The impact of nucleoside base modification in mRNA vaccine is influenced by the chemistry of its lipid nanoparticle delivery system.

The use of modified nucleosides is an important approach to mitigate the intrinsic immunostimulatory activity of exogenous mRNA and to increase its translation for mRNA therapeutic applications. However, for vaccine applications, the intrinsic immunostimulatory nature of unmodified mRNA could help induce productive immunity. Additionally, the ionizable lipid nanoparticles (LNPs) used to deliver mRNA vaccines can possess immunostimulatory properties that may influence the impact of nucleoside modification. Here we show that uridine replacement with N1-methylpseudouridine in an mRNA vaccine encoding influenza hemagglutinin had a significant impact on the induction of innate chemokines/cytokines and a positive impact on the induction of functional antibody titers in mice and macaques when MC3 or KC2 LNPs were used as delivery systems, while it impacted only minimally the titers obtained with L319 LNPs, indicating that the impact of nucleoside modification on mRNA vaccine efficacy varies with LNP composition. In line with previous observations, we noticed an inverse correlation between the induction of high innate IFN-α titers in the macaques and antigen-specific immune responses. Furthermore, and consistent with the species specificity of pathogen recognition receptors, we found that the effect of uridine replacement did not strictly translate from mice to non-human primates.

Anti-staphylococcus aureus adaptive immunity is impaired in end-stage renal disease patients on hemodialysis: one-year longitudinal study.

Introduction: Patients with end-stage renal disease (ESRD) display defects in adaptive and innate immunity, increasing susceptibility to infection. Staphylococcus aureus (S. aureus) is a major cause of bacteraemia in this population and is associated with increased mortality. More information on the immune response to S. aureus in these patients is needed to inform effective vaccine development. Methods: A longitudinal prospective study was carried out at two medical centers and included 48 ESRD patients who started chronic hemodialysis (HD) treatment ≤3 months before inclusion. Control samples were taken from 62 consenting healthy blood donors. Blood samples were obtained from ESRD patients at each visit, on month (M) 0 (beginning of HD), M6 and M12. Around 50 immunological markers of adaptive and innate immunity were assessed to compare immune responses to S. aureus in ESRD patients versus controls to document the changes on their immune profile during HD. Results: S. aureus survival in whole blood was significantly higher in ESRD patients than in controls at M0 (P=0.049), while impaired oxidative burst activity was observed in ESRD patients at all timepoints (P<0.001). S. aureus-specific immunoglobulin G (IgG) responses to iron surface determinant B (IsdB) and S. aureus α hemolysin (Hla) antigens were lower in ESRD patients than in healthy donors at M0 (P=0.003 and P=0.007, respectively) and M6 (P=0.05 and P=0.03, respectively), but were restored to control levels at M12. Moreover, S. aureus-specific T-helper cell responses were comparable to controls for IsdB but were impaired for Hla antigen at all timepoints: 10% of ESRD patients responded to Hla at M0, increasing to 30% at M12, compared with 45% of healthy donors. B-cell and T-cell concentrations in blood were significantly reduced (by 60% and 40%, respectively) compared with healthy controls. Finally, upregulation of Human Leucocyte Antigen-DR (HLA-DR) and C-C chemokine Receptor type 2 (CCR2) was impaired at M0 but was restored during the first year of HD. Conclusion: All together, these results show that adaptive immunity was largely impaired in ESRD patients, whereas innate immunity was less impacted and tended to be restored by HD.

Bacterial capsular polysaccharides with antibiofilm activity share common biophysical and electrokinetic properties.

