Antigenic and immunogenic properties of cyanogen bromide peptides from gonococcal outer membrane protein IB. Evidence for the existence of a surface-exposed conserved epitope.

Two distinct species of gonococcal porin proteins exist that differ with regard to surface exposure. Protein IB, expressed by strains of the WII/III serogroup, has both termini buried in the outer membrane, leaving a central region of the molecule exposed at the cell surface. We have attempted to define this region of protein IB in detail by studying the antigenic and immunogenic properties of peptides derived from protein IB. Treatment of gonococcal protein IB (serotype 5) with cyanogen bromide resulted in cleavage of protein IB into three major fragments of Mr of 15,000, 13,000, and 8,000. The location of these peptides in the intact protein was determined by analysis of partial cleavage products. The 8,000 Mr peptide (CB2) was found to be located in the central region of the protein. Chymotrypsin cleavage of protein IB revealed a cleavage site near one of the cyanogen bromide cleavage sites. Trypsin was found to cleave the protein, either in outer membranes complexes (OMC) or in detergent micelles, in the central CB2 fragment. These results suggest that CB2 is a part of the surface-exposed region of protein IB. Immunization of mice with purified protein IB (serotype 5) induced antibodies against all three CB-peptides. Absorption of the sera with homologous OMC resulted in a complete removal of antibodies against CB2, supplying further evidence for its surface-exposed nature. Antibodies against the 13,000 Mr peptide (CB1) could not be absorbed with intact OMC, suggesting that this peptide is buried within the outer membrane. Antisera raised against CB2 of serotype 5 demonstrated a considerable cross-reactivity with heterologous outer membranes. On the contrary, intact OMC induced mainly type-specific antibodies. These data demonstrate the presence of conserved epitopes on the surface-exposed CB2 peptide. These conserved epitopes are generally not very immunogenic when present in intact OMC.

Expression of phosphofructokinase in Neisseria meningitidis.

Neisseria meningitidis serogroup B is a pathogen that can infect diverse sites within the human host. According to the N. meningitidis genomic information and experimental observations, glucose can be completely catabolized through the Entner-Doudoroff pathway and the pentose phosphate pathway. The Embden-Meyerhof-Parnas pathway is not functional, because the gene for phosphofructokinase (PFK) is not present. The phylogenetic distribution of PFK indicates that in most obligate aerobic organisms, PFK is lacking. We conclude that this is because of the limited contribution of PFK to the energy supply in aerobically grown organisms in comparison with the energy generated through oxidative phosphorylation. Under anaerobic or microaerobic conditions, the available energy is limiting, and PFK provides an advantage, which explains the presence of PFK in many (facultatively) anaerobic organisms. In accordance with this, in silico flux balance analysis predicted an increase of biomass yield as a result of PFK expression. However, analysis of a genetically engineered N. meningitidis strain that expressed a heterologous PFK showed that the yield of biomass on substrate decreased in comparison with a pfkA-deficient control strain, which was associated mainly with an increase in CO(2) production, whereas production of by-products was similar in the two strains. This might explain why the pfkA gene has not been obtained by horizontal gene transfer, since it is initially unfavourable for biomass yield. No large effects related to heterologous expression of pfkA were observed in the transcriptome. Although our results suggest that introduction of PFK does not contribute to a more efficient strain in terms of biomass yield, achievement of a robust, optimal metabolic network that enables a higher growth rate or a higher biomass yield might be possible after adaptive evolution of the strain, which remains to be investigated.

A practical approach for exploration and modeling of the design space of a bacterial vaccine cultivation process.

