Fate and Dissemination of Bacillus subtilis Spores in a Murine Model

Bacterial spores are being consumed as probiotics, although little is known about their efficacy or mode of action. As a first step in characterizing spore probiotics, we have studied the persistence and dissemination of Bacillus subtilis spores given orally to mice. Our results have shown that spores do not appear to disseminate across the mucosal surfaces. However, we found that the number of spores excreted in the feces of mice was, in some experiments, larger than the original inoculum. This was an intriguing result and might be explained by germination of a proportion of the spore inoculum in the intestinal tract, followed by limited rounds of cell growth and then sporulation again. This result raises the interesting question of whether it is the spore or the germinated spore that contributes to the probiotic effect of bacterial spores.     

Novel C5a Agonist-Based Dendritic Cell Vaccine in a Murine Model of Melanoma

A novel method to improve targeting and presentation of poorly immunogenic tumor-related antigens was investigated. This was performed with a molecular adjuvant constructed by covalently linking a response selective peptide agonist of C5a (YSFKDMP(MeL)aR) to known melanoma tumor-related antigens. C57Bl/6J mice were injected subcutaneously with bone marrow derived dendritic cells (DCs) pulsed with a melanoma epitope (TRP2-P2/Agonist), melanoma epitope tyrosinase (TYR/Agonist), a nonfunctional reverse conformation C5a agonist bound to TYR(reverse peptide) or DMSO-PBS vehicle. Mice were injected with the pulsed DCs and cytokines IL-2 and GM-CSF three times prior to subcutaneous challenge with B16-F10 melanoma cells. All groups subsequently received DC vaccine boosters twice per week. Tumor growth was reduced and survival enhanced in mice immunized with the combination of TRP2-P2/Agonist and TYR/Agonist compared to mice receiving reverse peptide or vehicle.     

Cytochrome Pigments in Spores of Bacillus cereus T

Absorption spectra of dormant spores of Bacillus cereus T suspended in glycerol showed peaks characteristic of cytochrome pigments.     

Germination of Unactivated Spores of Bacillus cereus T

Heat-activated spores of Bacillus cereus T germinate rapidly in the presence of l-alanine alone or inosine alone. In contrast, unactivated spores can not germinate in the presence of either germinant alone but rapidly in the presence of both germinants. The highest level of cooperative action of l-alanine and inosine on the germination was observed when they were present in a ratio 1 :1. Preincubations of unactivated spores with l-alanine or inosine had opposite effects on the subsequent germination in the presence of both germinants: preincubation with l-alanine stimulated the initiation of subsequent germination, while preincubation with inosine inhibited it. These results suggest that germination of unactivated spores initiated by l-alanine and inosine includes two steps, the first initiated by l-alanine and the second prompted by inosine. The effect of preincubation of unactivated spores with l-alanine was not diminished by washings. The pH dependence of the preincubation of unactivated spores was not so marked as that of the subsequent germination in the presence of inosine.     

Bacillus subtilis Spores as Vaccine Adjuvants: Further Insights into the Mechanisms of Action

Bacillus subtilis spores have received growing attention regarding potential biotechnological applications, including the use as probiotics and in vaccine formulations. B. subtilis spores have also been shown to behave as particulate vaccine adjuvants, promoting the increase of antibody responses after co-administration with antigens either admixed or adsorbed on the spore surface. In this study, we further evaluated the immune modulatory properties of B. subtilis spores using a recombinant HIV gag p24 protein as a model antigen. The adjuvant effects of B. subtilis spores were not affected by the genetic background of the mouse lineage and did not induce significant inflammatory or deleterious effects after parenteral administration. Our results demonstrated that co-administration, but not adsorption to the spore surface, enhanced the immunogenicity of that target antigen after subcutaneous administration to BALB/c and C57BL/6 mice. Spores promoted activation of antigen presenting cells as demonstrated by the upregulation of MHC and CD40 molecules and enhanced secretion of pro-inflammatory cytokines by murine dendritic cells. In addition, in vivo studies indicated a direct role of the innate immunity on the immunomodulatory properties of B. subtilis spores, as demonstrated by the lack of adjuvant effects on MyD88 and TLR2 knockout mouse strains.     

