Lesley Bergmeier

Protective mucosal immunity elicited by targeted iliac lymph node immunization with a subunit SIV envelope and core vaccine in macaques.

Prevention of sexually transmitted HIV infection was investigated in macaques by immunization with a recombinant SIV (simian immunodeficiency virus) envelope gp120 and core p27 vaccine. In two independent series of experiments, we used the novel targeted iliac lymph node (TILN) route of immunization, aiming close to the iliac lymph nodes draining the genitorectal mucosa. Rectal challenge with the SIVmac 32H J5 molecular clone in two series induced total protection in four out of seven macaques immunized by TILN, compared with infection in 13 of 14 unimmunized macaques or immunized by other routes (P = 0.025). The remaining three macaques showed either a decrease in viral load (>90%) or transient viremia, indicating that all seven TILN–immunized macaques showed total or partial protection (P = 0.001). Protection was associated with significant increase in the iliac lymph nodes of lgA antibody–secreting cells to p27 (P < 0.02), CD8–suppressor factor (P< 0.01), and the chemokines RANTES and MIP–1β (P< 0.01).

CD40 Is a Cellular Receptor Mediating Mycobacterial Heat Shock Protein 70 Stimulation of CC-Chemokines

The 70 kDa mycobacterial heat shock protein (Mtb HSP70) stimulates mononuclear cells to release CC-chemokines. We now show that this function of Mtb HSP70, but not human HSP70, is dependent on the cell surface expression of CD40. Deletion of the CD40 cytoplasmic tail abolished, and CD40 antibody inhibited, Mtb HSP70 stimulation of CC-chemokine release. Mtb HSP70 stimulated THP1, KG1 cells, and monocyte-derived dendritic cells to produce RANTES. Specific binding of CD40-transfected HEK 293 cells to Mtb HSP70 was demonstrated by surface plasmon resonance. Coimmunoprecipitation of Mtb HSP70 with CD40 indicates a physical association between these molecules. The results suggest that CD40 is critical in microbial HSP70 binding and stimulation of RANTES production.

Stimulation of Th1-Polarizing Cytokines, C-C Chemokines, Maturation of Dendritic Cells, and Adjuvant Function by the Peptide Binding Fragment of Heat Shock Protein 70

The peptide binding C-terminal portion of heat shock protein (HSP)70 (aa 359–610) stimulates human monocytes to produce IL-12, TNF-α, NO, and C-C chemokines. The N-terminal, ATPase portion (HSP701–358) failed to stimulate any of these cytokines or chemokines. Both native and the truncated HSP70359–610 stimulation of chemokine production is mediated by the CD40 costimulatory molecule. Maturation of dendritic cells was induced by stimulation with native HSP70, was not seen with the N-terminal HSP701–358, but was enhanced with HSP70359–610, as demonstrated by up-regulation of CD83, CCR7, CD86, CD80, and HLA class II. In vivo studies in macaques showed that immunization with HSP70359–610 enhances the production of IL-12 and RANTES. Immunization with peptide-bound HSP70359–610 in mice induced higher serum IgG2a and IgG3 Abs than the native HSP70-bound peptide. This study suggests that the C-terminal, peptide-binding portion of HSP70 is responsible for stimulating Th1-polarizing cytokines, C-C chemokines, and an adjuvant function.

Identification of Stimulating and Inhibitory Epitopes within the Heat Shock Protein 70 Molecule That Modulate Cytokine Production and Maturation of Dendritic Cells

The 70-kDa microbial heat shock protein (mHSP70) has a profound effect on the immune system, interacting with the CD40 receptor on DC and monocytes to produce cytokines and chemokines. The mHSP70 also induces maturation of dendritic cells (DC) and thus acts as an alternative ligand to CD40L on T cells. In this investigation, we have identified a cytokine-stimulating epitope (peptide 407–426), by activating DC with overlapping synthetic peptides (20-mers) derived from the sequence of mHSP70. This peptide also significantly enhances maturation of DC stimulated by mHSP70 or CD40L. The epitope is located at the base of the peptide-binding groove of HSP70 and has five critical residues. Furthermore, an inhibitory epitope (p457–496) was identified downstream from the peptide-binding groove that inhibits cytokine production and maturation of DC stimulated by HSP70 or CD40L. The p38 MAP kinase phosphorylation is critical in the alternative CD40-HSP70 pathway and is inhibited by p457–496 but enhanced by p407–426.

