IL-7-Adjuvanted Vaginal Vaccine Elicits Strong Mucosal Immune Responses in Non-Human Primates

Mucosal immune responses are crucial in protecting against pathogens entering through mucosal surfaces. However, due to poor T-cell responsiveness upon mucosal antigenic stimulation, mucosal immunity remains difficult to obtain through vaccines and requires appropriate adjuvants. We previously demonstrated that administered systemically to healthy macaques or locally expressed in the intestinal mucosa of acutely SIV-infected macaques, interleukin-7 (IL-7) triggers chemokine expression and immune cell homing into mucosae, suggesting its important role in the development of mucosal immune responses. We therefore examined whether local delivery of recombinant glycosylated simian IL-7 (rs-IL-7gly) to the vaginal mucosa of rhesus macaques could prepare the lower female genital tract (FGT) for subsequent immunization and act as an efficient mucosal adjuvant. First, we showed that local administration of rs-IL-7gly triggers vaginal overexpression of chemokines and infiltration of mDCs, macrophages, NKs, B- and T-cells in the lamina propria while MamuLa-DR+ APCs accumulated in the epithelium. Subsequent mucosal anti-DT immunization in macaques resulted in a faster, stronger, and more persistent mucosal antibody response compared to DT-immunization alone. Indeed, we detected robust productions of DT-specific IgAs and IgGs in their vaginal secretions and identified cells secreting DT-specific IgAs in their vaginal mucosa and IgGs in draining lymph nodes. Finally, the expression of chemokines involved in the organization of tertiary lymphoid structures (TLS) was only increased in the vaginal mucosa of IL-7-adjuvanted immunized macaques. Interestingly, TLSs developed around PNAd+ high endothelial venules in their lower FGT sampled 2 weeks after the last immunization. Non-traumatic vaginal administration of rs-IL-7gly prepares the mucosa to respond to subsequent local immunization and allows the development of a strong mucosal immune response in macaques, through the chemokine-dependent recruitment of immune cells, the activation of mDCs and the formation of TLSs. The localization of DT-specific IgA+ plasma cells in the upper vaginal mucosa argues for their contribution to the production of specific immunoglobulins in the vaginal secretions. Our results highlight the potential of IL-7 as a potent mucosal adjuvant to stimulate the FGT immune system and elicit vaginal antibody responses to local immunization, which is the most promising way to confer protection against many sexually transmitted diseases.

0.07ug/mL) for 1 hour at 37°C. After several washes in PBS/0.1%Tween20, 3,3',5,5'-Tetramethylbenzidine/TMB peroxidase substrate solution was added (KPL, Maryland, USA) and incubated for 10 minutes at RT, then the reaction was stopped with 1M of H 3 PO 4 . Plates were read at 450nm (SpectraMaxTM384 PLUS ELISA Microplate Reader, Molecular Devices) and duplicate experiments were performed for each sample. Data were analyzed using SoftMax Pro software (5.0.1 version, Molecular Devices). Total IgA or IgG concentrations were determined by interpolation, using the calibration line of IgA or IgG standards, respectively. For quantification of specific Igs, ELISA plates were coated with DT (1µg/mL) and incubated overnight at 4°C, washed in PBS/0.1%Tween20, blocked with 2% BSA in PBS/0.1%Tween20 for 2 hours at 37°C, incubated with dilutions of each samples overnight at 4°C, washed in PBS/0.1%Tween20, then goat anti-monkey IgA-(Abnova) or IgG-HRP conjugated antibodies (Rockland Immunochemicals) were added for 1 hour at 37°C. Finally, after several washes in PBS/0.1%Tween20, peroxidase substrate was added (TMB from KPL) and incubated for 30 minutes at 37°C and the reaction was stopped with 1M of H 3 PO 4 . Plates were read and analyzed as mentioned above. Samples with a signal at least twice above the background were considered positive. Total IgA or IgG concentrations were determined by interpolation, using serial dilutions of monkey IgGs (Rockland Immunochemicals, Gilbertsville, USA) or human IgAs (Jackson ImmunoResearch, West Grove, USA). In contrast, the absolute quantification of the concentration of DT-specific IgAs and IgGs cannot be performed due to the lack of commercially available purified monkey DTspecific IgGs and IgAs that could be used as standards. Accordingly, we normalized DTspecific IgG and IgA data at each dilution (1/d) on the concentrations of IgGs and IgAs (mg/mL) in the CVLs and expressed DT-specific IgGs or IgAs in the CVLs as OD×d/[Ig]mg/mL (3 dilutions per sample; in duplicate experiments). For each sample, data are presented for the first dilution (1/d) giving an OD at least twice above the background. In addition, a few samples were systematically tested in all the ELISA plates in order to normalize the results obtained in the different experiments. Results are expressed as OD for specific anti-DT divided by the concentration of total IgA or IgG (mg/mL) in a given sample.

