Biochemical and biophysical characterization of in vitro folded outer membrane porin PorA of Neisseria meningitidis

Two subtypes of the outer membrane porin PorA of Neisseria meningitidis, P1.6 and P1.7,16, were folded in vitro after overexpression in, and isolation from Escherichia coli. The PorA porins could be folded efficiently by quick dilution in an appropriate buffer containing the detergent n-dodecyl-N, N-dimethyl-1-ammonio-3-propanesulphonate. Although the two PorA porins are highly homologous, they required different acidities for optimal folding, that is, a pH above the pI was needed for efficient folding. Furthermore, whereas trimers of PorA P1.7,16 were almost completely stable in 2% sodium dodecyl sulphate (SDS), those of P1.6 dissociated in the presence of SDS. The higher electrophoretic mobility of the in vitro folded porins could be explained by the stable association of the RmpM protein to the porins in vivo. This association of RmpM contributes to the stability of the porins. The P1.6 pores were moderately cation-selective and displayed a single-channel conductance of 2.8 nS in 1 M KCl. The PorA P1.6 pores, but not the PorA P1.7,16 pores, showed an unusual non-linear dependence of the single-channel conductance on the salt concentration of the subphase. We hypothesize that a cluster of three negatively charged residues in L5 of P1.6 is responsible for the higher conductance at low salt concentrations.     

Adjuvant Effects Elicited by Novel Oligosaccharide Variants of Detoxified Meningococcal Lipopolysaccharides on Neisseria meningitidis Recombinant PorA Protein: A Comparison in Mice

Neisseria meningitidis lipopolysaccharide (LPS) has adjuvant properties that can be exploited to assist vaccine immunogenicity. The modified penta-acylated LPS retains the adjuvant properties of hexa-acylated LPS but has a reduced toxicity profile. In this study we investigated whether two modified glycoform structures (LgtE and IcsB) of detoxified penta-acylated LPS exhibited differential adjuvant properties when formulated as native outer membrane vesicles (nOMVs) as compared to the previously described LgtB variant. Detoxified penta-acylated LPS was obtained by disruption of the lpxL1 gene (LpxL1 LPS), and three different glycoforms were obtained by disruption of the lgtB, lgtE or icsB genes respectively. Mice (mus musculus) were immunized with a recombinant PorA P1.7-2,4 (rPorA) protein co-administered with different nOMVs (containing a different PorA serosubtype P1.7,16), each of which expressed one of the three penta-acylated LPS glycoforms. All nOMVs induced IgG responses against the rPorA, but the nOMVs containing the penta-acylated LgtB-LpxL1 LPS glycoform induced significantly greater bactericidal activity compared to the other nOMVs or when the adjuvant was Alhydrogel. Compared to LgtE or IcsB LPS glycoforms, these data support the use of nOMVs containing detoxified, modified LgtB-LpxL1 LPS as a potential adjuvant for future meningococcal protein vaccines.     

A comparison of the immunogenicity of a pair of enantiomeric proteins

The immunogenicity of a folded, all D -amino acid protein- rubredoxin, has been compared with that for the corresponding L -protein enantiomer. Following multiple administrations with alum adjuvant, the L -protein induced a strong, specific lgG antibody response, whereas the D -protein did not. This relative lack of responsiveness to the D -protein cannot be attributed to rapid excretion, since it is retained at least 4 times longer than the natural L -protein. These observations provide the first direct evidence that a folded D -amino acid protein has low immunogenicity and is long lived in vivo. Proteins with such properties may be useful as molecular platforms in a variety of chemical and pharmaco-logical applications. © 1993 Wiley-Liss, Inc.     

FetA Antibodies Induced by an Outer Membrane Vesicle Vaccine Derived from a Serogroup B Meningococcal Isolate with Constitutive FetA Expression

