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Department of Biological and Technological Research and Infectious Diseases Clinic, San Raffaele Scientific Institute, Milano, Italy;
Istituto Biocatalisi e Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Milano, Italy;
Malattie Infettive, Ospedale S.M. Annunziata, Firenze, Italy;
§
Cattedra di Immunologia, Università di Milano, Milano, Italy; and
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Dipartimento di Biologia e Genetica per le Scienze Mediche, Milano, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-cell Abs and T cell-mediated immunity have been associated with protection in primate models of lentivirus infection (16, 17, 18), but the role of these immune responses in humans is still unclear. More recently, it was shown that the cells of some ESN couldn’t be infected in vitro by M-tropic (R5) strains of HIV because they lack the essential CCR5 coreceptor (19, 20). In the CCR5 gene, at least two mutations (20, 21) have been associated with total or partial resistance to infection by M-tropic R5 strains of HIV, and one mutation was associated with slowing progression of the disease (22). R5 strains (23) account for most of the transmission of HIV infections (particularly sexually transmitted infections) and are associated with the earlier phases of the disease (24). Because CCR5-defective individuals were described to have normal inflammatory and immune reactions, CCR5 has been interpreted as a redundant molecule in adults, and has thus become an important potential target for blocking drugs (25) and immune modulation (26, 27, 28). Therefore, we searched for anti-CCR5 autoantibodies in the sera of ESN individuals on the assumption that, if such Abs exist, they are unlikely to affect immune function but may interfere with the HIV coreceptor function of CCR5.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two different cohorts of monogamous couples discordant for HIV
serostatus were selected for the study. A first cohort was enrolled in
Milan (3 homosexual and 37 heterosexual couples); a second group was
enrolled in Florence (2 homosexual and 6 heterosexual couples). The 48
ESN subjects included in the study were 26 heterosexual females, 17
heterosexual males, and 5 homosexual males (passive recipients).
Written informed consent was obtained from all the participants. The
inclusion criterion was a history of penetrative sexual intercourse
without condom, at least twice per week and for at least 2 years with
no other known risk factors during that period or afterward. HIV-1
infection was excluded because of the absence of anti HIV-1 Abs (ELISA
and Western blot) and viral DNA (PCR). Seronegativity was confirmed 6
mo after enrollment. Forty-five serum samples from unexposed, sexually
active, seronegative individuals (USN) (29 females, 16 males) were used
as negative controls. Forty serum samples from HIV-seropositive
partners of ESN subjects were also analyzed. After enrollment in the
protocol, counseling was offered to all of the couples and many (but
not all) switched to safe-sex practices. The ESN were characterized for
the presence of CCR5-
32 alleles (courtesy of Drs. V. Nardese and P.
Lusso), anti-CD4 Abs, and HIV-specific IgA (3, 5).
Purification of CD4+ cells
PBMC from healthy blood donors were isolated by Ficoll-Hypaque
centrifugation and stimulated for 3 days with PHA (3 µg/ml)
(Sigma-Aldrich, Steinheim, Germany) and rIL-2 (100 U/ml) (Amersham,
Buchinghamshire, U.K.). CD4+ cells were purified
from activated PBMC by immunoadsorption to anti-CD4 magnetic beads
(Oxoid, Hampshire, U.K.). Purified CD4+ cells
were stimulated for 3 days with IL-2 before the macrophage inflammatory
protein 1ß (Mip1ß) binding assay. PBMC were characterized on the
basis of the presence of CCR5-32 alleles; only PBMC from donors not
carrying the CCR5-32 allele were used.
