Download A 32-bp Deletion within the CCR5 Locus Protects against

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A 32-bp Deletion within the CCR5 Locus Protects against Transmission of
Parenterally Acquired Human Immunodeficiency Virus but Does Not Affect
Progression to AIDS-Defining Illness
David A. Wilkinson, Eva A. Operskalski,
Michael P. Busch, James W. Mosley,
and Richard A. Koup
Department of Medicine, University of Texas Southwestern Medical
Center, Dallas; Department of Medicine, University of Southern
California, Los Angeles, and Irwin Memorial Blood Centers and
Department of Laboratory Medicine, University of California,
San Francisco
The b-chemokine receptor CCR5 is required as a coreceptor by non – syncytium-inducing (NSI)
strains of human immunodeficiency virus type 1 (HIV-1). NSI viruses predominate early during an
infection and are thought to be important for the transmission of HIV-1. The importance of CCR5
during parenteral transmission of HIV-1 was investigated. The distribution of the homozygous
deleted CCR5 genotype among 566 exposed persons with hemophilia and 97 exposed transfusion
recipients indicated that the lack of CCR5 expression protected persons from infection. This suggests
that the initial predominance of NSI viruses during an infection does not result from limited
availability of CXCR4-expressing cells within the mucosa but rather implies a more fundamental
requisite for CCR5-expressing cells early during an infection regardless of the route of transmission.
In addition, no difference in the rate of progression to AIDS (CDC 1987 definition) of infected
heterozygous compared with homozygous wild type subjects was observed.
Transmission of human immunodeficiency virus type 1
(HIV-1) is dependent on a number of viral and host parameters
that are not yet fully elucidated. Viruses having a non – syncytium-inducing (NSI) phenotype predominate early during infection, leading to the hypothesis that NSI viruses are instrumental
in establishing HIV-1 infection [1]. The key importance of NSI
viruses during transmission has recently been supported by
studies of the entry cofactors required by different HIV-1 isolates. In addition to the primary receptor, the CD4 molecule,
HIV-1 must also bind to a chemokine receptor before entering
a cell. NSI viruses require the b-chemokine receptor CCR5,
whereas T cell line – adapted virus strains use the a-chemokine
receptor CXCR4 [2]. In addition, some virus strains can use
other chemokine receptors, such as CCR2b, CCR3, Bonzo, and
BOB, but the relevance of extended coreceptor usage to events
in vivo has not yet been determined [2].
A nonfunctional allele of the CCR5 gene resulting from a
32-bp deletion has been identified (CCR5-D32). About 1% of
the Caucasian population are homozygous for this mutation
and roughly 14% are heterozygous [3 – 5]. Persons who are
Received 17 November 1997; revised 6 May 1998.
All patients gave written consent for participation, including specific authorization for HIV-1 serologic testing.
Financial support: NIH (contracts HB-47002, -47003, and -97074; grants
AI-35522, AI-42397, AI-42630). R.A.K. is an Elizabeth Glaser Scientist of
the Pediatric AIDS Foundation.
Reprints or correspondence: Dr. David A. Wilkinson, Dept. of Medicine,
University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd.,
Dallas, TX 75235-9113 ([email protected]).
The Journal of Infectious Diseases 1998;178:1163–6
q 1998 by the Infectious Diseases Society of America. All rights reserved.
0022–1899/98/7804–0035$02.00
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homozygous for the CCR5-D32 allele have been shown to be
protected from sexual transmission of HIV-1 infection, and in
one study, hemophilia patients were also shown to be protected
[3 – 5]. Peripheral blood mononuclear cells (PBMC) isolated
from such persons resisted infection in vitro [6, 7]. These results underscore the importance of NSI viruses during initial
infection, because the loss of CCR5 confers resistance to NSI,
but not SI, viruses. Such protection does not appear to be
absolute, however, because a few cases of infections in persons
homozygous for CCR5-D32 have been reported ([8] and references therein). In addition, several studies have found that
infected persons who are heterozygous for the CCR5-D32 allele progress to AIDS more slowly than do persons homozygous for the wild type allele ([9] and references therein). Heterozygous patients generally have lower virus loads and slower
rates of CD4 cell depletion, possibly because heterozygous
persons may express lower amounts of CCR5, resulting in
decreased viral replication and slower disease progression [10].
