Influence of Aerosol Delivered BCG Vaccination on Immunological and Disease Parameters Following SARS-CoV-2 Challenge in Rhesus Macaques

Abstract

The tuberculosis vaccine, Bacille Calmette-Guerin (BCG), also affords protection against non-tuberculous diseases attributable to heterologous immune mechanisms such as trained innate immunity, activation of non-conventional T-cells, and cross-reactive adaptive immunity. Aerosol vaccine delivery can target immune responses toward the primary site of infection for a respiratory pathogen. Therefore, we hypothesised that aerosol delivery of BCG would enhance cross-protective action against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and be a deployable intervention against coronavirus disease 2019 (COVID-19). Immune parameters were monitored in vaccinated and unvaccinated rhesus macaques for 28 days following aerosol BCG vaccination. High-dose SARS-CoV-2 challenge was applied by intranasal and intrabronchial instillation and animals culled 6-8 days later for assessment of viral, disease, and immunological parameters. Mycobacteria-specific cell-mediated immune responses were detected following aerosol BCG vaccination, but SARS-CoV-2-specific cellular- and antibody-mediated immunity was only measured following challenge. Early secretion of cytokine and chemokine markers associated with the innate cellular and adaptive antiviral immune response was detected following SARS-CoV-2 challenge in vaccinated animals, at concentrations that exceeded titres measured in unvaccinated macaques. Classical CD14+ monocytes and Vδ2 γδ T-cells quantified by whole-blood immunophenotyping increased rapidly in vaccinated animals following SARS-CoV-2 challenge, indicating a priming of innate immune cells and non-conventional T-cell populations. However, viral RNA quantified in nasal and pharyngeal swabs, bronchoalveolar lavage (BAL), and tissue samples collected at necropsy was equivalent in vaccinated and unvaccinated animals, and in-life CT imaging and histopathology scoring applied to pulmonary tissue sections indicated that the disease induced by SARS-CoV-2 challenge was comparable between vaccinated and unvaccinated groups. Hence, aerosol BCG vaccination did not induce, or enhance the induction of, SARS-CoV-2 cross-reactive adaptive cellular or humoral immunity, although an influence of BCG vaccination on the subsequent immune response to SARS-CoV-2 challenge was apparent in immune signatures indicative of trained innate immune mechanisms and primed unconventional T-cell populations. Nevertheless, aerosol BCG vaccination did not enhance the initial clearance of virus, nor reduce the occurrence of early disease pathology after high dose SARS-CoV-2 challenge. However, the heterologous immune mechanisms primed by BCG vaccination could contribute to the moderation of COVID-19 disease severity in more susceptible species following natural infection.

Keywords: Aerosol BCG vaccination; COVID-19; SARS-CoV-2; cross-protection; macaque; non-specific; trained immunity.

Figure 1

Study timeline relative to aerosol BCG vaccination and disease outcome measures following SARS-CoV-2 challenge. (A) Rhesus macaques received aerosol BCG vaccination (n = six) at study day zero or were left as unvaccinated controls (n = six, reducing to five after day 28). Animals received SARS-CoV-2 by split intranasal and intrabronchial challenge (target dose 5 × 10⁶ PFU) at day 28 and were monitored for up to 8 days post challenge (pc). Blue shaded circles represent procedures involving blood sample collection and application of immunological analyses; large circles represent key study events: vaccination and SARS-CoV-2 challenge, application of in vivo CT scanning is indicated. All animals were euthanized, and postmortem necropsies conducted upon completion of the study schedule (black circles) at days 34–36 (six–eight post challenge). (B) CT total score recorded at day five post-challenge. (C) Heatmap of pulmonary histopathology scores recorded in individual animals (males and females indicated). (D) Total lung histopathology scores. (E) Representative lung sections from animals from each group stained with either H&E or for expression of SARS-CoV-2 RNA. (F) Percentage area positive for SARS-CoV-2 staining in lung tissue sections. (G) SARS-CoV-2 viral RNA recovered from nasal swabs. Lower limits of detection (LLOD) and quantification (LLOQ) are indicated. (H–K) SARS-CoV-2 viral RNA recovered from (H) BAL samples collected at necropsy; (I) lung; (J) tonsil; and (K) trachea tissue samples. Plots show group median values (+/- IQR, plot G) with dots representing individual animals. Blue = BCG vaccinated, black = no vaccine.

Figure 2

Antigen-specific IFN-γ producing cell frequency as detected by ELISPOT assay in PBMCs, lung MNC and splenocytes after aerosol BCG vaccination and/or SARS-CoV-2 challenge. Frequencies of (A) tuberculin PPD-, (B) PPD avium-, (C) CMV-, (D) spike protein MP1-, (E) spike protein MP2-, (F) spike protein MP3-, (G) spike complete peptide pool (S)-, (H) membrane (M)-, and, (I) nucleocapsid (N)-specific IFN-γ SFU measured before and after aerosol BCG vaccination or SARS-CoV-2 challenge. Antigen-specific IFN-γ SFU measured in (J) PBMCs, (K) lung MNCs and (L) splenocytes collected at necropsy. Black = unvaccinated control group, blue = aerosol BCG vaccinated. Medians shown. Mann–Whitney U tests were used for comparisons between groups (p ≤ 0.05) and Wilcoxon matched pair test for comparisons of time points within groups (p ≤ 0.05).

Figure 3

Immunophenotyping applied to whole blood before, and after aerosol BCG vaccination and SARS-CoV-2 challenge. (A) CD3+ T-cells, (B) CD4+ T-cells, (C) CD8+ T-cells, (D) Tregs, (E) CD4+ CD95+ T-cells, (F) CD8+ CD95+ T-cells, (G) NK T-cells, (H) CD14+ CD16+ monocytes, (I) Vδ2 cells, (J) Vδ1 cells, (K) γδ+ CD4+ cells, (L) γδ+ CD4+ cells as a proportion of CD3+ cells. All cell populations shown as counts/µl. Group medians are shown with data from individual animals represented by a dot. Blue = BCG vaccinated (n = six), black = unvaccinated (n = 12 or six, reducing to 11 and five post challenge). Significant differences determined by the Wilcoxon signed-rank test for comparisons within groups (colour coded by group) and Mann–Whitney U-test for comparisons between groups are denoted by asterisks. *p ≤ 0.05, **p = 0.01.

Figure 4

Chemokine markers in serum by multiplex bead array assay. Plots show titres of selected chemokine markers measured in serum samples collected from individual animals with group median values plotted as lines. (A) MIP-1α, (B) MIP-1β, (C) I-TAC, (D) eotaxin, (E) MCP-1, (F) IL-8, (G) TNF-α, (H) IL-1β, (I) IL-18, (J) IL-6, (K) IFNα, (L) IL-12p70, (M) IFN-γ, (N) IL-2, (O) IL-10, (P) IL-1RA, (Q) IL-5. Aerosol BCG vaccination and SARS-CoV-2 challenge are indicated by dotted lines. Asterisks indicate significant differences between pre- and postinfection values (colour coded to vaccination group) measured by the Wilcoxon matched-pair test. Red asterisks indicate significant differences between the groups determined by the Mann–Whitney U-test (p ≤ 0.05).