Group A streptococcal vaccine delivery by immunization with a self-adjuvanting M protein-based lipid core peptide construct

Background & objectives: To develop a broad strain coverage GAS vaccine, several strategies have been investigated which included multi-epitope approaches as well as targeting the M protein conserved C-region. These approaches, however, have relied on the use of adjuvants that are toxic for human application. The development of safe and effective adjuvants for human use is a key issue in the development of effective vaccines. In this study, we investigated the lipid polylysine core peptide (LCP) system as a self-adjuvanting GAS vaccine delivery approach. Methods: An LCP-GAS construct was synthesised incorporating multiple copies of a protective peptide epitope (J8) from the conserved carboxy terminal C-repeat region of the M protein. B10.BR mice were immunized parenterally with the LCP-J8 construct, with or without conventional adjuvant, prior to the assessment of immunogenicity and the induction of serum opsonic antibodies. Results: Our data demonstrated immunogenicity of LCP-J8 when coadministered in complete Freund's adjuvant (CFA), or administered in the absence of conventional adjuvant. In both cases, immunization led to the induction of high-titre J8 peptide-specific serum IgG antibody responses, and the induction of heterologous opsonic antibodies that did not cross-react with human heart tissue proteins. Interpretation & conclusion: These data indicated the potential of a novel self-adjuvanting LCP vaccine delivery system incorporating a synthetic GAS M protein C-region peptide immunogen in the induction of broadly protective immune responses, and pointed to the potential application of this system in human vaccine development against infectious diseases.

Semliki Forest virus and Kunjin virus RNA replicons elicit comparable cellular immunity but distinct humoral immunity

RNA replicons offer a number of qualities which make them attractive as vaccination vectors. Both alphavirus and flavivirus replicon vaccines have been investigated in preclinical models yet there has been little direct comparison of the two vector systems. To determine whether differences in the biology of the two vectors influence immunogenicity, we compared two prototypic replicon vectors based on Semliki Forest virus (SFV) (alphavirus) and Kunjin virus (KUN) (flavivirus). Both vectors when delivered as naked RNAs elicited comparable CD8+ T cell responses but the SFV vectors elicited greater humoral responses to an encoded cytoplasmic antigen beta-galactosidase. Studies in MHC class II-deficient mice revealed that neither vector could overcome the dependence of CD4+ T cell help in the development of humoral and cellular responses following immunization. These studies indicate that the distinct biology of the two replicon systems may differentially impact the adaptive immune response and this may need to be considered when designing vaccination strategies. (c) 2005 Elsevier Ltd. All rights reserved.

A recombinant diheme SoxAX cytochrome - Implications for the relationship between EPR signals and modified heme-ligands

The multiheme SoxAX proteins are notable for their unusual heme ligation (His/Cys-persulfide in the SoxA subunit) and the complexity of their EPR spectra. The diheme SoxAX protein from Starkeya novella has been expressed using Rhodobacter capsulatus as a host expression system. rSoxAX was correctly formed in the periplasm of the host and contained heme c in similar amounts as the native SoxAX. ESI-MS showed that the full length rSoxA, in spite of never having undergone catalytic turnover, existed in several forms, with the two major forms having masses of 28 687 +/- 4 and 28 718 +/- 4 Da. The latter form exceeds the expected mass of rSoxA by 31 4 Da, a mass close to that of a sulfur atom and indicating that a fraction of the recombinant protein contains a cysteine persulfide modification. EPR spectra of rSoxAX contained all four heme-dependent EPR signals (LS1a, LS1b, LS2, LS3) found in the native SoxAX proteins isolated from bacteria grown under sulfur chemolithotrophic conditions. Exposure of the recombinant SoxAX to different sulfur compounds lead to changes in the SoxA mass profile as determined by ESI while maintaining a fully oxidized SoxAX visible spectrum. Thiosulfate, the proposed SoxAX substrate, did not cause any mass changes while after exposure to dimethylsulfoxide a + 112 +/- 4 Da form of SoxA became dominant in the mass spectrum. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Protection against group A streptococcal infection by vaccination with self-adjuvanting lipid core M protein peptides

We have investigated the lipid polylysine core peptide (LCP) system as a self-adjuvanting group A streptococcal (GAS) vaccine delivery approach. LCP constructs were synthesised incorporating peptides from the M protein conserved carboxy terminal C-repeat region, the amino terminal type-specific region and from both of these regions. Immunisation with the constructs without adjuvant led to the induction of peptide-specific serum IgG antibody responses, heterologous opsonic antibodies, and complete protection from GAS infection. These data indicate that protective immunity to GAS infection can be evoked using the self-adjuvanting LCP system, and point to the potential application of this system in human mucosal GAS vaccine development. (c) 2005 Elsevier Ltd. All rights reserved.

Microbial hyaluronic acid production

Hyaluronic acid (HA) is a commercially valuable medical biopolymer increasingly produced through microbial fermentation. Viscosity limits product yield and the focus of research and development has been on improving the key quality parameters, purity and molecular weight. Traditional strain and process optimisation has yielded significant improvements, but appears to have reached a limit. Metabolic engineering is providing new opportunities and HA produced in a heterologous host is about to enter the market. In order to realise the full potential of metabolic engineering, however, greater understanding of the mechanisms underlying chain termination is required.

