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The Journal of Immunology, 2006, 176: 463-471.
Copyright © 2006 by The American Association of Immunologists

Effects of Immunization with CCR5-Based Cycloimmunogen on Simian/HIVSF162P3 Challenge1

Shogo Misumi2,*, Daisuke Nakayama2,*, Masashi Kusaba*, Takaaki Iiboshi*, Ryouzaburo Mukai{dagger}, Kuniomi Tachibana{ddagger}, Tadashi Nakasone§, Mamoru Umeda{ddagger}, Hideaki Shibata{ddagger}, Masafumi Endo*, Nobutoki Takamune* and Shozo Shoji3,*

* Department of Pharmaceutical Biochemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan; {dagger} Tsukuba Primate Center for Medical Science, National Institute of Infectious Diseases, Tsukuba, Ibaraki, Japan; {ddagger} Research Laboratory, Nissui Pharmaceutical, Ibaraki, Japan; and § AIDS Research Center, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
A synthetic cycloimmunogen targeting the HIV-1 coreceptor CCR5 was evaluated for its capacity to induce CCR5-specific Abs with anti-HIV-1 activity in cynomolgus macaques. The cyclic closed-chain dodecapeptide (cDDR5) mimicking the conformation-specific domain of human CCR5 was chemically prepared, in which the Gly-Glu dipeptide links the amino and carboxy termini of the decapeptidyl linear chain (Arg168 to Thr177) derived from the undecapeptidyl arch (Arg168 to Cys178) of extracellular loop-2 in CCR5. The immunization of cynomolgus macaques with the cDDR5-conjugated multiple-Ag peptide (cDDR5-MAP) induced anti-cDDR5 serum production for ~15 wk after the third immunization. The antisera raised against cDDR5-MAP reacted with both human and macaque CCR5s, and potently suppressed infection by the R5 HIV-1 laboratory isolate (HIVJRFL), R5 HIV-1 primary isolates (clade A:HIV93RW004 and clade C:HIVMJ4), and a pathogenic simian/HIV (SHIVSF162P3) bulk isolate in vitro. To examine the prophylactic efficacy of anti-CCR5 serum Ab for acute HIV-1 infection, cynomolgus macaques were challenged with SHIVSF162P3. The cDDR5-MAP immunization attenuated the acute phase of SHIVSF162P3 replication. The geometric mean plasma viral load in the vaccinated macaques was 217.10 times lower than that of the control macaques at 1 wk postchallenge. Taken together, these results suggest that cDDR5-MAP immunization is an effective prophylactic vaccine strategy that suppresses and delays viral propagation during the initial HIV-1 transmission for the containment of HIV-1 replication subsequent to infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although numerous trials on preventive HIV-1 vaccines are ongoing (International AIDS Vaccine Initiative report, <www.iavi.org/iavireport>) or being planned, a major obstacle to the development of an HIV-1 vaccine is unfortunately the marked genetic diversity of HIV-1 (1). To contend with this issue, some attempts have been made to match candidate vaccines with strains prevalent in sites in which phase III efficacy trials are to be conducted (2, 3, 4). Other strategies include the use of mixture vaccines containing Ags representative of several genetic subtypes (5, 6), the design of candidate vaccines targeting conserved HIV-1 epitopes (7, 8), and the use of candidate vaccines based on the consensus or ancestor sequences selected to minimize genetic differences between vaccine strains and current isolates (2, 9).

As alternative approaches for developing HIV-1 vaccine, other attempts have also been made to induce anti-CCR5 Abs that can bind native CCR5 and block viral infection because CCR5 is genetically stable, unlike viral targets that may rapidly mutate during the course of infection (10, 11, 12, 13, 14), and has been considered important in HIV-1 transmission on the basis of the findings that individuals homozygous for a 32-bp deletion in the CCR5-coding region have a very low susceptibility to HIV-1 infection (15, 16, 17, 18, 19). Furthermore, CCR5 is also considered as a redundant molecule in adults because CCR5-defective individuals have normal inflammatory and immune reactions (20). In fact, it is reported that CCR5-specific autoantibodies that strongly block HIV infection are induced in the sera of HIV-seronegative individuals (referred to exposed seronegative (ESN)4 subjects) despite multiple exposures to HIV-1 (21). Therefore, CCR5 may be an important target for developing a more effective HIV-1 vaccine.

