Intranasal Lentiviral Vector-Mediated Antibody Delivery Confers Reduction of SARS-CoV-2 Infection in Elderly and Immunocompromised Mice

Abstract

Vaccines for COVID-19 are now a crucial public health need, but the degree of protection provided by conventional vaccinations for individuals with compromised immune systems is unclear. The use of viral vectors to express neutralizing monoclonal antibodies (mAbs) in the lung is an alternative approach that does not wholly depend on individuals having intact immune systems and responses. Here, we identified an anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibody, NC0321, which can efficiently neutralize a range of SARS-CoV-2 variants, including alpha, beta, delta, and eta. Both prophylactic and therapeutic NC0321 treatments effectively protected mice from SARS-CoV-2 infection. Notably, we adopted viral vector-mediated delivery of NC0321 IgG1 as an attractive approach to prevent SARS-CoV-2 infection. The NC0321 IgG1 expression in the proximal airway, expressed by a single direct in-vivo intranasal (I.N.) administration of a self-inactivating and recombinant lentiviral vector (rSIV.F/HN-NC0321), can protect young, elderly, and immunocompromised mice against mouse-adapted SARS-CoV-2 surrogate challenge. Long-term monitoring indicated that rSIV.F/HN-NC0321 mediated robust IgG expression throughout the airway of young and SCID mice, importantly, no statistical difference in the NC0321 expression between young and SCID mice was observed. A single I.N. dose of rSIV.F/HN-NC0321 30 or 180 days prior to SARS-CoV-2 challenge significantly reduced lung SARS-CoV-2 titers in an Ad5-hACE2-transduced mouse model, reconfirming that this vectored immunoprophylaxis strategy could be useful, especially for those individuals who cannot gain effective immunity from existing vaccines, and could potentially prevent clinical sequelae.

Keywords: COVID-19; SIV.F/HN vectored immunoprophylaxis; monoclonal neutralizing antibody; old and immunodeficient mice; passive immunoprophylaxis.

Figure 1

Isolation of the potent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) human neutralizing antibody NC0321. (A) Germline analysis of the mAb NC0321. V-(D)-J rearrangement summary for the isolated mAb NC0321. (B) NC0321 isolated from a COVID-19 convalescent patient was tested for binding to SARS-CoV-2 spike (S), S1, S2, receptor-binding domain (RBD), N-terminal domain NTD, C-terminal domain CTD, and Chikungunya virus (CHIKV) E2 (as control) proteins in ELISA. (C) Computational models of SARS-CoV-2 RBD in complex with monoclonal antibody NC0312. Heavy (VH in cyan) and light (VL in purple) chains were colored as in (B), and the structures of the complex between hACE2 and RBD were provided as a comparison. Residues that contribute substantially to interactions are colored in green. (D) Neutralization activity of mAb NC0321 against multiple live SARS-CoV-2 variants (wild type, alpha, beta, delta, and eta) in Vero E6 cells. (E) Passive transfer of NC0321 confers protection to mice prophylactically and therapeutically; we transferred 10 mg/kg NC0321 (200 µl in PBS) into Ad5-hACE2-transduced mice (6–8 weeks) intraperitoneally 1 day before or 1 day after the intranasal (I.N.) infection with 1 × 10⁵ FFU SARS-CoV-2 strain (SARS-CoV-2/human/CHN/IQTC01/2020, GenBank: MT123290.1). Virus titers in the lungs were measured at 3 dpi. **** indicate p-values of <0.0001, *** p-values of 0.0001<p<0.001.

Figure 2

Generation of mouse-adapted SARS-CoV-2 surrogate permits lung transduction in wild-type mice. (A) Mouse-adapted SARS-CoV-2 surrogate (maS-LV) transduction in the parental HEK293T/17, mouse ACE2+TMPRSS2 or human ACE2+TMPRSS2 co-expressing HEK293T/17 cell line was determined (symbols represent mean±SEM, n=3). TMPRSS2, transmembrane protease serine 2 precursor, is important for SARS-CoV-2 spike priming as a serine protease (23). (B) Neutralization activity of NC0321 against an maS-LV expressing EGFP was confirmed by flow cytometry at the serial dilutions as indicated (n=3). An anti-spike antibody (40592-R001, Sino Biological) acted as positive control. (C) Bioluminescence imaging of mice (5-8 week old, female, n=3/group) at day 2, 7, 14, 21 and 56 dpi with the indicated p24 doses of a maS-LV expressing luciferase. Mice were naïve or received 1E11 Genome copies (GC) rAAV9.hACE2 14 days prior to maS-LV infection. (D) Time-course of bioluminescence imaging data for the indicated treatment groups after maS-LV infection at the indicated p24 dose ±hACE2 as indicated. The dotted line indicates the mean naïve background signal. (E) Area Under Curve (AUC) of bioluminescence (photons/sec/cm²/sr) values for each animal in (C) was computed, symbols represent mean±SEM for each treatment group (ANOVA, Dunnett’s multiple comparison of AUC between naïve and hACE2-expressing BALB/c mice). The right panel indicates the AUC in naïve and hACE2-expressing mice following treatment with 400ng p24 maS-LV (ns represents p > 0.05).

Figure 3

rSIV.F/HN mediated NC0321 mAb expression in young, old, and SCID mice against maS-LV infection. (A) Bioluminescence imaging of young (5-9 weeks weeks), old (7-8 months weeks), and SCID mice (6 months weeks) dosed with 5E8 TU rSIV.F/HN-NC0321 or an rSIV.F/HN vector expressing an irrelevant isotype control mAb; at 21 dpi, mice were challenged with 100 ng maS-LV-expressing luciferase. Imaging was performed 7 days post-maS-LV challenge. Representative images of n = 4–8/group are shown. (B) The bioluminescent value (photons/s/cm²/sr) for each animal in (A) was normalized such that the isotype control value was 100%. Naive values are from animals that did not receive maS-LV. Bars represent mean ± SEM (t-test; ** represents p = 0.0095 and **** represents p < 0.0001, n = 4–8 per group). (C) The human IgG levels in sera and epithelial lining fluid (ELF) in each group with NC0321 or isotype expressing vector transduction were determined. Human IgG levels in ELF were computed by comparison of urea levels in BALF and serum in every single sample (mean ± SEM, ordinary one-way ANOVA. ns represents p > 0.05 and **** represents p < 0.0001). Naive value, without urea normalization, is indicated by the dotted line.

Figure 4

Protection against SARS-CoV-2 infection with vector-mediated NC0321 gene transfer. Viral load was measured in the lungs of groups (n = 3) of BALB/ c mice 1 and 3 dpi with the 1E5 FFU SARS-CoV-2 strain (SARS-CoV-2/human/CHN/IQTC01/2020, GenBank: MT123290.1). Groups had previously received 1E8 TU rSIV.F/HN-NC0321, rSIV.F/HN-NC0321DAAG, or rSIV.F/HN expressing an isotype control (A), as indicated, 30 (B) or 180 days (C) previously. All animals received Ad5-hACE2 5 days prior to SARS-CoV-2 infection. Lungs were harvested for viral titers at 1 and 3 dpi (n = 3 mice per group). Dotted line indicates limit of detection (LOD); gm indicates gram lung tissue. ns, **, *** and **** represent p > 0.05, p < 0.05, < 0.001 and < 0.0001, respectively.