Mucosal Vaccination with a Self-Adjuvanted Lipopeptide Is Immunogenic and Protective against Mycobacterium tuberculosis

Tuberculosis (TB) remains a staggering burden on global public health. Novel preventative tools are desperately needed to reach the targets of the WHO post-2015 End-TB Strategy. Peptide or protein-based subunit vaccines offer potential as safe and effective generators of protection, and enhancement of local pulmonary immunity may be achieved by mucosal delivery. We describe the synthesis of a novel subunit vaccine via native chemical ligation. Two immunogenic epitopes, ESAT61−20 and TB10.43−11 from Mycobacterium tuberculosis (Mtb), were covalently conjugated to the TLR2-ligand Pam2Cys to generate a self-adjuvanting lipopeptide vaccine. When administered mucosally to mice, the vaccine enhanced pulmonary immunogenicity, inducing strong Th17 responses in the lungs and multifunctional peripheral T-lymphocytes. Mucosal, but not peripheral vaccination, provided substantial protection against Mtb infection, emphasizing the importance of delivery route for optimal efficacy.NHMR

Synthetic protein conjugate vaccines provide protection against Mycobacterium tuberculosis in mice

The global incidence of tuberculosis remains unacceptably high, with new preventative strategies needed to reduce the burden of disease. We describe here a method for the generation of synthetic self-adjuvanted protein vaccines and demonstrate application in vaccination against Mycobacterium tuberculosis Two vaccine constructs were designed, consisting of full-length ESAT6 protein fused to the TLR2-targeting adjuvants Pam2Cys-SK4 or Pam3Cys-SK4 These were produced by chemical synthesis using a peptide ligation strategy. The synthetic self-adjuvanting vaccines generated powerful local CD4+ T cell responses against ESAT6 and provided significant protection in the lungs from virulent M. tuberculosis aerosol challenge when administered to the pulmonary mucosa of mice. The flexible synthetic platform we describe, which allows incorporation of adjuvants to multiantigenic vaccines, represents a general approach that can be applied to rapidly assess vaccination strategies in preclinical models for a range of diseases, including against novel pandemic pathogens such as SARS-CoV-2

Inhalation of Respirable Crystalline Rifapentine Particles Induces Pulmonary Inflammation

Rifapentine is an anti-tuberculosis (anti-TB) drug with a prolonged half-life, but oral delivery results in low concentrations in the lungs because of its high binding (98%) to plasma proteins. We have shown that inhalation of crystalline rifapentine overcomes the limitations of oral delivery by significantly enhancing and prolonging the drug concentration in the lungs. The delivery of crystalline particles to the lungs may promote inflammation. This <i>in vivo</i> study characterizes the inflammatory response caused by pulmonary deposition of the rifapentine particles. The rifapentine powder was delivered to BALB/c mice by intratracheal insufflation at a dose of 20 mg/kg. The inflammatory response in the lungs and bronchoalveolar lavage (BAL) was examined at 12 h, 24 h, and 7 days post-treatment by flow cytometry and histopathology. At 12 and 24 h post-treatment, there was a significant influx of neutrophils into the lungs, and this returned to normal by day 7. A significant recruitment of macrophages occurred in the BAL at 24 h. Consistent with these findings, histopathological analysis demonstrated pulmonary vascular congestion and significant macrophage recruitment at 12 and 24 h post-treatment. In conclusion, the pulmonary delivery of crystalline rifapentine caused a transient neutrophil-associated inflammatory response in the lungs that resolved over 7 days. This observation may limit pulmonary delivery of rifapentine to once a week at a dose of 20 mg/kg or less. The effectiveness of weekly dosing with inhalable rifapentine will be assessed in murine <i>Mycobacterium tuberculosis</i> infection

CXCR6-Deficiency Improves the Control of Pulmonary Mycobacterium tuberculosis and Influenza Infection Independent of T-Lymphocyte Recruitment to the Lungs

T-lymphocytes are critical for protection against respiratory infections, such as Mycobacterium tuberculosis and influenza virus, with chemokine receptors playing an important role in directing these cells to the lungs. CXCR6 is expressed by activated T-lymphocytes and its ligand, CXCL16, is constitutively expressed by the bronchial epithelia, suggesting a role in T-lymphocyte recruitment and retention. However, it is unknown whether CXCR6 is required in responses to pulmonary infection, particularly on CD4+ T-lymphocytes. Analysis of CXCR6-reporter mice revealed that in naïve mice, lung leukocyte expression of CXCR6 was largely restricted to a small population of T-lymphocytes, but this population was highly upregulated after either infection. Nevertheless, pulmonary infection of CXCR6-deficient mice with M. tuberculosis or recombinant influenza A virus expressing P25 peptide (rIAV-P25), an I-Ab-restricted epitope from the immunodominant mycobacterial antigen, Ag85B, demonstrated that the receptor was redundant for recruitment of T-lymphocytes to the lungs. Interestingly, CXCR6-deficiency resulted in reduced bacterial burden in the lungs 6 weeks after M. tuberculosis infection, and reduced weight loss after rIAV-P25 infection compared to wild type controls. This was paradoxically associated with a decrease in Th1-cytokine responses in the lung parenchyma. Adoptive transfer of P25-specific CXCR6-deficient T-lymphocytes into WT mice revealed that this functional change in Th1-cytokine production was not due to a T-lymphocyte intrinsic mechanism. Moreover, there was no reduction in the number or function of CD4+ and CD8+ tissue resident memory cells in the lungs of CXCR6-deficient mice. Although CXCR6 was not required for T-lymphocyte recruitment or retention in the lungs, CXCR6 influenced the kinetics of the inflammatory response so that deficiency led to increased host control of M. tuberculosis and influenza virus

