
showing a virological response.
These analyses were conducted using logistic regression controlling for race/
ethnicity, HCV infection, CD4?count, and HIV viral load at the visit prior to
HAART initiation, as well as the enrollment period (1994 to 1995 or 2001 to
2002). Analyses of virological response also controlled for self-reported adher-
ence (?95% or ?95%) to the prescribed HAART regimen during the first 12
months of HAART use. Lastly, we evaluated new “incident” occurrences of
AIDS-defining conditions following HAART initiation. These analyses were
conducted using multivariate Cox models with censoring and adjustment for
confounding variables as described above.
RESULTS
Demographic and clinical characteristics of the study pop-
ulation. Selected characteristics of the 860 HIV-seropositive
women in this study are shown in Table 1. Study women were
largely in their late thirties to early forties at enrollment and
were majority black, non-Hispanic. Thirty-one percent of study
women reported clinical AIDS at enrollment, and 30% had
received prior antiretroviral therapy (but not HAART). As
expected based on the study sampling design (see Materials
and Methods), HIV-seropositive women included in the cur-
rent investigation were more likely to be HCV seropositive
than those HIV-seropositive women who were not enrolled in
this substudy (65% versus 24%; P ? 0.01). Furthermore, study
women had higher baseline CD4?T cell counts (a median of
397 versus 325 cells/?l) and were more likely to be black,
non-Hispanic, and less likely to be Hispanic than the HIV-
seropositive women who were not included (all P ? 0.01).
Overall, the subjects enrolled in this substudy had a median
number of four visits or 1.5 years of follow-up (involving 2,615
person visits of data) prior to the widespread use of HAART
and post-HAART initiation follow-up time of 8.5 years (in-
volving 7,678 person visits).
HIV disease prior to HAART initiation. We examined HLA
alleles and allele groups with a frequency of ?3% in our
population. Table 2 shows the subset of these alleles with a
high prior probability of association based on earlier studies
and their relationships with HIV disease cross-sectionally at
enrollment and prospectively before women initiated HAART.
Results for all other class I and II alleles are shown in Table S1
in the supplemental material.
Our cross-sectional analyses of HIV viral load included all
subjects (n ? 860), while analyses of CD4?count at enroll-
ment excluded four of these women who lacked CD4?data. Of
15 alleles and allele groups with a high prior probability of
association with untreated HIV disease, 13 had cross-sectional
associations with CD4?count and/or HIV viral load at enroll-
ment that were in the predicted direction, showing that the
WIHS population was not dissimilar to those studied in earlier
investigations. The HLA-B*57 allele group, for example, had a
very strong and highly significant positive association with
the CD4?count at enrollment (? ? 226; 95% confidence
interval [CI] ? 166 to 285; P ? 6 ? 10?11) and a negative
association with the log10HIV viral load at enrollment (? ?
?0.7; 95% CI ? ?0.9 to ?0.5; P ? 5 ? 10?11) in models
that adjusted for race/ethnicity and HCV infection.
Additional alleles with associations in the predicted direc-
tion at enrollment included B*18:01, B*27:05, B*57:01, B*57:
03, B*58:02, the B*27 group, the B*35(Px) group, the Bw4
homozygous group, the Bw4-80I homozygous group, the
common HLA-B allele group, the rare HLA-B allele group,
TABLE 1. Characteristics of HIV-seropositive women at enrollmentd
CharacteristicValue
Median age in yrs (IQR)c...................................................38 (33–43)
Race/ethnicity (no. of women ?%?)
Black, non-Hispanic.........................................................
White, non-Hispanic........................................................
Hispanic.............................................................................
Other..................................................................................
538 (63)
130 (15)
170 (20)
22 (3)
Recruitment cycle (no. of women ?%?)
1994-1995...........................................................................
2002....................................................................................
832 (97)
28 (3)
CD4?T cell count (no. of women ?%?)a
?200 cells/?l....................................................................
200–500 cells/?l ................................................................
?500 cells/?l....................................................................
201 (23)
340 (40)
315 (37)
Median HIV log10viral load (IQR)................................... 4.1 (3.6–4.9)
No. of women with clinical AIDS (%)..............................
No. of women with prior
antiretroviral therapy (%)...........................................
HCV status (no. of women ?%?)........................................
HCV seropositive.............................................................
