The characteristics of the chemical composition and biological activity of Proteus antigens isolated using hydroxylamine

The comparative study of the chemical composition and biological properties of antigens isolated from Proteus vulgaris with the use of hydroxylamine and by two classical methods (Boivin's and Westphal's methods) has been made. As shown in this study, the treatment of bacteria with hydroxylamine makes it possible to obtain antigenic complexes with lower toxicity. At the same time hydroxylamine produces no denaturing effect on lipopolysaccharides and protein fractions of bacterial cells.     

Immunogenic complexes of the soluble surface antigens of Sh. sonnei]

The comparative study of the immunogenic properties of Sh. sonnei (phases I and II) soluble surface antigens obtained by the modified method of aqueous-saline extraction and Sh. sonnei (phase I) antigen obtained by Boivin's method was made with the use of the keratoconjunctival test in guinea pigs. The protective activity of a high molecular fraction obtained by the fractionation of phase I soluble surface antigens in Sepharose 4B was studied. Boivin's antigen, when used for immunization in optimum doses, was found to have pronounced protective properties, whereas phase II soluble surface antigens showed no protective activity. A high molecular fraction obtained from phase I soluble surface antigen was found to be the most immunogenic. Protective activity was largely connected with protein antigen. The question whether protein antigen was an independent protective antigen or whether it constituted a part of a complex which determined the protective activity of a high molecular fraction remained unsolved.     

The characteristics of the O-specific humoral immune response of mice vaccinated with Salmonella choleraesuis antigens]

The work deals with the results of the comparative enzyme immunoassay (EIA) of serum samples taken from (CBA X C57BL/6) F1 mice immunized with O-specific polysaccharides, O-antigens (O-Ag) obtained by Boivin's method and antigenic preparations isolated with hydroxylamine (HA) from S. choleraesuis and S. typhimurium. O-Ag and lipopolysaccharide (LPS) of the corresponding bacterial species were used as antigens for the sensitization of polystyrene plates. The primary and secondary humoral immune response was studied by means of EIA. As revealed in this investigation, the immunization of mice with HA-isolated antigenic preparations and O-Ag, obtained from S. typhimurium, in a single injection (in doses of 1-100 micrograms) led to the development of weak specific immune response to O-Ag. Response to LPS was absent. After the second immunization of the animals pronounced immune response to O-Ag and LPS was observed. It developed as a response of both IgM and IgG type. The immunization of mice, made in a single injection, with HA-isolated antigenic preparations and O-Ag, obtained from S. choleraesuis, did not lead to the development of O-specific immune response. After the immunization of mice with these antigens in two injections sharply pronounced nonspecific activity of IgM and IgG serum antibodies with respect to O-Ag and LPS of homologous and heterologous bacterial species was noted in EIA. Neither S. typhimurium O-polysaccharide, nor S. choleraesuis O-polysaccharide did not induce O-specific immune response even after the second immunization.     

Erythrocyte diagnostica made from salmonellal phagolysates]

For the first time O antigens obtained from phagolysates were proved to be suitable for use as material for the production of highly specific erythrocyte diagnostic preparations. O antigens obtained from Salmonella by two methods, i.e. phage disintegration and Grasset's method, were subjected to comparative chemical analysis and found to have no essential difference. Nevertheless, the sensitizing potency of O antigens obtained from phagolysates were experimentally shown to be 3 times greater than that of O antigens obtained by Grasset's method. The optimum sensitizing doses established in the passive hemagglutination test for O antigens obtained by both methods indicated that these antigens were highly sensitive and specific.     

Bivalent Vaccines Against Bacterial Enteropathogens: Construction of Live Attenuated Vaccine Strains With Two O-Serotype Specificities

Abstract. A considerable interest exists worldwide in the development of live attenuated oral vaccines against diarrhoeal diseases. In addition to vaccination against the corresponding pathogens, such vaccine strains can be used as carriers for the expression of protective antigens from other organisms. The antigenic repertoire of a given vaccine strain may thereby be extended, potentially leading to a bivalent vaccine. The lipopolysaccharide is known to be a major antigenic surface component of bacterial enteric pathogens. The feasibility of the development of combined vaccines based on live attenuated carriers expressing two O-serotype specificities is illustrated here by the development of candidate live oral vaccines against Shigella sonnei using Salmonella typhi and Vibrio cholerae as carriers. Various factors that may limit the potential of such hybrid strains as bivalent vaccines are discussed.     

