Induction of Immune Responses and Break of Tolerance by DNA against the HIV-1 Coreceptor CCR5 but No Protection from SIVsm Challenge

Virology 278, 400–411 (2000) doi:10.1006/viro.2000.0633, available online at http://www.idealibrary.com on

Induction of Immune Responses and Break of Tolerance by DNA against the HIV-1 Coreceptor CCR5 but No Protection from SIVsm Challenge Bartek Zuber,* ,† ,‡ ,1 Jorma Hinkula,* Dalma Vo¨dro¨s,† Peter Lundholm,* Charlotta Nilsson,* Andreas Mo¨rner,† Mikael Levi,* Reinhold Benthin,* and Britta Wahren* ,† *Swedish Institute for Infectious Disease Control, Solna, Sweden; †Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden; and ‡Medivir AB, Lunastigen 7, S-141 44 Huddinge, Sweden Received June 6, 2000; returned to author for revision July 24, 2000; accepted September 2, 2000; published online November 16, 2000 An inactivating mutation in the human CCR5 gene reduces the risk of HIV-1 infection in individuals with homozygous alleles. We explored whether genetic immunization would induce an immune response directed to CCR5 structures and if immunological tolerance toward endogenous CCR5 could be broken. We also studied whether this immunization approach could protect cynomolgus monkeys from an infection, with SIVsm, which primarily uses CCR5 as a coreceptor. Epidermal but not intramuscular delivery of the CCR5 gene to mice elicited strong IgG antibody binding responses to CCR5. Intramucosal immunization of cynomolgus macaques with CCR5 DNA followed by boosts with CCR5 peptides induced prominent IgG and IgA antibody responses in serum and vaginal washings. The CCR5-specific antibodies neutralized the infectivity of primary human R5 HIV-1 strains, and the macaque SIVsm but not that of a tissue culture-adapted X4 HIV-1 strain. The consecutive CCR5 gene and CCR5 peptide immunizations induced B- and T-cell responses to peptides representing both human and macaque amino acid sequences of the respective CCR5 proteins. This indicates that tolerance was broken against endogenous macaque CCR5, which has a 98% homology to the human CCR5 gene. After the final boost, the vaccinated monkeys together with two control monkeys were challenged with SIVsm. Neither protection against nor enhancement of SIVsm infection was achieved. © 2000 Academic Press

INTRODUCTION

on the cell surface, but this deletion has no obvious effect on the immunocompetence of the affected individuals. Persons homozygous for the 32-bp deletion in CCR5 are protected against primary HIV-1 infection (Huang et al., 1996), and individuals heterozygous for the deletion show a slow early course of infection (Bratt et al., 1997). These findings indicate that blockage of the CCR5 receptor by drugs or antibodies may confer in vivo protection against primary HIV-1 infection. The natural CCR5 binding chemokines RANTES, MIP-1 alpha, and MIP-1 beta are major HIV-1-suppressive factors (Cocchi et al., 1995). Chemokine derivatives like (AOP)-RANTES (Simmons et al., 1997) have been shown to block HIV-1 infection of cells in vitro. RANTES also contributes to protection from SIV infection in vivo (Ahmed et al., 1999). Monoclonal antibodies directed toward the CCR5 receptor can efficiently block the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro (Olson et al., 1999; Wu et al., 1997a,b). Similarly, inhibition of a macrophage-tropic HIV-1-derived envelope glycoprotein gp120 binding to CCR5 could be achieved with monoclonal antibodies recognizing either the second extracellular loop or the N-terminal region of the CCR5 protein (Wu et al., 1997a). The CCR5 receptor has also been reported to act as an alloantigen in individuals homozygous for the ⌬32 CCR5 deletion; serum from these individuals inhibited infection of peripheral

Human and simian immunodeficiency viruses (HIV-1 and SIV) use the CD4 receptor to enter their target cells. In addition, they use a number of different Gprotein-coupled chemokine receptors. HIV-1 mainly uses the coreceptors CCR5 and CXCR4. All HIV-1 strains described to date use one or both of these receptors. CCR5 is used by the macrophage-tropic (R5) HIV-1 isolates and CXCR4 serves as a fusion cofactor for entry of T-cell tropic (X4) HIV-1 isolates. Infection of macaques with SIV provides one of the best models for HIV pathogenesis in humans. In addition to CCR5, SIV has been shown to use the orphan receptors BOB/gpr15 and Bonzo/STRL33 (Deng et al., 1997; Edinger et al., 1998; Farzan et al., 1997). Recently it was demonstrated that some SIV isolates also utilize CXCR4 (Owen et al., 2000). CCR5 is the coreceptor preferentially used by HIV-1 in primary infection of human macrophages and lymphocytes (Deng et al., 1996). A 32-bp deletion in the CCR5 gene prevents the CCR5 protein from being expressed

1 To whom reprint requests should be addressed at Swedish Institute for Infectious Disease Control, Virology Department, 171 82 Solna, Sweden. Phone: ⫹ 46-8-457 26 95. Fax: ⫹ 46-8-33 72 72. E-mail: [email protected]

0042-6822/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

400

IMMUNE RESPONSES AGAINST THE HIV-1 CORECEPTOR CCR5

blood lymphocytes by R5 viruses using CCR5 but not X4 viruses using the CXCR4 receptor (Ditzel et al., 1998). Genetic immunization confers the advantage of presenting antigens in a conformationally native way and induces effective humoral and cellular immune responses against a variety of immunogens (Robinson and Torres, 1997; Wahren and Brytting, 1997). DNA-based immunization has been shown to induce anti-CD4 antibodies directed primarily to native epitopes (Attanasio et al., 1997). Furthermore, rhesus macaques immunized with recombinant soluble CD4 have been shown to develop anti-self CD4 antibody responses with anti HIV-1 activity (Watanabe et al., 1991), indicating that immunization with an HIV-1 receptor may be a feasible way to create an HIV-1 immune defense. Recently it was shown that a monoclonal antibody directed against the CD4 receptor complex had broad neutralizing activity against HIV-1 and provided postexposure prophylaxis to chimpanzees (Wang et al., 1999). The aim of this study was to investigate the use of the CCR5 gene as an immunogen. We wanted to induce systemic and local IgG and IgA antibody responses toward parts of the CCR5 protein that are important for inhibition of infection. We have recently shown that a DNA-based jet immunization induces both systemic and local IgG and IgA antibodies (Lundholm et al., 1999). Here we describe genetic immunization of cynomolgus macaques with the human CCR5 gene together with human GM-CSF protein. We show that DNA immunization with the human CCR5 gene induces antibodies that are reactive both with the human and macaque CCR5 and block HIV-1 infection in vitro but not in vivo. RESULTS Expression of CCR5 from pcDNA3-CCR5 and pcDNA3-tet.CCR5 Expression and localization of the CCR5 and tet.CCR5 proteins were analyzed in transfected CHO cells (Fig. 1). A strong membrane fluorescence was observed in the transfected cells, which confirms the protein expression from both plasmids. CCR5-specific antibody and cellular immune responses in mice To induce strong CCR5-specific antibody responses, we decided to try several adjuvants and immunization routes known to stimulate antibody production. The CCR5 gene alone or fused to a promiscuous T-helper cell epitope from the tetanus toxin (Chin et al., 1994; Kumar et al., 1992; Panina-Bordignon et al., 1989) was administered together with murine GM-CSF or IL-4 genes. After the third immunization, immune responses to the Nterminus of CCR5 (Peptide N1, Table 1) were strong in both groups of gene gun immunized mice (Groups G and

