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Immunogenicity of the Extracellular Domains of C-C Chemokine Receptor 5 and the In Vitro Effects on Simian Immunodeficiency Virus or HIV Infectivity¹

Thomas Lehner^2,*, Carl Doyle^*, Yufei Wang^*, Kaboutar Babaahmady^*, Trevor Whittall^*, Louisa Tao^*, Lesley Bergmeier and Charles Kelly

Departments of ^* Immunobiology and Oral Medicine and Pathology, Guy’s, King’s & St. Thomas’ Hospital Medical Schools, London, United Kingdom

	Abstract

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The C-C chemokine receptor CCR5 serves an important function in chemotaxis of lymphocytes, monocytes, and dendritic cells. CCR5 is also the major coreceptor in most macrophage-tropic HIV-1 infections. Immunization of rhesus macaques with a baculovirus-generated CCR5 construct or peptides derived from the sequences of the four extracellular domains of CCR5 elicited IgG and IgA Abs, inhibition of SIV replication, and CD4⁺ T cell proliferative responses to three of the extracellular domains of CCR5. The immune sera reacted with cell surface CCR5 expressed on HEK 293 cells. T and B cell epitope mapping revealed major and minor T and B cell epitopes in the N-terminal, first, and second loops of CCR5. The three C-C chemokines, RANTES, macrophage-inflammatory protein-1, and macrophage-inflammatory protein-1, were up-regulated by immunization with the CCR5-derived peptides, and the cell surface expression of CCR5 was decreased. The CCR5 Abs were complementary to the C-C chemokines in inhibiting HIV replication in vitro. Immunization with the four extracellular domains of CCR5 suggests that three of them are immunogenic, with maximal T cell responses being elicited by the second loop peptide. However, maximal Abs to the cell surface CCR5 or viral inhibitory Abs in vitro were induced by the N-terminal peptide. Up-regulation of the three C-C chemokines and down-modulation of cell surface CCR5 were elicited by the second loop, N-terminal, and first loop peptides. The data suggest that a dual mechanism of C-C chemokines and specific Abs may engage and down-modulate the CCR5 coreceptors and prevent in vitro HIV or SIV replication.

	Introduction

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C-C chemokine receptor 5 is a seven-transmembrane G protein-coupled molecule expressed on Th1 and Th0 cells (1), CD4⁺CD45 RO⁺ memory cells (2), macrophages, and immature dendritic cells (3, 4). CCR5 is an important cell surface receptor, regulating the traffic of mononuclear cells by binding C-C chemokines. The receptors play an essential role in inflammatory processes and autoimmunity, and they bind RANTES, macrophage-inflammatory protein (MIP)³-1, MIP-1, as well as viral MIP-I and II. The CCR5 cell surface molecule has acquired a central stage in HIV infection, as it functions as a major coreceptor to the CD4 glycoprotein receptor, for primary macrophage (M)-tropic HIV (5, 6) and M- as well as T-tropic SIV infections (7, 8, 9). Indeed, HIV-1 tropism is generated largely by coreceptor selection, and SIV, HIV-1, and HIV-2 strains can use coreceptors in the absence of CD4 for viral entry (10, 11, 12, 13, 14). Many primary SIV strains use CCR5 to infect simian cells in the absence of CD4, suggesting that CCR5 and not CD4 was the primordial SIV receptor (10).

Thus, there is a great deal of evidence that CCR5 is a major receptor in HIV and SIV infections, as well as in inflammation and autoimmunity. An investigation of the immunogenicity of CCR5 receptors was first conducted in rhesus macaques. Abs to human CCR5 were induced by xenoimmunization of macaques with human T cells (15, 16) and by alloimmunization in humans (Ref. 17 and Y. Wang, R. Vaughan, A. Harmer, P. Armstrong, J. Underwood, and T. Lehner, manuscript in preparation). There is 97.7% identity between the human and rhesus monkey CCR5, but 8 aa differ, and three of these are located in the N-terminal chain and second extracellular loop (7). Whether these differences can account for the immunogenicity of CCR5 in macaques is not clear. Functional assays showed that serum from xenoimmunized macaques inhibited MIP-1-generated CCR5-specific chemotaxis and SIV replication in a CCR5-dependent assay (16). In both assays, the serum Ab function was specifically inhibited by CCR5-transfected HEK 293 cells. Recently, Abs to CCR5 were reported in two subjects with homozygous 32-bp deletions of CCR5 (18) and in some seronegative women at risk from HIV infection (19). CCR5 is not expressed on the cell surfaces from these subjects, yet they do not suffer from ill health, so the presence of CCR5 is not essential for health (20, 21, 22).

