Polyethylenimine quantity and molecular weight influence its adjuvanting properties in liposomal peptide vaccines.

We recently reported that polyethylenimine (PEI; molecular weight of 600 Da) acted as a vaccine adjuvant for liposomal group A Streptococcus (GAS) vaccines, eliciting immune responses in vivo with IgG antibodies giving opsonic activity against five Australian GAS clinical isolates. However, to date, no investigation comparing the structure-activity relationship between the molecular weight of PEI and its adjuvanting activity in vaccine development has been performed. We hypothesized that the molecular weight and quantity of PEI in a liposomal vaccine will impact its adjuvanting properties. In this study, we successfully formulated liposomes containing different molecular weights of PEI (600, 1800, 10k and 25k Da) and equivalents of PEI (0.5, 1 and 2) of branched PEI. Outbred mice were administrated the vaccine formulations intranasally, and the mice that received a high ratio of PEI 600 reported a stronger immune response than the mice that received a lower ratio of PEI 600. Interestingly, mice that received the same quantity of PEI 600, PEI 10k and PEI 25k showed similar immune responses in vivo and in vitro. This comparative study highlights the ratio of PEI present in the liposome vaccines impacts adjuvanting activity, however, PEI molecular weight did not significantly enhance its adjuvanting properties. We also report that the stability of PEI liposomes is critical for vaccines to elicit the desired immune response.

Immunogenicity and in vitro and in vivo protective effects of antibodies targeting a recombinant form of the Streptococcus mutans P1 surface protein.

Streptococcus mutans is a major etiologic agent of dental caries, a prevalent worldwide infectious disease and a serious public health concern. The surface-localized S. mutans P1 adhesin contributes to tooth colonization and caries formation. P1 is a large (185-kDa) and complex multidomain protein considered a promising target antigen for anticaries vaccines. Previous observations showed that a recombinant P1 fragment (P1(39-512)), produced in Bacillus subtilis and encompassing a functional domain, induces antibodies that recognize the native protein and interfere with S. mutans adhesion in vitro. In the present study, we further investigated the immunological features of P1(39-512) in combination with the following different adjuvants after parenteral administration to mice: alum, a derivative of the heat-labile toxin (LT), and the phase 1 flagellin of S. Typhimurium LT2 (FliCi). Our results demonstrated that recombinant P1(39-512) preserves relevant conformational epitopes as well as salivary agglutinin (SAG)-binding activity. Coadministration of adjuvants enhanced anti-P1 serum antibody responses and affected both epitope specificity and immunoglobulin subclass switching. Importantly, P1(39-512)-specific antibodies raised in mice immunized with adjuvants showed significantly increased inhibition of S. mutans adhesion to SAG, with less of an effect on SAG-mediated bacterial aggregation, an innate defense mechanism. Oral colonization of mice by S. mutans was impaired in the presence of anti-P1(39-512) antibodies, particularly those raised in combination with adjuvants. In conclusion, our results confirm the utility of P1(39-512) as a potential candidate for the development of anticaries vaccines and as a tool for functional studies of S. mutans P1.

Microbially synthesized modular virus-like particles and capsomeres displaying group A streptococcus hypervariable antigenic determinants.

Effective and low-cost vaccines are essential to control severe group A streptococcus (GAS) infections prevalent in low-income nations and the Australian aboriginal communities. Highly diverse and endemic circulating GAS strains mandate broad-coverage and customized vaccines. This study describes an approach to deliver cross-reactive antigens from endemic GAS strains using modular virus-like particle (VLP) and capsomere systems. The antigens studied were three heterologous N-terminal peptides (GAS1, GAS2, and GAS3) from the GAS surface M-protein that are specific to endemic strains in Australia Northern Territory Aboriginal communities. In vivo data presented here demonstrated salient characteristics of the modular delivery systems in the context of GAS vaccine design. First, the antigenic peptides, when delivered by unadjuvanted modular VLPs or adjuvanted capsomeres, induced high titers of peptide-specific IgG antibodies (over 1 × 10(4) ). Second, delivery by capsomere was superior to VLP for one of the peptides investigated (GAS3), demonstrating that the delivery system relative effectiveness was antigen-dependant. Third, significant cross-reactivity of GAS2-induced IgG with GAS1 was observed using either VLP or capsomere, showing the possibility of broad-coverage vaccine design using these delivery systems and cross-reactive antigens. Fourth, a formulation containing three pre-mixed modular VLPs, each at a low dose of 5 µg (corresponding to <600 ng of each GAS peptide), induced significant titers of IgGs specific to each peptide, demonstrating that a multivalent, broad-coverage VLP vaccine formulation was possible. In summary, the modular VLPs and capsomeres reported here demonstrate, with promising preliminary data, innovative ways to design GAS vaccines using VLP and capsomere delivery systems amenable to microbial synthesis, potentially adoptable by developing countries.