Mip1ß binding assay
The assay was performed as described (29). Briefly, 106 purified CD4+ cells in 200 µl of RPMI 1640 (Life Technologies, Milan, Italy) (containing 0.05 M NaN3, 1% BSA, and 25 mM HEPES) were incubated with appropriate dilutions of sera and/or Ig-enriched fractions; after 45 min of incubation, 125I-Mip1ß (DuPont-NEN, Mechelem, Belgium) was added (final concentration, 0.1 nM, 0.2 µCi), and the cells were further incubated for 2 h on ice. Unbound radioactivity was separated by centrifugation on a two-step gradient (30) in 0.3-ml tubes (Nunc, Roskilde, Denmark) as follows: the lower layer consisted of FCS containing 10% sucrose; the upper layer consisted of 80% silicone (Sigma-Aldrich) and 20% mineral oil (Sigma-Aldrich). The bound radioactivity in the cell pellets was measured in a gamma counter. Serum samples were diluted 1:10 (five replicas for each sample). A specificity control consisting of a 100-fold excess of unlabeled Mip1ß was included in all experiments. The binding of the 125I-Mip1ß to activated CD4+ cells ranged between 1000 and 6000 cpm. The cut-off value was set at 12% (three SD above the mean value of the 45 USN serum samples).
Anti-CCR5 Ab assay
Anti-CCR5 Abs bound to CCR5-positive cells were revealed by 0.5 µCi/ml of 125I-sheep anti human Ig F(ab')2 Ab fragments (Amersham). Activated CD4+ T cells were treated with human sera diluted 1/10 and then processed as described for the Mip1ß binding assay. The cut-off value (1065 cpm) was established as 3 SD above the mean of 10 USN serum samples. Cellular suspensions from CXCR4- or CCR5-transfected U87 cells monolayers pretreated with human sera diluted 1/10 were processed as described above. A pool of 10 USN sera was used as negative control, while 0.1 nM 125I-Mip1ß and the anti-CCR5 mouse mAb 2D7 (31) were used as positive controls. The mAb was used at 0.3 µg/ml and revealed by 0.5 µCi/ml 125I-sheep anti-mouse IgG F(ab')2 Ab fragments (Amersham).
Chemotaxis assay
PBMC from one healthy donor (selected for high expression of CCR5) were activated with PHA and IL-2 for 3 days (see above) in the presence of two concentrations of purified Igs (250 and 62 µg/ml) from ESN34, ESN55, and USN5. Then, 3 x 105 activated PBMC in 50 µl of RPMI 1640 medium containing 0.3% human serum albumin were placed in the upper chamber of 5-µm pore size bare filter Transwell (Costar, Europe, Amsterdam, The Netherlands). Chemotaxis was conducted in the presence of 1.5 µg/ml of Mip1ß (placed in the lower chamber). The transwells were incubated for 2 h at 37°C; cells that migrated from the upper to the lower chamber were then quantified by FACS analysis. PBMC from ESN34 and ESN55 were also used in chemotaxis assays to evaluate the capacity of ESN PBMC to migrate in the presence of Mip1ß. The results were expressed as chemotaxis index, which represents the fold increase in the number of migrated cells in response to Mip1ß over the spontaneous cell migration in control medium.
C-C chemokines assay
Serum chemokines concentrations (RANTES, Mip1
, and Mip1ß)
were determined with commercial ELISA kits (R&D Systems,
Minneapolis, MN).
Ig purification
Anti-human polyvalent Ig-coupled Agarose (Sigma-Aldrich) was used to purify total Ig from the sera of ESN and USN. Briefly, 100 µl of serum were incubated overnight at 4°C in columns containing 5 ml of anti-human Ig-agarose. After recovering the column washout (Ig-depleted fraction), the columns were washed six times in phosphate buffer (0.01 M with 0.5 M NaCl). Bound Igs were eluted with glycine/NaCl 0.2 M, and the eluted fractions neutralized with 1 M Tris (Ig-enriched fraction). Ig-enriched and Ig-depleted fractions were concentrated on Ultrafree-15 Biomax 30 membranes (Millipore, Bedford, MA) with a cut-off of 30 kDa and dialyzed against RPMI 1640. Ig concentration was determined by ELISA using commercial Igs as standard and adjusted to 2.5 mg/ml (corresponding to a serum dilution of 1:10). The Ig-depleted fractions were diluted by the same factor.