In view of the significance of CCR5 receptors in inflammation, autoimmunity, and HIV transmission, we studied the immunogenicity and T and B cell epitopes of the extracellular domains of CCR5 in macaques. The aims of this study were first to express CCR5 in baculovirus and then to study the immunogenicity of the construct, as well as synthetic peptides derived from the sequences of the four extracellular domains of CCR5. The results suggest that both T and B cell responses are readily elicited by immunization with the CCR5 construct, as well as all CCR5 peptides except that derived from the third loop. This was observed not only in circulating blood, but also regional lymph nodes and spleen. Furthermore, immunization especially with peptides residing within the second loop of CCR5 induced up-regulation of three C-C chemokines and down-modulation of the cell surface expression of CCR5. Protection against SIV infection can be elicited in macaques by generating CD8-SF (CAF, cell antiviral factor), the three C-C chemokines, and Abs to the extracellular domains of CCR5, with or without any cognate immunity to the SIV Ags. These results suggest a novel strategy against SIV or HIV infection by using innate and cognate immunity directed to the CCR5 receptor.

	Materials and Methods

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Preparations of CCR5 and the peptide constituting the four extracellular domains

Baculovirus (Autographa californica) nuclear polyhedrosis virus was used to express CCR5 in an insect cell line, Spodoptera frugiperda (23). A DNA fragment comprising the full-length gene of human CCR5 was amplified by PCR using recombinant plasmid pcDNA 3.1 containing the human CCR5 gene (provided by J. Moore, Aaron Diamond AIDS Research Center, New York, NY) as template. Amplified DNA was cloned into the baculovirus transfer vector pAChis, and the construct was used to form baculovirus expressing CCR5 with a (His)₆ tag, as described earlier for SIV gp120 (23). Expression of the gene was confirmed by PCR and by immunostaining of infected cells. High titer (10⁹ PFU/ml) baculovirus stock was used to infect insect cells for 2 days at 28°C. A cell lysate of this preparation and a control baculovirus lysate were used.

The six peptides and a random unrelated 20 aa peptide were purchased from Neosystem Laboratories (Strasbourg, France), and the sequences of the peptides are shown as follows. N-terminal (aa 1–20), Met-Asp-Tyr-Gln-Val-Ser-Ser-Pro-ILe-Tyr-Asp-ILe-Asp-Tyr-Tyr-Thr-Ser-Glu-Pro-Cys; N-terminal 2 (aa11–31), Asp-ILe-Asp-Tyr-Tyr-Thr-Ser-Glu-Pro-Cys-Gln-Lys-ILe-Asn-Val-Lys-Gln-ILe-Ala-Ala-Arg (macaque differs from the human sequence in Thr⁹ (ILe) and Asp¹³ (Asn)); first loop (aa 89–102), His-Tyr-Ala-Ala-Ala-Gln-Trp-Asp-Phe-Gly-Asn-Thr-Met-Cys-Gln; second loop (168–187), Arg-Ser-Gln-Arg-Glu-Gly-Leu-His-Tyr-Thr-Cys-Ser-Ser-His-Phe-Pro-Tyr-Ser-Gln-Tyr (macaque differs from the human sequence in Lys 171 (Arg)); second loop (aa 178–197), Cys-Ser-Ser-His-Phe-Pro-Tyr-Ser-Gln-Tyr-Gln-Phe-Trp-Lys-Asn-Phe-Gln-Thr-Leu-Lys; third loop (aa 258–279), Asn Thr-Phe-Gln-Glu-Phe-Phe-Gly-Leu-Asn-Asn-Cys-Ser-Ser-Asn-Arg-Leu-Asp-Gln.

Western blotting

SF9 cells expressing CCR5 or I domain of _L integrin polypeptide (control) were lysed at a concentration of 2 x 10⁶/ml, and aliquots of the lysates were separated by SDS-PAGE using a gradient gel (4–20% acrylamide). After transfer, the blot was probed with anti-(His)₆ mAb conjugated to HRP (Amersham Pharmacia-Biotech, St. Albans, U.K.), and bands were revealed by chemiluminescence.

Immunization schedule

Four groups of a total of 12 rhesus macaques were immunized three times at about monthly intervals, and two control macaques were not immunized. Three macaques were immunized with 200 µg of the CCR5 preparation in alum (AluGel; Uniscience, London, U.K.) by the i.m. route. A group of four macaques was given 200 µg of the CCR5-derived N-terminal peptide in alum; three macaques received p1–20 and 1 p11–31 in alum by targeting s.c. inguinal lymph nodes (targeted lymph nodes), as described previously (24). The remaining five macaques were immunized by the targeted lymph node route, using synthetic peptides from the sequence of the first loop (aa 89–102) (n = 3) or second loop (aa 178–197) (n = 2). Prior attempts at immunization with the third loop peptide (aa 258–279) failed to elicit an immune response, so this peptide was not used further for immunization in the present series. Detailed autopsy examinations were conducted in four macaques with removal of lymphoid tissues, 2 mo after the last immunization.