An optimized caries model of Streptococcus mutans in rats and its application for evaluating prophylactic vaccines.

Dental caries is a prevalent oral disease that mainly results from Streptococcus mutans. Susceptibility to S. mutans decreased rapidly after weaning in a well-known rat model. However, owing to the lack of time to establish protective immunity ahead of challenge, the weaning rat model is suboptimal for assessing prophylactic vaccines against S. mutans infection. In this study, we found that, in adult rats, S. mutans cultured under air-restricted conditions showed dramatically increased colonization efficacy and accelerated development of dental caries compared with those cultured under air-unrestricted conditions. We propose that S. mutans cultured under air-restricted conditions can be used to develop an optimal caries model, especially for the evaluation of prophylactic efficacy against S. mutans. Therefore, we used the anti-caries vaccine, KFD2-rPAc, to reevaluate the protection against the challenge of S. mutans. In immunized rats, rPAc-specific protective antibodies were robustly elicited by KFD2-rPAc before the challenge. In addition to inhibiting the initial and long-term colonization of S. mutans in vivo, KFD2-rPAc immunization showed an 83% inhibitory efficacy against the development of caries, similar to that previously evaluated in a weaning rat model. These results demonstrate that culturing under air-restricted conditions can promote S. mutans infection in adult rats, thereby helping establish a rat infection model to evaluate the prophylactic efficacy of vaccines and anti-caries drugs.

Prime-Pull Immunization with a Bivalent M-Protein and Spy-CEP Peptide Vaccine Adjuvanted with CAF®01 Liposomes Induces Both Mucosal and Peripheral Protection from covR/S Mutant Streptococcus pyogenes.

Infections with Streptococcus pyogenes and their sequelae are responsible for an estimated 18 million cases of serious disease with >700 million new primary cases and 500,000 deaths per year. Despite the burden of disease, there is currently no vaccine available for this organism. Here, we define a combination vaccine P*17/K4S2 comprising of 20-mer B-cell peptide epitopes, p*17 (a mutant derived from the highly conserved C3-repeat region of the M-protein), and K4S2 (derived from the streptococcal anti-neutrophil factor, Spy-CEP). The peptides are chemically conjugated to either diphtheria toxoid (DT) or a nontoxic mutant form of diphtheria toxin, CRM197. We demonstrate that a prime-pull immunization regimen involving two intramuscular inoculations with P*17/K4S2 adjuvanted with a two-component liposomal adjuvant system (CAF01; developed by Statens Serum Institut [SSI], Denmark), followed by an intranasal inoculation of unadjuvanted vaccine (in Tris) induces peptide- and S. pyogenes-binding antibodies and protects from mucosal and skin infection with hypervirulent covR/S mutant organisms. Prior vaccination with DT does not diminish the response to the conjugate peptide vaccines. Detailed Good Laboratory Practice (GLP) toxicological evaluation in male and female rats did not reveal any gross or histopathological adverse effects.IMPORTANCE A vaccine to control S. pyogenes infection is desperately warranted. S. pyogenes colonizes the upper respiratory tract (URT) and skin, from where it can progress to invasive and immune-mediated diseases. Global mortality estimates for S. pyogenes-associated diseases exceeds 500,000 deaths per year. S. pyogenes utilizes antigenic variation as a defense mechanism to circumvent host immune responses and thus a successful vaccine needs to provide strain-transcending and multicompartment (mucosal and skin) immunity. By defining highly conserved and protective epitopes from two critical virulence factors (M-protein and Spy-CEP) and combining them with a potent immunostimulant, CAF®01, we are addressing an unmet clinical need for a mucosally and skin-active subunit vaccine. We demonstrate that prime-pull immunization (2× intramuscular injections followed by intranasal immunization) promotes high sustained antibody levels in the airway mucosa and serum and protects against URT and invasive disease.

