Analytical development to support manufacturing of a sustainable vaccine against Invasive Nontyphoidal Salmonellosis

GVGH is developing a candidate trivalent Salmonella vaccine to fight invasive nontyphoidal Salmonellosis (iNTS) and typhoid fever, especially aimed for sub-Saharan Africa to impact disease burden and to reduce anti-microbial resistance spread. This trivalent vaccine may be the only viable option for a sustainable iNTS vaccine in sub-Saharan Africa over the separate administration of Typhoid Conjugate Vaccines (TCV) and a vaccine against iNTS. GVGH generated the iNTS-TCV formulation by combining the GMMA technology for the iNTS components, S. Typhimurium (STm) and S. Enteritidis (SEn) GMMA adsorbed on Alhydrogel, and the Vi-CRM197 glycoconjugate, originally developed by GVGH and recently WHO prequalified as TCV TYPHIBEV by Biological E Ltd (Hyderabad, India). A set of analytical methods to support the vaccine lot release and characterization have been developed by GVGH. In particular, to quantify the key active ingredients of iNTS components a competitive ELISA-based method (FAcE, Formulated Alhydrogel competitive ELISA assay) has been setup and characterized in terms of specificity, accuracy and precision. Vi component is instead characterized by means of HPAEC-PAD method, able to specifically identify and quantify the total polysaccharide in the final drug product. With regard to safety assessment, a Monocyte Activation Test (MAT) has been developed as to monitor the intrinsic pyrogenicity of GMMA-based vaccines and applied as surveillance test for the Phase 1 clinical lot, with the plan to set release criteria based on clinical experience. In vivo potency assay has been set to characterize the immunogenicity of vaccine lots in comparison to freshly formulated material at the time of release and during real-time stability. A significant antibody response to each of the active ingredients of the trivalent vaccine is raised in mice and assessed by Parallel Line Assay. Overall, the applied analytical panel and the results support the development of an iNTS-TCV vaccine as a viable option for a sustainable iNTS vaccine in sub-Saharan Africa

Candida albicans Scavenges Host Zinc via Pra1 during Endothelial Invasion

The ability of pathogenic microorganisms to assimilate essential nutrients from their hosts is critical for pathogenesis. Here we report endothelial zinc sequestration by the major human fungal pathogen, Candida albicans. We hypothesised that, analogous to siderophore-mediated iron acquisition, C. albicans utilises an extracellular zinc scavenger for acquiring this essential metal. We postulated that such a “zincophore” system would consist of a secreted factor with zinc-binding properties, which can specifically reassociate with the fungal cell surface. In silico analysis of the C. albicans secretome for proteins with zinc binding motifs identified the pH-regulated antigen 1 (Pra1). Three-dimensional modelling of Pra1 indicated the presence of at least two zinc coordination sites. Indeed, recombinantly expressed Pra1 exhibited zinc binding properties in vitro. Deletion of PRA1 in C. albicans prevented fungal sequestration and utilisation of host zinc, and specifically blocked host cell damage in the absence of exogenous zinc. Phylogenetic analysis revealed that PRA1 arose in an ancient fungal lineage and developed synteny with ZRT1 (encoding a zinc transporter) before divergence of the Ascomycota and Basidiomycota. Structural modelling indicated physical interaction between Pra1 and Zrt1 and we confirmed this experimentally by demonstrating that Zrt1 was essential for binding of soluble Pra1 to the cell surface of C. albicans. Therefore, we have identified a novel metal acquisition system consisting of a secreted zinc scavenger (“zincophore”), which reassociates with the fungal cell. Furthermore, functional similarities with phylogenetically unrelated prokaryotic systems indicate that syntenic zinc acquisition loci have been independently selected during evolution

Frontiers in the Standardization of the Plant Platform for High Scale Production of Vaccines

The recent COVID-19 pandemic has highlighted the value of technologies that allow a fast setup and production of biopharmaceuticals in emergency situations. The plant factory system can provide a fast response to epidemics/pandemics. Thanks to their scalability and genome plasticity, plants represent advantageous platforms to produce vaccines. Plant systems imply less complicated production processes and quality controls with respect to mammalian and bacterial cells. The expression of vaccines in plants is based on transient or stable transformation systems and the recent progresses in genome editing techniques, based on the CRISPR/Cas method, allow the manipulation of DNA in an efficient, fast, and easy way by introducing specific modifications in specific sites of a genome. Nonetheless, CRISPR/Cas is far away from being fully exploited for vaccine expression in plants. In this review, an overview of the potential conjugation of the renewed vaccine technologies (i.e., virus-like particles—VLPs, and industrialization of the production process) with genome editing to produce vaccines in plants is reported, illustrating the potential advantages in the standardization of the plant platforms, with the overtaking of constancy of large-scale production challenges, facilitating regulatory requirements and expediting the release and commercialization of the vaccine products of genome edited plants

Analysis of morphogenesis and chlamydospore formation by Candida albicans and Candida dubliniensis