Bacterial biofilms are surface-attached communities that are difficult to eradicate due to a high tolerance to antimicrobial agents. The use of non-biocidal surface-active compounds to prevent the initial adhesion and aggregation of bacterial pathogens is a promising alternative to antibiotic treatments and several antibiofilm compounds have been identified, including some capsular polysaccharides released by various bacteria. However, the lack of chemical and mechanistic understanding of the activity of these polymers limits their use to control biofilm formation. Here, we screen a collection of 31 purified capsular polysaccharides and first identify seven new compounds with non-biocidal activity against Escherichia coli and/or Staphylococcus aureus biofilms. We measure and theoretically interpret the electrophoretic mobility of a subset of 21 capsular polysaccharides under applied electric field conditions, and we show that active and inactive polysaccharide polymers display distinct electrokinetic properties and that all active macromolecules share high intrinsic viscosity features. Despite the lack of specific molecular motif associated with antibiofilm properties, the use of criteria including high density of electrostatic charges and permeability to fluid flow enables us to identify two additional capsular polysaccharides with broad-spectrum antibiofilm activity. Our study therefore provides insights into key biophysical properties discriminating active from inactive polysaccharides. The characterization of a distinct electrokinetic signature associated with antibiofilm activity opens new perspectives to identify or engineer non-biocidal surface-active macromolecules to control biofilm formation in medical and industrial settings.

An optimized purified inactivated Zika vaccine provides sustained immunogenicity and protection in cynomolgus macaques.

The recent spread of Zika virus (ZIKV) through the Americas and Caribbean and its devastating consequences for pregnant women and their babies have driven the search for a safe and efficacious ZIKV vaccine. Among the vaccine candidates, a first-generation ZIKV purified inactivated vaccine (ZPIV), adjuvanted with aluminum hydroxide, developed by the Walter Reed Army Institute of Research (WRAIR), has elicited high seroconversion rates in participants in three phase-I clinical trials. In collaboration with the WRAIR, Sanofi Pasteur (SP) optimized the production scale, culture and purification conditions, and increased the regulatory compliance, both of which are critical for clinical development and licensure of this vaccine. Using a clinical batch of the first-generation ZPIV as a benchmark, we report that different doses of the optimized vaccine (ZPIV-SP) elicited sustained neutralizing antibodies, specific T- and memory B-cells, and provided complete protection against a ZIKV challenge in cynomolgus macaques. These data provide evidence that the ZPIV-SP vaccine performs at least as well as the ZPIV vaccine, and provide support for continued development in the event of future ZIKV outbreaks.

Glycosylation of Staphylococcus aureus cell wall teichoic acid is influenced by environmental conditions.

Wall teichoic acid (WTA) are major constituents of Staphylococcus aureus (S. aureus) cell envelopes with important roles in the bacteria's physiology, resistance to antimicrobial molecules, host interaction, virulence and biofilm formation. They consist of ribitol phosphate repeat units in which the ribitol residue is substituted with D-alanine (D-Ala) and N-acetyl-D-glucosamine (GlcNAc). The complete S. aureus WTA biosynthesis pathways was recently revealed with the identification of the two glycosyltransferases, TarM and TarS, respectively responsible for the α- and ß-GlcNAc anomeric substitutions. We performed structural analyses to characterize WTAs from a panel of 24 S. aureus strains responsible for invasive infections. A majority of the S. aureus strains produced the ß-GlcNAc WTA form in accordance with the presence of the tarS gene in all strains assessed. The ß-GlcNAc anomer was preferentially expressed at the expense of the α-GlcNAc anomer when grown on stress-inducing culture medium containing high NaCl concentration. Furthermore, WTA glycosylation of the prototype S. aureus Newman strain was characterized in vivo in two different animal models, namely peritonitis and deep wound infection. While the inoculum used to infect animals produced almost exclusively α-GlcNAc WTA, a complete switch to ß-glycosylation was observed in infected kidneys, livers and muscles. Overall, our data demonstrate that S. aureus WTA glycosylation is strongly influenced by environmental conditions and suggest that ß-GlcNAc WTA may bring competitive advantage in vivo.

Production, secretion and purification of a correctly folded staphylococcal antigen in Lactococcus lactis.