Most investigations of the role of CCR5 in transmission and
progression of HIV-1 have focused on homosexual men, for
whom the primary risk of infection is anal-receptive intercourse. The transmission rate associated with sexual exposure
ranges from õ0.1% to 7.0% per contact, depending primarily
on the prevalent virus load of the infected person responsible
for exposure [11]. In contrast, recipients of HIV-1 – contaminated blood components experience a much higher transmission rate, of 90% [11]. The principal factors responsible for
such a high incidence are probably the direct administration of
virus and virus-infected cells into the host’s circulation, as well
as the relatively large inoculum. These important differences
associated with parenteral transmission of HIV-1 prompted us
to investigate the importance of CCR5 in such cases. To do
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this, we analyzed the distribution of CCR5 alleles among two
study groups previously enrolled by the Transfusion Safety
Study, consisting of a cohort of persons who had been transfused with blood components subsequently found to have been
HIV-1 – contaminated and a cohort of patients with hemophilia
treated with clotting factor concentrates before heat-inactivation was initiated.
Table 1. Distribution of deleted CCR5 allele among recipients of
HIV-1 – positive blood products.
Hemophiliacs
Genotype
///
D//
D /D
Total
Subjects and Methods
The Transfusion Safety Study was a multicenter, cooperative
investigation of factors that determine the occurrence or modify
the expression of transfusion-transmitted infections. The study enrolled recipients of blood components given in late 1984 and early
1985 if the donor was subsequently found to be HIV-1–infected
when testing of stored specimens became possible [12]. Of the
136 enrolled recipients, 122 (90%) were infected. The study also
enrolled 1237 persons with congenital clotting disorders, of whom
684 were HIV-1–infected at entry into the study [13].
At 6-month intervals, blood was drawn for hematologic, immunologic, and serologic evaluation, and a history was taken that
included medical developments and AIDS-defining conditions. For
persons with hemophilia, a history of treatment with clotting factor
concentrates and blood components since January 1979 was obtained by checking medical charts, clinic and pharmacy records,
and individual treatment diaries. For all subjects at each visit, both
plasma (stored at 0707C) and buffy coat (stored in liquid nitrogen)
were placed in the Transfusion Safety Study/National Heart, Lung,
and Blood Institute Subject Repository.
To investigate the association of heterozygous or homozygous
CCR5-deletion genotypes with HIV-1 infection and progression,
we evaluated 97 transfusion recipients for whom buffy coats were
available, of whom 88 (91%) were HIV-1–infected and 9 (9%)
were uninfected despite having received a component from a
known HIV-1–positive donation. We also evaluated 566 patients
with hemophilia, of whom 543 (96%) were HIV-1–infected and
23 (4%) remained uninfected despite receiving at least 50,000 U
of factor VIII concentrate in 1981–1984, the period when clotting
factor concentrates had the highest risk of HIV-1 transmission. In
total, the racial makeup of the cohorts studied was 69% Caucasian,
13% Hispanic/Latino, 12% African-American, and 3.5% Asian.
For the analysis of progression to AIDS, we used the 1987
AIDS definition of the Centers for Disease Control and Prevention
(CDC). Date of infection for recipients was assumed to be the date
of transfusion with the known anti–HIV-positive component. For
hemophiliacs, we used July 1982 as the probable median date of
their infection, the median of the period when the majority of
hemophiliacs treated with factor VIII concentrates were infected.
This estimate is supported by the observed median time for progression to AIDS within the cohort (approaching 12 years), which
is similar to the progression rate reported for an independent cohort
of hemophilia patients [14].