HCV RNA positiveb........................................................
269 (31)
259 (30)
563 (65)
423 (82)
aAmong the 856 women with baseline CD4?cell counts.
bPercentage among 517 HCV-seropositive women tested for HCV RNA.
cIQR, interquartile range.
dA total of 860 women were analyzed.
10828KUNIHOLM ET AL.J. VIROL.

Page 6

and C*04:01. One exploratory allele, C*07:01, had signifi-
cant associations with high CD4?counts at enrollment (? ?
87; 95% CI ? 40 to 134; Pcorrected? 0.03) and low log10HIV
viral loads at enrollment (? ? ?0.3; 95% CI ? ?0.5 to
?0.2; Pcorrected? 0.01) after correction for the number of
tests performed (see Table S1 in the supplemental mate-
rial).
Although the pre-HAART follow-up was fairly short, we
observed inverse associations between rapid CD4?count de-
cline and B*57 alleles individually (B*57:01: odds ratio [OR] ?
0.1, 95% CI ? 0 to 0.9; B*57:03: OR ? 0.3, 95% CI ? 0.1 to
1.0) and as a group (B*57: OR ? 0.2, 95% CI ? 0.1 to 0.6),
consistent with the cross-sectional results. We also observed
positive associations of rapid CD4?count decline with the
B*35(Px) group (OR ? 2.6; 95% CI ? 1.2 to 5.9) and with
having a common HLA-B allele (OR ? 5.5; 95% CI ? 2.6 to
11.5). However, no alleles showed association with incident
AIDS during the short follow-up period.
HIV disease after HAART initiation. To study the occur-
rence of new AIDS events, we focused on the 503 women who
initiated HAART during follow-up and had CD4?counts and
HIV RNA levels measured within the year prior to initiating
therapy. A subset of 372 of these women had complete viro-
logical data for the 12 months (three consecutive visits) fol-
lowing HAART initiation and detectable plasma HIV RNA
levels prior to HAART initiation, and these data were used to
evaluate the short-term virological response to HAART. We
additionally examined the short-term immunological response
to HAART among the subset of 176 women who achieved a
virological response. Four women with undetectable plasma
HIV RNA levels prior to HAART initiation were not included
in our analyses of virological and immunological responses.
We first examined the enrollment characteristics of those
who did versus did not initiate HAART or were excluded from
these analyses for other reasons. Some women, for example,
died (n ? 74) or were lost to follow-up (n ? 51) prior to the
widespread use of HAART (as defined in Materials and Meth-
ods), while other women died (n ? 79) or were lost to fol-
low-up (n ? 26) in the HAART era without ever initiating
HAART. Still other women were excluded because they lacked
CD4?counts and HIV RNA levels measured within the year
prior to initiating therapy (n ? 54), because they started
HAART only on their last WIHS visit and thus had no fol-
low-up data (n ? 17) or because they are still being followed
but have not initiated HAART (n ? 56). Overall, women
included in our analyses of HAART initiators were less likely
to have a CD4?count of ?200 (P ? 0.01), to have clinical
AIDS (P ? 0.01), or to be HCV seropositive (P ? 0.04) than
excluded women.
Two alleles, HLA-B*57:01 (OR ? 0.2; 95% CI ? 0.0 to 0.9;
P ? 0.03) and B*58:01 (OR ? 0.3; 95% CI ? 0.1 to 0.9; P ?
0.03), and the Bw4-80I homozygous allele group (OR ? 0.3;
95% CI ? 0.1 to 1.0; P ? 0.04) were associated with low odds
of virological response to HAART; i.e., a reduction in the HIV
RNA level by ?90% or to undetectable levels, during at least
two sequential visits within 12 months of HAART initiation
(shown in Table 3). The broader Bw4 homozygous group had
a borderline association (OR ? 0.6; 95% CI ? 0.3 to 1.0; P ?
0.05) with virological response. In contrast, other alleles and
allele groups with a high prior probability of association with
TABLE 2. HLA associations with HIV disease progression before HAART initiation (pre-HAART)g
Allele/allele groupe
No. of
womenf
CD4?cell count at
enrollment
(? ?95% CI?) (n ? 856)a,b
Log10HIV RNA level at
enrollment
(? ?95% CI?) (n ? 860)b
Rapid CD4?decline
(OR ?95% CI?)
