Vi Antibody Research Articles

Solubility of Vi antigen of Salmonella typhi.

The observations reported here show that the T Vi antigen is effective in stimulating antibodies when it is attached to the cells. It is also clear that the Vi antigen is readily removed in aqueous solutions, but in such organic solvents as acetone, chloroform, ether, alcohol, carbon tetrachloride and benzene it is retained by the cell. In alcohol-water mixtures the Vi antigen is progressively more soluble in those solutions where the alcohol concentration is less than 70 %. However, its solubility in water is limited, and by making progressively heavier suspensions it was found that a point is reached at which Vi antigen is retained by the cell, the suspending solution apparently being saturated. Such an aqueous suspension is able to stimulate the production of Vi antibody.The administration of dried organisms in peanut oil resulted in the production of antibodies against both O and Vi antigens. This was in contradistinction to the results obtained when mineral oil, mineral oil + saline, or peanut oil + saline were used: in these cases the O antibody titre was lower and Vi antibodies were produced in low titre or not at all. It seems clear that the presence of water in the vaccine has the effect of removing the antigenicity of the Vi hapten; water-free, vegetable-oil vaccine is suggested by these studies as an effective agent for immunizing against all of the antigens ofSalmonella typhi.The use of such a vaccine in producing active and passive immunity in animals is discussed in the paper which follows.

The influence of storage on the antibody-stimulating properties of different typhoid vaccines.

SUMMARYThree typhoid vaccines were tested: an alcohol‐killed and alcohol‐preserved, a heat‐killed and phenol‐preserved, and a formalin‐killed and phenol‐preserved. The antibody‐stimulating properties of the vaccines were determined 1 month after the preparation and after storage for on an average nearly 2 years in the refrigerator.The antibody production was determined by means of Vi and O agglutination tests on immune rabbit sera.If the results achieved with different vaccine preparations are put together it was shown that fresh heat‐killed and formalin‐killed phenol‐preserved vaccines gave a significantly higher Vi antibody production than vaccines stored for on an average 21 months. Alcohol‐killed and alcohol‐preserved vaccine had lost none of its Vi antibody‐stimulating property after storage.The O‐antibody‐stimulating property was impaired only for the formalin‐killed and phenol‐preserved vaccine after storage.By the method of immunization employed we could expect demonstrable impairment of the O antibody stimulation only if this was very considerable.

Estimation of Vi antibody employing erythrocytes treated with purified Vi antigen.

Summary1. The preparation of a sensitive and specific Vi agglutination reagent by sensitization of human type O erythrocytes with Vi antigen isolated from E. colt has been described. 2. Vi antibody content of rabbit and human sera was determined by this method and the conventional bacterial agglutination test. The hemagglutination test provided a more sensitive measure of Vi antibody by a factor of 3 to 6 fold. Greater variability in Vi titers was encountered in testing the sera of typhoid carriers; however, both methods were in good agreement in the detection of Vi antibody in these sera. The advantages inherent in the Vi hemagglutination procedure are discussed.

The properties of different Salmonella Vi antigens.

