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IL-7 as an adjuvant for mucosal vaccine development
Magali Rancez, S Logerot, S. Figueiredo, B Charmeteau-De-Muylder, I Bourgault-Villada, A Couëdel-Courteille, R Cheynier, Carolina Moraes-Cabe
To cite this version:
Magali Rancez, S Logerot, S. Figueiredo, B Charmeteau-De-Muylder, I Bourgault-Villada, et al.. IL-7 as an adjuvant for mucosal vaccine development. 22nd International AIDS Conference (AIDS 2018), Jul 2018, Amsterdam, Netherlands. 2018. �hal-03000425�
IL-7 AS AN ADJUVANT FOR
MUCOSAL VACCINE DEVELOPMENT
M. Rancez
1, S. Logerot
1, S. Figueiredo
1, B. Charmeteau-de-Muylder
1, I. Bourgault-Villada
1, A.Couëdel-Courteille
2, R. Cheynier
1 1- Institut Cochin / IINSERM U 1016 / CNRS UMR 8104 / Université Paris Descartes - Sorbonne Paris Cité, Paris, France.2- Institut Cochin / IINSERM U 1016 / CNRS UMR 8104 / Université Paris Descartes - Sorbonne Paris Cité / Université Paris Diderot, Paris, France.
We here evidenced that 10µg of sIL-7, administered by spray on rhesus macaques vaginal mucosa, stimulates local transcription of CCL2, CCL5, CCL7, CCL17, CCL19, CCL20, CCL21,
CCL22, CXCL10, CXCL12, CXCL13, and CX3CL1 by 48H after administration. Local expression of these chemokines leads to massive mDC, macrophages, NK, CD4+ T-, CD8+ T-, and B-cells
infiltration into the vaginal mucosa.
Healthy rhesus macaques immunized with a cocktail of antigens (Diphtheria Toxoid + the HIV-1 gp41-P1 peptide) directly applied on the vaginal mucosa two days after vaginal sIL-7 (10µg) administration developed an earlier and stronger mucosal antigen-specific immune response as compared to animals receiving the antigenic cocktail alone. Indeed, during the immunization protocol, sIL-7 pre-treatment promoted then sustained a robust production of antigen-specific IgAs and IgGs in vaginal secretions, as detected by ELISA.
By attracting immune cells, local sIL-7 administration prepares the mucosal immune system, gathering conditions that result in enhanced antigen-specific vaginal immune responses. Moreover, throughout the immunization protocol, CXCL12, CXCL13, CCL19 and CCL21 known to contribute to tertiary lymphoid organ formation and organization (Nat Immunol 2006, 7:
344-53), which could support the induction of immune responses, in particular by sustaining productive immunoglobulin class switching in germinal centers, were increased only in the
vaginal mucosa of sIL-7 treated macaques, and sIL-7 pretreatment favored the formation of tertiary lymphoid organs genesis in the vaginal mucosa. Besides, high levels of IgAs in the vaginal secretions are produced by mucosally localized plasma cells as evidenced by reverse IHF. CXCL12 is known as mucosal chemoattractant molecule for IgA- and IgG-secreting plasma cells (J Exp Med 2002, 196(1):65-75), suggesting its role in mucosal infiltration of DT- and P1-specific plasma cells. Finally, we evidenced an Increased frequency of B-cell response in peripheral blood.
Altogether, these data demonstrate the capacity of sIL-7 to be used as a mucosal adjuvant leading to enhanced mucosal antibody response, a very promising strategy to confer protection to numerous sexually transmitted infections.
CONCLUSION
LOCAL IMPACT OF sIL-7 ADMINISTRATION BY SPRAY ON VAGINAL MUCOSA
Administration by vaginal spray of sIL-7 (10µg) induces the highest local chemokine transcription
sIL-7 administration in vaginal mucosa stimulates T-cells, B-cells, and antigen-presenting cells (macrophages and myeloid dendritic
cells) migration into the chorion
Fig 1: Impact of vaginal sIL-7 administration on chemokine transcription
(A) mRNAs coding for CXCL12 and CCL7, were quantified and normalized to HPRT mRNAs in each vaginal biopsies sampled one month before (PRE) and 48H after the
administration of 1µg (n=2), 5µg (n=2), 10µg (n=3) or 15µg (n=2) of sIL-7 (POST), by vaginal spray. Each point represents the median of a triplicate quantification in a given biopsy. (B) mRNAs coding for 19 chemokines, were quantified in triplicate and normalized to HPRT mRNAs in each vaginal biopsies sampled one month before (PRE) and 48H after the administration of 10µg (n=3) and 15µg
(n=2) of sIL-7 (POST), by vaginal spray. Each point represents the mean of all the quantifications performed per animal PRE and POST administration of the sIL-7. Four biopsies per animal PRE and POST administration. *: p<0.05 by the Wilcoxon Signed-Rank Test.