Oral tolerization with peptide 336–351 linked to cholera toxin B subunit in preventing relapses of uveitis in Behcet's disease

Behcets disease (BD) specific peptide (p336–351) was identified within the human 60 kD heat shock protein (HSP60). Oral p336–351 induced uveitis in rats which was prevented by oral tolerization with the peptide linked to recombinant cholera toxin B subunit (CTB). This strategy was adopted in a phase I/II clinical trial by oral administration of p336–351‐CTB, 3 times weekly, followed by gradual withdrawal of all immunosuppressive drugs used to control the disease in 8 patients with BD. The patients were monitored by clinical and ophthalmological examination, as well as extensive immunological investigations. Oral administration of p336–351‐CTB had no adverse effect and withdrawal of the immunosuppressive drugs showed no relapse of uveitis in 5 of 8 patients or 5 of 6 selected patients who were free of disease activity prior to initiating the tolerization regimen. After tolerization was discontinued, 3 of 5 patients remained free of relapsing uveitis for 10–18 months after cessation of all treatment. Control of uveitis and extra‐ocular manifestations of BD was associated with a lack of peptide‐specific CD4+ T cell proliferation, a decrease in expression of TH1 type cells (CCR5, CXCR3), IFN‐γ and TNF‐α production, CCR7+ T cells and costimulatory molecules (CD40 and CD28), as compared with an increase in these parameters in patients in whom uveitis had relapsed. The efficacy of oral peptide‐CTB tolerization will need to be confirmed in a phase III trial, but this novel strategy in humans might be applicable generally to autoimmune diseases in which specific antigens have been identified.

The role of γ δ T cells in generating antiviral factors and β‐chemokines in protection against mucosal simian immunodeficiency virus infection

In view of the role of γ δ+ T cells in mucosal protection against infection, the proportion of γ δ T cells was examined in cells eluted from lymphoid and mucosal tissues of macaques immunized with simian immunodeficiency virus (SIV) gp120 and p27 in alum and challenged with live SIV by the rectal mucosal route. This revealed a significant increase in γ δ T cells eluted from the rectal mucosa (p < 0.01) and the related iliac lymph nodes (p < 0.0001) in protected as compared with infected macaques. Preferential homing of PKH‐26‐labeled γ δ+ T cells from the primed iliac lymph nodes to the rectal and cervico‐vaginal mucosa was demonstrated after targeted iliac lymph node as compared with i. m. immunization. Investigations of the mechanism of protection revealed that γ δ+ T cells can generate antiviral factors, RANTES, macrophage inflammatory protein (MIP)‐1α and MIP‐1β which can prevent SIV infection by binding to the CCR5 coreceptors. Up‐regulation of γ δ+ T cells was demonstrated by immunization of macaques with heat shock protein (HSP)70 linked to peptides and with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). This was confirmed by in vitro studies showing that GM‐CSF can up‐regulate γ δ+ T cells from macaques immunized with HSP‐linked peptides but not those from naive animals. We suggest that a novel strategy of immunization with HSP70 linked to antigen may generate both cognate immunity to the antigen and innate immunity by virtue of up‐regulation of γ δ+ T cells. These cells generate antiviral factors and the three β‐chemokines that prevent binding and transmission of SIV or M‐tropic HIV by the CCR5 coreceptor.

A novel HIV-CCR5 receptor vaccine strategy in the control of mucosal SIV/HIV infection.

Objective: To develop a novel SIV-CCR5 receptor vaccine strategy that will protect macaques from SHIV infection by the vaginal mucosal route. Design: The rationale for this strategy is that humans who express the homozygous Δ32 CCR5 mutation and the associated upregulation of CC chemokines, the downmodulation of cell-surface expression of CCR5 and antibodies to CCR5 are protected against HIV infection. Methods: A vaccine was prepared consisting of three extracellular peptides of CCR5, an N-terminal HIV gp120 fragment generated in transgenic plants and recombinant SIV p27. These were linked to the 70 000 Mr microbial heat shock protein (HSP70) carrier. The vaccine was administered (×3) either by the vaginal mucosal route or by targeting the proximity of the draining iliac lymph nodes. Results: Serum and vaginal fluid IgG and IgA antibodies, IL-2 and IFN-γ-producing cells, and macrophage-inflammatory protein (MIP) 1β and MIP-1α (CCL4 and CCL3) were significantly raised in immunized macaques (P = 0.01–0.05). Vaginal challenge with SHIV89.6P infected all macaques, but sequential analysis over 24 weeks showed a significant variation in viral loads between the animals (P = 0.05). Whereas SHIV89.6P persisted in the four unimmunized macaques, in five of the eight immunized macaques the virus was cleared or became undetectable by reverse transcriptase–polymerase chain reaction. The CD4 cell counts in the immunized macaques were significantly higher than those in unimmunized animals (P < 0.05). Conclusion: An immunization strategy that targets both the virus and its CCR5 receptor has significantly inhibited SHIV89.6P infection and may serve as a novel strategy in the prevention of HIV transmission.