The labile Vi antigens ofSalmonella typhi, Salm. paratyphi AandB, and ofSalm. typhi-muriumwere compared, especially in their response to various chemical and physical agents.1. Inactivation by dilute acid:(a) The TVi antigen is the most resistant to acid treatment, the BVi antigen is most readily inactivated and the AVi antigen holds an intermediate position.(b) Pure Vi sera may be obtained by absorption with acid-treated bacilli, but this technique gives irregular results.(c) Contrary to Kauffmann's statements, the TVi antigen is not ‘destroyed’ by acid treatment.2. Inactivation by dilute alkali:(a) All the Vi antigens so far tested are susceptible to dilute alkali.(b) Complete inactivation, or extraction, of the Vi antigens is ensured when the yield of alkali-treated bacteria represents no more than 25% of the original bacterial count.(c) The most reliable method of preparing a pure Vi serum is by absorption of the Vi + O serum with alkali-treated organisms of the same strain as that employed in immunization.(d) Immunization with alkali-treated bacilli results in the elaboration of pure 0 antisera entirely devoid of Vi antibody.3. Inactivation by heat (in aqueous suspensions):(a) The TVi antigen is the one most readily inactivated, the BVi antigen is the one most resistant and the AVi antigen holds an intermediate position.(b) These differences are only of minor significance, since similar differences have been established for the TVi antigen itself when it is contained in the two varieties of Vi strain of Salm. typhi.4. Treatment with alcohol:(a) Most of the properties of the threeSalmonellaVi antigens remain unimpaired by alcohol treatment.(b) The physico-chemical behaviour of alcohol-treated or heated bacilli, as exemplified by agglutinability by pure Vi antiserum, depends on the presence of other constituents of the bacterial cell.5. Treatment with formalin:(a) The Vi agglutinability of the differentSalmonellais preserved undamaged for long periods of time. The 0 inagglutinability ofSalm. typhiis also well maintained.(b) It is not yet known whether formolized AVi and BVi antigens undergo an alteration that leads to ‘functional deficiency’ of the corresponding antibody similar to that of the altered T Vi antibody.6. Treatment with phenol produces a reversible inactivation of the agglutinogenic activity of the T Vi antigen but not of the A Vi and B Vi antigens.7. The inadequacy of the symbols employed by Kauffmann for expressing the changed reactivity of differently treated antigens is emphasized.

Studies on Vi Antigen

Summary A study was made of the immunological properties of the Vi antigen obtained from Escherichia coli 5396/38 by a NaCl-ethanol fractionation procedure. The purified Vi antigen could be absorbed on collodion particles or on erythrocytes which then became specifically agglutinable by Vi antisera. It possessed excellent serological activity and specificity in these tests and also in the quantitative complement-fixation and quantitative precipitin tests. In the range of antibody excess the Vi antigen precipitated in a uniform manner the Vi antibody in coli, ballerup, typhoid and purified Vi-antisera. Supernatant fluid analysis in the quantitative precipitin test with E. coli antiserum indicated that the purified antigen approached but did not quite meet this criterion of immunological homogeneity. The antigen was markedly effective in the protection of mice against challenge with virulent strains of Salmonella typhosa. It was antigenic for mice, rabbits and man, evoking the production of antibody reactive with both Vi antigen and with Vi strains of S. typhosa. The antibody produced in rabbits possessed mouse protective potency and in vitro activity essentially comparable to the Vi antibody obtained by immunization with Vi strains of S. typhosa, although certain differences in these activities were pointed out. The antigen was as effective as V form S. typhosa in the removal of mouse protective potency from various Vi antisera.

Studies on Vi Antigen

Summary A simple ethanol-salt fractionation procedure followed by acetic acid hydrolysis is described which yields a purified Vi antigen from saline extracts of acetone dried Escherichia coli, strain 5396/38. The various stages in the purification were accompanied by increased serological activity. The final product was the only fraction that was free from contamination with E. coli somatic antigen. This fraction showed a high degree of serological activity and specificity. The residue left after extracting the bacterial cells with saline contained only 1% of the original activity as determined by mouse protective potency tests. On a dry weight basis, however, the saline extract was consistently less active than the intact bacteria. It is suggested that the presence of other antigens in the whole organisms may have influenced the response to the specific Vi antigen. Alternatively, the insoluble cellular components may have functioned as a depot substance which indirectly stimulated the production of Vi antibody. The stages up to and including ethanol-salt concentration effected a 10-fold purification of the Vi antigen on a dry weight basis and a 25-fold purification on the basis of total nitrogen content. Chemical analysis at this stage revealed the presence of phosphorus, protein and carbohydrate. No differences were observed in the antibody response of rabbits to the various fractions up to this stage. The next stage, shaking with chloroform-ethanol, removed only trace amounts of inactive material, yet the antigenicity of the product for rabbits was significantly reduced. No comparable reduction in antigenicity for mice was observed. Acetic acid hydrolysis liberated the Vi antigen from its complex without further impairing its antigenicity. Chemical analysis of the purified Vi antigen showed that it is a fairly strong acid but that it does not contain uronic, sulphuric or phosphoric acid residues. Both nitrogen and acetyl groups are present, but apparently not in the usual form of N-acetylhexosamine.

