Preclinical studies on new proteins as carrier for glycoconjugate vaccines.

Glycoconjugate vaccines are made of carbohydrate antigens covalently bound to a carrier protein to enhance their immunogenicity. Among the different carrier proteins tested in preclinical and clinical studies, five have been used so far for licensed vaccines: Diphtheria and Tetanus toxoids, the non-toxic mutant of diphtheria toxin CRM197, the outer membrane protein complex of Neisseria meningitidis serogroup B and the Protein D derived from non-typeable Haemophilus influenzae. Availability of novel carriers might help to overcome immune interference in multi-valent vaccines containing several polysaccharide-conjugate antigens, and also to develop vaccines which target both protein as well saccharide epitopes of the same pathogen. Accordingly we have conducted a study to identify new potential carrier proteins. Twenty-eight proteins, derived from different bacteria, were conjugated to the model polysaccharide Laminarin and tested in mice for their ability in inducing antibodies against the carbohydrate antigen and eight of them were subsequently tested as carrier for serogroup meningococcal C oligosaccharides. Four out of these eight were able to elicit in mice satisfactory anti meningococcal serogroup C titers. Based on immunological evaluation, the Streptococcus pneumoniae protein spr96/2021 was successfully evaluated as carrier for serogroups A, C, W, Y and X meningococcal capsular saccharides.

Carrier priming with CRM 197 or diphtheria toxoid has a different impact on the immunogenicity of the respective glycoconjugates: biophysical and immunochemical interpretation.

Glycoconjugate vaccines play an enormous role in preventing infectious diseases. The main carrier proteins used in commercial conjugate vaccines are the non-toxic mutant of diphtheria toxin (CRM197), diphtheria toxoid (DT) and tetanus toxoid (TT). Modern childhood routine vaccination schedules include the administration of several vaccines simultaneously or in close sequence, increasing the concern that the repeated exposure to conjugates based on these carrier proteins might interfere with the anti-polysaccharide response. Extending previous observations we show here that priming mice with CRM197 or DT does not suppress the response to the carbohydrate moiety of CRM197 meningococcal serogroup A (MenA) conjugates, while priming with DT can suppress the response to DT-MenA conjugates. To explain these findings we made use of biophysical and immunochemical techniques applied mainly to MenA conjugates. Differential scanning calorimetry and circular dichroism data revealed that the CRM197 structure was altered by the chemical conjugation, while DT and the formaldehyde-treated form of CRM197 were less impacted, depending on the degree of glycosylation. Investigating the binding and avidity properties of IgGs induced in mice by non-conjugated carriers, we found that CRM197 induced low levels of anti-carrier antibodies, with decreased avidity for its MenA conjugates and poor binding to DT and respective MenA conjugates. In contrast, DT induced high antibody titers able to bind with comparable avidity both the protein and its conjugates but showing very low avidity for CRM197 and related conjugates. The low intrinsic immunogenicity of CRM197 as compared to DT, the structural modifications induced by glycoconjugation and detoxification processes, resulting in conformational changes in CRM197 and DT epitopes with consequent alteration of the antibody recognition and avidity, might explain the different behavior of CRM197 and DT in a carrier priming context.

Comparison of CRM197, diphtheria toxoid and tetanus toxoid as protein carriers for meningococcal glycoconjugate vaccines.

Glycoconjugate vaccines are among the most effective and safest vaccines ever developed. Diphtheria toxoid (DT), tetanus toxoid (TT) and CRM197 have been mostly used as protein carriers in licensed vaccines. We evaluated the immunogenicity of serogroup A, C, W-135 and Y meningococcal oligosaccharides conjugated to CRM197, DT and TT in naïve mice. The three carriers were equally efficient in inducing an immune response against the carbohydrate moiety in immunologically naïve mice. The effect of previous exposure to different dosages of the carrier protein on the anti-carbohydrate response was studied using serogroup A meningococcal (MenA) saccharide conjugates as a model. CRM197 showed a strong propensity to positively prime the anti-carbohydrate response elicited by its conjugates or those with the antigenically related carrier DT. Conversely in any of the tested conditions TT priming did not result in enhancement of the anti-carbohydrate response elicited by the corresponding conjugates. Repeated exposure of mice to TT or to CRM197 before immunization with the respective MenA conjugates resulted in a drastic suppression of the anti-carbohydrate response in the case of TT conjugate and only in a slight reduction in the case of CRM197. The effect of carrier priming on the anti-MenA response of DT-based conjugates varied depending on their carbohydrate to protein ratio. These data may have implications for human vaccination since conjugate vaccines are widely used in individuals previously immunized with DT and TT carrier proteins.

