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Summary The elderly are an important target for influenza vaccination, and the determination of factors that underlie immune responsiveness is clinically valuable. We evaluated the immune and metabolic profiles of 205 elderly Singaporeans administered with Vaxigrip. Despite high seroprotection rates, we observed heterogeneity in the response. We stratified the cohort into complete (CR) or incomplete responders (IR), where IR exhibited signs of accelerated T cell aging. We found a higher upregulation of genes associated with the B-cell endoplasmic-reticulum stress response in CR, where XBP-1 acts as a key upstream regulator. B-cells from IR were incapable of matching the level of XBP-1 upregulation observed in CR after inducing ER stress with tunicamycin in vitro. Metabolic signatures also distinguished CR and IR – as CR presented with a greater diversity of bile acids. Our findings suggest that the ER-stress pathway activation could improve influenza vaccination in the elderly., Graphical abstract, Highlights • Seroprotected elderly had heterogeneous titre responses to all three influenza strains • Multi-strain responses are linked to distinct transcriptomic and bile acid profiles • XBP-1 related pathways are specifically enriched in complete responders • XBP-1 upregulation is better preserved in vaccinated complete responders, Immunology; Virology; Clinical microbiology; Cell biology

Enterotoxigenic E. coli (ETEC) is a leading cause of moderate-to-severe diarrhoea. ETEC colonizes the intestine through fimbrial tip adhesin colonization factors and produces heat-stable and/or heat-labile (LT) toxins, stimulating fluid and electrolyte release leading to watery diarrhoea. We reported that a vaccine containing recombinant colonization factor antigen (CfaEB) targeting fimbrial tip adhesin of the colonization factor antigen I (CFA/I) and an attenuated LT toxoid (dmLT) elicited mucosal and systemic immune responses against both targets. Additionally, the toll-like receptor 4 ligand second-generation lipid adjuvant (TLR4-SLA) induced a potent mucosal response, dependent on adjuvant formulation. However, a combination of vaccine components at their respective individual optimal doses may not achieve the optimal immune profile. We studied a subunit ETEC vaccine prototype in mice using a response surface design of experiments (DoE), consisting of 64 vaccine dose-combinations of CfaEB, dmLT and SLA in four formulations (aqueous, aluminium oxyhydroxide, squalene-in-water stable nanoemulsion [SE] or liposomes containing the saponin Quillaja saponaria-21 [LSQ]). Nine readouts focusing on antibody functionality and plasma cell response were selected to profile the immune response of parenterally administered ETEC vaccine prototype. The data were integrated in a model to identify the optimal dosage of each vaccine component and best formulation. Compared to maximal doses used in mouse models (10 µg CfaEB, 1 µg dmLT and 5 µg SLA), a reduction in the vaccine components up to 37%, 60% and 88% for CfaEB, dmLT and SLA, respectively, maintained or even maximized immune responses, with SE and LSQ the best formulations. The DoE approach can help determine the best vaccine composition with a limited number of experiments and may accelerate development of multi-antigen/component ETEC vaccines.

Clostridium difficile infection (CDI) is a leading cause of nosocomial and antibiotic-associated diarrhea. A vaccine, based on formalin-inactivated toxins A and B purified from anaerobic cultures of C. difficile strain VPI 10463 (toxinotype 0), has been in development for the prevention of symptomatic CDI. We evaluated the breadth of protection conferred by this C. difficile toxoid vaccine in cross-neutralization assessments using sera from vaccinated hamsters against a collection of 165 clinical isolates. Hamster antisera raised against the C. difficile toxoid vaccine neutralized the cytotoxic activity of culture supernatants from several toxinotype 0 strains and heterologous strains from 10 different toxinotypes. Further assessments performed with purified toxins confirmed that vaccine-elicited antibodies can neutralize both A and B toxins from a variety of toxinotypes. In the hamster challenge model, the vaccine conferred significant cross-protection against disease symptoms and death caused by heterologous C. difficile strains from the most common phylogenetic clades, including the most prevalent toxinotypes.