Invasive meningococcal disease causes over 3500 cases each year in Europe, with particularly high incidence among young children. Among serogroup B meningococci, which cause most of the cases, high diversity in the outer membrane proteins (OMPs) is observed in endemic situations; however, comprehensive molecular epidemiological data are available for the diversity and distribution of the OMPs PorA and FetA and these can be used to rationally design a vaccine with high coverage of the case isolates. The aim of this study was to determine whether outer membrane vesicles (OMVs) derived from an isolate with constitutive FetA expression (MenPF-1 vaccine) could be used to induce antibodies against both the PorA and FetA antigens. The immunogenicity of various dose levels and number of doses was evaluated in mice and rabbits, and IgG antibody responses tested against OMVs and recombinant PorA and FetA proteins. A panel of four isogenic mutants was generated and used to evaluate the relative ability of the vaccine to induce serum bactericidal activity (SBA) against FetA and PorA. Sera from mice were tested in SBA against the four target strains. Results demonstrated that the MenPF-1 OMVs were immunogenic against PorA and FetA in both animal models. Furthermore, the murine antibodies induced were bactericidal against isogenic mutant strains, suggesting that antibodies to both PorA and FetA were functional. The data presented indicate that the MenPF-1 vaccine is a suitable formulation for presenting PorA and FetA OMPs in order to induce bactericidal antibodies, and that proceeding to a Phase I clinical trial with this vaccine candidate is justified.     

流感新型黏膜疫苗的研究

目的评价PorA、PorB和Class4对流感裂解疫苗的免疫增强作用,从中挑选出最有效的流感黏膜佐剂,为发展流感黏膜疫苗提供理论基础。方法流感三价裂解抗原按比例与PorA、PorB和Class4非共价结合,滴鼻免疫Balb／c小鼠3次,采取间接ELISA检测血清特异性IgG抗体及抗体亚型,检测鼻咽、肺、小肠和阴道冲洗液中IgA效价,采用血凝抑制试验检测血清中HAI效价。结果PorB重组蛋白佐剂组较无佐剂的流感裂解抗原组在提高小鼠早期免疫应答的同时诱导较强的系统免疫应答和黏膜免疫应答;PorA组也有黏膜佐剂的功能,但和无佐剂的流感裂解抗原组相比,差异无统计学意义。结论在蛋白体的三分子中,以PorB为佐剂的流感黏膜疫苗不仅提高了抗原的系统免疫应答,而且诱导了较强的小鼠呼吸道、生殖道的局部黏膜免疫应答,为流感黏膜疫苗的研制奠定了理论基础。     

Epitope specificity of murine and human bactericidal antibodies against PorA P1.7,16 induced with experimental meningococcal group B vaccines

Synthetic peptides derived from the predicted loops 1 and 4 of meningococcal PorA, sero-subtype P1.7,16, were used to study the epitope specificity of murine and human PorA P1.7,16 bactericidal antibodies. The predicted loops 1 and 4 are surface exposed and carry in their apices the sero-subtype epitopes P1.7 (loop 1) or P1.16 (loop 4), respectively. Peptides were synthesized as mono- and multimeric peptides. Murine monoclonal and polyclonal antibodies were induced with meningococcal whole cell preparations. Polyclonal antibodies were evoked in volunteers after one immunization with 50 μg or 100 μg protein of a hexavalent meningococcal PorA vesicle vaccine. The induction of PorA antibodies was determined in ELISA using purified PorA P1.7,16. The epitope specificity of anti-PorA antibodies for both murine and human antibodies could be demonstrated by direct peptide ELISA using overlapping multimeric peptides almost spanning the entire loops 1 or 4 of the protein. The capacity of peptides to inhibit the bactericidal activity of murine and human antibodies was investigated using meningococcal strain H44/76 (B:15:P1.7,16) as a target strain. Bactericidal activities could be inhibited with both monomeric and multimeric peptides derived from epitopes P1.7 and P1.16.     

Expression of the class 1 outer-membrane protein of Neisseria meningitidis in Escherichia coli and purification using a self-cleavable affinity tag

The class 1 protein (PorA) is a major component of the outer membrane of Neisseria meningitidis and functions as a cationic porin. The protein is particularly effective in generating a bactericidal immune response following infection and is therefore under investigation as a potential antigen for inclusion in new meningococcal vaccines. Studies on the vaccine potential of PorA would be facilitated by the production of pure protein, free from other components of the meningococcal outer membrane. In the current study, PorA was expressed from the heterologous host Escherichia coli as a C-terminal fusion to an inducible protein-splicing element (intein) with an N-terminal chitin-binding domain (CBD) (IMPACT-TWIN system). The CBD acted as an affinity tag and allowed binding of the fusion protein to a chitin bead column, after which self-cleavage of the intein at its C-terminus was induced, resulting in the release of mature PorA. Cleavage of the fusion protein was temperature- and time-dependent, and was optimal at pH 7.0 after 5 days of storage at 4 degrees C. Efficient cleavage was also dependent on the addition of a minimal amino acid sequence (Gly-Arg-Ala) to the N-terminus of the mature PorA protein. This represented a significant improvement on the large N-terminal sequences introduced by other expression systems previously used to prepare recombinant PorA, and the yields of PorA purified with the IMPACT-TWIN system were similar. Thus, the IMPACT-TWIN system provides a facile method for producing recombinant PorA and may also be useful for the production of other bacterial outer-membrane proteins for vaccine studies.     