Serum IgG and IgA Abs

Specific serum Abs to CCR5 and its extracellular peptides were assayed by ELISA, as described previously (16). Briefly, plates were coated with a predetermined optimal concentration of Ag (1 µg/ml) and with a random 20-residue peptide (R20) as a control Ag; they were incubated with doubling dilutions of test samples. Bound Ab was detected by incubation with rabbit IgG anti-monkey IgA (8 µg/ml; Nordic Immunological Laboratories, Tilburg, The Netherlands) or IgG (2 µg/ml; Sigma, Poole, U.K.), followed by affinity-purified goat anti-rabbit IgG-alkaline phosphatase conjugate (Sigma). The IgG and IgA Ab titers are presented as reciprocals before and after each immunization.

Serum Abs reacting with cell surface CCR5 expressed on HEK 293 cells

Culture of human embryonic kidney epithelial cells (HEK 293), with or without transfected CCR5, was performed as described before (16). The cells were incubated for 30 min at 4°C with sera taken before and after immunization with CCR5 or peptides. After washing to remove excess Ab, the cells were incubated with FITC-labeled Ab to human IgG (Sigma) known to cross-react with macaque IgG. After further 30-min incubation, the cells were washed and analyzed using a Coulter XL flow cytometer (Coulter Pharmaceutical, Palo Alto, CA) with XL software. Fluorescence for each serum was compared between the control HEK 293 and CCR5-expressing HEK 293 cells, and the results for pre- and postimmunization sera are presented at a dilution of 1/10. The sera were then titrated, and the highest dilution yielding greater than 2% binding, as compared with the preimmunization serum, was used as the end-point titer. The proportion of HEK 293 cells reacting with the sera was always lower than that reacting with the CCR5-transfected HEK 293 cells. Inhibition studies were conducted with the CCR5 preparation, and the three CCR5 peptides using up to 1 mg/ml of the peptides. The sera were preincubated with the CCR5 or peptides at 37°C for 30 min; these were then added to the CCR5-transfected HEK 293 cells and examined by flow cytometry. The control CCR5 mAb-2D7 was treated similarly with CCR5 and the peptides.

T cell proliferative responses to CCR5 and the peptides

T cell cultures were set up by separating mononuclear cells from defibrinated blood by Lymphoprep (Nycomed, Oslo, Norway) density-gradient centrifugation before and after immunization from all macaques (24). The cells were cultured without and with 1 and 10 µg/ml of CCR5 and its extracellular peptides, a control peptide (R20), or Con A in 96-well round-bottom plates (Costar, Cambridge, MA), containing RPMI 1640 (Life Technologies), as described before (24). The results were expressed as stimulation indices (SI, ratio of counts with and without Ag), as well as cpm before and after each immunization, for cultures stimulated with the optimum concentration of Ag. All cultures yielded high SI and counts with Con A, and no significant increase in counts was seen with the control peptide (data not presented).

T and B cell epitope mapping

The T cell proliferative responses and IgG Abs to the six overlapping peptides of the four extracellular domains of CCR5 were determined using PBMC and sera from the CCR5-immunized macaques. The methods are described above, and the results are presented as SI and reciprocal IgG Ab titers to each peptide, and compared with the responses to the CCR5 construct.

Lymphoid tissue examination at autopsy

Autopsies were conducted on four macaques to study the T cell proliferative responses to the CCR5 preparation and its extracellular domains in related and unrelated lymphoid tissues. After exsanguination, the spleen, internal and external iliac, superior and inferior mesenteric, bronchial, and axillary and submaxillary lymph nodes were removed. The tissues were cut into fragments, teased apart, and passed through a fine mesh and processed. The cell suspensions were collected, washed with RPMI 1640, and then cultured with CCR5 and the peptides, as described above.

Serum inhibition of SIV replication

To assay serum inhibition of SIV replication, PHA-stimulated human CD4⁺ T cells were infected with SIV J5 molecular clone in the presence of IgG Abs (10 and 100 µg/ml) separated from the sera by ion-exchange chromatography, as described before (16). A control mAb to CCR5 (2D7; PharMingen, San Diego, CA) was also used. The cells were then plated onto 96-well plates (2 x 10⁵ cells/well) and cultured in 20% IL-2 medium (Lymphocult-T-LF; Biotest, Solihull, U.K.) containing the serum. The cultures were refed at days 2 and 5 with the same medium, and by day 7 the supernatants of the cultures were removed to determine the reverse transcriptase (RT) activity by using the Quan-T-RT kits (Amersham, Buckinghamshire, Little Chalfont, U.K.).

The specificity of Ab-mediated inhibition of CCR5 was examined by inhibition with CCR5-transfected and untransfected HEK 293 cells. The cell line was cultured as described previously (16). The sera from macaques immunized with CCR5, N-terminal, or second loop CCR5 peptide were incubated with HEK 293 cells transfected with CCR5 or HEK 293 cells alone, for 30 min at a concentration of 100 µg/ml IgG per 1 x 10⁶ cells. The sera were collected after centrifugation, and the adsorption procedure was repeated three times. The human serum IgG was prepared and adsorbed in the same way.