Enhancing the immunogenicity of a DNA vaccine against Streptococcus mutans by attenuating the inhibition of endogenous miR-9.

DNA vaccine provides a promising method for preventing and treating diseases. However, the low immunogenicity restricts its application. New approaches are urgent to be explored to enhance the immune response of DNA vaccine. MicroRNAs are endogenous, small non-coding RNAs which play parts in gene expression inhibition. In this study, microRNA-9 (miR-9) was found to inhibit the expression of the GLU-A-P antigen protein encoded by the anti-caries DNA vaccine. Mutation of miR-9 binding sites in the gene fragment encoding GLU-A-P antigen protein significantly increased the expression of antigen protein. Moreover, miR-9 sponge can improve the expression of the GLU-A-P antigen protein. The co-immunization with miR-9 sponge and anti-caries DNA vaccine significantly enhanced the specific immune response in vivo. In conclusion, attenuating the inhibition of endogenous miR-9 enhanced the antigen expression and immunogenicity of the anti-caries DNA vaccine.

Enhanced immunogenicity of an anti-caries vaccine encoding a cell-surface protein antigen of Streptococcus mutans by intranasal DNA prime-protein boost immunization.

BACKGROUND: The present study aimed to enhance the specific anti-caries immunity induced by DNA prime-protein boost strategy for an A-P fragment of a cell-surface protein antigen of Streptococcus mutans (PAc). METHODS: BALB/c mice were immunized with DNA prime-protein boost, DNA-DNA or protein-protein regimens by the intranasal route, using combinations of plasmid vector (pCIA-P) that express PAc protein and a pure secretec recombinant PAc protein (rPAc). Then, a gnotobiotic mouse model was constructed 2 weeks after the last immunization, and specific immune responses in vivo and their protection against dental caries were observed. RESULTS: The present study revealed stronger antibody responses in the DNA prime-protein boost group compared to those elicited by either DNA-DNA vaccination or protein-protein vaccination. In particular, PAc-specific antibody concentrations were improved significantly after boosting the pCIA-P DNA-primed mice with rPAc. Moreover, protection against S. mutans challenge was obtained in the mice treated with the DNA prime-protein boost vaccination, as demonstrated by a significant reduction in S. mutans colonization compared to control mice and animals immunized with the DNA-DNA vaccination or protein-protein vaccination. CONCLUSIONS: The results obtained in the present study suggest that the intranasal DNA prime-protein boost vaccination regimen is a novel strategy for the practical application of DNA vaccine against dental caries.

Secretory immunity following mutans streptococcal infection or immunization.

Salivary IgA antibody responses to mutans streptococci can be observed in early childhood, sometimes even before permanent colonization of the oral biofilm occurs. Many of these early immune responses are directed to components thought to be essential for establishment and emergence of mutans streptococci in the oral biofilm. Initial responses are likely to be modulated by antigen dose, by immunological maturity, and by previous encounters with similar antigenic epitopes in the pioneer commensal flora. Our understanding of these modulating factors is modest and is an opportunity for continued investigation. Under controlled conditions of infection, experimental vaccine approaches have repeatedly shown that infection and disease can be modified in the presence of elevated levels of antibody in the oral cavity. Protection can be observed regardless of antibody isotype or method used to actively or passively provide the immune reagent. Limited clinical trials have supported the utility of both of these approaches in humans. Refinements in antigen formulation, delivery vehicles, enhancing agents and routes of application, coupled with approaches that are timed to intercept most vulnerable periods of infection of primary and permanent dentition may well provide the healthcare practitioner with an additional tool to maintain oral health.

[Protective efficacy of a new fusion anti-caries DNA vaccine encoding antigens of both Streptococcus mutans and Streptococcus sobrinus].

