Subcutaneous Immunization Of Mice Research Articles

Further Evaluation of Enterohemorrhagic Escherichia coli Gold Nanoparticle Vaccines Utilizing Citrobacter rodentium as the Model Organism.

Enterohemorrhagic E. coli (EHEC) is a group of pathogenic bacteria that is associated with worldwide human foodborne diarrheal illnesses and the development of hemolytic uremic syndrome, a potentially deadly condition associated with Shiga toxins (Stxs). Currently, approved vaccines for human prophylaxis against infection do not exist, and one barrier preventing the successful creation of EHEC vaccines is the absence of dependable animal models, including mice, which are naturally resistant to EHEC infection and do not manifest the characteristic signs of the illness. Our lab previously developed gold nanoparticle (AuNP)-based EHEC vaccines, and assessed their efficacy using Citrobacter rodentium, which is the mouse pathogen counterpart of EHEC, along with an Stx2d-producing strain that leads to more consistent disease kinetics in mice, including lethality. The purpose of this study was to continue evaluating these vaccines to increase protection. Here, we demonstrated that subcutaneous immunization of mice with AuNPs linked to the EHEC antigens EscC and intimin (Eae), either alone or simultaneously, elicits functional robust systemic humoral responses. Additionally, vaccination with both antigens together showed some efficacy against Stx2d-producing C. rodentium while AuNP-EscC successfully limited infection with non-Stx2d-producing C. rodentium. Overall, the collected results indicate that our AuNP vaccines have promising potential for preventing disease with EHEC, and that evaluation of novel vaccines using an appropriate animal model, like C. rodentium described here, could be the key to finally developing an effective EHEC vaccine that can progress into human clinical trials.

Development and Evaluation of an Immunoinformatics-Based Multi-Peptide Vaccine against Acinetobacter baumannii Infection.

Multi-drug-resistant (MDR) Acinetobacter baumannii is an opportunistic pathogen associated with hospital-acquired infections. Due to its environmental persistence, virulence, and limited treatment options, this organism causes both increased patient mortality and incurred healthcare costs. Thus, prophylactic vaccination could be ideal for intervention against MDR Acinetobacter infection in susceptible populations. In this study, we employed immunoinformatics to identify peptides containing both putative B- and T-cell epitopes from proteins associated with A. baumannii pathogenesis. A novel Acinetobacter Multi-Epitope Vaccine (AMEV2) was constructed using an A. baumannii thioredoxin A (TrxA) leading protein sequence followed by five identified peptide antigens. Antisera from A. baumannii infected mice demonstrated reactivity to rAMEV2, and subcutaneous immunization of mice with rAMEV2 produced high antibody titer against the construct as well as peptide components. Immunization results in increased frequency of IL-4-secreting splenocytes indicative of a Th2 response. AMEV2-immunized mice were protected against intranasal challenge with a hypervirulent strain of A. baumannii and demonstrated reduced bacterial burden at 48 h. In contrast, all mock vaccinated mice succumbed to infection within 3 days. Results presented here provide insight into the effectiveness of immunoinformatic-based vaccine design and its potential as an effective strategy to combat the rise of MDR pathogens.

A TonB dependent transporter YncD of Salmonella entericaSerovar Typhi possesses vaccine potential.