Inhibition of HIV replication by the combined effect of Abs to CCR5 and C-C chemokines

Human PBMCs were isolated on Ficoll-Hypaque gradient from normal blood. CD4⁺ cells were enriched by depletion of CD8⁺ cells by panning using mouse anti-human CD4 (OKT4) mAb and goat anti-mouse Ab (Serotec, Oxford, U.K.). The cells (1.5 x 10⁶/ml) were washed in 5% FCS-RPMI 1640 medium and then activated for 5 days with 10 µg/ml PHA in RPMI 1640 medium, supplemented with 2 mM glutamine, 100 mg/ml penicillin and streptomycin, 10% FCS, and 20% IL-2 (TLF; Biotest Diagnostics, Danville, NJ). The cells were washed (twice) and cultured in 20% IL-2/RPMI 1640 medium overnight. Aliquots of 2 x 10⁵ cells were centrifuged, and to these were added 100 µl of mAb to the second loop of CCR5 (2D7; PharMingen), at a concentration of 0.2–12.8 µg/ml. In a parallel assay, a constant amount of recombinant RANTES, MIP-1, and MIP-1 (total of 0.25 ng/ml) was added to the cells treated with the mAb to CCR5. A similar assay was conducted with the three C-C chemokines (R&D Systems, Minneapolis, MN) in concentrations of 0.5–32 ng/ml alone or with a constant amount of 1 µg/ml mAb to CCR5. The PHA-stimulated CD4⁺ cells were incubated with the C-C chemokines and/or the Abs for 30 min before infection with HIV1 bronchoalveolar lavage (BAL; obtained from the Medical Research Council, National Institute of Biological Standards Control, Potters Bar, U.K.). The M-tropic HIV-1 BAL was added to the cells at a concentration of 20,000 cpm RT activity per 10⁶ enriched CD4⁺ cells. The infected CD4⁺ cells were incubated at 37°C with 5% CO₂ for 3 h, washed three times, and plated out in 96-well round-bottom plates. Aliquots of 1 x 10⁵ cells/200 µl/well in duplicates of IL-2/RPMI 1640 were supplemented with C-C chemokines and/or mAb. Every 2 days, 100 µl of supernatant was removed and replaced with 100 µl of fresh medium with the corresponding concentrations of C-C chemokines and/or Abs. This was repeated three times, and on day 9, 70 µl of cell-free supernatant was used to assay the RT activity using Quan-T-RT assay system (Amersham Life Science, Little Chalfont, U.K.).

Generation of C-C chemokines

CD8 cell-derived culture supernatants were first prepared by enrichment of CD8⁺ cells by negative selection, which removed CD4 cells, B cells, and monocytes from PBMC (25, 26). The cells were suspended in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, and 100 µg/ml penicillin and streptomycin (3 x 10⁶ cells/ml), and stimulated for 3 days with 10 µg/ml PHA. The culture supernatant was discarded, and the cells were then cultured in 10% IL-2 RPMI 1640 medium with 10% FCS; after 2 days, the cultures were centrifuged and the supernatants were collected. The cells were passaged the same way three times, resulting in up to 10 ml supernatant. RANTES, MIP-1, MIP-1, and monocyte chemoattractant protein-1 (MCP-1) were assayed in the culture supernatants by the specific enzyme immunoassay for each chemokine (R&D Systems), and the results are expressed in pg/ml.

Assay of cell surface CCR5 by flow cytometry

Freshly isolated PBMC were incubated with mAbs to CCR5 (227), supplied by P. Gray (ICOS, Seattle, WA). The cells were incubated with FITC-labeled rabbit anti-mouse IgG (Dako, Glostrup, Denmark), or with the latter alone as a control, and flow cytometry was performed using a FACScan (BD Biosciences, Franklin Lakes, NJ), running LYSIS II software for both acquisition and analysis.

	Results

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CCR5 expression

Human CCR5, which shows 97% identity with rhesus macaque CCR5 (7), was expressed in the SF9 insect cell line using baculovirus. CCR5 expression was confirmed by immunostaining of infected cells (not presented). Western blotting of cell lysates, using an anti-(His)₆ tag Ab, revealed the bulk of the rCCR5 as an aggregate of high molecular mass (>210 kDa) (Fig. 1, lanes 1–3). An alternative interpretation is that the CCR5 preparation is hyperglycosylated. The presence of minor bands of lower molecular mass suggests limited proteolysis. In the control lysate from cells expressing the I domain of _L integrin polypeptide, the recombinant polypeptide also stained strongly.

View larger version (110K): [in this window] [in a new window]   FIGURE 1. Detection of recombinant polypeptide in insect cell lysates by Western blotting. Aliquots (lane 1, 2 µl; lane 2, 5 µl; lane 3, 10 µl) of lysates of baculovirus-infected SF9 cells expressing CCR5 or I domain of L integrin polypeptide (lane 4, 5 µl) were probed with anti-(His)6 mAb. Bands were revealed by chemiluminescence: A, 1-s exposure; B, 5-s exposure.

