An intranasal lentiviral booster reinforces the waning mRNA vaccine-induced SARS-CoV-2 immunity that it targets to lung mucosa

Abstract

As the coronavirus disease 2019 (COVID-19) pandemic continues and new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern emerge, the adaptive immunity initially induced by the first-generation COVID-19 vaccines starts waning and needs to be strengthened and broadened in specificity. Vaccination by the nasal route induces mucosal, humoral, and cellular immunity at the entry point of SARS-CoV-2 into the host organism and has been shown to be the most effective for reducing viral transmission. The lentiviral vaccination vector (LV) is particularly suitable for this route of immunization owing to its non-cytopathic, non-replicative, and scarcely inflammatory properties. Here, to set up an optimized cross-protective intranasal booster against COVID-19, we generated an LV encoding stabilized spike of SARS-CoV-2 Beta variant (LV::S_Beta-2P). mRNA vaccine-primed and -boosted mice, with waning primary humoral immunity at 4 months after vaccination, were boosted intranasally with LV::S_Beta-2P. A strong boost effect was detected on cross-sero-neutralizing activity and systemic T cell immunity. In addition, mucosal anti-spike IgG and IgA, lung-resident B cells, and effector memory and resident T cells were efficiently induced, correlating with complete pulmonary protection against the SARS-CoV-2 Delta variant, demonstrating the suitability of the LV::S_Beta-2P vaccine candidate as an intranasal booster against COVID-19. LV::S_Beta-2P vaccination was also fully protective against Omicron infection of the lungs and central nervous system, in the highly susceptible B6.K18-hACE2^IP-THV transgenic mice.

Keywords: SARS-CoV-2 emerging variants of concern; intranasal vaccination; lentiviral vaccine; mucosal booster vaccine; mucosal immunity; waning anti-COVID-19 immunity.

Graphical abstract

Figure 1

Down-selection of a S_COV-2 variant with the highest potential to induce cross-sero-neutralizing antibodies (A) Timeline of prime-boost vaccination with LV::S_Alpha, LV::S_Beta or LV::S_Gamma and (cross-) sero-neutralization assays in C57BL/6 mice (n = 4–5/group). (B) The median effective concentration (EC₅₀) of neutralizing activity of sera from vaccinated mice was evaluated before and after the boost, against pseudo-viruses carrying S_CoV-2 from D614G, Alpha, Beta, or Gamma variants. (C) The EC₅₀ of sera from C57BL/6 mice, vaccinated following the regimen detailed in (A) with LV encoding for S_D614G, either WT or carrying the K⁹⁸⁶P - V⁹⁸⁷P substitutions in the S2 domain. The EC₅₀ was evaluated before and after the boost, as indicated in (B).

Figure 2

Anti-S_CoV-2 humoral responses in mRNA-vaccinated mice, which were further i.n. boosted with LV::S_Beta-2P (A) Timeline of mRNA i.m.-i.m. prime-boost vaccination in C57BL/6 mice which were later immunized i.n. by escalating doses of LV::S_Beta-2P (n = 4–5/group) and the (cross) sero-neutralization follow-up. (B) Serum median effective concentration (EC₅₀) determined at the indicated time points against pseudo-viruses carrying S_CoV-2 from D614G, Alpha, Beta, Gamma, Delta, or Delta+ variants. (C) Anti-S_CoV-2 IgG (top) or IgA (bottom) titers in the sera 2 weeks after i.n. LV::S_Beta-2P boost. Statistical significance was determined by Mann-Whitney test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). (D) Sera EC₅₀, after the late boost given at week 15, and as determined at week 17. £ = not determined.

Figure 3

Lung B cell resident memory subset in mRNA-vaccinated mice that were further i.n. boosted with LV::S_Beta-2P The mice are those detailed in Figure 2. Mucosal immune cells were studied 2 weeks after LV::S_Beta-2P i.n. boost. (A) Cytometric gating strategy to detect lung Brm in mRNA-vaccinated mice which were further i.n. boosted with LV::S_Beta-2P. (B) Percentages of these cells among lung CD19⁺ surface IgM⁻/IgD⁻ B cells in mRNA-vaccinated mice that were further i.n. boosted with LV:S_Beta-2P. Statistical significance was determined by Mann-Whitney test (∗p < 0.05).

Figure 4

Systemic CD8⁺ T cell responses to S_COV-2 in mRNA-vaccinated mice that were further i.n. boosted with LV::S_Beta-2P The mice are those detailed in Figure 2. T-splenocyte responses were evaluated 2 weeks after LV::S_Beta-2P i.n. boost by IFN-γ ELISPOT after stimulation with S:256-275, S:536-550, or S:576-590 synthetic 15-mer peptides encompassing S_CoV-2 MHC-I-restricted epitopes. Statistical significance was evaluated by the Mann-Whitney test (∗p < 0.05).

Figure 5

Mucosal CD8⁺ T cell responses to S_COV-2 in mRNA-vaccinated mice that were further i.n. boosted with LV::S_Beta-2P The mice are those detailed in Figure 2. (A) Representative IFN-γ response by lung CD8⁺ T cells detected by intracellular cytokine staining after in vitro stimulation with a pool of S:256-275, S:536-550, and S:576-590 peptides. Cells are gated on alive CD45⁺ CD8⁺ T cells.

Figure 6

Lung T resident memory subset in mRNA-vaccinated mice that were further i.n. boosted with LV::S_Beta-2P The mice are those detailed in Figure 2. Mucosal immune cells were studied 2 weeks after an LV::S_Beta-2P i.n. boost. (A) Cytometric gating strategy to detect lung CD8⁺ T resident memory (CD44⁺CD69⁺CD103⁺), and (B) percentages of this subset among CD8⁺ CD44⁺ T cells in mRNA-vaccinated mice that were further i.n. boosted with LV::S_Beta-2P. Statistical significance was evaluated by the Mann-Whitney test (∗p < 0.05).

Figure 7

Full protective capacity of LV::S_Beta-2P i.n. boost against the Delta variant in initially mRNA-primed and boosted mice (A) Timeline of mRNA i.m.-i.m. prime-boost vaccination in C57BL/6 mice that were later immunized i.n. with 1 × 10⁸ TU/mouse of LV::S_Beta-2P (n = 4–5/group), pre-treated i.n. with Ad5::hACE-2 4 days before i.n. challenge with 0.3 × 10⁵ TCID₅₀ of SARS-CoV-2 Delta variant. (B) Comparative quantification of hACE-2 mRNA in the lungs of Ad5::hACE-2 pre-treated mice at 3 days after infection. (C) Lung viral RNA contents, evaluated by conventional E-specific (top) or sub-genomic Esg-specific (bottom) qRT-PCR at 3 days after infection. Red lines indicate the detection limits. Statistical significance was evaluated by the Mann-Whitney test (∗p < 0.05, ∗∗p < 0.01).

Figure 8

Full protective capacity of LV::S_Beta-2P used in a prime (i.m.) boost (i.n.) regimen against the Omicron variant (A) Timeline of prime-boost vaccination and i.n. challenge with 0.3 × 10⁵ TCID₅₀ of SARS-CoV-2 Omicron variant in B6.K18-hACE2^IP−THV transgenic mice (n = 5/group). (B) Lung viral RNA contents, evaluated by sub-genomic Esg-specific qRT-PCR at 5 days after infection. Red lines indicate the detection limits. Statistical significance was evaluated by the Mann-Whitney test (∗p < 0.05, ∗∗p < 0.01).