Fig 2: Immune cell recruitment into vaginal chorion of sIL-7 treated macaques
(A) Sections of monkey vaginal mucosa biopsies sampled
one month before (Ctrl), or 48H after (IL-7) the
administration of 10µg of sIL-7 by vaginal spray were labeled for CD3, CD11c, PM-2K and CD20 (Alexa-Fluor® 546)
in combinaison with CD4 or CD8, DC-SIGN, CD20 and MHC-II MamuLa-DR (Alexa-Fluor® 488), respectively. Nuclei were
stained with DAPI. EP= Pluristratified Epithelium. (B, C) Cell quantifications by image analysis of IHF staining (expressed as number of cells per 500,000 µm2 of chorion
± SEM) were realized from 20 to 32 vaginal mucosa
biopsies (8 macaques, except for PM-2K+ cells
quantification: 5 macaques) sampled one month before (Ctrl – white bars) and 48H after (IL-7 48H – black bars) the
administration of 10µg of sIL-7. (Mann-Whitney U test). A C C L 7 m R N A co p ie s/ H P R T m R N A co p y C X C L 1 2 m R N A co p ie s/ H P R T m R N A co p y Mac#1 Mac#2 Mac#3 Mac#4 Mac#8 Mac#9 Mac#5 Mac#6 Mac#7 0.0 0.1 0.2 0.3 0.4 0.0 0.1 0.2 0.3 0.4 0.0 0.1 0.2 0.3 0.4 0.0 0.1 0.2 0.3 0.4 PRE POST PRE POST PRE POST PRE POST 0 4 8 12 0 4 8 12 0 4 8 12 0 4 8 12 PRE POST PRE POST PRE POST PRE POST
IL-7: 1µg IL-7: 5µg IL-7: 10µg IL-7: 15µg Chemokine highly expressed in vaginal mucosa at baseline
Chemokine barely expressed in vaginal mucosa at baseline B 0 1 2 3 4 5 PRE POST CXCL12 CCL17 CCL5 CCL19 CXCL10 CXCL8 CCL2 ** * * * (p<0.08) 0.0 0.2 0.4 0.6 0.8 PRE POST CCL22 CCL21 CCL20 CXCL13 CX3CL1 CCL28 CCL7 * * * * * * C h e m o ki n e m R N A co p ie s/ H P R T m R N A co p y Ctrl (PRE) IL-7 48H (POST) B C CD3 -CD8+ NK CD20+ B-cells p<0.01 T-cells CD3+ CD4+ CD8+ 0 50 100 150 200 250 300 350 450 C e lls / 5 0 0 0 0 0 µ 2o f ch o ri o n 400 p<0.01 p<0.01 p<0.05 p<0.01 C e lls / 5 0 0 0 0 0 µ 2o f ch o ri o n CD11c+ PM-2K+ 0 100 200 300 400 500 600 CD209+ DR+ CD20 -mΦΦΦΦ mDC APCs p=0.076 p<0.03 p<0.01 p<0.04 CD3/CD4 200µm EP Chorion EP CD11c/DC-SIGN 200µm EP Chorion PM-2K/CD20 200µm Chorion EP CD20/DR 200µm EP Chorion CD3/CD8 Chorion EP EP 200µm Ctrl (PRE) CD3/CD4 200µm Chorion EP 200µm EP Chorion CD11c/DC-SIGN 200µm Chorion EP PM-2K/CD20 200µm EP EP Chorion CD20/DR CD3/CD8 Chorion EP 200µm IL-7 48H (POST) EP A
RESULTS
MUCOSAL ADJUVANT POTENTIAL OF sIL-7 in DT and P1 IMMUNIZATION
Preferential localization of DT-specific IgAs plasma
cells in the vaginal mucosa of sIL-7 treated macaques
Immunization protocol
Figure 3: Local sIL-7 treatment allows vaginal mucosa to mount a specific immune response and boost lead to its maintenance
Specific anti-DT IgGs (A, D) and IgAs (B, E), and anti-P1 IgAs (C, F), were quantified by ELISA in vaginal secretions of 6 rhesus macaques that received vaginal administration of either 10µg of sIL-7 (black bars, n=3) or PBS (white bars, n=3), followed at day 2 by antigen administration (DT and P1). Similar immunizations were repeated thrice, every 4 months. Results are expressed as optical density over IgG or IgA concentration at any time-point for the 3 animals from the same group in any given sample for specific anti-DT (A-B, D-E) or as ng of specific anti P1 per mg of total IgAs (C, F). Bars and error bars represent means ± SEM. Non interpretable data due to menstruations were excluded. (MANOVA with Fisher test). D0: Administration of sIL-7 or PBS; W: Week post-antigens administration.
