immunity to pneumococcal infections during immunological paralysis to type i pneumococcal...

IMMUNITY TO PNEUMOCOCCAL INFECTIONS DURING IMMUNOLOGICAL PARALYSIS TO TYPE I

PNEUMOCOCCAL POLYSACCHARIDE

William F. Pike* and David C. Lueker

Department of Microbiology Colorado State University

Fort Collins, Colo.

Assay of the immunological status of pneumococcal polysaccharide-treated mice has conventionally been accomplished utilizing challenge with homologous organisms. Absence of protection in mice receiving paralyzing doses of pneumococcal polysaccharide, followed by a known immunizing dose of the same antigen, to challenge with virulent homologous pneumococci was the criterion used by Felton and coworkers in demonstrating existence of the paralytic state. Apparent lack of an immune response in “paralyzed” mice has been confirmed by many workers utilizing essentially the same methodology and rationale. The concepts of totality of the paralytic state and its lengthy duration, as well as that of hypersusceptibility of paralyzed animals to pneumococcal infection, had their origins in such studies.

Failure to detect a response, however, does not necessarily imply its absence and may instead reflect an inherent imperfection or limitation of experimental design. The plausibility of this suggestion is demonstrated in the present work in which critical examination of the mouse-challenge method was undertaken and in which it was shown that classically paralyzed mice possess demonstrable immunity to challenge with virulent pneumococci.

METHODS AND MATERIALS

Mice. Female Swiss Webster mice obtained from Carworth Farms or maintained without inbreeding in our laboratory were used in each experiment. Each experimental and control group ordinarily consisted of ten to 12 mice. Unless otherwise specified, all animals were challenged when eight weeks old.

Pneumococci. Type I Diplococcus pneumoniae (PnI), obtained from a microbiology department stock culture, was plated on blood agar following serial passage through six mice. Growth was harvested and lyophilized as a lot in separate tubes.

Pneumococcal Polysaccharide. Type I pneumococcal polysaccharide (SI) was prepared in our laboratory using a modification of Heidelberger’s method.* Preliminary characterization of immunogenicity of the preparation in mice indicated that as little as 0.05 pg SI produced uniform immunity to intraperitoneal challenge with 1 0 5 virulent type I pneumococci.

Roure of Injection. With the exception of serum transfer experiments in

* Present Address: Division of Natural Sciences, New Mexico Junior College, Hobbs, N. M.

47

48 Annals New York Academy of Sciences

which injections were made intravneously via the lateral tail vein, all polysaccharide, murine spleen cell, and challenge injections were by the i.p. route.

Preparations for Challenge. An attempt was made to "standardize" viru- lence of the organism by using lyophilized preparations in each experiment. Single passage of the organism from lyophilized stock through mice was made in each experiment followed by plating on blood agar, harvest of growth, and suspension in the appropriate precooled diluent as described subsequently. Sus- pensions were agitated to separate short chains of pneumococci into individual diplococci. The thoroughness of separation was monitored microscopically. Suspensions were adjusted to a fixed optical density and dilutions made to yield desired cell concentrations for challenge purposes and plate counts. Confirma- tion of the number of viable pneumococci composing the challenge dose was made by triplicate plating aliquots of the final dilution on brain heart infusion in agar both before and after challenge injections were made, followed by incubation and colonial enumeration.

TIME PARALYZED IMMUNE NORMAL (DAYS) GROUP CONTROL CONTROL

0 500)rg SI - - I

7 0.5pg SI t 0.5 pg SI

I I t t

14 CHALLENGE CHALLENGE CHALLENGE FIGURE 1. Injection schedule for paralyzed and control groups. No treatment was

given at those positions denoted by a dash. Challenge refers to an i.p. injection of virulent type I Diplococcus pneumoniae. SI is a standard abbreviation for type I pneumococcal polysaccharide.

Challenge. Unless otherwise specified, experimental groups consisted of 1 ) a test group that had received an initial injection of SI known to produce the unresponsive state (500 pg) when six weeks old followed by a known immunizing dose (0.5 pg SI) one week subsequent to the paralyzing dose, 2) control animals immunized (0.5 pg SI) one week before challenge to demonstrate the immunizing capacity of the polysaccharide fraction used, and 3) normal, untreated controls (FIGURE 1). All animals were challenged at the same time. This injection schedule was used in most cases.

