X-ray Irradiated Vaccine Confers protection against Pneumonia caused by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a gram-negative bacterium and one of the leading causes of nosocomial infection worldwide, however, no effective vaccine is currently available in the market. Here, we demonstrate that inactivation of the bacteria by X-ray irradiation inhibits its replication capability but retained antigenic expression functionally thus allowing its use as a potential vaccine. Mice immunized by this vaccine were challenged by the parental strain, the O-antigen-homologous strain PAO-1 (O2/O5) and heterologous strain PAO-6 (O6) in an acute pneumonia model. We further measured the protective effect of the vaccine, as well as host innate and cellular immunity responses. We found immunized mice could protect against both strains. Notably, the antiserum only had significant protective role against similar bacteria, while adoptive transfer of lymphocytes significantly controlled the spread of the virulent heterologous serogroup PAO-6 infection, and the protective role could be reversed by CD4 rather than CD8 antibody. We further revealed that vaccinated mice could rapidly recruit neutrophils to the airways early after intranasal challenge by PAO-6, and the irradiated vaccine was proved to be protective by the generated CD4(+) IL-17(+) Th17 cells. In conclusion, the generation of inactivated but metabolically active microbes is a promising strategy for safely vaccinating against Pseudomonas aeruginosa.

Detecting and preventing reversion to toxicity for a formaldehyde-treated C. difficile toxin B mutant.

The toxicity of Clostridium difficile large clostridial toxin B (TcdB) can be reduced by many orders of magnitude by a combination of targeted point mutations. However, a TcdB mutant with five point mutations (referred to herein as mTcdB) still has residual toxicity that can be detected in cell-based assays and in-vivo mouse toxicity assays. This residual toxicity can be effectively removed by treatment with formaldehyde in solution. Storage of the formaldehyde-treated mTcdB as a liquid can result in reversion over time back to the mTcdB level of toxicity, with the rate of reversion dependent on the storage temperature. We found that for both the "forward" mTcdB detoxification reaction with formaldehyde, and the "reverse" reversion to toxicity reaction, mouse toxicity correlated with several biochemical assays including anion exchange chromatography retention time and appearance on SDS-PAGE. Maintenance of a low concentration of formaldehyde prevents reversion to toxicity in liquid formulations. However, when samples with 0.016% (v/v) formaldehyde were lyophilized and stored at 37 °C, formaldehyde continued to react with and modify the mTcdB in the lyophilized state. Lyophilization alone effectively prevented reversion to toxicity for formaldehyde-treated, formaldehyde-removed mTcdB samples stored at 37 °C for 6 months. Formaldehyde-treated, formaldehyde-removed lyophilized mTcdB showed no evidence of reversion to toxicity, appeared stable by several assays, and was immunogenic in mice, even after storage for 6 months at 37 °C.

Preserving immunogenicity of lethally irradiated viral and bacterial vaccine epitopes using a radio- protective Mn2+-Peptide complex from Deinococcus.

Although pathogen inactivation by γ-radiation is an attractive approach for whole-organism vaccine development, radiation doses required to ensure sterility also destroy immunogenic protein epitopes needed to mount protective immune responses. We demonstrate the use of a reconstituted manganous peptide complex from the radiation-resistant bacterium Deinococcus radiodurans to protect protein epitopes from radiation-induced damage and uncouple it from genome damage and organism killing. The Mn(2+) complex preserved antigenic structures in aqueous preparations of bacteriophage lambda, Venezuelan equine encephalitis virus, and Staphylococcus aureus during supralethal irradiation (25-40 kGy). An irradiated vaccine elicited both antibody and Th17 responses, and induced B and T cell-dependent protection against methicillin-resistant S. aureus (MRSA) in mice. Structural integrity of viruses and bacteria are shown to be preserved at radiation doses far above those which abolish infectivity. This approach could expedite vaccine production for emerging and established pathogens for which no protective vaccines exist.

Vaccine preparation by radiation processing.

A new radiation biotechnology for the acquirement of a commercial vaccine, designed for prophylaxis of ruminant infectious pododermatitis (IP), produced by gram negative bacteria Fusobacterium necrophorum (F.n.), is presented. Two different processes for preparing F.n. vaccine are used: a) the inactivation of F.n. bacteria exotoxins by microwave (MW) or/and electron beams (EB) irradiation; b) the isolation of exotoxins from F.n. cultures irradiated with MW or/and EB and the inactivation of isolated F.n. exotoxins with formalin. The EB irradiation of F.n. cultures produced simultaneously with the cells viability decrease an increasing of exotoxin quantity released in the culture supranatant as compared with classical methods. The MW irradiation is able to reduce the cells viability to zero but without an increase of exotoxin quantity in cultures supranatant. Instead of this MW irradiation, for certain conditions, is able to induce an important stimulation degree of the F.n. proliferation in cultures, from two to three log10. Two vaccine types were prepared: A1 vaccine that contains whole cell culture irradiated with MW/EB and A2 vaccine that contains cell-free culture supernatant of an MW/EB irradiated F.n. strain producing exotoxins. Also, other two vaccines are prepared: B1 and B2 that contain the same materials as A1 and A2 respectively, but without using MW/EB exposure. The vaccine efficiency is tested in ruminant farms in which IP evolves. It is expected that this new vaccine to offer a better protection, more than 60%, which is the best presently obtained result in ruminant farms.

