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

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

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.

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 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.

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.

Immunogenicity of trypsin treated type 2 and type 3 poliovirus in rats.

Oral polio vaccine will encounter the proteolytic enzyme trypsin during administration but inactivated polio vaccine not. To investigate the effect on the humoral immune response, rats were immunized intramuscularly with trypsin treated type 2 and type 3 poliovirus. IgG and IgM responses were determined as well as the neutralizing antibody titer. It is shown that the immunogenicity of type 2 poliovirus, unlike that of type 3, is hardly affected by trypsin treatment. For type 3, trypsin treatment results in an increase in IgM and neutralizing response. The IgG response decreases after trypsin treatment. The results indicate that IPV formulations may be improved by the addition of trypsin treated type 3, as suggested by Roivainen and Hovi (J Virol 1987; 61: 3749-3753) but not by the addition of trypsin treated type 2 poliovirus.

Glycosyl compositions and structural characteristics of the potential immuno-adjuvant active saponins in the Quillaja saponaria Molina extract quil A.

Fifty saponin components of Quil A, a commercially available extract from the bark of the South American tree Quillaja saponaria Molina, were partially structurally characterised. The molecular weights were determined by fast-atom bombardment mass spectrometry. The glycosyl and elemental composition of all the saponins was determined by applying our recently developed method, monomer mapping, consisting of a computer program and accurate mass measurements. Support for the presumed identity of the aglycone, i.e. quillaic acid, was found in the accurate mass determination, 1H NMR measurement and chemical reactions. The saponin composition of Quil A was shown to consist of pairs. Within the 3-O bound glycosyl moiety of a pair there was a structural difference: a pentose and rhamnose were interchanged. Structural differences between different pairs were located in the 28-O bound glycosyl moiety. A structural element, unknown to date and of which the elemental composition was deduced to be C8H12O5, was found in the 28-O bound glycosyl moiety of several saponins.

On the structure of immune-stimulating saponin-lipid complexes (iscoms).

Immune-stimulating complexes (iscoms) are stable complexes of cholesterol, phospholipid and Quil A, a triterpene saponin mixture in the size range from 40 to 100 nm. They can be used as antigen carriers in subunit vaccines. In this paper it is demonstrated that iscoms are rigid, negatively charged vesicles in which small water soluble molecules like carboxyfluorescein cannot be retained. The negative zeta-potential prevents iscoms from aggregation. The chemical composition of iscoms in one dispersion varied considerably. A typical example of the composition of iscoms is cholesterol/phospholipid/Quil A = 1.0:1.2:6.2 by weight for the iscom matrix, that is iscoms without antigen, and 1.0:1.3:5.1 for antigen-containing iscoms. A hypothetical model for the structure of the iscom matrix and related structures is presented, based on analytical chemical, physico-chemical and electronmicroscopic data. In this model iscoms are considered to be multi-micellar structures, shaped and stabilized by hydrophobic interactions, electrostatic repulsion, steric factors and possibly hydrogen bonds. The individual micelles are relatively flat, ring-shaped structures, the center offering space for one of the two bulky sugar chains of the saponins.

Immunogenicity of liposomes and iscoms containing the major outer membrane protein of Neisseria gonorrhoeae: influence of protein content and liposomal bilayer composition.

The influences of Neisseria gonorrhoeae protein IB content and bilayer composition of liposomes and protein content of iscoms on immunogenicity were investigated. Changes in the protein content of liposomes did not influence the immunoglobulin G response, whereas the response was lowered when the amount of protein in iscoms was increased. Bilayer composition only influenced the primary immunoglobulin G response; immunological memory was not affected.

Incorporation of the major outer membrane protein of Neisseria gonorrhoeae in saponin-lipid complexes (iscoms): chemical analysis, some structural features, and comparison of their immunogenicity with three other antigen delivery systems.

We incorporated the major outer membrane protein (PI) of Neisseria gonorrhoeae into immunostimulating complexes (iscoms) and examined some analytical, physicochemical, and immunological properties of these structures. The immunogenicity was compared with that of three other PI-containing structures, i.e., liposomes, outer membrane complexes produced by the bacterium, and protein-detergent-adjuvant complexes. AIPO4 and dioctadecyldimethylammonium bromide were used as adjuvants. Our results show that iscoms are much more immunogenic than liposomes and protein-detergent complexes but are also much more toxic. The localization of PI in iscoms was investigated. Therefore, the chymotrypsin susceptibility of PI in iscoms was tested, and the incorporation of fragments of PI was determined. Amphiphilic fragments of PI were incorporated in iscoms, but hydrophilic and hydrophobic fragments were not. Chymotrypsin degradation of PI in iscoms indicated that the protein is exposed to the environment in a similar manner as PI in outer membrane complexes, i.e., with both termini anchored in the iscom.