Mathematical Model of Thermal Destruction of Bacillus stearothermophilus Spores

The experimental survival curves of Bacillus stearothermophilus spores in aqueous suspension, for six constant temperatures ranging from 105 to 130°C, displayed an initial shoulder before a linear decline. To interpret these observations, we supposed that, before the heat treatment, the designated spore suspension contained a countable and mortal N₀ population of activated spores and an M₀ population of dormant spores which remained masked during spore counting and had to be activated before being destroyed by heat. We also hypothesized that the mechanisms of both activation and destruction are, at constant temperature, ruled by first-order kinetics, with velocity constants k_A and k_D, respectively. Mathematical analysis showed that this model could represent not only our experimental survival curves, but also all other shapes (linear and biphasic) of survival curves found in the literature; also, there is an inherent symmetry in the model formulation between the activation and destruction reactions, and we showed that the dormancy rate (τ = M₀/N₀) is the only parameter which permits a distinction between the two reactions. By applying the model to our experimental data and considering that the dormancy rate is not dependent on the treatment temperature, we showed that, for the studied suspension, the limiting reaction was the activation reaction.     

UV Resistance of Bacillus anthracis Spores Revisited: Validation of Bacillus subtilis Spores as UV Surrogates for Spores of B. anthracis Sterne

Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of Bacillus anthracis. A review of the literature revealed that B. anthracis Sterne spores may be three to four times more resistant to 254-nm-wavelength UV than are spores of commonly used indicator strains of Bacillus subtilis. To test this notion, B. anthracis Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of B. subtilis, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against B. subtilis spores are likely also sufficient to inactivate B. anthracis spores and that the spores of standard B. subtilis strains could reliably be used as a biodosimetry model for the UV inactivation of B. anthracis spores.     

IL-12增强流感血凝素DNA疫苗在小鼠中抗流感作用

流感病毒的表面抗原血凝素 (hemagglutinin ,HA)能作为DNA疫苗抗流感病毒攻击 ,在小鼠模型中检测白介素 12 (interleukin 12 ,IL 12 )能否作为HADNA疫苗佐剂增强小鼠抗流感病毒攻击。将IL 12和HA共同免疫小鼠 ,免疫 2次 ,间隔 3周 ,加强免疫后用致死量流感病毒攻击。共同免疫IL 12和HADNA与单独免疫HA相比 ,无论初免还是加强免疫后血清中抗HA的IgG抗体显著提高 ,小鼠体重丢失 (一种临床症状 )明显减少且提高了小鼠的存活率。这些结果表明了IL 12能作为一种佐剂提高流感DNA疫苗的免疫效价。     

Evaluation of a Stochastic Inactivation Model for Heat-Activated Spores of Bacillus spp.

