In addition to providing partial protection against pediatric tuberculosis, vaccination with bacille Calmette-Guérin (BCG) has been reported to confer nonspecific resistance to unrelated pulmonary pathogens, a phenomenon attributed to the induction of long-lasting alterations within the myeloid cell compartment. Here, we demonstrate that intravenous, but not subcutaneous, inoculation of BCG protects human-ACE2 transgenic mice against lethal challenge with SARS-CoV-2 (SCV2) and results in reduced viral loads in non-transgenic animals infected with an α variant. The observed increase in host resistance was associated with reductions in SCV2-induced tissue pathology, inflammatory cell recruitment, and cytokine production that multivariate analysis revealed as only partially related to diminished viral load. We propose that this protection stems from BCG-induced alterations in the composition and function of the pulmonary cellular compartment that impact the innate response to the virus and ensuing immunopathology. While intravenous BCG vaccination is not a clinically acceptable practice, our findings provide an experimental model for identifying mechanisms by which nonspecific stimulation of the pulmonary immune response promotes host resistance to SCV2 lethality.

Infection with Bacillus anthracis, the causative agent of anthrax, can lead to persistence of lethal secreted toxins in the bloodstream, even after antibiotic treatment. VHH single-domain antibodies have been demonstrated to neutralize diverse bacterial toxins both in vitro and in vivo, with protein properties such as small size and high stability that make them attractive therapeutic candidates. Recently, we reported on VHHs with in vivo activity against the protective antigen component of the anthrax toxins. Here, we characterized a new set of 15 VHHs against the anthrax toxins that act by binding to the edema factor (EF) and/or lethal factor (LF) components. Six of these VHHs are cross-reactive against both EF and LF and recognize the N-terminal domain (LF, EF) of their target(s) with subnanomolar affinity. The cross-reactive VHHs block binding of EF/LF to the protective antigen C-terminal binding interface, preventing toxin entry into the cell. Another VHH appears to recognize the LF C-terminal domain and exhibits a kinetic effect on substrate cleavage by LF. A subset of the VHHs neutralized against EF and/or LF in murine macrophage assays, and the neutralizing VHHs that were tested improved survival of mice in a spore model of anthrax infection. Finally, a bispecific VNA (VHH-based neutralizing agent) consisting of two linked toxin-neutralizing VHHs, JMN-D10 and JMO-G1, was fully protective against lethal anthrax spore infection in mice as a single dose. This set of VHHs should facilitate development of new therapeutic VNAs and/or diagnostic agents for anthrax.

Anthrax lethal toxin (LT) is a protease that activates the NLRP1b inflammasome sensor in certain rodent strains. Unlike better‐studied sensors, relatively little is known about the priming requirements for NLRP1b. In this study, we investigate the rapid and striking priming‐independent LT‐induced release of IL‐1β in mice within hours of toxin challenge. We find IL‐1β release to be a NLRP1b‐ and caspase‐1‐dependent, NLRP3 and caspase‐11‐independent event that requires both neutrophils and peptidyl arginine deiminiase‐4 (PAD4) activity. The simultaneous LT‐induced IL‐18 response is neutrophil‐independent. Bone marrow reconstitution experiments in mice show toxin‐induced IL‐1β originates from hematopoietic cells. LT treatment of neutrophils in vitro did not induce IL‐1β, neutrophil extracellular traps (NETs), or pyroptosis. Although platelets interact closely with neutrophils and are also a potential source of IL‐1β, they were unable to bind or endocytose LT and did not secrete IL‐1β in response to the toxin. LT‐treated mice had higher levels of cell‐free DNA and HMGB1 in circulation than PBS‐treated controls, and treatment of mice with recombinant DNase reduced the neutrophil‐ and NLRP1‐dependent IL‐1β release. DNA sensor AIM2 deficiency, however, did not impact IL‐1β release. These data, in combination with the findings on PAD4, suggest a possible role for in vivo NETs or cell‐free DNA in cytokine induction in response to LT challenge. Our findings suggest a complex interaction of events and/or mediators in LT‐treated mice with the neutrophil as a central player in induction of a profound and rapid inflammatory response to toxin.