Meningococcal X polysaccharide quantification by high-performance anion-exchange chromatography using synthetic N-acetylglucosamine-4-phosphate as standard.

A method for meningococcal X (MenX) polysaccharide quantification by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is described. The polysaccharide is hydrolyzed by strong acidic treatment, and the peak of glucosamine-4-phosphate (4P-GlcN) is detected and measured after chromatography. In the selected conditions of hydrolysis, 4P-GlcN is the prevalent species formed, with GlcN detected for less than 5% in moles. As standard for the analysis, the monomeric unit of MenX polysaccharide, N-acetylglucosamine-4-phosphate (4P-GlcNAc), was used. This method for MenX quantification is highly selective and sensitive, and it constitutes an important analytical tool for the development of a conjugate vaccine against MenX.

Differentiation of Staphylococcus aureus and Staphylococcus epidermidis by PCR for the fibrinogen binding protein gene.

Mastitis is one of the most common and burdensome diseases afflicting dairy animals. Among other causes of mastitis, staphylococci are frequently associated with clinical and subclinical mastitis. Although Staphylococcus aureus is the predominant species involved, Staphylococcus epidermidis and other coagulase-negative staphylococci are increasingly being isolated from cases of bovine mastitis. Although Staph. aureus and Staph. epidermidis can be easily differentiated based on their biochemical properties, such phenotypic identification is time consuming and laborious. This study aimed to rapidly identify Staph. aureus and Staph. epidermidis. Accordingly, a multiplex PCR was developed and we found that a single gene encoding the adhesin fibrinogen binding protein could be used to identify and differentiate the two species. Consequently, a multiplex reaction combining a triplex PCR for Staph. aureus and a duplex PCR for Staph. epidermidis was standardized, first using bacterial cultures and then with pasteurized milk spiked with live organisms or DNA extracted from the organisms. The test could specifically detect Staph. aureus and Staph. epidermidis even in the presence of a dozen other organisms. The limit of detection for detecting Staph. aureus and Staph. epidermidis separately was 10 to 100 cfu/mL for simplex PCR and 10(4)cfu/mL for multiplex PCR. Conversely, the limit was 10(6)cfu/mL by multiplex PCR for simultaneous detection of both the organisms when spiked into culture medium or pasteurized milk. Overnight enrichment enhanced the assay sensitivity 100-fold. The assay had a high diagnostic sensitivity and specificity. The application of the test was verified on 602 field isolates of staphylococci that had been characterized earlier by phenotypic methods. Importantly, 25 coagulase-negative isolates were identified as Staph. aureus by the multiplex PCR. The test could be adapted for use in clinical diagnostic laboratories.

Relative stability of meningococcal serogroup A and X polysaccharides.

Prior to the introduction of the MenAfriVac™ serogroup A glycoconjugate vaccine in September 2010, serogroup A was the major epidemic disease-causing meningococcal serogroup in the African meningitis belt. However, recently serogroup X meningococcal (MenX) disease has received increased attention because of outbreaks recorded in this region, with increased endemic levels of MenX disease over the past 2 years. Whereas polysaccharide-protein conjugate vaccines against meningococcal serogroups A, C, W and Y (MenA, MenC, MenW, MenY) are on the market, a vaccine able to protect against MenX has never been achieved. The structure of serogroup A, C, W and Y meningococcal polysaccharides has been already fully elucidated by NMR. MenX capsular polysaccharide (MenX CPS) structure is also documented but fewer characterization data have been published. We have applied here (1)H NMR, (31)P NMR and HPLC to evaluate the stability of MenX CPS in aqueous solution as compared to MenA capsular polysaccharide (MenA CPS). The stability study demonstrated that MenA CPS is more susceptible to hydrolytic degradation than MenX CPS. The different stereochemistry of the N-acetyl group at position C(2) of mannosamine (MenA CPS) and glucosamine (MenX CPS) respectively might play a fundamental role in this susceptibility to polysaccharide chain degradation. The satisfactory stability of MenX CPS predicts the possibility that a stable fully-liquid MenX polysaccharide or glycoconjugate vaccine could be developed.