DNA was extracted from 100 mL of previously frozen buffy
coat specimens by use of a commercially available kit (United
States Biolabs, Beverly, MA). The DNA was resuspended in 10
mL, of which 2 mL was used for polymerase chain reaction analysis
of the deleted region of the CCR5 gene. Oligonucleotide primers
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JID 1998;178 (October)
Transfusion recipients
Seronegative
Seropositive
Seronegative
Seropositive
19 (83%)
2 (9%)
2 (9%)
23
P Å .002
471 (87%)
72 (13%)
0
543
6 (67%)
2 (22%)
1 (11%)
9
P Å .036
78 (89%)
10 (11%)
0
88
NOTE. ///, homozygous wild type; //D, heterozygous; D/D, homozygous deleted.
flanking the deleted region (5*-CAAAAAGAAGGTCTTCATTACACC-3* upstream and 5*-CCTGTGCCTCTTCTTCTCATTTCG-3* downstream) amplified either a 189-bp fragment from the
wild type allele or a 157-bp fragment from the deleted allele, which
were subsequently resolved by electrophoresis on a 4% agarose
gel [5].
Results
The CCR5 genotypes by exposure group and HIV-1 status
are summarized in table 1. None of the patients homozygous for
the CCR5-D32 allele were infected in either cohort. Previous
studies have determined that 1.1% – 1.4% of the general population are homozygous for CCR5-D32 [3, 4]. In contrast, 9% and
11% of the exposed seronegative hemophiliacs and transfusion
recipients, respectively, were homozygous for CCR5-D32 alleles. In both cohorts, the frequency of the deleted allele among
uninfected subjects was significantly higher than among infected subjects (P Å .002 in patients with hemophilia and P
Å .036 in transfusion recipients). The 2 hemophilia patients
who were seronegative homozygous-deleted were among the
most heavily treated (ú400,000 U of clotting factor during
1981 – 1984) and hence at great risk of infection. The absence
of any CCR5 homozygous deleted subjects among 631 seropositive patients, together with the overrepresentation of the genotype among seronegative exposed subjects, show that lack of
expression of CCR5 is associated with protection from HIV-1
infection, even in cases of high levels of parenteral exposure.
Heterozygosity for CCR5-D32 has been reported to delay
progression of the onset of AIDS among cohorts of homosexually and heterosexually infected persons and hemophiliacs ([9]
and references therein). Heterozygous patients have been found
to have slower rates of CD4 cell decline as well as lower
initial virus levels in their serum [5]. In our study, however,
no difference in the progression to AIDS was observed between
the 471 homozygous wild type and 72 heterozygous infected
patients within the hemophilia cohort (figure 1). Similarly, no
difference in progression was observed among transfusion recipients, but the sample size was small (data not shown). There
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Concise Communications
Figure 1. Kaplan-Meier analysis of progression to AIDS for homozygous wild type subjects (n Å 471) compared with subjects heterozygous for the CCR5-D32 allele (n Å 72) within hemophilia cohort.
Progression to AIDS was defined by 1987 CDC criteria. Time of
seroconversion was estimated to be median of period when majority
of persons were infected.
was also no difference in progression to a CD4 cell count of
õ400 cells/mL in both cohorts.
Discussion
The failure of this study to observe a difference in the progression to disease in our parenterally exposed cohort compared
with earlier studies may indicate a difference in the role of
CCR5 in progression to disease by inoculum size and route of
infection. Both the amount of the initial inoculum and route
of exposure could have significant impact on the establishment
of an infection. For example, in vitro infectivity data show that
PBMC from patients homozygous for the CCR5 deletion can
be infected with NSI isolates if very high challenge titers are
used [6]. Alternatively, the route of infection may affect which
susceptible cell types the virus encounters initially. Hence, the
importance of NSI viruses during sexual transmission may
result from the availability of susceptible cell types within the
mucosa. The present study, however, suggests that the restriction that results in NSI predominance early in infection is not
explainable by simple target cell availability. Such restrictions
would not be encountered by parenterally transmitted virus,
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yet a protective effect in persons lacking CCR5 expression was
still observed. Thus, there appears to be a biologic requisite
for the CCR5-dependent infection of critical target cells for
the establishment of the infection. The early predominance of
NSI isolates does not occur in all persons, but this may be
explained by the fact that, in contrast to laboratory-adapted SI
viruses, many primary SI isolates are able to infect macrophages and use either CCR5 or CXCR4 as a coreceptor [15].
