Dominique Raze

Current pertussis vaccines protect against disease, but not against colonization by and transmission of Bordetella pertussis, whereas natural infection protects against both. The live attenuated vaccine BPZE1 was developed to mimic immunogenicity of natural infection without causing disease, and in preclinical models protected against pertussis disease and B. pertussis colonization after a single nasal administration. Phase 1 clinical studies showed that BPZE1 is safe and immunogenic in humans when administered as a liquid formulation, stored at ≤−70 °C. Although BPZE1 is stable for two years at ≤−70 °C, a lyophilized formulation stored at ≥5 °C is required for commercialization. The development of a BPZE1 drug product, filled and lyophilized directly in vials, showed that post-lyophilization survival of BPZE1 depended on the time of harvest, the lyophilization buffer, the time between harvest and lyophilization, as well as the lyophilization cycle. The animal component-free process, well defined in terms of harvest, processing and lyophilization, resulted in approximately 20% survival post-lyophilization. The resulting lyophilized drug product was stable for at least two years at −20 °C ± 10 °C, 5 °C ± 3 °C and 22.5 °C ± 2.5 °C and maintained its vaccine potency, as evaluated in a murine protection assay. This manufacturing process thus enables further clinical and commercial development of BPZE1.

Bordetella pertussis, the main causative agent of whooping cough, is a reemerging pathogen, and recent vaccine-resistant strain outbreaks and emergence of macrolides-resistant strains in China raised new concerns for control of the disease. New vaccines and potentially new antibiotics are thus needed. B. pertussis is tedious to culture and requires several days of growth to count isolated colonies on agar-based media, making large-scale screening of new anti-B. pertussis compounds or functional evaluation of large sample sizes of immune sera difficult. Here, we developed a scalable, rapid, high-throughput luminescence-based Bordetella growth inhibition assay (BGIA) to quantify surviving bacteria after treatment with anti-B. pertussis compounds. A strong correlation between luminescence and colony-forming units (r² = 0.9345, p < 0.0001) was found and the BGIA showed high sensitivity and reproducibility. We demonstrate here that the BGIA can be used to quantify resistance of B. pertussis to antibiotics, sensitivity to complement and to human serum in an easy-to-operate and fast manner. We have optimized the assay and tested the effects of different B. pertussis strains and growth conditions on serum and complement sensitivity. We also uncovered complement-independent antibody-mediated inhibition of B. pertussis growth. The BGIA can thus effectively be implemented for large-scale serum studies to further investigate anti-B. pertussis immune responses at a functional level, as well as for screening of B. pertussis strains for their resistance to antibiotics or complement, and for high-throughput screening of novel anti-B. pertussis compounds.

Summary: The heparin‐binding hemagglutinin (HBHA) is a multifunctional protein involved in adherence of Mycobacterium tuberculosis to non‐phagocytic cells and in the formation of intracytosolic lipid inclusions. We demonstrate that the expression of hbhA is regulated by a transcriptional repressor, named HbhR, in Mycobacterium marinum. The hbhR gene, located upstream of hbhA, was identified by screening a transposon insertion library and detailed analysis of a mutant overproducing HBHA. HbhR was found to repress both hbhA and hbhR transcription by binding to the promoter regions of both genes. Complementation restored production of HBHA. RNA‐seq analyses comparing the mutant and parental strains uncovered 27 genes, including hbhA, that were repressed and 20 genes activated by HbhR. Among the former, the entire locus of genes coding for a type‐VII secretion system, including esxA, esxB and pe‐ppe paralogs, as well as the gene coding for PspA, present in intracellular lipid vesicles, was identified, as was katG, a gene involved in the sensitivity to isoniazid. The latter category contains genes that play a role in diverse functions, such as metabolism and resistance to oxidative conditions. Thus, HbhR appears to be a master regulator, linking the transcriptional regulation of virulence, metabolic and antibiotic sensitivity genes in M. marinum.