In this study, we developed a CCR5-based cycloimmunogen that can elicit an anti-CCR5 autoantibody to reconstruct the immune response induced in ESN subjects, and examined the in vivo protective effects of vaccination on acute viral infection in cynomolgus macaques, as well as the duration and magnitude of autoantibody induction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Preparation of cyclic closed-chain dodecapeptide (cDDR5)-MAP and biotinylated cDDR5

A CCR5-derived linear dodecapeptide (linear DDR5, H2N-ERSQKEGLHYTG-COOH), in which all side-chain groups are protected, was synthesized using an automatic peptide synthesizer and cyclized, as previously described (10). The free {beta}-carboxyl group of Glu1 in the protected cDDR5 was separately conjugated to the MAP resin (Applied Biosystems) and 5-[5-(N-succinimidyloxycarbonyl)pentylamido]hexyl D-biotinamide through ethylenediamine. cDDR5 derivatives, in which all protected groups were removed using trifluoroacetic acid, were used for the following purposes. cDDR5-MAP was used to immunize cynomolgus macaques. MAP is commercially available (Applied Biosystems) and is composed of a 2-fold bifurcating polylysine core developed as a carrier of a peptide Ag. In contrast, biotinylated cDDR5 was used to confirm the specific binding of Abs to cDDR5 using a BIAcore biosensor. Unless otherwise specified, all of the peptides used were purified by HPLC (Waters), and the molecular masses of the compounds were determined by MALDI-TOF mass spectrometry (Burker Franzen Analytik).

Immunization schedule

All of the cynomolgus macaques were housed in individual cages and maintained according to the rules and guidelines of the National Institute for Infectious Diseases for experimental animal welfare. In the pilot test, a 4-year-old cynomolgus macaque was immunized i.p. at 0 and 1 wk with 300 µg of cDDR5-MAP in CFA and boosted s.c. at 6 wk with 300 µg of cDDR5-MAP in IFA. Furthermore, three 4- to 6-year-old cynomolgus macaques (nos. 11, 13, and 16) were also immunized with cDDR5-MAP according to the same schedule. Another three cynomolgus macaques (nos. 7–9) were immunized with MAP following the same immunization schedule as the controls. Immune sera were obtained at 0, 1, 2, 4, 6, 8, 10, and 21 wk postinitial immunization (wpim), and were then subjected to BIAcore analysis and MAGIC-5 assay.

Real-time biomolecular interaction analysis using surface plasmon resonance

The principle in analyzing Ag-Ab interactions has been described (10). Biotinylated cDDR5 was injected into a streptavidin-coated sensor chip (BIAcore KK). Binding experiments were performed by injecting immune sera diluted 1/9 with PBS (0.02% KH2PO4, 0.29% Na2HPO4'12H20, 0.8% NaCl, and 0.02% KCl).

Flow cytometry

CEM-CCR5 cells (22) were washed with PBS and then suspended in a cold washing buffer (PBS containing 2% FBS and 0.02% NaN3) at 1 x 106 cells/ml. The cells were incubated with preimmunization and 8 wpim sera, which were dialyzed using Spectra/Por (cutoff molecular mass, 100,000; Spectrum Laboratories), according to the manufacturer’s instructions, and diluted 1/5, 1/100, or 1/400 with PBS. Because immunization with the extracellular linear peptide of CCR5 up-regulates the concentrations of CCL5, CCL3, and CCL4, which are CCR5 ligands (11), the sera were dialyzed and then diluted with PBS for flow cytometry. The cells were resuspended in the washing buffer containing FITC-conjugated anti-monkey Ig (IgG, IgA, and IgM) Abs (H&L) (Rockland). After 30 min of incubation at 4°C, the cells were washed three times and then analyzed using an EPICS XL flow cytometer (Beckman Coulter).

The specificity of anti-cDDR5 serum for native CCR5 expressed on cynomolgus macaque PBMCs or HSC-F (23) was tested by examining the ability of anti-cDDR5 serum to block binding of a CCR5-specific mAb (3A9; BD Pharmingen). A total of 1 x 106 cells was incubated with preimmunization or 10 wpim serum, which was dialyzed and diluted (1/2) with PBS, from vaccinated macaques no. 16 for 15 min at room temperature. The cells were washed and then stained with FITC-labeled anti-CD95 (BD Pharmingen) and PE-labeled anti-CCR5 (3A9; BD Pharmingen) for 20 min at room temperature. Finally, the cells were washed again and then analyzed using an EPICS XL flow cytometer. Control experiments were conducted without preincubation with preimmunization or 10 wpim serum.

MAGIC-5 assay

The antiviral activity of the sera obtained before and after immunization with cDDR5-MAP was determined using MAGIC-5 cells, as previously described (10). MAGIC-5 cells were plated at 1 x 104 cells/well (96-well plates) and incubated overnight in RPMI 1640 containing 5% FCS (200 µl); the medium was then replaced with the preimmunization and 8 wpim sera (30 µl), which were dialyzed using Spectra/Por, and serially diluted (1/5–1/1280) with PBS. The cells were then separately incubated in suspensions of R5 and X4 clade B viruses, nonclade B primary isolates, or simian/HIV (SHIV) (10 µl: HIVLAV-1, 5077; HIVJRFL, 3749; HIV93RW004, 1406; HIVMJ4, 844; SHIVSF162P3, 713 tissue culture infective dose50 (TCID50)/ml in the presence of 20 µg/ml DEAE dextran for 2 h, and then cocultured in the medium (160 µl) for 48 h. The cells were fixed, and HIV-1-infected cells identified by their blue staining were counted by conventional methods.