Structural variation and immune recognition of the P1.2 subtype meningococcal antigen

Neisseria meningitidis is a globally important cause of bacterial meningitis and septicemia. No comprehensive antimeningococcal vaccine is available, largely as a consequence of the high sequence diversity of those surface proteins that could function as components of a vaccine. One such component is the protein PorA, a major surface porin of this Gram-negative organism that has been used in a number of experimental and licensed vaccines. Here we describe a series of experiments designed to investigate the consequences for antibody recognition of sequence diversity within a PorA antigen. The binding of a 14-residue peptide, corresponding to the P1.2 subtype antigen, to the MN16C13F4 monoclonal antibody was sensitive to mutation of five out of the six residues within the epitope sequence. The crystal structure of the antibody Fab fragment, determined in complex with the peptide antigen, shows a remarkably hydrophobic binding site and interactions between the antigen and antibody are dominated by apolar residues. Nine intrachain hydrogen bonds are formed within the antigen which maintain the beta-hairpin conformation of the peptide. These hydrogen bonds involve residues that are highly conserved amongst different P1.2 sequence variants, suggesting that some positions may be conserved for structural reasons in these highly polymorphic regions. The sensitivity of antibody recognition of the antigen towards mutation provides a structural explanation for the widespread sequence variation seen in different PorA sequences in this region. Single point mutations are sufficient to remove binding capability, providing a rationale for the manner in which different meningococcal PorA escape variants arise.     

The low-molecular-mass subunit of the cell wall channel of the Gram-positive Corynebacterium glutamicum. Immunological localization, cloning and sequencing of its gene porA.

The 5-kDa protein PorA of the Gram-positive bacterium Corynebacterium glutamicum is the subunit of the cell wall channel. Antibodies raised against PorA specifically detected the protein on the cell surface. PorA was sequenced using Edman degradation and a gas phase sequencer. The primary sequence was used to create degenerate oligonucleotide primers. The gene of the channel-forming protein and its flanking regions were obtained by PCR followed by inverse PCR. The gene porA comprises 138 bp and encodes a 45-amino-acid-long acidic polypeptide with an excess of four negatively charged amino acids in agreement with the high cation selectivity of the PorA cell wall channel. PorA does not contain an N-terminal extension. A ribosomal-binding site was recognized 6 bp before the start codon ATG of porA. It codes for the smallest subunit of a membrane channel known so far and for the first cell wall channel protein of a corynebacterium. Southern blots demonstrated that only the chromosomes of corynebacteria contain homologous sequences to porA; no hybridization could be detected with DNA from other mycolata.     

Self-adjuvanting vaccine against group A streptococcus: Application of fibrillized peptide and immunostimulatory lipid as adjuvant

Peptides are of great interest to be used as vaccine antigens due to their safety, ease of manufacturing and specificity in generating immune response. There have been massive discoveries of peptide antigens over the past decade. However, peptides alone are poorly immunogenic, which demand co-administration with strong adjuvant to enhance their immunogenicity. Recently, fibril-forming peptides such as Q11 and lipoamino acid-based carrier have been identified to induce substantial immune responses when covalently linked to peptide epitope. In this study, we have incorporated either Q11 or lipoamino acids to a peptide epitope (J14) derived from M protein of group A streptococcus to develop self-adjuvanting vaccines. J14, Q11 and lipoamino acids were also conjugated together in a single vaccine construct in an attempt to evaluate the synergy effect of combining multiple adjuvants. Physicochemical characterization demonstrated that the vaccine constructs folded differently and self-assembled into nanoparticles. Significantly, only vaccine constructs containing double copies of lipoamino acids (regardless in conjugation with Q11 or not) were capable to induce significant dendritic cells uptake and subsequent J14-specific antibody responses in non-sizes dependent manners. Q11 had minimal impact in enhancing the immunogenicity of J14 even when it was used in combination with lipoamino acids. These findings highlight the impact of lipoamino acids moiety as a promising immunostimulant carrier and its number of attachment to peptide epitope was found to have a profound effect on the vaccine immunogenicity.     

