Vaccine constructs and formulations

The αLang.Env vaccine constructs have been designed and produced as previously published³⁴. Briefly, the anti-Langerin mAb was developed in-house (clone 4C7³¹, GenBank sequences JX002668, JX002669) and exhibited cross-binding to both mouse and human Langerin receptors. These antibodies featured a mouse IgG2b constant region and had a dockerin (doc) domain (residues 551-625 of GenPept Sequence ID: P0C2S4.1) fused to the C-terminal end of the H-Chain³¹. αLang 4C7.doc was produced in stably transfected Expi-CHO-S cells (Thermo Fisher), captured from culture supernatant on protein A affinity matrix and purified by FPLC (Akta, GE Healthcare). Batches of αLang.doc were further purified by reverse chromatography using a SEC 26/600 column (Cytiva), to avoid any aggregates or degraded product (see Supplementary Fig S2). The HIV-1 Envelope Clade C 96ZM651 gp140 sequence (GenBank accession # AY181197.1 residues 94 to 2064) was inserted at the C-terminus of a Cohesin (Coh) domain (residues 150-169 appended to residues 165–314 of GenPept Sequence ID: CAA47806.1) via a flexV1 flexible linker spacer (residues 468–494 of GenPept Sequence ID: AJD85777.1)³¹ and produced independently, captured on EPEA C-tag column and purified by FPLC. All recombinant mAbs were stored in 1 M Arginine 100 mM Tris-HCl (pH 7; 4 mg/mL; −80 °C) and dialyzed into Dulbecco’s phosphate buffered saline (DPBS) before immunization. Quality controls were done by SDS-PAGE followed by Coomassie staining. All reagents and final products utilized in this study tested less than 0.5 ng lipopolysaccharide per milligram of protein (endotoxin kit, ThermoFisher). His-tagged trimeric HIV-1 Envelope proteins were produced in stably transfected Expi-CHO-S cells, purified on a HIS-capture column (ThermoFisher) and then biotinylated according to manufacturer’s instructions (Avidity, US).

The αLang.Env was prepared by an equimolar mix of Coh antigen and the doc vehicle, 1 h at 37 °C in DPBS containing calcium and magnesium. The OVA.CT.Alum formulation consisting of 10 µg per dose of ovalbumin imject^™ (Thermo Fisher Scientific), adjuvanted with Cholera Toxin (Sigma Aldrich) (0.1 µg/dose) adsorbed in Thermo Scientific Imject^™ Alum Adjuvant (200 µg/dose) was prepared in sterile DPBS 1x (Sigma-Aldrich) 24 hours prior to immunizations.

Mice

Xcr1^Cre-mTFP1 mice were generated as previously described³⁸ and were crossbred with Rosa26^LSL-DTA [B6.129P2-Gt(ROSA)26Sor^{tm1(DTA)Lky/J}] mice⁵³, generating heterozygous Xcr1^DTA mice where Cre-inducible deletion of the floxed-STOP cassette allows for the expression of diphtheria toxin subunit alpha gene (DTA*G128D) and the toxin produced in Xcr1-expressing cells causes a general ablation of cDC1 in those mice. OT-I⁵⁴ and OT-II⁵⁵ mice were kept on a CD45.1(Ly5.1)/CD45.2(Ly5.2) B6 background and were employed as donors in adoptive transfer and proliferation assays. C57BL/6^J (B6) mice were provided by Charles River. All mice were maintained on C57BL/6^J (B6) background and in a specific pathogen-free environment. All in vivo procedures were conducted in adherence to protocols approved by the Ethics Committee of Marseille (approval APAFIS number 28232-2021020512136337) in compliance with institutional, national, and European guidelines governing the ethical treatment and care of animals used in research.

Adoptive transfer of CTV-labelled OT-I and OT-II T-cells

Superficial lymph nodes (auricular, cervical, brachial, axillary and inguinal LN), mesenteric LN and spleen of transgenic OT-I or OT-II mice were harvested, and total cells suspensions were prepared by smashing the organs through a 70 µm nylon mesh. Single-cell suspensions were then washed with sterile PBS and spleen cells were subjected to red blood cells lysis using Red Blood Cell Lysis Buffer (eBioscience). OT-I and OT-II cells were isolated by negative selection and purified using CD8⁺ or CD4⁺ T cell enrichment Kits (Dynabeads Untouched mouse CD8⁺ or CD4⁺ T Cells, ThermoFisher Scientifics) according to the manufacturer’s guidelines. Purified T cells were subsequently counted and labelled with 5 µM CellTrace Violet (CTV; Molecular Probes) for 30 minutes at 37 °C, following the manufacturer’s instructions. CTV labelled OT-I or OT-II T cells were washed and resuspended in sterile DPBS at a concentration of 1.5 × 10⁷ cells/mL, and 200 μl (3 × 10⁶ cells) and were adoptively transferred into B6 mice by intravenous injection, as per standard procedures^56,57,58.