Productive infection assay using SHIVSF162P3

CEM-CCR5 cells (5 x 105) (22) were infected with SHIVSF162P3 (3.2 ng/ml as measured using p27 Ag) in the presence of the preimmunization and 8 wpim sera for 18 h. The cells were washed with PBS, and then plated onto 24-well plates and cultured in 3 ml of the RPMI 1640 medium containing 10% FBS. The culture supernatants 24, 48, 72, 96, and 120 h after infection were removed to detect SIV p27 Ag by RETRO-TEK SIV type 1 p27gag Ag ELISA, according to the manufacturer’s instructions.

HIV-1 and SHIV strains

Clade B laboratory-adapted strains HIV-1JRFL and HIV-1 LAV-1 were used. These clade B viruses were propagated in a chronically HIV-1JRFL-infected T cell line (Molt4-CCR5/JRFL) and a chronically HIV-1LAV-1-infected T cell line (CEM/LAV-1) grown in a complete medium consisting of RPMI 1640 supplemented with 10% heat-inactivated, defined FBS (HyClone), penicillin (100 IU/ml), and streptomycin (0.1 mg/ml). The nonclade B strains HIV93RW004 and HIVMJ4 (AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health) were propagated in 3-day-cultured, PHA-activated human PBMCs. Furthermore, SHIV-1SF162P3 (24, 25) (AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health) was similarly propagated in 3-day-cultured, PHA-activated cynomolgus macaque PBMCs. Each cell-free virus stock was prepared from tissue culture supernatants harvested from chronically and acutely infected cells. Virus-containing supernatants were pooled, filtered through a 0.45-µm membrane, aliquoted, and frozen to provide a uniform stock of infectious virus.

Macaque challenge

All of the six cynomolgus macaques were i.v. challenged with 1 ml of 10 TCID50/ml SHIVSF162P3. Blood, serum, and plasma samples were collected from the infected animals at regular intervals after infection.

Determination of SHIV RNA viral load by SYBR green-based quantitative real-time PCR assay

Viral RNA was extracted from plasma using a QIAamp viral RNA minikit (Qiagen), then retrotranscribed using the SuperScript III First-Strand Synthesis system (Invitrogen Life Technologies). cDNA duplicates were amplified by SYBR green real-time PCR assay previously described (26) with some modifications. Briefly, primers that recognize specific and highly conserved sequences on the gag region of SIV described by Ui et al. (27) were selected. The sequences of SIV gag primers were 5'-GGAAATTACCCAGTACAACAAATAGG-3' and 5'-TCTATCAATTTTACCCAGGCATTTA-3'. The SIV gag gene was amplified in 20 µl of a PCR mixture consisting of 10 µl of 2x master mix containing modified DyNAmo hot start DNA polymerase, SYBR green I, optimized PCR buffer, 5 mM MgCl2, a dNTP mix including dUTP (Finnzymes), 2 µl of each primer, and 8 µl of cDNA. PCR was conducted as follows: initial activation of hot start DNA polymerase at 95°C for 15 min; 40 cycles of four steps of 95°C for 10 s, 57°C for 20 s, 72°C for 20 s, and 76°C for 2 s. At the end of the amplification cycle, melting temperature analysis was conducted by gradually increasing the temperature (0.5°C/s) to 95°C. Amplification, data acquisition, and analysis were conducted with the DNA Engine Opticon 2 System (MJ Research) using Opticon Monitor version 2.02 software (MJ Research). The detection limit of this system was 1 x 103 copies/ml.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Design and synthesis of cDDR5

The hypothetical structural model of CCR5 was based on its homology with rhodopsin (28), and was energy minimized using the molecular operating environment (MOE; Chemical Computing Group) (Fig. 1A). The extracellular loop-2 region of CCR5 and its structure deduced on the basis of the Cys178 residue bound to the Cys101 residue of extracellular loop-1 by a disulfide bond using MOE showed a unique arch consisting of 11 aa residues (undecapeptidyl arch (UPA)) (Fig. 1B). The cDDR5 moiety designed to mimic the deduced conformational epitope of UPA was generated by the cyclization of a decapeptide (R168SQKEGLHYT177) derived from the UPA sequence by inserting a spacer-armed dipeptide (Gly-Glu). The deduced structure of cDDR5 (shown in cyan) was adopted in the construction of the hypothetical structural model of UPA in CCR5 (shown in yellow) using the MOE-Align tool (Chemical Computing Group) (Fig. 1C).



