Development of a Specific and Sensitive HPAEC-PAD Method for Quantification of Vi Polysaccharide Applicable to other Polysaccharides Containing Amino Uronic Acids.

Typhoid fever is a major cause of morbidity and mortality in developing countries. Vaccines based on the Vi capsular polysaccharide are licensed or in development against typhoid fever. Vi content is a critical quality attribute for vaccines release, to monitor their stability and to ensure appropriate immune response. Vi polysaccharide is a homopolymer of α-1,4-N-acetylgalactosaminouronic acid, O-acetylated at the C-3 position, resistant to the commonly used acid hydrolysis for sugar chain depolymerization before monomer quantification. We previously developed a quantification method based on strong alkaline hydrolysis followed by High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection analysis, but with low sensitivity and use for quantification of an unknown product coming from polysaccharide depolymerization. Here we describe the development of a method for Vi polysaccharide quantification based on acid hydrolysis with concomitant use of trifluoroacetic and hydrochloric acids. A Design of Experiment approach was used for the identification of the optimal hydrolysis conditions. The method is 100-fold more sensitive than the previous one, and specifically, resulting in the formation of a known product, confirmed to be the Vi monomer both de-O- and de-N-acetylated by mono- and bidimensional Nuclear Magnetic Resonance spectroscopy and mass spectrometry. Accuracy and precision were determined, and chromatographic conditions were improved to result in reduced time of analysis. This method will facilitate characterization of Vi-based vaccines. Furthermore, a similar approach has the potential to be extended to other polysaccharides containing 2-amino uronic acids, as already verified here for Shigella sonnei O-antigen, Streptococcus pneumoniae serotype 12F, and Staphylococcus aureus types 5 and 8 capsular polysaccharides.

Determination of free polysaccharide in Vi glycoconjugate vaccine against typhoid fever.

Glycoconjugate vaccines based on the Vi capsular polysaccharide directed against Salmonella enterica serovar Typhi are licensed or in development against typhoid fever, an important cause of morbidity and mortality in developing countries. Quantification of free polysaccharide in conjugate vaccines is an important quality control for release, to monitor vaccine stability and to ensure appropriate immune response. However, we found that existing separation methods based on size are not appropriate as free Vi non-specifically binds to unconjugated and conjugated protein. We developed a method based on free Vi separation by Capto Adhere resin and quantification by HPAEC-PAD. The method has been tested for conjugates of Vi derived from Citrobacter freundii with different carrier proteins such as CRM197, Tetanus Toxoid and Diphtheria Toxoid.

Diphtheria Antibodies and T lymphocyte Counts in Patients Infected with HIV-1

We assessed the IgG levels anti-diphtheria (D-Ab) and T cell counts (CD4+ and CD8+) in HIV-1 infected subjects undergoing or not highly active antiretroviral therapy (HAART). Approximately 70% of all HIV-1 patients were unprotected against diphtheria. There were no differences in D-Ab according to CD4 counts. Untreated patients had higher D-Ab (geometric mean of 0.62 IU/ml) than HAART-patients (geometric mean of 0.39 IU/ml). The data indicated the necessity of keeping all HIV-1 patients up-to-date with their vaccination.

