Investigation of Cysteine Modifications in Recombinant Protein Tetanus Toxoid Heavy Chain Fragment C.

For conjugated HIV-1 fusion peptide vaccine development, recombinant Tetanus toxoid heavy chain fragment C (rTTHC) was applied as a carrier protein to boost peptide immunogenicity. Understanding the characteristics of rTTHC is the first step prior to the peptide conjugation. A comprehensive mass spectrometry (MS) characterization was performed on E. coli expressed rTTHC during its purification process. Intact mass along with peptide mapping analysis discovered the existence of three cysteine modification forms: glutathionylation, trisulfide bond modification, and disulfide bond shuffling, in correlation to a three-peak profile during a hydrophobic interaction chromatography (HIC) purification step. Coexistence of these multiple oxidative forms indicated that the active thiols underwent redox reaction in the rTTHC material. Identity confirmation of the rTTHC carrier protein by MS analysis provided pivotal guidance to assess the purification step and helped ensure that vaccine development could proceed.

Improving Analytical Characterization of Glycoconjugate Vaccines through Combined High-Resolution MS and NMR: Application to Neisseria meningitidis Serogroup B Oligosaccharide-Peptide Glycoconjugates.

Conjugate vaccines are highly heterogeneous in terms of glycosylation sites and linked oligosaccharide length. Therefore, the characterization of conjugate vaccines' glycosylation state is challenging. However, improved product characterization can lead to enhancements in product control and product quality. Here, we present a synergistic combination of high-resolution mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) for the analysis of glycoconjugates. We use the power of this strategy to characterize model polysaccharide conjugates and to demonstrate a detailed level of glycoproteomic analysis. These are first steps on model compounds that will help untangle the details of complex product characterization in conjugate vaccines. Ultimately, this strategy can be applied to enhance the characterization of polysaccharide conjugate vaccines. In this study, we lay the groundwork for the analysis of conjugate vaccines. To begin this effort, oligosaccharide-peptide conjugates were synthesized by periodate oxidation of an oligosaccharide of a defined length, α,2-8 sialic acid trimer, followed by a reductive amination, and linking the trimer to an immunogenic peptide from tetanus toxoid. Combined mass spectrometry and nuclear magnetic resonance were used to monitor each reaction and conjugation products. Complete NMR peak assignment and detailed MS information on oxidized oligosialic acid and conjugates are reported. These studies provide a deeper understanding of the conjugation chemistry process and products, which can lead to a better controlled production process.

Mechanisms of Antigen Adsorption Onto an Aluminum-Hydroxide Adjuvant Evaluated by High-Throughput Screening.

The adsorption mechanism of antigen on aluminum adjuvant can affect antigen elution at the injection site and hence the immune response. Our aim was to evaluate adsorption onto aluminum hydroxide (AH) by ligand exchange and electrostatic interactions of model proteins and antigens, bovine serum albumin (BSA), ß-casein, ovalbumin (OVA), hepatitis B surface antigen, and tetanus toxin (TT). A high-throughput screening platform was developed to measure adsorption isotherms in the presence of electrolytes and ligand exchange by a fluorescence-spectroscopy method that detects the catalysis of 6,8-difluoro-4-methylumbelliferyl phosphate by free hydroxyl groups on AH. BSA adsorption depended on predominant electrostatic interactions. Ligand exchange contributes to the adsorption of ß-casein, OVA, hepatitis B surface antigen, and TT onto AH. Based on relative surface phosphophilicity and adsorption isotherms in the presence of phosphate and fluoride, the capacities of the proteins to interact with AH by ligand exchange followed the trend: OVA < ß-casein < BSA < TT. This could be explained by both the content of ligands available in the protein structure for ligand exchange and the antigen's molecular weight. The high-throughput screening platform can be used to better understand the contributions of ligand exchange and electrostatic attractions governing the interactions between an antigen adsorbed onto aluminum-containing adjuvant.