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  		FIGURE 1. Deduced structure of UPA in extracellular loop-2 of CCR5. A, The predicted model of CCR5 was constructed using the segmented approach, and MOE was used for actual calculation. The determined structure of rhodopsin was used as the template. Transmembrane helixes are in cyan. The extracellular loops of CCR5 are color coded: N terminus, blue; extracellular loop-1, green; extracellular loop-2, yellow-orange; UPA, yellow; and extracellular loop-3, magenta. The intercellular loops are in gray, and the C terminus is in red. B, Predicted model of UPA in CCR5. Because cysteine residues in extracellular loop-1 and extracellular loop-2 form a disulfide bond, the extracellular loop-2 region of CCR5 has a unique arch structure consisting of 11 aa residues (yellow). In this study, UPA was selected as the target of peptide immunogen. C, To mimic the deduced conformational epitope of UPA in CCR5, the decapeptide (R168SQKEGLHYT177) derived from the UPA sequence was cyclized by inserting the spacer-armed dipeptide (Gly-Glu), and the deduced structure of cDDR5 (in cyan) was adopted as the deduced structural model of UPA in CCR5 using the MOE-Align tool.

 
Induction of cDDR5-specific Abs after immunization with cDDR5-MAP in cynomolgus macaques

We pilot tested whether the immunization of cynomolgus macaques with cDDR5-MAP induces cDDR5-specific Abs. To examine the duration and magnitude of anti-cDDR5 autoantibody induction, a cynomolgus macaque was immunized i.p. at 0 and 1 wk with cDDR5-MAP in CFA and boosted s.c. at 6 wk with cDDR5-MAP in IFA. Ab responses against cDDR5 were measured by real-time biomolecular interaction analysis using surface plasmon resonance. As shown in Fig. 2, the titer of anti-cDDR5 sera measured 4 wk after the third immunization (10 wpim) was the highest in the cynomolgus macaques (titer of ~630 response units). Furthermore, the immunization with cDDR5-MAP induced anti-cDDR5 serum production for ~15 wk after the third immunization, although the titer of anti-cDDR5 sera declined over time until 21 wpim (Fig. 2).



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  		FIGURE 2. Induction of cDDR5-specific Abs after immunization with cDDR5-MAP in cynomolgus macaques. Serum samples obtained before and after immunization with cDDR5-MAP (at 0, 1, 2, 4, 6, 8, 10, and 21 wpim) were examined to investigate whether the antisera against the moiety of cDDR5 can be raised in cynomolgus macaques using real-time biomolecular interaction analysis using surface plasmon resonance with a biotinylated cDDR5-bound BIAcore biosensor.

 
Vaccination of cynomolgus macaques with cDDR5-MAP and induction of CCR5-specific Abs

To verify whether cDDR5-MAP could induce CCR5-specific Abs with anti-HIV-1 activity in nonhuman primates, an experiment was performed using cynomolgus macaques immunized following the time schedule shown in Fig. 3A. Three cynomolgus macaques were immunized with cDDR5-MAP in CFA or IFA by i.p. or s.c. injection, as described in Fig. 2. Another three cynomolgus macaques were immunized with MAP as the control. cDDR5-specific Abs were significantly induced in cynomolgus macaque nos. 11, 13, and 16 at 8 wpim (Fig. 3B). In contrast, the immunization of cynomolgus macaque nos. 7–9 with MAP did not elicit cDDR5-specific Abs (Fig. 3B). Furthermore, the sera from cynomolgus macaque nos. 11, 13, and 16 at 8 wpim with cDDR5-MAP were also examined by flow cytometry to determine whether they recognize intact cell surface-expressed CCR5 on CEM-CCR5 cells. The sera from no. 11 (diluted 1/5 and 1/100), no. 13 (diluted 1/5 and 1/100), and no. 16 (diluted 1/100 and 1/400) macaques showed the immunofluorescence staining of CEM-CCR5 cells, compared with preimmunization sera from these macaques (Fig. 3, C–H). In contrast, the immunization of cynomolgus macaque nos. 7–9 with MAP did not induce CCR5-specific Abs (data not shown).



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  		FIGURE 3. Immunization schedule and detection of anti-CCR5 Abs in cynomolgus macaques. A, Immunization schedule for cynomolgus macaques. Three cynomolgus macaques (nos. 11, 13, and 16) were immunized i.p. at 0 and 1 wk with 300 µg of cDDX4-MAP, and boosted s.c. at 6 wk with 300 µg of cDDR5-MAP. Another three cynomolgus macaques (nos. 7, 8, and 9) were immunized with MAP as the control. Blood sampling was performed at 0, 1, 2, 4, 6, 8, 10, 12, 14, 15, 17, and 21 wpim. B, Detection of anti-cDDR5-MAP serum Abs in cynomolgus macaques. Serum samples obtained before and after immunization with cDDR5-MAP (macaque nos. 11 ({circ}), 13 ({blacksquare}), and 16 ({blacktriangleup}) or MAP (macaque nos. 7 ({circ}), 8 ({square}), and 9 ({triangleup}) were examined to investigate whether the antisera against the moiety of cDDR5 can be raised in cynomolgus macaques. The anti-cDDR5 macaque Abs from each serum sample were detected by real-time biomolecular interaction analysis using surface plasmon resonance with a biotinylated cDDR5-bound BIAcore biosensor, as shown in Fig. 2. Sensorgrams from the serum samples taken before and after immunization were obtained, and the highest response units (R.Us.) in each sample were plotted. C–H, Flow cytometry of serum Abs from cynomolgus macaques (no. 11, C and D; no. 13, E and F; no. 16, G and H) immunized with cDDR5-MAP. CEM-CCR5 cells were subjected to flow cytometry, as described in Materials and Methods, in which the cells were separately incubated with the preimmunization (dark blue histogram) and 8 wpim (red histogram) sera diluted 1/5, 1/100, or 1/400 with PBS. The dilution folds are shown as 5, 100, or 400 dil in each figure. Significant binding to a cell was determined positive when the mean fluorescence intensity ratio of 8 wpim serum to 0 wpim serum is >1.5.