Multiple TonB dependent transporters (TBDTs) contribute to bacterial virulence due to the importance roles that their substrates play in bacterial growth, and possess vaccine potential. A putative TBDT, YncD, had been identified as one of in vivo induced antigens during human infection of typhoid fever, and is required for the pathogenicity of Salmonella enterica Serovar Typhi. The present study was aimed to determine the function and immunogenicity of YncD. Homologous recombination method was used to construct an yncD-deletion mutant and cirA-iroN-fepA-deletion mutant from the wild-type S. Typhi Ty2. The growth of mutants and the wild-type strain were assessed in iron-deficient medium, as well as in human macrophage cells. Recombinant YncD protein was expressed and purified using Ni-NTA affinity chromatography and anion exchange. A mouse model was then used to evaluate the immunogenicity and protection efficacy of the recombinant YncD. Antibody levels, serum bactericidal efficiency, passive immune protection, opsonophagocysis were assayed to analyse the immunoprotection mechanism of the recombinant YncD. Our results showed that YncD is associated with the iron-uptake of S. Typhi. The yncD-deletion mutant displayed impaired growth in iron-deficient medium, comparable to that the cirA-iroN-fepA-deletion mutant did. The mutation of yncD markedly decreased bacterial growth within human macrophage cells. Moreover, subcutaneous immunization of mice with recombinant YncD elicited high levels of specific anti-YncD IgG, IgG1 and IgG2a, which protected the immunized mice against the intraperitoneal challenge of S. Typhi, and decreased bacterial burdens in the livers and spleens of the infected mice. Passive immunization using the immunized sera also efficiently protected the mice from the challenge of S. Typhi. Moreover, the immunized sera enhanced in vitro bactericidal activity of complement, and opsonophagocytosis. Our results showed that YncD displays a role in the iron-uptake of S. Typhi and possesses immunogenicity.

Self-Assembled Multiepitope Nanovaccine Provides Long-Lasting Cross-Protection against Influenza Virus.

Seasonal influenza vaccines typically provide strain-specific protection and are reformulated annually, which is a complex and time-consuming process. Multiepitope vaccines, combining multiple conserved antigenic epitopes from a pathogen, can trigger more robust, diverse, and effective immune responses, providing a potential solution. However, their practical application is hindered by low immunogenicity and short-term effectiveness. In this study, multiple linear epitopes from the conserved stem domain of hemagglutinin and the ectodomain of matrix protein 2 are combined with the Helicobacter pylori ferritin, a stable self-assembled nanoplatform, to develop an influenza multiepitope nanovaccine, named MHF. MHF is prokaryotically expressed in a soluble form and self-assembles into uniform nanoparticles. The subcutaneous immunization of mice with adjuvanted MHF induces cross-reactive neutralizing antibodies, antibody-dependent cell-mediated cytotoxicity, and cellular immunity, offering complete protection against H3N2 as well as partial protection against H1N1. Importantly, the vaccine cargo delivered by ferritin triggers epitope-specific memory B-cell responses, with antibody level persisting for over 6 months post-immunization. These findings indicate that self-assembled multiepitope nanovaccines elicit potent and long-lasting immune responses while significantly reducing the risk of vaccine escape mutants, and offer greater practicality in terms of scalable manufacturing and genetic manipulability, presenting a promising and effective strategy for future vaccine development.

Plant-Produced Nanoparticles Based on Artificial Self-Assembling Peptide Bearing the Influenza M2e Epitope.

Despite advances in vaccine development, influenza remains a persistent global health threat and the search for a broad-spectrum recombinant vaccine against influenza continues. The extracellular domain of the transmembrane protein M2 (M2e) of the influenza A virus is highly conserved and can be used to develop a universal vaccine. M2e is a poor immunogen by itself, but it becomes highly immunogenic when linked to an appropriate carrier. Here, we report the transient expression of a recombinant protein comprising four tandem copies of M2e fused to an artificial self-assembling peptide (SAP) in plants. The hybrid protein was efficiently expressed in Nicotiana benthamiana using the self-replicating potato virus X-based vector pEff. The protein was purified using metal affinity chromatography under denaturing conditions. The hybrid protein was capable of self-assembly in vitro into spherical particles 15-30 nm in size. The subcutaneous immunization of mice with M2e-carrying nanoparticles induced high levels of M2e-specific IgG antibodies in serum and mucosal secretions. Immunization provided mice with protection against a lethal influenza A virus challenge. SAP-based nanoparticles displaying M2e peptides can be further used to develop a recombinant "universal" vaccine against influenza A produced in plants.