Analysis of outer membrane porin complexes of Neisseria meningitidis in wild‐type and specific knock‐out mutant strains

The structure of the porin complexes of Neisseria meningitidis was assessed in the vaccine strain H44/76 and its homologous mutants lacking the main porins (PorA and PorB) and other outer membrane (OM) components (RmpM and FetA). The analysis using 1‐D blue native (BN) electrophoresis, 2‐D BN/SDS‐PAGE and 2‐D diagonal electrophoresis, followed by LC/MS‐MS (for 1‐D gels) or MALDI‐TOF (for 2‐D gels) revealed at least six porin complexes in the wild‐type strain with molecular masses (MW) ranging from 145 to 195 kDa and variable composition: The two higher MW complexes are formed by PorA, PorB and RmpM, the following three are formed by PorA and PorB, and the lower MW one is formed by only PorB. Complexes in the mutants lacking either PorA, PorB or RmpM, but not those in the mutant lacking FetA, were alterered respect to those in the wild‐type strain. The most evident alteration was seen in the mutant lacking PorB, in which PorA formed only a high MW complex (≈?800 kDa). Our results suggest that PorA and PorB could form a ‘basic’ template for the transportation systems in the OM of the meningococci. Other proteins (such as RmpM) could be transiently associated to the porin complexes, depending on the specific tranport needs at different stages of the meningococcal life cycle, resulting in a dynamic net of pores of variable composition.     

Synthesis and biological properties of polysaccharide-peptide conjugates as potential antigens for a vaccine against meningococci of serogroups A and B

A new approach to the development of a vaccine against meningococci of serogroups A and B was proposed. It involves the synthesis of conjugates of high-molecular capsule polysaccharides of the serogroup A meningococcus (PsA) with earlier synthesized protective fragments of membrane proteins from serogroup B meningococci. The conjugates were synthesized using a method that consists of the generation of aldehyde groups by oxidizing free vicinal hydroxyl groups of PsA and subsequent reaction of these groups with amino groups of the peptide. The reaction proceeds with the intermediate formation of the Schiff base, which is reduced to the stable secondary amine. The main parameters of the reaction were optimized in the synthesis of a PsA conjugate with a model peptide and methods of their characterization were developed. The reproducibility and efficiency of the synthetic procedure were demonstrated by the example of synthesis of PsA conjugates with fragments of protein PorA from the outer membrane of the serogroup B meningococcus. It was shown that, when administered without adjuvant, a conjugate of PsA with a protective peptide, which represents an exposed conserved fragment 306–332 of protein PorA, stimulates the formation of antibodies to the peptide and polysaccharide moieties of the molecule and is also capable of decreasing the degree of bacteremia in animals infected with serogroup A and serogroup B meningococci. The approach can be applied to the development of a complex vaccine for serogroup A and serogroup B meningococci.     

Progressive Decrease in the Potential Usefulness of Meningococcal Serogroup B Vaccine (4CMenB,Bexsero?) in Gipuzkoa,Northern Spain

The effectiveness of a vaccine is determined not only by the immunogenicity of its components, but especially by how widely it covers the disease-causing strains circulating in a given region. Because vaccine coverage varies over time, this study aimed to detect possible changes that could affect vaccine protection during a specific period in a southern European region. The 4CMenB vaccine is licensed for use in Europe, Canada, and Australia and is mainly directed against Neisseria meningitidis serogroup B. This vaccine contains four main immunogenic components: three recombinant proteins, FHbp, Nhba and NadA, and an outer membrane vesicle [PorA P1.4]. The allelic distribution of FHbp, Nhba, NadA, and PorA antigens in 82 invasive isolates (B and non-B serogroups) isolated from January 2008 to December 2013 were analyzed. 4CMenB was likely protective against 61.8% and 50% of serogroup B and non-B meningococci, respectively, in the entire period, but between 2012 and 2013, the predicted protection fell below 45% (42.1% for serogroup B isolates).The observed decreasing trend in the predicted protection during the 6 years of the study (Χ ² for trend  = 4.68, p = 0.03) coincided with a progressive decrease of several clonal complexes (e.g., cc11, cc32 and cc41/44), which had one or more antigens against which the vaccine would offer protection.     

Crystal structure of an Fab fragment in complex with a meningococcal serosubtype antigen and a protein G domain.

