GMP-grade pneumococcal whole-cell vaccine injected subcutaneously protects mice from nasopharyngeal colonization and fatal aspiration-sepsis.

Mucosal immunization with a killed whole-cell pneumococcal vaccine, given with enterotoxin-related adjuvants, has been shown to confer multi-serotype protection against colonization of the nasopharynx and middle ear in mice. However, because novel mucosal immunization strategies may be difficult to implement, here we evaluated subcutaneous injection. Strain RM200 was engineered to be capsule-negative, autolysin-negative, and to express a non-toxic mutant pneumolysoid. Liter-scale and 60-l Good Manufacturing Practice (GMP) cultures were grown in bovine-free soy-based medium, killed with chloroform or beta-propiolactone, and injected into C57Bl/6 mice without or with aluminum adjuvant. The adjuvant Al(OH)(3) strongly increased responses, particularly if pre-treated with phosphate. Protection was found in several tested model infections: nasal colonization with a serotype 6B strain and fatal aspiration-sepsis with strains of serotype 3 and 5. Protection against colonization was mechanistically dependent on the presence of CD4+ T cells at the time of challenge; in contrast, in the type 3 aspiration-sepsis model, CD4+ T cells were not required for protection at the time of challenge, suggesting that antibody alone was sufficient to protect against death in this model. Rabbits receiving sequential intramuscular injections in a pilot toxicity study displayed local reactogenicity at injection sites but no clinical signs. The rabbit antiserum thus produced was active in an in vitro phagocytic killing assay and passively protected mice in the type 3 aspiration-sepsis model. Approval is being sought for human trials of this vaccine.

r-Sm14 - pRSETA efficacy in experimental animals

Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega). The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system

Chromatographic purification of antivenoms and antitoxins

Commercial preparations of antivenoms and antitoxins produced in horses, that are essentially pure F (ab)2 immunoglobulin preparations, were submitted to ion-exchange chromatography. For anticrotalic, anti-bothropic and anti-tetanic sera it is possible to remove 40-60% of the inactive globulins

The possible role of para-pepsin in the preparation of anti-venom sera

A quantidade de pepsina no tratamento do soro com vistas a obtençäo de um bom rendimento da antitoxina é aproximadamente 1000 vezes maior do que a requerida para a remoçäo da fraçäo FC. Comparando-se a atividade de várias preparaçöes de pepsinas comerciais, e fraçöes obtidas por cromatografia DEAE observou-se que o rendimento está relacionado à atividade em pH3.2, o que é devido a parapepsinas. Os resultados sugerem que o soro antitoxina pode ser processado com pepsina purificada, com um bom rendimento e menor contaminaçäo a partir de extrato gástrico de suínos

Detection of classic and invasive E.coli and Shigella serotypes in stools by indirect immunofluorescence.

A imunofluorescencia indireta (IFI) de sorotipos enteropatogenicos classicos e invasores de E. coli e de Shigella foi comparada com os metodos tradicionais de coprocultura e soroaglutinacao. Os resultados da IFI concordaram com os da coprocultura em 128 dos 140 casos testados para E.coli enteropatogenica (91%) e em 108 dos 112 testados para Shigella (96%). Todos os casos com reacoes positivas por coprocultura foram confirmados por IFI. No grupo controle, onde nao haviam sido isolados tais patogenos por coprocultura, foram evidenciados por IFI, 12 casos com reacoes positivas para E. coli enteropatogenica e 4 para Shigella, incluindo-se 2 com infeccao mista: E. coli 026/Sh. dysenteriae e E. coli 0124/Sh. dysenteriae. Foi discutida a alta sensibilidade e especificidade da IFI quando comparada aos metodos tradicionais, sendo sugerido o valor desta tecnica em estudos epidemiologicos envolvendo os microrganismos em questao e sua importancia no estabelecimento de diagnostico precoce na diarreia infantil aguda