Diphtheria toxoid dissolving microneedle vaccination: Adjuvant screening and effect of repeated-fractional dose administration.

In this study the effect of repeated-fractional intradermal administration of diphtheria toxoid (DT) compared to a single administration in the presence or absence of adjuvants formulated in dissolving microneedles (dMNs) was investigated. Based on an adjuvant screening with a hollow microneedle (hMN) system, poly(I:C) and gibbsite, a nanoparticulate aluminum salt, were selected for further studies: they were co-encapsulated with DT in dMNs with either a full or fractional DT-adjuvant dose. Sharp dMNs were prepared regardless the composition and were capable to penetrate the skin, dissolve within 20 min and deposit the intended antigen-adjuvant dose, which remained in the skin for at least 5 h. Dermal immunization with hMN in repeated-fractional dosing (RFrD) resulted in a higher immune response than a single-full dose (SFD) administration. Vaccination by dMNs led overall to higher responses than hMN but did not show an enhanced response after RFrD compared to a SFD administration. Co-encapsulation of the adjuvant in dMNs did not increase the immune response further. Immunization by dMNs without adjuvant gave a comparable response to subcutaneously injected DT-AlPO4 in a 15 times higher dose of DT, as well as subcutaneous injected DT-poly(I:C) in a similar DT dose. Summarizing, adjuvant-free dMNs showed to be a promising delivery tool for vaccination performed in SFD administration.

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

This corrects the article DOI: 10.1038/mi.2017.81.

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

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

Dissolving Microneedle Patches for Dermal Vaccination.

The dermal route is an attractive route for vaccine delivery due to the easy skin accessibility and a dense network of immune cells in the skin. The development of microneedles is crucial to take advantage of the skin immunization and simultaneously to overcome problems related to vaccination by conventional needles (e.g. pain, needle-stick injuries or needle re-use). This review focuses on dissolving microneedles that after penetration into the skin dissolve releasing the encapsulated antigen. The microneedle patch fabrication techniques and their challenges are discussed as well as the microneedle characterization methods and antigen stability aspects. The immunogenicity of antigens formulated in dissolving microneedles are addressed. Finally, the early clinical development is discussed.

Demonstrating Functional Equivalence of Pilot and Production Scale Freeze-Drying of BCG.

Process analytical technology (PAT)-tools were used to monitor freeze-drying of Bacille Calmette-Guérin (BCG) at pilot and production scale. Among the evaluated PAT-tools, there is the novel use of the vacuum valve open/close frequency for determining the endpoint of primary drying at production scale. The duration of primary drying, the BCG survival rate, and the residual moisture content (RMC) were evaluated using two different freeze-drying protocols and were found to be independent of the freeze-dryer scale evidencing functional equivalence. The absence of an effect of the freeze-dryer scale on the process underlines the feasibility of the pilot scale freeze-dryer for further BCG freeze-drying process optimization which may be carried out using a medium without BCG.

Trypsin diminishes the rat potency of polio serotype 3.

This study addresses observations made in view of testing in practice the guideline in the European Pharmacopoeia (EP) on omitting the rat potency test for release of polio containing vaccines. In general, use of the guideline is valid and the D-antigen ELISA can indeed be used as an in vitro alternative for the in vivo test. However, the set-up of the ELISA is crucial and should include detection of antigenic site 1 in polio serotype 3 as destruction of that site by trypsin results in a reduced rat potency. Antigenic site 1 in polio serotype 2 may also be modified by trypsin, but the cleavage of viral protein 1 (VP1) did not affect the rat potency. Therefore, any antigenic site, except site 1, can be used for detection of polio serotype 2. It is advised to include testing of the effect of trypsin treatment in the EP-guideline. This allows polio vaccine manufacturers to check whether their in-house ELISA needs improvement.

Improved storage stability and immunogenicity of hepatitis B vaccine after spray-freeze drying in presence of sugars.

The current hepatitis B vaccines need to be stored and transported under refrigerated conditions (2-8°C). This dependence on a cold-chain is highly challenging in areas where hepatitis B virus infections are endemic. To decrease the cold-chain dependency, powder formulations of the hepatitis B surface antigen (HBsAg) without aluminum were prepared by spray-freeze drying in the presence of either inulin or a combination of dextran and trehalose. The stability of HBsAg in the amorphous powder formulations was strongly improved during storage both at room temperature and at an elevated temperature (60°C), compared to a liquid plain and an aluminum hydroxide adjuvanted HBsAg formulation. Immunogenicity studies in mice showed that reconstituted powder formulations induced higher IgG immune responses after intramuscular administration than those induced after administration of unprocessed plain antigen. Although the immune response was not as high as after administration of aluminum adjuvanted HBsAg, the immune response to the reconstituted vaccines shifted towards a more balanced Th1/Th2 response compared to the aluminum containing HBsAg formulation.