 
Inhibition of HIV-1 infection

The anti-HIV-1 activities of the immune sera from cynomolgus macaque nos. 11, 13, and 16 were also determined using MAGIC-5 cells expressing CCR5. The cells were separately inoculated with two laboratory strains of clade B HIV-1 (R5 HIV-1, HIV-1JRFL; X4 HIV-1, HIV-1LAV-1) or R5 nonclade B HIV-1 primary isolates (clade A:HIV93RW004 and clade C:HIVMJ4) in the presence or absence of immune sera. As expected, 8 wpim sera from cDDR5-MAP-immunized cynomolgus macaque nos. 11, 13, and 16 markedly suppressed infection by HIV-1JRFL (R5 HIV-1) in a dose-dependent manner (Fig. 4, D–F). Furthermore, 10 wpim sera from macaque no. 16 suppressed infection by R5 nonclade B HIV-1 primary isolates (clade A:HIV93RW004 and clade C:HIVMJ4) (Fig. 4, G and H). In contrast, the immune sera did not prevent HIV-1LAV-1 (X4 HIV-1) infection as observed in the control experiment (Fig. 4, A–C).



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  		FIGURE 4. Inhibitory effects of pre- and postimmunization sera on HIV-1 R5 and X4 infections. MAGIC-5 cells were separately incubated with serially diluted 8 wpim serum from cDDR5-MAP-immunized macaques (A and D, no. 11; B and E, no. 13; C and F, no. 16). Preimmunization serum was used as control. The cells were further cocultured in a medium containing various HIV-1 strains (A–C, LAV-1; D–F, JRFL) for 48 h, as described in Materials and Methods. The infected cells, which were stained blue, were counted by a conventional method. The number of cells stained blue is expressed as percentage relative to the number of cells in the preimmunization serum (control) stained blue for each macaque. No significant cytotoxicity of the immunization serum-containing medium was observed. Values represent the mean of three determinations. G and H, MAGIC-5 cells were separately incubated with preimmunization and 10 wpim sera, which were dialyzed and diluted (1/2) with PBS, from vaccinated macaque no. 16. The cells were further cocultured in a medium containing nonclade B HIV-1 strains (HIV93RW004, HIVMJ4) for 48 h, as described in Materials and Methods.

 
Inhibition of CCR5-specific SHIVSF162P3 infection

The SHIV-1SF162P3 bulk isolate is a pathogenic CCR5-specific SHIV in rhesus macaques (25). The 8 wpim sera from macaque nos. 11, 13, and 16 were examined by infection assay using both MAGIC-5 and CEM-CCR5 cells to determine whether they inhibit SHIV-1SF162P3 bulk isolate infection. The 8 wpim sera significantly suppressed the infection of MAGIC-5 cells by the SHIV-1SF162P3 bulk isolate, compared with the preimmune sera (Fig. 5, A–C). To verify the antiviral activity of immune sera in another experiment, the inhibitory effect of immune sera from cynomolgus macaque nos. 11, 13, and 16 on SHIVSF162P3 replication in CEM-CCR5 cells was investigated. CEM-CCR5 cells, a CCR5-transfected human T cell line, were acutely infected with SHIVSF162P3 in the presence of preimmunization and 8 wpim sera from cynomolgus macaque nos. 11, 13, and 16, and the spread of infection was then monitored on the basis of the accumulation of p27 Ag in culture supernatants. The 8 wpim sera from macaque nos. 11, 13, and 16 effectively suppressed SHIVSF162P3 propagation (Fig. 5, D–F).