[The isolation and immunologic study of ribosomal preparations from Shigella flexneri]. / Poluchenie i immunologicheskoe issledovanie ribosomal'nykh preparatov iz Shigella flexneri.

S. flexneri ribosomal preparations were isolated by differential centrifugation or by fractionation with polyethylene glycol-6000. Their chemical composition and spectrophotometric properties were characteristic of ribosomes, and, as shown by the results of the serological assay, the content of O-specific component was, on the average, 1.4%. The ribosomal preparations were nontoxic for mice when injected intraperitoneally and intravenously in large doses and induced systemic O-antibody response in mice and rabbits. The parenteral administration of ribosomes to guinea pigs led to the increase of resistance to Shigella keratoconjunctivitis. The results of different tests with the use of this model greatly varied. According to the summary data of several tests, the ribosomal vaccine enhanced the resistance of the eyes from 11.3% to 48.5% and the effectiveness coefficient of immunization was 42 +/- 6. Ribosomes isolated from S. flexneri avirulent strain 2a 51.6 M (Iu. A. Belaia's vaccine) showed the same activity as those isolated from virulent strains. The results obtained in this study suggest the expediency of further experimental study of ribosomal preparations obtained from S. flexneri as potential vaccine.

Resistance to Klebsiella pneumoniae and the importance of two bacterial antigens.

The resistance of mice to a lethal infection with Klebsiella pneumoniae, type I, was investigated. Antibody of both IgG and IgM class produced good protection when given with an intra-nasal inoculum of the organism. While antibody directed against capsular antigens was highly protective, so too was antibody directed against a second, non-capsular and heat-labile antigen. This antigen does not appear to be related to either the K or O antigens of Klebsiella and is present in several strains of Klebsiella. It is suggested that this antigen may play a role in determining resistance to this organism by humans.

Effect of purification steps on the immunogenicity of Mycobacterium leprae.

In studies aimed at the development of an antileprosy vaccine for use in man, Mycobacterium leprae suspensions were prepared from livers of experimentally infected armadillos. The 2 methods of purification in chief use, carried out after irradiation of the tissue with 2.5 megarads of gamma irradiation from 60Co, involved treatment with 0.1N NaOH for 2 h at room temperature, trypsin and chymotrypsin digestion for 24h at 37 degrees, and separation in a 2-phase liquid polymer (dextran:polyethylene glycol) system. All vaccines were autoclaved and injected intradermally in mice. Earlier studies have shown that heat inactivation does not interfere with the immunogenicity of M. leprae. Immunogenicity was measured by foot-pad enlargement (FPE) after challenge with heat-killed M. leprae suspensions or by protection against infectious foot-pad challenge. The results indicated that the irradiation and 2-phase separation did not decrease immunogenicity but the NaOH treatment and enzyme digestion did.

Comparative tests with formolized and irradiated vaccines against leptospirosis.

The effectiveness of gamma-irradiated antileptospirosis vaccines and of vaccines killed with formol was compared in guineapigs. Irradiated vaccines proved no more effective than formolized vaccines in preventing either death or persistent kidney infection. Anti-pomona vaccines gave little protection against serotype icterohaemorrhagiae, and there was no significant difference between irradiated and formolized preparations. Vaccine prepared from a virulent strain showed little advantage over vaccine prepared from weak strains.

Immunogenicity of irradiated Salmonella typhimurium cells and endotoxin.

The effects of high doses of radiation (1, 5, or 20 Mrad) on the toxicity, pyrogenicity, and immunogenicity of Salmonella typhimurium cells and endotoxin were studied. Toxicity decreased progressively after exposure to 1, 5, or 20 Mrad. The lethal effect of 1-Mrad exposed cells was greater than that of heat-, acetone-, or alcohol-killed preparations. An amount of 5 Mrad is about a 50% end point in terms of inactivation of the lethal lipopolysaccharide or cell-associated determinants. The fever response to radiation-killed salmonellae decreased between 1- and 20-Mrad exposure. The immunogenicity of 1-Mrad-treated cells usually exceeded that of nonirradiated preparations in mouse-protection tests. With increasing radiation doses, there was a dramatic decrease in, but not an abolition of, immunogenicity. Preparations exposed to 20 Mrad which were nonlethal afforded significant protection. The results are interpreted as a reflection of a dissociation of the primary and secondary toxic determinants of endotoxin after irradiation. The data indicate the potential value of radiation sterilization as a means of production of Salmonella vaccine.