BACKGROUND: Lactococcus lactis, a lactic acid bacterium traditionally used to ferment milk and manufacture cheeses, is also, in the biotechnology field, an interesting host to produce proteins of medical interest, as it is "Generally Recognized As Safe". Furthermore, as L. lactis naturally secretes only one major endogenous protein (Usp45), the secretion of heterologous proteins in this species facilitates their purification from a protein-poor culture medium. Here, we developed and optimized protein production and secretion in L. lactis to obtain proteins of high quality, both correctly folded and pure to a high extent. As proteins to be produced, we chose the two transmembrane members of the HtrA protease family in Staphylococcus aureus, an important extra-cellular pathogen, as these putative surface-exposed antigens could constitute good targets for vaccine development. RESULTS: A recombinant ORF encoding a C-terminal, soluble, proteolytically inactive and tagged form of each staphylococcal HtrA protein was cloned into a lactococcal expression-secretion vector. After growth and induction of recombinant gene expression, L. lactis was able to produce and secrete each recombinant rHtrA protein as a stable form that accumulated in the culture medium in similar amounts as the naturally secreted endogenous protein, Usp45. L. lactis growth in fermenters, in particular in a rich optimized medium, led to higher yields for each rHtrA protein. Protein purification from the lactococcal culture medium was easily achieved in one step and allowed recovery of highly pure and stable proteins whose identity was confirmed by mass spectrometry. Although rHtrA proteins were monomeric, they displayed the same secondary structure content, thermal stability and chaperone activity as many other HtrA family members, indicating that they were correctly folded. rHtrA protein immunogenicity was established in mice. The raised polyclonal antibodies allowed studying the expression and subcellular localization of wild type proteins in S. aureus: although both proteins were expressed, only HtrA1 was found to be, as predicted, exposed at the staphylococcal cell surface suggesting that it could be a better candidate for vaccine development. CONCLUSIONS: In this study, an efficient process was developed to produce and secrete putative staphylococcal surface antigens in L. lactis and to purify them to homogeneity in one step from the culture supernatant. This allowed recovering fully folded, stable and pure proteins which constitute promising vaccine candidates to be tested for protection against staphylococcal infection. L. lactis thus proved to be an efficient and competitive cell factory to produce proteins of high quality for medical applications.

Impact of Anesthesia Protocols on In Vivo Bioluminescent Bacteria Imaging Results.

Infectious murine models greatly benefit from optical imaging using bioluminescent bacteria to non-invasively and repeatedly follow in vivo bacterial infection. In this context, one of the most critical parameters is the bioluminescence sensitivity to reliably detect the smallest number of bacteria. Another critical point is the anesthetic approaches that have been demonstrated to impact the bioluminescence flux emission in studies with luciferase-transfected tumor cells. However, this impact has never been assessed on bacteria bioluminescent models. To this end, we investigated the effects of four anesthesia protocols on the bioluminescence flux in a central venous catheter murine model (SKH1-hr(hr) mice) infected by a bioluminescent S. aureus Xen36 strain. Bioluminescence imaging was performed on mice anesthetized by either ketamine/xylazine (with or without oxygen supplementation), or isoflurane carried with air or oxygen. Total flux emission was determined in vivo daily for 3 days and ex vivo at the end of the study together with a CFU counting of the biofilm in the catheter. Bioluminescence flux differences appear between the different anesthetic protocols. Using a ketamine/xylazine anesthesia (with air), bacteria detection was impossible since the bioluminescence signal remains in the background signal. Mice anesthetized with isoflurane and oxygen led to a signal significantly higher to the background all along the kinetics. The use of isoflurane in air presents a bioluminescence signal similar to the use of ketamine/xylazine with oxygen. These data highlight the importance of oxygen to improve bioluminescence flux by bacteria with isoflurane as well as with ketamine/xylazine anesthetics. As a conclusion, we recommend the use of isoflurane anesthetic with oxygen to increase the bioluminescence sensitivity in this kind of study.

Improvement of immunogenicity of meningococcal lipooligosaccharide by coformulation with lipidated transferrin-binding protein B in liposomes: implications for vaccine development.