OBJECTIVE: To construct a new fusion anti-caries DNA vaccine pGJGAC/VAX encoding antigens of both S. mutans and S. sobrinus so as to enhance the protective effect of DNA vaccine against S. sobrinus infection. METHODS: The CAT fragment of S. sobrinus OMZ176 gtf-I was amplified by semi-nest PCR and then inserted into the plasmid pGJA-P/VAX to construct the recombinant plasmid pGJGAC/VAX. The CHO cell was transfected and the expression of fusion protein detected using cellular immunohistochemistry and Western blot. Mice were immunized with pGJGAC/VAX and control plasmids via the intramuscular (i.m) or intranasal (i.n) routes. During the experiment, blood and saliva samples were collected at a 2-week interval for antibody assay by ELISA. Rats were orally challenged with S. mutans Ingbritt or S. sobrinus 6715 and then immunized i.n with pGJGAC/VAX, pGJA-P/VAX or pVAX1. The Keyes method was used to determine the caries activity. RESULTS: (1) CAT sequence was identical to the related sequence of gtf-I (OMZ176) in GenBank. The recombinant plasmid pGJGAC/VAX encoded the genes of antigens of both S. mutans and S. sobrinus. The expressed protein could respond to specific anti-PAc, anti-GLU and anti-CAT antibodies respectively. (2) As for antibody reactions, mice in the experiment group had significantly higher levels of anti-PAc, anti-GLU and anti-CAT IgG antibodies than those in the pVAX1 group (P < 0.01). The peak responses of specific anti-CAT antibodies were observed at 8 weeks (GAC/i.m) and 10 weeks (GAC/i.n) and were approximately 62.13 microg/ml and 11.43 microg/ml respectively. The peak responses of specific anti-CAT IgA antibodies were seen at 8 weeks (GAC/i.m) and 10 weeks (GAC/i.n) and were approximately 0.67% and 0.80% respectively. (3) In the group infected with S. mutans or S. sobrinus, the pGJGAC/VAX-immunized rats showed significantly fewer E, Ds and Dm lesions than pVAX1-immunized rats (P < 0.05) and decreased Ds and Dm levels than pGJA-P/VAX-immunized rats (P < 0.05) while there was no obvious difference in E lesions between the two groups (P > 0.05). CONCLUSION: A new fusion anti-caries DNA vaccine pGJGAC/VAX encoding antigens of both S. mutans and S. sobrinus is constructed successfully and expressed correctly in eukaryotic cells. It induces effective mucosal and systematic humoral responses so as to provide a better protection against S. sobrinus.

Nasal vaccination with pneumococcal surface protein A in combination with cationic liposomes consisting of DOTAP and DC-chol confers antigen-mediated protective immunity against Streptococcus pneumoniae infections in mice.

Infectious diseases are the second leading cause of death worldwide, suggesting that there is still a need for the development of new and improved strategies for combating pathogens effectively. Streptococcus pneumoniae is the most virulent bacteria causing pneumonia with high mortality, especially in children and the elderly. Because of the emergence of antibiotic resistance in S. pneumoniae, employing a serotype-independent mucosal vaccine would be the best approach to prevent and treat the diseases caused by S. pneumoniae. In this study, we have developed a pneumococcal nasal vaccine, consisting of pneumococcal surface protein A (PspA) and cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and cholesteryl 3ß-N-(dimethylaminoethyl)-carbamate (DC-chol) (DOTAP/DC-chol liposome). The efficiency of this cationic liposome-based PspA nasal vaccine was examined in a murine model of S. pneumoniae infection. Intranasal vaccination with PspA and DOTAP/DC-chol liposomes conferred protective immunity against lethal inhalation of S. pneumoniae, improving the survival rate of infected mice. Moreover, intranasal immunization with PspA and DOTAP/DC-chol liposomes not only induced the production of PspA-specific IgA and IgG by both mucosal and systemic compartments but also elicited PspA-specific Th17 responses, which play a pivotal role in controlling S. pneumoniae infection by host innate immune response. We further demonstrated that DOTAP/DC-chol liposomes enhanced PspA uptake by nasal dendritic cells (DCs), which might be a mechanism for the induction of protective immune responses to S. pneumoniae infection. These results show that DOTAP/DC-chol liposome would be an efficient mucosal vaccine system for a serotype-independent universal nasal vaccine against pneumococcal infection.

Enhancement of salivary IgA response to a DNA vaccine against Streptococcus mutans wall-associated protein A in mice by plasmid-based adjuvants.

A specific salivary IgA (sIgA) response was obtained in mice by intranasal immunization with a naked DNA vaccine consisting of the Streptococcus mutans wall-associated protein A gene (wapA) inserted into the mammalian expression vector pcDNA3.1/V5/His-TOPO. In the present study, the vaccine, referred to as pcDNA-wapA, was administered with or without the cationic lipid DMRIE-C. No mucosal response was observed in mice immunized with the vaccine alone, whereas a weak and temporal sIgA response was obtained when the vaccine was mixed with DMRIE-C. To investigate the use of pcDNA containing the interleukin 5 (IL-5) gene (pcDNA-il-5) or the cholera toxin B gene (pcDNA-ctb) as genetic adjuvants, these constructs were used in co-immunization studies. The enhancement effect was transient with pcDNA-il-5, but longer lasting with pcDNA-ctb, thus supporting the use of the latter as a genetic adjuvant to DNA vaccine.