Polysaccharide-treated animals were considered paralyzed if they, along with untreated controls, succumbed to infection by homologous pneumococci used in the challenge dose, while immunized controls survived.

In each experiment observations were made and deaths recorded for individual groups at 24-hour intervals following challenge. Final results were tabu-

Pike & Lueker: Pneumococcal Infections 49

lated 72 hours after challenge. Both percent mortality and survival time in- dices were utilized in interpretation of data.

RESULTS

Eflect of Time, Temperature, and Diluent on Survival of Type I Pneumococci

Preliminary work in our laboratory indicated that the number of viable pneumococci constituting the challenge dose is a function of elapsed time following preparation of dilutions for challenge.

Reliance on the mouse-challenge test clearly necessitates standardization of conditions affecting survival of the challenge organisms. Factors such as diluent type, diluent temperature, and the time required for challenging large numbers of animals must be considered to ensure that each animal receives a uniform challenge dose. Review of the literature disclosed that, with few ex- ceptions, the type of diluent employed in challenge injections was not specified and in no case was diluent temperature specified.

Three diluents-phosphate-buffered saline (PBS) , Earle's balanced salt solution (EBSS) , and tryptose broth (TB)-were arbitrarily selected to com- pare the effect of diluent type, time, and temperature on survival of type I pneumococci. Each diluent was adjusted to pH 7.2 before use.

Suspensions were prepared as previously described using diluents precooled (4" C ) or at room temperature (20" C ) to yield cell concentrations of approximately 1,000 pneumococci per milliliter, One-tenth ml aliquots of this final dilution for each diluent and at each temperature were plated in triplicate on brain heart infusion in agar at time zero and at 15, 30, 45, and 60 minutes.

Percent survival was calculated by taking the mean of the triplicate plate counts at time zero as 100% for each temperature and diluent. The mean of plate counts at each 15-minute interval thereafter was divided by the mean of the counts at time zero for the respective diluent and temperature and multi- plied by 100 to obtain percent survival.

It should be noted that the initial mean for each diluent and temperature was reasonably close to the expected value of 100 pneumococci/O.l ml. The range of the means at time zero was from 91 to 108. Similar results have been repeatedly obtained.

Results (FIGURE 2) clearly indicate that diluent type, diluent temperature, and elapsed time are important in maintaining uniform challenge doses of type I pneumococci.

Relationship of Challenge Dose to the Paralytic State

Contrary to Felton's finding .* that paralyzed mice are hypersusceptible to pneumococcal infection, the fortuitous observation was made in our laboratory that although animals that had received a standard paralyzing dose (500 yg SI) did indeed succumb to challenge, they lived somewhat longer than did untreated controls receiving the same challenge dose. This observation was made while using a smaller challenge dose (10' PnI) than that employed by most investigators. Additional preliminary work indicated that demonstration


0 15 30 45 60 TIME (MINUTES)

- 4 C r = T B - 2 0 C A = TB .= EBSS O= EBSS

== PBS 00 PBS FIGURE 2. Survival of type I D. pneumoniue in tryptose broth (TB), phosphate-

buffered saline (PBS), and Earle's basal salt solution (EBSS). Solid lines indicate diluents maintained at 4" C. Broken lines represent diluents maintained at 20" C. Diluent symbols are denoted under the Figure.

of the paralytic state appeared to depend on the number of organisms constituting the challenge dose.

Experiments were therefore designed to determine what effect different challenge doses may have on the degree of paralysis exhibited in animals that had received standard paralyzing doses of type I pneumococcal polysaccharide.

For a given challenge dose three groups of mice were used as previously described (see METHODS AND MATERIALS). Dilutions were prepared and groups of mice were challenged with doses ranging from 10,000 to 50 PnI. The degree of paralysis, as measured by percent mortality, ranged from 100% at the 10,000 to 40% at the 50 pneumococci challenge level, thereby demonstrating a degree of immunity in classically paralyzed mice (FIGURE 3).