Among various meningococcal antigens, lipooligosaccharide (LOS) and recombinant lipidated transferrin-binding protein B (rlip-TbpB) are considered to be putative vaccine candidates against group B Neisseria meningitidis. In the present work, we report the development of a new liposome-based vaccine formulation containing both rlip-TbpB and L8 LOS. The endotoxic activity of the liposomal LOS was evaluated in vitro using the Limulus Amebocyte Lysate assay and compared to the endotoxic activity of free LOS. Above a 250:1 lipid/LOS molar ratio, liposomes were shown to effectively detoxify the LOS as the endotoxic activity of the LOS was reduced by more than 99%. Immunogenicity studies in rabbits showed that the presence of rlip-TbpB dramatically increased the immunogenicity of the LOS. While the formulation raised a strong anti-TbpB response, it elicited a higher anti-LOS IgG level than the liposomal LOS alone. Sera from rabbits immunized with rlip-TbpB/liposomal LOS displayed increased ability to recognize LOS on live bacteria expressing the L8 immunotype and increased anti-LOS-specific bactericidal activity compared to sera from rabbits immunized with liposomal LOS alone. Measurement of interleukin-8 (IL-8) produced by HEK293 cells transfected with Toll-like receptor (TLR) after stimulation with rlip-TbpB showed that the protein is a TLR2 agonist, which is in accordance with the structure of its lipid. Furthermore, an in vivo study demonstrated that the lipid moiety is not only required for its adjuvant effect but also has to be linked to the protein. Overall, the rlip-TbpB/LOS liposomal formulation was demonstrated to induce an effective anti-LOS response due to the adjuvant effect of rlip-TbpB on LOS.

CD4 T cell antigens from Staphylococcus aureus Newman strain identified following immunization with heat-killed bacteria.

Staphylococcus aureus is a commensal bacterium associated with the skin and mucosal surfaces of humans and animals that can also cause chronic infection. The emergence of antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA) and strains causing chronic intramammary infections (IMI) in cows results in severe human and livestock infections. Conventional approaches to vaccine development have yielded only a few noneffective vaccines against MRSA or IMI strains, so there is a need for improved vaccine development. CD4 T lymphocytes are required for promoting gamma interferon (IFN-γ) mediated immunoglobulin isotype switching in B lymphocytes to produce high-affinity IgG antibodies and IFN-γ-mediated phagocyte activation for an effective resolution of bacterial infection. However, the lack of known CD4 T cell antigens from S. aureus has made it difficult to design effective vaccines. The goal of this study was to identify S. aureus proteins recognized by immune CD4 T cells. Using a reverse genetics approach, 43 antigens were selected from the S. aureus Newman strain. These included lipoproteins, proteases, transcription regulators, an alkaline shock protein, conserved-domain proteins, hemolysins, fibrinogen-binding protein, staphylokinase, exotoxin, enterotoxin, sortase, and protein A. Screening of expressed proteins for recall T cell responses in outbred, immune calves identified 13 proteins that share over 80% sequence identity among MRSA or IMI strains. These may be useful for inclusion in a broadly protective multiantigen vaccine against MRSA or IMI.

Genetic and structural characterization of L11 lipooligosaccharide from Neisseria meningitidis serogroup A strains.

The lipooligosaccharide (LOS) of immunotype L11 is unique within serogroup A meningococci. In order to resolve its molecular structure, we conducted LOS genotyping by PCR analysis of genes responsible for alpha-chain sugar addition (lgtA, -B, -C, -E, -H, and -F) and inner core substituents (lgtG, lpt-3, and lpt-6). For this study, we selected seven strains belonging to subgroup III, a major clonal complex responsible for meningococcal meningitis epidemics in Africa. In addition, we sequenced the homopolymeric tract regions of three phase-variable genes (lgtA, lgtG, and lot-3) to predict gene functionality. The fine structure of the L11 LOS of each strain was determined using composition and glycosyl linkage analyses, NMR, and mass spectrometry. The masses of the dephosphorylated oligosaccharides were consistent with an oligosaccharide composed of two hexoses, one N-acetyl-hexosamine, two heptoses, and one KDO, as proposed previously. The molar composition of LOS showed two glucose residues to be present, in agreement with lgtH sequence prediction. Despite phosphoethanolaminetransferase genes lpt-3 and lpt-6 being present in all seven Neisseria meningitidis strains, phosphoethanolamine (PEtn) was found at both O-3 and O-6 of HepII among the three ST-5 strains, whereas among the four ST-7 strains, only one PEtn was found and located at O-3 of the HepII. The L11 LOS was found to be O-acetylated, as was indicated by the presence of the lot-3 gene being in-frame in all of the seven N. meningitidis strains. To our knowledge, these studies represent the first full genetic and structural characterization of the L11 LOS of N. meningitidis. These investigations also suggest the presence of further regulatory mechanisms affecting LOS structure microheterogeneity in N. meningitidis related to PEtn decoration of the inner core.