A licensed pharmaceutical process is required to be executed within the validated ranges throughout the lifetime of product manufacturing. Changes to the process, especially for processes involving biological products, usually require the manufacturer to demonstrate that the safety and efficacy of the product remains unchanged by new or additional clinical testing. Recent changes in the regulations for pharmaceutical processing allow broader ranges of process settings to be submitted for regulatory approval, the so-called process design space, which means that a manufacturer can optimize his process within the submitted ranges after the product has entered the market, which allows flexible processes. In this article, the applicability of this concept of the process design space is investigated for the cultivation process step for a vaccine against whooping cough disease. An experimental design (DoE) is applied to investigate the ranges of critical process parameters that still result in a product that meets specifications. The on-line process data, including near infrared spectroscopy, are used to build a descriptive model of the processes used in the experimental design. Finally, the data of all processes are integrated in a multivariate batch monitoring model that represents the investigated process design space. This article demonstrates how the general principles of PAT and process design space can be applied for an undefined biological product such as a whole cell vaccine. The approach chosen for model development described here, allows on line monitoring and control of cultivation batches in order to assure in real time that a process is running within the process design space.

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.

Human IgM antibodies do not activate guinea-pig complement after interaction with soluble antigen.

Activation of guinea-pig complement by human IgM antibodies after interaction with a particulate antigen is well established. Human IgM antibodies directed against meningococcal group-C capsular polysaccharide, however, were not able to fix guinea-pig complement in a classical complement fixation test with soluble antigen. In the same test, human complement was readily activated by these antibodies. This inability of human IgM antibodies to activate guinea-pig complement after interaction with soluble antigen was confirmed in other antigen systems. In contrast, efficient activation of both human and guinea-pig complement was found with the same antigen in a particulate form.

Comparison of four bifunctional reagents for coupling peptides to proteins and the effect of the three moieties on the immunogenicity of the conjugates.

Peptide-carrier conjugates are widely used to raise antipeptide antibodies. In a model system using angiotensin and tetanus toxoid as the peptide and the carrier protein respectively, four cross-linking reagents were employed to study their effect on the immunogenicity of the conjugates. Optimization of the conjugation method for these heterobifunctional reagents, all succinimidyl active esters, resulted in well-defined conjugates of predictable composition. ELISA assays were performed to compare the antigenicity and the immunogenicity of the conjugates. The antipeptide antibody titres were of the order of 2 X 10(4)-2 X 10(5). The anti-carrier antibody titres were high, in spite of the modification of the protein. Three of the four coupling reagents used for conjugation were of the 'maleimide' type: succinimidyl 6-(N-maleimido)-n-hexanoate (MHS), succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) and succinimidyl m-maleimidobenzoate (MBS). One coupling reagent contained an activated disulphide: succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The constrained linkers originating from SMCC and MBS induced very high linker-specific antibody levels. The more flexible non-aromatic linkers originating from MHS and SPDP showed almost no reactivity. For this reason and since the thioether linkage is more stable than the disulphide bond, we recommend MHS as the crosslinking reagent of choice.

Analysis of glycoforms present in two mouse IgG2a monoclonal antibody preparations.

Lectins have been used for the determination of the oligosaccharide structures expressed by two monoclonal IgG antibodies, MN12 and RIV6. Dot blot experiments revealed the presence of terminal Fuc alpha (1-->6)GlcNAc, Gal beta (1-->3)GalNAc, Gal beta (1-->4)GlcNAc, Man alpha (1-->6, 1-->3)Man, NeuAc alpha (2-->6)Gal and NeuAc alpha (2-->6)GalNAc on both monoclonal antibodies. MN12 was shown to contain a carbohydrate moiety within the Fc region only. RIV6 contained carbohydrate moieties within both the Fc and Fab regions. Additional O-glycosidic linked carbohydrate chains were detected within the Fc region of both monoclonal antibodies. High mannose structures were also detected on both Mabs.

An isotype-specific spot-ELISA for the enumeration of antibody-secreting hybridomas and the determination of isotype switch variants.