A novel DNA vaccine targeting macrophage migration inhibitory factor protects joints from inflammation and destruction in murine models of arthritis.

OBJECTIVE: Previous studies have demonstrated that neutralization of macrophage migration inhibitory factor (MIF) by anti-MIF antibodies decreases joint inflammation and destruction in a type II collagen-induced arthritis model in mice. The aim of this study was to develop and describe a simple and effective method of active immunization that induces anti-MIF autoantibodies, which may neutralize MIF bioactivity. METHODS: We developed a MIF DNA vaccine by introducing oligonucleotides encoding a tetanus toxoid (TTX) Th cell epitope into the complementary DNA sequence of murine MIF. Mice were injected with this construct in conjunction with electroporation. The ability of this immunization to inhibit the development of collagen antibody-induced arthritis (CAIA) in BALB/c mice and spontaneous autoimmune arthritis in interleukin-1 receptor antagonist (IL-1Ra)-deficient mice was then evaluated. RESULTS: Mice that received the MIF/TTX DNA vaccine developed high titers of autoantibodies that reacted to native MIF. Compared with unvaccinated mice, vaccinated mice also produced less serum tumor necrosis factor alpha after receiving an intravenous injection of lipopolysaccharide. In addition, vaccination with MIF/TTX DNA resulted in significant amelioration of both CAIA in BALB/c mice and symptoms of autoimmune arthritis in IL-1Ra-knockout mice. CONCLUSION: These results suggest that MIF/TTX DNA vaccination may be useful for ameliorating the symptoms of rheumatoid arthritis.

Pertusis en lactantes, niños y adolescentes: diagnóstico, tratamiento y prevención / Whooping Cough in infants, children, and adolescents: diagnosis, treatment, and prevention

La vacuna celular contra pertusis, asociada a toxoide tetánico y diftérico (DPT) ha disminuido sustancialmente la enfermedad. El alto porcentajede reacciones adversas: reacción local, fiebre y síntomas sistémicos, han dado origen al desarrollo de una vacuna menos reactogénica llamada vacuna antipertúsica acelular (DaPT), cuya eficacia es comparable a la celular, aunque la duración de la protección después de la inmunización, no ha sido establecida definitivamente. La DaPT se aplica a los 2, 4 y 6 meses, un refuerzo entre los 15 y 18 meses y uno último entre 4 y 6 años de edad. A partir del año 2005 se licenció la vacuna Tdap para adultos y adolescentes entre 11 y 18 años.

Diagnosis of tetanus immunization status: multicenter assessment of a rapid biological test.

Diagnosis of tetanus immunization status by medical interview of patients with wounds is poor. Many protected patients receive unnecessary vaccine or immunoglobulin, and unprotected patients may receive nothing. The aim of this study is to evaluate the feasibility and accuracy of the Tetanos Quick Stick (TQS) rapid finger prick stick test in the emergency department for determining immunization status. We designed a prospective multicenter study for blinded comparison of TQS with an enzyme-linked immunosorbent assay (ELISA). Adults referred for open wounds in 37 French hospital emergency departments had the TQS after receiving standard care (emergency-TQS). TQS was also performed in the hospital laboratory on total blood (blood/lab-TQS) and serum (serum/lab-TQS). ELISA was performed with the same blood sample at a central laboratory. We assessed concordance between emergency-TQS and blood/lab-TQS by the kappa test and the diagnostic accuracy (likelihood ratios) of medical interview, emergency-TQS, and lab-TQS. ELISA was positive in 94.6% of the 988 patients included. Concordance between blood/emergency-TQS and blood/lab-TQS results was moderate (kappa=0.6), with a high proportion of inconclusive blood/emergency-TQS tests (9.8%). Likelihood ratios for immunization were 3.0 (95% confidence interval [CI], 1.8 to 5.1), 36.6 (95% CI, 5.3 to 255.3), 89.1 (95% CI, 5.6 to 1,405.0), and 92.7 (95% CI, 5.9 to 1,462.0) for medical interview, blood/emergency-TQS, blood/lab-TQS, and serum/lab-TQS, respectively. The sensitivity of the blood/emergency-TQS was 76.7%, and the specificity was 98% by reference to the ELISA. TQS use in the emergency room could make tetanus prevention more accurate if its technical feasibility were improved, and our assessment will be supplemented by a cost effectiveness study.

