Oral vaccination against diphtheria using polyacryl starch microparticles as adjuvant

Oral vaccination offers the advantage of eliciting both a mucosal and a systemic immune response. This study investigated the use of polyacryl starch microparticles as adjuvant for oral vaccination against diphtheria. Diphtheria toxin or cross-reacting material (CRM197) were covalently conjugated to the microparticles and fed to mice by oral gavage. Investigation of formaldehyde treatment as a means of either detoxifying (diphtheria toxin) or stabilising (CRM197) these formulations were also made. We show that all our formulations given orally or parenterally to mice induced a strong systemic immune response. Only formulations given orally induced a mucosal IgA-response. Furthermore, our formulations given parenterally or orally induced a strong diphtheria toxin-neutralising antibody response.     

Mucosal vaccination against diphtheria using starch microparticles as adjuvant for cross-reacting material (CRM197) of diphtheria toxin

Mucosal vaccination has the advantage of eliciting a local mucosal immune response as well as a systemic response. In this investigation, polyacryl starch microparticles were conjugated to diphtheria toxin cross-reacting material (CRM197) as a mucosal adjuvant for oral or intranasal immunisation of mice. Various methods of stabilising CRM197 with formaldehyde were investigated. A good systemic and local mucosal immune response was attained with oral immunisation when CRM197 was treated with a relatively low formaldehyde concentration prior to conjugation to the microparticles. No immune response was seen after intranasal immunisation.     

Lipid microparticles for mucosal immunization against hepatitis B

Parenteral administration of vaccines often does not lead to optimal or long lasting protection against disease causing organisms particularly those that are inhaled, ingested or sexually transmitted. For optimal mucosal protection induction of immune response via mucosal routes is therefore highly desirable. Double emulsion-solvent evaporation (w/o/w) method best suited for water-soluble bioactives was selected for the preparation of hepatitis B surface antigen (HBsAg) loaded lipid microparticles. Intranasal route was considered for mucosal administration and hence to prepare the delivery system biocompatible and least irritable, soyalecithin (phospholipid) was taken instead of polymer because phosphatidylcholine is the major component of endogenous lung surfactant. The studies performed in present work included antigen characterization, development of lipid microparticles, stability studies of the prepared lipid microparticle formulations, percent mucoadhesion, ex vivo cellular uptake studies and in vivo studies. The general order obtained from in vivo studies for mucosal immune response (IgA) followed the sequence: LMST-HBsAg (IN)>LM-HBsAg (IN)>alum-HBsAg (IN)>LMST-HBsAg (IM)>alum-HBsAg (IM)>or=LM-HBsAg (IM)>plain HBsAg (IN)>plain HBsAg (IM).     

Polyacryl starch microparticles as adjuvant in oral immunisation,inducing mucosal and systemic immune responses in mice

A new vaccine adjuvant for oral administration has been examined in mice. Polyacryl starch microparticles (2-3 microm in diameter) are prepared from a water-in oil-emulsion with stabilising hydrocarbon chains. A model antigen, human serum albumin (HSA), which is not binding to the gut epithelium, was covalently coupled to the highly porous starch particles. Upon oral administration, the HSA-microparticles induced a good, diversified immune response without any signs of tolerance development. A strong cellular response can be detected as a delayed-type hypersensitivity reaction. The Th1/Th2 ratio increased with the number of doses and time during the immunisation procedure as indicated by the subclass distribution of the systemic, humoral response. Furthermore, the mucosal response was very strong in the groups that received HSA-microparticles, while the groups receiving soluble HSA did not have any detectable s-IgA in faeces. The specificity was confirmed in an ELISPOT assay. These findings indicate that starch microparticles can be an interesting candidate as an oral vaccine adjuvant. The possible causes of the strong immune responses and the possible role of the dendritic cells in the diversified immune response are discussed.     

Multifunctional liposomes constituting microneedles induced robust systemic and mucosal immunoresponses against the loaded antigens via oral mucosal vaccination

To develop effective, convenient and stable mucosal vaccines, mannose-PEG-cholesterol (MPC)/lipid A-liposomes (MLLs) entrapping model antigen bovine serum albumin (BSA) were prepared by the procedure of emulsification–lyophilization and used to constitute microneedles, forming the proMLL-filled microneedle arrays (proMMAs). The proMMAs were rather stable and hard enough to pierce porcine skin and, upon rehydration, dissolved rapidly recovering the MLLs without size and entrapment change. The proMMAs given to mice via oral mucosal (o.m.) route, rather than routine intradermal administration, elicited robust systemic and mucosal immunoresponses against the loaded antigens as evidenced by high levels of BSA-specific IgG in the sera and IgA in the salivary, intestinal and vaginal secretions of mice. Enhanced levels of IgG2a and IFN-γ in treated mice revealed that proMMAs induced a mixed Th1/Th2 immunoresponse. Moreover, a significant increase in CD8⁺ T cells confirmed that strong cellular immunity had also been established by the immunization of the proMMAs. Thus, the proMMAs can be immunized via o.m. route to set up an effective multiple defense against pathogen invasion and may be an effective vaccine adjuvant-delivery system (VADS) applicable in the controlled temperature chain.     