A solid-phase spot enzyme-linked immunosorbent assay (spot-ELISA) using rat monoclonal antibodies (MAbs) and an image-processing system is described. This isotype-specific spot-ELISA permits the enumeration of antibody-secreting cells irrespective of the specificity of the secreted antibodies. When used in combination with an ELISA, the antibody production per cell can also be evaluated. In addition, isotype switch variants, which arise spontaneously in antibody-producing cell lines, can be determined. This study compared four assays: three antigen-specific spot-ELISAs, using enzyme-conjugated polyclonal antibodies as well as rat MAbs; and an isotype-specific spot-ELISA using rat MAbs. There were no significant differences between these four spot-ELISA systems. For one tested cell line (alpha huIgA1/gamma 1), the number of antibody-secreting cells fluctuated between 60% and 95% during several passages. For the other tested cell line (alpha huIgA1/gamma 2b), the number of antibody-secreting cells decreased from 90% to 70% after several passages. The results of the spot-ELISA were in agreement with flow cytometric (FC) analysis of cytoplasmic IgG. This indicates that for these two cell lines, the synthesized IgG was also secreted into the culture fluid. Using the isotype-specific spot-ELISA, the switch frequency of five murine hybridomas (alpha huIgA1/gamma 1, alpha huIgA1/gamma 2b, alpha HRP, RIV6, MN12) was determined. The switch frequencies varied from 1/82,000 for the alpha HRP cell line to 1/660,000 for the alpha huIgA1/gamma 2b cell line.

Purification and stabilisation of a poorly soluble mouse IgG3 monoclonal antibody.

The anti-T cell monoclonal antibody (Mab) RIV9 (mouse IgG3, kappa) has been developed for clinical use in the treatment of allograft rejection. In order to obtain a clinical grade Mab preparation, RIV9 was purified from cell culture supernatants by protein A affinity and anion exchange chromatography. Reasonable yields of highly purified product could only be obtained if stabilising compounds were added and Tween 80 was used in all stages of the purification process. Prior to anion exchange chromatography, dextran sulphate (MW 5000) was added to keep the Mab in solution. Many other additives were tested but did not solubilise RIV9 under the low salt strength conditions required for ion exchange chromatography. The complex character of the solubility-determining factors was demonstrated by the influence of buffer composition, buffer concentration, pH, and sodium chloride concentration on the solubility of RIV9.

Preparation of clinical grade monoclonal antibodies from serum-containing cell culture supernatants.

Three mouse monoclonal antibodies (Mab), RIV6, MN12, and WT31, were purified from cell culture supernatants containing foetal bovine serum (FBS) by two-step purification protocols, involving protein A affinity and ion exchange chromatography. Provided that the purification conditions were adapted to the physico-chemical properties of the individual Mab, clinical grade products could be obtained. The residual levels of bovine IgG originating from FBS were below 1% on a protein basis. Endotoxin levels were below 1 ng/ml. The contents of other serum proteins, DNA, and protein A were below or near the detection limits. The final products met the requirements for therapeutic Mab. Special attention was paid to the behaviour of foetal bovine IgG in the different purification steps. Large variations in the IgG contents of different batches of FBS were observed. However, the properties of the IgG fractions of the batches were very similar. A major IgG fraction with a low affinity for protein A and with components with relatively acidic isoelectric points (pIs) was distinguished from a minor fraction exhibiting a high affinity for protein A and a more diverse pI pattern. The impact of these findings on the purification strategy used for the Mab is discussed.

Two-step purification of a murine monoclonal antibody intended for therapeutic application in man. Optimisation of purification conditions and scaling up.

The murine hybridoma cell line WT31, which produces a monoclonal antibody (Mab) of the IgG1 isotype with specificity for the human T cell receptor, was grown in batch-suspension cultures in the presence of foetal bovine serum (FBS). To acquire a clinical grade product for the reversal of allograft rejection, the clarified and concentrated cell culture supernatant was purified by a two-step chromatographic procedure, involving protein A affinity chromatography and Q Sepharose anion exchange chromatography. After choosing the appropriate conditions on a small scale, the purification process was scaled up. A BioPilot system was used for automated purification of 1 g WT31 Mab in a closed system. In spite of a relatively high initial ratio of bovine IgG to mouse IgG, the residual level of bovine IgG could be reduced to 1% or less with respect to the Mab content. No other serum proteins nor DNA were detected in the purified product. The efficacy of the purification procedure was demonstrated by a combination of several analytical techniques: ELISA (mouse and bovine IgG contents, protein A content), countercurrent immunoelectrophoresis (bovine serum albumin content), fluorescence activated cell sorter analysis (potency), DNA assay, sodium dodecylsulphate polyacrylamide gel electrophoresis, immunoblotting, isoelectric focusing, and gel permeation chromatography.