Adjuvant effect of mesoporous silica SBA-15 on anti-diphtheria and anti-tetanus humoral immune response

Routine immunization against diphtheria and tetanus has drastically reduced the incidence of these diseases worldwide. Anti-diphtheria/tetanus vaccine has in general aluminum salt as adjuvant in its formulation that can produce several adverse effects. There is a growing interest in developing new adjuvants. In this study, we evaluated the efficiency of SBA-15 as an adjuvant in subcutaneous immunization in mice with diphtheria (dANA) and tetanus (tANA) anatoxins as well as with the mixture of them (dtANA). The tANA molecules and their encapsulation in SBA-15 were characterized using Small-Angle X-ray Scattering (SAXS), Dynamical Light Scattering (DLS), Nitrogen Adsorption Isotherm (NAI), Conventional Circular Dichroism (CD)/Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy, and Tryptophan Fluorescence Spectroscopy (FS). The primary and secondary antibody response elicited by subcutaneous immunization of High (HIII) and Low (LIII) antibody responder mice with dANA, tANA, or dtANA encapsulated in the SBA-15 were determined. We demonstrated that SBA-15 increases the immunogenicity of dANA and tANA antigens, especially when administered in combination. We also verified that SBA-15 modulates the antibody response of LIII mice, turning them into high antibody responder. Thus, these results suggest that SBA-15 may be an effective adjuvant for different vaccine formulations.

Comparison of adjuvant properties of chitosan during oral and subcutaneous immunization of mice with BSA

Vaccination is the best method to prevent the spread of infectious diseases, its disadvantages are side effects. Potentially safe DNA, RNA or protein molecules possess antigenic properties, but are low-immunogenic and therefore require conjugation with an adjuvant. The aim of the research was to evaluate Chitosan (CS) potency as an adjuvant and compare its effectiveness depending on the route of drug administration. The experiments were carried out on 3 groups of BALB/c mice. Mice of the first group were injected subcutaneously with 20 µl of a mixture of CS (3.3 mg/kg) and BSA (1.7 mg/kg). The mixture of CS and BSA at the same doses and volume was administered orally to mice of the second experimental group. The third group – control – unvaccinated mice. Anti-BSA antibody levels were measured by ELISA. Aspartate aminotransferase, alanine aminotransferase activity and cholesterol, creatinine and urea levels were determined in the serum. It was found that both subcutaneous and mucosal immunizations provided a 2-fold increase in anti-BSA antibody titers against the background of maintaining all biochemical blood parameters at the level of the physiological norm. However, AST activity in the serum of oral-immunized mice was elevated as compared to subcutaneous-immunized mice. Serum cholesterol level in the group of subcutaneously immunized mice and creatinine and urea levels in both experimental groups were reduced compared to the control. It is concluded that oral immunization with CS is the optimal route for antigen-specific IgG antibody response induction.

Protein antigen conjugated with cholesteryl amino-pullulan nanogel shows delayed degradation in dendritic cells and augmented immunogenicity

Carriers that augment delivery, immunogenicity or both are crucial in the development of vaccines especially component vaccines as components of pathogens are often poorly immunogenic. Cholesteryl pullulan (CHP) that forms nano-sized hydrogel (nanogel) and encapsulates proteins was shown to be useful in the delivery of vaccines. Here we demonstrate that subcutaneous immunization of mice with bovine serum albumin (BSA) chemically conjugated to NH2-CHP nanogel induces strong antibody production. This augmented antibody production requires covalent conjugation between BSA and CHP, but does not require nanogel formation. Conjugation of NH2-CHP nanogel induces persistence of BSA in dendritic cells (DCs) in vivo. As resistance to lysosomal degradation was previously shown to augment antigen presentation by DCs, conjugation of antigens with CHP nanogel may enhance antibody production to antigens by delaying lysosomal degradation. Therefore, delayed degradation of antigens by covalent conjugation with nanoparticles may be a good strategy for the development of effective vaccines.

Targeting human langerin promotes HIV-1 specific humoral immune responses.