Immunizations and study design

Between n = 3–5 mice (8–12 weeks old) were sorted into different groups comprising different skin routes of immunization, and each experiment was repeated 2–3 times. Four different routes and sites of application were compared: i. subcutaneous (s.c.), to access hypodermis at the axillary flank of mice; ii. intradermal (i.d.) to access preferably dermis of the dorsal side of the ear pinna skin; iii. transcutaneous (t.c.) to access both the epidermis and dermis of the dorsal side of ear pinna skin, and iv. topical route to access preferably the epidermis of the lower ventral abdomen skin. Prior to all procedures, mice underwent anesthesia via an intraperitoneal injection containing zolazepam hydrochloride (Zoletil 20, Laboratoires Virbac, France) and 6 mg/kg of tiletamine along with 3 mg/kg of xylazine (Xilor 2%, Bio 98 Srl, Italy). Both s.c. and i.d. injections were done using 0.3 mL 30 G insulin syringes (BD Micro-Fine+ Demi 0.3 mm x 8 mm). For the t.c. groups, prior to vaccine application, the dorsal side of the ear pinna skin was electroporated using the P.L.E.A.S.E. device (Pantec Biosolutions)⁵⁹ and vaccines were topically applied on the electroporated skin. P.L.E.A.S.E. configurations were set as follows: pulse duration: 75 microseconds, number of pulses per pore: 2, pore array size: 14 square millimeters, pore density: 8 percent, fluence: 11.9 Joules per square centimeter and RepRate: 200 Hertz, as described previously³⁹. The Hilltop chamber system (Cliantha Research) was used for topical application. Mice were shaved in the lower ventral surface of abdomen, the skin was tape-stripped 25 times (Scotch 3 M Magic #810, 19 mm×33 mm) and the vaccine, embed in the cotton pad of the Hilltop, was applied onto the treated skin and left in contact for 24 hours. All groups received the same dose of vaccines (20 µg of αLang.Env or 10 µg of OVA) but the volumes were adjusted for each route of immunization. The volumes of injection for s.c., i.d. and t.c. application were equally divided between both right and left sides of axillary flank (50 µL each for s.c.) and the skin of the dorsal side of both ear pinna (10 µl each for i.d. and t.c.), respectively. The volume of vaccine used in the Hilltop device was 100 µL (embedded in the device cotton pad) applied at one site of mice abdomen skin. Mock groups, injected with the same volume of sterile DPBS at each site of immunization, were used as Negative Control in all experiments. Two different immunization schemes were used depending on the goal of each experiment and the antigen used, either OVA or αLang.Env. Twenty-four hours after adoptive transfer of CTV-labelled OT-I or OT-II T cells, B6 mice were immunized with OVA.CT.Alum formulations through the aforementioned skin routes (s.c., i.d., t.c. and topical). 72 h after immunization (day 3) skin-dLNs (auricular, brachial, axillary and inguinal) were harvested for each mouse and filtered through a 70 μm cell strainer. Cells were then rinsed with sterile DPBS and stained to follow OT-I and OT-II proliferation by FACS (Fig. 1A; Sup. Fig. S1). B6, Xcr1^Cre-mTFP1 and Xcr1^DTA were primed at day 0 with the HIV-1-gp140z targeting Langerin (αLang.Env) through the aforementioned skin routes (s.c., i.d., t.c. and topical). Three weeks later (day 21) mice were boosted with the same dose of the vaccine candidate. Around one week after the boost (day 27) mice were sacrificed and samples (blood and dLN) were collected for immunophenotyping of lymphocytes (GC, Tfh and T memory cells) as well as for measurement of Env-specific IgG levels in plasma (Fig. 2, Sup. Fig. 3).

Cell extraction and preparation

After each protocol of immunization, mice were sacrificed via a CO₂ chamber and samples including blood and lymph nodes (LN) were harvested. Total cells suspensions were prepared by smashing the LN through a 70 µm nylon mesh. Cells were washed, resuspended in DPBS/2% FCS (5 mM EDTA), counted and adjusted for 3 × 10⁶/mL for FACS staining. Approximately 100 μl of fresh blood was collected on EDTA capillary tubes via the ocular plexus of isoflurane-anesthetized animal, then RBC lysis was performed for 10 minutes and the PBMC were washed twice with DPBS/2% FCS and then stained for FACS analysis.

