Monophosphoryl lipid A-adjuvanted nucleoprotein-neuraminidase nanoparticles improve immune protection against divergent influenza viruses.

Universal influenza vaccines are urgently needed to prevent recurrent influenza epidemics and inevitable pandemics. We generated double-layered protein nanoparticles incorporating two conserved influenza antigens-nucleoprotein and neuraminidase-through a two-step desolvation-crosslinking method. These protein nanoparticles displayed immunostimulatory properties to antigen-presenting cells by promoting inflammatory cytokine (IL-6 and TNF-α) secretion from JAWS II dendric cells. The nanoparticle immunization induced significant antigen-specific humoral and cellular responses, including antigen-binding and neutralizing antibodies, antibody- and cytokine (IFN-γ and IL-4)-secreting cells, and NP147-155 tetramer-specific cytotoxic T lymphocyte (CTL) responses. Co-administration of monophosphoryl lipid A (MPLA, a toll-like receptor 4 agonist) with the protein nanoparticles further improved immune responses and conferred heterologous and heterosubtypic influenza protection. The MPLA-adjuvanted nanoparticles reduced lung inflammation post-infection. The results demonstrated that the combination of MPLA and conserved protein nanoparticles could be developed into an improved universal influenza vaccine strategy.

Tumor-Infiltrating Lymphocytes (TILs) as a Biomarker of Abscopal Effect of Cryoablation in Breast Cancer: A Pilot Study.

BACKGROUND: Morphological evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer is gaining momentum as an immunological biomarker. This experiment evaluates the role of TILs in distant tumors as a measure of abscopal effect from cryoablation of breast cancer. METHODS: BALB/c mice underwent bilateral orthotopic transplant with 4T1-12B (triple-negative) cells. At 2 weeks, left tumors were treated by either resection (standard of care group) or cryoablation (intervention group) followed by resection of the distant right tumors 1 week posttreatment. TIL scores were calculated from hematoxylin and eosin-stained sections and phenotyped for cytotoxic T-lymphocyte (CTL) markers by immunofluorescence. Primarily resected tumors served as baseline (Tbaseline), whereas resected distant right-sided served as the readout for abscopal effect (AbsRes or AbsCryo). Mice were monitored for tumor recurrence and metastasis. RESULTS: The AbsCryo had a significant mean (SD) increase in stromal (2.8 [1.1]%; p = 0.015) and invasive margin TILs (50 [12]%; p = 0.02) compared with TBaseline (1.0 [0]% and 31 [4.9]%, respectively). CTL phenotyping revealed a significant increase in mean (SD) CD8+ T cells (15.7 [12.1]; p = 0.02) and granzyme B (4.8 [3.6]; p = 0.048) for the AbsCryo compared with TBaseline (5.2 [4.7] and 2.4 [0.9], respectively). Posttreatment, the cryoablation group had no recurrence or metastasis, whereas the resected group showed local recurrence and lung metastasis in 40% of the mice. Postprocedure increase in TIL score of distant tumors was associated with decrease in tumor relapse (p = 0.02). CONCLUSIONS: Cryoablation induced a robust tumor-specific TIL response compared with resection, suggesting an abscopal effect leading to the prevention of cancer recurrence and metastasis.

Role of Sporopollenin Shell Interfacial Properties in Protein Adsorption.

Sporopollenin shells isolated from natural pollen grains have received attention in translational and applied research in diverse fields of drug delivery, vaccine delivery, and wastewater remediation. However, little is known about the sporopollenin shell's potential as an adsorbent. Herein, we have isolated sporopollenin shells from four structurally diverse pollen species, black walnut, marsh elder, mugwort, and silver birch, to study protein adsorption onto sporopollenin shells. We investigated three major interfacial properties, surface area, surface functional groups, and surface charge, to elucidate the mechanism of protein adsorption onto sporopollenin shells. We showed that sporopollenin shells have a moderate specific surface area (<12 m2/g). Phosphoric acid and potassium hydroxide treatments that were used to isolate sporopollenin shells from natural pollen grains also result in the functionalization of sporopollenin shell surfaces with ionizable groups of carboxylic acid and carboxylate salt. As a result, sporopollenin shells exhibit a negative ζ potential in the range of -75 to -82 mV at pH 10 when dispersed in water. The ζ potentials of sporopollenin shells remain negative in the pH range of 2.5-11, with the absolute value of ζ potential showing a decrease with the decrease in pH. The negative surface charge promotes the adsorption of protein onto the sporopollenin shell via electrostatic interaction. Despite having a moderate surface area, sporopollenin shells adsorb a significant amount of lysozyme (145-340 µg lysozyme per mg of sporopollenin shells). Lysozyme adsorption onto sporopollenin shells alters the surface, and the surface charge becomes positive at acidic pH. Overall, this study demonstrates the potential of sporopollenin shells to adsorb proteins, highlights the critical role of sporopollenin shell's interfacial properties in protein adsorption, and identifies the mechanism of protein adsorption on sporopollenin shells.