(n ? 253; 82
events)c
Incident AIDS
(HR ?95% CI?)
(n ? 523; 86 events)d
B*18:01
B*27:05
B*27
B*51:01
B*57:01
B*57:03
B*57
B*58:01
B*58:02
C*04:01
B*35(Px)
Bw4 homozygosity
Bw4-80I homozygosity
B common
B rare
49
33
44
55
29
64
99
65
46
246
142
156
48
207
207
?119 (?204, ?34)
103 (0, 206)
94 (5, 183)
?79 (?159, 1)
151 (41, 261)
240 (167, 314)
226 (166, 285)
66 (?9, 140)
?87 (?175, 2)
?78 (?122, ?35)
?74 (?128, ?20)
53 (2, 104)
132 (47, 217)
?59 (?106, ?13)
10 (?38, 57)
0.1 (?0.2, 0.4)
?0.5 (?0.9, ?0.2)
?0.4 (?0.7, ?0.1)
0.1 (?0.2, 0.3)
?0.8 (?1.2, ?0.4)
?0.7 (?1.0, ?0.5)
?0.7 (?0.9, ?0.5)
?0.1 (?0.4, 0.1)
0.4 (0.1, 0.7)
0.2 (0.1, 0.4)
0.2 (0.0, 0.4)
?0.2 (?0.4, ?0.1)
?0.5 (?0.7, ?0.2)
0.3 (0.1, 0.4)
?0.2 (?0.3, 0)
0.4 (0.1, 2.0)
0.6 (0.1, 3.6)
1.0 (0.2, 4.4)
1.5 (0.4, 4.5)
0.1 (0, 0.9)
0.3 (0.1, 1.0)
0.2 (0.1, 0.6)
1.2 (0.4, 4.0)
2.4 (0.6, 8.7)
0.9 (0.4, 1.7)
2.6 (1.2, 5.9)
1.1 (0.6, 2.2)
0.3 (0.1, 1.1)
5.5 (2.6, 11.5)
0.7 (0.3, 1.4)
0.6 (0.2, 1.7)
0.8 (0.2, 3.7)
1.5 (0.6, 3.8)
1.8 (0.9, 3.5)
2.2 (0.8, 6.4)
0.5 (0.2, 1.6)
1.0 (0.5, 2.0)
1.0 (0.4, 2.5)
1.2 (0.6, 2.7)
0.9 (0.6, 1.5)
1.0 (0.5, 1.8)
1.0 (0.5, 1.7)
0.3 (0.0, 1.8)
0.6 (0.3, 1.1)
1.2 (0.7, 2.0)
a? estimates were obtained from analyses of untransformed CD4?counts to facilitate interpretation. Statistical significance, however, was obtained from analyses
using square-root-transformed values (see Table S1 in the supplemental material). Four women lacked baseline CD4?T cell data.
bLinear regression models adjusted for race/ethnicity and HCV infection.
cLogistic regression models adjusted for race/ethnicity, HCV infection, and baseline CD4?count.
dCox regression models adjusted for race/ethnicity, HCV infection, baseline CD4?count, and baseline HIV RNA level.
eIn addition to individual alleles, the analyses included HLA-B and -C alleles that act as ligands for killer immunoglobulin-like receptors (KIR), namely, the Bw4
and Bw4-80I groups (30) and groups C1 and C2 (24), allele zygosity (HLA-B homozygosity was too rare ??3%? for inclusion) (10, 39), and allele frequency at the
HLA-B locus (7, 33, 40). Allele frequency was examined by comparing alleles with moderately common genotypes (second and third quartiles of allele frequency) to
those with rare (first quartile) or common (fourth quartile) genotypes (7, 33). This table shows the results from alleles and allele groups with a high prior probability
of association with untreated HIV progression. Other exploratory results are shown in Table S1 in the supplemental material.
fThe number of women homozygous or heterozygous for a given allele or allele group in the total study population (n ? 860). Analysis-specific allele frequencies
are shown in Table S1 in the supplemental material.
gHR, hazard ratio. Note that shading indicates statistically significant associations.