Virus isolation and titration
HIV was isolated from the PBMC of HIV-seropositive partners by cocultivation with PHA-stimulated PBMC of two USN. Cultures were maintained until increasing levels of HIV-p24 Ag were detected in two consecutive determinations. The infectivity (ID50) of each virus isolate was determined on PBMC from one single donor as follows: six replicas (150 µl) of 5-fold serial dilutions (from 1:5 to 1:3, 125) of virus were added to six wells of a round-bottom Microtiter plate (Nunc) containing 105 resting PBMC in 75 µl of medium, incubated for 2 h, washed, and resuspended in RPMI 1640 medium containing PHA (3 µg/ml) and 10 U/ml rIL-2. HIV-1 p24 Ag was titrated after 5, 7, and 9 days of culture by standard assays (32). ID50 titers were defined as the reciprocal of the virus dilution yielding 50% positive wells (Reed-Muench calculation).
Phenotypic characterization of viral coreceptor usage
Each virus isolate was used to infect U87 human glioma cell lines (provided by Dr. P. Lusso) expressing one of the following chemokine receptors: CCR1, CCR2B, CCR3, CCR5, or CXCR4. Then, 1 ml of virus-containing culture supernatant was incubated with 8 x 104 U87 cells in 12-wells plates for 4 h at 37°C. Cells were washed twice and incubated in RPMI 1640 medium supplemented with 10% FCS. Cell cultures were observed daily for cytopathic effect. Culture supernatants, collected from each well 2, 5, and 7 days after infection, were analyzed for HIV-1 p24 Ag.
Virus neutralization assays
The "resting cell assay" was performed according to Zolla-Pazner (33). Briefly, 2 x 105 resting PBMC were added to 75 µl of serial dilutions of Ig-enriched fractions from ESN or USN; after 1 h incubation, 75 µl of a virus dilution (ID50 adjusted to 20) was added. The cultures were incubated for another 2 h, washed, and resuspended in PHA and IL-2-containing medium. HIV-1 p24 Ag in the supernatants was determined on days 7 and 9. Percent neutralization was calculated relatively to a nontreated control.
For the "activated PBMC assay," the cells were cultured in medium containing PHA and IL2 for 48 h before the neutralization assay.
Mip1ß ELISA assay
First, 100 µl of recombinant human Mip1ß (1 mg/ml) (R&D Systems) was coated on 96 Microplate wells in 50 mM NaHCO3-Na2CO3 buffer (2 h at 37°C). After overcoating with PBS buffer plus 10% BSA and 1% Tween 20 (1 h at 37°C), serum from ESN and USN in various dilutions (from 1/5 to 1/40) were added (1 h at 37°C). Goat anti-human Mip1ß IgG Abs at 300–200-100–50 pg/ml were used as positive control. After four washes in PBS buffer, 100 µl of 1:1000 peroxidase-conjugated rabbit anti-human IgG or rabbit anti-goat IgG (Dako, Copenhagen, Denmark) were added to the wells and incubated (30 min at 37°C). The wells were washed seven times in PBS; substrate buffer containing o-phenylenediamine plus H2O2 was then added, and absorbance was determined at 492 nm.
Adsorption of purified Igs on U87 cell lines
Ig-enriched fractions from ESN and USN were adsorbed on monolayers of CXCR4- or CCR5-transfected U87 cell lines obtained by seeding 8 x 104 cells in Microtiter wells; 100 µl of Ig-enriched fractions (2.5 mg Ig/ml) were immunoadsorbed on the cell monolayers (10 min at 37°C); the liquid was then collected and passed to a second cell monolayer (10 min at 37°C). The procedure was repeated 10 times.
Synthesis of peptides and preparation of peptide/beads
Peptides were synthesized by the solid phase F-moc method (34) using an Applied Biosystem model 433 A peptide synthetizer (Foster City, CA). After the peptide assembly, the side chain-protected peptidyl resin was deblocked as previously described (35) and purified to apparent homogeneity by reverse-phase chromatography. An extra-sequence cysteine was added to peptides 1, 3, and 4 to obtain conformationally cyclic peptides. These peptides were treated overnight with a 5-fold excess of oxidized glutathione and purified by reverse-phase chromatography.
Coupling of CCR5 peptides to tosyl-activated Dynabeads M280 (Dynal, Oslo, Norway) was obtained following standard procedures. Briefly, 3 x 107 beads were incubated with 9 µg of CCR5 peptides in 50 mM borate buffer pH 9.5 (16 h at 37°C). After four washes in PBS, peptide/beads were ready for use.