The immunizing potency of alcohol-killed and alcohol-preserved typhoid vaccine after storage for ten years.

1. Alcohol-killed and alcohol-preserved typhoid vaccine retains its power of stimulating Vi- and O-antibody formation unimpaired during storage at 1–2° C. for at least 10 years.2. The Vi antibody induced by immunization with the 10-year-old vaccine possesses full ‘functional efficacy’ in passive-immunity tests in mice.3. The expiry date of this type of vaccine can be extended to a period of 10 years, provided the vaccine is kept in cold storage.4. Alcoholized vaccine can serve as ‘standard vaccine’ in any future attempt at standardization of the potency of typhoid vaccine.

Thermostability and Alcohol Solubility of Vi Antigen

The present investigation was suggested by an unpublished observation made by one of us several years ago while working on Vi antigens and antibodies. An antiserum prepared against a motile Vi positive typhoid strain, H-484, was adsorbed 3 times with Salmonella ballerup. No drop in titer occurred for either O-901, H-484 or its nonmotile Vi positive variant, O-484. However, a marked prozone, increasing with the number of adsorptions, developed against 0-484 (Table I). When S. ballerup was washed in several changes of saline before being used for adsorption, the prozone against 0-484 failed to appear. It would seem that the Vi antigen was related to this prozone since it was the only component common to 0-484 and S. ballerup. The present work is an attempt to explain this reaction.The growth of S. ballerup was removed from plates using 5 ml of saline for every 2 plates. The mixture was shaken several times, centrifuged after 30 minutes and the supernatant fluid used to harvest more plates. The procedure was...

A study of the serological type differentiation ofStaphylococcus pyogenes

1. Of 1552 strains ofStaph. pyogenestested by the slide-agglutination technique, using the absorbed and unabsorbed sera suggested by Cowan, 74% of strains were identified while 26% remained untyped. Of untyped strains, half were inagglutinable while the remainder gave ‘indefinite’ results and remained unclassified. The introduction of two new sera, used diluted but unabsorbed, considerably reduced the number of untyped organisms but made little difference to the proportion of ‘indefinite’ strains. The method was simple and quick, but the variety of cross-reactions often prevented a clearcut differentiation of strains and the number of types identifiable was limited.2. Of three methods of agglutination, titrations on glass slides were preferred because by this technique end-points were clearer than those obtained in a water-bath or by the method for determination of Vi antibody.3. Agglutination of seventeen strains ofStaph. pyogeneswith homologous and heterologous sera showed that the use of unabsorbed sera was of limited value for typing because of the marked cross-reactions.4. Christie & Keogh's division ofStaph. pyogenesinto nine types was promising but their absorbing methods allowed much antigenic overlap.5. Experiments with absorbed sera showed that no single strain ofStaph. pyogenescould be used successfully to absorb all sera. There appeared to be groups or pairs of types in which serologically the individual members resembled each other very closely and yet were not identical. In these instances the absorption of a serum by a heterologous strain nearly related to the homologous strain proved of most value in the preparation of specific sera.6. A set of seventeen absorbed sera was prepared from Cowan's three types ofStaph. pyogenes, from six of Christie & Keogh's type strains and from eight additional strains freshly isolated from staphylococcal lesions. With some absorbed sera there was specific agglutination with the homologous strain and no heterologous reactions, other sera reacted with several strains and could not be made specific.7. Seven specific serological types were recognized while a further eight types showed less specific reactions and were identifiable by a pattern of reactions. A number of strains differed slightly one from another in their agglutination reactions, and it was not possible to decide whether these were additional types or variants within types.8. In a small series of experiments the precipitin reaction, using unabsorbed sera, seemed of little value in the differentiation of strains ofStaph. pyogenes.9. Of 259 coagulase-positive strains examined during 1946 and 1947, all were identified by agglutination reactions, although they varied in their specificity; 67.5% of these strains were identified by staphylococcal bacteriophage reactions.10. A preliminary comparison of serological and bacteriophage types showed a fairly regular but not complete correlation between the results obtained by the two methods. A number of serological types were regularly lysed by the same bacteriophage filtrates; a few were consistently untypable by phage indicating that filtrates had not as yet been found for them or that the strains were phage resistant.Others which were not clear-cut in their reactions and which were recognized by patterns of reactions with both sera and phage filtrates did not always show a close correlation in the typing results.11. The application of both serological and bacteriophage typing to the investigation of outbreaks of staphylococcal infection has proved to be of value in tracing sources and paths of spread of infection. In several instances the use of both methods of typing has led to conclusions which would not have been warranted if one method alone had been used.