VOL. 85, 2011 HLA AND HIV DISEASE PROGRESSION BEFORE AND AFTER HAART10829

Page 7

untreated HIV disease progression were not significantly as-
sociated with virological response, nor did any associations
with exploratory alleles retain statistical significance after ad-
justment for the number of tests performed. In sensitivity anal-
yses that used only undetectable HIV RNA to define virolog-
ical response, the Bw4-80I homozygous group remained
significantly associated with virological response to HAART
(OR ? 0.2; 95% CI ? 0.1 to 0.8; P ? 0.02), whereas B*57:01
and B*58:01, while retaining their inverse associations, became
nonsignificant (see Table S1 in the supplemental material).
We also studied immunological response to HAART. While
the Bw4 homozygous and Bw4-80I groups had similar associ-
ations with low odds of immunological response (Table 3), and
B*57:01 and B*58:01 also had an inverse association with odds
of immunologic response, only the association with the broad
Bw4 homozygous group reached statistical significance (OR ?
0.4; 95% CI ? 0.1 to 1.0; P ? 0.04). No other significant
associations with immunological response were observed, ei-
ther among alleles with a high prior probability of association
with untreated HIV disease progression or among the ex-
ploratory alleles, after correction for the number of tests
performed (Table 3; see also Table S1 in the supplemental
material).
Because B*57:01 and B*58:01 are members of the Bw4 and
Bw4-80I allele groups, we studied the extent to which these two
individual alleles might have accounted for the Bw4 and Bw4-
80I findings. After excluding women with B*57:01 and B*58:
01, the Bw4-virological response relationship became null
(OR ? 1.0; 95% CI ? 0.5 to 1.9; P ? 0.92), as did the
Bw4-80I-virological response relationship. In contrast, the re-
lation of Bw4 with immunological response was not meaning-
fully altered by these exclusions (OR ? 0.4; 95% CI ? 0.1 to
1.0; P ? 0.06) or even by additionally excluding women with
B*27 (OR ? 0.3; 95% CI ? 0.1 to 0.9; P ? 0.04). Too few
subjects with Bw4-80I without B*57:01 and B*58:01 were
available to study in relation to immunological response.
Prior studies of HAART-naïve patients found that B*57
alleles had among the strongest and most consistent associa-
tions with the rate of HIV disease progression (18). Therefore,
we additionally examined whether poor outcomes in B*57:01-
positive women who initiated HAART could relate to a high
prevalence of characteristics associated with HIV disease pro-
gression. B*57:01-positive women who did and did not initiate
HAART were similar in relation to age, race/ethnicity, recruit-
ment cohort, HIV viral load, HCV status, and the prevalence
of other alleles associated with higher rates of HIV disease
progression [e.g., B*58:02, B*35(Px), and C*04:01]. While
B*57:01-positive HAART initiators did have lower CD4?
counts at enrollment than those who did not initiate HAART
(median CD4?counts ? 424 and 682, respectively; P ? 0.04),
as mentioned previously, our statistical models addressed this
by appropriately adjusting for CD4?and HIV RNA levels at
the visit prior to HAART initiation (see “Statistical methods”).
B*57:01-positive HAART initiators were also more likely than
noninitiators to have received prior antiretroviral therapy
(33% versus 0%; P ? 0.04), but inclusion of this as a covariate
in our models had no meaningful effect on the findings (data
not shown).
Lastly, we examined HLA associations with long-term risk of
new AIDS-defining conditions following HAART initiation.
TABLE 3. HLA associations with HIV disease progression after HAART initiation (post-HAART)h
Allele/allele groupf
No. of womeng
Response to HAART (OR ?95% CI?)
New AIDS events (HR ?95% CI?)