Pike & Lueker : Pneumococcal Infections 51

Effect o f Deleting Immunizing Dose Normally Administered Subsequent to Paralyzing Dose

The slight degree of immunity in paralyzed mice suggested the possibility that the small increment of polysaccharide composing the immunizing dose (normally used to demonstrate that the paralytic state exists) could affect the previously described level of immunity. An experiment was therefore designed to study the possible effect of an immunizing dose subsequent to a paralyzing dose at the lower end of the challenge dose spectrum.

It should be noted (FIGURE 4) that when the customary injection schedule was altered, i.e., when an immunizing dose was not administered one week subsequent to the paralyzing injection, a transition to uniform immunity was effected in supposedly paralyzed animals at the 50 pneumococci challenge level. The effect of the immunizing dose is also demonstrable, although to a lesser degree, at the 100 pneumococci challenge level. Little difference was noted in the effect of the amount of immunizing dose (0.5 or 5.0 pg SI), however. Similar results have been obtained in other experiments.

Spleen Cell Transfer to Normal Recipients

The potential value of techniques involving transfer of lymphoid cells in determining whether the unresponsive state is due to inhibition of antibody production or to neutralization of antibody by excess residual antigen suggested their application in the present work, although adoptive transfer experiments

5000 1000 500 250 100 50 OR

MORE CHALLENGE DOSE PNEUMOCOCCI

FIGURE 3. Relationship of challenge dose of type I D. pneumoniae to incidence of paralysis as determined by percent mortality. Only paralyzed groups are included in this histogram. Immune controls were uniformly immune and normal controls experienced 100% mortality in each challenge group.

52 Annals New

p2 p3

York Academy of Sciences

Ij--

p2 p3 100 PNEUMOCOCCI 50 PNEUMOCOCCI CHALLENGE DOSE CHALLENGE DOSE

FIGURE 4, Effect on incidence of paralysis of an immunizing dose subsequent to a paralyzing dose. Only paralyzed groups are shown. Immune controls were uniformly immune and normal controls experienced 100% mortality. Abbreviations: P1=500 pg SI+O.5 pg SI; Pn=500 pg SI+S.O pg SI; Ps=500 pg SI with no subsequent immunizing dose.

by others G-7 appeared to support the concept of absence of antibody production in paralyzed mice.

An experiment was designed to determine if the immunity previously observed in paralyzed animals could be adoptively transferred to normal animals and if, in doing so, the level of immunity could be increased by freeing such isolated lymphoid cells from their antigen-loaded environment.

Paralyzed donors received a paralyzing dose (500 pg SI) and no subsequent immunizing dose two weeks prior to sacrifice and cell transfer to normal recipients. Immune donors received an immunizing dose (0.5 pg SI) one week prior to sacrifice and cell transfer. Normal donors received no treatment prior to sacrifice.

Cell suspensions were prepared using a stainless steel screen expulsion method and were separated from coarse debris. Cells were washed twice in the cold and resuspended in cold Eagle’s basal medium. The volume was adjusted to that of the original suspension. Normal recipient groups received the equivalent of 1.6 spleens ( 150 x 1 O6 nucleated cells) from paralyzed donors, 1 .O spleen (95 x loa nucleated cells) from immune donors, or 2.0 spleens (185 x lo6 nucleated cells) from normal donors. Approximately 92% of the transferred cells were viable, as determined by trypan blue exclusion.

All recipients were challenged 48 hours after cell transfer in an attempt to avoid an active response by the recipient to any polysaccharide carried over in the transfer. Results (FIGURE 5 ) clearly established that protective antibody was being produced by the transferred cells from paralyzed donors. This was indicated by the fact that no animal that received the lysed (six freeze-thaw


cycles) preparation from paralyzed donors survived. Indeed, there appeared to be some indication of an increased response by virtue of the fact that although mortality rates are comparable to those of previous experiments, the recipient acquired (in 1.6 spleens) only 3540% of the total “lymphoid-type tissue’’ found in an intact paralyzed donor.

Serum Transfer to Normal Recipients

A serum transfer experiment was designed to determine if the low level of immunity previously observed in supposedly paralyzed mice was, in fact, due to a humoral factor.