The data presented here corroborate those of Dean et al. [4],
who reported a protective effect of CCR5 homozygous deletion
among persons with hemophilia exposed to untreated preparations of clotting factor. We extended this finding to transfusion
recipients and, importantly, now present our findings for the
hemophilia cohort within the context of the relative exposures
of the group as a whole. This study did not detect a difference
in the rate of progression to AIDS (1987 CDC criteria) among
heterozygous compared with homozygous wild type persons.
This is consistent with previously reported findings [4]. However, an effect was reported for a combined analysis of several
cohorts, including a hemophilia cohort [4]. It is possible that
a difference within a given cohort could be masked when analyzed in combination with other cohorts of different composition. A delay in progression to AIDS among heterozygous
persons in a hemophilia cohort was reported when the 1993
CDC definition of AIDS was used as the outcome [4]. The
1993 definition included a CD4 T cell count õ200 cells/mL as
one of the criteria. That finding is consistent with studies that
have found a slower rate of CD4 T cell depletion among heterozygous persons [5, 16]. However, the slower loss of CD4 T
cells among heterozygous persons has not been associated with
a concomitant delay in the development of clinical manifestations of AIDS, as shown by this study.
In addition to the 3 homozygous persons, a total of 31 heavily
treated hemophiliacs and exposed transfusion recipients with
heterozygous or wild type CCR5 genotypes did not seroconvert
(table 1). The factors contributing to their lack of infection
remain to be determined but may involve novel host determinants of infection or result from immunologic control of the
virus. Studies are in progress to investigate whether additional
mechanisms of resistance to HIV-1 can be discovered among
these persons.
References
1. Zhu T, Mo H, Wang N, et al. Genotypic and phenotypic characterization
of HIV-1 in patients with primary infection. Science 1993; 261:
1179 – 81.
2. Clapham PR, Weiss RA. Immunodeficiency viruses. Spoilt for choice of
co-receptors. Nature 1997; 388:230 – 1.
3. Samson M, Libert F, Doranz BJ, et al. Resistance to HIV-1 infection in
caucasian individuals bearing mutant alleles of the CCR-5 chemokine
receptor gene. Nature 1996; 382:722 – 5.
4. Dean M, Carrington M, Winkler C, et al. Genetic restriction of HIV-1
infection and progression to AIDS by a deletion allele of the CKR5
structural gene. Science 1996; 273:1856 – 62.
UC: J Infect
1166
Concise Communications
5. Huang Y, Paxton WA, Wolinsky SM, et al. The role of a mutant CCR5
allele in HIV-1 transmission and disease progression. Nature Med 1996;
2:1240 – 3.
6. Paxton WA, Martin SR, Tse D, et al. Relative resistance to HIV-1 infection
of CD4 lymphocytes from persons who remain uninfected despite multiple high-risk sexual exposures. Nature Med 1996; 2:412 – 7.
7. Connor RI, Paxton WA, Sheridan KE, Koup RA. Macrophages and CD4/
T lymphocytes from two multiply exposed, uninfected individuals resist
infection with primary non-syncytium- inducing isolates of human immunodeficiency virus type 1. J Virol 1996; 70:8758 – 64.
8. Balotta C, Bagnarelli P, Violin M, et al. Homozygous delta 32 deletion
of the CCR-5 chemokine receptor gene in an HIV-1 – infected patient.
AIDS 1997; 11:F67 – 71.
9. Meyer L, Magierowska M, Hubert JB, et al. Early protective effect of
CCR-5 delta 32 heterozygosity on HIV-1 disease progression: relationship with viral load. AIDS 1997; 11:F73 – 8.
10. Wu L, Paxton WA, Kassam N, et al. CCR5 levels and expression pattern
correlate with infectability by macrophage-tropic HIV-1, in vitro. J Exp
Med 1997; 185:1681 – 91.
JID 1998;178 (October)
11. Royce RA, Sena A, Cates W Jr, Cohen MS. Sexual transmission of HIV.
N Engl J Med 1997; 336:1072 – 8.
12. Busch MP, Operskalski EA, Mosley JW, et al. Factors influencing human
immunodeficiency virus type 1 transmission by blood transfusion. J
Infect Dis 1996; 174:26 – 33.