401

FIG. 1. Transient expression of CCR5 (A) and tet.CCR5 (B). Indirect immunofluorescence was performed on CHO cells transfected with the pcDNA3-CCR5 or pcDNA3-tet.CCR5 constructs. CHO cells constitutively expressing the human CCR5 protein (CHO-CCR5) were used as a positive control (C).

H, Fig. 2). Moderately strong responses were measured toward the second extracellular loop of CCR5 (peptide X2.1, Table 1) in both groups of gene gun immunized mice. Intramuscular immunization (Groups A–F) did not induce any significant CCR5-specific antibody responses even after three immunizations with 100 ␮g of tet.CCR5 DNA and GM-CSF (represented by Group D, Fig. 2). A similar response pattern was seen when induction of T-cell responses against regions of CCR5 were measured (Table 2). Epidermal immunization by gene gun induced moderate but significant T-cell proliferative responses together with pCI-GM-CSF against the N-terminus, first, second, and third extracellular loops of the CCR5 protein (group G, Table 2). When the IL-4 plasmid was added, response was measurable to the N-terminus and first extracellular loop of CCR5 (Group H, Table 2). Intramuscular immunization did not induce positive T-cell proliferative responses, regardless of whether the pcDNA-CCR5 plasmid was coinjected with the plasmid pCI-GM-CSF (Group D, Table 2) or with pCI-IL-4 (data not shown). Neutralization capacity of sera from CCR5 immunized mice Pooled sera from mice in Groups G, H, and D were tested in a neutralization assay. Surprisingly, sera from these groups did not show any neutralizing effect (data not shown), despite the high CCR5-specific antibody binding titers in Groups G and H. CCR5-specific antibody and cellular immune responses in cynomolgus macaques We reasoned that a strong local CCR5-specific IgG and IgA mucosal response is optimal for the prevention of primary HIV-1 infection at mucosal sites. Furthermore, we wanted to break tolerance toward the endogenous ma-

402

ZUBER ET AL. TABLE 1 Aminoacids of Peptides Used in the Study CCR5 position N-terminus human CCR5 N-terminus cynomolgus macaque CCR5 N-terminus human CCR5 N-terminus human CCR5 1st extracellular loop human CCR5 2nd extracellular loop human CCR5 2nd extracellular loop cynomolgus macaque CCR5 2nd extracellular loop human CCR5 3rd extracellular loop human CCR5 Control peptides

a b

CCR5 peptides a

Aminoacid sequence b

N1 N1c N2 N1/N2 X1 X2.1 X2.1c X2.2 X3

MDYQVSSPIYDINYYTSEPC MDYQVSSPTYDIDYYTSEPC DINYYTSEPCQKINVKQIAA MDYQVSSPIYDINYYTSEPCQKINVKQIAA HYLAAQWDFGNTMC FTRSQKEGLHYTCSSHFPYS FTRSQREGLHYTCSSHFPYS YTCSSHFPYSQYQFWKNFQT QEFFGLNNCSSSNRLDQAM

CXCR4 N CXCR4 I1 Rev 3 Rev 5

MEGISIYTSDNYTEEMGSGD LVMGYQKKLRSMTDK PEGTRQARRNRRRRWRERQR STYLGRSAEPVPLQLPPLER

Peptides used for boosting are shown in bold and underlined letters. The letter c indicates macaque peptides. Amino acids that differ between the human and cynomolgus macaque CCR5 sequences are shown in bold letters.

caque CCR5 receptor. Mucosal and systemic CCR5-specific antibody responses were therefore studied. Two cynomolgus macaques were immunized in the rectal mucosa with pcDNA3-CCR5 together with recombinant human GMCSF protein, then boosted twice with the same combination. The second boost contained pcDNA3-tet.CCR5. To enhance CCR5-specific antibody responses, the macaques were further boosted twice with peptides representing the N-terminus, the first and the second extracellular loops of the human CCR5 protein.

were analyzed. After the first DNA boost, both immunized animals developed significant IgG titers toward the peptides N1, N1c, X2.1, and X2.1c (Figs. 3 and 4A). The response toward N1 decreased 4 weeks after the DNA immunization, while responses toward N1c, X2.1, and X2.1c persisted (Fig. 3). Following the first peptide boost, both animals reacted toward all four tested peptides. By the second peptide boost, the titers increased by one log against the X2.1 and X2.1c peptides and by several logs against the N-terminal peptides (Fig. 3).

IgG reactivity to human and macaque CCR5 sequences

In vitro IgG synthesis, IgA, and neutralization responses

Antibody responses toward the human and the macaque CCR5 proteins were measured using peptides and by neutralization of HIV-1 (Figs. 3 and 4). Antibody responses directed to the human and macaque CCR5 peptides (N1, X2.1, N1c, and X2.1c) (Table 1)

The in vitro IgG synthesis from antigen-stimulated peripheral lymphocytes was measured after the last immuTABLE 2 T-Cell Proliferation Mapped in Vitro with Peptides of CCR5: Frequency of Responding Mice (SI Range) a Group H Group G

CCR5 peptides

FIG. 2. Time course of antibody response against the human CCR5 protein in three groups of immunized BALB/c mice (n ⫽ 5). Stars indicate immunization events. Sera were diluted 1/50, the response against the N-terminal part of CCR5 (aa 1–20) represented by a linear peptide (N1, Table 1) was measured by ELISA.