The blunting effect of pertussis immunization during pregnancy on infant antibody responses induced by whole-cell pertussis (wP) vaccination is not well-defined. This randomized controlled trial (NCT02408926) followed term infants born to mothers vaccinated with tetanus, diphtheria, and acellular pertussis (Tdap) vaccine during pregnancy in Thailand. Infants received either acellular pertussis (aP)- or wP-containing vaccine at 2, 4, 6, and 18 months of age. A comparison group comprised wP-vaccinated children born to mothers not vaccinated during pregnancy. Antibodies against pertussis toxin (PT), filamentous hemagglutinin (FHA), and pertactin (PRN) were evaluated using commercial enzyme-linked immunosorbent assays. Functionality of antibodies against Bordetella pertussis was measured using Bordetella pertussis growth inhibition assay. After maternal Tdap vaccination, 158 infants vaccinated with aP-containing vaccines possessed higher antibody levels (P < .001) against all tested B. pertussis antigens postpriming compared to 157 infants receiving wP-containing vaccines. At 1 month postbooster, only anti-FHA and anti-PRN antibodies were still significantly higher (P < .001) in the aP group. Significantly higher anti-PT and anti-FHA (P < .001), but not anti-PRN immunoglobulin G, were observed among 69 wP-vaccinated infants born to control mothers compared with wP-vaccinated infants of Tdap-vaccinated mothers after primary and booster vaccination. The antibody functionality was higher in all wP-vaccinated infants at all times. Maternal Tdap vaccination inhibited more pertussis-specific responses in wP-vaccinated infants compared to aP-vaccinated infants, and the control group of unvaccinated women had highest PT-specific responses, persisting until after the booster dose. Antibody functionality was better in the wP groups. NCT02408926. Infant whole-cell pertussis (wP) vaccine responses are blunted after maternal Tdap vaccination. Pertussis antibody titers are higher in acellular pertussis (aP)– than wP-vaccinated infants of immunized mothers, yet quality of antibodies, measured as serum-mediated bacterial growth inhibition, is better after wP than aP vaccination.

The heparin-binding hemagglutinin adhesin (HBHA) is an important virulence factor of Mycobacterium tuberculosis. It is a surface-displayed protein that serves as an adhesin for non-phagocytic cells and is involved in extra-pulmonary dissemination of the tubercle bacillus. It is also an important latency antigen useful for the diagnosis of latently M. tuberculosis-infected individuals. Using fluorescence time-lapse microscopy on mycobacteria that produce HBHA-green fluorescent protein chimera, we show here that HBHA can be found at two different locations and dynamically alternates between the mycobacterial surface and the interior of the cell, where it participates in the formation of intracytosolic lipid inclusions (ILI). Compared to HBHA-producing mycobacteria, HBHA-deficient mutants contain significantly lower amounts of ILI when grown in vitro or within macrophages, and the sizes of their ILI are significantly smaller. Lipid-binding assays indicate that HBHA is able to specifically bind to phosphatidylinositol and in particular to 4,5 di-phosphorylated phosphatidylinositol, but not to neutral lipids, the main constituents of ILI. HBHA derivatives lacking the C-terminal methylated, lysine-rich repeat region fail to bind to these lipids and these derivatives also fail to complement the phenotype of HBHA-deficient mutants. These studies indicate that HBHA is a moonlighting protein that serves several functions depending on its location. When surface exposed, HBHA serves as an adhesin, and when intracellularly localized, it participates in the generation of ILI, possibly as a cargo to transport phospholipids from the plasma membrane to the ILI in the process of being formed.

Heparin-binding haemagglutinin (HBHA) is a surface-exposed virulence factor of Mycobacterium tuberculosis and is involved in the binding of mycobacteria to non-phagocytic cells, allowing for extra-pulmonary dissemination of the bacilli. Despite its surface exposure, HBHA is not produced as a pre-protein containing a typical cleavable N-terminal signal peptide and is thus likely secreted by a Sec-independent, as of yet unknown mechanism. Here, we used the bacterial adenylate cyclase two-hybrid system to identify the proteins encoded by rv0613c and mmpL14 as being able to interact with HBHA. Our study was focused on Rv0613c, as it showed more consistent interactions with HBHA than MmpL14. Deletion of its orthologous gene MSMEG_1285 in recombinant Mycobacterium smegmatis producing HBHA from M. tuberculosis resulted in the loss of proper surface exposure of HBHA, as evidenced by atomic force microscopy. Furthermore, the lack of MSMEG_1285 also abolished the clumping phenotype and rough colony morphology of the recombinant M. smegmatis and reduced its adherence to A549 epithelial cells. These phenotypes have previously been associated with surface-exposed HBHA. Thus, MSMEG_1285 is directly involved in the proper cell-surface exposure of HBHA. These observations identify MSMEG_1285/Rv0613c as the first accessory protein involved in the cell surface exposure of HBHA.