A dilemma for mucosal vaccination: efficacy versus toxicity using enterotoxin-based adjuvants

In the development of mucosal vaccines, cholera toxin (CT) has been shown to be an effective adjuvant and to induce both mucosal and systemic immune responses via a Th2 cell-dependent pathway. However, a major concern for use of mucosal adjuvants such as CT is that this molecule is not suitable for use in humans because of its innate toxicity. Recent vaccine development efforts have emphasized nasal application of antigen and CT for the induction of mucosal IgA responses. When we examined potential toxicity of CT for the central nervous system (CNS), both CT and CT-B accumulated in the olfactory nerves/epithelium and olfactory bulbs of mice when given by the nasal route. The development of effective mucosal vaccines for the elderly is also an important issue; however, only limited information is available. When mucosal adjuvanticity of CT was evaluated in aged mice, an early immune dysregulation was evident in the mucosal immune system. The present review discusses these potential problems for effective mucosal vaccine development.     

Experiments on a sub-unit vaccine encapsulated in microparticles and its efficacy against Brucella melitensis in mice

The aim of this study was to evaluate the effect of the excipients used to facilitate the encapsulation of high hydrophobic antigenic complex extracted from Brucella ovis (HS) on the physico-chemical properties of the resulting microparticles. Poly(epsilon-caprolactone) (PEC) microparticles containing HS were prepared by the solvent extraction/evaporation method using total recirculation one-machine system (TROMS). Different excipients, beta-cyclodextrin (beta-CD), Pluronic F68, Tween 20 or Tween 80, were used in order to facilitate the encapsulation and conserve the bioactivity of the encapsulated antigenic complex. HS was efficiently loaded in all the different PEC-microparticle formulations, although the combined use of beta-cyclodextrin and Pluronic F68 permitted an increase in the amount of antigenic extract in the core of the resulting microparticles without loss of its antigenic properties. Finally, the protective ability of this F68-CD-MP formulation was evaluated against an experimental challenge with the virulent Brucella melitensis H38 strain in BALB/c mice. This innocuous subcellular vaccine induced a similar protection to that of the live attenuated Rev 1 vaccine; these are promising results that would merit further investigation in target animals.     

Chitosan/alginate microparticles for the oral delivery of fowl typhoid vaccine: Innate and acquired immunity

Oral fowl typhoid (FT) vaccine is necessary for improved flock vaccinations and economic growth. This study was undertaken to evaluate the immune responses of birds given oral fowl typhoid vaccine coated with chitosan/alginate microparticles and comparing it with the conventional subcutaneous route of administration. Preliminary studies were done to evaluate the particle size, encapsulation efficiency and agglutination. Sixty day-old chicks were divided into three groups of twenty birds each. This comprised a negative control group NEG 451 (non-vaccinated and non-challenged used as control for cytokine quantification), SC 634 (live 9R vaccine by the injection route) and OCV 567 (live 9R vaccine coated with chitosan/alginate microparticles). Vaccination was done at 10 weeks and 14 weeks of age followed by challenge at 16 weeks of age. IgG was measured using ELISA. mRNA fold expression of IFN-γ in spleen was calculated using qRT-PCR. Particle sizes ranged between 0.55 µm and 10 µm. Encapsulation efficiency was above 60%. ELISA showed E-values of 0.10 ± 0.14, 0.07 ± 0.01 and 0.02 ± 0.01 for OCV 567, SC 634 and NEG 451 respectively after primary vaccination. Also E-values were 0.25 ± 0.16, 0.19 ± 0.04 and 0.0008 ± 0.005 for SC 634, OCV 567 and NEG451 respectively after boost vaccination. The expression of IFN-$\gamma$in spleen using 2−ΔΔ CT calculation was upregulated with values of 1.97 and 0.75 for OCV 567 and SC 634 respectively. After challenge with the 85-kb virulence plasmid SG9, there was 100% protection of the birds in both OCV 567 and SC 634 groups with no mortality. In conclusion, there was no significant difference at p < 0.05 of the means ± SD in immune responses between the oral fowl typhoid vaccine coated with chitosan/alginate microparticles and the subcutaneous route of administration. However, it is noteworthy to mention that the protective efficacy of the oral route is due to the chitosan/alginate biopolymers which coated the vaccine preventing destruction in the gastrointestinal tract.     