Development of a fast ELISA for quantifying polio D-antigen in in-process samples.

A fast ELISA was developed and qualified for analysis of polio D-antigen. The original 20 h-protocol was optimized by minimizing the total incubation time to 1 h, and by replacing the signal reagent 3,3',5,5'-tetramethylbenzidine by a chemiluminogenic signal reagent with a theoretical low intrinsic background and high dynamic range.

Microneedle arrays for the transcutaneous immunization of diphtheria and influenza in BALB/c mice.

Transcutaneous immunization (TCI) is limited by poor permeation of macromolecules across the skin. Microneedle arrays form transient conduits and enhance the transport of vaccine molecules across the skin barrier without pain sensation. Here we investigated in mouse the immune responses after TCI using two model antigens, diphtheria toxoid (DT) and influenza subunit vaccine. The electric applicator enabled shorter microneedle (300 microm) to pierce mouse skin effectively, as shown by Trypan blue staining and trans-epidermal water loss measurement. The vaccines were topically applied with and without cholera toxin (CT) on microneedle-treated skin. In DT TCI, microneedle array pretreatment of the skin was essential to achieve substantial IgG and toxin-neutralizing antibody titers. Addition of CT further boosted the immune response to similar levels as observed after subcutaneous injection of AlPO4-adsorbed DT (DT-alum). In contrast, microneedle array pretreatment showed no effect on the immune response to plain influenza vaccine. This response was strongly improved by inclusion of CT, independent of microneedle treatment. These results indicate that TCI of DT and CT with microneedle treatment results in comparable protection as injection of DT-alum, and TCI of influenza vaccine adjuvanted with CT is superior to the injection of plain vaccine.

Immune modulation by adjuvants combined with diphtheria toxoid administered topically in BALB/c mice after microneedle array pretreatment.

PURPOSE: In this study, modulation of the immune response against diphtheria toxoid (DT) by various adjuvants in transcutaneous immunization (TCI) with microneedle array pretreatment was investigated. METHODS: TCI was performed on BALB/c mice with or without microneedle array pretreatment using DT as a model antigen co-administrated with lipopolysaccharide (LPS), Quil A, CpG oligo deoxynucleotide (CpG) or cholera toxin (CT) as adjuvant. The immunogenicity was evaluated by measuring serum IgG subtype titers and neutralizing antibody titers. RESULTS: TCI with microneedle array pretreatment resulted in a 1,000-fold increase of DT-specific serum IgG levels as compared to TCI. The immune response was further improved by co-administration of adjuvants, showing a progressive increase in serum IgG titers when adjuvanted with LPS, Quil A, CpG and CT. IgG titers of the CT-adjuvanted group reached levels comparable to those obtained after DT-alum subcutaneous injection. The IgG1/IgG2a ratio of DT-specific antibodies decreased in the following sequence: plain DT, Quil A, CT and CpG, suggesting that the immune response was skewed towards the Th1 direction. CONCLUSIONS: The potency and the quality of the immune response against DT administered by microneedle array mediated TCI can be modulated by co-administration of adjuvants.

An in vitro approach in quality control of toxoid vaccines.

For routine immunogenicity testing of traditionally produced vaccines, animal tests are required by regulatory authorities, with potency estimated in International Units. A new concept focuses on assuring immunogenicity by monitoring batch-to-batch consistency in production. This concept is used for well-defined biologicals such as hormones. Through the use of immunochemical and bio- and physiochemical techniques the traditional products can be characterised as completely as possible. Developments in in vitro methodologies offer opportunities for immunogenicity testing in vitro. This study describes the possibilities for applying the consistency concept to the traditional products, tetanus and diphtheria toxoids. The sources of variation in these products were studied by flocculation time, SDS-PAGE, biosensor analysis, gel permeation chromatography and in vitro cytokine production studies. Batch-to-batch variation was shown using these in vitro techniques. Results indicate that it is possible to apply the consistency concept in the quality control of traditional vaccines like tetanus and diphtheria toxoids.

Outer membrane protein purification.

The major outer membrane proteins (OMPs) from Neisseria meningitidis, which are expressed at high levels, are subdivided in five classes based on molecular weight (1,2) (see Table 1). Table 1 Major Meningococcal Outer-Membrane Proteins Outer-membrane proteins Name Molecular maass Function/characteristics Class 1 PorA 44-47 kDa Porin Class 2/3 PorB 37-42 kDa Porin Class 4 Rmp Reductionmodifiableprotein, unknown Class 5 Opa 26-30 kDa Adhesion,opacity protein Opc 25 kDa Invasion, opacity protein Iron-regulated proteins Mirp 37 kDa Iron acquisition (?);majoriron-regulatedprotein FrpB 70 kDa Ferric enterobactin receptor (also FetA) Adapted from ref. (1).