Vacunas adsorbidas: ¡no congelar! / Vaccines inactived: ¡not to freeze!

Las vacunas inactivadas son vacunas a base de gérmenes muertos, enteros o fraccionados, que generalmente requieren la presencia de un agente adyuvante que adsorbe los antígenos y los libera lentamente para garantizar una respuesta inmunológica adecuada. Los adyuvantes más utilizados son las sales de aluminio como el hidróxido y el fosfato de aluminio, de los cuales se conoce su eficacia y seguridad. Sin embargo, su presencia es indicativo de que la vacuna deberá ser conservada a una temperatura de entre 2 y 8° C y evitar su congelación, ya que pierden su estructura coloidal, cristalizando, lo cual ocasiona además de la pérdida de potencia de la vacuna, severas reacciones locales. En este trabajo se evaluó la relación que existe entre el tiempo de congelación, el aspecto macro y microscópico de diferentes tipos de vacunas adsorbidas y la posibilidad de determinar a simple vista si la vacuna sufrió congelación. Los resultados señalan que no se puede establecer relación entre la velocidad de congelación, el tipo de vacuna, envase en que se encuentra y la aparición de cristales y partículas. Todas las muestras desarrollaron cristalización en apenas 1 hora de estar sometidas a temperatura de -20°C, aun cuando la mayoría se encontraba en estado líquido y sin presencia de partículas visibles a simple vista. La cristalización fue fácilmente observada en microscopio óptico, siendo una forma rápida y confiable para determinar si una vacuna adsorbida sufrió congelación y evitar así su administración

Monitoring of diphtheria, pertussis and tetanus toxoids by circular dichroism, fluorescence spectroscopy and size-exclusion chromatography.

A combination of spectroscopic and chromatographic methods has been used to monitor the quality and integrity of diphtheria, pertussis and tetanus toxoids (DTxd, PTxd and TTxd) which have been prepared from the toxins by formaldehyde treatment. Different processes for detoxifying all three toxins have yielded toxoids varying in their molecular size, including oligomers (associated monomers) and aggregates (high molecular weight complexes of non-specifically associated monomers). Changes in the intrinsic fluorescence spectra of the polypeptides have been observed in some sized fractions of DTxd and PTxd. Some physico-chemical changes have been observed to correlate with a loss of antigenicity. Spectroscopic and chromatographic methods are useful not only in monitoring the stability and consistency of vaccine starting materials, but can also be used to dissect heterogeneous toxoid preparations.

Profilaxis Antitetánica Posterior a Trauma

El tétanos es una enfermedad grave con alta mortalidad, causada por una toxina producida por el bacilo Gram positivo anaerobio Clostridium tetani. Se produce cuando las esporas que habitan en el medio ambiente penetran en el organismo por cualquier herida, permitiendo que estas eclosionen, proceso en el que se genera la tetanospasmina, sustancia proteica responsable del cuadro clínico. Este se produce por inhibición de la sinaptobrevina II, metaloproteína enzimática que participa en el proceso de degranulación de los axones inhibitorios motores. Esta inhibición es irreversible y solo desaparece con la generación de un nuevo axón. La prevención es posible con la práctica de inmunización previa y sistemática de la población con toxoide tetánico y la aplicación de antitoxina tetánica en los casos en los que el estado inmune del paciente es incierto, o las heridas sean potencialmente tetanógenas

Estabelecimento da concentracao antimicrobiana eficaz de tiomersal em toxoide tetanico / Determination of effective antimicrobial concentration of thimerosal in tetanus toxoid