13. Gjerset GF, Pike MC, Mosley JW, et al. Effect of low- and intermediatepurity clotting factor therapy on progression of human immunodeficiency virus infection in congenital clotting disorders. Blood 1994; 84:
1666 – 71.
14. Sabin C, Phillips A, Elford J, Griffiths P, Janossay G, Lee C. The progression of HIV disease in a haemophilic cohort followed for 12 years. Br
J Haematol 1993; 83:330 – 3.
15. Simmons G, Wilkinson D, Reeves JD, et al. Primary, syncytium-inducing
human immunodeficiency virus type 1 isolates are dual-tropic and most
can use either Lestr or CCR5 as coreceptors for virus entry. J Virol
1996; 70:8355 – 60.
16. Eugen-Olsen J, Iversen AK, Garred P, et al. Heterozygosity for a deletion
in the CKR-5 gene leads to prolonged AIDS- free survival and slower
CD4 T-cell decline in a cohort of HIV-seropositive individuals. AIDS
1997; 11:305 – 10
Changes in Lymphocyte Subsets in Human Immunodeficiency Virus–Positive
Persons with õ5 CD4 T Lymphocytes/mm3
Caroline A. Sabin, Amanda Mocroft, Margarita Bofill,
George Janossy, Christine A. Lee, Margaret Johnson,
and Andrew N. Phillips
HIV Research Unit, Department of Primary Care and Population
Sciences, Department of Clinical Immunology, Haemophilia Centre and
Haemostasis Unit, and Departments of Haematology and of Thoracic
Medicine, Royal Free Hospital and School of Medicine, London,
United Kingdom
All patients seen at the Royal Free Hospital, London, who had at least one CD4 T lymphocyte
count of õ5 cells/mm3 (n Å 166) were prospectively followed to assess changes in their total T
lymphocyte and CD8 T lymphocyte counts over time. While overall there were no clear trends
towards a drop or increase in either count, persons who died during the study experienced a rapid
drop in both CD8 T lymphocyte and total T lymphocyte levels in the months preceding death.
Multivariate Cox proportional hazards models revealed that both the total T lymphocyte count and
CD8 T lymphocyte count provided important prognostic information for survival. Despite almost
a complete absence of CD4 T lymphocytes, lymphocyte subset monitoring is useful in identifying
decreasing CD8 T lymphocyte levels that predict short-term prognosis.
After a rapid CD8 T cell rise during primary human immunodeficiency virus (HIV) infection, CD8 lymphocytosis is almost
invariably maintained throughout HIV infection, leading to inverted CD4:CD8 ratios [1 – 3]. The increase in the CD8 lymphocyte count after infection is prognostic for the development
Received 10 November 1997; revised 1 May 1998.
Financial support: Medical Research Council of the United Kingdom.
Reprints or correspondence: Dr. C. A. Sabin, HIV Research Unit, Department of Primary Care and Population Sciences, Royal Free Hospital School
of Medicine, Rowland Hill Street, London NW3 2PF, UK (caroline@rfhsm.
ac.uk).
The Journal of Infectious Diseases 1998;178:1166–9
q 1998 by the Infectious Diseases Society of America. All rights reserved.
0022–1899/98/7804–0036$02.00
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of AIDS in the long term [4, 5]. However, at later stages of
infection, prior to the onset of AIDS, CD8 T lymphocytes drop
[2, 6], and their prognostic value is limited [4, 7]. At this
stage, a drop in CD8 T lymphocytes, rather than an increase,
is associated with poorer prognosis [4, 8]. Previously it was
not possible to study the true natural history of CD8 T cell
loss at this late stage of disease because many persons develop
AIDS and die before a complete loss of CD4 T lymphocytes.
However, the introduction of antiretroviral and prophylactic
therapies has meant that increasing numbers of persons are
seen after total loss of circulating CD4 T lymphocytes, and the
prognostic significance of CD8 cells can be studied. We have
followed a group of 166 patients with undetectable CD4 T
lymphocytes. In these subjects, a lower CD8 T lymphocyte
count at the time that CD4 cells become undetectable is associ-
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