Self-assembling anticaries mucosal vaccine containing ferritin cage nanostructure and glucan-binding region of S. mutans glucosyltransferase effectively prevents caries formation in rodents.

Anticaries protein vaccines that induce a mucosal immune response are not effective. Therefore, development of effective and convenient anticaries vaccines is a priority of dental research. Here we generated self-assembling nanoparticles by linking the glucan-binding region of Streptococcus mutans glucosyltransferase (GLU) to the N-terminal domain of ferritin to determine whether these novel nanoparticles enhanced the immunogenicity of an anticaries protein vaccine against GLU in rodents. We constructed the expression plasmid pET28a-GLU-FTH and purified the proteins from bacteria using size-exclusion chromatography. BALB/c mice were used to evaluate the ability of GLU-ferritin (GLU-FTH) nanoparticles to induce GLU-specific mucosal and systemic responses. The protective efficiency of GLU-FTH nanoparticles was compared with that of GLU alone or a mixture of GLU and poly(I:C) after administering an intranasal infusion to Wistar rats. The phagocytosis and maturation of dendritic cells (DCs) exposed in vitro to the nanoparticles were assessed using flow cytometry. The GLU-FTH nanoparticle vaccine elicited significantly higher levels of GLU-specific antibodies compared with GLU or a mixture of GLU and poly(I:C). Immunization with GLU-FTH achieved lower caries scores compared with those of the other vaccines. Administration of GLU-FTH nanoparticles enhanced phagocytosis by DCs and their maturation. Thus, self-assembling GLU-FTH is a highly effective anticaries mucosal vaccine that enhanced antibody production and inhibited S. mutans infection in rodents.

Development of a single-dose recombinant CAMP factor entrapping poly(lactide-co-glycolide) microspheres-based vaccine against Streptococcus agalactiae.

Streptococcus agalactiae is an important contagious bovine mastitis pathogen. Although it is well controlled and even eradicated in most Northern European and North American dairy herds, the prevalence of this pathogen remains very high in China. However, research on development of a vaccine against S. agalactiae mastitis is scarce. The aims of the present study were to: (1) develop a single-dose vaccine against S. agalactiae based on poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) encapsulated CAMP factor, a conserved virulent protein encoded by S. agalactiae's cfb gene; and (2) evaluate its immunogenicity and protective efficacy in a mouse model. The cfb gene was cloned and expressed in a recombinant Escherichia coli strain Trans1-T1. The CAMP factor was tested to determine a safe dose range and then encapsulated in MS of PLGA (50:50) to assess its release pattern in vitro and immune reaction in vivo. Furthermore, a mouse model and a histopathological assay were developed to evaluate bacterial burden and vaccine efficacy. In the low dosage range (<100µg), CAMP factor had no obvious toxicity in mice. The release pattern in vitro was characterized by an initial burst release (44%), followed by a sustained and slower release over 7wk. In mice immunized with either pure CAMP factor protein or PLGA-CAMP, increased antibody titers were detected in the first 2wk, whereas only PLGA-CAMP immunization induced a sustained increase of antibody titers. In mice vaccinated with PLGA-CAMP, mortality and bacteria counts were lower (compared to a control group) after S. agalactiae challenge. Additionally, no pathological lesions were detected in the vaccinated group. Therefore, PLGA-CAMP conferred protective efficacy against S. agalactiae in our mouse model, indicating its potential as a vaccine against S. agalactiae mastitis. Furthermore, the slow-release kinetics of PLGA MS warranted optimism for development of a single-dose vaccine.

Immunogenicity and persistence of a targeted anti-caries DNA vaccine.