Amostras de taxoide tetanico, contendo quatro concentracoes de tiomersal (0,005; 0,010; 0,015 e 0,020%) dentro dos limites recomendados oficialmente,foram submetidos ao teste de eficacia antimicrobiana do conservante. Com relacao as exigencias da Farmacopeia Americana XXII, apenas Staphylococcus aureus se mostrou resistente as quatro concentracoes, enquanto que para Pseudomonas aeruginosa, Candida albicans e Aspergillus niger o sistema conservador atendeu as exigencias oficiais, mesmo na menor concentracao. O teor de tiomersal em toxoide tetanico podera ser de 0,005%

[A rapid method of measuring the level of aluminum in biological preparations using aluminon]. / Szybka metoda oznaczania glinu w biopreparatach za pomoca aluminonu.

The aim of this study was to adapt aluminon for determination of Al3+ content in biopreparations adsorbed on Al(OH)3. Aluminum + aluminon complex was identified by spectrophotometry at A535. It was found that the method applied allows to obtain reproducible results. Its sensitivity for Al3+ contains between 85 to 680 micrograms. This method is less laborious in comparison with so far used versenian method.

[Physico-chemical studies of aluminum hydroxide in biological preparations preserved in different conditions]. / Ocena fizyko-chemicznego stanu wodorotlenku glinu w biopreparatach przechowywanych w róznych warunkach.

An observation of physico-chemical properties of 0.5% Al(OH)3 and vaccines adsorbed to it (Di, Te, Di-Te, Di-Te-Per) stored for 3-24 months in various pH (5, 7, 8) and temperatures -18 degrees, +37 degrees, +45 degrees and +65 degrees was carried out. For this purpose an analysis of sedimentation rate, microscopic observation and ++roentgenographic analysis were performed. It was found that a decrease of storage temperature of Al(OH)3 gel and vaccines down to -18 degrees C resulted in some changes in structure and morphology of this absorbent (big precipitates) and it led to a significant increase of sedimentation rate.

The aluminum content of biological products containing aluminum adjuvants: determination by atomic absorption spectrometry.

Aluminum compounds are used as adjuvants in certain types of vaccines, toxoids and allergenic extracts for human use. The most common Al compounds used in biological products to enhance the immune response are aluminum potassium sulphate (alum), aluminum hydroxide and aluminum phosphate. This study describes an atomic absorption spectrometric method for the determination of the Al content of Al adsorbed toxoid preparations and allergenic extracts at levels of less than 0.85 mg of Al per half millilitre human dose. Aliquots of the samples which contained Al suspensions were acid digested with nitric and sulphuric acid and analysed in the nitrous oxide-acetylene flame of an atomic absorption spectrometer. The 396.2 nm Al line was used for analysis. The Al content of the National Bureau of Standards (NBS) Standard Reference Material No. 1075a aluminum 2- ethylhexanoate was determined to within 1% of the NBS certificate value by this method. Atomic absorption results for the Al content of tetanus toxoids containing aluminum potassium sulphate and aluminum phosphate were compared with polarographic and inductively coupled argon plasma (ICP) emission spectrometry results. Reproducibility and recovery data for Al are tabulated for a variety of biological products containing aluminum phosphate, aluminum potassium sulphate and aluminum hydroxide adjuvants. In addition, ICP has been used to characterize the Al and P compositions of the precipitates and supernatant solutions which resulted from centrifuging toxoid suspensions that contained the three different Al adjuvants.

Tetanus toxin and antigenic derivatives. II. Effect of protein and formaldehyde concentration on toxoid formation.

Tetanus toxoid formation was examined under varying conditions. Products of the reaction depended upon the concentration of both formaldehyde and toxin. High concentrations of protein and formaldehyde favored the formation of large polymers, whereas low concentrations yielded smaller polymers and monomer. The monomer had an observed S value of 7.1, whereas the polymers ranged from 10.1S to 110S. The cross-linking between toxin molecules to form toxoid polymers appeared to be random.