Microneedle-Mediated Allergen-Specific Immunotherapy for the Treatment of Airway Allergy in Mice.

Subcutaneous allergen-specific immunotherapy (SCIT) qualifies as a promising approach for the permanent cure of IgE-mediated airway allergies, which can often manifest into allergic rhinitis and other allergic respiratory diseases. SCIT entails repeated administration of a high allergen dose into the subcutaneous (sc) region using a hypodermic needle for many (3-5) years, which is inconvenient and painful and reduces patient compliance. To overcome these limitations, we hypothesized that microneedles (MNs), which are minimally invasive and painless, could provide a novel approach for allergen desensitization by depositing the allergen into the superficial layers of the skin. To test this hypothesis, we compared MNs and SCIT for allergen desensitization in a mouse model of ovalbumin (Ova)-induced airway allergy. Mice were first made allergic to Ova and then treated with MNs coated with Ova (with or without CpG as an adjuvant) or via SCIT-Ova + alum (subcutaneous Ova + alum injections) for comparison. Treatment with coated MNs significantly induced Ova-specific serum IgG antibodies in a manner comparable to SCIT-Ova + alum-treated group. To test the efficacy against allergen challenge, treated mice were challenged with Ova via the nasal route. Coated MNs with Ova and CpG (MN-Ova + CpG) considerably suppressed the airway inflammation in allergic mice, evidenced by downregulation of proinflammatory cytokines (IL-5 and IL-13), upregulation of anti-inflammatory cytokine IL-10, and activation of Ova-specific immune response in bronchoalveolar (BAL) fluid. The therapeutic capacity of MN-based allergy treatment was further validated by the reduction in eosinophil and mast cell infiltration in the lung tissues of mice treated with MN-Ova + CpG, and low deposition of mucus inside their lung bronchioles. Overall, coated MNs ameliorated the symptoms of airway allergy in mice similar to SCIT and could provide a novel means of painless allergen-specific immunotherapy.

Microneedle Coating Methods: A Review with a Perspective.

A coated microneedle array comprises sharp micrometer-sized needle shafts attached to a base substrate and coated with a drug on their surfaces. Coated microneedles are under investigation for drug delivery into the skin and other tissues, and a broad assortment of active materials, including small molecules, peptides, proteins, deoxyribonucleic acids, and viruses, have been coated onto microneedles. To coat the microneedles, different methods have been developed. Some coating methods achieve selective coating of just the microneedle shafts, whereas other methods coat not only microneedle shafts but also the array base substrate. Selective coating of just the microneedle shafts is more desirable since it provides control over drug dosage, prevents drug waste, and offers high delivery efficiency. Different excipients are added to the coating liquid to modulate its viscosity and surface tension in order to achieve uniform coatings on microneedles. Coated microneedles have been used in a broad range of biomedical applications. To highlight these different applications, a table summarizing the different active materials and the amounts coated on microneedles is provided. We also discuss factors that should be considered when deciding suitability of coated microneedles for new-drug delivery applications. In recent years, many coated microneedles have been investigated in human clinical trials, and there is now a strong effort to bring the first coated microneedle-based product to market.

Microneedles Coated with Tramadol Exhibit Antinociceptive Effect in a Rat Model of Temporomandibular Hypernociception.