Vaccination against fowl cholera with acapsular Pasteurella multocida A : 1

We have previously constructed an acapsular Pasteurella multocida X-73 (serogroup A) mutant strain which was attenuated in virulence for chickens (Chung JY, Wilkie IW, Boyce JD, Townsend KM, Frost AJ, Ghodussi M, Adler B. Role of capsule in the pathogenesis of fowl cholera caused by Pasteurella multocida serogroup A. Infect. Immun. 2001;69:2487-2492). In this study, we have assessed the ability of this acapsular strain (PBA930) to induce protection against wild-type challenge in mice and the natural host chickens. Intramuscular administration of PBA930 to mice stimulated significant protection against X-73 and the heterologous strain P-1059 (A:3), but not against challenge with P-1662 (A:4). No protection was observed when PBA930 was introduced by the intraperitoneal or subcutaneous routes in mice. Significantly, the acapsular strain PBA930 was able to induce protection against challenge with wild type X-73 in chickens. (c) 2004 Elsevier Ltd. All rights reserved.

Characterization of a strong, constitutive mung bean (Vigna radiata L.) promoter with a complex mode of regulation in planta

We report the cloning and characterization in tobacco and Arabidopsis of a Vigna radiata L. (mung bean) promoter that controls the expression of VR-ACS1, an auxin-inducible ACC synthase gene. The VR-ACS1 promoter exhibits a very unusual behavior when studied in plants different from its original host, mung bean. GUS and luciferase in situ assays of transgenic plants containing VR-ACS1 promoter fusions show strong constitutive reporter gene expression throughout tobacco and Arabidopsis development. In vitro quantitative analyses show that transgenic plants harboring VR-ACS1 promoter-reporter constructs have on average 4-6 fold higher protein and activity levels of both reporter genes than plants transformed with comparable CaMV 35S promoter fusions. Similar transcript levels are present in VR-ACS1 and CaMV 35S promoter lines, suggesting that the high levels of gene product observed for the VR-ACS1 promoter are the combined result of transcriptional and translational activation. All tested deletion constructs retaining the core promoter region can drive strong constitutive promoter activity in transgenic plants. This is in contrast to mung bean, where expression of the native VR-ACS1 gene is almost undetectable in plants grown under normal conditions, but is rapidly and highly induced by a variety of stimuli. The constitutive behavior of the VR-ACS1 promoter in heterologous hosts is surprising, suggesting that the control mechanisms active in mung bean are impaired in tobacco and Arabidopsis. The 'aberrant' behavior of the VR-ACS1 promoter is further emphasized by its failure to respond to auxin and cycloheximide in heterologous hosts. VR-ACS1 promoter regulatory mechanisms seem to be different from all previously characterized auxin-inducible promoters.

Isolates of Candida albicans that differ in virulence for mice elicit strain-specific antibody-mediated protective responses

Three distinct isolates of Candida albicans were used to establish systemic and oral infections in inbred mice that are genetically resistant or susceptible to tissue damage. Patterns of infection differed significantly between both yeasts and mouse strains. Systemic infection conferred significant protection against re-challenge with the homologous, but not the heterologous yeast; however, the protective effect was more evident in the tissue-susceptible CBA/CaH mice than in the resistant BALB/c strain. In contrast, oral infection induced protection against both homologous and heterologous oral challenge, although this was significant only in the CBA/CaH mice. CBA/CaH mice produced antibodies of both IgG1 and IgG2a subclasses, whereas BALB/c mice produced predominantly IgG1. Western blotting demonstrated considerable differences between epitopes recognised by serum antibodies from mice of both strains after immunisation with each of the three yeasts. Thus, different strains of yeast show considerable specificity in antibody responses elicited by either systemic or oral infection. (c) 2005 Elsevier SAS. All rights reserved.

A novel conotoxin inhibitor of Kv1.6 channel and nAChR subtypes defines a new superfamily of conotoxins

Using assay-directed fractionation of the venom from the vermivorous cone snail Conus planorbis, we isolated a new conotoxin, designated p114a, with potent activity at both nicotinic acetylcholine receptors and a voltage-gated potassium channel subtype. p114a contains 25 amino acid residues with an amidated C-terminus, an elongated N-terminal tail (six residues), and two disulfide bonds (1-3, 2-4 connectivity) in a novel framework distinct from other conotoxins. The peptide was chemically synthesized, and its three-dimensional structure was demonstrated to be well-defined, with an R-helix and two 3(10)-helices present. Analysis of a cDNA clone encoding the prepropeptide precursor of p114a revealed a novel signal sequence, indicating that p114a belongs to a new gene superfamily, the J-conotoxin superfamily. Five additional peptides in the J-superfamily were identified. Intracranial injection of p114a in mice elicited excitatory symptoms that included shaking, rapid circling, barrel rolling, and seizures. Using the oocyte heterologous expression system, p114a was shown to inhibit both a K+ channel subtype (Kv1.6, IC50) 1.59 mu M) and neuronal (IC50 = 8.7 mu M for alpha 3 beta 4) and neuromuscular (IC50 = 0.54 mu M for alpha 1 beta 1 is an element of delta) subtypes of the nicotinic acetylcholine receptor ( nAChR). Similarities in sequence and structure are apparent between the middle loop of p114a and the second loop of a number of alpha-conotoxins. This is the first conotoxin shown to affect the activity of both voltage-gated and ligand-gated ion channels.

