The Network aims to promote multi-disciplinary approaches to address challenging vaccine-related questions. This page contains a curated list of publications that highlight high-impact and collaborative approaches.
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Journal articleFischetti L, Zhong Z, Pinder CL, et al., 2017,
The synergistic effects of combining TLR ligand based adjuvants on the cytokine response are dependent upon p38/JNK signalling., Cytokine, Vol: 99, Pages: 287-296, ISSN: 1043-4666
Toll like receptor (TLR) ligands are important adjuvant candidates, causing antigen presenting cells to release inflammatory mediators, leading to the recruitment and activation of other leukocytes. The aim of this study was to define the response of human blood derived dendritic cells and macrophages to three TLR ligands acting singly or in combination, Poly I:C (TLR3), GLA (TLR4) and R848 (TLR7/8). Combinations of TLR agonists have been shown to have a synergistic effect on individual cytokines, here we look at the global inflammatory response measuring both cytokines and chemokines. Using a custom Luminex assay we saw dose responses in several mediators including CCL3 (MIP1α), IL-1α, IL-1β, IL-12, CXCL10 (IP-10) and IL-6, all of which were significantly increased by the combination of R848 and GLA, even when low dose GLA was added. The synergistic effect was inhibited by specific MAP kinase inhibitors blocking the kinases p38 and JNK but not MEK1. Combining TLR adjuvants also had a synergistic effect on cytokine responses in human mucosal tissue explants. From this we conclude that the combination of R848 and GLA potentiates the inflammatory profile of antigen presenting cells. Since the pattern of inflammatory mediators released can alter the quality and quantity of the adaptive immune response to vaccination, this study informs vaccine adjuvant design.
Journal articleLynskey NN, Reglinski M, Calay D, et al., 2017,
Multi-functional mechanisms of immune evasion by the streptococcal complement inhibitor C5a peptidase, PLOS Pathogens, Vol: 13, Pages: 1-29, ISSN: 1553-7366
The complement cascade is crucial for clearance and control of invading pathogens, and as such is a key target for pathogen mediated host modulation. C3 is the central molecule of the complement cascade, and plays a vital role in opsonization of bacteria and recruitment of neutrophils to the site of infection. Streptococcal species have evolved multiple mechanisms to disrupt complement-mediated innate immunity, among which ScpA (C5a peptidase), a C5a inactivating enzyme, is widely conserved. Here we demonstrate for the first time that pyogenic streptococcal species are capable of cleaving C3, and identify C3 and C3a as novel substrates for the streptococcal ScpA, which are functionally inactivated as a result of cleavage 7 amino acids upstream of the natural C3 convertase. Cleavage of C3a by ScpA resulted in disruption of human neutrophil activation, phagocytosis and chemotaxis, while cleavage of C3 generated abnormally-sized C3a and C3b moieties with impaired function, in particular reducing C3 deposition on the bacterial surface. Despite clear effects on human complement, expression of ScpA reduced clearance of group A streptococci in vivo in wildtype and C5 deficient mice, and promoted systemic bacterial dissemination in mice that lacked both C3 and C5, suggesting an additional complement-independent role for ScpA in streptococcal pathogenesis. ScpA was shown to mediate streptococcal adhesion to both human epithelial and endothelial cells, consistent with a role in promoting bacterial invasion within the host. Taken together, these data show that ScpA is a multi-functional virulence factor with both complement-dependent and independent roles in streptococcal pathogenesis.
Journal articleWang S, Chen R, 2017,
pH-Responsive, Lysine-Based, Hyperbranched Polymers Mimicking Endosomolytic Cell-Penetrating Peptides for Efficient Intracellular Delivery, Chemistry of Materials, Vol: 29, Pages: 5806-5815, ISSN: 0897-4756
The insufficient delivery of biomacromolecular therapeutic agents into the cytoplasm of mammalian cells remains a major barrier to their pharmaceutical applications. Cell-penetrating peptides (CPPs) are considered as potential carriers for cytoplasmic delivery of macromolecular drugs. However, due to the positive charge of most CPPs, strong nonspecific cell membrane bindings may lead to relatively high toxicity. In this study, we report a series of anionic, CPP-mimicking, lysine-based hyperbranched polymers, which caused complete membrane disruption at late endosomal pH while remaining nonlytic at physiological pH. The pH-responsive conformational alterations and the multivalency effect of the hyperbranched structures were demonstrated to effectively facilitate their interaction with cell membranes, thus leading to significantly enhanced membrane-lytic activity compared with their linear counterpart. The unique structures and pH-responsive cell-penetrating abilities make the novel hyperbranched polymers promising candidates for cytoplasmic delivery of biomacromolecular payloads.