Paralyzed donors received the paralyzing dose (500 pg SI) two weeks prior to sacrifice, with no intervening immunizing dose. Immune donors were immunized one week prior to sacrifice. Normal donors were untreated prior to sacrifice.

Sera were collected from the various donor groups two days before serum transfer and stored at - 10” C. Normal five-week-old recipient groups were injected i.v. via the lateral tail vein with 0.5 cc of the corresponding donor serum per animal. The following day each animal received an additional 0.5 cc of the corresponding donor serum i.p. All groups were challenged 24 hours

w e

FIGURE 5 . Murine spleen cell transfer from paralyzed to normal recipients. Only recipients of cells from paralyzed donors are represented here. Recipients of cells from immune donors survived challenge, whereas recipients of cells from normal donors succumbed. Abbreviations: R1= recipients of cells of 1.6 spleens from paralyzed donors followed by challenge with 50 type I pneumococci (PnI); Rp= recipients of cells of 1.6 spleens from paralyzed donors followed by challenge with 100 PnI; &=recipients of equivalent of cells of 1.6 spleens from paralyzed donors and challenged with 50 PnI.


following completion of serum transfer. Results (FIGURE 6) indicate that the immunity in supposedly paralyzed mice is due to a humoral factor presumed to be homologous antibody.

Duration of the Paralytic State

Pneumococcal polysaccharides were long considered extremely resistant to alteration and to persist in an unchanged form after injection. Later, Stark * I

determined that alteration, as measured by loss of antigenicity, was indeed occurring and that the greatest alteration occurred between the third and eighth weeks following injection of a paralyzing dose.

If, as had been indicated in earlier experiments, the mechanism of the paralytic state involves neutralization of antibody by excess antigen and if polysaccharide alteration was continuously occurring following injection, there might well be an increase in the immunity seen in classically paralyzed mice with the passage of time. Such an increase in immunity could conceivably be detected by the ability of paralyzed mice to withstand greater challenge doses with elapsed time.

A preliminary experiment demonstrated that a number of paralyzed mice were capable of withstanding challenge with as many as 1,000 pneumococci if one month elapsed between injection of the paralyzing dose and challenge.

F~GURE 6. Serum transfer from paralyzed, immune, or normal donors to normal recipients. Abbreviations: P1=recipients of 1.0 cc serum from paralyzed donors followed by challenge with 50 type I pneumococci (PnI); Pz=recipients of 1.0 cc Serum from paralyzed donors followed by challenge with 100 PnI; krecipients of 1.0 cc serum from immune donors followed by challenge with 1,000 PnI; N=recipients of 1.0 cc serum from normal donors followed by challenge with 50 PnI.


6.0 w

4.0

2 3.0 2.0

z w J

I 0

(3 5!

3 1.0 2 4 6 8

TIME(WEEKS SUBSEQUENT TO PARALYZING DOSE)

FIGURE 7. Duration of “paralysis” as determined by increased resistance to challenge with type I pneumococci. The Figure represents maximum challenge doses at which uniform immunity in a group was obtained. Higher challenge doses than those shown produced different degrees of mortality in other groups at the respective time.

A more detailed experiment was designed to determine the immunological status of paralyzed mice at two-week intervals over a period of eight weeks. It is apparent (FIGURE 7) that recovery from the paralytic state induced by SI occurs at a comparatively rapid rate. Eight weeks following administration of the paralyzing dose, supposedly paralyzed animals were capable of withstanding approximately 2,000 times the challenge dose that they could withstand two weeks subsequent to a paralyzing dose. Such mice, however, do not possess as great a degree of immunity as that observed in mice receiving an immunizing dose of 0.5 pg SI alone and undoubtedly could be shown to be “paralyzed” provided the challenge dose was sufficiently great. It seems probable that sufficient residual polysaccharide is present in antibody-forming tissue to produce this effect.

Residual Polysaccharide Detection in Serum

Although another experiment demonstrated the presence of immunogenic SI in spleens of paralyzed mice two weeks subsequent to the paralyzing dose (unpublished results), it appeared unlikely, due to the immunity previously observed in paralyzed mice, that immunogenic SI was present in serum at the end of a comparable period of time.