Two SERK genes are markers of pluripotency in Cyclamen persicum Mill.

The genetic basis of stem cell specification in somatic embryogenesis and organogenesis is still obscure. SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes are involved in embryogenesis and organogenesis in numerous species. In vitro culture of Cyclamen persicum immature ovules provides a system for investigating stem cell formation and maintenance, because lines forming either organs or embryos or callus without organs/embryos are available for the same cultivar and plant growth regulator conditions. The present aim was to exploit this property of cyclamen cultures to understand the role of SERK(s) in stem cell formation and maintenance in somatic embryogenesis and organogenesis in vitro, in comparison with expression in planta. CpSERK1 and CpSERK2 were isolated from embryogenic callus. CpSERK1 and CpSERK2 levels by RT-PCR showed that expression is high in embryogenic, moderate in organogenic, and null in recalcitrant calli. in situ hybridizations showed that the expression of both genes started in clumps of pluripotent stem cells, from which both pre-embryogenic aggregates and organ meristemoids derived, and continued in their trans-amplifying, meristem-like, derivatives. Expression declined in organ meristemoids, in parallel with a partial loss of meristematization. In mature somatic embryos, and in shoot and root primordia, CpSERK1 and CpSERK2 were expressed in meristems, and similar patterns occurred in zygotic embryo and primary meristems in planta. The results point to SERK1 and SERK2 as markers of pluripotency in cyclamen. It is proposed that the high expression of these genes in the trans-amplifying derivatives of the stem cells maintains a pluripotent condition leading to totipotency and, consequently, somatic embryogenesis.

Production of a conjugate vaccine for Salmonella enterica serovar Typhi from Citrobacter Vi.

A conjugate vaccine for Salmonella enterica serovar Typhi was produced by chemically linking Vi, purified from Citrobacter, to the non-toxic mutant diphtheria toxin CRM(197) via an adipic dihydrazide spacer using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide coupling chemistry. The polysaccharide purification process was developed based on Vi precipitation from culture supernatant with cetyl trimethylammonium bromide (CTAB), solubilization of the CTA-polysaccharide salt with ethanol followed by exchange of the CTA(+) counter ion with Na(+). The purified Vi polysaccharide was fully O-acetylated and with high purity. The conjugation process was optimized to obtain a scalable process that has been used for GMP production at pilot scale of vaccine currently in clinical trials.

Vi-CRM 197 as a new conjugate vaccine against Salmonella Typhi.

An efficacious, low cost vaccine against typhoid fever, especially for young children, would make a major impact on disease burden in developing countries. The virulence capsular polysaccharide of Salmonella Typhi (Vi) coupled to recombinant mutant Pseudomonas aeruginosa exoprotein A (Vi-rEPA) has been shown to be highly efficacious. We investigated the use of carrier proteins included in infant vaccines, standardized the conjugation process and developed key assays required for routine lot release at production scale. Vi from a BSL1 organism, Citrobacter freundii, strain WR7011, was used as an alternative to Vi from S. Typhi. We showed that Vi conjugated to CRM(197), a non-toxic mutant of diphtheria toxin, widely used in commercial vaccines, was produced at high yield. Vi-CRM(197) proved immunogenic in animal studies, even without adjuvant. Thus, Vi-CRM(197) appears to be a suitable candidate for the development of a commercially viable, effective typhoid vaccine for developing countries.

Rationally designed strings of promiscuous CD4(+) T cell epitopes provide help to Haemophilus influenzae type b oligosaccharide: a model for new conjugate vaccines.