Journal articleLima Keesen TS, de Almeida RP, Gois BM, et al., 2017,
Journal articlede Silva TI, Gould V, Mohammed NI, et al., 2017,
Comparison of mucosal lining fluid sampling methods and influenza-specific IgA detection assays for use in human studies of influenza immunity, Journal of Immunological Methods, Vol: 449, Pages: 1-6, ISSN: 0022-1759
We need greater understanding of the mechanisms underlying protection against influenza virus to develop more effective vaccines. To do this, we need better, more reproducible methods of sampling the nasal mucosa. The aim of the current study was to compare levels of influenza virus A subtype-specific IgA collected using three different methods of nasal sampling. Samples were collected from healthy adult volunteers before and after LAIV immunization by nasal wash, flocked swabs and Synthetic Absorptive Matrix (SAM) strips. Influenza A virus subtype-specific IgA levels were measured by haemagglutinin binding ELISA or haemagglutinin binding microarray and the functional response was assessed by microneutralization. Nasosorption using SAM strips lead to the recovery of a more concentrated sample of material, with a significantly higher level of total and influenza H1-specific IgA. However, an equivalent percentage of specific IgA was observed with all sampling methods when normalized to the total IgA. Responses measured using a recently developed antibody microarray platform, which allows evaluation of binding to multiple influenza strains simultaneously with small sample volumes, were compared to ELISA. There was a good correlation between ELISA and microarray values. Material recovered from SAM strips was weakly neutralizing when used in an in vitro assay, with a modest correlation between the level of IgA measured by ELISA and neutralization, but a greater correlation between microarray-measured IgA and neutralizing activity. In conclusion we have tested three different methods of nasal sampling and show that flocked swabs and novel SAM strips are appropriate alternatives to traditional nasal washes for assessment of mucosal influenza humoral immunity.
Journal articleBerger CN, 2017,
The Enterohemorrhagic Escherichia coli Effector EspW Triggers Actin Remodeling in a Rac1-Dependent Manner, Infection and Immunity, Vol: 85, ISSN: 1098-5522
Enterohemorrhagic Escherichia coli (EHEC) is a diarrheagenic pathogen that colonizes the gut mucosa and induces attaching-and-effacing lesions. EHEC employs a type III secretion system (T3SS) to translocate 50 effector proteins that hijack and manipulate host cell signaling pathways, which allow bacterial colonization and subversion of immune responses and disease progression. The aim of this study was to characterize the T3SS effector EspW. We found espW in the sequenced O157:H7 and non-O157 EHEC strains as well as in Shigella boydii. Furthermore, a truncated version of EspW, containing the first 206 residues, is present in EPEC strains belonging to serotype O55:H7. Screening a collection of clinical EPEC isolates revealed that espW is present in 52% of the tested strains. We report that EspW modulates actin dynamics in a Rac1-dependent manner. Ectopic expression of EspW results in formation of unique membrane protrusions. Infection of Swiss cells with an EHEC espW deletion mutant induces a cell shrinkage phenotype that could be rescued by Rac1 activation via expression of the bacterial guanine nucleotide exchange factor, EspT. Furthermore, using a yeast two-hybrid screen, we identified the motor protein Kif15 as a potential interacting partner of EspW. Kif15 and EspW colocalized in cotransfected cells, while ectopically expressed Kif15 localized to the actin pedestals following EHEC infection. The data suggest that Kif15 recruits EspW to the site of bacterial attachment, which in turn activates Rac1, resulting in modifications of the actin cytoskeleton that are essential to maintain cell shape during infection.
Journal articleBassano I, Ong SH, Lawless N, et al., 2017,
Accurate characterization of the IFITM locus using MiSeq and PacBio sequencing shows genetic variation in Galliformes., BMC Genomics, Vol: 18
BACKGROUND: Interferon inducible transmembrane (IFITM) proteins are effectors of the immune system widely characterized for their role in restricting infection by diverse enveloped and non-enveloped viruses. The chicken IFITM (chIFITM) genes are clustered on chromosome 5 and to date four genes have been annotated, namely chIFITM1, chIFITM3, chIFITM5 and chIFITM10. However, due to poor assembly of this locus in the Gallus Gallus v4 genome, accurate characterization has so far proven problematic. Recently, a new chicken reference genome assembly Gallus Gallus v5 was generated using Sanger, 454, Illumina and PacBio sequencing technologies identifying considerable differences in the chIFITM locus over the previous genome releases. METHODS: We re-sequenced the locus using both Illumina MiSeq and PacBio RS II sequencing technologies and we mapped RNA-seq data from the European Nucleotide Archive (ENA) to this finalized chIFITM locus. Using SureSelect probes capture probes designed to the finalized chIFITM locus, we sequenced the locus of a different chicken breed, namely a White Leghorn, and a turkey. RESULTS: We confirmed the Gallus Gallus v5 consensus except for two insertions of 5 and 1 base pair within the chIFITM3 and B4GALNT4 genes, respectively, and a single base pair deletion within the B4GALNT4 gene. The pull down revealed a single amino acid substitution of A63V in the CIL domain of IFITM2 compared to Red Jungle fowl and 13, 13 and 11 differences between IFITM1, 2 and 3 of chickens and turkeys, respectively. RNA-seq shows chIFITM2 and chIFITM3 expression in numerous tissue types of different chicken breeds and avian cell lines, while the expression of the putative chIFITM1 is limited to the testis, caecum and ileum tissues. CONCLUSIONS: Locus resequencing using these capture probes and RNA-seq based expression analysis will allow the further characterization of genetic diversity within Galliformes.