To test this supposition, serum was collected from mice receiving the standard paralyzing dose of SI at several intervals up to, and including, the 16th day following administration of the paralyzing dose. Normal recipients were injected i.p. with either undiluted or diluted sera from grouped donors. All recipients were challenged one week later using a range of challenge doses for the various groups. Unexpectedly, recipients of diluted or undiluted sera from paralyzed animals possessed near uniform immunity, thereby indicating the presence of immunogenic SI in sera of paralyzed mice at the end of 16 days at dilutions as great as 1 : 100. This anomaly is considered subsequently.

DISCUSSION

Central to elucidation of the mechanism involved in the paralytic state is the question whether excess residual antigen neutralizes antibody as it is produced or, alternatively, whether the unresponsive state is a result of inhibition, i.e., functional elimination, of immunologically competent cells.

Assertion of “hypersusceptibility” to pneumococcal i n fe~ t ion ,~ lack of SUC- cess by many workers in detecting immunity in paralyzed mice by the challenge method, failure to detect antibody-containing cells in paralyzed mice with immunofluorescent methods,lo lack of detectable histological response in lymph nodes of paralyzed animals,” and negative results associated with adoptive transfer studies 5-7 led to general acceptance of the concept that no antibody production takes place in pneumococcal polysaccharide-paralyzed mice.

As previously noted, however, failure to detect an immune response does not necessarily imply its absence. Indeed, validity of conclusions based exclusively on negative results would be contingent upon infinite sensitivity of the techniques employed. It therefore appears relevant to examine the aforementioned experimental findings since they represent distinct contrasts to results of the present investigation.

Sercarz and Coons 10 found no antibody-containing cells in spleens of mice paralyzed with SII. It should be noted, however, that even in control animals that had received a known immunizing dose of SII the number of positive cells was quite small. Sensitivity of immunofluorescent techniques in detecting antibody production in polysaccharide-treated mice is questionable in view of our investigation and the accomplishment of Howard and colleagues l2 in demonstrating large numbers of antibody-forming cells in spleens of SIII-paralyzed mice by an immunocytoadherence method.

Gitlin and coworkers 11 found no significant signs of antibody production in lymph nodes of mice receiving paralyzing doses of pneumococcal polysaccharide. The significance of this observation is somewhat doubtful since an increment in plasma cell production was used as a measure of antibody production. As noted by other workers,1Z the majority of rosette-forming cells in the spleens of paralyzed mice appear to be blast cells o r large lymphocytes.

Adoptive transfer techniques are of critical importance in the study of phenomena associated with an immunologically unresponsive state for they provide a means of investigating the immunological function of isolated lymphoid cells which have been freed from an antigen-loaded environment or, con- versely, of immune cells transferred to an antigen-loaded environment. Brooke and Karnovsky,6 with SII, and Neeper and Seastone,6 with SI, .concluded that normal recipients showed no immunity two days following injection of spleen


cells of paralyzed donors, as detected by challenge with homologous pneumococci. Brooke reached the same conclusion with SIII-paralyzed mice and assay of immunological status by his hemagglutination technique.13 Similar experiments by the same workers in which paralyzed mice were successfully adoptively immunized by i.p. injection of spleen cells from immune donors appeared to further substantiate the case for an inhibition mechanism.

Successful adoptive transfer in our work and indications of an increased response by cells freed from their antigen-loaded environment was undoubtedly due to the fact that low challenge doses were used. This may be contrasted with the negative results previously cited by examination of experimental design. Brooke and Karnovsky employed 1,000 lethal doses in evaluating the status of normal recipients of spleen cells from paralyzed donors. Neeper and Seastone 6

demonstrated adoptive immunity in normal recipients of spleen cells from SI-immune animals by challenge with 70 virulent homologous pneumococci. The immunological status of recipients of spleen cells from SI-paralyzed animals, however, was evaluated using about six times as many organisms in the challenge dose.