PLGA particulate subunit tuberculosis vaccines promote humoral and Th17 responses but do not enhance control of <i>Mycobacterium tuberculosis</i> infection

<div><p>Tuberculosis places a staggering burden on human health globally. The new World Health Organisation End-TB Strategy has highlighted the urgent need for more effective TB vaccines to improve control of the disease. Protein-based subunit vaccines offer potential as safe and effective generators of protective immunity, and the use of particulate vaccine formulation and delivery by the pulmonary route may enhance local immunogenicity. In this study, novel particulate subunit vaccines were developed utilising biodegradable poly(lactic-<i>co</i>-glycolic acid) (PLGA) slow-release particles as carriers for the <i>Mycobacterium tuberculosis</i> lipoprotein MPT83, together with the adjuvants trehalose-dibehenate (TDB) or Monophosphoryl lipid A (MPL). Following delivery by the pulmonary or subcutaneous routes, the immunogenicity and protective efficacy of these vaccines were assessed in a murine model of <i>M</i>. <i>tuberculosis</i> infection. When delivered peripherally, these vaccines induced modest, antigen-specific Th1 and Th17 responses, but strong anti-MPT83 antibody responses. Mucosal delivery of the PLGA(MPT83) vaccine, with or without TDB, increased antigen-specific Th17 responses in the lungs, however, PLGA-encapsulated vaccines did not provide protection against <i>M</i>. <i>tuberculosis</i> challenge. By contrast, peripheral delivery of DDA liposomes containing MPT83 and TDB or MPL, stimulated both Th1 and Th17 responses and generated protection against <i>M</i>. <i>tuberculosis</i> challenge. Therefore, PLGA-formulated vaccines primarily stimulate strong humoral immunity, or Th17 responses if used mucosally, and may be a suitable carrier for vaccines against extracellular pathogens. This study emphasises the critical nature of the vaccine carrier, adjuvant and route of delivery for optimising vaccine efficacy against TB.</p></div

GRAPPA 2020 Research Award Recipients

At the 2021 Group for Research and Assessment of Psoriasis and Psoriatic Arthritis (GRAPPA) annual meeting, a summary of the research conducted by the recipients of the 2020 GRAPPA Research Awards was presented by the awardees. The summary of the 4 presentations is provided here

PLGA vaccination induces systemic antigen-specific cytokine responses.

<p>Frequency of cytokine-producing CD4⁺ T-lymphocytes in the spleens of mice immunised (A) i.n and (B) s.c with TDB adjuvanted vaccines, and (C) s.c with MPL adjuvanted vaccines, as assessed at four weeks following final immunisation. Antigen-specific cells were detected by intra-cellular immunostaining and flow cytometry after recall with MPT83 (10 μg/ml) for 12 hr followed by addition of Brefeldin A and further incubation for 4 hr. Data are the means ± SEM (n = 3) and are representative of two independent experiments. Statistically significant differences were determined by ANOVA with post-hoc Bonferroni comparison to adjuvant only control (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).</p

Antigen-specific IFNγ-secreting cells were induced systemically following immunisation with PLGA particulate vaccines.

<p>C57BL/6 mice were immunised with 1 mg PLGA vaccine either i.n or s.c as indicated, three times at two-weekly intervals. At four weeks after the last vaccination, lymphocytes of vaccinated mice were stimulated ex vivo with MPT83 (10 μg/ml). The numbers of MPT83-specific IFNγ-secreting cells/10⁶ lymphocytes in the spleen or DLN of mice immunised with PLGA vaccines adjuvanted with (A) TDB or (B) MPL were enumerated by ELISPOT after 20 hr antigen stimulation. The data are the means ± SEM (n = 3) and are representative of two independent experiments. Statistical significance was calculated by ANOVA with Bonferroni post-hoc comparison to adjuvant only control (*p<0.05, ***p<0.001).</p

PLGA particulate vaccination failed to induce protection against <i>M</i>. <i>tuberculosis</i> infection.

<p>C57BL/6 mice were immunised three times at two-weekly intervals with PLGA particles (n = 6) or DDA liposomes (n = 3–6), containing MPT83 adjuvanted with TDB, (A) lungs and (B) spleen, or adjuvanted with MPL, (C) lungs and (D) spleen. Six weeks following the final vaccination, mice were challenged with a low-dose aerosol of <i>M</i>. <i>tuberculosis</i> H37Rv (100 CFU). Additional mice (n = 6) were immunised with 5×10⁵ CFU BCG once by s.c injection 12 weeks before challenge. After 4 weeks the bacterial loads were enumerated in mice immunised either i.n or s.c as indicated. The data are the means ± SEM. Statistical significance was determined by Student’s <i>t</i>-test, comparing the vaccinated group to the relevant unimmunised/adjuvant only control (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).</p