293T Lenti-X cells (Clontech, Mountain View, CA, USA) were kept in Dulbecco’s modified Eagles medium, with high glucose 4.5 g/L (Gibco, Life Technologies Italia, Monza, Italy) supplemented with 100 units/ml penicillin/streptomycin (Gibco) and 10% fetal bovine serum (Corning, Merk Life Science S.r.l., Milan, Italy), and were utilized for the production of SIV-based IDLV expressing the optimized Spike (IDLV-S-2PFGC, hereinafter referred to as IDLV-S) or the control GFP (IDLV-Mock) by transient transfection, as described (33, 34). In brief, cells (3.5 × 10⁶ cells) were plated on 10 cm Petri dishes (Corning) and transfected with i) the self-inactivating lentiviral transfer vector plasmid pGAE-S2PFGC, encoding the Wuhan-Hu-1 Spike ORF stabilized by the introduction of two consecutive prolines in S2 (2P, K986P and V987P), by functional mutation of RRAR into GSAS in the furin cleavage site (FCS), by inclusion of the D614G mutation and by deletion of the terminal 21 amino acid (aa) of the cytoplasmic tail (delta21) (33) or pGAE-GFP, encoding the control antigen GFP, ii) the SIV-based integrase-defective packaging plasmid pAdSIVD64V (33, 35) and iii) the phCMV-VSV.G plasmid, expressing the pseudotyping vesicular stomatitis virus envelope glycoprotein G (VSV.G) (33, 35), using JetPrime transfection kit (Polyplus Transfection, Illkirch, France) following the procedure of the manufacture. Forty-eight hours post transfections, the cell culture supernatants containing the vectors were harvested, filtered through a 0.45 μm pore size filter (Millipore Corporation, Billerica, MA, USA) and ultracentrifuged at 65,000 × g at 4°C for 2.5 h on a 20% sucrose cushion. Vector particles were resuspended in 1× phosphate buffered saline (PBS, Gibco) and stored at −80 °C. Each stock of IDLV was titered using the reverse transcriptase (RT) activity assay and the corresponding transducing units (TU) were determined by comparing the recovered RT activity with that of IDLV-GFP virions with known infectious titers (36).

Female and male BALB/c mice were acquired from Envigo RMS (San Pietro al Natisone, Udine, Italy) and subsequently kept in the animal facility at the Istituto Superiore di Sanità (ISS, Rome, Italy) under pathogen-free conditions. Procedures involving all murine protocols were always performed in accordance with the Italian legislation and European Union guidelines for animal care. Studies were authorized by the Italian Ministry of Health (Authorization n. 731/2020-PR, 21/7/20, prot. D9997.107) and were reviewed by the Service for Animal Welfare at ISS. Seven weeks old female and male mice were immunized once intramuscularly with 10 × 10⁶ RT units/mice of IDLV-S or IDLV-Mock or left untreated. Sampling of retro orbital blood was performed with glass Pasteur pipettes and sera were collected prior the immunization and monthly thereafter and stored at -80 °C. One week or 24 weeks after immunization, mice were euthanized by cervical dislocation and draining lymph nodes (LNs) and spleens were collected and processed for the analysis of cellular responses, including quantification of germinal center (GC) B cells and T follicular helper (Tfh) cells by flow cytometry, and Spike-specific T cells by FluoroSpot.

To evaluate the GC activation in the IDLV-S immunized mice, draining inguinal and popliteal LNs were collected 1 week after the immunization and homogenized into a single-cell suspension. Cells were stained with live/dead near IR (Thermo Fisher Scientific, Waltham, MA, USA) and Fc blocked with anti-CD16/CD32 monoclonal antibody (cat. number 156604, BioLegend, San Diego, CA, USA, 1:200) prior to staining. The cells were afterward stained with a mixture of monoclonal antibodies, including anti-CD19 Brilliant Violet 421™ (cat. number 152421, 1:500), anti-GL7 PE (cat. number 144608, 1:200), anti-Fas AF647 (cat. number 152620, 1:500), anti-CD4 FITC (cat. number 100510, 1:500), anti-PD-1 Brilliant Violet 421™ (cat. number 135217, 1:200), anti-CXCR5 PE/Cyanine7 (cat. Number 145516, 1:50), which were purchased from BioLegend and anti-CD3 BB700 (cat. Number 745836, 1:200) purchased from BD Bioscences (Franklin Lakes, NJ, USA). Next, the cells were fixed, resuspended in PBS, and analyzed on a Cytoflex S flow cytometer (Beckman Coulter Life Sciences, Brea, CA, USA), using CytExpert analysis software (Beckman Coulter Life Sciences). Gating strategies are showed in Supplementary Figure 1.