Many pathogens present highly variable surface proteins to their host as a means of evading immune responses. The structure of a peptide antigen corresponding to the subtype P1.7 variant of the porin PorA from the human pathogen Neisseria meningitidis was determined by solution of the X-ray crystal structure of the ternary complex of the peptide (ANGGASGQVK) in complex with a Fab fragment and a domain from streptococcal protein G to 1.95 A resolution. The peptide adopted a beta-hairpin structure with a type I beta-turn between residues Gly4P and Gly7P, the conformation of the peptide being further stabilised by a pair of hydrogen bonds from the side-chain of Asn2P to main-chain atoms in Val9P. The antigen binding site within the Fab formed a distinct crevice lined by a high proportion of apolar amino acids. Recognition was supplemented by hydrogen bonds from heavy chain residues Thr50H, Asp95H, Leu97H and Tyr100H to main-chain and side-chain atoms in the peptide. Complementarity-determining region (CDR) 3 of the heavy chain was responsible for approximately 50 % of the buried surface area formed by peptide-Fab binding, with the remainder made up from CDRs 1 and 3 of the light chain and CDRs 1 and 2 of the heavy chain. Knowledge of the structures of variable surface antigens such as PorA is an essential prerequisite to a molecular understanding of antigenic variation and its implications for vaccine design.     

Effect of trypsin and ribonuclease on the immunogenic activity of ribosomes and ribonucleic acid isolated from Mycobacterium tuberculosis

Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Effect of trypsin and ribonuclease on the immunogenic activity of ribosomes and ribonucleic acid isolated from Myobacterium tuberculosis. J. Bacteriol. 91:2146-2154. 1966.-The ribosomal fraction of the attenuated strain, H37Ra, of Mycobacterium tuberculosis was treated with trypsin alone, ethylenediaminetetraacetic acid (EDTA) alone, EDTA and pancreatic ribonuclease, or with trypsin and ribonuclease. After each of these treatments, the ribosomal fractions were injected intraperitoneally into male CF-1 mice to test their capacity to produce an immune response to infection with virulent tubercle bacilli, strain H37Rv. Removal of protein with trypsin left the immunogenicity unchanged; EDTA alone reduced immunogenicity in the smaller vaccinating doses; EDTA plus ribonuclease reduced the immunogenicity by approximately 50% in the highest (1.0 mg) vaccinating dose; ribonuclease alone, after treatment with trypsin, reduced immunogenicity also approximately 50%. A crude mycobacterial ribonucleic acid (RNA) was prepared by extraction of the ribosomal fraction with alcohol. This RNA preparation was as effective in producing an immune response as the ribosomal fraction from which it was prepared, unless the RNA was partially or completely degraded during the preparation. The effect of ribonuclease on the immunogenicity of the RNA was similar to that obtained with the ribosomal fractions, except that ribonuclease completely destroyed the immunogenicity of a partially degraded RNA. RNA appears to be an essential part of an immunizing substance in attenuated tubercle bacilli, which produces a high degree of immunity in mice; 50 mug (dry weight) will protect approximately 80% of the mice, and as little as 0.5 mug will protect approximately 30% of the mice. Mycobacterial RNA not incorporated in Freund's incomplete adjuvant was nonimmunogenic. Yeast RNA incorporated in Freund's incomplete adjuvant was not immunogenic.     

Immunogenicity of peptide-vaccine candidates predicted by molecular dynamics simulations

We present an in silico, structure-based approach for design and evaluation of conformationally restricted peptide-vaccines. In particular, we designed four cyclic peptides of ten or 11 residues mimicking the crystallographically observed beta-turn conformation of a predicted immunodominant loop of PorA from Neisseria meningitidis. Conformational correctness and stability of the peptide designs, as evaluated by molecular dynamics simulations, correctly predicted the immunogenicity of the peptides. We observed a peptide-induced functional antibody response that, remarkably, exceeded the response induced by the native protein in outer membrane vesicles, without losing specificity for related strains. The presented approach offers tools for a priori design and selection of peptide-vaccine candidates with full biological activity. This approach could be widely applicable: to outer membrane proteins of Gram-negative bacteria, and to other epitopes in a large range of pathogens.     