Vacunación contra la fiebre tifoidea / Vaccination against typhoid fever

La fiebre tifoidea es una enfermedad infecciosa aguda y febril causada por Salmonella typhi. La infección se adquiere por medio de la ingestión de alimentos o agua masivamente contaminados con la bacteria. Debido a que este microorganismo afecta sólo al ser humano y no hay reservorios animales, el elemento más importante en la cadena de transmisión son los individuos que no presentan sintomatología clínica pero son portadores y excretores activos del agente (portadores asintomáticos). En México, la enfermedad tiene características endémico-epidémicas relacionadas con deficiencias en el saneamiento ambiental y el aprovisionamiento de agua potable. La tasa de letalidad es de alrededor del 1 por ciento, sin embargo en los casos complicados puede ascender hasta el 30 por ciento. La vacuna que se utiliza más ampliamente en la actualidad es la inactivada por calor y fenol, la cual tiene efectos colaterales debido al LPS que siempre lo contamina. Además, confiere protección parcial y de corta duración, por lo tanto su empleo se ha limitado a grupos considerados como de alto riesgo y no está indicada en niños. La vacuna de Germanier -mutante avirulenta de Salmonella que se administra por vía oral- proteje hasta un 95 por ciento, siempre y cuando antes de administrarse se neutralice el jugo gástrico, aunque su utilización general en Salud Pública es impráctica. Ya que las porinas son buenos inmunógenos en modelos animales, la obtención de un conjugado antígenico Vi-porinas puede ser una excelente opción como vacuna contra la fiebre tifoidea

[Characteristics of preparations of typhoid antigens obtained by ultrafiltration]. / Kharakteristika preparatov briushno-tifoznykh antigenov, poluchaemykh s primeneniem ul'trafiltratsii.

The properties of the preparations of the total typhoid antigens isolated by various methods, purified and concentrated by ultrafiltrations, were studied, and the technique of the preservation of their high solubility on drying (the neutralization and removal of disperse components prior to drying) was developed. Preparations obtained by ultracentrifugation were characterized by lower ash content and, when compared with alcohol tryptic preparations, had better antigenic properties, inducing the synthesis of not only O- and Vi-, but also H-antibodies in high titers.

[Parathyphoid B-antigen containing a complex of protective surface antigens]. / Issledovanie paratifoznogo B-antigena, soderzhashchego kompleks protektivnykh poverkhnostnykh antigenov.

The authors obtained a complex antigen from paratyphoid B bacilli containing complete O-, K- and H-antigens. The preparation was nontoxic and was characterised by marked antigenic properties. In intravenous and oral administration it stimulated production of specific O-, K-, and H-antibodies in high titres. Complex paratyphoid B antigen possessed a marked protective activity and provided intense immunity in subcutaneous and oral administration to experimental animals.

[The presence of a hypothetical extracellular fraction in integral typhoid vaccines prepared on a semi-synthetic medium (author's transl)]. / La presenza di una ipotetica frazione extracellulare in vaccini antitifici allestiti su terreno semisintetico.

The Authors have carried out a research on the purpose of explain the action of a typhoid vaccines prepared on a semi-synthetic medium, which in preliminary tests on mice and men evinced good immunizing power. The tests now performed demonstrate that an acellular filtrate of the vaccine (obtained from 24 h old culture) showed have the characteristics of an exotoxin and developes a strong immunizing power.

[Design of new types of chemical vaccines for the prevention of intestinal infections and problems of their qualitative evaluation. Report 2. Immunochemical characteristics of the experimental preparations]. / Konstruirovanie novykh tipov khimicheskikh vaktsin dlia profilaktiki kishechnykh infektsii i voprosy ikh kachestvennoi otsenki. Soobshchenie 2. Immunokhimicheskaia kharakteristika éksperimental'nykh preparatov

A study was made of chemical composition and immunochemical properties of 36 samples of the antigenic preparations from typhoid, paratyphoid (A and B) and dysentery (Flexner and Sonne) cultures obtained at the Leningrad, Moscow and Tashkent Institutes of Vaccine and Sera by various methods. The preparations isolated by the method of tryptic proteolysis had a polysaccharide-protein nature, and preparations isolated by the action of chemical reagents (hydroxylamine, hydrogen peroxide, detergents) -- a protein-polysaccharide one. The former contained more nucleic acids, this indicating the presence of components of cytoplasmic and nuclear origin. The preparations were characterized by molecular heterogeneity and contained high- and low-molecular components. The preparations had complex antigenic spectra by the immunodiffusion data; the preparations obtained had complex antigenic spectra by the immunodiffusion data; the preparations obtained had complex antigenic spectra by the immunodiffusion data; the preparations obtained by different method displayed identity reactions, and group specificity was expressed in some of them.