The relationship of the antigenic characteristics of the tick-borne encephalitis virus to the level of the protective activity of inactivated cultured vaccines]

As shown in this study, the immunization of animals with killed vaccines against tick-borne encephalitis (TBE) leads to the formation of specific immunity, depending on the antigenic structure of the vaccine strain and the test strains used for challenge. Vaccines obtained on the basis of the TBE virus strain of the Eastern antigenic variant induced the development of a wider spectrum of specific protective activity than vaccines obtained on the basis of the TBE virus strain of the Western antigenic variant.     

Role of antibodies against outer-membrane proteins in murine resistance to infection with encapsulated Klebsiella pneumoniae

In the assessment of immunity to the encapsulated virulent strain of Klebsiella pneumoniae and its avirulent mutant defective for capsular polysaccharide (CPS), killed bacterial vaccine of both strains could protect mice equally against challenge with 100 x LD50 of encapsulated wild strain. Antisera to each strain conferred the same level of protection on naive mice upon transfer; the protective anti-mutant serum was highly capable of opsonizing the encapsulated bacteria. In addition to the common antigenic components shared by both strains, the wild strain had antigen(s) unrelated to the mutant since the protective capacity of the anti-wild serum was not affected by preabsorption with the mutant strain; the protection conferred by the anti-mutant serum was mediated by antibodies against non-capsular antigens since the antiserum did not contain antibodies against purified CPS detectable by ELISA. As possible candidates among the non-capsular antigens, outer-membrane proteins (OMPs) extracted from the mutant strain were examined for their immunogenicity. Immunoblotting of the protein-containing fraction and ELISA using LPS-free OMP suggested that a number of proteins were involved in the immune response evoked by K. pneumoniae. Furthermore, mice immunized with OMP or anti-OMP serum could overcome a lethal challenge with the wild strain. These results indicated that OMPs of K. pneumoniae are implicated as the protective antigens and may pave the way for the development of non-capsular, proteinaceous vaccines.     

Antigenic competition among different 'O' antigens of Salmonella enterica subspecies enterica serovars during hyperimmunization in pony mares

The present study on antigenic competition among somatic 'O' antigens of different Salmonella groups (A, B, C1, C2, D and E1) in mares revealed that the immune response to most of the antigens was not (A, B, C2) or little (C1, D) affected by antigenic competition. However, E1 group antigen, which induced high antibody titres (Avg. 12967.3) when given alone, produced almost 3.5 log2 lower antibody titres on giving with other antigens, indicating the antigenic competition among some Salmonella group antigens. The antigenic competition varied for different antigens even of the similar chemical nature. Therefore, antigens belonging to different somatic groups should not be given together for the purpose of raising polyvalent serum or for immunization using multivalent Salmonella vaccines prepared from strains of different 'O' groups revealing antigenic competition.     

Immunochemical characteristics and serologic properties of lipopolysaccharides isolated from various Brucella species using various extraction methods

The results of the comparative analysis of LPS isolated by different methods of extraction from the cultures of several Brucella species differing in their virulence are presented. Purified LPS preparations have been obtained from Brucella virulent and vaccine strains by using such methods as water-phenol extraction, Boivin's method, mild alkaline hydrolysis of the antigen according to Boivin's procedure. The presence of certain relationship between the method used for the extraction of Brucella LPS to be compared and their chemical composition, immunological characteristics and serological activity has been established. As shown in this investigation, in the process of the preparation of a highly sensitive diagnosticum for the passive hemagglutination test the use of LPS obtained from Brucella virulent strains, but not from the vaccine strain, by the method of mild alkaline hydrolysis according to Boivin's procedure is expedient. The data presented in this work indicate that the soluble complex of lipid A obtained from Brucella LPS has been found to possess serological activity. The results of the study of the serological properties of lipid A indicate that the lipid component may also play a certain role in the manifestation of the serological activity of Brucella LPS.     

