Biology subjects

Counoupas, C.

Publications and source records attributed to Counoupas, C..

10 recordsLinked to original sources

bioRxiv2026

A mobile ESX type VII secretion system enhances intracellular persistence in globally distributed Mycobacterium abscessus

Mycobacterium abscessus are non-tuberculous mycobacteria that are widespread in the environment and of increasing global clinical significance. Accumulating evidence shows that M. abscessus has emerged as an important pathogen, driven by highly drug-resistant lineages, enhanced transmissibility and the acquisition of specific virulence factors. In this study, we describe a previously uncharacterised ESX secretion system encoded on a 123-kbp plasmid identified in a clinical isolate of M. abscessus. This ESX system, termed ESX-pMA07, is distinct from ESX systems previously reported in M. abscessus in both sequence composition and locus organisation, characterised by a unique arrangement of core ESX components and low sequence identity to ESX-3, ESX-4 and plasmid-borne ESX-P systems. ESX-pMA07 was detected in geographically diverse clinical isolates but was restricted to particular genotypes within the global M. abscessus phylogeny. Transcriptional profiling revealed expression of ESX-pMA07 components in artificial cystic fibrosis media and during intracellular growth in macrophage cell lines. Using CRISPR interference, we show that inducible silencing of eccC, encoding the ATPase component of ESX-pMA07, significantly reduced intracellular survival of M. abscessus within macrophages. To our knowledge, this is the first characterisation of a functional, plasmid-borne ESX secretion system in M. abscessus, demonstrating that mobile genetic elements contribute to the pathogens intracellular persistence and may influence its evolving virulence. Author SummaryMycobacterium abscessus is a rapidly emerging, highly drug-resistant bacterium that causes chronic infections, particularly in people with underlying lung disease such as cystic fibrosis. The factors that enable certain M. abscessus strains to persist inside host cells are not fully understood. In this study, we identified a previously unrecognised type VII secretion system (ESX) encoded on a large plasmid in a clinical isolate of M. abscessus. This plasmid-borne ESX system, which we termed ESX-pMA07, is genetically distinct from the ESX systems normally found on the chromosome and was detected in geographically diverse clinical isolates, but restricted to specific lineages within the global M. abscessus population. We show that ESX-pMA07 genes are expressed under conditions relevant to lung infection and during intracellular growth in macrophages. Using inducible CRISPR interference to silence the ESX ATPase gene eccC, we demonstrate that ESX-pMA07 contributes to intracellular survival of M. abscessus in macrophages. These findings reveal that mobile genetic elements can encode functional secretion systems that enhance intracellular persistence, providing a mechanism for the emergence and spread of virulence traits in this important pathogen.

microbiology↗

bioRxiv2024

A Single Chimeric Spike Antigen Induces Pan-Sarbecovirus Immunity

Next-generation vaccines are required to address the evolving nature of SARS-CoV-2 and to protect against emerging pandemic threats from other coronaviruses. These vaccines should aim to elicit broad-protection, provide long-lasting immunity and facilitate equitable access for all populations. In this study, a panel of chimeric, full-length spike antigens were developed that incorporate mutations from previous, circulating and predicted SARS-CoV-2 variants. The lead candidate (CoVEXS5) was obtained from a high-yield production process in stable CHO cells with purity of >95%, long-term stability and elicitation of broadly cross-reactive neutralising antibodies when delivered to mice in a squalene emulsion adjuvant (Sepivac SWE). In both mice and hamsters, CoVEXS5 immunisation reduced clinical disease signs, lung inflammation and organ viral titres after SARS-CoV-2 infection, including challenge with the highly immunoevasive Omicron XBB.1.5 subvariant. In mice previously primed with a licenced protein vaccine (NVX-CoV2373), CoVEXS5 could boost T cell immunity, as well as neutralising antibodies levels against viruses from three sarbecoviruses clades. The breadth of sarbecovirus cross-reactivity elicited by CoVEXS5 exceeded that observed after boosting with the NVX-CoV2373 vaccine. These findings highlight the potential of a chimeric spike antigen, formulated in an open-access adjuvant, as a next-generation vaccine candidate to enhance cross-protection against emerging sarbecoviruses in vaccinated populations globally.

immunology↗

bioRxiv2024

Optimisation of a multiplexed, high throughput assay to measure neutralising antibodies against SARS-CoV-2 variants.