N-terminus 1 1st extracellular loop 2 2nd extracellular loop 3 3rd extracellular loop 4 Concanavalin-A a

Group D

pcDNA3pcDNA3tet.CCR5 ⫹ tet.CCR5 ⫹ pCI-GM-CSF pCI-GM-CSF ⫹ pCI-IL-4 Gene gun Gene gun

pcDNA3tet.CCR5 ⫹ pCI-GM-CSF intramuscularly

2/5 (2.6–4.0) 2/5 (2.8–3.7)

1/5 (3.5) 2/5 (2.5)

0/5 0/5

2/5 (2.2–4.4) 1/5 (2.7) 5/5 (310–13)

0/5 0/5 5/5 (55–10)

0/5 0/5 5/5 (922–21)

A more than twofold SI was considered positive. Pooled results from stimulations with peptides N1, N2, and N1/N2, 2 peptide X1, 3 peptides X2.1 and X2.2, and 4 peptide X3. 1

IMMUNE RESPONSES AGAINST THE HIV-1 CORECEPTOR CCR5

403

creased to 30 for B189 and to 600 for U9. IgA in vaginal washings from both macaques was measurable after three DNA immunizations and two peptide boosts (Fig. 4C). In conclusion, immunization of macaques with the human CCR5 gene induced new antibody responses to both the human CCR5 protein and the endogenous macaque CCR5 protein. Neutralization capacity of sera from CCR5-immunized monkeys

FIG. 3. Time course of serum antibody response directed toward the N-terminus or second extracellular loop of human and macaque CCR5. Arrows indicate immunization events. Antibody titers toward peptides (Table 1) were determined using peptide ELISA, with a cutoff of 4 times the reactivity of proimmunization serum.

nization (Fig. 4D). The antibody responses could be mapped to peptides representing the whole CCR5 protein. Both macaques responded strongly against the Nterminal peptide and the first- and second-extracellular loop peptides of human CCR5. The strongest response was mapped to the first part of the Nalterminus (aa 1–20). Two weeks after the initial injection of DNA, both animals had significant IgG and IgA titers toward peptide N1 in serum (Figs. 4A and 4B). Ten days after the first DNA boost, IgG and IgA responses against N1 increased in both monkeys. IgA titers against N1decreased below cutoff levels after the second DNA boost for Monkey U9, but persisted in Monkey B189. IgG titers in both animals decreased after the second DNA boost. To increase the CCR5 binding titers, we boosted the monkeys 13 weeks after the second DNA boost with peptides representing external parts of CCR5. Following both peptide boosts, the IgG and IgA titers toward all tested peptides increased substantially for both macaques (Fig. 4). After the second peptide boost, Monkeys U9 and B189 had IgG binding titers against N1 of 130,000 and 18,000, respectively. The IgA titers against N1 in-

The capacity of serum to block HIV-1 infection of human peripheral blood lymphocytes (PBL) was analyzed by using two primary R5 HIV-1 strains (J2234 and J562) derived from asymptomatic HIV-1 patients and the laboratory-adapted but PBL-passaged X4 Strain III B. The neutralizing abilities of monkey serum before immunization, after two DNA immunizations, and after peptide boosts were compared (Figs. 4A and 4B and Fig. 5). No neutralization was seen before immunization. Sera from both monkeys neutralized isolate J2234 effectively after two DNA immunizations (Fig. 5A); over 90% neutralization of 81 ID50 of J2234 was achieved with serum from Monkey U9 at this time point (Fig. 5A). U9 serum after two DNA immunizations also neutralized the R5 isolate J562 (Fig. 5B) but failed to neutralize the X4 strain HIV-1 III B (Fig. 5D). Isolate J562 was not neutralized by serum from Monkey B189. Neutralizing responses were enhanced by the first peptide boost in both monkeys (Fig. 4A). No enhancement was seen after the second peptide boost. IgG was purified from serum after two DNA immunizations of Macaque U9. Serum and purified IgG neutralized HIV-1, while the IgG-depleted serum had no effect (Fig. 5C). This clearly shows that IgG was of major importance for neutralization. The capacity of serum from immunized monkeys to block SIVsm (the virus used for challenge) infection of cynomolgus macaque PBL was analyzed. No neutralization was seen before immunization. After two DNA immunizations, over 90% neutralization was achieved with sera from both immunized monkeys (U9 and B189, Fig. 5E). T-cell proliferative responses The T-cell proliferative responses were mapped against human CCR5 peptides and against membrane lysates of CHO cells expressing human CCR5 at the surface. Macaque B189 mounted a broad and strong response already after the first immunization (Fig. 6A). This was retained after the second immunization. At Day 82 of follow-up, the response narrowed to a moderately strong response directed toward the N-terminus of CCR5. A specific response of Macaque U9 was not identified until late after the second immunization. At Day 82, we identified a strong and narrow CCR5-specific response directed against the N-terminal peptide of the

404

ZUBER ET AL.

FIG. 4. Time course of neutralization (NT) responses against the R5 HIV-1 isolate J2234 and serum antibody directed toward the N-terminus (peptide N1, Table 1) of CCR5. Arrows indicate immunization events. Neutralization is presented as the dilution at which 50% NT was achieved. Neutralization responses shown together with serum IgG toward the CCR5 N-terminus (A). Neutralization responses shown together with serum IgA toward the CCR5 N-terminus (B). IgA directed toward the N-terminus of CCR5 in vaginal washings after the first DNA immunization (Day 13) and after the second peptide boost (Day 260) (C). In vitro IgG synthesis by B cells after the last immunization (D). The response was mapped to different parts of CCR5 using peptides (Table 1). D79 is a nonimmunized control monkey.

CCR5 protein and very similar to the late response of Macaque B189. The T-cell proliferative responses against all peptides used for immunization were enhanced in both monkeys after the second peptide boost (Fig. 6B). Challenge of immunized macaques One month after the last peptide boost, the vaccinees together with two naive control monkeys (D69 and 73–7)

were challenged intrarectally with 10 MID 50 of SIVsm (Quesada-Rolander et al., 1996). This SIVsm virus stock uses CCR5 with high efficiency. BOB and Bonzo are also used but with less efficiency (D. Vo¨dro¨s, R. Thorstensson, G. Biberfeld, D. Schols, E. De Clerq, E. M. Fenyo¨, unpublished data). The control monkeys as well as the two vaccinees became infected as determined by virus isolation, DNA PCR, and anamnestic antibody response (data not shown). No enhancement of viral replication

FIG. 5. Neutralization of two primary R5 HIV-1 isolates grown on human PBL: J2234 (A) and J562 (B) and one X4 isolate: III B (D) was compared. The neutralization effect of whole serum was compared to the purified IgG fraction and to the same serum depleted of IgG (C). Mab 2D7 which recognizes the second extracellular loop of CCR5 and RANTES was used as a positive control (C). Neutralization of the challenge virus SIVsm grown on cynomolgus macaque PBL was also performed (E).

IMMUNE RESPONSES AGAINST THE HIV-1 CORECEPTOR CCR5

405

406

ZUBER ET AL.