Vaccination and the population structure of antigenically diverse pathogens that exchange genetic material.

Populations of antigenically diverse pathogens undergoing genetic exchange may be categorized into strains on the basis of a set of principal protective antigens. The extent to which polyvalent vaccines based on these protective antigens can alter the population structure of the pathogen is determined by the degree of cross-protection between strains. In the case where there is no cross-protection, vaccinating against a particular strain will have no effect on the others. As cross-protection increases, the strains containing the antigenic variants included in the vaccine will be diminished in prevalence, and those that do not will increase in prevalence. The rise in prevalence of the latter will become more and more exaggerated as cross-protection increases. However, beyond a critical level of cross-protection, in the absence of vaccination, the steady state of the system is asymmetric in that a certain subset of strains (with non-overlapping repertoires of antigenic variants) will dominate over the others in terms of prevalence. Under these circumstances, a vaccine consisting of the most immunogenic combinations of antigenic variants can cause a dramatic increase in frequency of a subset of rare strains.     

Revealing the potential of DNA-based vaccination: lessons learned from the hepatitis B virus surface antigen

DNA-based vaccination is a novel technique to efficiently stimulate humoral (antibody) and cellular (T cell) immune responses to protein antigens. In DNA-based vaccination, immunogenic proteins are expressed in in vivo transfected cells of the vaccine recipients in their native conformation with correct posttranslational modifications from antigen-encoding expression plasmid DNA. This ensures the integrity of antibody-defined epitopes and supports the generation of protective (neutralizing) antibody titers. Plasmid DNA vaccination is furthermore an exceptionally potent strategy to stimulate CD8+ cytotoxic T lymphocyte (CTL) responses because antigenic peptides are efficiently generated by endogenous processing of intracellular protein antigens. These key features make DNA-based immunization an attractive strategy for prophylactic and therapeutic vaccination against extra- and intracellular pathogens. In this brief review, we summarize the current state of expression vector design, DNA delivery strategies, priming immune responses to intracellular or secreted antigens by DNA vaccines and unique advantages of DNA- versus recombinant protein-based vaccines using the hepatitis B surface antigen (HBsAg) as a model antigen.     

Role of pertussigen (pertussis toxin) on the mouse protective activity of vaccines made from Bordetella species

Pertussigen [pertussis toxin (Ptx)] from Bordetella pertussis, when detoxified, induces protection in mice to intracerebral challenge (ic) with virulent B. pertussis. In its native form, minute nonprotective doses promote the development of immunity induced by other antigens of B. pertussis. As little as 4 ng of Ptx, given with a nonprotective dose of 8 X 10(7) killed cells of the phase III Sakairi strain, promoted detectable protection to ic challenge. Native Ptx in doses of 0.4 to 400 ng did not protect mice, and vaccines made from strains not producing Ptx induced only weak protection. The marked enhancing action of Ptx was also observed with 5 micrograms of purified filamentous hemagglutinin and with vaccines made from other species of the Bordetella genus, such as B. parapertussis and B. bronchiseptica, but it was not observed with B. pertussis endotoxin. In addition, Ptx was still effective when given as late as 7 days after the vaccine. Antibodies to surface antigens of the challenge strain were demonstrated in sera of mice immunized with vaccines prepared with the different Bordetella species tested, but antibodies to Ptx were detected only in the sera of mice immunized with the wild-type B. pertussis strains. Glutaraldehyde detoxified Ptx does not have this action. Pretreatment of normal mice with Ptx, also enhanced the protective action of a mouse antiserum to a wild-type strain of B. pertussis. These observations show that antigens other than Ptx are responsible for the protection, and that Ptx acts non-specifically to enhance the mouse protective action of those antigens.     