The main avenue for the development of an HIV-1 vaccine remains the induction of protective antibodies. A rationale approach is to target antigen to specific receptors on dendritic cells (DC) via fused monoclonal antibodies (mAb). In mouse and non-human primate models, targeting of skin Langerhans cells (LC) with anti-Langerin mAbs fused with HIV-1 Gag antigen drives antigen-specific humoral responses. The development of these immunization strategies in humans requires a better understanding of early immune events driven by human LC. We therefore produced anti-Langerin mAbs fused with the HIV-1 gp140z Envelope (αLC.Env). First, we show that primary skin human LC and in vitro differentiated LC induce differentiation and expansion of naïve CD4+ T cells into T follicular helper (Tfh) cells. Second, when human LC are pre-treated with αLC.Env, differentiated Tfh cells significantly promote the production of specific IgG by B cells. Strikingly, HIV-Env-specific Ig are secreted by HIV-specific memory B cells. Consistently, we found that receptors and cytokines involved in Tfh differentiation and B cell functions are upregulated by LC during their maturation and after targeting Langerin. Finally, we show that subcutaneous immunization of mice by αLC.Env induces germinal center (GC) reaction in draining lymph nodes with higher numbers of Tfh cells, Env-specific B cells, as well as specific IgG serum levels compared to mice immunized with the non-targeting Env antigen. Altogether, we provide evidence that human LC properly targeted may be licensed to efficiently induce Tfh cell and B cell responses in GC.

Protective role of PhtD and its amino and carboxyl fragments against pneumococcal sepsis

The implementation of polysaccharide-based vaccines has massively reduced the incidence of invasive pneumococcal diseases. However, there is great concern regarding serotype replacement and the increase in antibiotic resistant strains expressing non-vaccine capsular types. In addition, conjugate vaccines have high production costs, a limiting factor for their implementation in mass immunization programs in developing countries. These limitations have prompted the development of novel vaccine strategies for prevention of Streptococcus pneumoniae infections. The use of conserved pneumococcal antigens such as recombinant proteins or protein fragments presents an interesting serotype-independent alternative. Pht is a family of surface-exposed proteins which have been evaluated as potential vaccine candidates with encouraging results. The present work investigated the immune responses elicited by subcutaneous immunization of mice with the polyhistidine triad protein D (PhtD) and its amino and carboxyl terminal fragments. The proteins were immunogenic and protective against pneumococcal sepsis in mice. Antibodies raised against PhtD increased complement C3b deposition on the pneumococcal surface, mainly mediated by the alternative pathway. Sera from mice immunized with PhtD and PhtD_Cter promoted an increase in bacterial uptake by mouse phagocytes. The interaction of PhtD with the complement system regulator factor H was investigated in silico and in vitro by ELISA and western blot, confirming PhtD as a factor-H binding protein. Our results support the inclusion of PhtD and more specifically, its C-terminal fragment in a multicomponent serotype independent vaccine and suggests a role for the complement system in PhtD-mediated protection.

Long-Term Anti-Bacterial Immunity against Systemic Infection by Salmonella enterica Serovar Typhimurium Elicited by a GMMA-Based Vaccine.

Salmonella Typhimurium (STm) represents the most prevalent cause of invasive non-typhoidal Salmonella (iNTS) disease, and currently no licensed vaccine is available. In this work we characterized the long-term anti-bacterial immunity elicited by a STm vaccine based on Generalized Modules of Membrane Antigens (GMMA) delivering O:4,5 antigen, using a murine model of systemic infection. Subcutaneous immunization of mice with STmGMMA/Alhydrogel elicited rapid, high, and persistent antigen-specific serum IgG and IgM responses. The serum was bactericidal in vitro. O:4,5-specific IgG were also detected in fecal samples after immunization and positively correlated with IgG observed in intestinal washes. Long-lived plasma cells and O:4,5-specific memory B cells were detected in spleen and bone marrow. After systemic STm challenge, a significant reduction of bacterial load in blood, spleen, and liver, as well as a reduction of circulating neutrophils and G-CSF glycoprotein was observed in STmGMMA/Alhydrogel immunized mice compared to untreated animals. Taken together, these data support the development of a GMMA-based vaccine for prevention of iNTS disease.

Carboxymethyl-β-glucan/chitosan nanoparticles: new thermostable and efficient carriers for antigen delivery.