Progress in Bacteriological Technique

PROF. J. CRUICKSHANK has prepared for the British Council an account of recent advances in bacteriological methods (Brit. Med. Bull., 1, No. 8; 1943). The principal advances in the last ten years have, in his opinion, been made in the discovery of more efficient selective culture media for the isolation of bacteria, in the determination of the stable subgroups or types of bacteria and in the development of typing methods which make it possible to trace the probable source of an infection or an epidemic. Antigenic analysis has resulted in such valuable discoveries as the Vi or virulence antigen of the typhoid bacillus. Antityphoid serum made for therapeutic use should contain Vi antibodies. The blood of typhoid carriers almost always contains these, so that the Vi agglutination test has become a valuable means of helping to trace the sources of the infection. The discovery of a Vi bacteriophage, which has a specific action on Vi strains of typhoid bacilli, can be used for the identification of particular strains of these bacilli. Epidemiologists have used this means of tracing the source of isolated infections or epidemics. The typing of diphtheria bacilli has also produced valuable results.

A Precipitin Test for Typhoid Fever

TREATMENT of typhoid fever with the serum from convalescents of this disease is a well-recognized therapeutic procedure. The good results obtained are believed to be due to passive immunization, as in other forms of serum therapy. From the serological analysis of a large number of convalescent typhoid sera it was, however, observed that their antibody content, both of O and Vi antibodies, was universally poor. On the other hand, in spite of their paucity in antibodies, such sera had proved to be very efficacious therapeutic agents in our hands during the last few years. This experience could not, therefore, be reconciled with the statements of Felix and his co-workers on the important role of Vi antibody, and its antigenic counterpart, in typhoid immunity and infection ; in fact, this antibody forms the principal constituent of Felix's therapeutic antityphoid serum. Further experiments were, therefore, undertaken which revealed that when a convalescent serum was injected into human beings, as well as laboratory animals, it led to the production of an appreciable amount of Vi antibody and that its therapeutic efficacy was directly proportional to the amount of antibody so produced. These observations could only be explained by assuming that the convalescent serum either stimulated the antibody-forming apparatus in some unknown manner or that it contained an antigen responsible for Vi antibody formation. It has now been ascertained that the latter explanation is correct and the typho-precipitin reaction reported here is based on the presence of a Vi precipitinogen in the sera of typhoid patients both during infection and during convalescence. The qualitative and quantitative determination of this antigen has been found to be of definite significance in the diagnosis, the prognosis and the treatment of typhoid fever.