A multiplexed, lentivirus-based pseudovirus neutralisation assay (pVNT) was developed for high-throughput measurement of neutralising antibodies (nAbs) against three distinct SARS-CoV-2 spike variants. Intra-assay variability was minimised by optimising the plate layout and determining an optimal percentage transduction for the pseudovirus inoculum. Comparison of monoclonal antibody EC50 titres between single and multiplexed pVNT assays showed no significant differences, indicating reliability of the multiplexed assay. Evaluation of convalescent human sera confirmed assay validity, with consistent fold drops in EC50 for variant pseudoviruses relative to the ancestral strain observed across single and multiplexed assays. This multiplexed pVNT provides a reliable tool for assessing nAb responses against SARS-CoV-2 variants and could be used to accelerate preclinical vaccine assessment in preparation for the next coronavirus pandemic.

immunology↗

bioRxiv2024

An LNP-mRNA vaccine provides potent immunogenicity and protection against Mycobacterium tuberculosis

Mycobacterium tuberculosis remains the largest infectious cause of mortality worldwide, even with over a century of widespread administration of the only licensed tuberculosis (TB) vaccine, Bacillus Calmette-Guerin (BCG). mRNA technology remains an underexplored approach for combating chronic bacterial infections such as TB. We have developed a lipid nanoparticle (LNP)-mRNA vaccine encoding for a fusion protein of two immunogenic TB antigens, termed mRNACV2. In C57BL/6 mice intramuscularly vaccinated with mRNACV2, high frequencies of polyfunctional, antigen-specific Th1 CD4+ T cells were observed in the blood and lungs, which was associated with the rapid recruitment of both innate and adaptive immune cells to lymph nodes draining the site of immunisation. mRNACV2 vaccination provided significant pulmonary protection in M. tuberculosis-infected mice, reducing bacterial load and inflammatory infiltration in the lungs. As BCG is widely administered in infants in TB endemic countries, new TB vaccines should be able to boost the effects of BCG. Importantly, mRNACV2 enhanced immune responses and long-term protection when used to boost BCG-primed mice. These findings, which provide the first report of a highly protective LNP-mRNA vaccine for TB, highlight the potential of the LNP-mRNA platform for TB control and support further research to facilitate translation to humans.

immunology↗

bioRxiv2021

Neutralising antibodies against the SARS-CoV-2 Delta variant induced by Alhydroxyquim-II-adjuvanted trimeric spike antigens

Global control of COVID-19 will require the deployment of vaccines capable of inducing long-term protective immunity against SARS-CoV-2 variants. In this report, we describe an adjuvanted subunit candidate vaccine that affords elevated, sustained and cross-variant SARS-CoV-2 neutralising antibodies (NAbs) in multiple animal models. Alhydroxiquim-II is a TLR7/8 small-molecule agonist chemisorbed on aluminium hydroxide. Vaccination with Alhydroxiquim-II combined with a stabilized, trimeric form of the SARS-CoV-2 spike protein (termed CoVac-II) resulted in high-titre NAbs in mice, with no decay in responses over an 8-month period. NAbs from sera of CoVac-II-immunized mice, horses and rabbits were broadly neutralising against SARS-CoV-2 variants. Boosting long-term CoVac-II-immunized mice with adjuvanted spike protein from the Beta variant markedly increased levels of NAb titres against multiple SARS-CoV-2 variants; notably high titres against the Delta variant were observed. These data strongly support the clinical assessment of Alhydroxiquim-II-adjuvanted spike proteins to protect against SARS-CoV-2 variants of concern.

microbiology↗

bioRxiv2020

A single dose, BCG-adjuvanted SARS-CoV-2 vaccine induces Th1-polarized immunity and high-titre neutralizing antibodies in mice