Heat activates the dormant spores of certain Bacillus spp., which is reflected in the “activation shoulder” in their survival curves. At the same time, heat also inactivates the already active and just activated spores, as well as those still dormant. A stochastic model based on progressively changing probabilities of activation and inactivation can describe this phenomenon. The model is presented in a fully probabilistic discrete form for individual and small groups of spores and as a semicontinuous deterministic model for large spore populations. The same underlying algorithm applies to both isothermal and dynamic heat treatments. Its construction does not require the assumption of the activation and inactivation kinetics or knowledge of their biophysical and biochemical mechanisms. A simplified version of the semicontinuous model was used to simulate survival curves with the activation shoulder that are reminiscent of experimental curves reported in the literature. The model is not intended to replace current models to predict dynamic inactivation but only to offer a conceptual alternative to their interpretation. Nevertheless, by linking the survival curve''s shape to probabilities of events at the individual spore level, the model explains, and can be used to simulate, the irregular activation and survival patterns of individual and small groups of spores, which might be involved in food poisoning and spoilage.Heat inactivation kinetics of bacterial spores is a well-researched field. Much of the work on its relation to foods has focused on the heat-resistant spores of Clostridia, particularly those of Clostridium botulinum, which to this date serves as the reference organism in sterility calculations of low-acid foods (8, 32). The thermal resistance of Bacilli spores, although also extensively studied, has received less attention in the literature on food preservation. This is primarily because they are unlikely to germinate and produce cells that will survive and divide under the anaerobic conditions in a sterilized food container. Yet the mere possibility of viable Bacillus spores being present in processed foods has become an issue of food safety and a security concern. For this reason, there is a renewed interest in these spores'' heat resistance (2, 3, 6, 7, 16, 30). One of the peculiarities of certain Bacillus spores, like those of Bacillus sporothermodurans or Bacillus stearothermophilus, is that many of them can remain dormant unless activated by heat. The result is a survival curve that exhibits an “activation shoulder,” as shown schematically in Fig. ​Fig.11 and with published data in Fig. ​Fig.2.2. Thus, modeling this survival pattern, where the number of spores initially grows rather than declines, must account for the heat''s dual role of being a lethal agent and activator at the same time.Open in a separate windowFIG. 1.A schematic view of a survival curve having an activation shoulder. S(t) is the ratio between the number N(t) of viable spores at time t and the initial number N₀. Notice the discrepancy between the two ways to estimate the number of dormant spores, represented by the dashed and dotted gray lines.Open in a separate windowFIG. 2.Demonstration of the fit of equation 1 (solid line) and equation 2 (dashed line) to survival curves of B. stearothermophilus spores at two temperatures. Notice the postpeak concavity of the curves. In such cases, the estimated number of dormant spores reached by the tangent method will depend on the experiment duration. The original experimental data are from Sapru et al. (25).Traditionally, the thermal inactivation of both Clostridia and Bacilli spores has been thought to follow first-order kinetics (9, 12, 31), an assumption that has been frequently challenged in recent years (18, 21, 33, 35). The most publicized model of the simultaneous heat activation and inactivation of Bacillus spores in food is that proposed by Sapru et al. (24, 25), which is an improved version of models proposed earlier by Shull et al. (29) and Rodriguez et al. (23). All of these authors and others (1, 17) assumed that the activation of dormant spores follows first-order kinetics and so does their inactivation before and after activation. The temperature dependence of the corresponding exponential rate constants was assumed to follow the Arrhenius equation.Peleg (18, 20) and van Boekel (33, 35) have shown that none of the above assumptions was necessary and that the same survival data on Bacillus stearothermophilus reported by Sapru et al. (25) and other investigators (5) can be described by different kinds of alternative four-parameter empirical models, which have a slightly better fit. This was evident not only visually (Fig. ​(Fig.2)2) but also as judged by statistical criteria (34). Fig. ​Fig.22 shows the fit of the “double Weibullian” model proposed by van Boekel (33). It has the following form: (1) where S(t) = N(t)/N₀ is the survival/activation ratio, N₀ and N(t) are the initial and momentary number of countable spores, respectively, and b₁, b₂, n₁, and n₂ are adjustable temperature-dependent constants. Figure 2 also shows the fit of an ad hoc empirical model, a hybrid between the double Weibullian model and one previously proposed (20) that can be written in the following form: (2) or (3) where a₁, b₁, t_c2, and m₂ are adjustable temperature-dependent parameters. According to this model, a₁ is the asymptote of the first term on the right, b₁ is a time characteristic of the activation, t_c2 is a characteristic time of the inactivation, and m₂ is a parameter that represents the curve''s postpeak concavity. The structure of equation 2 or 3 dictates that the number of dormant spores must be finite and cannot exceed N₀ × 10^a1, if the logarithm is base 10, or N₀ × exp(a₁), if it is base e. (A demonstration that generates realistic-looking activation/inactivation curves using equation 3 as a model is available from Wolfram Research [http://demonstrations.wolfram.com/SurvivalCurvesOfBacilliSporesWithAnActivationShoulder/].)Corradini and Peleg (5) proposed a way to estimate the initial number of dormant spores from survival curves having an activation shoulder using a similar model, which was originally described in Peleg (20). They suggested that the intersection of a tangent to the survival curve drawn at its postpeak region with the time axis (Fig. ​(Fig.1)1) is not a recommended method to estimate the number of dormant spores and that it can render unrealistically high values if used. Also, where there is no evidence that the survival curve in the postpeak region ever becomes a straight line; the same survival curve will yield different estimates of the dormant spores'' initial number depending on the experiment''s duration. Moreover, if in the postpeak region the survival ratio drop rate progressively increases, as it most probably does (Fig. ​(Fig.2)2) (20, 33), then the number of dormant spores estimated by the tangent extrapolation method will grow indefinitely, despite the fact that it must be finite (1). Also, since the exponential inactivation rate can be a function of time as well as of temperature, the applicability of the Arrhenius equation as a secondary model might come into question. The same can also be said about the log-linearity of the D value''s temperature dependence if used instead of the Arrhenius equation.The question that arises in light of all the above is whether one can construct a conceptual population dynamic model of the activation/inactivation of spores without assuming any fixed kinetic order. The biochemical and biophysical mechanisms that govern bacterial spore germination, activation, and inactivation have been thoroughly investigated (11, 13, 14, 15, 22, 26-28). Still, it is not clear how processes within an individual spore can be translated into activation and survival patterns at the population level and how their manifestation can be expressed in a mathematical model. Whenever a system has inherent variability and knowledge of its working is incomplete or merely insufficient to develop a model from basic principles, one can, and sometimes must, resort to a probabilistic modeling approach. The general objective of this work has been to explore the merits and limitations of this option by developing a stochastic model of Bacilli spores'' heat activation and inactivation and examining its properties. The goal has not been to develop a new method to predict the spores'' survival under dynamic conditions—rate versions of the existing empirical models such as equation 1, 2, or 3 seem to be quite suitable for that—but to offer an alternative interpretation of the patterns reported and discussed in the literature.     