Lentiviral vectors expressing luciferase (LV-Luc) and pseudotyped with Spike variants were generated by transient transfection of 293T Lenti-X cells, using pGAE-LucW transfer vector expressing luciferase, the integrase competent packaging plasmid pAdSIV3+, and each pseudotyping plasmid expressing the Spike proteins from SARS-CoV-2 VoC, as previously described (33, 37). In brief, plasmids pSpike-C3, pSpike-INC3, pSpike-BA.1C3, pSpike-BA.2C3 and pSpike-BA.4/5C3, encoding respectively the codon optimized cytoplasmic-truncated Spike ORF from Wuhan-Hu-1, B.1.617.2 (Delta), B.1.1.529 (Omicron) BA.1, BA.2 and BA.4/5 were used for pseudotyping the LV-Luc. All preparations of LV-Luc pseudoviruses were titered on Vero E6 cells (Cercopithecus aethiops derived epithelial kidney, ATCC C1008) as previously described (37).

For neutralization assay, serial 2-fold dilutions of sera starting from 1:80 were incubated in duplicate with each LV-Luc pseudotype (3 × 10⁵ relative light units, RLU) at 37 °C for 30 min in 96-deep well plates (Resnova, Roma, Italy). The mixture was then added to Vero E6 cells (2.2 × 10⁴ cells/well) seeded in a 96-well Isoplate (Revvity, Inc, Waltham, MA, USA). Cell-only and virus-only controls were included. After forty-eight hours, luciferase expression was measured by using the Britelite plus Reporter Gene Assay System (Revvity, Inc.). The inhibitory dilution (ID) 50 reported in the results corresponds to the dilution of serum showing 50% inhibition of the infection (corresponding to neutralization), calculated from virus-only control wells (100% infection). ID50 was calculated using a linear interpolation method (31).

Suspensions of splenocytes were obtained following mechanical disruption of spleens in the presence of 3 mL of Ammonium-Chloride-Potassium (ACK) lysing buffer followed by passage through cell strainers (Corning). Splenocytes were then washed using complete medium (RPMI 1640 medium supplemented with 1% penicillin/streptomycin, 2 mM L-glutamine, 10% FBS, and 50 mM 2-mercaptoethanol), centrifuged at 1500 rpm for 10 min at 4 °C, and then counted. The assay was performed using Mabtech reagents and protocol (FluoroSpot Plus, Mabtech AB, Sweden). Splenocytes were seeded in 96-well plates, previously coated with anti-IFNγ and anti-TNFα antibodies, at a density of 2.5 × 10⁵/well and thereafter stimulated overnight either with 1 µg/mL of SARS-CoV-2 Wuhan Spike peptide pool of 15-mer sequences with 11 amino acids overlap (PepTivator^® SARS-CoV-2 Prot_S complete, Miltenyi Biotec, Bologna, Italy) or with 5 µg/ml of a H2-D^d-restricted SARS-CoV-2-Spike 9-mer peptide (KNKCVNFNF, ProImmune Ltd., Oxford, UK (38, 39), as specific stimulation. Positive control included Concanavalin A (5 µg/mL, Sigma Chemicals; St. Louis, MO, USA), while the complete medium was used as negative control. Wells were washed and incubated with anti-IFNγ and anti-TNFα detection antibodies and then with fluorophore-conjugates for 60 min. The number of Spot Forming Cells (SFC) was counted using a FluoroSpot reader (AID iSpot, AID GmbH, Strassberg, Germany) and results were expressed as SFC/10⁶ cells. The number of SFC counted in the wells treated with the medium (background) was subtracted from the number of SFC counted in the wells treated with the specific peptides. Samples were recorded positive when the number was equivalent to at least 50 specific SFC/10⁶ cells and was at least two-fold higher than the background values.

Data and graphs were prepared using GraphPad Prism 9.4.1 (GraphPad Software Inc., San Diego CA, USA). The normality of data distributions was verified by the Shapiro-Wilk test. Data were analyzed by repeated-measure (RM) one-way ANOVA or unpaired t test, as indicated. Values of P < 0.05 were considered to infer statistical significance.

