Induction of murine mucosal CCR5-reactive antibodies as an anti-human immunodeficiency virus strategy - PubMed (original) (raw)

Induction of murine mucosal CCR5-reactive antibodies as an anti-human immunodeficiency virus strategy

C Barassi et al. J Virol. 2005 Jun.

Abstract

The genital mucosa is the main site of initial human immunodeficiency virus type 1 (HIV-1) contact with its host. In spite of repeated sexual exposure, some individuals remain seronegative, and a small fraction of them produce immunoglobulin G (IgG) and IgA autoantibodies directed against CCR5, which is probably the cause of the CCR5-minus phenotype observed in the peripheral blood mononuclear cells of these subjects. These antibodies recognize the 89-to-102 extracellular loop of CCR5 in its native conformation. The aim of this study was to induce infection-preventing mucosal anti-CCR5 autoantibodies in individuals at high risk of HIV infection. Thus, we generated chimeric immunogens containing the relevant CCR5 peptide in the context of the capsid protein of Flock House virus, a presentation system in which it is possible to engineer conformationally constrained peptide in a highly immunogenic form. Administered in mice via the systemic or mucosal route, the immunogens elicited anti-CCR5 IgG and IgA (in sera and vaginal fluids). Analogous to exposed seronegative individuals, mice producing anti-CCR5 autoantibodies express significantly reduced levels of CCR5 on the surfaces of CD4+ cells from peripheral blood and vaginal washes. In vitro studies have shown that murine IgG and IgA (i) specifically bind human and mouse CD4+ lymphocytes and the CCR5-transfected U87 cell line, (ii) down-regulate CCR5 expression of CD4+ cells from both humans and untreated mice, (iii) inhibit Mip-1beta chemotaxis of CD4+ CCR5+ lymphocytes, and (iv) neutralize HIV R5 strains. These data suggest that immune strategies aimed at generating anti-CCR5 antibodies at the level of the genital mucosa might be feasible and represent a strategy to induce mucosal HIV-protective immunity.

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Figures

FIG. 1.

(A) Homology between human and mouse CCR5 sequences and sequence of the synthetic peptide. (B) Schematic representation of the FHV capsid protein. The structure displays five different superficial peptide loops (L1, L2, L3, I2, and I3), in each of which a foreign peptide sequence can be inserted and presented on the protein surface in a stable, structurally constrained form. Numbers in parentheses refer to the original amino acid residues replaced by the foreign epitope.

FIG. 2.

Comparison between the immune responses elicited by the four different CCR5-FHV recombinant immunogens (FHV-L1, -L2, -I2, and -I3) and negative (FHV-WT) and positive (synthetic cyclic CCR589-102 peptide) controls. (A) Fractions of CCR5-binding total Igs elicited by FHV recombinant immunogens versus FHV-WT negative control. (B) CCR5 down-regulation on human CD4+ lymphocytes by purified Igs (20 ng/assay) derived from antisera elicited by FHV recombinant immunogens or FHV-WT. (C) Fractions of CCR5-binding total Igs elicited by FHV-I2, FHV-WT, and the synthetic cyclic CCR589-102 peptide administered either via systemic or mucosal route. (D) Fractions of CCR5-binding IgA and IgG purified from the total Ig fractions of panel C.

FIG. 3.

Flow cytometry analysis of the specificities and biological activities of anti-CCR5 total Igs or IgG- or IgA-enriched fractions from antisera elicited by the recombinant immunogen FHV-I2. (A) Binding of either IgA or IgG fractions (20 ng/assay) to CCR5 receptors on mouse CD4+ lymphocytes. Positive controls were lymphocytes treated with an anti-mouse CCR5 MAb (M20), and negative controls were lymphocytes treated only with the secondary Ab (RAM-FITC). (B) Kinetics of CCR5 down-regulation on mouse CD4+ lymphocytes of untreated mice by total Ig fractions (20 ng/assay) purified from FHV-I2 antisera. Lymphocytes were incubated with purified Igs for 12 and 48 h before flow analysis. Positive and negative controls were as in panel A. (C) CCR5 down-regulation obtained by incubating mouse CD4+ lymphocytes with purified Igs (20 ng/assay) for 48 h before flow analysis. As negative controls, target cells were incubated either with Igs purified from NMS or with only the secondary Ab (RAM-FITC). (D) Dose-response curves of CCR5 down-regulation obtained by incubating mouse CD4+ lymphocytes for 48 h with the indicated concentrations of Igs purified from pools of antisera obtained by either systemic or mucosal immunizations. As negative controls, mouse CD4+ lymphocytes were also incubated with Igs purified from FHV-WT antisera or with only the secondary Ab (RAM-FITC). These data were obtained with serum samples drawn 1 week after the third immunization.