In the last few decades, nanotechnology has emerged as an important tool aimed at enhancing the immune response against modern antigens. Nanocarriers designed specifically for this purpose have been shown to provide protection, stability, and controlled release properties to proteins, peptides, and polynucleotide-based antigens. Polysaccharides are particularly interesting biomaterials for the construction of these nanocarriers given their wide distribution among pathogens, which facilitates their recognition by antigen-presenting cells (APCs). In this work, we focused on an immunostimulant beta-glucan derivative, carboxymethyl-β-glucan, to prepare a novel nanocarrier in combination with chitosan. The resulting carboxymethyl-β-glucan/chitosan nanoparticles exhibited adequate physicochemical properties and an important protein association efficiency, with ovalbumin as a model compound. Moreover, thermostability was achieved through the optimization of a lyophilized form of the antigen-loaded nanoparticles, which remained stable for up to 1 month at 40 ºC. Biodistribution studies in mice showed an efficient drainage of the nanoparticles to the nearest lymph node following subcutaneous injection, and a significant co-localization with dendritic cells. Additionally, subcutaneous immunization of mice with a single dose of the ovalbumin-loaded nanoparticles led to induced T cell proliferation and antibody responses, comparable to those achieved with alum-adsorbed ovalbumin. These results illustrate the potential of these novel nanocarriers in vaccination.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s13346-021-00968-9.

Protective efficacy of mucosal and subcutaneous immunization with DnaJ-ΔA146Ply against influenza and Streptococcus pneumoniae co-infection in mice

Respiratory tract coinfections, specifically involving influenza A virus (IAV) and Streptococcus pneumoniae (S. pneumoniae), remain a major health problem worldwide. Secondary bacterial pneumonia is a common complication and an important cause of mortality related to seasonal and pandemic influenza infections. Vaccination is a basic control strategy against influenza and S. pneumoniae. The fusion protein DnaJ-ΔA146Ply is a vaccine candidate which can induce immune responses against pneumococcal infections via mucosal and subcutaneous immunization in mice. In the present study, we established a co-infection model using mouse-adapted laboratory strains of IAV (PR8) and S. pneumoniae (19F) in mice intranasally and subcutaneously immunized with DnaJ-ΔA146Ply. Our results showed that vaccinated mice suffered decreased weight loss compared with control mice. The survival rates were higher in intranasally and subcutaneously immunized mice than in control mice. In addition, the bacterial loads in nasal washes and lung homogenates were lower in vaccinated mice than in control mice. Furthermore, lung damage was alleviated in vaccinated mice compared with control mice, with less broken alveoli and less proinflammatory cytokine production. Taken together, these results indicate that vaccination with DnaJ-ΔA146Ply shows protective potential against influenza and S. pneumoniae co-infection in mice.

Poly(amino acids) as a potent self-adjuvanting delivery system for peptide-based nanovaccines.

To be optimally effective, peptide-based vaccines need to be administered with adjuvants. Many currently available adjuvants are toxic, not biodegradable; they invariably invoke adverse reactions, including allergic responses and excessive inflammation. A nontoxic, biodegradable, biocompatible, self-adjuvanting vaccine delivery system is urgently needed. Herein, we report a potent vaccine delivery system fulfilling the above requirements. A peptide antigen was coupled with poly-hydrophobic amino acid sequences serving as self-adjuvanting moieties using solid-phase synthesis, to produce fully defined single molecular entities. Under aqueous conditions, these molecules self-assembled into distinct nanoparticles and chain-like aggregates. Following subcutaneous immunization in mice, these particles successfully induced opsonic epitope-specific antibodies without the need of external adjuvant. Mice immunized with entities bearing 15 leucine residues were able to clear bacterial load from target organs without triggering the release of soluble inflammatory mediators. Thus, we have developed a well-defined and effective self-adjuvanting delivery system for peptide antigens.

Evaluation of inactivated Bordetella pertussis as a delivery system for the immunization of mice with Pneumococcal Surface Antigen A.