Coated microneedles have emerged as a promising drug delivery system for inflammatory pain treatment. We have previously shown that tramadol injection into the rat temporomandibular joint (TMJ) induces an antinociceptive and anti-inflammatory effect. In this study, microneedles coated with tramadol were investigated as a platform to treat TMJ pain. Male Wistar rats were administered tramadol using an intra-TMJ injection or with microneedles coated with tramadol, followed by 1.5% formalin nociceptive challenge administered 15 minutes later. The nociceptive behavior of rats was evaluated, and their periarticular tissues were removed after euthanasia for analysis. The duration of antinociceptive effect was determined by performing the formalin challenge at different time points extending up to 6 days post tramadol administration. Microneedles coated with tramadol produced an antinociceptive effect similar to injection of tramadol into the rat TMJ. Surprisingly, tramadol delivery using coated microneedles produced a more durable antinociceptive effect lasting as much as 2 days post tramadol delivery as compared with an antinociceptive effect lasting under 2 hours from intra-TMJ injection of tramadol. The proinflammatory cytokines tumor necrosis factor-α and interleukin-1ß (IL-1ß) were found to be reduced, whereas the anti-inflammatory cytokine IL-10 was found to be elevated in tramadol-treated groups. In conclusion, microneedles coated with tramadol can offer a therapeutic option for pain control of inflammatory disorders in the TMJ.

Assessment of Th1/Th2 Bias of STING Agonists Coated on Microneedles for Possible Use in Skin Allergen Immunotherapy.

Microneedle-based skin allergen-specific immunotherapy (AIT) can benefit from adjuvants that can stimulate a stronger Th1 response against the allergen. We evaluated two stimulator of interferon genes (STING) agonists, namely, cyclic diguanylate monophosphate (c-di-GMP) and cyclic diadenylate monophosphate (c-di-AMP), as skin adjuvants using coated microneedles (MNs). For comparison, the approved subcutaneous (SC) hypodermic injection containing alum was used. Ovalbumin (Ova) was used as a model allergen. Ova-specific IgG2a antibody in serum, which is a surrogate marker for Th1 type immune response was significantly higher when STING agonists were used with coated MNs as compared to SC injection of Ova+alum in mice. In contrast, IgG1 antibody, a surrogate marker for Th2 type immune response, was at comparable levels in the MN and SC groups. Restimulation of splenocytes with Ova produced higher levels of Th1 cytokines (IFN-γ and IL-2) in the STING agonists MN groups as compared to the SC group. In conclusion, delivery of STING agonists into the skin using coated MNs activated the Th1 pathway better than SC- and MN-based delivery of alum. Thus, STING agonists could fulfill the role of adjuvants for skin AIT and even for infectious disease vaccines, where stimulation of the Th1 pathway is of interest.

Generation of Broad Protection against Influenza with Di-Tyrosine-Cross-Linked M2e Nanoclusters.

Tyrosine cross-linking has recently been used to produce nanoclusters (NCs) from peptides to enhance their immunogenicity. In this study, NCs were generated using the ectodomain of the ion channel Matrix 2 (M2e) protein, a conserved influenza surface antigen. The NCs were administered via intranasal (IN) or intramuscular (IM) routes in a mouse model in a prime-boost regimen in the presence of the adjuvant CpG. After boost, a significant increase in anti-M2e IgG and its subtypes was observed in the serum and lungs of mice vaccinated through the IM and IN routes; however, significant enhancement in anti-M2e IgA in lungs was observed only in the IN group. Analysis of cytokine concentrations in stimulated splenocyte cultures indicated a Th1/Th17-biased response. Mice were challenged with a lethal dose of A/California/07/2009 (H1N1pdm), A/Puerto Rico/08/1934 (H1N1), or A/Hong Kong/08/1968 (H3N2) strains. Mice that received M2e NCs + CpG were significantly protected against these strains and showed decreased lung viral titers compared with the naive mice and M2e NC-alone groups. The IN-vaccinated group showed superior protection against the H3N2 strain as compared to the IM group. This research extends our earlier efforts involving the tyrosine-based cross-linking method and highlights the potential of this technology in enhancing the immunogenicity of short peptide immunogens.

Development of a mini pig model of peanut allergy.