FIG. 4.

Radio-binding assay analysis of the specificities and biological activities of anti-CCR5 mouse Abs on human CD4+ lymphocytes. (A) CCR5-binding activities of antibodies elicited by systemic or mucosal immunizations with FHV-WT, synthetic cyclic CCR589-102 peptide, or FHV-I2. (B) Radio binding to human CCR5 of serum (Se) IgG, serum IgA, or mucosal (Mu) IgA elicited by mucosal immunization with FHV-WT or FHV-I2. The negative control (Cntr) was an Ig fraction from NMS (20 ng/assay). The positive control was the anti-human CCR5 MAb 2D7. (C) Dose-response curves of CCR5 down-regulation obtained by incubating human CD4+ lymphocytes for 48 h with the indicated dilutions of FHV-I2 or FHV-WT antisera. Negative controls were target cells incubated only with the secondary Ab (RAM-FITC).

FIG. 5.

Inhibition of Mip-1β-induced chemotaxis of human CD4+ T lymphocytes by anti-CCR5 mouse antibodies. Purified lymphocytes were incubated with Ig fractions (20 ng/ml) from antisera elicited by FHV-I2, FHV-WT, or the synthetic cyclic CCR589-102 peptide. Controls (Cntr) included lymphocytes treated with Ig fractions from NMS and lymphocytes without added antibodies. Means and standard errors from three replicates.

FIG. 6.

Neutralization of the infectivity of HIV-1 primary isolates by anti-CCR5 mouse antibodies. Isolates HIV no. 36 and HIV no. 40 were subtype B, R5 tropic; isolate HIV no. 45 was subtype B, R5, R3, X4 tropic. Antibodies were affinity purified on peptide-coupled beads from mucosal IgA- and IgG-enriched fractions derived from FHV-I2 or FHV-WT antisera. In panels A, B, and C, 8 ng/ml of Igs were used. The positive control (Cntr) was the anti-human CD4 MAb SIM4. In the negative control, no antibody was added. The error bars indicate standard deviations. (D and E) Dose-response curves of infectivity reduction obtained by treating lymphocytes with the indicated concentrations of IgA or IgG antibodies from FHV-I2 or FHV-WT antisera. Since only small serum samples could be collected, the antibodies for the neutralization assay were purified from a pool of samples drawn after the fourth and fifth immunizations.

FIG. 7.

Antibody-mediated CCR5 down-regulation in vivo. The CCR5 phenotype of CD4+ cells was evaluated by two-color flow cytometry of cells derived from peripheral blood or from vaginal fluids of mice immunized by mucosal or systemic immunizations with FHV-WT, the synthetic cyclic CCR589-102 peptide, or FHV-I2. The double staining was obtained using M20 (anti-mouse CCR5 polyclonal antibody) and GK1.5 (anti-mouse CD4 MAb). (A) CCR5 expression by CD4+ cells of mice subjected to mucosal immunizations. The error bars indicate standard deviations. (B) CCR5 expression by CD4+ cells of mice subjected to systemic immunizations. (C) Kinetics of CCR5 reappearance on the surfaces of cultured PBMC obtained from mice immunized by either route with FHV-WT or FHV-I2.

FIG. 8.

Long-term kinetic assays of in vivo CCR5 down-regulation by FHV-I2 immunization. CCR5 expression by CD4+ cells of mice subjected to mucosal (panel A) or systemic (panel B) immunization with FHV-I2, FHV-WT, or the synthetic cyclic CCR589-102 peptide. The lowest levels of CCR5 expression are seen in the samples drawn 1 week after the seventh immunization (groups I). Expression of CCR5 receptors was gradually recovered in the samples obtained after 2 and 4 weeks of rest (groups II and III). The samples of groups IV were obtained 1 week after a further immunization.

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Induction of murine mucosal CCR5-reactive antibodies as an anti-human immunodeficiency virus strategy - PubMed (original) (raw)