Preparation of cells for ELISPOT assay.
Iliac LN and vagina were samples at necropsy from macaques of the IL-7+DT and PBS+DT groups. LN cells were isolated by crushing the organs through a 40 µm cell strainer to obtain a single cell suspension. The cells were then washed in RPMI 1640 medium containing 100 U/mL penicillin, 100 µg/mL streptomycin and 10% fetal calf serum (RPMI/PS/10% FCS), centrifuged, and cell numbers and viability were determined by trypan blue exclusion.
Vaginal cells were prepared from the upper region of the vaginal walls and from the fornix. Small pieces of tissues (<10mm 3 ) were incubated in RPMI/PS/Hepes 20mM supplemented with dispase II (2.6 U/ml, Sigma) 2h at 37°C, then washed in RPMI/PS/Hepes 20mM supplemented with 5mM EDTA, then in RPMI/PS/Hepes 20mM and each time the supernatant was removed through a 100µm cell strainer. The tissue pieces were then digested in RPMI/PS supplemented with collagenase VIII (2 mg/mL, Sigma) and DNase (20 U/mL, ROCHE) for 1h30 on a magnetic stirrer at 37°C, the supernatant decanted through a 100µm cell strainer was saved, and the collagenase/DNase treatment was repeated once with fresh medium. The cells were washed in RPMI/PS/10% FCS, passed through 70µm then 40µm cell strainers, centrifuged, and the numbers and viability of cells were determined by trypan blue exclusion.

Quantification of antibody secreting cells by B-cells ELISPOT.
Antibody secreting cells (ASCs) were assayed in Multiscreen HA plates (Merck Milipore, Molsheim, France) coated with DT (10µg/mL), overnight at 4°C. After washes with PBS, the reactive sites were blocked by incubation with RPMI/PS/10% FCS for 2 hours at 37°C. After washes, PBMCs or cell suspensions recovered from tissue were transferred into plates at 1.10 6 , 4.10 5 , 1.10 5 cells per well in RPMI/PS/10% FCS and incubated for 40 hours at 37°C in 5% CO 2 . After removal of the cells, the plates were washed again and the DT-specific antibodies were detected with either goat anti-monkey IgG-HRP (Rockland Immunochemicals) or goat anti-monkey IgA-HRP (Abnova) and incubated 4 hours at 37°C. Plates were washed and spots were detected by addition of AEC (ImmPACT AEC, VECTOR Laboratories). Spots were counted in an ELISPOT Reader (BIOREADER ® -5000-F, BioSys GmbH, Serlabo, Entraigues, France). Spot numbers were reported as DT-specific ASCs per million of PBMCs.

Supplementary Figure 1. Topical rs-IL-7gly administration induces local chemokine transcription in the vaginal mucosa.
The transcription of CXCL12 (top panels), CCL7 (middle panels) or CXCL10 (bottom panels) was quantified in vaginal biopsies sampled from macaques (n=9) one month before (PRE) and 48 hours after (POST) administration, by vaginal spray, of 1µg (light gray symbols), 5µg (dark gray symbols), 10µg (red symbols) or 15µg (black symbols) of rs-IL-7gly. Each point represents the median of 4 vaginal biopsies. For each sample, the data were normalized to HPRT mRNAs simultaneously quantified with the chemokines.

Supplementary Figure 2. Topical administration of rs-IL-7gly induces local transcription of cytokines in the vaginal mucosa.
The transcription of IL-17A and TSLP (Thymic stromal lymphopoietin) was quantified in vaginal biopsies (n=4 per macaque and time point) taken from macaques (n=5) one month before (Ctrl, white bars) and 48 hours after (IL-7 48 H , black bars) the administration of 10µg (n=3 macaques) or 15µg (n=2 macaques) of rs-IL-7gly, by vaginal spray, and normalized to HPRT mRNAs quantified simultaneously with the cytokines (mRNA copies/HPRT mRNA copy). Means and SEM of the data obtained for each of the 5 macaques are presented. Statistical differences are shown (Wilcoxon Signed-Rank Test). (A) mRNAs coding for CD132 (γc chain) and CD127 (IL-7Rα chain) were quantified in primary vaginal epithelial cells (Vag EC) obtained after Dispase/EDTA treatment of the vaginal epithelial layer, circulating T-cells, T-cells and B-cells purified from secondary lymphoid organ (SLO: spleen and lymph node) gathered from 3 healthy macaques and were normalized to HPRT mRNAs simultaneously quantified together with IL-7R mRNAs (IL-7R mRNA copies/HPRT mRNA copy). Bars and error bars represent means and SEM, respectively. Statistically significant differences are shown (Mann-Whitney U test). (B) Example of primary simian vaginal epithelial cells cultured for 2 weeks after isolation, on flask coated with collagen I in SmGM2 medium (Lonza).

IgG-and IgA-producing DT-specific plasma cells (ASC) were quantified by B-cell ELISPOT on isolated cells from the vaginal upper part and the vaginal fornix of macaques immunized
with PBS+DT (White bars, n=2 macaques) or IL-7+DT (Black bars, n=2 macaques), sampled at necropsy. Results are expressed as IgG or IgA anti-DT-specific plasma cells per 10 6 cells. Bars and error bars represent means and SD, respectively (4 replicates for Mac#1 and Mac#6, one replicate for Mac#2 and Mac#5). ASC: antibody secreting cells.

Supplementary Table 1. Oligonucleotides used for real-time PCR quantification of chemokines, cytokines and IL-7R mRNA.
Outer 3'/5' primer pairs for first amplification Inner 3'/5' primer pairs for qPCR Name Sequence Name Sequence