We have previously reported that a targeted anti-caries DNA vaccine, pGJA-P, induced accelerated and increased antibody responses compared with a non-targeted anti-caries DNA vaccine. Recently, pGJA-P/VAX, a new targeted anti-caries DNA vaccine for human trials, was constructed by replacing the pCI vector used in the construction of pGJA-P with pVAX1, the only vector authorized by the US Food and Drug Administration in clinical trials. Here, we report on our exploration of the kinetics of the antibody responses generated following pGJA-P/VAX immunization and the persistence of pGJA-P/VAX at both the inoculation site and the draining lymph nodes. Intranasal vaccination of mice with pGJA-P/VAX induced strong antibody responses that lasted for more than 6 months. Furthermore, pGJA-P/VAX could still be detected at both the inoculation site and the draining cervical lymph nodes 6 months after immunization. Thus, the persistent immune responses are likely due to the DNA depot in the host, which acts as a booster immunization.

Liposome-based intranasal delivery of lipopeptide vaccine candidates against group A streptococcus.

UNLABELLED: Group A streptococcus (GAS), an exclusively human pathogen, causes a wide range of diseases ranging from trivial to life threatening. Treatment of infection is often ineffective following entry of bacteria into the bloodstream. To date, there is no vaccine available against GAS. In this study, cationic liposomes encapsulating lipopeptide-based vaccine candidates against GAS have been employed for intranasal vaccine delivery. Cationic liposomes were prepared with dimethyldioctadecylammonium bromide (DDAB) using the film hydration method. Female Swiss mice were immunized intranasally with the liposomes. In contrast to unmodified peptides, lipopeptides entrapped by liposomes induced both mucosal and systemic immunity, IgA and IgG (IgG1 and IgG2a) production in mice, respectively. High levels of antibody (IgA and IgG) titres were detected even five months post immunization. Thus, the combination of lipopeptides and liposomes generates a very promising delivery system for intranasal vaccines. STATEMENT OF SIGNIFICANCE: Group A streptococcus, causing rheumatic heart diseases, kills approximately half a million people annually. There is no vaccine available against the infection. Mucosal immunity is vital in ensuring an individual is protected as this gram positive bacteria initially colonizes at the throat. Herein, we demonstrated that lipopeptides entrapped by liposomes induced both mucosal and systemic immunity. High levels of antibody (IgA and IgG) titres were detected even five months post immunization and lead vaccine candidate was able to induce humoral immune responses even after single immunization. Thus, the combination of lipopeptides and liposomes generates a very promising delivery system for intranasal vaccines.

The Role of Size in Development of Mucosal Liposome-Lipopeptide Vaccine Candidates Against Group A Streptococcus.

BACKGROUND: Group A streptococcus (GAS) is an exclusively human pathogenic bacteria. A delay in treatment of GAS infection often lead to severe diseases such as rheumatic heart disease which attributes to hundreds of thousands deaths annually. For the past few decades, the quest for a commercial GAS vaccine has been futile. Currently one of the most investigated strategies to develop vaccine against GAS includes the use of conserved epitopes from major virulent factor of GAS, M-protein. METHODS: In this study, cationic liposomes of various sizes (70 nm to 1000 nm) were prepared with dimethyldioctadecylammonium bromide (DDAB) encapsulating lipopeptide bearing M-protein derived B-cell epitope (J14). RESULTS: Smaller liposomes induced higher antibody titres, though the differences between groups were not statistically significant. CONCLUSION: Nonetheless, all mice which were immunized with liposome-lipopeptide delivery system elicited high levels of systemic (IgG) and mucosal antibodies (IgA), which were discernably higher than those induced with the help of commercial adjuvant (cholera toxin B subunit).

Multilayer engineered nanoliposomes as a novel tool for oral delivery of lipopeptide-based vaccines against group A Streptococcus.

AIM: To develop an oral nanovaccine delivery system for lipopeptide-based vaccine candidate against group A Streptococcus. MATERIALS & METHODS: Lipid-core peptide-1-loaded nanoliposomes were prepared as a template and coated with opposite-charged polyelectrolytes to produce particles with size <200 nm. Efficacy of this oral nanovaccine delivery system was evaluated in mice model. RESULTS: Polymer-coated liposomes produced significantly higher antigen-specific mucosal IgA and systemic IgG titers in comparison to vaccine formulated with a strong mucosal adjuvant upon oral immunization in mice. Moreover, high levels of systemic antibody titers were retained even at day 185 postprimary immunization. CONCLUSION: Efficient oral delivery platform for lipopeptide-based vaccines has been developed.

Flagellin-rPAc vaccine inhibits biofilm formation but not proliferation of S. mutans.