Introduction: The prevalence of peanut allergies is increasing, emphasizing the need for an animal model to enhance our understanding of peanut allergy pathogenesis and to advance diagnostic tools and therapeutic interventions. While mice are frequently used as model organisms, their allergic responses do not fully mirror those observed in humans, warranting the exploration of a higher animal model. The porcine gastrointestinal system closely resembles that of humans, and exhibits allergy symptoms akin to human responses, making pigs a promising model for peanut allergy research. Methods: In this study we compared two allergen sensitization protocols involving either topical allergen application after repeated tape stripping (TS) or intraperitoneal (IP) injections to induce peanut-specific allergy and anaphylaxis reactions in mini pigs. Mini pigs sensitized with a combination of peanut protein extract (PE) and cholera toxin (CT) through either the IP or the TS route. Results: Sensitized pigs via both methods developed systemic PE-specific IgG and IgE responses. Following peanut challenge via the IP route, both TS- and IP-sensitized pigs displayed allergy symptoms, including lethargy, skin rashes, vomiting, and a drop in body temperature. However, respiratory distress was observed exclusively in pigs sensitized through the TS route and not in those sensitized through the IP route. However, it is noteworthy that both groups of sensitized pigs maintained peanut hypersensitivity for up to two months post-sensitization, albeit with a reduction in the severity of allergy symptoms. Importantly, both groups exhibited sustained levels of PE-specific IgG, IgE, and elevated concentrations of mast cell protease in their blood following the IP challenges. Discussion: Overall, this study reports TS and IP as two different modes of sensitization leading to onset of peanut specific allergic reactions in mini pigs, but only the TS-sensitization led to systemic anaphylaxis (simultaneous presence of symptoms: breathing difficulty, intense skin rash, and impaired mobility). A distinctive feature of these sensitization protocols is the 100% success rate (N = 4 pigs per group) in sensitizing the subjects.

Influenza immune imprinting synergizes PEI-HA/CpG nanoparticle vaccine protection against heterosubtypic infection in mice.

The first influenza virus infection (imprinting) can lead to long-term immune memory and influence subsequent vaccinations and infections. Previously, we reported a polyethyleneimine (PEI)-Aichi hemagglutinin (HA)/CpG (PHC) nanoparticle with cross-protective potential against homologous and heterologous influenza strains. Here we studied how influenza immune imprinting influences the antibody responses to the PHC vaccination and the protection against heterosubtypic virus challenges. We found that pre-existing virus immunity can maintain or synergize the vaccine-induced antibody titers, depending on the imprinting virus HA phylogenetic group. The HA group 1 virus (PR8, H1N1)-imprinted mice displayed comparable antigen-specific antibody responses to those without imprinting post-PHC vaccination. In contrast, the group 2 virus (Phi, H3N2)-imprinted mice showed significantly more robust and balanced antibodies post-vaccination, conferring complete protection against body weight loss and lung inflammation upon heterosubtypic reassortant A/Shanghai/2/2013 (rSH, H7N9) virus challenge. Our findings suggest that influenza imprinting from the same HA phylogenetic group can synergize subsequent vaccination, conferring heterosubtypic protection.

Microprecision delivery of oligonucleotides in a 3D tissue model and its characterization using optical imaging.

Despite significant potential of oligonucleotides (ONs) for therapeutic and diagnostic applications, rapid and widespread intracellular delivery of ONs in cells situated in tissues such as skin, head and neck cavity, and eye has not been achieved. This study was aimed at evaluating the synergistic combination of microneedle (MN) arrays and biochemical approaches for localized intratissue delivery of oligonucleotides in living cells in 3D tissue models. This synergistic combination was based on the ability of MNs to precisely deliver ONs into tissues to achieve widespread distribution, and the ability of biochemical agents (streptolysin O (SLO) and cholesterol conjugation to ONs) to enhance intracellular ON delivery. The results of this study demonstrate that ON probes were uniformly coated on microneedle arrays and were efficiently released from the microneedle surface upon insertion in tissue phantoms. Co-insertion of microneedles coated with ONs and SLO into 3D tissue models resulted in delivery of ONs into both the cytoplasm and nucleus of cells. Within a short incubation time (35 min), ONs were observed both laterally and along the depth of a 3D tissue up to a distance of 500 µm from the microneedle insertion point. Similar widespread intratissue distribution of ONs was achieved upon delivery of ON-cholesterol conjugates. Uniformity of ON delivery in tissues improved with longer incubation times (24 h) postinsertion. Using cholesterol-conjugated ONs, delivery of ON probes was limited to the cytoplasm of cells within a tissue. Finally, delivery of cholesterol-conjugated anti-GFP ON resulted in reduction of GFP expression in HeLa cells. In summary, the results of this study provide a novel approach for efficient intracellular delivery of ONs in tissues.

Delivery of gold nanoparticle-conjugated M2e influenza vaccine in mice using coated microneedles.