(n ? 503; 219 events)e
Virological
(n ? 372; 176 events)a,b
Immunological
(n ? 176; 45 events)c,d
B*18:01
B*27:05
B*27
B*51:01
B*57:01
B*57:03
B*57
B*58:01
B*58:02
C*04:01
B*35(Px)
Bw4 homozygosity
Bw4-80I homozygosity
B common
B rare
29
23
30
35
15
33
53
36
22
148
80
91
29
134
127
1.9 (0.7, 5.5)
1.4 (0.4, 4.2)
1.2 (0.4, 3.4)
0.9 (0.4, 2.3)
0.2 (0.0, 0.9)
1.7 (0.7, 4.3)
0.9 (0.4, 1.9)
0.3 (0.1, 0.9)
1.0 (0.3, 2.7)
1.4 (0.8, 2.3)
1.5 (0.8, 2.8)
0.6 (0.3, 1.0)
0.3 (0.1, 1.0)
0.7 (0.4, 1.2)
1.3 (0.7, 2.2)
0.7 (0.2, 2.5)
0.8 (0.2, 3.7)
0.5 (0.1, 2.1)
1.6 (0.4, 7.2)
0.2 (0.0, 2.4)
0.4 (0.1, 1.6)
0.6 (0.2, 1.7)
0.5 (0.1, 2.3)
1.8 (0.2, 18.0)
1.3 (0.6, 2.8)
1.4 (0.4, 4.2)
0.4 (0.1, 1.0)
0.4 (0.1, 2.3)
1.5 (0.6, 4.0)
1.0 (0.4, 2.3)
1.2 (0.6, 2.2)
0.6 (0.2, 1.4)
0.7 (0.4, 1.4)
1.6 (1.0, 2.6)
1.2 (0.5, 2.8)
0.7 (0.4, 1.3)
0.8 (0.5, 1.3)
1.1 (0.6, 1.8)
1.2 (0.6, 2.2)
1.0 (0.7, 1.3)
1.0 (0.7, 1.4)
1.0 (0.7, 1.4)
0.8 (0.4, 1.5)
1.1 (0.8, 1.5)
1.2 (0.9, 1.6)
aDefined as a reduction in the HIV RNA level by ?90%, or to undetectable levels, for at least two sequential visits within 12 months of HAART initiation.
bLogistic regression models adjusted for race/ethnicity, HCV serostatus, HCV viremia status, pre-HAART CD4?count, pre-HAART HIV viral load, enrollment
period, and self-reported adherence.
cDefined as a CD4?count increase of ?50 cells/?l following at least two sequential visits of virologic suppression.
dLogistic regression models adjusted for race/ethnicity, HCV serostatus, HCV viremia status, pre-HAART CD4?count, pre-HAART HIV viral load, and enrollment
period.
eCox regression models adjusted for race/ethnicity, HCV serostatus, HCV viremia status, pre-HAART CD4?count, pre-HAART HIV viral load, enrollment period,
and self-reported adherence as a time-dependent covariate.
fAlleles and allele groups with a high prior probability of association with untreated HIV progression. Other exploratory results are shown in Table S1 in the
supplemental material. Allele group definitions are described in Table 2.
gThe number of women homozygous or heterozygous for a given allele or allele group in the population of women who initiated HAART (n ? 503). Analysis-specific
allele frequencies are shown in Table S1 in the supplemental material.
hNote that shading indicates statistically significant associations.
10830KUNIHOLM ET AL.J. VIROL.

Page 8

This analysis involved all 503 women, and there was consider-
able follow-up time and a large number of AIDS-defining
events. However, no significant associations were observed,
with either high prior probability alleles or exploratory alleles
(Table 3; see also Table S1 in the supplemental material).
DISCUSSION
We conducted high-resolution HLA class I and II genotyp-
ing in a large, long-term prospective cohort of HIV-seroposi-
tive women, many of whom were enrolled in 1994, prior to the
widespread use of HAART. This design allowed us to study
HLA associations with HIV disease both before and after the
introduction of HAART in a single population. Our results in
women before they initiated HAART confirmed many of the
previously reported associations between HLA and HIV dis-
ease in untreated patients, showing that our population was
not dissimilar from those reported in previous studies of pa-
tients not using HAART. However, in treated patients, we
observed few HLA associations. In fact, we detected only three
significant HLA associations with virological response to
HAART, each of them opposite to those that would be pre-
dicted based on prior results from untreated patients.
Specifically, B*57:01, B*58:01, and the Bw4-80I group were
strongly associated with failure to control HIV replication fol-
lowing HAART initiation. Similar associations between these
alleles and immunological response to HAART were also ob-
served, although these relationships were nonsignificant, pos-
sibly because we had less extensive data to examine this end-
point. That is, the immunological response was only assessed
among the subset of 176 women who were using effective
HAART (i.e., those who had a virological response). The
larger Bw4 allele group, which includes the Bw4-80I group (30)
and other HLA-B alleles with the Bw4 serological epitope (22),
also showed inverse associations with virological and immuno-
logical responses to HAART. In exploring these results fur-
ther, however, we found that associations of B*57:01 and
B*58:01 might have accounted for the associations of Bw4 and
Bw4-80I with virological response. In contrast, though, we
found no evidence that B*57:01 and B*58:01 accounted for the
association of Bw4 with immunological response. We cannot
therefore exclude the possibility that epistatic interactions be-
tween HLA and killer immunoglobulin-like receptor (KIR)
genes may influence the response to HAART, as Bw4 alleles
can act as a ligand for KIR.
