Intranasal immunization with outer membrane vesicle pertussis vaccine confers broad protection through mucosal IgA and Th17 responses

A vaccine based on outer membrane vesicles of pertussis (omvPV) is protective in a mouse-challenge model and induces a broad antibody and mixed Th1/Th2/Th17 response against multiple antigens following subcutaneous immunization. However, this route did not result in mucosal immunity and did not prevent nasopharyngeal colonization. In this study, we explored the potential of intranasal immunization with omvPV. Only intranasal immunization induced strong mucosal immune responses that encompasses enhanced pulmonary and nasal IgA antibody levels, mainly directed against Vag8 and LPS. Furthermore, high numbers of IgA- and IgG-producing plasma cells were detected as well as lung-resident IgA memory B-cells. Finally, only intranasal immunization induced pulmonary Th1/Th17-related cytokine responses. The magnitude and type of systemic immunity was comparable between both routes and included high systemic IgG antibody levels, strong IgG-producing plasma cell responses, memory B-cells residing in the spleen and systemic Th1/Th2/Th17-related cytokine responses. Importantly, only intranasal immunization prevented colonization in both the lungs and the nasal cavity. In conclusion, intranasal omvPV immunization induces mucosal IgA and Th17-mediated responses without influencing the systemic immunity profile. These responses resulted in prevention of Bordetella pertussis colonization in the respiratory tract, including the nasal cavity, thereby potentially preventing transmission.Drug Delivery Technolog

Development of a Multivalent Subunit Vaccine against Tularemia Using Tobacco Mosaic Virus (TMV) Based Delivery System

Francisella tularensisis a facultative intracellular pathogen, and is the causative agent of a fatal human disease known as tularemia. F. tularensis is classified as a Category A Biothreat agent by the CDC based on its use in bioweapon programs by several countries in the past and its potential to be used as an agent of bioterrorism. No licensed vaccine is currently available for prevention of tularemia. In this study, we used a novel approach for development of a multivalent subunit vaccine against tularemia by using an efficient tobacco mosaic virus (TMV) based delivery platform. The multivalent subunit vaccine was formulated to contain a combination of F. tularensis protective antigens: OmpA-like protein (OmpA), chaperone protein DnaK and lipoprotein Tul4 from the highly virulent F. tularensisSchuS4 strain. Two different vaccine formulations and immunization schedules were used. The immunized mice were challenged with lethal (10xLD100) doses of F. tularensisLVS on day 28 of the primary immunization and observed daily for morbidity and mortality. Results from this study demonstrate that TMV can be used as a carrier for effective delivery of multiple F. tularensisantigens. TMV-conjugate vaccine formulations are safe and multiple doses can be administered without causing any adverse reactions in immunized mice. Immunization with TMV-conjugated F. tularensisproteins induced a strong humoral immune response and protected mice against respiratory challenges with very high doses of F. tularensis LVS. This study provides a proof-of-concept that TMV can serve as a suitable platform for simultaneous delivery of multiple protective antigens of F. tularensis. Refinement of vaccine formulations coupled with TMV-targeting strategies developed in this study will provide a platform for development of an effective tularemia subunit vaccine as well as a vaccination approach that may broadly be applicable to many other bacterial pathogens

Acid Phosphatases Do Not Contribute to the Pathogenesis of Type A Francisella tularensis▿ †

The intracellular pathogen Francisella tularensis is the causative agent of tularemia, a zoonosis that can affect humans with potentially lethal consequences. Essential to Francisella virulence is its ability to survive and proliferate within phagocytes through phagosomal escape and cytosolic replication. Francisella spp. encode a variety of acid phosphatases, whose roles in phagosomal escape and virulence have been documented yet remain controversial. Here we have examined in the highly virulent (type A) F. tularensis strain Schu S4 the pathogenic roles of three distinct acid phosphatases, AcpA, AcpB, and AcpC, that are most conserved between Francisella subspecies. Neither the deletion of acpA nor the combination of acpA, acpB, and acpC deletions affected the phagosomal escape or cytosolic growth of Schu S4 in murine and human macrophages, despite decreases in acid phosphatase activities by as much as 95%. Furthermore, none of these mutants were affected in their ability to cause lethality in mice upon intranasal inoculation. Hence, the acid phosphatases AcpA, AcpB, and AcpC do not contribute to intracellular pathogenesis and do not play a major role in the virulence of type A Francisella strains

Structure-Function Analysis of DipA, a Francisella tularensis Virulence Factor Required for Intracellular Replication

Francisella tularensis is a highly infectious bacterium whose virulence relies on its ability to rapidly reach the macrophage cytosol and extensively replicate in this compartment. We previously identified a novel Francisella virulence factor, DipA (FTT0369c), which is required for intramacrophage proliferation and survival, and virulence in mice. DipA is a 353 amino acid protein with a Sec-dependent signal peptide, four Sel1-like repeats (SLR), and a C-terminal coiled-coil (CC) domain. Here, we determined through biochemical and localization studies that DipA is a membrane-associated protein exposed on the surface of the prototypical F. tularensis subsp. tularensis strain SchuS4 during macrophage infection. Deletion and substitution mutagenesis showed that the CC domain, but not the SLR motifs, of DipA is required for surface exposure on SchuS4. Complementation of the dipA mutant with either DipA CC or SLR domain mutants did not restore intracellular growth of Francisella, indicating that proper localization and the SLR domains are required for DipA function. Co-immunoprecipitation studies revealed interactions with the Francisella outer membrane protein FopA, suggesting that DipA is part of a membrane-associated complex. Altogether, our findings indicate that DipA is positioned at the host-pathogen interface to influence the intracellular fate of this pathogen

Early-life rotavirus and norovirus infections in relation to development of atopic manifestation in infants.