FIG. 6. Kinetics of T-cell proliferation in vitro with PBL from vaccinated monkeys. Arrows indicate immunization events. Cells were stimulated with peptides (Table 1) representing different parts of the CCR5 protein; control peptides CXCR4 N and CXCR4 I1 derived from the HIV-1 coreceptor CXCR4; Rev 3 is a part of the HIV-1 Rev protein (A). Comparison of T-cell proliferations at Day 260 after the last peptide boost. Monkeys U9, B189, and D82 (nonimmunized control monkey) are shown (B). CHO–CCR5 ⫽ membrane lysates from CHO cells which have been transfected with the human CCR5 gene and express CCR5 on the surface; CHO ⫽ membrane lysates from CHO cells. The stimulation index (SI) was calculated by dividing the mean counts/min in triplicate antigen-stimulated wells by the mean counts/min in three medium control wells. In some cases the SI becomes low because of high background in the medium wells. If the net CPM (CPM antigen ⫺ CPM medium) is calculated, the PHA values become quite similar. net CPM for PHA is 21416 (U9), 46259 (B189), and 47357 (D82).

was seen in the immunized compared to control macaques. MATERIALS AND METHODS Plasmids, reagents, and virus isolates CCR5 gene constructs. pcDNA3-CCR5 was obtained from M. Samson, University of Brussels. To construct pcDNA3-tet.CCR5, pcDNA3-CCR5 served as a template for PCR using a upper 89-bp primer containing a HindIII cloning site and 45 bases encoding a 15 amino acid epitope (QYIKANSKFIGITEL) from the tetanus toxin. Cloning sites are underlined; the tetanus epitope is shown in bold letters: 5⬘-CCCAAGCTTCATGCAGTACATCAAGGCCAACTCCAAGTTCATCGGCATCACCGAGCTGAAGAAGCTGGAGATTATCAAGTGTCAAGTCC-3⬘ and the lower 31-bp primer low5tet containing a XbaI cloning site, 5⬘-GATTCTAGAATGAGTCCGTGTCACAAGCCCA-3⬘. A human codon usage table based on 5,251,149 codons was used to change the bacterial gene sequence of the

tetanus epitope into triplets favored by eukaryotes. The PCR product was cloned into pcDNA3 as a HindIII–XbaI fragment. The new plasmid pcDNA3-tet.CCR5 contained the tetanus epitope upstream of the CCR5 N-terminal encoding region. E. coli DH5␣ was then transformed with pcDNA3-tet.CCR5. After bacterial lysis, plasmid DNA was prepared using Plasmid MegaPrep kit (Qiagen, Hilden, Germany). The tet.CCR5 genes were sequenced using ABI 310 (Perkin–Elmer). Reagents. The plasmids expressing murine GM-CSF (pCI-GM-CSF) and murine IL-4 (pCI-IL-4) were kindly provided by Cecilia Svanholm (Svanholm et al., 1997). Recombinant human GM-CSF was Leucomax (ScheringPlough, Sandoz), kindly provided by Håkan Mellstedt. The CCR5-specific mouse Mab 2D7 was purchased from Pharmingen (San Diego, CA). Recombinant human RANTES was purchased from R&D Systems (Minneapolis, MN). Virus isolates. The primary R5 HIV-1 isolates J2234 and J562 obtained from asymptomatic patients (Jansson et

IMMUNE RESPONSES AGAINST THE HIV-1 CORECEPTOR CCR5

al., 1996) were kindly provided by Marianne Jansson. The X4 isolate III B passaged once in human peripheral blood lymphocytes was kindly provided by Kajsa Aperia. The SIVsm isolate used for challenge has been described earlier (Quesada-Rolander et al., 1996). Virus ID50 titration was performed essentially as described (Albert et al., 1990). Briefly, PBL from human blood donors were PHA stimulated for 3–5 days. A mix of PBL from two blood donors was used in all ID50 titrations and virus neutralization assays. Seventy-five microliters of medium and 1 ⫻ 10 5 PBL in 75 ␮l medium were seeded in each well in a 96-well culture plate which was incubated for 1 h at 37°C. Virus aliquots were thawed and serially diluted in threefold steps; 75 ␮l of each dilution was added to five parallel wells containing medium and PBL and incubated overnight at 37°C. The culture medium was changed at Days 2 and 4 postinfection, and on Day 7 cell-culture medium was harvested and HIV-1 p24 was determined using an in-house ELISA (Hinkula et al., 1990). The ID50 value was defined as the reciprocal of the virus dilution resulting in 50% positive wells (Reed– Muench calculation). Expression of CCR5 and tet.CCR5 in vitro Chinese hamster ovary (CHO) cells were grown in chamber slides (Nunc, Aahus, Denmark). The cells were transfected with 2 ␮g of pcDNA3-CCR5 or pcDNA3tet.CCR5 and 3 ␮l of FuGENE 6 transfection reagent (Roche Diagnostics Scandinavia, Bromma, Sweden). After 48 h, the transfected cells were fixed with 2% paraformaldehyde. Transfected cells were visualized using a primary mouse anti-CCR5 monoclonal antibody (Mab 181, R&D Systems), followed by fluorescein-isothiocyanate (FITC)-labeled rabbit anti-mouse IgG (FO313, Dako). CHO cells constitutively expressing CCR5 (CHO-CCR5), kindly donated by M. Samson, were used as controls. Immunization BALB/c mouse strain. Six groups (A–F) of five 6–8 week old BALB/c mice were immunized with 100 ␮g (2 ␮g/␮l) of plasmid in 50 ␮l water, injected in the quadriceps muscle. The injected plasmids were as follows: pcDNA3 ⫹ pCI-GM-CSF (100 ␮g ⫹ 100 ␮g) (A), pcDNA3CCR5 ⫹ pCI-GM-CSF (100 ␮g ⫹ 100 ␮g) (B), pcDNA3tet.CCR5 (100 ␮g) (C), pcDNA3-tet.CCR5 ⫹ pCI-GM-CSF (100 ␮g ⫹ 100 ␮g) (D), pcDNA3-tet.CCR5 ⫹ pCI-IL-4 (100 ␮g ⫹ 100 ␮g) (E), and pcDNA3-CCR5 ⫹ pCI-GM-CSF ⫹ pCI-IL-4 (100 ␮g ⫹ 50␮g ⫹ 50 ␮g) (F). Two groups of five mice (G–H) were inoculated epidermally at four adjacent sites of the shaved abdomen with a total of 1 mg of gold particles coated with 4 ␮g of: pcDNA3-tet.CCR5 ⫹ pCI-GM-CSF (2 ␮g ⫹ 2 ␮g) (G) or pcDNA3-tet.CCR5 ⫹ pCI-GM-CSF ⫹ pCI-IL-4 (2 ␮g ⫹ 1 ␮g ⫹ 1 ␮g) (H) using the helium-pulsed Accell delivery device (Geniva Inc., Middletown, WI). The mice were