We are aware of only three prior studies of HLA genotype
and HIV disease in treated patients (2, 7, 35). As in the current
investigation, few associations between HLA and HIV disease
were observed, and in two of these studies, B*57 was associ-
ated with poor CD4?recovery following HAART initiation (2,
35). Bw4 homozygosity was also associated with poor CD4?
recovery in one of these studies (35).
The current investigation was the first to examine the rela-
tion of HLA genotype with the long-term risk of AIDS events
in treated patients and to examine HLA alleles other than
those already reported to be significant in untreated patients.
The cohort involved extensive person-years of observation and
included a substantial number of AIDS events. Thus, if a re-
lationship between HLA genotype and risk of AIDS-defining
conditions in treated patients had been present, we would
likely have detected it, but no such associations were found.
Overall, it increasingly appears that most of the HLA asso-
ciations with HIV disease in untreated patients are not ob-
served in treated individuals, and in fact, some of these rela-
tionships may be opposite one another. The reasons there may
be different HLA genotype associations in treated versus un-
treated HIV disease progression are unknown. One possible
explanation for the limited number of HLA associations in
treated patients may reflect a reduced role for immune genes
in the inhibition of HIV replication, because HAART has very
strong antiviral effects regardless of a patient’s HLA genotype.
Second, HAART exerts strong selective pressure on the HIV
quasispecies, and HIV variants associated with drug resistance,
largely in the pol and env genes (21), are common in patients
exposed to antiretroviral therapies (17). CTLs recognize
epitopes in gag, nef, and pol (6), so it is possible that therapy-
associated selection pressures on pol could change the distri-
bution of HIV antigens presented to T cells, thereby altering
established HLA associations. Even if this hypothesis is cor-
rect, though, it remains unclear why the few HLA associations
in HAART users that we and others observed involved specific
HLA genotypes that are highly protective in HAART-naïve
patients but are high risk in treated patients.
Important limitations of this study must also be considered
in the interpretation of the findings. First, we must consider the
possibility that a bias related to study design explains the ob-
served inverse associations. While our analyses adjusted for
cofactors associated with both HLA genotype and HIV disease
progression (i.e., conventional confounders), we cannot ex-
clude the possibility that other factors unrelated to HLA could
also have influenced the results through less conventional
pathways (e.g., common effects) (see a recent review of this
topic [20]). It is also possible that women with HLA genotypes
associated with slow untreated disease progression had de-
layed HAART initiation compared to other women; that is,
women with protective HLA genotypes may have been HIV
infected for longer periods of time prior to HAART initiation.
While we controlled for the CD4?count prior to HAART
initiation, it is possible that other, independent factors associ-
ated with long-term HIV infection (e.g., chronic immune acti-
vation) may explain the observed inverse associations between
protective HLA genotype and response to HAART. As in a
prior study (35), though, we could not directly examine this
issue because the dates of HIV seroconversion for the vast
majority of WIHS women are unknown. Cohorts in which
duration of HIV infection is known or can be accurately esti-
mated would be best suited to address this issue. One might
additionally ask whether hypersensitivity to abacavir could ex-
plain why some patients with the B*57:01 genotype (and the
Bw4-80I genotype, which includes B*57:01) do not respond to
HAART (29). In the current cohort, however, only three
women with B*57:01 received abacavir during the time period
studied, and exclusion of these women from the data set did
not meaningfully change the results (data not shown).
Another important consideration in the interpretation of
these results is the impact of survival bias. That is, women with
rapid HIV disease progression may be underrepresented in the
WIHS cohort because they did not live long enough to be
enrolled and initiate HAART. The impact of this bias is that
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Page 9

HLA genotypes that predispose to rapid disease progression
with too little prevalence for analysis may have been under-
represented (we studied only HLA alleles with ?3% preva-
lence). While this may have limited our ability to detect asso-
ciations with certain alleles related to rapid progression, we
note that all of the HLA alleles found to be strongly associated
with HIV progression in prior studies were included in our
analysis, having been found in ?3% of women in our cohort.