The preservation of Vi antigen in T.A.B.C. vaccine: With a note on combined active immunization with T.A.B.C. vaccine in tetanus formol-toxoid

1. V strains of Bact. typhosum can be sterilized by means of merthiolate or colloidal silver without destructive effect on either the Vi or O antigens.2. Suspensions in physiological saline sterilized and preserved by the addition of either merthiolate or silver retain their property of stimulating Vi antibody formation in the rabbit for at least one year if stored at 0–2° C, but when stored at 23–25° C. this property is lost after 4 months' storage.3. The Vi antibody-stimulating property of a merthiolate-killed and preserved suspension in physiological saline is almost completely destroyed by an exposure of 5 days at 37° C. or 4 hr. at 60° C.4. Bact. typhosum bacilli killed with merthiolate or alcohol, and dried with acetone, show no loss of Vi antigen when stored in the dry state for 1 year at 23–25° C, or after 3 months at 37° C.5. Suspensions sterilized and preserved with merthiolate in 32% saline will withstand 50 days' storage at 37° C, or 7 months at 23–25° C, without significant loss of their Vi antibody-stimulating properties.6. Suspensions in 32% saline can be sterilized by an exposure to 60° C. for 2 hr., and even after 4 hr. exposure at this temperature, show no loss of Vi antigen content.7. Suspensions killed by merthiolate or alcohol, and preserved by the addition of 25% alcohol in physiological saline, failed to stimulate Vi antibody formation in the rabbit after being stored for 20 days at 37° C.8. The application of these findings to the production of a stable T.A.B.C. vaccine of enhanced immunological qualities is discussed.9. The effect of mixing Bact. typhosum with tetanus formol-toxoid has been examined, and the advisability of employing heat-killed-phenolized vaccine mixed with tetanus formol-toxoid for combined immunization against tetanus and typhoid infections is discussed.10. A suggested technique for the preparation, supply, and administration of two new types of T.A.B.C. vaccine is described.

Further studies on the sterilization of T.A.B.C. vaccine

1. Silver nitrate or sodium merthiolate can be used for the sterilization of suspensions of B. typhosus and neither have any immediate destructive effect on the Vi, O or H antigens.2. Two methods of employing silver nitrate as a sterilizing agent are described.3. Physiological saline solution with silver nitrate added is bactericidal for B. typhosus and the staphylococcus but has no inhibitory action on the growth of moulds.4. Vaccines sterilized and preserved with silver nitrate after storage for 6 months at 7° C. were found to have retained their Vi antibody stimulating properties.5. A vaccine sterilized and preserved with “katadyn” after storage for 18 months at 7° C. still contained some Vi antigen and was superior as an immunogenic agent to a freshly prepared heat-killed-phenolized vaccine.6. It would appear that the stability of Vi antigen is increased by the addition of silver within certain limits, but this effect is only obtained if low temperature storage conditions are maintained.7. A vaccine sterilized and preserved with sodium merthiolate after 6 months' storage at 7°C. failed to stimulate any Vi antibody in the rabbit.

The sterilization of T.A.B.C. vaccine

1. Horgan's (1936) findings with regard to heat sterilization of B. typhosus vaccine are not confirmed.2. Silver in the form of katadyne or electro-katadyne appears to be an effective sterilizing agent for B. typhosus suspensions.3. Katadyne and electro-katadyne have no destructive action on Vi, O or H agglutinogens of B. typhosus.4. Electro-katadyne, even when used in excessive doses, has no detrimental effect on the immunizing activities of Vi antigen.5. Electro-katadyne sterilized suspensions of V strains of B. typhosus are more effective immunogenic agents than those sterilized by heat and phenol.6. The bactericidal properties of electro-katadyne, though impaired in 0·85 per cent saline, are adequate as a means for preserving vaccine.7. It is suggested that katadyne sterilized suspensions of V strains might be of value in testing for the presence of Vi agglutinins in human sera since they are hypersensitive to Vi antibody while retaining most of their resistance to O antibody.8. In view of the suggestion by Felix that additional antigens with activities similar to those of Vi antigen may exist in bacteria other than B. typhosus, it may be found that the electro-katadyne process has an application in the preparation of vaccines generally, particularly those which have hitherto proved ineffectual in prophylaxis.