A Sendai Virus-Derived RNA Agonist of RIG-I as a Virus Vaccine Adjuvant

The innate immune system is responsible for recognizing invading pathogens and initiating a protective response. In particular, the retinoic acid-inducible gene 1 protein (RIG-I) participates in the recognition of single- and double-stranded RNA viruses. RIG-I activation leads to the production of an appropriate cytokine and chemokine cocktail that stimulates an antiviral state and drives the adaptive immune system toward an efficient and specific response against the ongoing infection. One of the best-characterized natural RIG-I agonists is the defective interfering (DI) RNA produced by Sendai virus strain Cantell. This 546-nucleotide RNA is a well-known activator of the innate immune system and an extremely potent inducer of type I interferon. We designed an in vitro-transcribed RNA that retains the type I interferon stimulatory properties, and the RIG-I affinity of the Sendai virus produced DI RNA both in vitro and in vivo. This in vitro-synthesized RNA is capable of enhancing the production of anti-influenza virus hemagglutinin (HA)-specific IgG after intramuscular or intranasal coadministration with inactivated H1N1 2009 pandemic vaccine. Furthermore, our adjuvant is equally effective at increasing the efficiency of an influenza A/Puerto Rico/8/34 virus inactivated vaccine as a poly(I·C)- or a squalene-based adjuvant. Our in vitro-transcribed DI RNA represents an excellent tool for the study of RIG-I agonists as vaccine adjuvants and a starting point in the development of such a vaccine.     

Protective Immunity against Trichinella spiralis Infection Induced by a Multi-Epitope Vaccine in a Murine Model

Trichinellosis is one of the most important food-borne parasitic zoonoses throughout the world. Because infected pigs are the major source of human infections, and China is becoming the largest international producer of pork, the development of a transmission-blocking vaccine to prevent swine from being infected is urgently needed for trichinellosis control in China. Our previous studies have demonstrated that specific Trichinella spiralis paramyosin (Ts-Pmy) and Ts-87 antigen could provide protective immunity against T. spiralis infection in immunized mice. Certain protective epitopes of Ts-Pmy and Ts-87 antigen have been identified. To identify more Ts-Pmy protective epitopes, a new monoclonal antibody, termed 8F12, was produced against the N-terminus of Ts-Pmy. This antibody elicited significant protective immunity in mice against T. spiralis infection by passive transfer and was subsequently used to screen a random phage display peptide library to identify recognized epitopes. Seven distinct positive phage clones were identified and their displayed peptides were sequenced. Synthesized epitope peptides conjugated to keyhole limpet hemocyanin were used to immunize mice, four of which exhibited larval reduction (from 18.7% to 26.3%, respectively) in vaccinated mice in comparison to the KLH control. To increase more effective protection, the epitope 8F7 that was found to induce the highest protection in this study was combined with two other previously identified epitopes (YX1 from Ts-Pmy and M7 from Ts-87) to formulate a multi-epitope vaccine. Mice immunized with this multi-epitope vaccine experienced a 35.0% reduction in muscle larvae burden after being challenged with T. spiralis larvae. This protection is significantly higher than that induced by individual-epitope peptides and is associated with high levels of subclasses IgG and IgG1. These results showed that a multi-epitope vaccine induced better protective immunity than an individual epitope and provided a feasible approach for developing a safer and more effective vaccine against trichinellosis.     