We first investigated the GC responses after IDLV vaccine injection in both female and male mice. Groups of female and male BALB/c mice were immunized intramuscularly with IDLV-S (N = 12 per sex) or left untreated (naïve, N = 9 per sex). The draining inguinal and popliteal lymph nodes (LNs) were collected 1 week after the immunization (Figure 1A). The total number of the LN derived lymphocytes was significantly higher in vaccinated mice compared to naïve, as expected (p<0.0001 and p<0.05 in females and males, respectively), as well as in females compared to male mice (p<0.01) (Figure 1B). To detect GC B cells and Tfh cells, lymphocytes were stained and analyzed by flow cytometry. GC B cells were evaluated as CD3- CD19+ GL7+ CD95+ and the Tfh cells as CD3+ CD4+ CXCR5+ PD1 +. The gating strategy for the identification of the indicated cell populations is depicted in Supplementary Figure 1. One week after immunization percentages of GC B cells and Tfh cells were significantly increased in the IDLV-S vaccinated groups compared to naïve mice, irrespective of sex (Figure 1C). In particular, GC B cells ranged from 7.4 to 16.3% of total CD19+ cells and the Tfh cells ranged from 0.4 to 2.4% of the CD4+ T cells, while the percentage of GC B cells and of Tfh cells in naïve mice were much lower, ranging between 0.25-0.99 and 0.03-0.05, respectively. Importantly, vaccinated females showed significantly higher GC B cells compared to males (p<0.05). Tfh cells were higher in females, but a large variability observed in both sexes probably hindered statistical significance (Figure 1C). Flow cytometry analysis from a representative experiment is shown in Figure 1D. Overall, IDLV-S immunization elicits a robust GC response characterized by coordinated GC B-cell and Tfh-cell activation, with a preferential expansion of GC B cells in females, suggesting that sex-dependent differences in early GC dynamics may contribute to the magnitude and durability of humoral immunity.

To test the immunogenicity of IDLV-S and the durability of the vaccine induced responses, groups of female or male mice were vaccinated once with IDLV-S or IDLV-Mock and the Spike-specific immune response was monitored for up to 6 months (Figure 2A).

The kinetics of nAbs in serum of immunized mice were evaluated with a pseudovirus assay based on lentiviral vector expressing luciferase (LV-Luc) pseudotyped with Spike derived from Wuhan-Hu-1 and VoC, as previously described (31, 33, 37, 40, 41). IDLV-S vaccine induced high levels of nAbs against the Wuhan-Hu-1 Spike, homologous to the vaccine strain (Figure 2B). In females the level of nAbs peaked at 3 months and remained stable up to 6 months, while immunized males showed a peak at 3 months which significantly decreased thereafter (p<0.01 between 12 and 24 weeks within males), determining a significant difference of nAb levels between females and males 24 weeks post-immunization (p<0.05). The difference was more pronounced when we analyzed the kinetics of nAbs against the Omicron BA.1 Spike protein. As shown in Figures 2C the levels of heterologous nAbs were higher in females than in males in almost all the assayed time points (p<0.05; p<0.01). No specific anti-Spike neutralizing activity was detected in the IDLV-Mock immunized mice, as expected.

Serum samples from all IDLV-S vaccinated mice collected at 20 weeks post-immunization were assayed for cross-neutralization activity against the Spike protein from Delta and Omicron BA.1, BA.2 and BA.4/5 VoC. As shown in Figure 3A, nAb titers against VoC were significantly lower compared to those measured against Wuhan-Hu-1 Spike, homologous to the vaccine sequence, and ID50 values were Delta > Omicron BA.1 > BA.2 > BA.4/5, showing a similar pattern in both sexes. However, we observed higher cross-neutralizing titers against VoC in females, with a statistically significant difference between sexes for anti-Delta and anti-BA.1 nAb titers (p<0.05 and p<0.001, respectively) (Figure 3B). Overall, these data demonstrate that a single IDLV-S immunization elicits durable anti-Spike nAb responses with distinct sex-dependent features.

IFNγ/TNFα FluoroSpot was performed on splenocytes 24 weeks after immunization using a pool of 15-mer peptides covering the entire Wuhan Spike protein and an immunodominant MHC-I-restricted Spike-derived peptide (38, 39). As shown in Figure 4, Spike-specific IFNγ, TNFα and double positive T cells were induced in all mice vaccinated with IDLV-S. In particular, the response was higher in females, as shown by the number of cytokine-producing T cells upon stimulation with ConA and Spike pool, whereas the stimulation with MHC-I-restricted peptide did not induce a statistically significant different response between females and males. Mice immunized with IDLV-Mock did not show any Spike-specific T cell response and ConA stimulation induced higher, but not significant, number of SFC in females (data not shown). These findings confirm that IDLV-S vaccination induces a durable Spike-specific T cell immunity which is detectable at late time points, with females displaying a higher magnitude of multifunctional cytokine-producing T cell responses.