407

immunized at 0, 4, and 8 weeks. Blood was collected at 2-week intervals. Cynomolgus macaques. Two cynomolgus macaques (macaca fascicularis) (Monkeys B189 and U9), 4–5 years old, were immunized with the plasmids pcDNA3-CCR5, pcDNA3-tet.CCR5, and human GM-CSF protein (Leucomax, Schering-Plough, Sandoz) three times and then boosted with a peptide mix twice. The plasmid/GM-CSF mix was administered at several sites in the rectal mucosa by high-pressure injection using a jet injector (SyriJet markII, Mizzy Inc.). The animals were immunized at 0 (500 ␮g pcDNA3-CCR5 ⫹ 75 ␮g GM-CSF), 8 (500 ␮g pcDNA3-CCR5 ⫹ 75 ␮g GM-CSF), and 18 weeks (1000 ␮g pcDNA3-tet.CCR5 ⫹ 75 ␮g GM-CSF) with DNA. At Weeks 31 and 35, they were boosted with a peptide mix at several sites in the rectal mucosa using a jet injector and intramuscularly using a syringe. A mix of four CCR5 peptides (Table 1) (100 ␮g of each) representing the N-terminus (peptides N1 and N1/N2), first (peptide X1), and second extracellular loops (peptide X2.2) of CCR5 ⫹ 75 ␮g GM-CSF protein was administered intrarectally. The same mix of peptides was administered intramuscularly together with the Ribilike (MPL ⫹ TDM ⫹ CWS) adjuvant system (Sigma); a 1-ml dose in saline solution was administered in 500 ␮l into each hind leg. D79 and D82 are nontreated control monkeys. The macaques were housed at the Primate Research Center at the Swedish Institute for Infectious Disease Control. Animal handling was carried out in accordance with the guidelines of the Swedish Ethical Committee for Animal Protection. Antibody assays ELISA. Serological responses were measured by enzyme-linked immunosorbent assay (ELISA). For all antibody titer determinations, plates coated with 10 ␮g/ml of peptide (Table 2) were used. The peptides were synthesized by the 9-fluorenylmethoxycarbonyl method (Sallberg et al., 1991). ELISA was performed essentially as described (Hinkula et al., 1997). CCR5 reactive antibodies in sera were detected by horseradish peroxidase (HRP)-labeled goat anti-human IgG or IgA (Bio-Rad, Richmond, CA) or HRP-labeled goat anti-mouse IgG (BioRad). Antibody titers were calculated as the dilution which gave an optical density (OD) that equaled the cutoff. For cynomolgus macaques, the cutoff value was determined for each animal as the equivalent of 3 (IgA in serum) or 4 (IgG in serum) times the OD of sera obtained before the primary injection. Total IgG determination. IgG was purified from serum and quantified essentially as described (Lundholm et al., 1999). Briefly, IgG was purified from serum using protein A coated beads (Biotech IgG, Denmark). IgG was quantified using an ELISA.

408

ZUBER ET AL.

B-cell stimulation in vitro. CCR5-vaccinated monkeys were tested for circulating B cells capable of secreting HIV-1-specific antibodies when stimulated in vitro with the antigens used for immunization. PBL (100,000/well in 200 ml RPMI 1640 supplemented with 5% inactivated fetal calf serum) were cultured for 72 h in triplicate wells at 37°C in 96-well, flat-bottomed cell culture plates (Nunclon, Nunc) coated with peptides at concentrations of 2 ␮g/well. After washing, the bound Ig was assayed by the ELISAs described above. Neutralization assay. Sera were inactivated at 56°C for 30 min and subsequently diluted in 75 ␮l medium in twofold dilution steps, starting from the final dilution 1:30. 1 ⫻ 10 5 monkey or human PBL in 75 ␮l medium were added to the cells and incubated at 37°C for 1 h. Then virus in three different concentrations was added and the plates were incubated at 37°C. After overnight incubation the cells were washed by centrifugation; this procedure was repeated at day three. At day six, 100 ␮l of supernatants were tested in an in-house HIV-1 p24 antigen capture ELISA or an in-house SIV p26 capture ELISA (Thorstensson et al., 1991). An ID50 determination was made in parallel with each neutralization test. The mean OD value of positive control wells was considered to represent 100% of p24 release. Percentage neutralization was calculated according to the formula: 100 ⫺ [100 ⫻ (OD sample wells/OD positive control wells)]. T-cell stimulation T-cell stimulation was performed as described (Hinkula et al., 1997). Briefly, mouse spleen cells (200,000/well) were cultured in the presence of 5 ␮g of antigen. Then, 3H-labeled thymidine was added and thymidin incorporation was measured. The stimulation index (SI) was calculated by dividing the mean counts/min in triplicate antigen-stimulated wells by the mean counts/ min in three medium control wells. A more than twofold increase of SI was considered positive. T-cell stimulation was performed on monkey PBL as described, except that 5 ␮g of antigen were used per well (Putkonen et al., 1998). An SI over two was considered positive. In all tests, phytohemagglutinin (PHA) or concanavalin-A (Con-A) were included as controls of cell viability. DISCUSSION The major role of the HIV-1 coreceptor CCR5 in primary HIV-1 infection prompted us to choose this receptor gene as an antigen. As the CCR5 protein is difficult to produce in a native form for immunization, we used the human CCR5 gene and a modified tet.CCR5 containing a promiscuous T-helper cell epitope in frame with the CCR5 gene. Peptide boosts were used to enhance antibody responses. To successfully use the CCR5 vaccination approach in humans, tolerance to the endogenous