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  		FIGURE 5. Inhibitory effects of pre- and postimmunization sera on CCR5-specific SHIVSF162P3 infection. Upper and bottom figures, Show the results of MAGIC-5 assay (A, no. 11; B, no. 13; C, no. 16) and productive infection assay (D, no. 11; E, no. 13; F, no. 16), respectively. In MAGIC-5 assay, MAGIC-5 cells were separately incubated with preimmunization and 8 wpim sera, which were dialyzed and diluted (1/10) with PBS, and cocultured in the SHIVSF162P3-containing medium for 48 h, as described in Materials and Methods. The infected cells, which were stained blue, were counted. {blacksquare} and {cjs2113}, Represent the results from preimmunization and 8 wpim sera, respectively. No significant cytotoxicity of the immunization serum-containing medium was observed. Diluted virus stocks that infected 100–300 cells, as identified by their blue staining, were used. The number of cells stained blue was expressed as percentage relative to the number of cells in the preimmunization sera (control) stained blue. All data represented means obtained from three separate experiments. Statistically significant differences compared with preimmunization serum are indicated by asterisks (*, p < 0.05; **, p < 0.01). In the productive infection assay, CEM-CCR5 cells (5 x 105) were infected with the SHIVSF162P3-containing medium in the presence of preimmunization and 8 wpim sera for 18 h. The cells were washed with PBS, plated onto 24-well plates, and then cultured in the RPMI 1640 medium containing 10% FBS. The p27 Ag in culture supernatants obtained at 24, 48, 72, 96, and 120 h postinfection was detected, as described in Materials and Methods. Cynomolgus macaque no. 11, Pre (•) and 8 wk ({circ}); no. 13, Pre ({blacktriangleup}) and 8 wk ({triangleup}); no. 16, Pre ({blacksquare}) and 8 wk ({square}).

 
Ability of macaque anti-cDDR5 sera to bind to macaque CCR5

The UPA sequence in human and macaque CCR5s differs in 1 aa (Lys171Arg). Anti-human CCR5 Ab, 2D7, binds to the epitope that includes Lys171, and this binding ability was prevented by the sequence substitution in CCR5 (Lys171Arg). To rule out whether anti-cDDR5 serum cross-reacts with native CCR5 as expressed on macaque lymphocytes, we examined the ability of anti-cDDR5 sera to block the binding of a commercially available CCR5-specific mAb (3A9). The 3A9 can bind to macaque CCR5. Cynomolgus macaque PBMC or the lymphocytic cell line HSC-F (23) was incubated with the anti-CD95 Ab with either the preimmunization or 10 wpim serum from cynomolgus macaque no. 16 and then reacted with a limiting amount of PE-labeled 3A9 according to the protocol of Chackerian et al. (13). The ability of anti-cDDR5 serum to block 3A9-PE binding was assessed by flow cytometry, gating lymphocytes, and examining the CD95+ population. The incubation of PBMC or HSC-F with 10 wpim serum reduced the percentage of CD95+ lymphocytes that were 3A9 positive (Fig. 6, D and H). In addition, the mean fluorescence index of cells that remained 3A9 positive was lower than that of cells that were preincubated with preimmunization serum. The 10 wpim serum did not decrease the binding of a control mAb (anti-CD95-FITC), suggesting that the observed inhibition of 3A9 binding was specific. In addition, the incubation of PBMC or HSC-F with preimmunization serum from macaque no. 16 had no effect on 3A9-PE binding (Fig. 6, C and G). Taken together, these results support the conclusion that anti-cDDR5 serum cross-reacts with native CCR5 as expressed on macaque lymphocytes.



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  		FIGURE 6. Inhibition of binding of a mAb against CCR5 (3A9) to cynomolgus macaque PBMC or HSC-F by anti-cDDR5 serum. Macaque PBMC (A–D) or HSC-F (E–H) was incubated with FITC-labeled IgG1 and PE-labeled IgG2 (A and E), FITC-labeled anti-CD95 and PE-labeled anti-human CCR5 mAb (3A9) (B and F), and FITC-labeled anti-CD95 and PE-labeled anti-human CCR5 mAb (3A9) after preincubation with preimmunization (C and G) or 10 wpim sera (D and H). Staining was assessed by flow cytometry, and fluorescence was measured after gating the lymphocyte population. Inhibition of 3A9 binding was determined by two methods: first, by quantitating the percentage of CD95+ cells that were also 3A9-PE positive; second, by determining the mean fluorescence index (MFI) of CD95+ cells that were bound by 3A9. These values are displayed in the quadrant.

 
Macaque challenge with SHIVSF162P3

Five weeks after the final boost (11 wpim), all of the MAP- and cDDR5-MAP-immunized macaques were i.v. challenged with 1 ml of 10 TCID50/ml SHIVSF162P3, which is a chimeric virus that contains the env, tat, and rev genes from HIVSF162, an R5 virus, in the background of SIVmac239. The course of acute viral infection was monitored by measuring plasma viral RNA load and CD4+ T cell number in acutely infected macaques (Fig. 7). All of the three control macaques developed detectable plasma viremia, as demonstrated by viral peaks between 1 x 109 and 3 x 109 viral RNA copies/ml plasma with a moderate decrease in peripheral CD4+ T cell number (%), as previously described (25), and sustained plasma viremia >108 viral RNA copies/ml plasma for 3 wk (1–4 wk postchallenge). The actual percentage of surviving peripheral CD4+ T cells inversely correlated with viral load (Fig. 7). Furthermore, we compared the geometric mean plasma viral RNA loads of the vaccinated and control groups to monitor the effectiveness of vaccination. The difference between viral loads of the two groups was statistically significant (p = 0.03) at one time point (week 1) by 4 wk after infection (Table I), and there were differences of ~19.95- to 217.10-fold in the geometric mean viral loads of the two groups between 1 and 4 wk postchallenge. The vaccinated macaques had a lower ~3-wk delayed peak viremia than the controls, so that the vaccinated macaques sustained plasma viremia between 104 and 3 x 106 viral RNA copies/ml plasma at 6 and 10 wk postchallenge in contrast to control macaques (between 103 and 105 viral RNA copies/ml plasma). Furthermore, we investigated whether the high in vitro anti-HIV and in vitro anti-SHIV activities are associated with low viral loads during the acute phase of SHIV infection. The in vitro anti-HIV and in vitro anti-SHIV activities before challenge were compared with the peak plasma viral RNA load at 1 wk postchallenge (Table II). Macaque no. 16 with the highest anti-HIV and anti-SHIV activities had the lowest viral load among vaccinated macaques at 1 wk postchallenge (2.0 x 106 viral RNA copies/ml plasma), suggesting that the higher in vitro anti-HIV and in vitro anti-SHIV activities in vaccinated macaques were responsible for the low viral loads (Table II). Taken together, these results suggest that viral loads in vaccinated macaques following a challenge with SHIVSF162P3 are controlled for a longer time if the anti-CCR5 Ab continues to be strongly induced by vaccination for a longer time.