The age-related and T cell-independent immunological properties of most capsular polysaccharides limit their use as vaccines, especially in children under 2 years of age. To overcome these limitations, polysaccharide antigens have been successfully conjugated to a variety of carrier proteins, such as diphtheria toxoid or tetanus toxoid (TT) and the diphtheria mutant (CRM197) to produce very successful glycoconjugate vaccines. The increasing demand for new conjugate vaccines requires the availability of additional carriers providing high and long-lasting T helper cell immunity. Here we describe the design and construction of three recombinant carrier proteins (N6, N10, N19) constituted by strings of 6, 10 or 19 human CD4(+) T cell epitopes from various pathogen-derived antigens, including TT and proteins from Plasmodium falciparum, influenza virus and hepatitis B virus. Each of these epitopes is defined as universal in that it binds to many human MHC class II molecules. When conjugated to Haemophilus influenzae type b (Hib) oligosaccharide, these carriers elicit a potent anti-Hib antibody response in mice. In the case of the N19-Hib conjugate, this response is at least as good as that observed with CRM197-Hib, a conjugate vaccine currently used for mass immunization. We also show that some of the universal epitopes constituting the recombinant carriers are specifically recognized by two human in vitro systems, suggesting that T cell memory is provided by the selected epitopes. The data indicate that rationally designed recombinant polyepitope proteins represent excellent candidates for the development and clinical testing of new conjugate vaccines.

The plant oncogene rolD encodes a functional ornithine cyclodeaminase.

The plant oncogene rolD stimulates the reproductive phase transition in plants. We define here the function of its gene product. We show that the RolD protein bears sequence homology with ornithine cyclodeaminase, an uncommon enzyme of specialized-niche eubacteria and archaea that catalyzes the unusual NAD(+)-dependent conversion of ornithine to proline. To confirm the prediction of the bioinformatic analysis, the RolD protein was expressed in Escherichia coli and purified. An ornithine-dependent NAD(+) reduction that can be ascribed only to ornithine cyclodeaminase (OCD) activity was detected both in bacterial extracts containing RolD and in assays on the purified RolD protein. Furthermore, OCD activity was observed in soluble extracts from plants overexpressing rolD. The role of rolD in plant pathogenesis and its effect on plant reproductive development are discussed in light of the newly demonstrated enzymatic activity of its gene product.

Tympanic membrane repair with a dermal allograft.

OBJECTIVES: To assess the use of an acellular dermal allograft in the repair of chronic tympanic membrane perforations. Chronic tympanic membrane perforations are a common problem in otolaryngology, and although surgical tympanoplasty using either temporalis fascia or rice paper has proven to be highly successful, these materials are not without their own limitations. The search has continued for a simpler, yet equally effective, means of repairing persistent tympanic membrane perforations in an office setting. In this study we experimentally evaluated the use of an acellular dermis (AlloDerm, (LifeCell Corporation, The Woodlands, TX) as an alternative to traditional tympanoplasty materials. STUDY DESIGN: Prospective study using 28 adult chinchillas. METHODS: Subtotal tympanic membrane perforations were created bilaterally in 28 adult chinchillas. Animals with noninfected, stable perforations that showed no signs of epithelial regeneration after 5 to 8 weeks were used to compare the use of rice paper patch with AlloDerm in patch tympanoplasties. RESULTS: Eighteen of 23 tympanoplasties (78%) that were performed using AlloDerm showed no signs of perforation after 5 to 6 weeks. In those performed using rice paper control, 14 of 21 (66%) showed no signs of perforation after 5 to 6 weeks. In addition, histological evaluation of the healed tympanic membranes demonstrated that the acellular dermis had been incorporated within the middle fibrous layer of the tympanic membrane. CONCLUSIONS: The results and histological studies suggest that acellular dermis may be a suitable alternative to traditional materials currently used for patch tympanoplasty. Future studies to evaluate the efficacy of acellular dermis in humans are warranted.

Sphenoethmoid cerebrospinal fluid leak repair with hydroxyapatite cement.

Despite advances in neurological, reconstructive, and endoscopic sinus surgery, sphenoethmoid cerebrospinal fluid (CSF) fistulae continually pose difficult management problems. Standard surgical techniques for fistulae closure succeed approximately 78% to 90% of the time. To improve this success rate, hydroxyapatite cement (HAC), a Food and Drug Administration-approved substance for cranial defect repair, was applied to this problem in a clinical setting. Twenty-one patients with spontaneous, posttraumatic, or postoperative CSF leaks of the sphenoid sinus, cribriform plate, or ethmoid region were treated with HAC. Study participants were prospectively accrued at 5 tertiary care medical centers in the eastern United States. The CSF leaks of all 21 patients treated with HAC were successfully sealed by its initial application. The sites of CSF leakage included the nasal cavity (n = 2) and sphenoid sinus (n = 19). Fifteen of the patients had previously undergone a failed repair by standard methods. There have been no recurrent CSF leaks with a maximum follow-up of 72 months, and an average follow-up of 36 months. All patients have survived to date. The only HAC-related morbidity was the extrusion of the HAC when placed in the nasal cavity. Hydroxyapatite cement is an effective method of repair for postoperative, posttraumatic, and spontaneous sphenoid CSF leaks. The efficacy of HAC in sealing the CSF leak was unaffected by previous attempts at leak closure by standard methods or by its origin. Hydroxyapatite cement should not be applied transnasally for the treatment of an ethmoid region fistula owing to its high probability of extrusion. Correct patient selection and technical familiarity with HAC are necessary for successful application.