protein must be broken and we therefore co-immunized with GM-CSF (Samanci et al., 1998). Clinical trials have shown that DNA vaccines against HIV-1 and malaria induce cellular and humoral responses in humans without any obvious side effects (Calarota et al., 1998; MacGregor et al., 1998; Wang et al., 1998). We report that naked DNA shows promise as a way to vaccinate against the HIV-1 coreceptor CCR5. Our results demonstrate that vaccination with cDNA representing the human CCR5 gene together with the murine GM-CSF gene elicited humoral and cellular immune responses in BALB/c mice. Cynomolgus macaques responded to immunization with the human CCR5 gene together with human GM-CSF protein even though the homology between the human CCR5 protein and CCR5 from cynomolgus macaques is as high as 98%. The monkeys that developed antibody and cellular responses responded both to the human CCR5 protein and to cynomolgus CCR5 protein. Furthermore, sera from immunized monkeys neutralized SIVsm infection of cynomolgus macaque PBL. We thus suggest that tolerance has been broken to the macaque CCR5 protein in macaques. This is important when it comes to human applications. One approach to break tolerance to the endogenous receptor in humans could be to immunize them with macaque CCR5. No side effects were noted clinically in these macaques which received both mucosal and intramuscular immunogens. BALB/c mice were immunized by gene gun or intramuscularly with the tet.CCR5 cDNA and GM-CSF cDNA. Gene gun covaccination with the tet.CCR5 plasmid and GM-CSF or GM-CSF and IL-4 plasmids induced strong antibody responses to a linear peptide representing the N-terminus of CCR5 and moderately strong responses to a peptide representing the second extracellular loop of CCR5. Furthermore, we found that significant T-cell proliferative responses to CCR5 peptides were induced only by gene gun immunization. Surprisingly, CCR5 reactive sera from the gene gun immunized mice failed to show neutralization in a cellbased assay. This phenomenon has been demonstrated by others. In one study concerning DNA vaccination against hepatitis C virus, both mice and small primates were immunized with the same plasmids. Although antibody titers in immunized mice were 10 times higher than those in the primates, the mouse sera did not have any neutralizing effect, while three of four immunized primates had neutralizing antibodies (Fournillier et al., 1999). This finding suggests that in addition to the form of the expressed antigen, host specificity is a critical component in the induction of biologically relevant antibodies. The antibody response was much stronger toward the N-terminus of CCR5 than to the second extracellular loop. These findings indicate that antibodies directed to the second extracellular loop may be more important for neutralization than antibodies directed to the N-terminus.

IMMUNE RESPONSES AGAINST THE HIV-1 CORECEPTOR CCR5

To enhance immune responses in macaques, the human CCR5 gene was administered together with human GM-CSF protein. We chose to immunize the macaques intrarectally with a novel high-pressure jet-gun delivery technique that has been shown to induce mucosal IgA antibodies (Lundholm et al., 1999). In both the immunized macaques, IgG and IgA responses in serum toward a peptide representing the N-terminal of CCR5 were detected already after the first DNA immunization. Both IgG and IgA responses toward the N-terminal peptide increased several logs after the second peptide boost. The strongest immune responses measured by in vitro B-cell stimulation and T-cell proliferation were directed toward the N-terminus followed by the first, second, and third extracellular loops of human CCR5. Strong antibody responses toward the macaque CCR5 protein represented by peptides were also demonstrated. Antibodies directed toward the Nalterminus and the second extracellular loop of CCR5 can neutralize HIV-1 infection of cells in vitro; monoclonal antibodies directed toward the second extracellular loop seem to be more effective in neutralization assays than antibodies recognizing the N-terminus (Olson et al., 1999). The most potent neutralizing Mab identified so far, 2D7, recognizes the first part of the second extracellular loop which seems to carry a good neutralizing epitope (Lee et al., 1999). Although there is 98% identity between human and cynomolgus monkey CCR5, eight amino acids differ and four of these are located in the extracellular N-terminal chain and second loop. The second-loop sequences of human and macaque CCR5 differ in amino acid 171 (Table 1), which is crucial for the recognition of CCR5 by 2D7 and several other Mabs. These differences may be crucial for the macaque to recognize human CCR5 as foreign and to elicit antibodies to the N-terminus and second loop of CCR5. Strong neutralizing responses toward HIV-1 R5 strains were detected in both immunized macaques after two DNA immunizations. Furthermore, infection of macaque lymphocytes by SIVsm was inhibited. Thus, immunization with DNA alone was sufficient to induce neutralizing responses in the nonhuman primate. In that DNA immunization is known to induce antibodies to native epitopes, we consider that a major part of the neutralizing effect may come from antibodies directed to conformational epitopes of CCR5, especially epitopes in the second extracellular loop. It was also noted that the titer of neutralization appeared to be optimal after two DNA immunizations and before peptide boost. It has also been demonstrated that induction of CCR5 inhibitory antibodies is important in preventing SIV infection in macaques (Lehner et al., 1999). Based on these data, we reasoned that blocking CCR5 with antibodies would prevent infection with SIVsm or change the course of the disease.

409

The monkeys were challenged intrarectally with SIVsm 1 month after the last immunization. We found that our challenge SIVsm virus uses CCR5 more efficiently than either BOB or Bonzo (Vo¨dro¨s et al., unpublished data). Others have shown that BOB plays a minor role for the pathogenicity of the HIV-2/SIVmac/SIVsm group of primate lentiviruses (Pohlmann et al., 1999). After challenge, the control monkeys as well as the immunized monkeys became infected by SIVsm, the viral load at set point appeared quite similar. Thus, neither protection nor enhancement was found in these two macaques. Interestingly though, we have preliminary data indicating that the virus isolates from the monkey with the highest CCR5-specific antibody titers differs in coreceptor usage from the virus of nonimmunized monkeys. SIVsm isolated post challenge from Monkey U9 uses less CCR5 and CCR3 and more of CXCR4 and BOB than the isolates from control monkeys. These data indicate a selective pressure from the CCR5-specific antibodies. In retrospect, SIVsm may not have been the optimal challenge virus. In the next study, challenge should be made with a SHIV containing the envelope of a R5 HIV-1. Immunization against chemokine receptors may be of general use in the regulation of inflammatory responses, allergic responses, and viral diseases other than HIV-1. A number of viruses like human cytomegalovirus and human herpesvirus Type 6 express chemokine receptor analogues which are feasible vaccine targets. It was recently found that CCR5-reactive antibodies in seronegative partners of HIV-1-seropositive individuals downmodulate surface CCR5 in vivo and neutralize the infectivity of R5 strains of HIV-1 in vitro (Lopalco et al., 2000). This finding indicates that therapeutic strategies aimed at preventing HIV-1 infection by means of antibodies to CCR5 with a modified CCR5 gene may be feasible. In conclusion, our results show that the human CCR5 gene is immunogenic in both mice and cynomolgus macaques and that tolerance to endogenous CCR5 could be overcome by the use of a homologous gene for CCR5. Antibodies induced by immunization with the human CCR5 gene could block infection of human lymphocytes by R5 but not X4 HIV-1 isolates. ACKNOWLEDGMENTS We thank Deborah Fuller and Joel Haynes for providing the gene gun device. We thank Dr. Cecilia Svanholm for kindly providing the pCI-GMCSF and pCI-IL-4 plasmids. Håkan Mellstedt is acknowledged for providing Leucomax. Dr. Marianne Jansson is acknowledged for providing virus isolate J2234. Åsa Bjo¨rndal is acknowledged for valuable discussions. This work was supported by Medivir AB, the Foundation for Knowledge and Competence Development, and the Swedish Medical Research Council.