Results of our investigation, demonstrating immunity in supposedly paralyzed mice, are in opposition to Felton’s conclusion that paralyzed mice are hypersusceptible to pneumococcal infection when compared to normal controls. This contrast in experimental findings warrants limited speculation in an attempt to account for the differing observations. It appears likely that animals were challenged with culture dilutions containing only a few encapsulated organisms of the “relatively avirulent strain” Felton used for challenge purposes. In view of evidence demonstrating a brief inductive phase to pneumococcal polysaccharide antigens,I4 it seems quite possible that some normal animals were capable of mustering an immune response, perhaps to pneumococcal polysaccharide present in the diluent, and that this response arrested the infection. Response of paralyzed animals, however, that received a large dose of polysaccharide simultaneously with the challenge dose was probably abrogated due to the large pool of circulating antigen, which neutralized any antibody that may have been produced. Consequently, a fatal infection ensued.

Lack of success in detecting immunity in paralyzed mice by the mouse- challenge method may well be due to the fact that in most instances the importance of the number of organisms composing the challenge dose has been overlooked. Challenge doses have been, in general, of sufficient magnitude to mask the slight degree of immunity present in mice two weeks following administration of the paralyzing dose. Indeed, even mice treated with known immunizing doses of 0.5 or 5.0 pg SI may be shown to be “paralyzed” provided the challenge dose is sufficiently great (unpublished results).

It should also be noted that study of the paralytic state utilizing the mouse- challenge test has been generally impeded by the fact that the significance of the effect of diluent type, diluent temperature, and elapsed time following preparation of dilutions on maintenance of uniform challenge doses of pneumococci has been overlooked by most workers.

Pertinent to consideration of the mechanism involved is the question whether the paralytic state is of permanent or finite duration. Although much importance has been attached to Felton’s report that the paralytic state is of extended duration,’ it has been predicted l6 and repeatedly demonstrated 16-19 that spontaneous transition to immunity occurs in from two to four months. The short- term nature of paralysis is confirmed in the present work in which it was shown


that immunity is detectable two weeks following administration of the paralyzing dose and that such mice are capable of increased resistance to pneumococcal infection with the passage of time. Eight weeks following administration of the paralyzing dose mice withstood challenge with 2,000 times as many pneumococci as they were capable of withstanding two weeks subsequent to the paralyzing injection.

Other than spontaneous transition to immunity,16 the earliest indication that injected pneumococcal polysaccharides were incompletely resistant to alteration was Felton's observation *O that type I pneumococcal polysaccharide extracted from spleens of SI-paralyzed mice one month after injection of the paralyzing dose retained only about one percent of its original activity. Stark,8 with 14C-SI, also noted a marked decrease in antigenicity of type I pneumococcal polysaccharide extracted from spleens of paralyzed mice. He observed that the greatest alteration of SI, as measured by loss of antigenicity, occurred between the third and eighth week postinjection. Radioactivity of splenic extracts, however, remained essentially unchanged, indicating persistence of altered antigen.

If alteration of pneumococcal polysaccharide results from antigen-antibody interaction, antigen alteration should bear a relationship to the amount of antibody present. Stark s tested this hypothesis in two ways. He found a decrease in the rate of loss of SI antigenicity of splenic extracts from paralyzed mice when antibody formation was inhibited by whole body irradiation. In a varia- tion of this experiment he reported an accelerated decrease of antigenic SI in paralyzed mice receiving rabbit anti31 serum.

The importance of antibody neutralization by a circulating pool of the corresponding antigen was further illustrated by Stark's determination 9 that immune mice injected i.p. with as little as 15,g SI succumbed when challenged one hour later. The counterpart to this phenomenon was observed in the present work when it was determined that an immunizing dose of 0.5 or 5.0 ,g subsequent to the paralyzing dose had a negative effect on the slight degree of immunity in paralyzed mice. This finding, coupled with similar results obtained later, is consistent with and indicative of an in vivo neutralization mechanism.

In view of the preceding, it appeared that demonstration of immunity in paralyzed mice depended upon removal of the circulating pool of pneumococcal polysaccharide. That depletion and sequestration of the circulating pool of antigen do occur was suggested by the work of other investigators s, in which paralyzed mice were adoptively immunized by spleen cells from immune donors.

Such work may be contrasted with the observation 21 that detectable immunogenic SII remained in sera of paralyzed mice as long as ten days postinjection and with the present work in which immunogenic SI was present in sera up to, and including, the 16th day following administration of the paralyzing dose. The apparent contradiction in the latter observation bears closer examination for it occurs at a time when paralyzed mice possess demonstrable immunity to pneumococcal infection.