Global control of COVID-19 requires broadly accessible vaccines that are effective against SARS-CoV-2 variants. In this report, we exploit the immunostimulatory properties of bacille Calmette-Guerin (BCG), the existing tuberculosis vaccine, to deliver a vaccination regimen with potent SARS-CoV-2-specific protective immunity. Combination of BCG with a stabilized, trimeric form of SARS-CoV-2 spike antigen promoted rapid development of virus-specific IgG antibodies in the blood of vaccinated mice, that was further augmented by the addition of alum. This vaccine formulation, BCG:CoVac, induced high-titre SARS-CoV-2 neutralizing antibodies (NAbs) and Th1-biased cytokine release by vaccine-specific T cells, which correlated with the early emergence of T follicular helper cells in local lymph nodes and heightened levels of antigen-specific plasma B cells after vaccination. Vaccination of K18-hACE2 mice with a single dose of BCG:CoVac almost completely abrogated disease after SARS-CoV-2 challenge, with minimal inflammation and no detectable virus in the lungs of infected animals. Boosting BCG:CoVac-primed mice with a heterologous vaccine further increased SARS-CoV-2-specific antibody responses, which effectively neutralized B.1.1.7 and B.1.351 SARS-CoV-2 variants of concern. These findings demonstrate the potential for BCG-based vaccination to protect against major SARS-CoV-2 variants circulating globally.

microbiology↗

bioRxiv2020

TCR affinity controls the dynamics but not the functional specification of the Th1 response to mycobacteria

The quality of T cell responses depends on the lymphocytes ability to undergo clonal expansion, acquire effector functions and traffic to the site of infection. Although TCR signal strength is thought to dominantly shape the T cell response, by using TCR transgenic CD4+ T cells with different pMHC binding affinity, we reveal that TCR affinity does not control Th1 effector function acquisition nor the functional output of individual effectors following mycobacterial infection. Rather, TCR affinity calibrates the rate of cell division to synchronize the distinct processes of T cell proliferation, differentiation and trafficking. By timing cell division-dependent IL-12R expression, TCR affinity controls when T cells become receptive to Th1-imprinting IL-12 signals, determining the emergence and magnitude of the Th1 effector pool. These findings reveal a distinct yet cooperative role for IL-12 and TCR signalling in Th1 differentiation and suggests that the temporal activation of clones with different TCR affinity is a major strategy to coordinate immune surveillance against persistent pathogens.

immunology↗

bioRxiv2020

Advax adjuvant formulations promote protective immunity against aerosol Mycobacterium tuberculosis in the absence of deleterious inflammation and reactogenicity

The development of safe and effective adjuvants is a critical goal of vaccine development programs. In this report, we defined the immunostimulatory profile and protective effect against aerosol Mycobacterium tuberculosis infection of vaccine formulations incorporating the semi-crystalline adjuvant {delta}-inulin (Advax). Advax formulated with CpG oligonucleotide and the QS-21 saponin (AdvaxCpQS) was the most effective combination, demonstrated by the capacity of CysVac2/AdvaxCpQS to significantly reduce the bacterial burden in the lungs of M. tuberculosis-infected mice. CysVac2/AdvaxCpQS protection was associated with rapid influx of neutrophils, macrophages and monocytes to the site of vaccination and the induction of antigen-specific IFN-{gamma}+/IL-2+/TNF+ polyfunctional CD4+ T cells in the lung. When compared to the highly potent adjuvant combination of monophosphoryl lipid A and dimethyldioctadecylammonium bromide (MPL/DDA), AdvaxCpQS imparted a similar level of protective efficacy yet without the profound stimulation of inflammatory cytokines and vaccination site ulceration observed with MPL/DDA. Addition of DDA to CysVac2/ AdvaxCpQS further improved the protective effect of the vaccine, which correlated with increased polyfunctional CD4+ T cells in the lung but with no increase in vaccine reactogenicity. The data demonstrate that Advax formulations can decouple protective tuberculosis immunity from reactogenicity, making them ideal candidates for human application. HighlightsO_LIAdvax adjuvant formulations improve pulmonary protection against aerosol Mycobacterium tuberculosis infection C_LIO_LIDifferent combinations of adjuvant components markedly influence the level of protection observed C_LIO_LIProtection is associated with the rapid influx of myeloid cells to the site of vaccination and the induction of antigen-specific polyfunctional CD4+ T cells in the lung. C_LIO_LIAdvax formulations abrogate vaccine-site ulceration and inflammatory cytokine production C_LI