Adjuvanticity of an Ornithine-Containing Lipid of Flavobacterium meningosepticum as a Candidate Vaccine Adjuvant

Ornithine-containing lipids (OrnL) extracted from Flavobacterium meningosepticum have been reported to have various biological activities such as B-cell mitogenicity and macrophage activation to generate interleukin-1 and prostaglandin E₂. We, using ovalbumin (OVA) as an antigen, evaluated the adjuvant activity of OrnL as an immunological adjuvant in BALB/c mice. OrnL showed the function of forming liposome-like vesicles retaining biological activities when prepared as either small unilamellar or dehydration-rehydration vesicles. Although OrnL was not shown to have enough entrapping efficacy for use as a vaccine adjuvant, phosphatidylglycerol (PG) and cholesterol (CHOL) added to stabilize the vesicle membrane increased the entrapping efficacy to the same extent as that of conventional liposomes. Furthermore, the stabilized OrnL vesicles tolerated centrifugation to remove non-entrapped antigens. Completely antigen-entrapped OrnL vesicles including PG and CHOL induced a significantly greater enhancement of IgG antibody production than did aluminum hydroxide gel in BALB/c mice from week 6. These results indicate that OrnL can be utilized as an immunological adjuvant for vaccines.     

Ultrastructural Changes Associated with Activation and Germination of Bacillus cereus T Spores

The ultrastructural changes occurring during defined stages of the transition of dormant Bacillus cereus T spores into heat-sensitive forms were investigated. The coat of the heat-activated spores displayed conspicuous striations across its middle layer. Electron microscopy of thin sections of heat-activated spores revealed the presence in the spore of a layer consisting of hexagonally oriented subunits. It was demonstrated that the subcoat region, but not the cortex, disappears rapidly during germination of B. cereus T spores. The fibrous structures apparently associated with the spore coat remain virtually unchanged during the entire course of activation and germination.     

Sensitization by 5-Bromouracil of DNA in Bacillus subtilis Spores to Ionizing Radiation

Quantitative determination by fluorescence spectroscopy is possible because of the linear relationship between the intensity of emitted fluorescence and the fluorophore concentration. However, concentration quenching may cause the relationship to become nonlinear, and thus, the optimal dilution ratio has to be determined. In the case of fluorescence fingerprint (FF) measurement, fluorescence is measured under multiple wavelength conditions and a method of determining the optimal dilution ratio for multivariate data such as FFs has not been reported. In this study, the FFs of mixed solutions of tryptophan and epicatechin of different concentrations and composition ratios were measured. Principal component analysis was applied, and the resulting loading plots were found to contain useful information about each constituent. The optimal concentration ranges could be determined by identifying the linear region of the PC score plotted against total concentration.     

Role of Calcium in Biphasic Germination of Bacillus cereus T Spores Sensitized to Lysozyme

Biphasic germination induced by inosine in the presence of Ca²⁺ was examined using Bacillus cereus T spores treated with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) at pH 10. The first phase of the germination was stimulated by Ca²⁺ in the concentration-dependent manner, showing the optimal concentration at 0.5-1.0 mM. The second phase appeared to be insensitive to the cation. The optimal temperatures for the first and the second phase were 25 C and 40 C, respectively; the optimal pHs for the two phases were 7-9 and around 7.5, respectively. Heat resistance and dipicolinic acid of the SDS-DTT-treated spores were lost mostly during the first phase. A Ca²⁺-specific chelator, glycoletherdiamine-N,N,N',N'-tetraacetic acid (GEDTA), inhibited the first phase evoked by Ca²⁺, while it had no inhibitory effect on the second phase. In contrast, the divalent cations examined, except Mg²⁺ and Sr²⁺, affected not only the first phase but also the second phase. The order of inhibitory effect on the first phase was Hg²⁺ > Zn²⁺ > Ba²⁺, Co²⁺, Cu²⁺ > Mn²⁺; on the second phase, it was Hg²⁺ > Cu²⁺ > Zn²⁺ > Co²⁺ > Mn²⁺ > Ba²⁺.     

Pasteurella haemolytica Ultraviolet-Irradiated Vaccine by Parenteral and Aerosol Routes Compared in the Goat Model