We also note that AIDS-defining conditions in WIHS are
ascertained by participant self-report, and so it is possible that
there was some misclassification in AIDS diagnoses, which may
have attenuated the statistical power for our analyses of AIDS
events. Even if this were the case, it is unlikely that misclassi-
fication completely explains the lack of HLA associations with
AIDS in HAART users, since there were many AIDS events
and a long duration of follow-up, as described above.
The current findings regarding HLA and HAART predom-
inantly reflect data on HAART initiation from the late 1990s.
HAART regimens have improved in recent years, and we
cannot exclude the possibility that the results would be differ-
ent in women using current regimens. Furthermore, most
(77%) HAART initiators had used nucleoside reverse trans-
criptase inhibitor (NRTI) monotherapy or combination ther-
apy prior to initiating HAART, and thus, the prevalence of
NRTI resistance mutations may have been higher in the stud-
ied HAART initiators than would be expected in an antiret-
roviral naïve population. To address this issue, we controlled
for prior antiretroviral use in additional sensitivity analyses of
virological and immunological responses (as described above),
but inclusion of this additional covariate did not meaningfully
change the results (data not shown). Our study was also nec-
essarily limited by the number of different analytic approaches
that could be presented for outcomes for which there is no
universally accepted definition (e.g., virological response to
HAART). In the current study, for example, we a priori chose
two definitions that we and others have used for virological
response and found the results to be equivalent. Lastly, we
could not conduct extensive subset analyses given the modest
prevalences of many alleles.
In conclusion, the paucity of HLA associations with HIV
disease progression in treated patients may reflect the effec-
tiveness of HAART in suppressing viral replication irrespec-
tive of host genotype. However, virological and immunological
nonresponses to HAART are not uncommon, and further
study is warranted to determine the relation of this with HLA
genotype and other host factors. Understanding why several
alleles (notably alleles that are protective against HIV dis-
ease progression in untreated women) are associated with a
greater risk of virological and immunological nonresponses
to HAART could provide new insights into this important
clinical issue.
ACKNOWLEDGMENTS
Funding for this project was provided in part by grants from the
National Institute of Allergy and Infectious Diseases (5R01AI057006
to H.D.S. and R01A1052065 to A.K.). The WIHS is funded by the
National Institute of Allergy and Infectious Diseases (grants UO1-
AI-35004, UO1-AI-31834, UO1-AI-34994, UO1-AI-34989, UO1-
AI-34993, and UO1-AI-42590) and by the Eunice Kennedy Shriver
National Institute of Child Health and Human Development (grant
UO1-HD-32632). The study is cofunded by the National Cancer In-
stitute, the National Institute on Drug Abuse, and the National Insti-
tute on Deafness and Other Communication Disorders. Funding was
also provided by the National Center for Research Resources (UCSF-
CTSI grant UL1 RR024131). This project has also been funded in part
with federal funds from the National Cancer Institute, National Insti-
tutes of Health, under contract HHSN261200800001E. This research
was supported in part by the Intramural Research Program of the
NIH, National Cancer Institute, Center for Cancer Research, and
by the Einstein-Montefiore Center for AIDS Research (grant
5P30AI051519-08).
We have no conflicts of interest to declare.
Data in the manuscript were collected by the Women’s Interagency
HIV Study (WIHS) Collaborative Study Group, with centers (principal
investigators) at the New York City/Bronx Consortium (Kathryn Anas-
tos); Brooklyn, NY (Howard Minkoff); Washington, DC, Metropolitan
Consortium (Mary Young); The Connie Wofsy Study Consortium of
Northern California (Ruth Greenblatt); Los Angeles County/Southern
California Consortium (Alexandra Levine); Chicago Consortium
(Mardge Cohen); and Data Coordinating Center (Stephen Gange).
The content of this publication does not necessarily reflect the views
or policies of the Department of Health and Human Services, nor does
mention of trade names, commercial products, or organizations imply
endorsement by the U.S. Government. The contents of this publication
are solely our responsibility and do not necessarily represent the offi-
cial views of the National Institutes of Health.
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