Abs to the immunizing CCR5 and the extracellular peptides. Serum Abs were readily elicited in macaques to the baculovirus-expressed human CCR5 administered with alum by the i.m. route. Both IgG and IgA Abs to CCR5 were found in the three macaques by ELISA, with mean (±SEM) titers of 3200 (±1306) and 2667 (±1524), respectively (Fig. 2). In view of the poor immunogenicity of synthetic peptides and the weak adjuvanticity of alum, peptides derived from the extracellular domains of CCR5 were administered by the s.c. route in the proximity of the inguinal and external iliac lymph nodes, which enhances the immune responses, but avoids the deep injection targeting the internal iliac lymph nodes (24, 27). Indeed, raised serum IgG and IgA Ab titers of 1600 (±754) were elicited by the immunizing N-terminal peptide 1–20 (Fig. 2), but no Abs were detected with the overlapping peptide 11–31 (not shown). The IgG and IgA titers induced by the first loop (aa 89–102) were 2267 (±762) and 1367 (±766), and by the second loop (aa 178–197) were 2000 and 1800, respectively (Fig. 2). The N-terminal peptide 1–20 failed to elicit Abs in one of the three macaques, although the peptide induced a T cell proliferative response in that animal. Abs induced by immunization with the three peptides failed to recognize the baculovirus CCR5 preparation, and this may be either due to the lack of a correct conformation of the synthetic peptides or because the CCR5 construct had the human sequence, whereas the synthetic peptides had the rhesus macaque sequence. The latter interpretation would not apply to the first and second loop, which share the same amino acid sequences between human and rhesus macaque CCR5, and yet were immunogenic and were recognized by Abs and T cells derived from the CCR5 construct-immunized macaques. Rectal washings were tested for Abs to CCR5, but these were not detected in any one of the immunized macaques.

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Serum IgG and IgA Abs to CCR5 or its extracellular peptides after i.m. immunization after targeted iliac-inguinal immunization with peptides in alum; the mean (±SEM) Ab titers (reciprocals) are given to the immunizing CCR5 or its peptides. The N-terminal peptide 11–31 failed to elicit a detectable Ab titer (not shown).

View larger version (41K): [in this window] [in a new window]   FIGURE 3. Serum Abs from macaques immunized with CCR5 or its extracellular domains reacting with CCR5 expressed on HEK 293 cells. Abs were evaluated by flow cytometry, subtracting the percentage of cells reacting with the preimmune sera from those of the postimmune sera (data presented at 1/10 dilution). The sera were titrated, and the end-point (>2%) is given in parentheses.

As with Abs, strong T cell proliferative responses were elicited with the CCR5 baculovirus in alum after two or three immunizations, with SI, mean (±SEM) of 13.6 (±4.8) (Fig. 4). The second loop peptide (178–197) elicited T cell proliferative responses after the third immunization, with SI of 13.5, which was comparable with that induced by the CCR5 preparation (Fig. 4). The N-terminal peptide (1–20) yielded moderate SI (4.4 ± 0.5), but peptide 11–31 failed to stimulate T cell proliferative or Ab responses. The first loop peptides failed to elicit T cell proliferation, unless the macaques were boosted with peptides covalently linked to heat-shock protein 70 by glutaraldehyde (SI 4.3 ± 0.8, Fig. 4). T cell proliferative responses were not elicited to the CCR5 preparation by PBMC from any of the synthetic peptide-immunized macaques.

View larger version (17K): [in this window] [in a new window]   FIGURE 4. T cell proliferation stimulated by CCR5 after i.m. immunization (3x) or its extracellular peptides after targeted iliac-inguinal immunization with CCR5 peptides (3x) in alum; the SI are given to the immunizing CCR5 or its peptides as mean (±SEM). Mitogenic stimulation with Con A showed the following SI:

The four extracellular domains, with the six overlapping synthetic peptides, were examined for T cell proliferation and B cell Ab binding after immunization with the baculovirus CCR5 preparation (Fig. 5). T and B cell epitopes were identified in the N-terminal peptide 1–20, but not 11–31, although the latter overlaps by 10 residues with peptide 1–20. A T cell, but weak B cell epitopes were detected with the first loop peptide (p89–102). However, the second loop peptide 178–197 expressed strong T and B cell epitopes, and this was also found with the overlapping peptide 168–187, but only for the B cell epitope (Fig. 5). Surprisingly, neither T nor B cell epitopes were recognized by the third loop peptide 258–279. The identification of these epitopes is largely consistent with the immunogenicity of the extracellular domains of CCR5, as demonstrated with the synthetic peptide (Figs. 2, 3, and 4).