Positive control 1 (PC1) (n = 9) goats were injected transthoracically into the left lung with live Pasteurella haemolytica biovar A, serovar 1 (PhA1) in polyacrylate (PA) beads on days 0 and 21. Positive control 2 (PC2) (n = 6) goats were nebulized with live PhA1 and PA beads on days 0 and 21. Negative control (NC) goats (n = 6) were each injected transthoracically into the left lung with PA beads alone on days 0 and 21. Four groups (n = 6) were administered PA beads mixed with ultraviolet (UV) killed PhA1 on days 0 and 21. The treatment doses of bacteria for these groups were principal group 1 (PR1) injected into the left lung (7.7 × 10¹⁰ cfu); PR2, 7.7 × 10¹⁰ UV-killed PhA1 injected subcutaneously (SC); PR3, 7.7 × 10¹⁰ UV-killed PhA1 injected SC only on day 21; PR4, nebulized with PA beads mixed with 5.6 × 10¹⁰ cfu of UV-killed PhA1; and PR5, nebulized with PA beads mixed with 5 × 10⁸ cfu of UV-killed PhA1. All goats were challenged transthoracically in the right lung with 1 × 10⁸ cfu of live PhA1 on day 42 and necropsied on day 46. The sizes of consolidated lung lesions at the challenge site were used as a measure of immunity. The data show that the introduction of live PhA1 into the lungs of goats, either by injection or aerosolization, offers excellent protection against a subsequent homologous challenge. The data also demonstrate that two transthoracic injections (21 days apart) of UV-killed PhA1 (PR1), and subcutaneous injection of UV-killed PhA1(PR2) also offer excellent protection against a subsequent homologous live PhA1 challenge. One SC injection of UV-killed PhA1 (PR3) appears to offer only partial protection against a subsequent homologous live PhA1 challenge. Inhalation of UV-killed PhA1 mixed with PA beads (PR4 and PR5) induced no protection in goats against a subsequent live PhA1 transthoracic challenge. Received: 3 February 1998 / Accepted: 18 March 1998     

Small, Acid-Soluble Proteins as Biomarkers in Mass Spectrometry Analysis of Bacillus Spores

The use of 1 N HCl for extraction of small, acid-soluble proteins (SASP) from different Bacillus spore species was examined. The extracts were analyzed by high-performance liquid chromatography and matrix-assisted laser desorption mass spectrometry and were found to be both qualitatively and quantitatively superior to extraction by acetonitrile-5% trifluoroacetic acid (70:30, vol/vol). Both major and minor α/β- and γ-type SASP were characterized by their molecular masses or tryptic peptide maps and by searches of both protein and unannotated genome databases. For all but 1 pair (B. cereus T and B. thuringiensis subsp. Kurstaki) among the 11 variants studied the suites of SASP masses are distinctive, consistent with the use of these proteins as potential biomarkers for spore identification by mass spectrometry.     

The Chemotherapeutic Agent DMXAA as a Unique IRF3-Dependent Type-2 Vaccine Adjuvant

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a potent type I interferon (IFN) inducer, was evaluated as a chemotherapeutic agent in mouse cancer models and proved to be well tolerated in human cancer clinical trials. Despite its multiple biological functions, DMXAA has not been fully characterized for the potential application as a vaccine adjuvant. In this report, we show that DMXAA does act as an adjuvant due to its unique property as a soluble innate immune activator. Using OVA as a model antigen, DMXAA was demonstrated to improve on the antigen specific immune responses and induce a preferential Th2 (Type-2) response. The adjuvant effect was directly dependent on the IRF3-mediated production of type-I-interferon, but not IL-33. DMXAA could also enhance the immunogenicity of influenza split vaccine which led to significant increase in protective responses against live influenza virus challenge in mice compared to split vaccine alone. We propose that DMXAA can be used as an adjuvant that targets a specific innate immune signaling pathway via IRF3 for potential applications including vaccines against influenza which requires a high safety profile.     

Evaluation of the Protective Immunity of a Novel Subunit Fusion Vaccine in a Murine Model of Systemic MRSA Infection

Staphylococcus aureus is a common commensal organism in humans and a major cause of bacteremia and hospital acquired infection. Because of the spread of strains resistant to antibiotics, these infections are becoming more difficult to treat. Therefore, exploration of anti-staphylococcal vaccines is currently a high priority. Iron surface determinant B (IsdB) is an iron-regulated cell wall-anchored surface protein of S. aureus. Alpha-toxin (Hla) is a secreted cytolytic pore-forming toxin. Previous studies reported that immunization with IsdB or Hla protected animals against S. aureus infection. To develop a broadly protective vaccine, we constructed chimeric vaccines based on IsdB and Hla. Immunization with the chimeric bivalent vaccine induced strong antibody and T cell responses. When the protective efficacy of the chimeric bivalent vaccine was compared to that of individual proteins in a murine model of systemic S. aureus infection, the bivalent vaccine showed a stronger protective immune response than the individual proteins (IsdB or Hla). Based on the results presented here, the chimeric bivalent vaccine affords higher levels of protection against S. aureus and has potential as a more effective candidate vaccine.