Inhibition of measles virus-induced cell-cell fusion with a monoclonal antibody directed against the haemagglutinin.

A neutralizing monoclonal antibody (C26-15) against the haemagglutinin (H protein) of measles virus was generated which caused cell-cell fusion inhibition in cultures of measles virus-infected cells. It was shown that this phenomenon coincided with a down-regulation of the expression of both the H protein and the fusion (F) protein. We also showed cell-cell fusion inhibition with a polyclonal rabbit serum directed against Tween-ether inactivated measles virus, which did not contain biologically active antibodies against the F protein. Cell-cell fusion inhibition caused by anti-H antibodies is distinct from cell-cell fusion inhibition induced by a direct interaction of anti-F antibody with the F protein in the membrane of infected cells. Since both mechanisms may also be involved in the in vivo situation, the exclusive role for the generation of anti-F antibody to prevent virus spread by cell-cell fusion in vivo is questioned. It is speculated that the observed down-regulation of both glycoproteins may lead to a less efficient killing of infected cells by cytotoxic T-lymphocytes, which may constitute an alternative explanation for the insufficient protection after vaccination with an inactivated measles vaccine.

Immunisation against bacterial meningitis.

The vaccine potential of different surface antigens of encapsulated bacteria is considered. It is concluded that only the capsular polysaccharides provide good protection. Polysaccharides are, however, thymus-independent antigens and the antibody response is strongly influenced by the age of the recipient and his naturally acquired immunity. For a long-lasting immunity the induction of mainly IgG antibodies is essential. Such a response can easily be induced by conjugates in which the polysaccharides have been attached to thymus-dependent carriers.

Vaccine potential of meningococcal group C polysaccharide-tetanus toxoid conjugate.

The antibody response in mice to Neisseria meningitidis (meningococcal) group C polysaccharide could be modified by its conjugation to proteins, i.e. tetanus toxoid. Whereas the pure polysaccharide behaved as a T-independent antigen, the polysaccharide-protein conjugate was clearly a T-dependent antigen, as shown by the pronounced IgG response after the first dose and by the booster effect after the second dose. In comparison, group C polysaccharide-outer membrane protein complexes isolated from the cell-free culture liquid of a meningococcal culture induced only a low level of IgG antibodies. Conjugation did not result in a reversion of tetanus toxoid to toxin. Tetanus toxoid present in the conjugate was as immunogenic as tetanus vaccine.

Instability of a hybridoma cell line in a homogeneous continuous perfusion culture system.

In this study several analytical techniques were applied to obtain information about the stability of expression, the yield, and the integrity of a monoclonal antibody (Mab) produced by hybridoma cell line RIV6 in a homogeneous continuous perfusion culture system. The total antibody as well as the isotype-specific antibody contents decreased continuously during the course of cultivation, while the viable cell concentration remained constant. The origin of the discrepancy between the Mab contents observed by two enzyme-linked immuno sorbent assay (ELISA) systems during the steady state was due to fragmentation of the IgG molecule, either cytoplasmic or in the culture fluid, as determined by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The IgG-secreting cells as well as the fraction of cells containing a high cytoplasmic IgG content decreased continuously during cultivation. The isoelectric focusing (IEF) pattern showed the appearance of two additional bands after five days of cultivation. This work indicates that cell line RIV6 is unstable in the culture system used, with respect to cell properties and product formation.

Antigenic and immunogenic properties of cyanogen bromide peptides from a serotype 5 gonococcal outer membrane protein I.