View larger version (17K): [in this window] [in a new window]   FIGURE 5. T and B cell epitope mapping of the extracellular domains after immunization with the CCR5 preparation in three macaques. The T cell epitopes were determined by T cell proliferation of PBMC stimulated with six peptides (20 aa) derived from the sequences of the four extracellular domains of CCR5 and the B cell epitopes by ELISA with sera using the above peptides. The mitogenic Con A response was 51.8 (±5.7). The results are given as mean (±SEM).

To increase T cell responses to the peptide-immunized macaques, they were boosted by the targeted iliac lymph node (TILN) route, with the peptide covalently linked to heat-shock protein 70 or 65 kDa, before the animals were killed (28). Autopsies were conducted on representative macaques from each of the four groups, removing most of the related and unrelated lymphoid tissues (Fig. 6). The eluted mononuclear cells from these tissues were stimulated with each of the six peptides, the baculovirus-grown CCR5 lysate, control baculovirus lysate, Con A, or without any Ag. The results in each of the four groups showed largely specific T cell proliferative responses only to the immunizing CCR5 or its peptides by PBMC, splenic, internal iliac, and inferior mesenteric lymph node cells (Fig. 6). No or minimal responses were induced by the superior mesenteric or axillary lymph node cells (Fig. 6), or by the bronchial, submandibular, or the tonsillar cells (data not shown). The lymphoid tissue results were similar to those obtained by stimulation with SIV p27 particulate Ag after TILN immunization (24). Immunization with CCR5 elicited T cell proliferative responses with CCR5 lysate, as well as some or all of the peptides from the extracellular domains of CCR5 (except the third loop) by PBMC, iliac, and inferior mesenteric, but the splenic cells responded only to the CCR5 lysate (Fig. 6). In contrast, the N-terminal, first, and second loop peptides elicited specific T cell proliferation only to the immunizing peptides, of which the second loop peptide appeared to be the most immunogenic (Fig. 6), but the T cells failed to respond to the CCR5 lysate. Cells from all the tissues responded normally to mitogenic stimulation with Con A (positive control) and failed to respond to the control baculovirus lysate (data not presented).

View larger version (24K): [in this window] [in a new window]   FIGURE 6. T cell proliferative responses to CCR5 and its four extracellular domains in PBMC, spleen, and lymph nodes after immunization of four macaques, each with CCR5 or one of the three extracellular peptides. The results are given in SI to each peptide and CCR5. The Con A responses were as follows:

CD4⁺ T cells infected with SIV were treated with the IgG fractions of sera from macaques immunized with CCR5 or the extracellular domains of CCR5. The serum IgG fractions at dilutions of 1/100 showed increases in inhibition of SIV replication after immunization with CCR5, N-terminal peptide 1–20, first loop, and second loop peptides (aa 178–197), but not serum IgG from a macaque immunized by the N-terminal peptide 11–30 (Table I). A control mAb to CCR5 showed a mean (±SEM) inhibition of 51.2 (±8.7)% of SIV replication. This functional assay is consistent with the concept that serum Abs to CCR5 may inhibit SIV replication by blocking access of SIV to the CCR5 coreceptor. It is unlikely that RANTES released from platelets has contributed to blocking the coreceptor, as the IgG fraction of serum was used.

Inhibition of HIV or SIV replication has been demonstrated in vitro by the three C-C chemokines (29, 30) and by monoclonal and polyclonal Abs to CCR5 (16). Sera from macaques immunized with CCR5 or its extracellular domains also inhibited HIV replication (Tables I and II). We have then examined the possibility that C-C chemokines can enhance CCR5 Ab inhibition, or conversely, CCR5 Abs can enhance C-C chemokine inhibition of HIV replication. Indeed, a dose-dependent inhibition of HIV replication resulted from increased concentration of mAb to CCR5 added to a suboptimal inhibitory dose of C-C chemokines (Fig. 7A). Conversely, increased concentrations of C-C chemokines added to a suboptimal inhibitory dose of CCR5 also elicited a dose-dependent inhibition of HIV replication (Fig. 7B). A mouse serum isotype control and MCP-1, a C-C chemokine control, had no effect on HIV replication. Dose-dependent inhibition of HIV BAL replication was also found with the three sera from macaques immunized with the CCR5 N-terminal, first, and second loop peptides, when added to a constant suboptimal concentration of the three C-C chemokines (Fig. 7C). This suggests that HIV inhibition with low concentrations of the three C-C chemokines can be enhanced with Abs to CCR5, and conversely low titers of Abs can be enhanced by the three C-C chemokines.

View larger version (17K): [in this window] [in a new window]   FIGURE 7. The effect on HIV replication of: A, increasing concentrations of the mAb to CCR5 (2D7) added to a suboptimal concentration of the three C-C chemokines (0.25 ng/ml); B, increasing concentrations of the three C-C chemokines on a suboptimal concentration of mAb to CCR5 (1 µg/ml); and C, polyclonal Abs to the three extracellular peptides of CCR5 added to 0.25 ng/ml of the three C-C chemokines.