Studies on the significance of “Vi Antigen” in the Mechanism of Typhoid Infection in Mice

SummaryWe have been able to confirm all the experimental results of Felix and his collaborators in regard to the Vi antigen and its significance in mouse infections. In the interpretation of some of the findings we are, however, not quite in agreement with Felix.When large doses of living V forms of typhoid bacilli are injected into the mouse they cause acute intoxication followed by death within 2 days—if the animals die. Surviving animals are clinically healthy after this time, often earlier, but they are nevertheless found to be massively infected in the organs and blood for several days.When smaller, non-intoxicating, doses are used it is not possible to demonstrate any difference between the V form and the W form of the same strain in regard to the severity of the infection they cause; so that we may conclude that the Vi antigen plays no part in the virulence of the typhoid bacillus in the mouse. (Remember in this connexion that the dysentery bacillus is also very toxic for mice injected in large doses, while its virulence, i.e. the ability to grow in the mouse is = 0; but when rapidly fatal doses are used a massive growth of dysentery bacilli can also be demonstrated here, the cause being that the natural immunity has become partly neutralized by the intoxication.)Like Felix we find that only the V form can be used both for active vaccination and the production of sera which protect against lethal doses of V forms in mouse experiments. We consider that this is due to the antitoxic effect. Our experiments show that the anti-bacterial effect in vaccination experiments differs little whether the V or the W form is used as vaccine, so long as the vaccinal effect is tested with non-toxic doses of bacilli. As shown in earlier experiments, the bactericidal effect in actively vaccinated animals is often a relatively slow process.Only the Vi antibody containing sera can protect prophylactically with certainty against the toxic effect of living typhoid bacilli. On the other hand; the bactericidal effect prophylactically of 0 and 0 + Vi sera is very similar when tested against non-toxic doses of “virulent” typhoid bacilli.The therapeutic value of potent Vi sera under the experimental conditions given was nil.

A Contribution to the Problem of the Virulence (“VI”) Antigen of B. typhosus

SUMMARY1. The strains of B. typhosus, which are inagglutinable, or non-sensitive to somatic O agglutinins, are virulent, whereas the agglutinable, or sensitive strains, show a low virulence.2. Virulence and inagglutinability of strains seem to depend upon the presence in them of a virulence (Vi) antigen.3. A considerable protective action, observed by means of active and passive immunisation, is exerted by the inagglutinable strain used (205 D), because it contains Vi antigen.4. When the O and H agglutinins are eliminated through absorption, the agglutination reaction may serve to demonstrate in vitro the presence of the Vi antibody.

Virulence and Immunogenic Activities ofB. Typhosusin Relation to its Antigenic Constituents

1. Rough variants, derived from strains ofB. typhosuswhich possess the Vi antigen, may still contain this antigen though the smooth O antigen has been lost.2. Such variants, which are non-virulent, are yet capable of inducing active and passive immunity.3. The virulence ofB. typhosusdepends on the combined activity of the smooth O and the Vi antigen. Nevertheless the Vi antibody alone is sufficient to protect against infection with strains of the highly virulent type.4. The use of avirulent, rough, but Vi-containing variants as vaccines and in the preparation of therapeutic sera is discussed.

The Occurrence of Typhoid Bacilli Containing Vi Antigen in Cases of Typhoid Fever and of Vi Antibody in their Sera

The investigation recorded in the present paper was undertaken as a corollary to the clinical trials with a new antityphoid serum, the results of which were recently published (Felix, 1935). Its object was to enquire into:(i) the occurrence of Vi antigen in strains of B. typhosus freshly isolated from cases of typhoid fever;(ii) the occurrence of Vi antibody in the blood serum of typhoid patients and convalescents.