Chitosan nanoparticles containing plasmid DNA encoding house dust mite allergen,Der p 1 for oral vaccination in mice

Our previous studies indicated that intramuscular (i.m.) immunisation with full length Der p 1 cDNA induced significant humoral response to the left domain (approximately corresponding to amino acids 1-116) but not to the right domain (approximately corresponding to amino acids 117-222) of Der p 1 allergen. This study explored the use of chitosan-DNA nanoparticles for oral immunisation to induce immune responses specific to both the left and right domains of Der p 1. DNA constructs pDer p 1 (1-222) and pDer p 1 (114-222) were complexed with chitosan and delivered orally followed by an i.m. injection of pDer p 1 (1-222) 13 weeks later. Such approach has successfully primed Th1-skewed immune responses against both domains of Der p 1. This strategy could be further optimised for more efficacious gene vaccination for full length Der p 1.     

Type I IFN is a powerful mucosal adjuvant for a selective intranasal vaccination against influenza virus in mice and affects antigen capture at mucosal level

In view of the increasing interest in mucosal vaccination, we investigated whether type I IFN could act as adjuvant of an intranasally administered influenza vaccine. A single intranasal administration of IFNalphabeta-adjuvanted vaccine in anesthetized C3H/HeN mice was capable of protecting the totality of animals against virus challenge, while vaccine alone was only partially effective. To mimic intranasal vaccine administration in man and to limit vaccine delivery strictly to nasal mucosa, we used a second method of vaccination based on vaccine fractionation in six doses and intranasal instillation in non-anesthetized mice. By using this vaccination schedule, IFNalphabeta-adjuvanted vaccine also prevented mice from disease development and induced an efficient long lasting immune response. Further experiments showed that IFNalphabeta increased the percentage of antigen-associated phagocytes in the nasal mucus layer, thus suggesting a new possible mechanism of action for type I IFN as an adjuvant.     

Compound 48/80 acts as a potent mucosal adjuvant for vaccination against Streptococcus pneumoniae infection in young mice

Streptococcus pneumoniae, a major respiratory pathogen, is a leading cause of death among children worldwide. Mucosal vaccination is a recommended method to prevent respiratory infection. However, development of mucosal vaccination is usually hindered due to the lack of safe and effective mucosal adjuvants. Mast cell activator compound 48/80 (C48/80) has been used as a mucosal adjuvant in immunization of adult mice, but its adjuvanticity is not clear in the immunization of young mice. In this study, the adjuvanticity of C48/80 was evaluated when intranasally co-administrated with a pneumococcal vaccine candidate strain SPY1 in a young mice model in comparison with a classical mucosal adjuvant cholera toxin (CT) and a relatively safe mucosal adjuvant Pam2CSK4. All three adjuvants enhanced antibody responses, whereas serum IgG titers were maintained at a stable level during the 3 months after the last immunization only in the SPY1 + C48/80 and SPY1 + CT groups. Furthermore, both the SPY1 + CT group and the SPY1 + C48/80 group induced strong Th17 immune response. Notably, C48/80 showed the exceptional ability to promote the clearance of nasal pneumococcal colonization which CT and Pam2CSK4 did not show. We found that C48/80's ability to induce protection against nasal pneumococcal colonization depended on B cells and IL-17A. Additionally, C48/80, as a mucosal adjuvant, showed a greater ability to protect young mice against lethal pneumococcal infection than CT. In comparison with CT, C48/80 also showed a favorable safety. These results reveal a promising perspective for using C48/80 as a mucosal adjuvant to improve protection against pneumococcal diseases early in life.     

Mouse models for the study of mucosal vaccination against otitis media

Otitis media (OM) is one of the most common infectious diseases in humans. The pathogenesis of OM involves nasopharyngeal (NP) colonization and retrograde ascension of the pathogen up the Eustachian tube into the middle ear (ME). Due to increasing rates of antibiotic resistance, there is an urgent need for vaccines to prevent infections caused by the most common causes of bacterial OM, including nontypeable Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis. Current vaccine strategies aim to diminish bacterial NP carriage, thereby reducing the likelihood of developing acute OM. To be effective, vaccination should induce local mucosal immunity both in the ME and in the NP. Studies in animal models have demonstrated that the intranasal route of vaccination is particularly effective at inducing immune responses in the nasal passage and ME for protection against OM. The mouse is increasingly used in these models, because of the availability of murine reagents and the existence of technology to manipulate murine models of disease immunologically and genetically. Previous studies confirmed the suitability of the mouse as a model for inflammatory processes in acute OM. Here, we discuss various murine models of OM and review the applicability of these models to assess the efficacy of mucosal vaccination and the mechanisms responsible for protection. In addition, we discuss various mucosal vaccine antigens, mucosal adjuvants and mucosal delivery systems.     