An automatable screen for the rapid identification of proteins amenable to refolding

Insoluble expression of heterologous proteins in Escherichia coli is a major bottleneck of many structural genomics and high-throughput protein biochemistry projects. Many of these proteins may be amenable to refolding, but their identification is hampered by a lack of high-throughput methods. We have developed a matrix-assisted refolding approach in which correctly folded proteins are distinguished from misfolded proteins by their elution from affinity resin under nondenaturing conditions. Misfolded proteins remain adhered to the resin, presumably via hydrophobic interactions. The assay can be applied to insoluble proteins on an individual basis but is particularly well suited for high-throughput applications because it is rapid, automatable and has no rigorous sample preparation requirements. The efficacy of the screen is demonstrated on small-scale expression samples for 15 proteins. Refolding is then validated by large-scale expressions using SEC and circular dichroism.

A DHA14 drug efflux gene from Xanthomonas albilineans confers high-level albicidin antibiotic resistance in Escherichia coli

Aims: Identification of a gene for self-protection from the antibiotic-producing plant pathogen Xanthomonas albilineans, and functional testing by heterologous expression. Methods and Results: Albicidin antibiotics and phytotoxins are potent inhibitors of prokaryote DNA replication. A resistance gene (albF) isolated by shotgun cloning from the X. albilineans albicidin-biosynthesis region encodes a protein with typical features of DHA14 drug efflux pumps. Low-level expression of albF in Escherichia coli increased the MIC of albicidin 3000-fold, without affecting tsx-mediated albicidin uptake into the periplasm or resistance to other tested antibiotics. Bioinformatic analysis indicates more similarity to proteins involved in self-protection in polyketide-antibiotic-producing actinomycetes than to multi-drug resistance pumps in other Gram-negative bacteria. A complex promoter region may co-regulate albF with genes for hydrolases likely to be involved in albicidin activation or self-protection. Conclusions: AlbF is the first apparent single-component antibiotic-specific efflux pump from a Gram-negative antibiotic producer. It shows extraordinary efficiency as measured by resistance level conferred upon heterologous expression. Significance and Impact of the Study: Development of the clinical potential of albicidins as potent bactericidial antibiotics against diverse bacteria has been limited because of low yields in culture. Expression of albF with recently described albicidin-biosynthesis genes may enable large-scale production. Because albicidins are X. albilineans pathogenicity factors, interference with AlbF function is also an opportunity for control of the associated plant disease.

Live Attenuated Chimeric Yellow Fever Dengue Type 2 (Chimeri Vax -DEN2) Vaccine: Phase 1 clinical trial for safety and immunogenicity

A randomized double-blind Phase I Trial was conducted to evaluate safety, tolerability, and immunogenicity of a yellow fever (YF)-dengue 2 (DEN2) chimera (ChimeriVax™-DEN2) in comparison to that of YF vaccine (YF-VAX®). Forty-two healthy YF naïve adults randomly received a single dose of either ChimeriVax™-DEN2 (high dose, 5 log plaque forming units [PFU] or low dose, 3 log PFU) or YF-VAXâ by the subcutaneous route (SC). To determine the effect of YF pre-immunity on the ChimeriVaxTM-DEN2 vaccine, 14 subjects previously vaccinated against YF received a high dose of ChimeriVax™-DEN2 as an open-label vaccine. Most adverse events were similar to YF-VAX® and of mild to moderate intensity, with no serious side-effects. One hundred percent and 92.3% of YF naïve subjects inoculated with 5.0 and 3.0 log10 PFU of ChimeriVaxTM-DEN2, respectively, seroconverted to wt DEN2 (strain 16681); 92% of subjects inoculated with YF-VAX® seroconverted to YF 17D virus but none of YF naïve subjects inoculated with ChimeriVax-DEN2 seroconverted to YF 17D virus. Low seroconversion rates to heterologous DEN serotypes 1, 3, and 4 were observed in YF naïve subjects inoculated with either ChimeriVax™-DEN2 or YF-VAX®. In contrast, 100% of YF immune subjects inoculated with ChimeriVax™-DEN2 seroconverted to all 4 DEN serotypes. Surprisingly, levels of neutralizing antibodies to DEN 1, 2, and 3 viruses in YF immune subjects persisted after 1 year. These data demonstrated that 1) the safety and immunogenicity profile of the ChimeriVax™-DEN2 vaccine is consistent with that of YF-VAX®, and 2) pre-immunity to YF virus does not interfere with ChimeriVaxTM-DEN2 immunization, but induces a long lasting and cross neutralizing antibody response to all 4 DEN serotypes. The latter observation can have practical implications toward development of a dengue vaccine.