Affinity-purification of Abs on peptide/beads
Binding of anti-CCR5 specific Igs to peptide/beads was obtained by incubating 9 µg Igs to 9 µg peptide/beads for 1 h at 4°C. Igs were eluted in 0.5 M acetic acid, dialyzed against RPMI 1640 medium, then tested in Mip1ß binding assays and/or in HIV neutralization assays. To establish if the region recognized by anti-CCR5 Abs corresponds to a conformational epitope, the specific peptide/beads were incubated with 10 mM of 2-ME, and subsequently with 30 mM of N-ethyl maleimide (final concentration, 30 mM) for 60 min before Ab binding.
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Results |
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Unlike RANTES and Mip1 (the other two CCR5-binding
ß-chemokines that inhibit infection by R5 strains of HIV), Mip1ß
binds exclusively to CCR5. For this reason, a Mip1ß radio-binding
assay to measure the presence and function of CCR5 on cell surfaces was
recently introduced (29). We used a similar radio-binding
inhibition assay to search for anti-CCR5 Abs in sera of ESN and USN
individuals as well as in sera of HIV-infected patients. A total of 133
serum samples from 45 USN, 40 HIV-seropositives, and 48 ESN were
assayed (at a 1:10 dilution; five replicas for each serum) to
investigate whether they could inhibit Mip1ß binding to purified
CD4+ cells. The ability of ESN sera to inhibit
Mip1ß binding to purified CD4+ cells was higher
than that observed with sera from either USN or HIV-positives, and the
difference was statistically significant (t =
1.939; p < 0. 05). The arithmetic means ± SE of
the inhibition values were 1.42 ± 0. 7 (USN), 1.23 ± 0.06
(HIV-seropositives), and 5.2 ± 2.2 (ESN). The sera of six ESN
(ESN9, 31, 32, 34, 53, 55) had values well above the cut-off (mean + 3
SE of USN controls) (Fig. 1
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, and Mip1ß in the six ESN sera in
which Mip1ß-inhibiting activity was detected and in the
CD4+ sera of 10 USN control. The concentrations
of all three chemokines were comparable between ESN and USN sera (data
not shown). Inhibition of Mip1ß binding to CCR5 by sera of ESN is mediated by Abs
The sera of the six ESN that inhibited Mip1ß binding to CCR5 and
of three USN were purified by affinity chromatography (on agarose-bound
anti-human Ig Abs) to obtain Ig-enriched and Ig-depleted fractions.
Both fractions were then tested in the radio-binding assay. Fig. 2A shows that Mip1ß-binding
inhibition was associated with the Ig-enriched fraction in all ESN
samples, whereas no activity was observed in the Ig-depleted fractions.
Both the Ig-enriched and Ig-depleted fractions from USN sera were
devoid of inhibitory activity. The data suggest the Mip1ß-binding
inhibition to be due to Abs. The dose-dependence of the binding
inhibition is shown in Fig. 2B. In all but one case (ESN55),
significant inhibition was observed at a concentration of 62.5 µg
(corresponding to a serum dilution of 1:40); all fractions lost their
inhibitory activity at a concentration of 15.6 µg (corresponding to a
serum dilution of 1:160).
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To exclude the possibility that the inhibitory Abs could be directed against Mip1ß (rather than against CCR5), we set up an ELISA to detect anti-Mip1ß Abs. Mip1ß-binding Abs could not be detected in either the whole sera or Ig-enriched fractions of either ESN or 10 USN (data not shown), although the ELISA was sensitive enough to detect concentrations as little as 100 pg/ml of anti-Mip1ß Abs. Therefore, it is likely that the observed inhibition is due to Abs interacting with CCR5 and blocking Mip1ß binding to its receptor, possibly by steric hindrance.
Anti-CCR5 Abs were demonstrated in five of five of the
Mip1ß-inhibiting sera by a similar radio-binding assay, which employs
CD4+ T cells or CCR5-transfected U87 cells and
radiolabeled anti-human Ig secondary Abs. A strong increase in
binding was observed when CCR5-transfected U87 cells were compared with
CD4+ T cells, as shown in Fig. 3, A and B.