In vivo human immune response to transferrin-binding protein 2 and other iron-regulated proteins of Neisseria meningitidis

Abstract When grown under iron restriction, Neisseria meningitidis expresses new outer-membrane proteins, some of which are antigenic and potentially useful as vaccine components. This is particularly relevant to N. meningitidis serogroup B, against which neither polysaccharide nor conjugate vaccines are effective. We investigated recognition of N. meningitidis serogroup B outer-membrane antigens by three sera from patients recovered from meningitis. Recognition of antigens from the homologous strain provided information on in vivo expression during infection and immunogenicity, while cross-reactivity with outer membrane proteins from the other two strains and from another five strains in our collection allowed evaluation of antigenic heterogeneity. Our results demonstrate that transferrin-binding protein 2 (TBP2) is immunogenic in humans, to varying degrees depending on the strain, and that TBP2s (like the equivalent proteins of Haemophilus influenzae type b) are among the most important iron-regulated outer membrane antigens expressed during infection. Other immunogenic outer membrane proteins (some iron-regulated) are also expressed during infection; in a previous study in mouse, three of these proteins (with M _r of 50, 70 and 77 kDa) did not induce an immune response. Our cross-reactivity data provide some support for Robki et al.'s two-group classification of N. meningitidis strains, and provide evidence against the possibility that the antigenic domains shared by the TBP2s of all N . meningitidis strains induce immune responses in vivo.     

Immunodominant Francisella tularensis antigens identified using proteome microarray

Stimulation of protective immune responses against intracellular pathogens is difficult to achieve using non-replicating vaccines. BALB/c mice immunized by intramuscular injection with killed Francisella tularensis (live vaccine strain) adjuvanted with preformed immune stimulating complexes admixed with CpG, were protected when systemically challenged with a highly virulent strain of F. tularensis (Schu S4). Serum from immunized mice was used to probe a whole proteome microarray in order to identify immunodominant antigens. Eleven out of the top 12 immunodominant antigens have been previously described as immunoreactive in F. tularensis. However, 31 previously unreported immunoreactive antigens were revealed using this approach. Twenty four (50%) of the ORFs on the immunodominant hit list belonged to the category of surface or membrane associated proteins compared to only 22% of the entire proteome. There were eight hypothetical protein hits and eight hits from proteins associated with different aspects of metabolism. The chip also allowed us to readily determine the IgG subclass bias, towards individual or multiple antigens, in protected and unprotected animals. These data give insight into the protective immune response and have potentially important implications for the rational design of non-living vaccines for tularemia and other intracellular pathogens.     

Delineation of epitopes on the Py235 rhoptry antigen of Plasmodium yoelii YM

The 235-kDa antigenic rhoptry protein Py235 of Plasmodium yoelii is encoded by a large, highly polymorphic gene family. Monoclonal antibodies to some of these antigens have been shown to attenuate the virulence of the lethal YM strain of the parasite, converting a potentially fatal YM infection to a fulminating one typical of the nonlethal 17X strain, by inducing a switch in target cell preference from mature red blood cells to reticulocytes. The reason for this is not known but would suggest that antigenic determinants of Py235 may be useful in or as subunit vaccines. To identify such determinants, we constructed an epitope expression library of one Py235 variant and screened the library with the antibodies. Thus, we mapped 5- and 12-amino acid epitopes to the C-terminus of the antigen. Both epitopes were more reactive with protective than with nonprotective monoclonal antibodies. This may explain the differential protection conferred by these antibodies upon their passive transfer into mice.     

Toxoplasma gondii: chimeric Dr fimbriae as a recombinant vaccine against toxoplasmosis

Toxoplasma gondii is responsible for fetopathy in farm animals and humans and severe disease in immunocompromised individuals (i.e. AIDS patients). Effective vaccines, inducing protective and long-lasting immunity to this global parasite, are still desired. In the work, we evaluated the immunogenic and immunoprotective activity of Escherichia coli chimeric Dr fimbriae bearing selected antigenic epitopes of three T. gondii antigens (SAG1, GRA1 and MAG1), in comparison with conventional recombinant antigens obtained in E. coli expression system. Our data demonstrate a very high protective efficacy of recombinant antigens supplemented with Freund's adjuvants, whereas chimeric Dr fimbriae as a vaccine proved non-protective. The recombinant antigen vaccine induced a strong specific antibody response and prevented the brain cysts formation by 89%. The results are promising and should be confirmed in further study on farm animals by use of less aggressive than Freund's adjuvant preparations.     