In vivo antibody response and in vitro CTL activation induced by selected measles vaccine candidates, prepared with purified Quil A components.

Semipurified Quil A and purified Quil A were used to prepare well-characterized subunit vaccine candidates against measles. Variation in the relative amounts of the measles virus (MV) fusion (F) protein, Quil A-components and lipids did not influence induction of antibody responses in mice, but had a pronounced effect on the capacity to induce cytotoxic T cell (CTL) activity of a CD8(+) MV F-protein specific human T cell clone in vitro. A characteristic MV iscom preparation based on the combined use of HPLC-purified Quil A-components QA-3 and QA-22 (QA-3/22) efficiently induced CTL activity in vitro. Comparable results were obtained by mixing beta-propiolactone inactivated MV with iscom-matrix QA-3/22 or free QA-22. On the basis of the data presented it was concluded that these three preparations are interesting MV vaccine candidates for further evaluation in pre-clinical experiments in a primate model.

Immunogenicity of outer membrane proteins in a lipopolysaccharide-deficient mutant of Neisseria meningitidis: influence of adjuvants on the immune response.

The immunogenicity of outer membrane complexes (OMCs) or heat-inactivated bacteria of a lipopolysaccharide (LPS)-deficient mutant derived from meningococcal strain H44/76 was studied. The immune response in BALB/c mice to the major outer membrane proteins was poor compared to the immune response elicited by wild-type immunogens. However, addition of external H44/76 LPS to mutant OMCs entirely restored the immune response. By using an LPS-deficient mutant, it may be possible to substitute a less toxic compound as adjuvant in meningococcal outer membrane vaccines. Therefore, a broad panel of adjuvants were tested for their potential to enhance the immunogenicity of LPS-deficient OMCs. AlPO(4), Rhodobacter sphaeroides LPS, monophosphoryl lipid A and alkali-hydrolyzed meningococcal LPS showed significantly lower adjuvant activity than did H44/76 LPS. Adjuvant activity similar to H44/76 LPS was found for Escherichia coli LPS, meningococcal icsB and rfaC LPS, QuilA, subfractions of QuilA, and MF59. Good adjuvant activity was also found with meningococcal htrB1 LPS, containing penta-acylated lipid A. Antisera elicited with the less active adjuvants showed relatively high immunoglobulin G1 (IgG1) titers, whereas strong adjuvants also induced high IgG2a and IgG2b responses in addition to IgG1. Antisera with the IgG2a and IgG2b isotypes showed high bactericidal activity, indicating that adjuvants promoting the IgG2a and IgG2b response contribute most to the protective mechanism. Thus, this study demonstrates that the immunogenicity of meningococcal LPS-deficient OMCs can be restored by using less toxic adjuvants, which opens up new avenues for development of vaccines against meningococcal disease.

Immunogenicity of various presentation forms of PorA outer membrane protein of Neisseria meningitidis in mice.

In this study we compare different vaccine formulations containing meningococcal PorA outer membrane protein; purified PorA, outer membrane vesicles (OMV) and immune-stimulating complexes (iscom). Bactericidal antibodies could be generated by the OMV and iscom formulation but not with purified PorA using either A1PO4 or Quil-A as adjuvant. OMV and iscom formulations revealed similar immunogenicity when tested in a dose response manner, with respect to bactericidal as well as OMV-binding antibodies. The anti-OMV IgG subclass response induced by PorA in OMV formulation was found in all subclasses IgG1, IgG2a, IgG2b, IgG3. OMP-iscoms induced very high IgG1 anti-OMV antibodies but almost no IgG3 response. Also, OMP-iscoms appeared to be a potent inducer of antibodies directed against linear peptides corresponding to surface exposed loops of PorA. In addition, iscoms as well as purified PorA with Quil-A as adjuvant (but not with A1PO4) induced high levels of antibodies against purified PorA. In summary, in addition to the OMV formulation, only iscoms containing PorA are able to generate an anamnestic and bactericidal antibody response.

Antigenic and immunogenic properties of inactivated polio vaccine made from Sabin strains.

Inactivated polio vaccine (IPV) was prepared with the Sabin strains, normally used for the attenuated live vaccine. The vaccine was characterized with respect to its antigenicity as determined by ELISA and biosensor analysis and its immunogenicity in rats. Compared with the vaccine prepared with virulent strains (Mahoney, MEF and Saukett) some distinct differences were found with regard to the interaction with glass vials (types 1 and 2) and antigenicity (type 3). The most profound difference however, was the immunogenicity of types 1 and 2. Standardized on the amount of virus, type 1 Sabin-IPV was about 3 times more immunogenic as compared to Mahoney-IPV. The immunogenicity of type 2 Sabin-IPV on the other hand was reduced approximately 10-fold, compared to MEF-IPV. Type 3 Sabin and Saukett IPV were comparable in immunogenicity but differences in antigenicity were evident. The lack of correlation between antigenicity and immunogenicity demonstrate that current IPV standards are not suitable to quantify IPV made from Sabin strains. The results indicate that future Sabin-IPV vaccines may have to contain virus amounts that are very different as compared to current IPV vaccines.