REFERENCES Ahmed, R. K., Nilsson, C., Wang, Y., Lehner, T., Biberfeld, G., and Thorstensson, R. (1999). ␤-chemokine production in macaques vac-

410

ZUBER ET AL.

cinated with live attenuated virus correlates with protection against simian immunodeficiency virus (SIVsm) challenge. J. Gen. Virol. 80(Pt. 7), 1569–1574. Albert, J., Abrahamsson, B., Nagy, K., Aurelius, E., Gaines, H., Nystro¨m, G., and Fenyo, E. M. (1990). Rapid development of isolate-specific neutralizing antibodies after primary HIV-1 infection and consequent emergence of virus variants which resist neutralization by autologous sera. AIDS 4(2), 107–112. Attanasio, R., Pehler, K., and Scinicariello, F. (1997). DNA-based immunization induces anti-CD4 antibodies directed primarily to native epitopes. FEMS Immunol. Med. Microbiol. 17(4), 207–215. Bratt, G., Sandstro¨m, E., Albert, J., Samson, M., and Wahren, B. (1997). The influence of MT-2 tropism on the prognostic implications of the delta32 deletion in the CCR-5 gene. AIDS 11(12), 1415–9. Calarota, S., Bratt, G., Nordlund, S., Hinkula, J., Leandersson, A. C., Sandstro¨m, E., and Wahren, B. (1998). Cellular cytotoxic response induced by DNA vaccination in HIV-1- infected patients. Lancet 351(9112), 1320–1325. Chin, L. T., Hinkula, J., Levi, M., Ohlin, M., Wahren, B., and Borrebaeck, C. A. (1994). Site-directed primary in vitro immunization: production of HIV-1 neutralizing human monoclonal antibodies from lymphocytes obtained from seronegative donors. Immunology 81(3), 428–434. Cocchi, F., DeVico, A. L., Garzino-Demo, A., Arya, S. K., Gallo, R. C., and Lusso, P. (1995). Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV- suppressive factors produced by CD8⫹ T cells. Science 270(5243), 1811–1815. Deng, H., Liu, R., Ellmeier, W., Choe, S., Unutmaz, D., Burkhart, M., Di Marzio, P., Marmon, S., Sutton, R. E., Hill, C. M., Davis, C. B., Peiper, S. C., Schall, T. J., Littman, D. R., and Landau, N. R. (1996). Identification of a major co-receptor for primary isolates of HIV-1. Nature 381(6584), 661–666. Deng, H. K., Unutmaz, D., KewalRamani, V. N., and Littman, D. R. (1997). Expression cloning of new receptors used by simian and human immunodeficiency viruses. [See comments] Nature 388(6639), 296– 300. Ditzel, H. J., Rosenkilde, M. M., Garred, P., Wang, M., Koefoed, K., Pedersen, C., Burton, D. R., and Schwartz, T. W. (1998). The CCR5 receptor acts as an alloantigen in CCR5Delta32 homozygous individuals: Identification of chemokine and HIV-1-blocking human antibodies. Proc. Natl. Acad. Sci. USA 95(9), 5241–5245. Edinger, A. L., Hoffman, T. L., Sharron, M., Lee, B., O’Dowd, B., and Doms, R. W. (1998). Use of GPR1, GPR15, and STRL33 as coreceptors by diverse human immunodeficiency virus type 1 and simian immunodeficiency virus envelope proteins. Virology 249(2), 367–378. Farzan, M., Choe, H., Martin, K., Marcon, L., Hofmann, W., Karlsson, G., Sun, Y., Barrett, P., Marchand, N., Sullivan, N., Gerard, N., Gerard, C., and Sodroski, J. (1997). Two orphan seven-transmembrane segment receptors which are expressed in CD4-positive cells support simian immunodeficiency virus infection. J. Exp. Med. 186(3), 405–411. Fournillier, A., Depla, E., Karayiannis, P., Vidalin, O., Maertens, G., Trepo, C., and Inchauspe, G. (1999). Expression of noncovalent hepatitis C virus envelope E1–E2 complexes is not required for the induction of antibodies with neutralizing properties following DNA immunization. J. Virol. 73(9), 7497–7504. Hinkula, J., Rosen, J., Sundqvist, V. A., Stigbrand, T., and Wahren, B. (1990). Epitope mapping of the HIV-1 gag region with monoclonal antibodies. Mol. Immunol. 27(5), 395–403. Hinkula, J., Svanholm, C., Schwartz, S., Lundholm, P., Brytting, M., Engstrom, G., Benthin, R., Glaser, H., Sutter, G., Kohleisen, B., Erfle, V., Okuda, K., Wigzell, H., and Wahren, B. (1997). Recognition of prominent viral epitopes induced by immunization with human immunodeficiency virus type 1 regulatory genes. J. Virol. 71(7), 5528–5539. Huang, Y., Paxton, W. A., Wolinsky, S. M., Neumann, A. U., Zhang, L., He, T., Kang, S., Ceradini, D., Jin, Z., Yazdanbakhsh, K., Kunstman, K., Erickson, D., Dragon, E., Landau, N. R., Phair, J., Ho, D. D., and Koup, R. A. (1996). The role of a mutant CCR5 allele in HIV-1 transmission and disease progression. Nat. Med. 2(11), 1240–1243.