Plasmodium berghei XAT: protective 155/160 kDa antigens are located in parasitophorous vacuoles of schizont-stage parasite

Effective blood-stage malaria vaccine candidates have been mainly developed from the proteins in exposed locations on the parasite such as the surface of free merozoites or infected red blood cells. In the present study, we identified and localized novel protective antigens derived from the blood-stage of Plasmodium berghei XAT after establishment of hybridomas producing protective monoclonal antibodies (mAbs) against the parasites. The protective antigens were expressed in schizonts but not in trophozoites, and located in the parasitophorous vacuoles in the infected erythrocyte cytoplasm. The antigens, with molecular weight of 155/160 kDa, were not identical to any merozoite/schizont antigens that have been reported as target molecules recognized by mAbs developed to rodent malaria parasites. The characterization of new malarial antigenic targets of potentially protective antibody responses following infection would give us new insights for the selection of candidate antigens for malaria vaccine.     

Sequential release of antigens from chloroform-treated Staphylococcus epidermidis: application towards a possible vaccine

A hmad , A., C larke , S., B uchan , A. & S kinner , G.R.B. 1990. Sequential release of antigens from chloroform-treated Staphylococcus epidermidis : application towards a possible vaccine. Journal of Applied Bacteriology 69 , 676–685.
This study describes the properties of two potential Staphylococcus epidermidis vaccines prepared by chloroform treatment of bacteria and release of antigen from these chloroform-treated organisms. Both vaccines were antigenic on testing with homologous hyperimmune serum and induced immune reactivity in immunized rabbits. There was protective efficacy in mice against intraperitoneal challenge by Staph, epidermidis .     

Ribonuclease-sensitive ribosomal vaccines

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 fromPseudomonas aeruginosa and fromListeria 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 forL. monocytogenes. The results suggest that the observed macrophage activation is probably T-cell-independent. With regard to the ribosomal vaccine ofP. 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.     

Low virulent strains of Candida albicans: unravelling the antigens for a future vaccine

Several low virulent Candida albicans mutant strains: CM1613 (deleted in the Mitogen Activated Protein (MAP) Kinase MKC1), CNC13 (deleted in the MAP-kinase HOG1) and the morphological mutant 92' were used as vaccines employing a murine model of systemic candidiasis. In this vaccination trial, only the CNC13 strain was able to induce protection against a subsequent infection with a lethal dose of the wild-type strain. The protection induced by CNC13 vaccinated animals resulted in 60-70% percent of survival. These results demonstrate that collaboration between cellular and humoral responses, induced by the CNC13 mutant, elicited a long lasting and effective protection. Using a proteomic approach (two-dimensional gel electrophoresis followed by Western blotting), twenty-five C. albicans immunogenic proteins were detected and identified by matrix-assisted laser desorption/ionization and/or tandem mass spectrometry. We were able to define an antibody pattern in the sera from the nonvaccinating strains (92' and CM1613), which was different from the profile detected in the sera from surviving animals (vaccinated with the CNC13 mutant). The utility of this proteomic approach has allowed us to identify antigens that induce protective IgG2a antibody isotype in the sera from vaccinated animals: enolase (Eno1p), pyruvate kinase (Cdc19p), pyruvate decarboxylase (Pdc11p), a component from the 40S ribosomal subunit (Bel1p), triosephosphate isomerase (Tpi1p), DL-glycerol phosphatase (Rhr2p), fructose-bisphosphate aldolase (Fba1p) and two new protective antigens: IMP dehydrogenase (Imh3p), and acetyl-CoA synthetase (Acs2p). The antigenic proteins that promote protective antibodies described in this work are excellent candidates for a future fungal vaccine; their heterologous expression and vaccine design is currently underway.