Intravenous Bacillus Calmette-Guérin (BCG) Induces a More Potent Airway and Lung Immune Response than Intradermal BCG in Simian Immunodeficiency Virus-infected Macaques

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the leading causes of death due to an infectious agent. Coinfection with HIV exacerbates M. tuberculosis infection outcomes in people living with HIV. Bacillus Calmette-Guérin (BCG), the only approved TB vaccine, is effective in infants, but its efficacy in adolescents and adults is limited. In this study, we investigated the immune responses elicited by BCG administered via i.v. or intradermal (i.d.) routes in SIV-infected Mauritian cynomolgus macaques (MCM) without the confounding effects of M. tuberculosis challenge. We assessed the impact of vaccination on T cell responses in the airway, blood, and tissues (lung, thoracic lymph nodes, and spleen), as well as the expression of cytokines, cytotoxic effectors, and key transcription factors. Our results showed that i.v. BCG induces a robust and sustained immune response, including tissue-resident memory T cells in lungs, polyfunctional CD4+ and CD8αβ+ T cells expressing multiple cytokines, and CD8αβ+ T cells and NK cells expressing cytotoxic effectors in airways. We also detected higher levels of mycobacteria-specific IgG and IgM in the airways of i.v. BCG-vaccinated MCM. Although i.v. BCG vaccination resulted in an influx of tissue-resident memory T cells in lungs of MCM with controlled SIV replication, MCM with high plasma SIV RNA (>105 copies/ml) typically displayed reduced T cell responses, suggesting that uncontrolled SIV or HIV replication would have a detrimental effect on i.v. BCG-induced protection against M. tuberculosis.

Figure 1:. Study design and SIV plasma viremia.

a) Study design indicating SIV infection, BCG vaccination, HRE treatment, serial BAL and blood collection, and PET-CT time points. b) Quantification of plasma viral copy equivalents over time in MCM vaccinated with ID BCG (top panel, purple; n=6) or IV BCG (bottom panel, green; n=5). The horizontal dashed line represents the LOQ of ~62 copies/mL. Plasma viral load of each group was assessed by comparing two weeks before BCG vaccination to two- or 12-weeks after BCG vaccination using mixed-effects model with uncorrected Fisher’s LSD p-values reported. Each symbol represents an individual animal. SIV controllers and non-controllers (baseline >10⁵ copies/mL) are indicated by circles and stars, respectively.

Figure 2:. Immune cell composition in airways during SIV infection and BCG vaccination.

a) Numbers of total leukocytes, T and NK cells in BAL. b) Numbers of T cell subsets in BAL. Each point represents an individual BAL with SIV non-controllers indicated by stars. Mixed-effects model with uncorrected Fisher’s LSD p-values (Table 1) are shown.

Figure 3:. T cell cytokines in airways following BCG vaccination.

(a and b) Number of CD4+ (a) and CD8αβ+ (b) T cells making mycobacterial-specific Th1- and Th17-associated cytokines in airways.

Figure 4:. CD8αβ+ T cells and NK cells cytotoxic effector in airways.

(a and b) Number of CD8αβ+ T cells (a) and NK cells (b) producing GzmB and GzmK in airways.

Figure 5:. Mycobacteria-specific antibodies in BAL and plasma following BCG vaccination.

IgG and IgM that bind WCL or LAM in plasma (a) and BALF (b). SIV non-controllers are indicated by stars. Mixed-effects model with uncorrected Fisher’s LSD p-values in Table 2 represents group comparison.

Figure 6:. T_RM cell responses in lungs 20 weeks after BCG vaccination.

Tissue-resident T cells (ivCD45-) are delineated from vascular-resident cells (ivCD45+). a) T_RM gating schematic_, b) The number of T_RM cells in lung tissue harvested 20 weeks after IV or ID BCG vaccination. c) The number of T_RM cell subsets in lung tissue. SIV non-controllers are depicted by stars. Each symbol represents the mean value from 3 lung lobes per animal. Mann-Whitney p-values are reported.

Figure 7:. Cytokines responses in lungs 20 weeks after BCG vaccination.

The number of CD4+ and CD8αβ+ T cells in the lung that make any a) Th1- and Th1-(IFNγ, IL-2, TNF), and, b) Th17- and Th17- (IFNγ, IL-2, IL-17, TNF) associated cytokines. SIV non-controllers are depicted by stars. Each symbol represents the mean value from 3 lung lobes per animal. Mann-Whitney p-values are reported.