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  		FIGURE 7. Intravenous challenge with SHIVSF162P3. Whole-blood samples were collected at –1, 1, 3, 4, 6, and 10 wk postchallenge, and the samples were examined for plasma viremia ({circ}) and peripheral CD4+ T cell count (%) (•). Viral RNA was extracted from macaque plasma and then reverse transcribed. The resulting cDNA duplicates were amplified by quantitative real-time PCR, as described in Materials and Methods.

 

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  		Table I. Statistical analysis of geometric mean viral loads

 

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  		Table II. Relationship among anti-cDDR5 Ab response, antiviral activity, and viral load in cynomolgus macaques

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
CCR5 is considered important in HIV-1 transmission on the basis of the findings that individuals homozygous for a 32-bp deletion in the CCR5-coding region have a very low susceptibility to HIV-1 infection (15, 16, 17, 18), and CCR5-reactive Abs in ESN subjects down-modulate surface CCR5 in vivo and neutralize the infectivity of R5 strains (21). Therefore, the induction of CCR5-specific autoantibody-based immunity specific to the infection by R5 HIV-1 from various HIV-1 clades may be a strategy for the development of a prophylactic HIV-1 vaccine.

Some vaccination strategies of inducing CCR5-specific autoantibodies have been reported. One of our previous attempts was to induce CCR5-specific autoantibodies with anti-R5 HIV-1 activity by the inoculation of cDDR5-MAP from the UPA (from Arg168 to Cys178) of extracellular loop-2 in CCR5 into BALB/c mice (10). Other attempts include the induction of CCR5-specific autoantibodies with anti-R5 HIV-1 activity by the inoculation of recombinant papillomavirus-like particles, which represent an extracellular loop of CCR5, into C57BL/6 mice and pig-tail macaques (12, 13), by the genetic immunization of cynomolgus macaques with the DNA of CCR5 (14), and by the immunization of rhesus macaques with synthetic linear peptides (N-terminal peptide 1–20, first-loop peptide 89–102, and second loop peptide 178–197) derived from the N terminus, first loop, and second loop in CCR5 (11). Results of these previous studies indicate that vaccines aimed at inducing CCR5-specific autoantibodies can be developed, as well as conventional viral protein-based vaccines.

In our present study, a cyclopeptide immunogen strategy was used to induce CCR5-specific autoantibodies in cynomolgus macaques. The advantages of a cyclopeptide immunogen are as follows: 1) it can induce Abs against a very restricted region that includes a biologically active conformational epitope; 2) its immunogenicity can be controlled by polymerization or conjugation with small carrier molecules such as MAP; and 3) its chemical purity can be exactly defined (29, 30). In particular, the conformational stability of a peptide immunogen in vivo is a key factor for generating Abs against a native protein. In general, an Ab induced by a linear peptide can recognize a denatured protein, but not a native protein. In contrast, an Ab that recognizes the conformational epitope of an Ag cannot recognize a denatured protein. Indeed, the commercially available anti-CXCR4 Ab 12G5 can recognize the conformational epitope of cell surface CXCR4, but cannot detect denatured CXCR4. Therefore, cDDR5-MAP was designed on the basis of the deduced conformation of UPA (Arg168 to Cys178) in CCR5 to induce Abs that can recognize native CCR5. In addition, the reason for selecting UPA as the target in our study is that the conformational heterogeneity of UPA is unlikely to arise, except for other extracellular domains because cysteine residues (Cys101 and Cys178) in extracellular loop-1 and extracellular loop-2 form a rigid disulfide bond. Consequently, cDDR5 was prepared by the cyclization of the decapeptide (R168SQKEGLHYT177) derived from the UPA sequence by the insertion of the spacer-armed dipeptide (Gly-Glu). cDDR5 can induce CCR5-specific autoantibodies capable of significantly inhibiting infection by the R5 laboratory-adapted strain (HIV-1JRFL), R5 HIV-1 primary isolates (clade A:HIV93RW004 and clade C:HIVMJ4), and SHIVSF162P3 bulk isolates (Figs. 4 and 5). These results suggest that UPA in CCR5 is a good target for preventing CCR5-dependent viral infection. Thompson et al. (31) found that in contrast to clade B isolates, a cluster of residues in the second extracellular loop of CCR5 significantly affects the fusion and entry of all the nonclade B isolates tested. Therefore, cDDR5-MAP derived from UPA may be a more useful immunogen than that derived from other domains of CCR5, except UPA as a HIV-1 coreceptor-based vaccine candidate for use in worldwide AIDS epidemics.