FACS cellular immunophenotyping

Prior to staining, lymph nodes and blood single cell suspensions were treated Fc block (clone 2.4G2) (Thermo-Fisher) in FACS buffer (DPBS at 2% of FCS and 5 mM of EDTA) at 4 °C for 15 minutes to prevent non-specific binding via Fc receptors. Cells were washed with DPBS and stained for viability with Zombie UV dye (BioLegend) at 4 °C in DPBS for 20 min. Cells were washed, and incubated for 30 min at 4 °C with in-house biotinylated trimeric HIV-1-gp140z³⁴. After washes, cells were stained for surface markers. Fluorochrome-conjugated mouse antibodies CD3 (145-2C11 or 17AZ), CD4 (GK1.5 or H129.19), CD5 (53-7.3), CD8b (H35-17.2), CD11b (MI/70), CD19 (1D3), CD38 (90/CD38 Ab90), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD62L (MEL-14), CD95 or FAS (29 F.1A12), CD138 (281-2), CD161.1c (PK136), CD279 or PD-1 (11/41), CXCR5 (2G8), GL7 (GL7), IgD (11-26c2a), IgG (Poly4053) (BioLegend), IgM (R6-60.2), MHCII (M5/114.15.2) were used for surface staining. Anti-Biotin-APC (REA76, Miltenyi Biotec) and anti-Biotin-PE (104-C5, Miltenyi Biotec) were used to detect the binding of the biotinylated trimeric HIV-1-gp140z to specific B cells. For CXCR5 staining, cells were pre-incubated with the antibody for 30 min at 4 °C in PBS/2% FCS. The remaining antibodies were mixed in FACS buffer, added following this step, and incubated at the same conditions for further 30 min. Following extra-cellular staining, cells were fixed with CytoFix solution from the Kit mouse/rat FoxP3 (eBioscience) at room temperature for 40 minutes and stained for intracellular marker Bcl6 (K112-91) diluted in CytoPerm solution for 40 minutes and subsequently rinsed and resuspended in FACS buffer. Unless specified, all antibodies were procured from BD Biosciences. 1 × 10⁴ Truecount absolute counting beads (BD) were added to the tubes before FACS acquisition allowing accurate quantification of the cells. Fluorescence was analyzed using a FACS Fortessa or FACS Symphony flow cytometers (BD Bioscience) and FlowJo software. The gating strategies for OT-I and OT-II T cell proliferation are described in Supplementary Fig. S1 and for GC B, Tfh and T memory cells in dLN and blood are described in Supplementary Fig. S3.

Plasma preparation

Blood was collected and allowed to coagulate for one hour, followed by centrifugation at 1000 x g to isolate plasma. Samples were frozen at −80 °C until analysis of humoral responses.

Humoral response measurements

Plasma levels of HIV-1-specific IgG were assessed using a specialized multivariate Luminex® assay, as detailed in prior descriptions³⁴. MagPlex beads (Bio-Rad, France), were activated by rinsing with DPBS and subsequently incubated with 6.3 μg of HIV-1 Env gp140z antigen, produced in-house, at 4 °C overnight with agitation, following the manufacturer’s guidelines. Beads bound to Env antigen were washed with DPBS, suspended in blocking buffer, and finally in 150 μl of storage buffer, counted using an Auto-2000 cell counter (Nexcelom) and stored at 4 °C in the dark until use. At the day of the experiment, the prepared beads were diluted to a concentration of 50,000 beads/mL in PBS, and 50 μl of this solution was dispensed into Bio-Plex Pro 96-well Flat Bottom Plates (Bio-Rad). After two washes with 0.05% tween PBS using a magnetic plate washer (Bio-Rad), 50 μl of individual serum samples diluted at 1:100 in DPBS were added to each well. Env-specific IgG of mice plasma beads binding occurred at room temperature for 30 minutes with agitation, followed by the addition of an anti-mouse IgG-PE secondary antibody (45 minutes at 0.5 μg/mL, ThermoFisher). The beads, suspended in 80 μl of Sheath fluid (Bio-Rad), were agitated for 5 minutes at 700 rpm on a plate shaker before being directly read on a Bioplex-200 plate reader (Bio-Rad) with an acquisition volume of 50 μl and DD gate settings of 5000–25,000. Median fluorescence intensities (MFI) were extracted using Bioplex Manager 6.1 software.

Statistical analysis

Normal distribution of the data was assessed using the Anderson-Darling test, D’Agostino and Pearson test, and Shapiro-Wilk test. Statistical analyses were conducted using either one-way ANOVA with Tukey’s post-test for normally distributed data or the Kruskal-Wallis non-parametric test with Dunn’s post-test. For two-groups analysis, non-parametric Mann-Whitney t-tests were used. A significance level of P < 0.05 was employed for determining statistical significance. Graphs and statistical analyses were generated using GraphPad Prism software (version 10.3). Figures were assembled using Adobe Illustrator (version 29.51), and schematic representations of mouse experiments were created with BioRender. Protein structures and the interaction between αLang.doc and Coh.Env were modeled using the AlphaFold3 software⁶⁰.