Distribution of transferrin binding protein B gene (tbpB) variants among Neisseria species.

BACKGROUND: Transferrin binding protein B (tbpB), an outer membrane lipoprotein, is required for the acquisition of iron from human transferrin. Two tbpB families have been documented in Neisseria meningitidis: an isotype I tbpB gene of 1.8 kb and an isotype II tbpB gene of 2.1 kb, the former expressed by meningococci in the disease-associated ST-11 clonal complex and the latter found among meningococci belonging to the hyper-invasive clonal complexes including ST-8, ST-18, ST-32, ST-41/44 as well as N. gonorrhoeae isolates. The origin of the isotype I tbpB gene is unknown, however several features in common with non-pathogenic Neisseria and the ST-11 clonal complex N. meningitidis isolate FAM18 have been documented leading to the hypothesis that the isotype I tbpB gene may also be shared between non-pathogenic Neisseria and ST-11 meningococci. As a result, the diversity of the tbpB gene was investigated in a defined collection of Neisseria species. RESULTS: Two families of isotype I tbpB were identified: family A containing conserved genes belonging to ST-11 meningococci, N. polysaccharea and N. lactamica isolates and family B including more diverse isotype I tbpB genes from N. sicca, N. mucosa, N. flava, N. subflava as well as N. cinerea, N. flavescens and N. polysaccharea isolates. Three isotype II tbpB families were identified with: family C containing diverse tbpB genes belonging to N. polysaccharea, N. lactamica, N. gonorrhoeae and N. meningitidis isolates, family D including another subset of isotype II tbpB genes from N. lactamica isolates and family E solely composed of N. gonorrhoeae tbpB genes. CONCLUSION: This study reveals another instance of similarity between meningococci of the ST-11 clonal complex and non-pathogenic Neisseria with the origin of the isotype I tbpB gene resulting from a horizontal genetic transfer event occurring between these two populations.

Molecular characterization of Clostridium tetani strains by pulsed-field gel electrophoresis and colony PCR.

Pulsed-field gel electrophoresis and PCR were applied for the first time to the molecular characterization of Clostridium tetani. Among five strains tested, one (CN1339) turned out to contain a mixture of two genetically different clones and two (D11 and G761) to contain bacteria differing by the presence or absence of the 74-kb plasmid harboring the tetX gene.

Helicobacter pylori infection induces interleukin-8 receptor expression in the human gastric epithelium.

The gastric pathogen Helicobacter pylori is known to activate multiple proinflammatory signaling pathways in epithelial cells. In this study, we addressed the question of whether expression of the interleukin-8 receptors IL-8RA (CXCR1) and IL-8RB (CXCR2) is upregulated in H. pylori-infected human gastric biopsy samples. Biopsy samples from patients infected with H. pylori strains harboring the cag pathogenicity island (PAI) expressed larger amounts of both receptors. In addition, IL-8RB expression was induced in the gastric epithelial cell line AGS upon infection with a clinical isolate containing the cag PAI, while a strain lacking the cag PAI did not. Our finding suggests that gastric epithelial cells express IL-8R in response to H. pylori infection.

Gastric mucosal recognition of Helicobacter pylori is independent of Toll-like receptor 4.

Little is known about the interactions between Helicobacter pylori, which specializes in colonizing the mucin layer that covers the gastric mucosa, and primary gastric epithelial cells. The expression pattern of Toll-like receptors (TLRs) in primary gastric epithelial cells and cell lines was compared. Primary cells did not express TLR4, whereas all cell lines expressed a nonsignaling form of TLR4. Because other cells within the mucosa expressed TLR4, it was next investigated whether H. pylori can be recognized by TLR4--they cannot. Moreover, H. pylori infection of primary cells induced a regulated production of interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha, whereas infection of cell lines only resulted in IL-8 production. The cytokine production in all cell types was strictly cag dependent. These findings indicate that, although the epithelium is important in directing the immune response against H. pylori infections, the response is independent of TLR4.