Journal articleKratochvil S, McKay PF, Kopycinski JT, et al., 2017,
A phase 1 human immunodeficiency virus vaccine Trial for cross-profiling the kinetics of serum and mucosal antibody responses to CN54gp140 modulated by two homologous prime-boost vaccine regimens, Frontiers in Immunology, Vol: 8, ISSN: 1664-3224
A key aspect to finding an efficacious human immunodeficiency virus (HIV) vaccine is the optimization of vaccine schedules that can mediate the efficient maturation of protective immune responses. In the present study, we investigated the effect of alternate booster regimens on the immune responses to a candidate HIV-1 clade C CN54gp140 envelope protein, which was coadministered with the TLR4-agonist glucopyranosyl lipid A-aqueous formulation. Twelve study participants received a common three-dose intramuscular priming series followed by a final booster at either 6 or 12 months. The two homologous prime-boost regimens were well tolerated and induced CN54gp140-specific responses that were observed in both the systemic and mucosal compartments. Levels of vaccine-induced IgG-subclass antibodies correlated significantly with FcγR engagement, and both vaccine regimens were associated with strikingly similar patterns in antibody titer and FcγR-binding profiles. In both groups, identical changes in the antigen (Ag)-specific IgG-subclass fingerprint, leading to a decrease in IgG1 and an increase in IgG4 levels, were modulated by booster injections. Here, the dissection of immune profiles further supports the notion that prime-boost strategies are essential for the induction of diverse Ag-specific HIV-1 responses. The results reported here clearly demonstrate that identical responses were effectively and safely induced by both vaccine regimens, indicating that an accelerated 6-month regimen could be employed for the rapid induction of immune responses against CN54gp140 with no apparent impact on the overall quality of the induced immune response. (This study has been registered at http://ClinicalTrials.gov under registration no. NCT01966900.)
Journal articleGould VMW, Francis JN, Anderson KJ, et al., 2017,
Nasal IgA provides protection against human influenza challenge in volunteers with low serum influenza antibody titre, Frontiers in Microbiology, Vol: 8, ISSN: 1664-302X
In spite of there being a number of vaccines, influenza remains a significant global cause of morbidity and mortality. Understanding more about natural and vaccine induced immune protection against influenza infection would help to develop better vaccines. Virus specific IgG is a known correlate of protection, but other factors may help to reduce viral load or disease severity, for example IgA. In the current study we measured influenza specific responses in a controlled human infection model using influenza A/California/2009 (H1N1) as the challenge agent. Volunteers were pre-selected with low haemagglutination inhibition (HAI) titres in order to ensure a higher proportion of infection; this allowed us to explore the role of other immune correlates. In spite of HAI being uniformly low, there were variable levels of H1N1 specific IgG and IgA prior to infection. There was also a range of disease severity in volunteers allowing us to compare whether differences in systemic and local H1N1 specific IgG and IgA prior to infection affected disease outcome. H1N1 specific IgG level before challenge did not correlate with protection, probably due to the pre-screening for individuals with low HAI. However, the length of time infectious virus was recovered from the nose was reduced in patients with higher pre-existing H1N1 influenza specific nasal IgA or serum IgA. Therefore, IgA contributes to protection against influenza and should be targeted in vaccines.
Journal articleWitcomb LA, Czupryna J, Francis KP, et al., 2017,
Non-invasive three-dimensional imaging of Escherichia coli K1 infection using diffuse light imaging tomography combined with micro-computed tomography, Methods, Vol: 127, Pages: 62-68, ISSN: 1046-2023
In contrast to two-dimensional bioluminescence imaging, three dimensional diffuse light imaging tomography with integrated micro-computed tomography (DLIT-μCT) has the potential to realise spatial variations in infection patterns when imaging experimental animals dosed with derivatives of virulent bacteria carrying bioluminescent reporter genes such as the lux operon from the bacterium Photorhabdus luminescens. The method provides an opportunity to precisely localise the bacterial infection sites within the animal and enables the generation of four-dimensional movies of the infection cycle. Here, we describe the use of the PerkinElmer IVIS SpectrumCT in vivo imaging system to investigate progression of lethal systemic infection in neonatal rats following colonisation of the gastrointestinal tract with the neonatal pathogen Escherichia coli K1. We confirm previous observations that these bacteria stably colonize the colon and small intestine following feeding of the infectious dose from a micropipette; invading bacteria migrate across the gut epithelium into the blood circulation and establish foci of infection in major organs, including the brain. DLIT-μCT revealed novel multiple sites of colonisation within the alimentary canal, including the tongue, oesophagus and stomach, with penetration of the non-keratinised oesophageal epithelial surface, providing strong evidence of a further major site for bacterial dissemination. We highlight technical issues associated with imaging of infections in new born rat pups and show that the whole-body and organ bioburden correlates with disease severity.
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