Dengue is an important threat for world-wide public health. Different vaccines are under development, which are currently assessed using a battery of in vitro and in vivo assays before moving on to humans. It is also important to assess vaccine characteristics on human primary cells; among them, dendritic cells, the most efficient antigen-presenting cells, are the first targets of dengue virus infection. In this study, we used flow cytometry to compare the consequences of such an infection by dengue serotype 2 live-attenuated vaccine (LAV2) or its parental strain DEN2 16681 (DEN2). Optimal conditions of infection have first been defined by a mathematical approach, and flow cytometry allowed studying modifications induced in both infected and noninfected dendritic cell populations after surface and intracellular labeling. Both DEN2 and LAV2 increased the expression of the phenotypic markers CD80, CD86, CD40, CD1a, HLA ABC and CD83, demonstrating cellular activation. Stimulated dendritic cells produced tumor necrosis factor-alpha in particular, and, to a lower extent, interleukin 6. Of importance, whereas DEN2 induced cytokine production both in the infected and noninfected populations, LAV2-induced cytokine production was restricted to the infected population. This limited activation triggered by LAV2 would be in agreement with its attenuation. In conclusion, these in vitro experiments using primary human dendritic cells may participate, in combination with other assays, to the evaluation of the immunogenicity and safety of dengue vaccine candidates.

Outbred OF1 mice infected in a first step with a mouse-adapted Helicobacter pylori strain were immunized in a second step by systemic or mucosal routes: systemic immunizations were performed subcutaneously with adjuvanted urease either in the infra or supra-diaphragmatic region of the body, while mucosal immunization was done with urease in the presence of E. coli heat Labile toxin. Mucosal and subcutaneous immunizations induced in infected mice a significant reduction in bacterial density whatever the site of injection but complete eradication was preferentially observed in mice immunized subcutaneously in the back. Systemic immunization with appropriate schedules and formulations could constitute a valuable approach to cure Helicobacter pylori infection.

A previous study showed that filamentous phage could be efficiently transfected into mammalian cells in the presence of the cationic lipid Transfectam. In the present study, we used an experimental plan based on a uniform network (Doehlert) matrix to estimate optimal transfection conditions in two different cell lines, CHO and Cos-7. Using the cationic lipid RPR120535b as a model, we show that optimal conditions can be determined much more readily than with standard response curves. Under optimal conditions as analyzed by FACS, up to 60% of Cos-7 and 50% of CHO cells can be transfected. Furthermore, a comparison of different lipids (Transfectam®, RPR120535b, TC1-12 and GAP-DLRIE/DOPE) suggests that lipids with multiple amine groups are more efficient for the transfection of filamentous phage.

Outbred OF1 mice were immunized subcutaneously with flu vaccine, either in the neck or in the lumbar region (back), in combination with adjuvants inducing either a Th1- or a Th2-type response, referred to as adjuvants A1 and A2, respectively. After two parenteral immunizations, the mice were boosted intranasally with nonadjuvanted vaccine. The serum response was analyzed after each immunization by measuring specific immunoglobulin A (IgA), IgG1, and IgG2a antibody levels, while the local response (same isotypes) was measured in the salivary glands after the mucosal boost by ELISPOTs. We observed that systemic priming at any of the two sites with a Th2 rather than a Th1 adjuvant dramatically enhanced the mucosal IgG1 and IgA responses following a mucosal boost with unadjuvanted vaccine. In addition, as judged by the IgG2a/IgG1 ratios and serum IgA levels, immunization of mice in the back induced a rise in Th2 response compared to neck immunization with adjuvant A1. In contrast, such back immunization with adjuvant A2 reversed the Th1-Th2 balance in favor of the Th1 response compared to neck immunization. Similar differences were observed in mucosal antibody levels according to the site of priming with one given adjuvant; priming in the back with adjuvant A1 increased the mucosal IgA and IgG1 responses compared to neck priming, while the local IgG2a levels were decreased. The reverse was true for adjuvant A2. Back versus neck priming with this latter adjuvant decreased the mucosal IgG1 response, while local IgG2a levels were increased. The different lymphatic drainages of the two sites of parenteral immunization may explain these differences, due to the targeting of particular lymphoid inductive sites. Some of these sites may represent crossroads between systemic and mucosal immunity.

The ability of systemic immunization to induce protection against Helicobacter pylori infection has been evaluated in a mouse model. It was observed that if appropriate formulations and adjuvants were used such immunization elicited in outbred Swiss mice levels of protection similar or better than those induced by the oral route in the presence of cholera toxin or Escherichia coli heat labile toxin. Recombinant urease mixed with adjuvants, which induced strong Th1 and Th2 responses elicited better protection than urease mixed with adjuvants which induced a predominant Th2 type response only. These experiments demonstrate the feasibility of parenteral immunization against H. pylori and suggest that an appropriate balance between Th1 and Th2 type responses is required to achieve complete protection.