A mucosal vaccine against diphtheria: formulation of cross reacting material (CRM(197)) of diphtheria toxin with chitosan enhances local and systemic antibody and Th2 responses following nasal delivery

The development of new generation vaccines against diphtheria is dependent on the identification of antigens and routes of immunization that are capable of stimulating immune responses similar to, or greater than, those obtained with the parenterally-delivered toxoid vaccine, while reducing the adverse effects that have been associated with the traditional vaccine. In this study, we examined the cellular and humoral immune responses in mice generated after both parenteral and mucosal immunizations with cross-reacting material (CRM(197)) of diphtheria toxin. We found that both native and mildly formaldehyde-treated CRM(197) and conventional diphtheria toxoid (DT) induced mixed Th1/Th2 responses and similar levels of anti-DT serum IgG following parenteral immunization. In contrast, CRM(197) preparations were poorly immunogenic when administered intranasally in solution. However, formulation of the antigens with chitosan significantly enhanced their immunogenicity, inducing high levels of antigen-specific IgG, secretory IgA, toxin-neutralizing antibodies and T cell responses, predominately of Th2 subtype. Furthermore, intranasal immunization with CRM(197) and chitosan induced protective antibodies against the toxin in a guinea pig passive challenge model. We also found that priming parenterally with DT in alum and boosting intranasally with CRM(197) was a very effective method of immunization in mice, capable of inducing high levels of anti-DT IgG and neutralizing antibodies in the serum and secretory IgA in the respiratory tract. Our findings suggest that boosting intranasally with CRM(197) antigen may be very effective in adolescents or adults who have previously been parenterally immunized with a conventional diphtheria toxoid vaccine.     

Evaluation of novel aerosol formulations designed for mucosal vaccination against influenza virus

Influenza viruses are among the most significant human pathogens, responsible for increased seasonal morbidity and mortality particularly in immunodepressed and chronically ill. Conventional vaccination with non-replicative vaccine is currently performed by injection. In the present study, we explore simple spray-dried lipid formulations containing whole inactivated virus or split-subunit vaccine that allow aerosolization and thus, mucosal vaccination of the pulmonary tract. We show that by using biocompatible excipients already approved for human use, one could engineer microparticles that induce substantial local and systemic immunity subsequent to pulmonary administration. Exposure of the bronchial-associated lymphoid tissue (BALT) to vaccine was more effective than parenteral or nasal administration in triggering specific immunity. Co-formulation of a biocompatible surfactant detergent greatly ameliorated the immune profile of microparticles containing a whole inactivated virus vaccine. In addition, mere formulation of a licensed split-subunit vaccine significantly enhanced its immunogenicity. Together, our data underline a simple strategy to convert conventional parenteral vaccination of currently available non-replicative vaccines against influenza virus, into one that is more effective and practical upon respiratory administration.     

The efficacy of oral vaccination of mice with alginate encapsulated outer membrane proteins of Pasteurella haemolytica and One-Shot

The goal of this study was to examine the efficacy of oral delivery of alginate encapsulated outer membrane proteins (OMP) of Pasteurella haemolytica and a commercial One-Shot vaccine in inducing protection in mice against lethal challenge with virulent P. haemolytica. We examined two alginate microsphere formulations and compared them with oral unencapsulated and subcutaneously administered vaccines. Alginate microspheres were made by the emulsion-cross-linking technique. They were examined for size, hydrophobicity, and antigen loading efficiency before they were used in the study. Mice were vaccinated by administering 200 microg of antigens in 200 microl of microspheres suspension orally or subcutaneously. One group of mice received blank microspheres and a second group was given unencapsulated antigen orally. A third and a fourth group received different formulations of alginate encapsulated antigens by oral administration. Three groups received subcutaneous inoculations (alginate encapsulated, non-adjuvanted and unencapsulated antigens, and adjuvanted One-Shot), and one group received water (na?ve group). Mice were vaccinated orally for four consecutive days and challenged with P. haemolytica 5 weeks after the first vaccination. Weekly serum and feces samples were assayed for antigen specific antibodies. The number of dead mice in each group 4 days post challenge was used to compare the efficacy of the various vaccination groups. The mean volume sizes of blank alginate microsphere formulations A, and AA were 15.9, 16 and 9.2 microm, respectively. Hydrophobicity of the microspheres was evaluated by measuring contact angle on a glass slide coated with the microspheres. The contact angles on A and AA were 37.8 and 74.3 degrees, respectively. Antigen concentration in a 1:1 w/w suspension of microspheres in water was 0.9 mg/ml. Rate of death for the blank group was 42.8% whereas for groups vaccinated with antigens encapsulated in A and AA the death rates were 40 and 33.33%, respectively. The death rate in mice vaccinated with unencapsulated antigens was 55.6%. Groups vaccinated by subcutaneous inoculation showed the lowest death rate. These results show that encapsulating OMP and One-Shot in alginate microspheres improves their performance as an oral vaccine.     