Figure 4: DT specific IgGs plasma cells are more abundant in vaginal draining lymph nodes while DT specific IgAs plasma cells are more abundant in the vaginal mucosa of sIL-7 treated macaques at necropsy
IgG and IgA anti-DT-specific ASC were quantified by B-cells ELISPOT assay on isolated cells from iliac LN from 3 macaques per group (A) and on isolated cells from vaginal chorion from 1 animal per group (C), sampled at necropsy (group IL-7+Ags, black bars; or PBS+Ags, white bars). Results are expressed as IgG or IgA anti-DT-specific ASC per 106
cells. Bars and error bars represent means ± SEM. LN: Lymph nodes; Ags: Antigens; ASC: antibody secreting cells. (Mann-Whitney U test). Sections of monkey iliac LN (B) and vaginal fornix (D), sampled at necropsy, were labeled after incubation with DT, thanks to an anti-DT antibody. Representative examples of the draining LN of sIL-7-treated (IL-7+Ags) or ctrl (PBS+Ags) macaques co-labeled with DT (green) and anti-IgG (red, B) or anti-IgA (red, D) are shown. Nuclei were stained with DAPI. (B, D) Arrowheads indicate single-stained cells. (D) Arrows identify IgA anti-DT-specific ASC.
Local administration of sIL-7 have an adjuvant effect on mucosal DT & P1 immunization
sIL-7-dependent immunization protocol induces ectopic lymphoid follicles genesis in vaginal mucosa
DTIgG 100µm PBS+Ags B DTIgG 100µm IL-7+Ags D DTIgA 100µm DTIgA 100µm PBS+Ags IL-7+Ags A – Draining LN 0 20 40 60 80 100 120 140 IgG anti-DT ASC IgA anti-DT ASC D T -s p e ci fi c A S C p e r 1 0 6ce lls p<0.05 C – Vaginal mucosa 0 200 400 600 800 1000 IgG anti-DT ASC IgA anti-DT ASC D T -s p e ci fi c A S C p e r 1 0 6ce lls PBS+Ags IL-7+Ags
Fig 5: Ectopic lymphoid follicles in vaginal mucosa of sIL-7 treated macaques at necropsy
Sections of monkey vaginal mucosa, lower part (A1, B1), upper part (A2, B2), and vaginal fornix (A3, B3), sampled at necropsy (2 weeks after the 3rd boost)
from sIL-7-treated (A, IL-7+Ags) or control (B, PBS+Ags) macaques were labeled for CD3 (Alexa-Fluor® 546), CD20 (Alexa-Fluor® 488) and CD31
(Alexa-Fluor® 633), respectively. Nuclei were stained with DAPI. EP= Pluristratified
Epithelium; Ch= Chorion.
RESULTS
Development of mucosal immune responses, while certainly essential to allow protection against numerous sexually transmitted infections, is still hard to induce in most models. Despite some progresses in the development of adjuvants possibly helping for the setting of this kind of immunity, results in terms of protection as for induction of the local antibody production and specific T-cells remain largely disappointing.
We have showed that systemic injection of IL-7 stimulates chemokine-induced recruitment of circulating T-cells into the mucosa (Blood 2009, 114(4):816–25), and evidenced local IL-7 expression in acutely infected mucosae (Front Immunol 2017, 19;8:588).
In this study, we aimed at using recombinant glycosylated simian IL-7 (sIL-7) as an immunostimulating molecule for vaginal immune responses in vaccine protocols. We administrated sIL-7 by spray directly on rhesus macaque’s vaginal mucosa surface and quantified local transcription of 19 chemokines by qRT-PCR and cell infiltration by immunochemistry plus image analysis to define the optimal dose of sIL-7 to be sprayed on mucosal surface. Then, six macaques were immunized with antigens (Diphtheria Toxoid (DT) and the HIV-1 gp41-P1 peptide) applied directly on the vaginal mucosa, two days after either IL-7 or PBS administration. The immunizations were repeated thrice, four months apart, and the macaques were euthanized 2 weeks after the last immunization. Antigen-specific IgA and IgG productions were quantified in vaginal secretions by ELISA. Antigen-specific plasma cells were detected by reverse immunohistochemistry and by B-cell ELISPOT.