Effects of protraction mechanics on the midface.

Forty patients with Class III maxillary deficiencies were each treated with a bonded maxillary palatal expansion appliance followed by protraction. Nineteen of the 40 patients were retained with a Frankel III appliance. This group was compared with 24 Class I patients treated solely with bonded expansion appliance mechanotherapy. To determine at which level protraction mechanics affects the maxilla, the Walker's analysis and other cephalometric measurements were used. The protraction group showed significant increases (p <.05) in the following measurements: ANB angle, Wits, A perpendicular to nasion and in sella to A point. Anterior molar movement, without changes in posterior nasal spine or upper incisor to SN, was evident (p <.05). Favorable change in the facial profile was noted. There were no changes in the angles between sella-nasion and its relationship with the Frankfurt, occlusal, palatal, and mandibular planes. Walker's analysis showed no change in the position of orbitale. The control group did not demonstrate any significant changes in the position of the maxillary complex as a result of expansion mechanics. The retention group maintained the position of the maxilla postprotraction. Facial contour was maintained and other profile related variables improved.

In vitro determination of antigen quality: biosensor analysis and fluorescence spectroscopy.

We are probing the potential of two techniques to monitor the quality of antigens in vitro. Structural and conformational differences between diphtheria toxin and toxoid are detected via biosensor analysis (BIA-core) and fluorescence spectrometry. With BIA-core the interaction kinetics between toxin and toxoid and a monoclonal antibody were established. The fluorescence properties of both antigens were determined with respect to fluorescence intensity and emission maximum as a function of guanidinium hydrochloride concentration. In all cases clear differences were found between toxin and toxoid. Antibody affinity of the toxoid was lower compared with toxin, caused by lower binding and higher release rates. Fluorescence intensity of toxoid was reduced by about 50%. Toxoid was less sensitive to guanidinium hydrochloride-induced denaturation, reflected in a diminished shift of the emission maximum.

A new WHO International Reference Reagent for use in potency assays of inactivated poliomyelitis vaccine.

Assays of the potency of inactivated poliovirus vaccine require the use of an appropriate reference reagent. Preparation 91/574 was shown by international collaborative study to be suitable for determination of antigenic content and immunogenicity of inactivated poliovirus vaccines by in vitro and in vivo assays, respectively. The reagent is a trivalent blend of formaldehyde-inactivated monovalent pools of poliovirus type 1 (Mahoney) poliovirus type 2 (MEF-1) and poliovirus type 3 (Saukett). Studies by antigen-capture ELISA showed that the component monovalent pools contained high titres of D antigen and trace amounts of C antigen. Sucrose gradient analysis showed that the D antigenicity was almost exclusively associated with 150S virus particles. Low levels of procapsids (75S particles with D antigenicity) were detected in the type 1 and 2 monovalent pools. The profile of intact virions and procapsids in 91/574 in sucrose gradients was very similar to PU78-02, a previously used inactivated trivalent poliovirus vaccine reference. The World Health Organization (WHO) Expert Committee on Biological Standardization at its 1994 meeting established preparation 91/574 as the 2nd WHO international Reference Reagent for poliomyelitis vaccine (inactivated). A potency of 430, 95, 285 D antigen units per ml was assigned to poliovirus type 1, 2 and 3, respectively. A separate aliquot of this preparation, established by the European Pharmacopeia Commission as a Biological Reference Preparation, has an identical assigned titre. The 2nd WHO International Reference Reagent 91/574 is intended for calibration of secondary reference reagents.

Single shot with tetanus toxoid in biodegradable microspheres protects mice despite acid-induced denaturation of the antigen.

Tetanus toxoid encapsulated in microspheres consisting of biodegradable polyesters, prepared by four different manufacturers were evaluated with respect to antigenic load, in vitro release pattern, antigen integrity and immunogenicity. In vitro release studies over periods up to 140 days indicated that only during the first days tetanus toxoid was released. Although some preparations were designed to release their antigen content in a pulsatile manner, this was never observed in vitro. A single immunization with 0.3 Lf tetanus toxoid in microspheres induced substantial humoral responses, in most cases higher than one immunization with plain tetanus toxoid, sometimes higher than one dose of alum-adsorbed toxoid but always lower than booster immunizations. It is shown that the moderate (no booster effect) performance of the microsphere preparations is probably due to acid induced denaturation of the antigen. Despite this drawback, protection level in mice after challenge with 50 LD50 1 year after one immunization with microspheres was, on average, substantially higher than mice receiving plain tetanus toxoid.