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To evaluate whether the cells of ESN in whom anti-CCR5 Abs are
detected are functionally CCR5-negative, purified Ig fractions from
ESN34, ESN55, and from one control (USN5) were used to inhibit the
chemotactic response induced by Mip1ß on PBMC from healthy controls.
The results show that anti-CCR5 Abs from ESN34 and ESN55 reduced
chemotaxis in a dose-dependent manner (Fig. 3
C).
Additionally, whereas control PBMCs migrated in response to Mip1ß,
PBMCs from ESN34 and ESN55 were not responsive to Mip1ß (Fig. 3C). These results indicate that anti-CCR5 Abs are
functionally active as they suppress chemotaxis and suggest that, in
vivo, these Abs down-regulate CCR5 expression.
Lack of correlation between anti-CCR5 Abs and other immune correlates of protection
No correlation was found between the presence of anti-CCR5 Abs
and other immune correlates of protection previously associated with
the ESN status. ESN were tested for the following correlates and
resulted negative (data not shown): 1) the presence of a homozygous
CCR5 deletion (CCR5-32), 2) anti-CD4 Abs (anti-CD4 Abs were
only detected in ESN53), or 3) serum HIV-specific IgA. Moreover, no
correlation was found between anti-CCR5 Abs and gender: ESN55, 32,
and 53 were males from heterosexual couples, the other three were
females. Interestingly, the anti-CCR5 response is persistent in
time: in the four cases tested (ESN34, 32, 53, and 55), anti-CCR5
titers persisted in sera obtained after 2 years of safe sex (data not
shown).
Anti-CCR5 Abs inhibit the infectivity of R5 strains of HIV
The data reported above demonstrate that anti-CCR5 Abs can be
found in the sera of ESN, and that these Abs prevent binding of Mip1ß
to CCR5. Because CCR5 is the main coreceptor for R5 strains of HIV, we
verified whether anti-CCR5 Abs could also inhibit HIV-1 binding to
CCR5. Thus, we assayed the activity of Ig fractions from five of the
ESN in whom anti-CCR5 Abs were observed (ESN31, 32, 34, 53, and 55)
in HIV-1 neutralization assays using primary virus isolates derived
from seropositive patients. A number of primary isolates were analyzed
for coreceptor usage; three isolates were chosen: HIV36 (R5 strain that
uses CCR5 as its coreceptor), HIV26 (X4 strain that uses CXCR4 as its
coreceptor), and HIV45 (amphitropic R3, R5, X4 strain that uses CCR3,
CCR5, and CXCR4 as coreceptors). Additionally, the isolates from four
seropositive partners (all using CCR5 as coreceptor) were used in the
neutralization assay. Ig-enriched fractions from all five ESN inhibited
infection by the R5 strain in a dose-dependent manner (Fig. 4A); these fractions had no
inhibitory effect on the other two HIV-1 strains (data not shown). The
purified Ig fractions of four ESN (ESN34, 53, 31, and 55) inhibit the
replication of the primary isolate derived from the corresponding four
HIV-seropositive partners (Fig. 4, B–E).
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Evidence that anti-CCR5 Abs are also responsible for the
neutralizing activity on the R5 strains was sought by immunoadsorption:
Ig-enriched fractions from sera of three ESN and three USN were
adsorbed on either CCR5- or CXCR4-transfected U87 cells. Adsorption on
CCR5-positive cells (but not on CXCR4-positive cells) reduced both the
virus-neutralizing activity (Fig. 5A) and the Mip1ß-binding
inhibitory activity (Fig. 5B). In particular, 1) in the
neutralization assay, the concentration of unabsorbed fractions giving
50% inhibition (IC50), as well as that of
CXCR4-adsorbed fractions, was 
30 µg/assay, whereas the
IC50 of CCR5-adsorbed fractions was >200
µg/assay; 2) Mip1ß-binding inhibition was 82% in both
unabsorbed and CXCR4-adsorbed fractions, but was 57% in CCR5-adsorbed
fractions. ESN inhibiting sera could also neutralize another primary R5
isolate (HIV53) in both resting and activated PBMC neutralization
assays (data not shown). These data are consistent with the hypothesis
that the Abs removed by adsorption on CCR5 are the same responsible for
the neutralization of HIV-1 R5 strains.