INTRODUCTION and OBJECTIVES
Blood, vaginal and rectal washes sampled (every week and before each spray)
Group IL-7+Ags (n=3) Group PBS+Ags (n=3) Biopsies Biopsies DT + P1 D2 DT + P1 IL-7 or PBS W16 W16 + D2 Boost #1 Biopsies Necropsy DT + P1 IL-7 or PBS W31 W31 + D2 Boost #2 DT + P1 W55 W55 + D2 Boost #3 IL-7 or PBS IL-7 or PBS D0 DT: Diphtheria Toxoid P1: HIV-1 gp41-P1 peptide 0 5 10 15 20 D -2 W 2 W 3 W 4 W 5 W 7 W 9 W 1 1 W 1 3 W 1 5 D -2 W 2 W 3 W 4 W 5 W 7 W 9 W 1 1 W 1 3 W 1 5 O D / [ V a g in a l Ig A s] B– Anti-DT IgA p<0.035 p<0.007 0 5 10 15 20 25 D 0 W 2 W 3 W 4 W 8 W 1 0 D 0 W 3 W 4 W 7 W 9 D 0 W 2 D 0 W 2 W 3 W 4 W 8 W 1 0 D 0 W 3 W 4 W 7 W 9 D 0 W 2 E– Anti-DT IgA O D / [ V a g in a l Ig A s] Boost n°3 Boost n°1 Boost n°2 Boost n°1 Boost n°2 Boost n°3 O D / [ V a g in a l Ig G s] O D / [ V a g in a l Ig G s] 0 20 40 60 80 100 D -2 W 2 W 3 W 4 W 5 W 7 W 9 W 1 1 W 1 3 W 1 5 D -2 W 2 W 3 W 4 W 5 W 7 W 9 W 1 1 W 1 3 W 1 5 A– Anti-DT IgG p<0.001 p<0.001 0 10 20 30 40 D 0 W 2 W 3 W 4 W 8 W 1 0 D 0 W 3 W 4 W 7 W 9 D 0 W 2 D 0 W 2 W 3 W 4 W 8 W 1 0 D 0 W 3 W 4 W 7 W 9 D 0 W 2 D – Anti-DT IgG p<0.001 Boost n°3 Boost n°1 Boost n°2 Boost n°1 Boost n°2 Boost n°3 0 5 10 15 20 25 30 D -2 W 2 W 3 W 4 W 5 W 7 W 9 W 1 1 W 1 3 W 1 5 D -2 W 2 W 3 W 4 W 5 W 7 W 9 W 1 1 W 1 3 W 1 5 C – Anti-P1 IgA n g / m g t o ta l Ig A s p<0.018 0 5 10 15 20 D 0 W 2 W 3 W 4 W 8 W 1 0 D 0 W 3 W 4 W 7 W 9 D 0 W 2 D 0 W 2 W 3 W 4 W 8 W 1 0 D 0 W 3 W 4 W 7 W 9 D 0 W 2 F– Anti-P1 IgA n g / m g t o ta l Ig A s Boost n°3 Boost n°1 Boost n°2 Boost n°1 Boost n°2 Boost n°3 p<0.030 p<0.023 IL-7+Ags PBS+Ags D 0 D 0 D 0 D 0 D 0 D 0 PBS+Ags
Lower part Upper part Vaginal fornix
CD3CD20 CD31 250µm Ch A1 IL-7+Ags Ch CD3CD20 CD31 250µm A2 EP CD3CD20 CD31 250µm Ch A3 CD3CD20 CD31 250µm Ch B1 Ch CD3CD20 CD31 250µm B2 Ch EP CD3CD20 CD31 250µm B3
Lower part Upper part Vaginal fornix
The authors thank the staffs of the Institut Pasteur (Paris, France) and Infectious Disease Models and Innovative Therapies (IDMIT, Fontenay-aux-Roses, France) Primate Centers. The authors greatly acknowledge Maryline Favier, Franck Letourneur and Pierre Bourdoncle, respectively heads of HistIM, GENOM’IC and IMAG’IC core facilities of the Institut Cochin. The authors thank Thomas Guilbert from IMAG’IC platform at the Institut Cochin for writing the routine for ImageJ image analysis software. The authors acknowledge Cytheris S.A. (now Revimmune Inc.), for R-sIL-7gly (sIL-7). This work was supported by ANRS, INSERM and Université Paris Descartes.
ACKNOWLEDGMENT
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