Immunization with the CCR5 preparation induced increased concentrations of RANTES, MIP-1, and MIP-1, but not MCP-1 (Fig. 8). Immunization with the extracellular peptides of CCR5 also up-regulated the concentrations of RANTES, MIP-1, and MIP-1, with the exception of MIP-1 by immunization with the first loop. However, the kinetics differed, especially with the N-terminal peptide 1–20 and the first loop. The CCR5 construct and second loop peptide (178–197) elicited the most consistent increase in the concentrations of the three C-C chemokines, and it is noteworthy that these also induced the highest T cell proliferative responses (Fig. 4).

View larger version (22K): [in this window] [in a new window]   FIGURE 8. Concentrations of RANTES (•), MIP-1 (), MIP-1 (), and MCP-1 () in picograms per milliliter after immunization (3x) with N-terminal (aa 1–20), first loop (aa 89–102), and second loop (aa 178–197) peptides or CCR5 lysate in alum, in four representative macaques.

Up-regulation of the C-C chemokines in vitro (31, 32, 33, 34) or in vivo (27) down-modulates the cell surface expression of CCR5. In this study, we have studied by flow cytometry the effect of immunization with CCR5 or its extracellular peptides on the cell surface expression (mean fluorescence intensity, MFI) or proportion of CCR5⁺ cells (Table III). Immunization with the N-terminal, first, and second loop peptides elicited a decrease in the MFI in all six macaques between the pre- and postimmunization CCR5, with a range of 91.6–335 (median 189) (Table III). The proportion of cells expressing CCR5 was also decreased, although to a lesser extent in five/six macaques, with a range of 2.3–21.9% (median 10.2%). The presence of C-C chemokines that bind CCR5 and Abs to CCR5 might be a dual mechanism that down-modulates cell surface CCR5. However, immunization with the CCR5 preparation induced no consistent effect on the MFI or proportion of CCR5.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In addition to ELISA, we used a functional assay to study serum Abs to CCR5. An increase in inhibition of SIV replication was demonstrable with serum Abs (at 1/100 dilution) to the CCR5, from 15.7 (±6.4)% to 39.3 (±13.6)%, N-terminal peptide (aa 1–20) from 8.1 (±3.4)% before to 49.2 (±8)% after immunization, and first loop from 16.9 (±6.9)% before to 38 (±11.6)% (mean ± SEM) after immunization, but to a limited extent to the second loop peptide (from 23.3 to 30.5%). It is of considerable interest that serum Ab titers to the native CCR5 expressed on the surface of HEK 293 cells and evaluated by flow cytometry showed similar quantitative differences to those found by the inhibition assay of SIV replication. Abs to the N-terminal peptides showed a net increase of 11.1% and a titer of 1:30, and those to the first and second loop were 4.2% (titers 1:30 or 1:10). Thus, serum Abs to the native CCR5 were associated with the functional virus inhibition assay, but not with the ELISA-binding assay. The specificity of SIV-inhibitory serum Abs for CCR5 was demonstrated by a significant decrease of SIV replication with CCR5-transfected, but not with untransfected HEK 293 cells. Furthermore, Abs recognizing cell surface-expressed CCR5 by immunofluorescence were also inhibited by the CCR5 baculovirus preparation. There is evidence that the N-terminal and second loop extracellular domains of CCR5 are predominantly involved in M-tropic HIV and SIV binding and internalization (36, 37). These findings raise the possibility that Abs may affect function of the extracellular domains of CCR5 differently from those reported for C-C chemokines or the virus, as will be discussed below.

Examination of T cell proliferative responses to the extracellular domains of CCR5 in the lymphoid tissues showed that the specificity of the three immunizing peptides stimulating PBMC was largely maintained in the iliac and inferior mesenteric lymph nodes, and the spleen. In contrast, immunization with the whole CCR5 preparation elicited T cell responses to the first and second loop, and to a lesser extent the N-terminal (aa 1–20) in the iliac and inferior mesenteric lymph nodes and PBMC. Cells from the unrelated superior mesenteric, axillary, submandibular, and bronchial lymph nodes failed to respond to any of the peptides or CCR5. The responses from the anatomically associated lymph nodes were consistent with the TILN immunization reported previously with SIV p27 particulate Ag (24).

Investigation of C-C chemokine concentrations revealed that immunization with the CCR5 preparation and peptides derived from the sequences of the extracellular domains of CCR5 up-regulated the concentrations of CD8 cell-derived RANTES, MIP-1, and MIP-1. Whereas up-regulation of the C-C chemokines was found with each immunization of the CCR5 preparation and the second loop, this was delayed with the first loop and the N-terminal peptide of CCR5 up to the third immunization for MIP-1 and MIP-1. The kinetics of generation of the C-C chemokines after immunization with whole CCR5 and the second loop was similar to those of T cell proliferative responses and to a lesser extent the IgA and IgG Abs. However, the delay on immunization with the N-terminal and first loop raises the possibility that MIP-1 and MIP-1 might be up-regulated in response to CCR5-Ab complexes.

