Native CGRP Neuropeptide and Its Stable Analogue SAX, But Not CGRP Peptide Fragments, Inhibit Mucosal HIV-1 Transmission

Abstract

BackgroundThe vasodilator neuropeptide calcitonin gene-related peptide (CGRP) plays both detrimental and protective roles in different pathologies. CGRP is also an essential component of the neuro-immune dialogue between nociceptors and mucosal immune cells. We previously discovered that CGRP is endowed with anti-viral activity and strongly inhibits human immunodeficiency virus type 1 (HIV-1) infection, by suppressing Langerhans cells (LCs)-mediated HIV-1 trans-infection in-vitro and mucosal HIV-1 transmission ex-vivo. This inhibition is mediated via activation of the CGRP receptor non-canonical NFκB/STAT4 signaling pathway that induces a variety of cooperative mechanisms. These include CGRP-mediated increase in the expression of the LC-specific pathogen recognition C-type lectin langerin and decrease in LC-T-cell conjugates formation. The clinical utility of CGRP and modalities of CGRP receptor activation, for inhibition of mucosal HIV-1 transmission, remain elusive.MethodsWe tested the capacity of CGRP to inhibit HIV-1 infection in-vivo in humanized mice. We further compared the anti-HIV-1 activities of full-length native CGRP, its metabolically stable analogue SAX, and several CGRP peptide fragments containing its binding C-terminal and activating N-terminal regions. These agonists were evaluated for their capacity to inhibit LCs-mediated HIV-1 trans-infection in-vitro and mucosal HIV-1 transmission in human mucosal tissues ex-vivo.ResultsA single CGRP intravaginal topical treatment of humanized mice, followed by HIV-1 vaginal challenge, transiently restricts the increase in HIV-1 plasma viral loads but maintains long-lasting higher CD4+ T-cell counts. Similarly to CGRP, SAX inhibits LCs-mediated HIV-1 trans-infection in-vitro, but with lower potency. This inhibition is mediated via CGRP receptor activation, leading to increased expression of both langerin and STAT4 in LCs. In contrast, several N-terminal and N+C-terminal bivalent CGRP peptide fragments fail to increase langerin and STAT4, and accordingly lack anti-HIV-1 activities. Finally, like CGRP, treatment of human inner foreskin tissue explants with SAX, followed by polarized inoculation with cell-associated HIV-1, completely blocks formation of LC-T-cell conjugates and HIV-1 infection of T-cells.ConclusionOur results show that CGRP receptor activation by full-length CGRP or SAX is required for efficient inhibition of LCs-mediated mucosal HIV-1 transmission. These findings suggest that formulations containing CGRP, SAX and/or their optimized agonists/analogues could be harnessed for HIV-1 prevention.

CGRP inhibits human Langerhans cells infection with HSV by differentially modulating specific HSV-1 and HSV-2 entry mechanisms

“…Similarly, inhibition of MDLCs-mediated HSV-1 and HSV-2 infection at 24hr pi by SAX, a metabolically stable peptide analogue of CGRP that has a longer half-life 39,40 , is more potent compared to CGRP. Hence, while SAX has lower potency than CGRP in inducing beneficial cardiovascular effects 40,41 , and inhibiting LCs-mediated HIV-1 transfer to CD4 + T-cells as we recently reported 31 , SAX might be preferable for potential clinical applications in the context of mucosal HSV infection.…”

“…To investigate the potential impact of CGRP on HSV infection, MDLCs were left untreated or pretreated for 24hr with different molar concentration of CGRP, within its effective concentration range that significantly inhibits MDLCs-mediated HIV-1 transfer to CD4 + T-cells, as we previously showed 29 . MDLCs were also pre-treated with SAX, a metabolically stable analogue of CGRP, which also inhibits MDLCs-mediated HIV-1 transfer, as we recently reported 31 . The cells were then pulsed with primary isolates of HSV-1 or HSV-2 for 2hr and infection was examined 24-48hr pi by flow were infected with different HSV-1/2 primary isolates at different MOIs (as shown in Figure 1C), infection was normalized to that of untreated cells serving as the 100% set point and averaged.…”

CGRP inhibits SARS-CoV-2 infection of bronchial epithelial cells and its pulmonary levels correlate with viral clearance in critical COVID-19 patients

“…Calu-3 cells were plated in 48-wells plate (2.5X10 5 cells/well), incubated overnight at 37°C, and pre-treated for 24h with the indicated molar concentrations of CGRP (Sigma) or its stable analogue SAX (40), as we previously described (29, 39). When indicated, the CGRP receptor antagonist BIBN4096 (Sigma) was added 15min before addition of agonists, and the antiviral drug RDV (MedChemExpress) was added at 10μM overnight before infection.…”

“…Next, Calu-3 cells were pre-treated for 24h at 37°C with CGRP within its concentration range that inhibits LCs-mediated HIV-1 and HSV-1/2 infection, as we reported (29,30). The cells were also pre-treated with the CGRP metabolically stable analogue SAX (serinyl-CGRP 2-37amide with an albumin binding fatty acid moiety in the N-terminus) that, like CGRP, exerts protective cardiovascular effects (37)(38)(39)(40) and anti-viral functions (29,30), as we reported. The cells were next washed, pulsed with SARS-CoV-2 variants for 4h, and infection was determined by flow cytometry as above.…”

Evaluation of serum calcitonin gene related peptide (CGRP) Level in HIV infected patients as an indicator of disease activity

“…4 Calcitonin gene related peptide (CGRP) is a 37-amino acid neuropeptide 5 , secreted by the sensory peripheral neurons that innervate all mucosal epithelial cells. 6 Langerhans cells (LCs) the resident antigen presenting immune cells which are interspersed in the mucosa of genital tract represents the cellular targets for HIV following sexual exposure and play a crucial role in HIV dissemination. 7 CGRP plays an essential role in immunomodulation by regulating antigen presenting cells (APCs) and their capacity to express costimulatory molecules and inhibiting antigen presentation for generation of Th1 immunity while enhancing Th2-type immunity.…”