To assess vaccine efficiency, we infected the control and cDDR5-MAP-immunized macaques with an R5-tropic SHIVSF162P3. All of the three control macaques developed detectable plasma viremia, as demonstrated by viral peaks between 1 x 109 and 3 x 109 viral RNA copies/ml plasma with a moderate decrease in peripheral CD4+ T cell number (%) and sustained plasma viremia >108 viral RNA copies/ml plasma for 3 wk (1–4 wk postchallenge). Reyes et al. (32) have recently demonstrated that i.v. SHIVSF162P3 inoculation moderately decreases the absolute number of peripheral blood CD4+ T cells at 2–3 wk postinfection, as we have observed. In contrast, all of the vaccinated macaques also developed detectable plasma viremia at 1 wk postchallenge, and the plasma viremia levels in the three vaccinated macaques peaked ~3 wk later than those in the control macaques (Fig. 7), so that the vaccinated macaques sustained plasma viremia levels between 104 and 3 x 106 viral RNA copies/ml plasma at 6 and 10 wk postchallenge in contrast to the control macaques (between 103 and 105 viral RNA copies/ml plasma). However, Table I shows there are differences of ~19.95- to 217.10-fold in the geometric mean viral loads of the two groups between 1 and 4 wk postchallenge, and at one time point (week 1), the difference (217.10-fold) was statistically significant (p = 0.03). Furthermore, Table II suggests that the higher in vitro anti-HIV and in vitro anti-SHIV activities of anti-cDDR5 sera are responsible for the low viral loads. These results suggest that the high induction of the anti-CCR5 Ab can suppress viral propagation during acute HIV-1 transmission, but only high induction of the anti-CCR5 Ab is not sufficient to clear detectable plasma-associated viruses because the anti-CCR5 Ab does not directly neutralize SHIVSF162P3. It seems difficult to completely eliminate or inhibit HIV-1 acute infection in vivo even if only the anti-CCR5 Ab delays viral propagation during the initial HIV-1 transmission. Lopalco et al. (33) have recently reported that anti-virus Abs such as IgA to gp41 are simultaneously induced with IgG to CCR5 and IgG to CD4 in some Italian ESN subjects. These humoral immune responses contribute to an extremely low level of viral replication below the detection limit of a standard assay in ESN subjects. Moreover, there is a possibility that more than one type of immunity such as anti-CCR5 and anti-HIV humoral responses must be induced by a vaccine if that vaccine is to be effective for HIV.

In conclusion, immunization with cDDR5-MAP induces CCR5-specific Abs in cynomolgus macaques and decreases viral load at peak viremia. Our results suggest that the CCR5-based cycloimmunogen strategy using cDDR5-MAP induces CCR5-specific autoantibodies capable of inhibiting R5 HIV-1 infection. With the basic knowledge of the induction of CCR5-specific Abs using cDDR5-MAP, we are currently developing cDDR5-MAP conjugated to the HIV env protein to reconstruct the immune response in ESN subjects.


   Acknowledgments
 
We thank Drs. Y. Maeda and S. Harada (Kumamoto University) for providing the CEM-CCR5 cell lines. We also thank Dr. M. Tatsumi (National Institute of Infectious Diseases, Tokyo, Japan) for providing the MAGIC-5 cells. BIAcore analysis was supported by the Center for AIDS Research, Kumamoto University. HIV93RW004, HIVMJ4, and SHIVSF162P3 were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health. We thank K. Tokunaga for excellent technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and a Health Science Research Grant from the Ministry of Health, Labour, and Welfare of Japan. Back

2 S.M. and D.N. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Shozo Shoji, Kumamoto University, Department of Pharmaceutical Biochemistry, Faculty of Medical and Pharmaceutical Sciences, 5-1 Oe-Honmachi, Kumamoto 862-0973, Japan. E-mail address: shoji{at}gpo.kumamoto-u.ac.jp Back

4 Abbreviations used in this paper: ESN, exposed seronegative; cDDR5, cyclic closed-chain dodecapeptide; MOE, molecular operating environment; SHIV, simian/HIV; TCID, tissue culture infective dose; UPA, undecapeptidyl arch; wpim, weeks postinitial immunization. Back

Received for publication March 10, 2005. Accepted for publication October 17, 2005.


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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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