It appears reasonable to assume that this phenomenon is the result of one or more of three conditions: 1) that antibody production occurs at a rate somewhat greater than the rate of neutralization by the small amount of antigen remaining in circulation; 2) that slight alteration of antigen remaining in the circulation results in reduced avidity of the antigen for the antibody present, even though the antigen remains immunogenic; and 3) that two types of antibody are produced, perhaps as a result of a minor molecular species in the purified antigen, a possibility suggested by work of other investigators.22- 23


In any event, immunity is detectable in paralyzed mice two weeks subsequent to the paralyzing injection,** and dramatic manifestation of an increased response occurs with elapsed time. In view of the slight degree of immunity in paralyzed animals two weeks postinjection, however, it is unlikely that immunity could be detected before this time by the mouse-challenge method. Develop- ment of a technique 25 of comprehensive applicability for investigation of early response at the cellular level, however, is indicative of resolution of this impediment.

Results of the present work should not be extrapolated without qualifica- tion to include pneumococcal polysaccharides other than SI. The greater paralytic potency associated with other pneumococcal polysaccharides leads us to predict that increased time will be necessary for detection of an immune response using the mouse-challenge method, but that similar observations will be made subsequent to that time.

1.

2.

3.

4.

5.

6.

10.

11.

12.

13.

REFERENCES

FELTON, L. D., G. KAUFFMANN, B. F‘RESCO-IT & B. O ~ N G E R . 1955. Studies on the mechanism of the immunological paralysis induced in mice by pneumococcal polysaccharides. J. Immunol. 7 4 17.

HEIDELBERGER, M., F. KENDALL & H. SCHERP. 1936. The specific polysaccharides of types I, 11, and 111 pneumococcus. J. Exp. Med. 64: 559.

NEEPER, C. A. & C. V. SEASTONE. 1964. Mechanisms of immunologic paralysis by pneumococcal polysaccharide. N. Comparison of polysaccharide and whole organisms. J. Immunol. 93: 867.

FELTON, L. D. & G. H. BAILEY. 1926. Biologic significance of the soluble specific substances of pneumococci. J. Infect. Dis. 38: 131.

BROOKE, M. S. & M. J. KARNOVSKY. 1961. Immunological paralysis and adoptive immunity. J. Immunol. 87: 205.

NEEPER, C. A. & C. V. SFASTONE. 1963. Mechanism of immunologic paralysis by pneumococcal polysaccharides. I. Studies of adoptively acquired immunity to pneumococcal infection in immunologically paralyzed and normal mice. J. Immunol. 91: 374.

BROOKE, M. S. 1966. Studies on the induction, specificity, prevention, and break- ing of immunologic paralysis and immunity to pneumococcal polysaccharides. J. Immunol. 9 6 364.

STARK, 0. K. 1955. Studies on pneumococcal polysaccharide. Mechanism involved in production of “immunological paralysis” by type I pneumococcal polysaccharide. J. Immunol. 74: 130.

STARK, 0. K. 1959. Further observations on immunologic unresponsiveness induced by type I pneumococcal polysaccharide. In The Mechanism of Hyper- sensitivity. J. H. ShafTer, G. A. Lo Grippo, & M. W. Chase, Eds. : 519. Little, Brown. Boston, Mass.

SERCARZ, E. & A. H. COONS. 1959. Specific inhibition of antibody formation during immunological paralysis and unresponsiveness. Nature 184: 1080.

GITLIN, D., F. MONCKEBERG & J. M. CRAIG. 1958. The nature of “immunological paralysis” induced by pneumococcal polysaccharides. Amer. J. Dis. Child. 9 6 496.

HOWARD, J. G., J. ELSON, G. H. CHRISTIE & R. G. KINSKY. 1969. Studies on immunological paralysis. 11. The detection and significance of antibody- forming cells in the spleen during immunological paralysis with type 111 pneumococcal polysaccharide. Clin. Exp. Immunol. 4: 41.

BROOKE, M. S. 1966b. A hemagglutination test and a specific depolymerase for studying immunologic paralysis in mice. J. Immunol. 9 6 358.