As a prospective influenza vaccination platform, a microneedle patch offers advantages such as self-administration and reduction of needle-phobia-associated vaccination avoidance. In an effort to design a broadly protective influenza vaccine we have previously developed a vaccine formulation containing the highly conserved ectodomain sequence of the M2 influenza protein (M2e) attached to the surface of gold nanoparticles (AuNPs) with CpG as a soluble adjuvant (AuNP-M2e + sCpG). Our previous studies have used the intranasal route for vaccination and demonstrated broad protection from this vaccine. Here we asked the question whether the same formulation can be effective when administered to mice using microneedles. We demonstrate that the microneedles can be coated with AuNP-M2e + sCpG formulation, and the AuNPs from the coating can be readily resuspended without aggregation. The AuNPs were delivered with high efficiency into murine skin, and the AuNPs cleared the skin within 12 h of microneedle treatment. After vaccination, strong M2e-specific humoral and cellular responses were stimulated, and the vaccinated mice were 100% protected following a lethal challenge with influenza A/PR/8/34 (H1N1).

Drug-Coated Floss to Treat Gum Diseases: In Vitro and In Vivo Characterization.

Treatment of gum disease often requires antibiotic treatment. In this study, our objective was to advance the practicality of drug-coated floss as an intra gum pocket drug delivery system. The initial design of this delivery system has been previously reported by us. Here, we advance the concept further through in vitro and in vivo evaluation. A floss piece was dip coated in the middle section with model molecules leaving free ends for holding. Porcine gum tissues were used ex vivo and in vivo to evaluate the coated floss, including effect of coating thickness on delivery efficiency, ability to deliver more than one type of molecule (one hydrophilic and one hydrophobic), mechanical properties using a scratch test, and finally retention of delivered material in vivo in the porcine model. After reaching a certain coating thickness, the delivery efficiency of the coated floss decreased, indicating the presence of an optimal coating thickness. Hydrophobic and hydrophilic molecules were successfully coated and delivered with high efficiency into gum pockets. The scratch test indicated that the coatings were resilient. Lastly, the in vivo analysis showed that the drug coating was delivered into the porcine gum pocket with about 65% efficiency, and the coatings could maintain extended residency within the gum pocket despite the native adverse environment of the oral cavity. Overall, this data shows that drug-coated floss can act as a drug delivery vehicle and has potential to provide a minimally invasive and practical method for the delivery of drugs into the gum pockets.

Peptide Cross-Linking Using Tyrosine Residues Facilitated by an Exogenous Nickel-Histidine Complex: A Facile Approach for Enhancing Vaccine-Specific Immunogenicity.

An improved method for the generation of peptide vaccines using di-tyrosine cross-linking is described. The conserved ion channel peptide, M2e, of influenza A virus was modified with the addition of small tyrosine-rich regions (GYGY-) at both the N- and C-termini and extensively cross-linked via tyrosine-tyrosine linkages to form peptide nanoclusters. The cross-linking was catalyzed using exogenous nickel(II) ions complexed to an exogenous glycine-glycine-histidine peptide in the presence of an oxidizer. Mice that were intranasally or intramuscularly immunized with the M2e-vaccine nanoclusters induced comparable levels of M2e-specific serum antibodies. Vaccination via the intranasal or intramuscular route protected mice from subsequent lethal challenge with an influenza A virus. In comparison to our previous approach, where a histidine-rich tag was added into the peptide structure, the use of exogenous histidine reduced irrelevant off-target immune response. Additionally, the purity of the resulting nanoclusters is an attractive feature, making this approach appealing for vaccine development.

Adjuvant Effects of a New Saponin Analog VSA-1 on Enhancing Homologous and Heterosubtypic Protection by Influenza Virus Vaccination.

Adjuvants can increase the magnitude and durability of the immune response generated by the vaccine antigen. Aluminum salts (Alum) remain the main adjuvant licensed for human use. A few new adjuvants have been licensed for use in human vaccines since the 1990s. QS-21, a mixture of saponin compounds, was included in the AS01-adjuvanted Shingrix vaccine. Here, we investigated the adjuvant effects of VSA-1, a newly developed semisynthetic analog of QS-21, on promoting protection in mice after vaccination with the inactivated split virus vaccine. The adjuvant effects of VSA-1 on improving vaccine efficacy after prime immunization were evident as shown by significantly higher levels of hemagglutination-inhibiting antibody titers and enhanced homologous protection compared to those by QS-21 and Alum adjuvants. The adjuvant effects of VSA-1 on enhancing heterosubtypic protection after two doses of adjuvanted vaccination were comparable to those of QS-21. T cell immunity played an important role in conferring cross-protection by VSA-1-adjuvanted vaccination. Overall, the findings in this study suggest that VSA-1 exhibits desirable adjuvant properties and a unique pattern of innate and adaptive immune responses, contributing to improved homologous and heterosubtypic protection by inactivated split influenza vaccination in mice.