Nanoparticle-based vaccine for mucosal protection against Shigella flexneri in mice

Shigellosis is one of the leading causes of diarrhea worldwide with more than 130 million cases annually. Hence, the research of an effective vaccine is still a priority. Unfortunately, a safe and efficacious vaccine is not available yet. We have previously demonstrated the capacity of outer membrane vesicles (OMVs) to protect mice against an experimental infection with Shigella flexneri. Now, we present results on the capacity of this antigenic complex to confer a longer-term protection by oral or nasal routes when encapsulated into nanoparticles. OMVs were encapsulated in poly(anhydride) nanoparticles (NP) prepared by a solvent displacement method with the copolymer poly methyl vinyl ether/maleic anhydride. OMVs loaded into nanoparticles (NP-OMVs) were homogeneous and spherical in shape, with a size of 148 nm (PdI = 0.2). BALB/c mice were immunized with OMVs either free or encapsulated in nanoparticles by nasal (20 μg or 10 μg of OMVs) or oral route (100 μg or 50 μg of OMVs). All immunized animals remained in good health after administration. Challenge infection was performed intranasally on week 8th with a lethal dose of 5 × 10⁷ CFU/mouse of S. flexneri 2a. The number of dead mice after challenge was recorded daily. Results confirmed the value of OMVs as a vaccine. By oral route, the OMV-vaccine was able to protect independently either the dose or the formulation. When vaccine was delivered by nasal route, encapsulation into NPs resulted beneficial in increasing protection from 40% up to 100% when low dose was administered. These results are extraordinary promising and put in relevance the positive effect of nanoencapsulation of the OMV subcellular vaccine.     

Self-adjuvanting modular virus-like particles for mucosal vaccination against group A streptococcus (GAS)

Group A streptococcus (GAS) causes a wide range of diseases, some of them related to autoimmune diseases triggered by repeated GAS infections. Despite the fact that GAS primarily colonizes the mucosal epithelium of the pharynx, the main mechanism of action of most vaccine candidates is based on development of systemic antibodies that do not cross-react with host tissues, neglecting the induction of mucosal immunity that could potentially block disease transmission. Peptide antigens from GAS M-surface protein can confer protection against infection; however, translation of such peptides into immunogenic mucosal vaccines that can be easily manufactured remains a challenge. In this work, a modular murine polyomavirus (MuPyV) virus-like particle (VLP) was engineered to display a GAS antigenic peptide, J8i. Heterologous modules containing one or two J8i antigen elements were integrated with the MuPyV VLP, and produced using microbial protein expression, standard purification techniques and in vitro VLP assembly. Both modular VLPs, when delivered intranasally to outbred mice without adjuvant, induced significant titers of J8i-specific IgG and IgA antibodies, indicating significant systemic and mucosal responses, respectively. GAS colonization in the throats of mice challenged intranasally was reduced in these immunized mice, and protection against lethal challenge was observed. This study shows that modular MuPyV VLPs prepared using microbial synthesis have potential to facilitate cost-effective vaccine delivery to remote communities through the use of mucosal immunization.     

Potential pathogenicity for rodents of vaccines intended for oral vaccination against rabies: a comparison.

Different oral vaccines intended to control fox rabies were administered to 271 wild rodents. Vaccines were administered orally or by the mucosal route to four different European species belonging to the genera Apodemus, Arvicola, Clethrionomys and Microtus. These rodents are likely to consume baits and to have contact with the vaccine. Two genetically engineered vaccines were tested: SAG1 (an avirulent mutant of the rabies virus) and V-RG (vaccinia recombinant virus expressing the rabies glycoprotein gene). Both were found to be completely innocuous when administered orally or by the mucosal route. The residual pathogenicity of conventional modified live vaccines derived from the SAD strain was confirmed.