A rational basis for mucosal vaccination against HIV infection.

Summary: The lack of success in the development of an effective conventional vaccine against HIV has focused attention on mucosal immunity. This is a rational move, since HIV is transmitted mostly by the mucosal route. The mucosal strategy is based on the concept that: a) HIV/SIV has to cross the mucosal‐regional lymph node‐blood barriers, each of which can prevent viral transmission or decrease the viral load, b) Immunization has to target directly the mucosal tissues or indirectly the regional lymph nodes, in order to prevent or control viral replication. This strategy is consistent with antigen localization and effective entry into the lymph nodes, driving the immune response, c) A dual immune mechanism may be necessary for effective mucosal protection, mediated by specific CD4 and CD8 T‐cell and antibody responses to the immunizing antigens, and innate antiviral factors and [i‐chemokines which downmodulate CCR5 co‐receptors. Targeted iliac lymph node immunization with SIVgp120 and p27 in alum prevents SIV infection or significantly decreases the viral load when challenged by the rectal route. Indeed, in addition to specific immunity, including significant sIgA antibody‐forming cells in the iliac lymph nodes, CD8‐suppressor factor and the three β‐chemokines (RANTES, macrophage inflammatory protein (MIP)‐ lα and MlP‐ 1β) are significantly associated with protection against rectal mucosal SIV infection.

Stimulation of Cell Surface CCR5 and CD40 Molecules by Their Ligands or by HSP70 Up-Regulates APOBEC3G Expression in CD4+ T Cells and Dendritic Cells

Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like-3G (A3G) is an intracellular innate antiviral factor that deaminates retroviral cytidine to uridine. In an attempt to harness the anti-HIV effect of A3G, we searched for an agent that would up-regulate A3G and identify the receptors involved. Stimulation of cell surface CCR5 with CCL3 and CD40 with CD40L or both molecules with microbial 70-kDa heat shock protein (HSP)70 up-regulated A3G mRNA and protein expression in human CD4+ T cells and monocyte-derived dendritic cells (DC), demonstrated by real-time PCR and Western blots, respectively. The specificity of CCR5 and CD40 stimulation was established by inhibition with TAK 779 and mAb to CD40, as well as using human embryonic kidney 293 cells transfected with CCR5 and CD40, respectively. A dose-dependent increase of A3G in CCL3- or HSP70-stimulated CD4+ T cells was associated with inhibition in HIV-1 infectivity. To differentiate between the inhibitory effect of HSP70-induced CCR5 binding and that of A3G, GFP-labeled pseudovirions were used to infect human embryonic kidney 293 cells, which showed inhibition of pseudovirion uptake, consistent with A3G being responsible for the inhibitory effect. Ligation of cell surface CCR5 receptors by CCL3 or CD40 by CD40L activated the ERK1/2 and p38 MAPK signaling pathways that induced A3G mRNA expression and production of the A3G protein. These in vitro results were corroborated by in vivo studies in rhesus macaques in which A3G was significantly up-regulated following immunization with SIVgp120 and p27 linked to HSP70. This novel preventive approach may in addition to adaptive immunity use the intracellular innate antiviral effect of A3G.

Interaction between the CCR5 chemokine receptors and microbial HSP70

Evidence is presented that the microbial 70‐kD heat shock protein (HSP70) binds to CCR5 chemokine receptors in CCR5‐transfected cell lines and in primary human cells. Significant CCR5‐mediated calcium mobilization was stimulated by HSP70 and inhibited with TAK 779, which is a specific CCR5 antagonist. HSP70‐mediated activation of the p38 MAPK phosphorylation signaling pathway was also demonstrated in CCR5‐transfected HEK 293 cells. Direct binding of three extracellular peptides of CCR5 to HSP70 was demonstrated by surface plasmon resonance. Functional evidence of an interaction between HSP70, CCR5 and CD40 was shown by enhanced production of CCL5 by HEK 293 cells transfected with both CD40 and CCR5. Primary monocyte‐derived immature DC stimulated with HSP70 produced IL‐12 p40, which showed dose‐dependent inhibition of >90% on treatment with both TAK 779 and anti‐CD40 mAb. Stimulation of IL‐12 p40 or TNF‐α by HSP70 was related to the differential cell surface expression of CCR5 in primary human immature and mature DC, and those with the homozygous ▵▵32 CCR5 mutation. These findings may be of significance in the interaction between HSP70 and immune responses of CCR5+ T cells in HIV‐1 infection, as well as in inflammatory bowel disease.