The development of a new adjuvant lipid-saponin complex and its use at experimental immunization by bacterial antigen

Immunostimulating complexes (ISCOM) have been modified by replacement of structural components of ISCOM; phosphatidylcholine has been replaced, by glycolipid of monogalactosyldiacylglycerol from sea macrophytes, saponin QuilA has been replaced by triterpene glycoside, Cucumarioside A₂-2. The resultant complex includes morphological structures of two types: the ISCOM-like structures with the characteristic morphology and sizes and also the tubular structures with diameter of about 40 nm and length of 150–400 nm. We have defined these structures as TI-complexes. These TI-complexes exhibit considerably lower toxicity than ISCOM. They may include an amphiphilic protein antigen and provide immunoadjuvant effect during experimental vaccination. Under conditions of the experimental immunization of mice with a weak immunogen (subunit membrane pore protein from Y. pseudotuberculosis), TI-complexes with antigen provided stronger humoral immune response to antigen than the complexes of porin with classical ISCOM, liposomes and Freund’s adjuvant. Thus, the TI-complexes represent a new type of adjuvant carriers for antigens.     

A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune responses in volunteers of diverse HLA types

This open-labeled phase I study provides the first demonstration of the immunogenicity of a precisely defined synthetic polyoxime malaria vaccine in volunteers of diverse HLA types. The polyoxime, designated (T1BT(*))(4)-P3C, was constructed by chemoselective ligation, via oxime bonds, of a tetrabranched core with a peptide module containing B cell epitopes and a universal T cell epitope of the Plasmodium falciparum circumsporozoite protein. The triepitope polyoxime malaria vaccine was immunogenic in the absence of any exogenous adjuvant, using instead a core modified with the lipopeptide P3C as an endogenous adjuvant. This totally synthetic vaccine formulation can be characterized by mass spectroscopy, thus enabling the reproducible production of precisely defined vaccines for human use. The majority of the polyoxime-immunized volunteers (7/10) developed high levels of anti-repeat Abs that reacted with the native circumsporozoite on P. falciparum sporozoites. In addition, these seven volunteers all developed T cells specific for the universal epitope, termed T(*), which was originally defined using CD4(+) T cells from protected volunteers immunized with irradiated P. falciparum sporozoites. The excellent correlation of T(*)-specific cellular responses with high anti-repeat Ab titers suggests that the T(*) epitope functioned as a universal Th cell epitope, as predicted by previous peptide/HLA binding assays and by immunogenicity studies in mice of diverse H-2 haplotypes. The current phase I trial suggests that polyoximes may prove useful for the development of highly immunogenic, multicomponent synthetic vaccines for malaria, as well as for other pathogens.     

Cutting edge: inflammasome activation by alum and alum's adjuvant effect are mediated by NLRP3

Alum is the only adjuvant approved for routine use in humans, although the basis for its adjuvanticity remains poorly understood. We have recently shown that alum activates caspase-1 and induces secretion of mature IL-1beta and IL-18. In this study we show that, in human and mouse macrophages, alum-induced secretion of IL-1beta, IL-18, and IL-33 is mediated by the NLR (nucleotide-binding domain leucine-rich repeat-containing) protein NLRP3 and its adaptor ASC, but not by NLRC4. Other particulate adjuvants, such as QuilA and chitosan, induce inflammasome activation in a NLRP3-dependent fashion, suggesting that activation of the NLRP3-inflammasome may be a common mechanism of action of particulate adjuvants. Importantly, we demonstrate that Ag-specific Ab production elicited by vaccines that contain alum is significantly impaired in NLRP3-deficient mice. Our results demonstrate for the first time a role for the NLRP3-inflammasome during development of the immune response elicited by alum-enhanced vaccination and suggest that therapeutic intervention aimed at NLRP3 may improve adjuvant efficacy.     

Antigenic features of protein carriers commonly used in immunisation trials

An aluminium hydroxide adjuvant induced a more elevated and rapid immune responses against short peptides conjugated to the Keyhole Lympet Hemocyanin carrier than immuneasy adjuvant. Furthermore, since carrier proteins may compete with the fused or chemically linked polypeptides in eliciting antigen-specific immune response, we classified the immunogenicity of the most common carrier proteins used in molecular biology for antigen expression and mouse immunisation. The disulfide isomerase protein A gave a carrier with the lowest immunogenicity whilst disulfide isomerase protein C gave the highest immunogenicity and therefore should be avoided as a fusion partner. Using this protein as a model, we identified and located the immunodominant epitopes along its sequence. These results now enable the combination of carrier and immunisation conditions to be optimized.