Pneumococcal Surface Protein A (PspA) has been successfully tested as vaccine candidate against Streptococcus pneumoniae infections. Vaccines able to induce PspA-specific antibodies and Th1 cytokines usually provide protection in mice. We have shown that the whole cell pertussis vaccine (wP) or components from acellular pertussis vaccines, such as Pertussis Toxin or Filamentous Hemagglutinin (FHA), are good adjuvants to PspA, suggesting that combined pertussis-PspA vaccines would be interesting strategies against the two infections. Here, we evaluated the potential of wP as a delivery vector to PspA. Bordetella pertussis strains producing a PspA from clade 4 (PspA4Pro) fused to the N-terminal region of FHA (Fha44) were constructed and inactivated with formaldehyde for the production of wPPspA4Pro. Subcutaneous immunization of mice with wPPspA4Pro induced low levels of anti-PspA4 IgG, even after 3 doses, and did not protect against a lethal pneumococcal challenge. Prime-boost strategies using wPPspA4Pro and PspA4Pro showed that there was no advantage in using the wPPspA4Pro vaccine. Immunization of mice with purified PspA4Pro induced higher levels of antibodies and protection against pneumococcal infection than the prime-boost strategies. Finally, purified Fha44:PspA4Pro induced high levels of anti-PspA4Pro IgG, but no protection, suggesting that the antibodies induced by the fusion protein were not directed to protective epitopes.

Alum impairs tolerogenic properties induced by allergoid‐mannan conjugates inhibiting mTOR and metabolic reprogramming in human DCs

BackgroundPolymerized allergoids conjugated to mannan (PM) are suitable vaccines for allergen‐specific immunotherapy (AIT). Alum remains the most widely used adjuvant in AIT, but its way of action is not completely elucidated. The better understanding of the mechanisms underlying alum adjuvanticity could help to improve AIT vaccine formulations.ObjectiveWe sought to investigate the potential influence of alum in the tolerogenic properties imprinted by PM at the molecular level.MethodsFlow cytometry, ELISAs, cocultures, intracellular staining and suppression assays were performed to assess alum and PM effects in human dendritic cells (DCs). BALB/c mice were immunized with PM alone or adsorbed to alum. Allergen‐specific antibodies, splenocyte cytokine production and splenic forkhead box P3 (FOXP3)+ regulatory T (Treg) cells were quantified. Metabolic and immune pathways were also studied in human DCs.ResultsAlum decreases PD‐L1 expression and IL‐10 production induced by PM in human DCs and increases pro‐inflammatory cytokine production. Alum impairs PM‐induced functional FOXP3+ Treg cells and promotes Th1/Th2/Th17 responses. Subcutaneous immunization of mice with PM plus alum inhibits in vivo induction of Treg cells promoted by PM without altering the capacity to induce functional allergen‐specific blocking antibodies. Alum inhibits mTOR activation and alters metabolic reprogramming by shifting glycolytic pathways and inhibiting reactive oxygen species (ROS) production in PM‐activated DCs, impairing their capacity to generate functional Treg cells.ConclusionWe uncover novel mechanisms by which alum impairs the tolerogenic properties induced by PM, which might well contribute to improve the formulation of novel vaccines for AIT.

Adjuvant activity of multimolecular complexes based on Glycyrrhiza glabra saponins, lipids, and influenza virus glycoproteins.

Numerous studies have shown that immunostimulatory complexes containing Quil-A saponin and various antigens are effective in stimulating the immune response and can be used as vaccine preparations for animals and humans. However, Quil-A saponin possesses toxicity and haemolytic activity. In the present work, a saponin-containing preparation named "Glabilox" was isolated from the roots of a Glycyrrhiza glabra L. plant by high-performance liquid chromatography (HPLC). The results showed that Glabilox has no toxicity or haemolytic activity and can form stable immunostimulatory complexes. Subcutaneous immunization of mice with an immunostimulating complex containing Glabilox and H7N1 influenza virus antigens stimulated high levels of humoral and cellular immunity. Vaccination of chickens with the same immunostimulating complex protected 100% of the animals after experimental infection with a homologous virus. Comparative studies showed that the immunogenic and protective activity of immunostimulatory complexes containing Quil-A and immunostimulatory complexes containing Glabilox are comparable to each other. The results of these studies indicated that Glycyrrhiza glabra saponins show great promise as safe and effective adjuvants.

