In mice, active protection against Pseudomonas aeruginosa could be induced with two fractions derived from a crude preparation of ribosomes from P. aeruginosa. The two fractions were obtained by gel filtration chromatography of the crude ribosomal preparation on Sepharose CL-2B. In fraction I, less than 1% of the ribonucleic acid (RNA) applied to the column was recovered. Fraction II contained RNA and protein in a ratio of 1.94. The presence of ribosomes in this fraction was confirmed by analysis on a sucrose density gradient. The protection by fraction I was not affected by treatment with ribonuclease; in contrast, incubation of fraction II with ribonuclease completely abolished active protection. Fraction I contained lipopolysaccharide (LPS) as was indicated by the presence of 2-keto-3-deoxyoctonic acid. No LPS was found in fraction II. The adjuvant dimethyl dioctadecyl ammonium bromide enhanced the protection by fraction II; however, immunity by a low dose of fraction I was abolished by dimethyl dioctadecyl ammonium bromide. Protection by fractions I and II appeared to be restricted to the homologous serotype of P. aeruginosa. These results indicate that RNA is required for protection by fraction II. Active protection by fraction I is likely due to LPS.
A ribosomal vaccine of Pseudomonas aeruginosa and a vaccine containing purified lipopolysaccharide (LPS) were compared with respect to their capacity to protect mice against a lethal challenge with P. aeruginosa. The route of vaccination appeared to be important for the protective activity of the ribosomal vaccine. Optimal protection was measured if both the immunizing and the challenge injection were given intraperitoneally. The ribosomal vaccine protected mice as early as 1 day after vaccination, and the protection lasted at least 6 days. LPS-specific antibodies were detectable 6 but not 2 days after vaccination. The ribosomal vaccine protected mice also against a heterologous serotype of Pseudomonas. Injection of purified LPS did not protect mice earlier than at day 3, and the protection induced by LPS was serotype specific. Ribonucleic acid (RNA) isolated from the ribosomal vaccine had the same protective properties as the ribosomes. RNA induced serotype-nonspecific protection as quickly as 1 day after injection, and the protection lasted at least 6 days. However, the capacity to induce antibodies to LPS was lost or reduced. It is concluded that the serotypenonspecific protection induced by RNA and the serotype-specific protection induced by LPS are due to different mechanisms. Experiments with combined
Crude ribosomes were isolated from Listeria monocytogenes serotype 4b and separated into two fractions by molecular sieve chromatography. Chemical analysis indicated that fraction I contained cell envelope components while fraction II contained the ribosomes. Both fractions protected mice against Listeria, but only in combination with the adjuvant dimethyldioctadecylammonium bromide (DDA). RNase-treatment, but not proteinase K-treatment destroyed the protective properties of fraction II, and RNA purified from fraction II also induced protection. Protection induced by fraction I was not affected by either RNase- or proteinase K-treatment. Both subcutaneous and intraperitoneal, but not intravenous administration of fraction I, fraction II, or purified RNA induced significant protection against intraperitoneal infection, the intraperitoneal route of administration being the most effective. All preparations induced high levels of protection 3 to 7 days after administration, but protection was already decreased after 14 days. Protection induced with RNA appeared to be biphasic, because it also protected mice 1 day, but not 2 days after administration. Protection induced with both fraction I and RNA was at least in part non-specific, because both preparations also protected mice against L. monocytogenes serotype 3, Streptococcus pneumoniae and Pseudomonas aeruginosa. Results are discussed in relation to previous work with analogous preparations from P. aeruginosa.
This paper presents an analysis of the protective properties of the components in ribonuclease (RNase)-sensitive ribosomal vaccines, in particular the ribonucleic acid (RNA). The protective activities in mice of purified ribosomes derived from Pseudomonas aeruginosa and from Listeria monocytogenes were compared. Both ribosomal vaccines had to be combined with the adjuvant dimethyldioctadecylammonium bromide (DDA) in order to be protective, and both lost their activity after RNase treatment. The ribosomal vaccines as well as RNA purified from the ribosomes induced non-specific protection. Intraperitoneal injection of RNA with DDA induced an influx of peritoneal cells. Furthermore, RNA with DDA activated macrophages as shown by, a.o., enhanced phagocytic activity and killing capacity for L. monocytogenes. The results suggest that the observed macrophage activation is probably T-cell-independent. With regard to the ribosomal vaccine of P. aeruginosa it is concluded that RNA also contributed to the protective activity by increasing the humoral response against suboptimal concentrations of contaminating cell surface antigens. In conclusion, it is proposed that ribosomal vaccines may be considered as a combination of a non-specific immunomodulator (RNA) with pathogen-specific cell surface antigens. This concept of ribosomal vaccines is discussed in relation to the literature concerning RNase-sensitive ribosomal vaccines.
To obtain information about the nature of the immunogens in the ribosomal vaccine (fraction II) of Pseudomonas aeruginosa, we studied the specificity of rabbit antibodies to fraction II. Crossed immunoelectrophoresis demonstrated the presence of antibodies which precipitated with ribosomal antigens, but not with lipopolysaccharide (LPS). By means of an enzyme-linked immunosorbent assay, antibodies to LPS were detected in antibodies to fraction II. Antibodies to fraction II could protect mice against a lethal challenge with P. aeruginosa. Absorption experiments demonstrated that the protective ability of antibodies to
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