There is evidence both in vitro (29, 30, 31, 32) and in vivo (27) that raised concentrations of C-C chemokines down-modulate the cell surface expression of CCR5. Whereas in vitro down-modulation of CCR5 treated with RANTES lasted 20 min, alloimmunization of women up-regulated the three C-C chemokines, induced Abs to CCR5, and down-modulated the cell surface expression of CCR5, which was evident up to 1 year (Ref. 17 and Y. Wang, R. Vaughan, A. Harmer, P. Armstrong, J. Underwood, and T. Lehner, manuscript in preparation). In this study, we suggest that Abs to CCR5 may have a similar effect on C-C chemokines in down-modulating CCR5 receptors, although this will need to be confirmed in a system in which only Abs are added; in the present experiment, both anti-CCR5 Abs and C-C chemokines bind to CCR5. Immunization with the four extracellular domains of CCR5 suggests that three of them are immunogenic, with maximal T cell responses being elicited by the second loop peptide (5- to 15-fold). However, maximal viral inhibitory Abs were induced by the N-terminal peptide, whereas up-regulation of the three C-C chemokines and down-modulation of cell surface CCR5 were elicited by the second loop, N-terminal, and first loop peptides. The second loop of CCR5 is functionally endowed with coreceptor function, ligand specificity (38, 39, 40, 41, 42), binding of M-tropic HIV and SIV, as well as T-tropic SIV (5, 6, 7, 8, 9) and the 32-bp deletion (20, 21, 22). However, the N-terminal is required for HIV-gp120 binding and viral entry (37) and is important for CCR5-mediated fusion and entry of R5 and R5 x 4 HIV-1 strains (43, 44, 45). These findings suggest that T cell responses generated by immunization with the N-terminal and second loop of CCR5 might prove to be of some significance in immunomodulation of inflammatory processes, autoimmunity, and prevention of HIV transmission. However, we cannot account for the lack of down-modulation of the cell surface expression of CCR5 after immunization with the CCR5 construct.

The inhibitory effect on HIV replication of a suboptimal concentration of RANTES, MIP-1, and MIP-1 was enhanced in a dose-dependent manner with mAb to CCR5, as well as polyclonal Abs to three of the four extracellular domains of CCR5. Conversely, the effect of suboptimal titer of these Abs on HIV inhibition was enhanced by the three C-C chemokines. Thus, a dual mechanism of blocking and possibly down-modulating CCR5 may operate, in which the three C-C chemokines and Abs to CCR5 may bind different or the same extracellular domains of CCR5, block the receptor function, and prevent HIV or SIV transmission. Preliminary results with mAb to the N-terminal and second loop of CCR5 suggest that the latter is more effective in dose-dependent inhibition of HIV replication.

The mechanism of CCR5-mediated inhibition of HIV infection has not been investigated, and there are four possibilities. First, C-C chemokines binding to CCR5 block interaction between HIV-env/CD4 complex and CCR5 by steric hindrance (33, 41). Second, C-C chemokines induce receptor desensitization, thereby preventing viral interaction (32); autodesensitization of dendritic cell surface CCR5 by activation of the cells to secrete C-C chemokines may result in loss of surface CCR5 (1). Third, CCR5 undergoes conformational changes due to dimerization induced by Abs to CCR5 or C-C chemokines (34); indeed, some mAb to the second loop of CCR5 prevent HIV infection by dimerization of CCR5, while others by steric hindrance (36). Fourth, CCR5 is down-modulated by agents such as LPS and enters an alternate route of intracellular trafficking, which inhibits cell surface recycling (46).

We suggest that immunization with CCR5 or some of its extracellular peptides may stimulate CD4⁺ and CD8⁺ T cells to elicit immune responses that result in specific anti-CCR5 Abs, CD4 cell proliferation, and CD8 cell generation of C-C chemokines. These may have significant general effects on chemotaxis of naive and memory T cells, immature dendritic cells, macrophages, and B cells, as well as specific effects on HIV or SIV infection.

	Footnotes

 
¹ This work was supported by the European Community Biomed Grant (BMH4 CT97-2345), National Institute of Allergy and Infectious Diseases, National Institutes of Health, and the Guy’s & St. Thomas’ Charitable Trust.

² Address correspondence and reprint requests to Dr. Thomas Lehner, Department of Immunobiology, 3rd Floor New Guy’s House, Guy’s Hospital, London SE1 9RT, United Kingdom. E-mail address: thomas.lehner{at}kcl.ac.uk

³ Abbreviations used in this paper: MIP, macrophage-inflammatory protein; BAL, bronchoalveolar lavage; MCP-1, monocyte chemoattractant protein-1; MFI, mean fluorescence intensity; RT, reverse transcriptase; SI, stimulation index; TILN, targeted iliac lymph node; M-tropic, macrophage-tropic.

Received for publication October 12, 2000. Accepted for publication April 10, 2001.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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