THESIS 9337Candida dubliniensis is the species that is most closely related to Candida albicans. Despite their close phylogenetic relatedness, epidemiological and infection model data suggest that C. albicans is a far more successful pathogen. The reasons for this disparity in virulence are still unclear, however, it has been shown that C. dubliniensis is less able than C. albicans to produce hyphae under a wide range of in vitro and in vivo conditions. The purpose of this study was to investigate the comparative effects of glucose/galactose and methionine on morphogenesis in the two species

Outer membrane vesicles: moving within the intricate labyrinth of assays that can predict risks of reactogenicity in humans

Outer membrane vesicles (OMV) are exosomes naturally released from the surface of Gram-negative bacteria. Since the ’80s, OMVs have been proposed as powerful vaccine platforms due to their intrinsic self-adjuvanticity and ability to present multiple antigens in natural conformation. However, the presence of several pathogen-associated molecular patterns (PAMPs), especially lipid A, has raised concerns about potential systemic reactogenicity in humans. Recently, chemical and genetic approaches allowed to efficiently modulate the balance between reactogenicity and immunogenicity for the use of OMV in humans. Several assays (monocyte activation test, rabbit pyrogenicity test, limulus amebocyte lysate, human transfectant cells, and toxicology studies) were developed to test, with highly predictive potential, the risk of reactogenicity in humans before moving to clinical use. In this review, we provide a historical perspective on how different assays were and can be used to successfully evaluate systemic reactogenicity during clinical development and after licensure

Purification and germination of Candida albicans and Candida dubliniensis chlamydospores cultured in liquid media

peer-reviewedCandida albicans and Candida dubliniensis are the only Candida species that have been observed to produce chlamydospores. The function of these large, thick-walled cells is currently unknown. In this report we describe the production and purification of chlamydospores from these species in defined liquid media. Staining with the fluorescent dye FUN-1 indicated that chlamydospores are metabolically active cells, but that metabolic activity is undetectable in chlamydospores that are greater than 30 days old. However, 5-15 day old chlamydospores could be induced to produce daughter chlamydospores, blastospores, pseudohyphae and true hyphae depending on the incubation conditions used. Chlamydospores that were pre-induced to germinate were also observed to escape from murine macrophages following phagocytosis, suggesting that these structures may be viable in vivo. Mycelium-attached and purified chlamydospores rapidly lost their viability in water and when subjected to dry stress, suggesting that are unlikely to act as long-term storage structures. Instead, our data suggest that chlamydospores represent an alternative specialised form of growth by C. albicans and C. dubliniensis

Candida albicans-epithelial interactions: dissecting the roles of active penetration, induced endocytosis and host factors on the infection process

International audienceCandida albicans frequently causes superficial infections by invading and damaging epithelial cells, but may also cause systemic infections by penetrating through epithelial barriers. C. albicans is a remarkable pathogen because it can invade epithelial cells via two distinct mechanisms: induced endocytosis, analogous to facultative intracellular enteropathogenic bacteria, and active penetration, similar to plant pathogenic fungi. Here we investigated the contributions of the two invasion routes of C. albicans to epithelial invasion. Using selective cellular inhibition approaches and differential fluorescence microscopy, we demonstrate that induced endocytosis contributes considerably to the early time points of invasion, while active penetration represents the dominant epithelial invasion route. Although induced endocytosis depends mainly on Als3-E–cadherin interactions, we observed E–cadherin independent induced endocytosis. Finally, we provide evidence of a protective role for serum factors in oral infection: human serum strongly inhibited C. albicans adhesion to, invasion and damage of oral epithelial cell

Draft genomes of Shigella strains used by the STOPENTERICS consortium.

International audienceDespite a significant global burden of disease, there is still no vaccine against shigellosis widely available. One aim of the European Union funded STOPENTERICS consortium is to develop vaccine candidates against Shigella. Given the importance of translational vaccine coverage, here we aimed to characterise the Shigella strains being used by the consortium by whole genome sequencing, and report on the stability of strains cultured in different laboratories or through serial passage. We sequenced, de novo assembled and annotated 20 Shigella strains being used by the consortium. These comprised 16 different isolates belonging to 7 serotypes, and 4 derivative strains. Derivative strains from common isolates were manipulated in different laboratories or had undergone multiple passages in the same laboratory. Strains were mapped against reference genomes to detect SNP variation and phylogenetic analysis was performed. The genomes assembled into similar total lengths (range 4.14-4.83 Mbp) and had similar numbers of predicted coding sequences (average of 4,400). Mapping analysis showed the genetic stability of strains through serial passages and culturing in different laboratories, as well as varying levels of similarity to published reference genomes. Phylogenetic analysis revealed the presence of three main clades among the strains and published references, one containing the Shigella flexneri serotype 6 strains, a second containing the remaining S. flexneri serotypes and a third comprised of Shigella sonnei strains. This work increases the number of the publically available Shigella genomes available and specifically provides information on strains being used for vaccine development by STOPENTERICS. It also provides information on the variability among strains maintained in different laboratories and through serial passage. This work will guide the selection of strains for further vaccine development