immunology↗

bioRxiv2020

Microbial activation converts neutrophils into anti-tumor effectors

Neutrophils infiltrate most solid tumors and their presence is usually correlated with suppression of anti-tumor responses, metastasis and poor prognosis. Here we used microbial bioparticles administered into the tumor microenvironment to transform neutrophils into anti-tumor effectors. Microbially activated neutrophils acquired an effector phenotype associated with pathogen killing and lost vascular endothelial growth factor expression associated with tumor growth and metastasis. They became the dominant immune cell infiltrating the tumor and inhibited tumor growth. Using intravital two-photon microscopy microbially activated neutrophils could be seen forming close contacts with tumor cells resulting in tumor tissue remodeling and tumor cell death. Thus, microbial bioparticle treatment can endow neutrophils with anti-tumor properties, suggesting that neutrophil plasticity in cancer could be exploited for tumor killing. These data highlight a pathway for the rational development of neutrophil-based cancer therapy.

immunology↗

bioRxiv2020

Mucosal vaccination with CysVac2/Advax induces Th17 lung-resident CD4+ T cells and interleukin-17-dependant protection against Mycobacterium tuberculosis infection

The development of effective vaccines against bacterial lung infections requires the induction of protective, pathogen-specific immune responses without deleterious inflammation within the pulmonary environment. Here, we made use of a polysaccharide-adjuvanted vaccine approach to elicit resident pulmonary T cells to protect against aerosol Mycobacterium tuberculosis infection. Intratracheal administration of the multistage fusion protein CysVac2 and the delta-inulin adjuvant Advax (formulated with a TLR9 agonist) provided superior protection against aerosol M. tuberculosis infection in mice, compared to parenteral delivery. Surprisingly, removal of the TLR9 agonist did not impact vaccine protection despite a reduction in cytokine-secreting T cell subsets, particularly CD4+ IFN-{gamma}+IL-2+TNF+ multifunctional T cells. CysVac2/Advax-mediated protection was associated with the induction of lung-resident, antigen-specific memory CD4+ T cells that expressed IL-17 and ROR{gamma}t, the master transcriptional regulator of Th17 differentiation. IL-17 was identified as a key mediator of vaccine efficacy, with blocking of IL-17 during M. tuberculosis challenge reducing phagocyte influx, suppressing priming of pathogen-specific CD4+ T cells in local lymph nodes and ablating vaccine-induced protection. These findings suggest that tuberculosis vaccines such as CysVac2/Advax that are capable of eliciting Th17 lung-resident memory T cells are promising candidates for progression to human trials. ImportanceMycobacterium tuberculosis, the causative agent of tuberculosis (TB), kills more individuals each year than any other single pathogen. The only approved vaccine, BCG, administered intradermally, is unreliable in protecting against pulmonary TB, therefore a more effective vaccine is critical for global control of the disease. Vaccination in the lung would be a rational way of inducing a local memory immune response to TB, however vaccine platforms would need to deliver antigens to delicate mucosal surfaces without inducing deleterious inflammatory responses. We developed a safe mucosal vaccine which induced protection against TB lung infection in mice by inducing high levels of lung-resident T cells expressing the cytokine IL-17. Removal of IL-17 limited the influx of phagocytic cells to the lung and completely ablated protection afforded by the vaccine. This study provides new insights into mechanisms of protection against M. tuberculosis and provides a promising candidate to protect against TB in humans.

immunology↗