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The issue of in vivo coexistence of anti-CCR5 Abs and CCR5 Ag
on the surface of cells was addressed by studying the CCR5 phenotype of
purified CD4+ cells from two patients (ESN55 and
ESN34) and from four USN controls using the Mip1ß binding assay. No
binding could be detected for the two patients, while all the USN
exhibited normal binding values (range, 2000–3500 cpm; data not
shown). To evaluate CCR5 expression on the cell surface, purified,
preactivated CD4+ cells from ESN55 and ESN34 and
one USN control were stained with a CCR5-specific Ab (mAb 2D7) and
tested in a flow cytometry assay, which employs FITC-labeled
anti-mouse Ig secondary Abs. Results (Fig. 6) showed that, whereas CCR5 was normally
detected on lymphocytes of the USN, no CCR5 expression was observed on
the cells of the two ESN individuals.
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Anti-CCR5 Abs were tested on a panel of synthetic peptides
covering the complete sequence of the extra membrane region of CCR5
(Table I). Specific Ab binding to peptide
3 (aa 89–102), corresponding to the second external domain (first
cystein loop) of CCR5, was observed. As shown in Fig. 7A, the Abs are highly
selective for this epitope as no binding was observed on a panel of
other peptides (peptides 1, 2, 4, and 5). Binding of the anti-CCR5
Abs from ESN sera to peptide 3 was abolished by addition of 2-ME and
N-ethyl maleimide, which cause reduction and alkylation of
the cystein loop (Fig. 7A), suggesting that Ab recognition
requires the maintenance of the epitope tertiary structure.
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Abs to peptide 3 were affinity-purified from ESN55 and ESN32 sera
and tested in HIV neutralization assays. The neutralizing titers
obtained with Igs eluted from peptide 3 (Fig. 7B) were
higher (IC50 of 1 µg/ml for ESN55 and 5 µg/ml
for ESN32) than those of total serum Igs (IC50 of
13 µg/ml for ESN55 and 82.7 µg/ml for ESN32) (Fig. 4A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Given that 1) the specific ligand for CCR5 is Mip1ß, 2) a functional CCR5 coreceptor is a requirement for infection by HIV-1 R5 strains (36, 37), 3) R5 strains are responsible for the establishment of most new infections (24), and 4) the occupancy of CCR5 receptor by its ligands (38, 39) or by Abs (31, 40) prevents infection by HIV, we investigated whether binding of Mip1ß to CCR5 could be inhibited by sera of ESN. Sera from neither controls nor HIV-infected individuals could interfere with the binding of Mip1ß to CCR5; in contrast, inhibitory activity was detected in sera of a number of ESN: binding of Mip1ß to CCR5 was inhibited by sera of 6/48 (12.5%) ESN, but not by sera of either 45 USN or 40 HIV+ seropositive individuals (p < 0.001). Characterization of this inhibitory activity indicated that anti-CCR5 Abs 1) are present in the sera of some ESN, 2) recognize a conformational epitope in the first cysteine loop of CCR5, 3) inhibit Mip1ß binding to CCR5, 4) down-modulate CCR5 expression on PBMC in vivo, 5) inhibit Mip1ß-induced chemotaxis of control PBMC, and 6) block the HIV coreceptor function of CCR5, neutralizing the infectivity of R5 strains of HIV-1.
Although the potentially protective role of such Abs is evident, their origin is a matter of speculation: anti-CCR5 Abs 1) may be "naturally" present in some rare individuals (<1/85, which is the sum of the two control populations), 2) may be elicited by exposure to HIV or by particular and still unidentified factor(s) involved in HIV exposure, or 3) may be elicited by some HIV-unrelated antigenic stimulation. In this context, it is interesting that a recent report showed that autoantibodies to CCR5 could be induced in C57BL/6 mice by inoculation with a papilloma virus modified to express CCR5 peptides (41). Such Abs could inhibit binding of ß-chemokines to CCR5, as well as block infection with HIV-1 (41). Interestingly, the CCR5 epitope used to immunize mice and the epitope recognized by the CCR5-specific Igs detected in ESN sera show >90% homology.