Treatment of a gonococcal major outer membrane protein IB (serotype 5) with cyanogen bromide (CNBr) resulted in cleavage of PIB into three major fragments of apparent molecular weight of 15, 13, and 8 kD. The location of these peptides in the intact protein was determined by analysis of partial cleavage products. The 8 kD peptide (CB2) was found to be located in the central region of the protein. Chymotrypsin cleavage of PIB revealed a cleavage site near one of the CNBr cleavage sites. Trypsin was found to cleave the protein, either in outer membranes (OMC) or in detergent micelles, in the central CB2 fragment. These result suggest that CB2 is a part of the surface exposed region of PIB. Immunization of mice with purified PIB (serotype 5) induced antibodies against all three CB-peptides. Absorption of the sera with homologous intact OMC resulted in a complete removal of antibodies against CB2, supplying further evidence for its surface exposed nature. Antibodies against the 13 kD peptide (CB1) could not be absorbed with intact OMC, suggesting that this peptide is buried within the outer membrane. Antisera raised against CB2 of serotype 5 demonstrated a considerable cross-reactivity with heterologous outer membranes. On the contrary, intact OMC induced mainly type-specific antibodies. These data demonstrate the presence of conserved epitopes on the surface exposed CB2 peptide. These conserved epitopes are generally not very immunogenic when present in intact OMC.

Induction of mucosal immunoglobulin A immune response by preparations of Neisseria gonorrhoeae porin proteins.

The aim of this study was to develop an immunization scheme appropriate for the induction of an immunoglobulin A (IgA) response against Neisseria gonorrhoeae at the mucosae. For several reasons, the major outer membrane protein of N. gonorrhoeae (gonococcal PI) is attractive as a component of a gonococcal vaccine. Purified PI obtained from strain B2 was used in Zwittergent 3-14 for immunization. Rats received the antigen subcutaneously, intraintestinally, or directly in Peyer's patches with or without the adjuvant N-acetylmuramyl dipeptide (MDP) or AlPO4. The immune response was studied in situ by a newly developed antigen-specific three-step immunoperoxidase method, whereas specific antibodies in the serum were measured by an enzyme-linked immunosorbent assay procedure. Subcutaneous immunizations triggered peripheral lymphoid organs, whereas intraintestinal and intra-Peyer's patch immunizations triggered mucosa-associated lymphoid organs. This was reflected not only in the lymph nodes involved, popliteal versus mandibular and mesenteric lymph nodes, but also in the isotypes of the produced anti-PI antibodies, IgG versus IgA. The adjuvants AlPO4 and MDP appeared to act differently during subcutaneous and intraintestinal immunizations. AlPO4 augmented subcutaneous immune responses, whereas MDP had no effects. In contrast, intraintestinal immune responses increased most with the adjuvant MDP. In summary, we concluded that PI is capable of inducing a mucosa-associated IgA response when administered intraintestinally and that this response can be augmented by the adjuvant MDP.

Comparison of the induction of immunoglobulin M and G antibodies in mice with purified pneumococcal type 3 and meningococcal group C polysaccharides and their protein conjugates.

The nature and kinetics of the serum antibody response to pneumococcal type 3 and meningococcal group C polysaccharides and their protein conjugates were studied in mice. Bovine serum albumin and diphtheria and tetanus toxoids were used as carrier proteins. The purified polysaccharides induced only immunoglobulin M (IgM) antibodies in thymus-bearing as well as congenic athymic (nude) mice. The polysaccharides covalently conjugated to proteins produced IgM and IgG antibodies in normal mice, but only IgM antibodies in nude mice. A second dose of the polysaccharide-protein conjugates resulted in a booster effect in the IgG response to the polysaccharides. Moreover, memory B-cells, generated after a primary injection with the polysaccharide-protein conjugates, could be triggered to the production of IgG antibodies after a second injection with the pure polysaccharides alone. These data indicate that the antibody response to the pure polysaccharides is thymus independent and that this response can be changed into a thymus-dependent response by covalent conjugation of the polysaccharide to a thymus-dependent protein.