Jansson, M., Popovic, M., Karlsson, A., Cocchi, F., Rossi, P., Albert, J., and Wigzell, H. (1996). Sensitivity to inhibition by ␤-chemokines correlates with biological phenotypes of primary HIV-1 isolates. Proc. Natl. Acad. Sci. USA 93(26), 15382–15387. Kumar, A., Arora, R., Kaur, P., Chauhan, V. S., and Sharma, P. (1992). “Universal” T helper cell determinants enhance immunogenicity of a Plasmodium falciparum merozoite surface antigen peptide. J. Immunol. 148(5), 1499–1505. Lee, B., Sharron, M., Blanpain, C., Doranz, B. J., Vakili, J., Setoh, P., Berg, E., Liu, G., Guy, H. R., Durell, S. R., Parmentier, M., Chang, C. N., Price, K., Tsang, M., and Doms, R. W. (1999). Epitope mapping of CCR5 reveals multiple conformational states and distinct but overlapping structures involved in chemokine and coreceptor function. J. Biol. Chem. 274(14), 9617–9626. Lehner, T., Wang, Y., Doyle, C., Tao, L., Bergmeier, L. A., Mitchell, E., Bogers, W. M., Heeney, J., and Kelly, C. G. (1999). Induction of inhibitory antibodies to the CCR5 chemokine receptor and their complementary role in preventing SIV infection in macaques. Eur. J. Immunol. 29(8), 2427–2435. Lopalco, L., Barassi, C., Pastori, C., Longhi, R., Burastero, SE, Tambussi, G., Mazzotta, F., Lazzarin, A., Clerici, M., and Siccardi, A. G. (2000). CCR5-Reactive antibodies in seronegative partners of HIV-seropositive individuals down-modulate surface CCR5 In vivo and neutralize the infectivity of R5 strains of HIV-1 In vitro. J. Immunol. 164(6), 3426–3433. Lundholm, P., Asakura, Y., Hinkula, J., Lucht, E., and Wahren, B. (1999). Induction of mucosal IgA by a novel jet delivery technique for HIV-1 DNA. Vaccine 17(15–16), 2036–2042. MacGregor, R. R., Boyer, J. D., Ugen, K. E., Lacy, K. E., Gluckman, S. J., Bagarazzi, M. L., Chattergoon, M. A., Baine, Y., Higgins, T. J., Ciccarelli, R. B., Coney, L. R., Ginsberg, R. S., and Weiner, D. B. (1998). First human trial of a DNA-based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host response. J. Infect. Dis. 178(1), 92–100. Olson, W. C., Rabut, G. E., Nagashima, K. A., Tran, D. N., Anselma, D. J., Monard, S. P., Segal, J. P., Thompson, D. A., Kajumo, F., Guo, Y., Moore, J. P., Maddon, P. J., and Dragic, T. (1999). Differential inhibition of human immunodeficiency virus type 1 fusion, gp120 binding, and CC-chemokine activity by monoclonal antibodies to CCR5. J. Virol. 73(5), 4145–4155. Owen, S. M., Masciotra, S., Novembre, F., Yee, J., Switzer, W. M., Ostyula, M., and Lal, R. B. (2000). Simian immunodeficiency viruses of diverse origin can use CXCR4 as a coreceptor for entry into human cells. J. Virol. 74(12), 5702–5708. Panina-Bordignon, P., Tan, A., Termijtelen, A., Demotz, S., Corradin, G., and Lanzavecchia, A. (1989). Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur. J. Immunol. 19(12), 2237–2242. Pohlmann, S., Stolte, N., Munch, J., Ten Haaft, P., Heeney, J. L., StahlHennig, C., and Kirchhoff, F. (1999). Co-receptor usage of BOB/GPR15 in addition to CCR5 has no significant effect on replication of simian immunodeficiency virus in vivo. J. Infect. Dis. 180(5), 1494–1502. Putkonen, P., Quesada-Rolander, M., Leandersson, A. C., Schwartz, S., Thorstensson, R., Okuda, K., Wahren, B., and Hinkula, J. (1998). Immune responses but no protection against SHIV by gene-gun delivery of HIV-1 DNA followed by recombinant subunit protein boosts. Virology 250(2), 293–301. Quesada-Rolander, M., Ma¨kitalo, B., Thorstensson, R., Zhang, Y. J., Castanos-Velez, E., Biberfeld, G., and Putkonen, P. (1996). Protection against mucosal SIVsm challenge in macaques infected with a chimeric SIV that expresses HIV type 1 envelope. AIDS Res. Hum. Retroviruses 12(11), 993–999. Robinson, H. L., and Torres, C. A. (1997). DNA vaccines. Semin. Immunol. 9(5), 271–283. Sa¨llberg, M., Ruden, U., Magnius, L. O., Norrby, E., and Wahren, B. (1991). Rapid “tea-bag” peptide synthesis using 9-fluorenylmethoxy-

IMMUNE RESPONSES AGAINST THE HIV-1 CORECEPTOR CCR5 carbonyl (Fmoc) protected amino acids applied for antigenic mapping of viral proteins. Immunol. Lett. 30(1), 59–68. Samanci, A., Yi, Q., Fagerberg, J., Strigard, K., Smith, G., Ruden, U., Wahren, B., and Mellstedt, H. (1998). Pharmacological administration of granulocyte/macrophage-colony-stimulating factor is of significant importance for the induction of a strong humoral and cellular response in patients immunized with recombinant carcinoembryonic antigen. Cancer Immunol. Immunother. 47(3), 131–142. Simmons, G., Clapham, P. R., Picard, L., Offord, R. E., Rosenkilde, M. M., Schwartz, T. W., Buser, R., Wells, T. N. C., and Proudfoot, A. E. (1997). Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist. Science 276(5310), 276–279. Svanholm, C., Lo¨wenadler, B., and Wigzell, H. (1997). Amplification of T-cell and antibody responses in DNA-based immunization with HIV-1 Nef by co-injection with a GM-CSF expression vector. Scand. J. Immunol. 46, 298–303. Thorstensson, R., Walther, L., Putkonen, P., Albert, J., and Biberfeld, G. (1991). A capture enzyme immunoassay for detection of HIV-2/SIV antigen. J. Acquir. Immune Defic. Syndr. 4(4), 374–379. Wahren, B., and Brytting, M. (1997). DNA increases the potency of vaccination against infectious diseases. Curr. Opin. Chem. Biol. 1(2), 183–189. Wang, C. Y., Sawyer, L. S., Murthy, K. K., Fang, X., Walfield, A. M., Ye, J., Wang, J. J., Chen, P. D., Li, M. L., Salas, M. T., Shen, M., Gauduin, M. C., Boyle, R. W., Koup, R. A., Montefiori, D. C., Mascola, J. R., Koff,

411

W. C., and Hanson, C. V. (1999). Postexposure immunoprophylaxis of primary isolates by an antibody to HIV receptor complex. Proc. Natl. Acad. Sci. USA 96(18), 10367–10372. Wang, R., Doolan, D. L., Le, T. P., Hedstrom, R. C., Coonan, K. M., Charoenvit, Y., Jones, T. R., Hobart, P., Margalith, M., Ng, J., Weiss, W. R., Sedegah, M., de Taisne, C., Norman, J. A., and Hoffman, S. L. (1998). Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine. Science 282(5388), 476– 480. Watanabe, M., Levine, C. G., Shen, L., Fisher, R. A., and Letvin, N. L. (1991). Immunization of simian immunodeficiency virus-infected rhesus monkeys with soluble human CD4 elicits an antiviral response. Proc. Natl. Acad. Sci. USA 88(11), 4616–4620. Wu, L., LaRosa, G., Kassam, N., Gordon, C.J., Heath, H., Ruffing, N., Chen, H., Humblias, J., Samson, M., Parmentier, M., Moore, J. P., and Mackay, C. R. (1997a). Interaction of chemokine receptor CCR5 with its ligands: multiple domains for HIV-1 gp120 binding and a single domain for chemokine binding. J. Exp. Med. 186(8), 1373–1381. Wu, L., Paxton, W. A., Kassam, N., Ruffing, N., Rottman, J. B., Sullivan, N., Choe, H., Sodroski, J., Newman, W., Koup, R. A., and Mackay, C. R. (1997b). CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro. J. Exp. Med. 185(9), 1681–1691.