As the main etiologic bacterium of dental caries, Streptococcus mutans (S. mutans) has been considered as the primary object of vaccine research. We previously constructed a recombinant flagellin-rPAc fusion protein (KF-rPAc) that consists of an alanine-rich region to proline-rich region fragment of PAc (rPAc) from S. mutans and flagellin KF from E.coli K12 strain. Intranasal (i.n) immunization of KF-rPAc could induce high level of rPAc-specific antibody responses and offer robust protection against dental caries. In caries development, biofilm formation was considered as the necessary process involved. As PAc possesses other activities besides affecting adherence of S. mutans to salivary glycoproteins, we wondered whether rPAc-specific antibody responses induced by KF-rPAc could inhibit biofilm formation. Hence, in the present study, a simple and convenient in vitro biofilm model of S. mutans was constructed without saliva pre-coated. Both serum and saliva from KF-rPAc immunized rats significantly inhibited biofilm formation. Moreover, with the presence of serum or saliva, the biofilm formation is negatively correlated with the level of rPAc-specific antibody, and positively correlated with caries scores in rat. Moreover, in immunized mice, the level of rPAc-specific antibody also negatively correlated with the biofilm formation. Unlike ampicillin, serum of KF-rPAc immunized mice only inhibited biofilm formation but not proliferation. All together, we discovered that besides the well known blocking adherence of S. mutans to salivary glycoproteins by rPAc-specific antibody, flagellin-rPAc vaccine could also protects tooth from caries by inhibiting biofilm structure formation in between bacteria.

Novel platform technology for modular mucosal vaccine that protects against streptococcus.

The upper respiratory tract (URT) is the major entry site for human pathogens and strategies to activate this network could lead to new vaccines capable of preventing infection with many pathogens. Group A streptococcus (GAS) infections, causing rheumatic fever, rheumatic heart disease, and invasive disease, are responsible for substantial morbidity and mortality. We describe an innovative vaccine strategy to induce mucosal antibodies of significant magnitude against peptide antigens of GAS using a novel biocompatible liposomal platform technology. The approach is to encapsulate free diphtheria toxoid (DT), a standard vaccine antigen, within liposomes as a source of helper T-cell stimulation while lipidated peptide targets for B-cells are separately displayed on the liposome surface. As DT is not physically conjugated to the peptide, it is possible to develop modular epitopic constructs that simultaneously activate IgA-producing B-cells of different and complementary specificity and function that together neutralize distinct virulence factors. An inflammatory cellular immune response is also induced. The immune response provides profound protection against streptococcal infection in the URT. The study describes a new vaccine platform for humoral and cellular immunity applicable to the development of vaccines against multiple mucosal pathogens.

Modular virus-like particles for sublingual vaccination against group A streptococcus.

Infection with Group A streptococcus (GAS)-an oropharyngeal pathogen-leads to mortality and morbidity, primarily among developing countries and indigenous populations in developed countries. The development of safe and affordable GAS vaccines is challenging, due to the presence of various unique GAS serotypes, antigenic variation within the same serotype, and potential auto-immune responses. In the present study, we evaluated the use of a sublingual freeze-dried (FD) formulation based on immunogenic modular virus-like particles (VLPs) carrying the J8 peptide (J8-VLPs) as a potential safe and cost-effective GAS vaccine for inducing protective systemic and mucosal immunity. By using in vivo tracing of the sublingual J8-VLPs, we visualized the draining of J8-VLPs into the submandibular lymph nodes, in parallel with its rapid absorption into the systemic circulation, which support the induction of effective immune responses in both systemic and mucosal compartments. The sublingual administration of J8-VLPs resulted in a high serum IgG antibody level, with a good balance of Th1 and Th2 immune responses. Of note, sublingual vaccination with J8-VLPs elicited high levels of IgA antibody in the saliva. The co-administration of mucosal adjuvant cholera toxin (CT) further enhanced the increase in salivary IgA antibody levels induced by the J8-VLPs formulation. Moreover, the levels of salivary IgA and serum IgG observed following the administration of the CT-adjuvanted FD formulation of J8-VLPs (FD-J8-VLPs) and non-FD formulation of J8-VLPs were comparable. In fact, the saliva isolated from mice immunized with J8-VLPs and FD-J8-VLPs with CT demonstrated opsonizing activity against GAS in vitro. Thus, we observed that the sublingually delivered FD formulation of microbially produced modular VLPs could prevent and control GAS diseases in endemic areas in a cost-effective manner.