14. BAKER, P. J. & M. LANDY. 1967. Brevity of the induction phase in the immune response of mice to capsular polysaccharide antigens. J . Immunol. 99: 687.

15. LUDWIG, K. A., F. J. MURRAY & E. J . STAUBACB. 1949. A note on the assay of antigenic polysaccharides and vaccines. J. Immunol. 62: 257.

16. DOWNIE, A. W. 1937. The specific capsular polysaccharide of pneumococcus type I and the immunity which it induces in mice. J. Path. Bacteriol. 45: 131.

17. BAER, H., J. K. BRINGAZE & M. MCNAMEE. 1953. The effect of the dose of bacterial polysaccharide antigen on antibody produced in mice. J. Bacteriol. 67: 123.

18. SISKIND, G. W., P. Y. PATERSON & L. THOMAS. 1963. Induction of unresponsiveness and immunity in newborn and adult mice with pneumococcal polysaccharide. J. Immunol. 90: 929.

19. NEEPER, C. A. 1964. Mechanisms of immunologic paralysis by pneumococcal polysaccharide. 111. Immunologic paralysis in relation to maturation of the immunologic response of mice. J . Immnuol. 93: 860.

20. FELTON, L. D. 1949. The significance of antigen in animal tissues. J. hmunol. 61: 107.

21. HOWARD, J. G. & G. W. SISKIND. 1969. Studies on immunological paralysis. I. A consideration of macrophage involvement in the induction of paralysis and immunity by type I1 pneumococcal polysaccharide. Clin. Exp. Immunol. 4: 29.

22. R. KEARNEY & W. J. HALLDAY. 1970. Humoral and cellular responses of mice to a pneumococcal polysaccharide antigen. Circulating antibodies. Aust. J . Exp. Biol. Med. Sci. 48: 215.

23. EkIKsEN, J. 1g69. Antibodies against degradation products of polysaccharide antigens. Immunology 17: 33.

24. PIKE. W. F., D. C. LUEKER & I. R. COLLIER. 1969. Demonstrable immunity in paralyzed.mice. Bact. Proc. 90.

25. BAKER, P. J., P. W. STASHAK & B. PRESCOTT. 1969. Use of tized with purified pneumococcal polysaccharides for the and antibody-producing cells. Appl. Microbiol. 17: 422.

erythrocytes sensi- assay of antibody

DISCUSSION

DR. CHASE: I would like to ask two questions for clarification. I think two slides were projected and taken off before my comprehension was complete. I want to ask you when after the administration of serum did you make the challenge? I don’t think you told us the time interval.

DR. PIKE: I think that I did mention that it was the day following completion of serum transfer.

DR. CHASE: What do you mean by completion of serum transfer? DR. PIKE: This was done over a period of two days, and the last challenge

DR. CHASE: How many injections were given in the two days. DR. PIKE: A total of three injections, counting the challenge injection. DR. CHASE: No, I mean the injection of serum. DR. PIKE: Two injections, then. DR. CHASE: One each day? DR. PIKE: Yes. DR. CHASE: Now, you had some data that puzzled me. What interpretation

have you placed upon the fact that the recipients challenged with 1,000 pneumococci are still as resistant as are the animals given 50 bacteria? Those given intermediate doses show so much mortality.

injection was made at the end of 24 hours.


DR. PIKE: Do you mean for the differences between higher and lower challenge groups?

DR. CHASE: Yes. DR. PIKE: I think that the level of circulating antibody is quite small and

extrapolation from the data of other workers leads me to believe that approximately 1 ng of antibody is present.

DR. CHASE: Would this experiment be repeatable? DR. PIKE: I assume that it would be, although it hasn’t as yet been repeated.

I would expect something very similar to this. We’ve seen, in other experiments, that the difference between 50 and 100 bacterial cells is reproducible, as far as percent mortality.

DR. CHASE: What do you mean by “recipients of equivalent cells of 1.6 spleens challenged with 50 pneumococci”?

DR. PIKE: The difference there is that the mice received lysed cells rather than intact cells.

DR. CHASE: Thank you. I was a little confused and I thought others might also have missed some of the points.

immunity to pneumococcal infections during immunological paralysis to type i pneumococcal...

Documents