Item does not contain fulltextBACKGROUND: The increase in incidence of atopic diseases (ADs) in the developed world over the past decades has been associated with reduced exposure of childhood infections. OBJECTIVE: To investigate the relation between early intestinal viral infections in relation to the development of atopic symptoms (eczema, wheeze and atopic sensitization) in the first and second year(s) of life. METHODS: In the KOALA Birth Cohort Study, we assessed IgG seropositivity for rota- and norovirus (GGI.1 and GGII.4) at 1 year of age. This was related to allergic sensitization [specific immunoglobulin E (IgE)] at 1 and 2 years, and parent reported eczema and wheeze in the first 2 years, using logistic regression analysis adjusted for confounders. RESULTS: Rotavirus seropositivity (39%) was associated with an unexpected higher risk of recurrent wheeze in the first and second year of life [adjusted odds ratio (OR) 3.1 and 95% confidence intervals (CI) 1.1-9.1] and persistent and new recurrent wheeze (adjusted OR 2.7 and 95% CI 1.1-6.2). No further associations were found between intestinal viral seropositivity and atopic manifestations. CONCLUSION: Our data did not show a clear protection by enteric viral infections in young children on development of IgE response to allergens, but rotavirus infection in the first year was a risk factor for wheeze. However, this needs to be followed up to older ages in order to establish the true importance of intestinal viral infections and especially cumulative effects in AD aetiology. Exposure to rotavirus may offer a new and interesting focus on infant wheeze and later asthma development

LD50 of SchuS4ΔFTT0369c and protective efficacy following increased challenge dose of wild type SchuS4.

<p>(A) Balb/c mice (n = 10/group) were infected intradermally with the indicated number of <i>F. tularensis</i> ΔFTT0369c. Mice were regularly monitored for up to 45 days after infection and euthanized at the first signs of irreversible illness. (B and C) Mice (n = 10/group) were challenged with approximately 5×10⁴ CFU intradermally. Forty-five days after infection mice were challenged intranasally with approximately 50 CFU (B) or 200 CFU (C) wild type SchuS4. Mice were regularly monitored for up to 30 days after infection and euthanized at the first signs of irreversible illness. * = p<0.05 compared to all other groups. ** = p<0.05 compared to mice receiving 7×10⁵, 1×10⁶ and 3×10⁶.</p

<i>In vitro</i> and <i>in vivo</i> attenuation of SchuS4ΔFTT0369c, ΔFTT1676 and ΔFTT0369cΔFTT1676 mutants.

<p>(<b>A</b>) Intracellular growth of <i>F. tularensis</i> SchuS4, ΔFTT0369c, ΔFTT1676 and ΔFTT0369cΔFTT1676 strains in murine BMMs derived from C57Bl/6J mice. BMMs were infected with either strains, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037752#s2" target="_blank">Materials and Methods</a> section, and intracellular viable bacteria were enumerated at the indicated times points after infection. Values are means ± SD of a representative experiment performed in triplicate. Data is representative of 2 experiments of similar design. (B–C) Balb/c mice (n = 5–10/group) were infected intradermally (B) or intranasally (C) with approximately 50 CFU or 10 CFU, respectively, of the indicated strains of <i>F. tularensis</i> SchuS4. Mice were regularly monitored up to 30 days after infection and euthanized at the first sign of irreversible illness.</p

Predicted structure of DipA.

<p>(A) Schematic representations of DipA and domain deletion mutants generated in this study. The predicted N-terminal 20 amino acid signal peptide is denoted is red, the Sel1-like repeat domains are denoted in blue and the coiled-coil (CC) domain is denoted in green. Domain deletion mutants were designed to encompass deletion of Sel1a and Sel1b domains (DipAΔSel1ab), Sel1c and Sel1d domains (DipAΔSel1cd), and the CC domain (DipAΔCC). (B) Helical wheel representations of the CC domain corresponding to amino acid residues of DipA. Heptad-repeat positions are denoted <i>a</i> to <i>g</i>. Two DipA CC domain substitution mutants where three hydrophobic residues in core positions <i>a</i> and <i>d</i> were mutated to aspartate [DipACC(AIL ³D) and DipACC(LAL ³D)]. Mutations are indicated in red. (C) Three-dimensional ribbon model of DipA predicted by I-TASSER and visualized using Chimera software. The Sel1a domain corresponding to residues 96-132 is indicated in red, the Sel1b domain corresponding to residues 133-169 is indicated in blue, the Sel1c domain corresponding to residues 193-229 is indicated in green, the Sel1d domain corresponding to residues 231-262 is indicated in purple, and the CC domain corresponding to residues 311-343 is indicated in cyan.</p

<i>In</i> vivo proliferation and dissemination of SchuS4ΔFTT0369c, ΔFTT1676 and ΔFTT0369cΔFTT1676 mutants following parenteral or pulmonary inoculations.

<p>Balb/c mice (n = 5–10/group) were infected intradermally (A) or intranasally (B) with approximately 50 CFU or 10 CFU, respectively, of the indicated strains of <i>F. tularensis</i> SchuS4. Mice were regularly monitored up to 30 days after infection and euthanized at the first sign of irreversible illness. At the indicated time points, organs were harvested and assessed for bacterial burdens as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037752#s2" target="_blank">materials and methods</a>. Error bars represent SEM. Data is representative of two experiments of similar design. * indicates a p value<0.05 compared to SchuS4. ** indicates a p value<0.05 compared to all other groups. Asterisks colors refer to the strain tested for statistically significant difference.</p