Immunogenicity of Conjugates of Protective Antigen Complexes of Tularemia Microbe with Gold Nanoparticles

Conjugates of gold nanoparticles with two isolated tularemia microbe antigens, a protective antigen complex and a glycosylated protein complex, are used to obtain anti-tularemia sera and to vaccinate animals. A conjugate of gold nanoparticles with the glycosylated protein complex during the subcutaneous immunization of mice is more effective than the unconjugated antigen, which is evident from the increase in protectiveness and antibody titers. The use of conjugates of both antigens with gold nanoparticles during the immunization of rabbits makes it possible to obtain sera with a high titer of specific antibodies (1/64–1/128 titer in the diffusion precipitation reaction and 1/5120–1/10240 in the reaction of indirect hemagglutination) during a relatively short period of time and with minimal antigen consumption (1.8–10 mg). The use of immunoglobulins extracted from sera during the enzyme-linked immunosorbent assay makes it possible to detect Francisella tularensis cells of different subspecies in the amount of (5.2 ± 0.5) × 105 MC/mL with 100% specificity for heterologous strains at a concentration of 108 MC/mL, which enables their subsequent application in the production of preparations for the diagnostics of tularemia.

Expression of Toll-Like Receptor 2 by Dendritic Cells Is Essential for the DnaJ-ΔA146Ply-Mediated Th1 Immune Response against Streptococcus pneumoniae.

The fusion protein DnaJ-ΔA146Ply could induce cross-protective immunity against pneumococcal infection via mucosal and subcutaneous immunization in mice in the absence of additional adjuvants. DnaJ and Ply are both Toll-like receptor 4 (TLR4) but not TLR2 ligands. However, we found that TLR2-/- mice immunized subcutaneously with DnaJ-ΔA146Ply showed significantly lower survival rates and higher bacterial loads in nasal washes than did wild-type (WT) mice after being challenged with pneumococcal strain D39 or 19F. The gamma interferon (IFN-γ) level in splenocytes decreased in TLR2-/- mice, indicating that Th1 immunity elicited by DnaJ-ΔA146Ply was impaired in these mice. We explored the mechanism of protective immunity conferred by DnaJ-ΔA146Ply and the role of TLR2 in this process. DnaJ-ΔA146Ply effectively promoted dendritic cell (DC) maturation via TLR4 but not the TLR2 signaling pathway. In a DnaJ-ΔA146Ply-treated DC and naive CD4+ T cell coculture system, the deficiency of TLR2 in DCs resulted in a significant decline of IFN-γ production and Th1 subset differentiation. The same effect was observed in adoptive-transfer experiments. In addition, TLR2-/- DCs showed remarkably lower levels of the Th1-polarizing cytokine IL-12p70 than did WT DCs, suggesting that TLR2 was indispensable for DnaJ-ΔA146Ply-induced IL-12 production and Th1 proliferation. Thus, our findings illustrate that dendritic cell expression of TLR2 is essential for optimal Th1 immune response against pneumococci in mice immunized subcutaneously with DnaJ-ΔA146Ply.

Molecular and cellular signatures underlying superior immunity against Bordetella pertussis upon pulmonary vaccination

Mucosal immunity is often required for protection against respiratory pathogens but the underlying cellular and molecular mechanisms of induction remain poorly understood. Here, systems vaccinology was used to identify immune signatures after pulmonary or subcutaneous immunization of mice with pertussis outer membrane vesicles. Pulmonary immunization led to improved protection, exclusively induced mucosal immunoglobulin A (IgA) and T helper type 17 (Th17) responses, and in addition evoked elevated systemic immunoglobulin G (IgG) antibody levels, IgG-producing plasma cells, memory B cells, and Th17 cells. These adaptive responses were preceded by unique local expression of genes of the innate immune response related to Th17 (e.g., Rorc) and IgA responses (e.g., Pigr) in addition to local and systemic secretion of Th1/Th17-promoting cytokines. This comprehensive systems approach identifies the effect of the administration route on the development of mucosal immunity, its importance in protection against Bordetella pertussis, and reveals potential molecular correlates of vaccine immunity to this reemerging pathogen.