Acellular dermis for facial soft tissue augmentation: preliminary report.

OBJECTIVE: To evaluate the efficacy of acellular dermis as a viable alternative for soft tissue augmentation in facial reconstruction. DESIGN: A prospective, nonrandomized observational study consisting of 10 patients who underwent soft tissue augmentation with acellular dermis. SETTING: A tertiary care university medical center in an urban setting. PATIENTS: Ten patients who had undergone soft tissue augmentation using acellular dermis participated in this study. Postimplantation follow-up was 17 to 36 months. INTERVENTION: The amount and location for placement of the acellular dermis was left to the discretion of the surgeon. All implants were placed in the subdermal tissues. MAIN OUTCOME MEASURES: The adequacy of acellular dermis for soft tissue augmentation was assessed by subjective evaluation of implant volume persistence, postoperative complications, and the restoration of normal contour. RESULTS: Of 10 patients who underwent implantation, 9 had no complications and 1 had a recurrent sterile abscess or mucocele at the implantation site. A 22-month postimplantation tissue sampling of acellular dermis in a patient with recurrent tumor revealed approximately 80% to 85% volume persistence. CONCLUSION: Preliminary experience with acellular dermis indicates that it shows promise in soft tissue augmentation.

Morphological and phase characterizations of retrieved calcium phosphate cement implants.

A self-hardening calcium phosphate cement (CPC), consisting of equimolar amounts of tetracalcium phosphate and dicalcium phosphate anhydrous, hardens when mixed with water and forms a resorbable hydroxyapatite (HA) as the end-product. The objective of this study was to investigate the changes of the phase and morphology of the CPC during hardening and aging under in vivo conditions. CPC samples retrieved 12 h after hardening in vivo had already contained carbonated HA (type B), even though the initial cement mixture did not contain carbonate as one of the solid components. The mass fraction of carbonate in the 12-h sample was about 1%. The results suggested that under in vivo conditions carbonate is readily available and this allows formation of carbonated HA in favor of carbonate-free HA. The carbonate content of the CPC samples retrieved 3 months after implantation was similar to that of the 12-h samples, and the exterior surfaces of the 3-month samples appeared less crystalline than that of the 12-h samples.

Expression of rolB in apomictic Hieracium piloselloides Vill. causes ectopic meristems in planta and changes in ovule formation, where apomixis initiates at higher frequency.

The effects of the Agrobacterium rhizogenes rolB oncogene on apomixis were examined in the facultative apomictic plant Hieracium piloselloides because the oncogene has been shown to alter plant growth, morphogenesis and cellular sensitivity to auxin. Introduction of rolB under the control of either its own promoter or the CaMV35S promoter induced ectopic meristem formation from the inflorescence, confirming in planta a meristem-inducing role for this oncogene previously observed only in tissue culture. These ectopic meristems formed vegetative rosettes and floral plant organs. Upon immersion in water these meristems generated roots, suggesting that meristem commitment towards the generation of a specific organ type is a separate and later event that is dependent upon the developmental context. Ovule identity and form was altered in ectopically induced florets in plants expressing the CaMV35S::rolB construct. In contrast to the ovules of untransformed apomictic plants, the sexual process ceased earlier, prior to meiosis, yet surprisingly, apomixis initiated from a greater number of cells, and embryos and endosperm continued to develop in the structurally altered ovules. The alternative possibilities that the effects on reproduction might result from rolB influencing cellular response to auxin, or from alterations in cell signaling caused by changes in ovule morphology that are induced because of the expression of the oncogene are discussed.