Targeted allergen-specific immunotherapy within the skin improves allergen delivery to induce desensitization to peanut.

Aim: Epicutaneous immunotherapy (EPIT) with peanut has been demonstrated to be safe but efficacy may be limited by allergen uptake through the skin barrier. To enhance allergen uptake into the skin, the authors used peanut-coated microneedles and compared them with EPIT in a peanut allergy mouse model. Methods: Sensitized mice were treated with peanut-coated microneedles or peanut-EPIT and then challenged with peanut to determine protection. Results: Treatment with peanut-coated microneedles was safe and showed enhanced desensitization to peanut compared with peanut-EPIT administered via a similar schedule. Protection was associated with reduced Th2 immune responses and mast cell accumulation in the intestine. Conclusion: Peanut-coated microneedles have the potential to present a safe method of improving allergen delivery for cutaneous immunotherapy.

Epicutaneous immunotherapy (EPIT) with peanut has been demonstrated to be safe but efficacy has been varied. The tight barrier provided by the skin may limit the amount of allergen taken up through the skin and thus reduce efficacy. The authors evaluated a microneedle-based approach to improve the amount of allergen deposited into the skin to improve efficacy. Mice were made allergic to peanut and then treated with peanut-coated microneedles or peanut-EPIT. Mice were challenged with peanut to determine suppression of allergic reactivity. In mice, treatment with peanut-coated microneedles was safe and enhanced desensitization to peanut compared with peanut-EPIT administered via a similar schedule. Peanut-coated microneedles may present a novel method of improving allergen immunotherapy delivered through the skin.

Coated floss for drug delivery into the gum pocket.

The purpose of this study was to develop a drug-coated floss to allow delivery of therapeutics into diseased gum pocket. Periodontal (gum) disease affects nearly 45% of adults over 30 years of age. Bacterial persistence makes treatment challenging. Drug-coated floss is expected to provide a self-administrable and targeted method for drug delivery into the diseased gum pockets. We investigated various types of floss and sutures as potential candidates to coat drug. An un-waxed nylon braided floss was selected and dip-coated with model hydrophilic and hydrophobic drugs either in free form or after encapsulation in poly(lactic-co-glycolic acid) particles. By tuning the drug concentration or the number of times a floss is dipped into the coating solution we were able to coat from as little as 0.4 µg to as high as 1.6 mg of drug. Coated floss was passed within the gum pocket of excised porcine mandibles to demonstrate delivery efficiency up to 91%. Utilizing the porcine jaw in an ex-vivo condition we illustrated the ability of the delivered drug to diffuse into the tissue. Overall, the data illustrates the potential of coated floss as an innovative modality for drug delivery to the gum pocket.

Demystifying particle-based oral vaccines.

Introduction: The oral route of vaccination is pain- and needle-free and can induce systemic and mucosal immunity. However, gastrointestinal barriers and antigen degradation impose significant hurdles in the development of oral vaccines. Live attenuated viruses and bacteria can overcome these barriers but at the risk of introducing safety concerns. As an alternative, particles have been investigated for antigen protection and delivery, yet there are no FDA-approved oral vaccines based on particle-based delivery systems. Our objective was to discover underlying determinants that can explain the current inadequacies and identify paradigms that can be implemented in future for successful development of oral vaccines relying on particle-based delivery systems.Areas covered: We reviewed literature related to the use of particles for oral vaccination and placed special emphasis on formulation characteristics and administration schedules to gain an insight into how these parameters impact production of antigen-specific antibodies in systemic and mucosal compartments.Expert opinion: Despite the long history of vaccines, particle-based oral vaccination is a relative new field with the first study published in 1989. Substantial variability exists between different studies with respect to dosing schedules, number of doses, and the amount of vaccine per dose. Most studies have not used adjuvants in the formulations. Better standardization in vaccination parameters is required to improve comparison between experiments, and adjuvants should be used to enhance the systemic and mucosal immune responses and to reduce the number of doses, which will make oral vaccines more attractive.