Preparation and immunological properties of detoxified bacterial endotoxins
Creators: name: Martin, William Jeffery
Publisher: name: University of Utah
Year: 2012
Subjects: Toxicity
Fever Vaccine

description: Chemical modification of a crude endotoxin prepared by the RE procedure from a strain of S^, typhimurium yielded certain significant changes in biological activity. The chemical procedures employed were oxidation by boron trifluoride and potassium periodate, acety-lation, and methylation. The results obtained with these endotoxoids, with the exception of the potassium periodate RE preparation, compared favorably with those reported by other investigators with regard to yield, pyrogenicity, lethality, and mouse immunogenicity. Where differences occurred these may have been due to the different strains of gram-negative organisms employed as well as the purity" of the respective endotoxins used as starting materials.  A main purpose for the use of chemically altered crude endotoxins was the possible development of a less toxic enteric fever vaccine, Each endotoxoid preparation was tested for pyrogenicity in rabbits as well as mouse lethality and immunogenicity, and these data were compared with similarly obtained parent RE preparation data as well as standard vaccine.  The pyrogen test results with Acet-RE and the potassium periodate RE preparations indicated that these, on a weight basis, were approximately 100 times less pyrogenic than the preparations of Boivin and parent RE. Under similar conditions the potassium methylate RE preparation showed an approximate 10 fold reduction in fever effects in rabbits, and the boron trifluoride RE preparation showed no reduction in pyrogenicity.  No enhancement or diminution in mouse lethality of the potassium periodate, potassium methylate, Acet-RE, and boron trifluoride preparations was noted when compared to comparable doses of the parent RE preparation. That is, none of these preparations approached a mouse LDcQ with the amounts tested (2,500 ug).  Studies were also undertaken to determine immunogenicity in mice by comparing Boivin, RE, and the previously mentioned endotoxoid preparations with a heat-killed, phenol-preserved (HP) vaccine prepared from the same strain of S. typhimurium. With the exception of using IOLD50 instead of an LD50 challenge, the immunogenicity of the respective vaccines was determined for the most part by active-immunized mouse protection tests as outlined by the Division of Biological Standards, Public Health Service? Although two 100 ug immunizing doses of the Boivin, RE, and the respective endotoxoid preparations varied in mouse protection (potassium methylate RE &gt;Boivin &gt; RE &gt; Acet-RE &gt;boron trifluoride RE &gt; potassium periodate RE), it was obvious, with the exception of the potassium methylate preparation, that the HP vaccine yielded greatest protection against the IOLD50 challenge with S. typhimurium. Further mouse protection studies indicated the minimal immunogenic dose (MID) of the potassium methylate RE vaccine preparation to be approximately 50 ug. These data suggested an approximate five fold difference between the minimal pyrogenic dose (MPD) of 10 ug and the MID (50 ug).  The procedure of acetylation, which apparently has the greatest effect on reducing rabbit pyrogenicity, received major experimental attention in the effort to develop a less toxic enteric fever vaccine, Active-immunized mouse protection experiments in mice employing the Acet-RE vaccine preparation in two 200 ug and 500 ug immunizing (pyrogenic) doses indicated a definite protective effect against a lOLD50 challenge when compared to the HP vaccine. Furthermore, the apparent similarity between the parent RE and Acet-RE vaccine preparations in their protective ability in mice suggested that acetylation had not altered immunogenically the antigenic sites to any great extent on the original RE preparation. These results indicated at most a five fold difference between the HH) in the rabbit and the approximate effective immunogenic dose (EID) in the mouse. Results with both the Acet-RE and potassium raethylate RE vaccine preparations should be considered further in the search for a less toxic enteric fever vaccine.