The autoantibodies produced during the course of viral infections have
been attributed to virus-induced self-alterations that become
autoimmunogenic (42). Therefore, the infrequent
anti-CCR5 response might be due to a particular antigenic
stimulation or to the particular reactivity of rare individuals. Lehner
et al. (43) have shown that xenogenic immunization of
macaques with SIV grown in human cells induces protective immunity and
the generation of Abs reactive with simian CCR5 that are capable of
neutralizing SIV in in vitro assays. Allogenic, rather than xenogenic,
immunization might have produced the same result in our ESN
individuals. The recent report that CCR5 can act as an alloantigen in
CCR5-32 homozygous individuals eliciting Abs that compete with
RANTES and inhibit infection by R5, but not X4, primary isolates of
HIV-1 supports this hypothesis (44). Along the same lines,
therapeutic strategies aimed at preventing HIV-1 infection by means of
Abs to CCR5 elicited via immunization with a modified CCR5 gene are
currently being developed (45). In this context, it is
interesting that, whereas interruption of at-risk sex in ESN is
followed by a sharp drop in the titers of HIV-specific IgA and in the
loss of HIV-specific cell-mediated immunity (5), no
modifications were observed in the concentration of anti-CCR5 Abs.
These observations indicate that, whereas HIV-specific immunity in ESN
is dependent on a continuous exposure to the virus, anti-CCR5 Abs
could be maintained independently of exposure to HIV. These results
also suggest that 1) immune protection against HIV infection may be
secondary to the activation of multiple mechanisms that, beside potent
HIV-specific cell-mediated immunity in peripheral blood and mucosal
immunity in the genital tract, include anti-CCR5-specific Abs; 2)
anti-CCR5 Ab-mediated protection against HIV infection could be
maintained even in the absence of HIV-specific immune responses in ESN;
3) vaccine strategies aimed at elicitation of anti-CCR5 Abs could
be less dependent on the need for repeated vaccine boost, compared with
vaccine strategies based on the induction of HIV-specific immune
responses.
In conclusion, our results provide further information on the heterogeneous mechanisms underlying resistance to HIV infection in ESN individuals and reinforce the concept that immune protection can be mediated by multiple mechanisms, one of which could be anti-CCR5 Abs. Because other, still unclear mechanisms, including complement, chemokines, and other antiviral factors, are likely to be involved in the phenomenon of resistance to HIV infection, more research is necessary to shed further light on the immunologic correlates of this fortunate condition.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lucia Lopalco, Centro San Luigi, San Raffaele Scientific Institute, via Stamira D’Ancona 20, 20127 Milano, Italy. E-mail address:
3 Abbreviations used in this paper: ESN, exposed seronegative; USN, unexposed seronegative; Mip1ß, macrophage inflammatory protein 1ß.
Accepted for publication January 5, 2000.
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 P. I. Lobo, K. H. Schlegel, W. Yuan, G. C. Townsend, and J. A. White Inhibition of HIV-1 Infectivity through an Innate Mechanism Involving Naturally Occurring IgM Anti-Leukocyte Autoantibodies J. Immunol., February 1, 2008; 180(3): 1769 - 1779. [Abstract] [Full Text] [PDF]
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T. C. Friedrich, L. E. Valentine, L. J. Yant, E. G. Rakasz, S. M. Piaskowski, J. R. Furlott, K. L. Weisgrau, B. Burwitz, G. E. May, E. J. Leon, et al. Subdominant CD8+ T-Cell Responses Are Involved in Durable Control of AIDS Virus Replication J. Virol., April 1, 2007; 81(7): 3465 - 3476. [Abstract] [Full Text] [PDF]
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) ± 3 SE of the values obtained
with the sera of 45 USN controls (used to establish the cut-off), 48
ESN, and 40 HIV-seropositives are shown. Sera were employed at 1:10
dilution.
