Induction of HIV-Blocking Anti-CCR5 IgA in Peyers's Patches without Histopathological Alterations

Abstract

The chemokine receptor CCR5 is essential for HIV infection and is thus a potential target for vaccine development. However, because CCR5 is a host protein, generation of anti-CCR5 antibodies requires the breaking of immune tolerance and thus carries the risk of autoimmune responses. In this study, performed in mice, we compared 3 different immunogens representing surface domains of murine CCR5, 4 different adjuvants, and 13 different immunization protocols, with the goal of eliciting HIV-blocking activity without inducing autoimmune dysfunction. In all cases the CCR5 sequences were presented as fusions to the Flock House virus (FHV) capsid precursor protein. We found that systemic immunization and mucosal boosting elicited CCR5-specific antibodies and achieved consistent priming in Peyer's patches, where most cells showed a phenotype corresponding to activated B cells and secreted high levels of IgA, representing up to one-third of the total HIV-blocking activity. Histopathological analysis revealed mild to moderate chronic inflammation in some tissues but failed in reporting signs of autoimmune dysfunction associated with immunizations. Antisera against immunogens representing the N terminus and extracellular loops 1 and 2 (Nter1 and ECL1 and ECL2) of CCR5 were generated. All showed specific anti-HIV activity, which was stronger in the anti-ECL1 and -ECL2 sera than in the anti-Nter sera. ECL1 and ECL2 antisera induced nearly complete long-lasting CCR5 downregulation of the receptor, and especially, their IgG-depleted fractions prevented HIV infection in neutralization and transcytosis assays. In conclusion, the ECL1 and ECL2 domains could offer a promising path to achieve significant anti-HIV activity in vivo. IMPORTANCEThe study was the first to adopt a systematic strategy to compare the immunogenicities of all extracellular domains of the CCR5 molecule and to set optimal conditions leading to generation of specific antibodies in the mouse model. There were several relevant findings, which could be translated into human trials. (i) Prime (systemic) and boost (mucosal) immunization is the best protocol to induce anti-self antibodies with the expected properties. (ii) Aluminum is the best adjuvant in mice and thus can be easily used in nonhuman primates (NHP) and humans. (iii) The Flock House virus (FHV) system represents a valid delivery system, as the structure is well known and is not pathogenic for humans, and it is possible to introduce constrained regions able to elicit antibodies that recognize conformational epitopes. (iv) The best CCR5 vaccine candidate should include either extracellular loop 1 or 2 (ECL1 or ECL2), but not N terminus domains.

ERK1-Based Pathway as a New Selective Mechanism To Modulate CCR5 with Natural Antibodies

“…ECL1 and ECL2 gave rise to stronger responses than did the N terminus; they achieved almost total CCR5 downregulation, and they blocked HIV infection (38). These findings possibly suggest that the CCR5 receptor-induced ERK1 signaling via crosstalk with b-arrestin1/2 does not exclusively depend on the ECL1 domain but on other specific stimuli, as well.…”

“…Nucleofection was performed with siRNA specific to CCR5 and NS-siRNA as a negative control; the cells were then treated with CCR5 Ab pos, CCR5 Ab neg, or RANTES, and collected at 150 min and 48 h. The 48-h incubation period was chosen on the basis of published data showing that preincubation of CD4 + T cells with CCR5 Ab pos induces complete long-lasting CCR5 downregulation. In contrast to all CCR5 ligands, which require only a few hours to achieve the downregulation, CCR5 Ab pos requires 48 h to obtain a complete CCR5 internalization (12,20,37,38). The presence of CCR5-specific puncta was analyzed by fluorescence microscopy.…”

Class B β-arrestin2-dependent CCR5 signalosome retention with natural antibodies to CCR5

“…We believe that different properties of each CCR5 extramembrane region, as revealed by the use of monoclonal antibodies to the N-terminus and to the second loop of CCR5, differentially modulate and influence receptor activity35. Moreover, in an experimental BalB/c mouse model, we recently found that ECL1 and ECL2 but not N-terminus antisera induced nearly complete long-lasting CCR5 downregulation of the receptor, thus suggesting that epitopes within each CCR5 extramembrane regions may be responsible of the different trafficking36. Indeed, we previously demonstrated that the substitution of some aminoacids within ECL1 elicits antibodies, which do not induce the long lasting internalization of CCR537, thus confirming that the epitope is extremely relevant to drive the different trafficking pathway.…”

Tackling HIV: Genetic vs. Immune CCR5 targeting

“…A recent preclinical study performed in mice have systematically addressed all aspects of anti-CCR5 immunization, including the use of adjuvants, in order to define the optimal schedule to elicit strong and long-lasting systemic and especially mucosal responses [106]. Three different immunogens, i.e.…”