Correlation of the Duration and Magnitude of Protection

Transcription

1 INFECTION AND IMMUNITY, Feb. 1980, p /80/ /09$02.00/0 Vol. 27, No. 2 Correlation of the Duration and Magnitude of Protection Against Salmonella Infection Afforded by Various Vaccines with Antibody Titers CARL R. ANGERMANt AND TOBY K. EISENSTEIN* Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania Groups of mice were immunized with optimal doses of the following vaccines of Salmonella typhimurium W118-2: acetone-killed cells, lipopolysaccharide, ribosomes, and live cells. At 3 weeks, 1 month, 2 months, 4 months, or 6 months postimmunization, sera were collected from control and vaccinated animals, and the anti-lipopolysaccharide and whole-cell agglutination titers of the sera were determined. Other groups of similarly vaccinated mice were tested for resistance to infection by challenging with live W118-2 and scoring the number of survivors 30 days postinfection. It was found that only ribosomes and live cells afforded significant protection 6 months after immunization. Thus, in duration of protection ribosomes were superior to the other nonviable vaccines tested. At all time intervals tested, purified lipopolysaccharide was the least effective vaccine. Protection afforded by the acetone-killed cell and ribosomal vaccines correlated better with the whole-cell agglutination titers than with the anti-lipopolysaccharide titers. However, the longer duration of protection afforded by the ribosomal vaccine, as compared with the acetone-killed vaccine, could not be accounted for by differences in whole-cell agglutination titers. These studies show that ribosomal vaccines are equal in all parameters to acetone-killed cells and have the advantage of providing longer-lasting immunity. The experiments reported in this paper are part of a larger study in which we have attempted to systematically compare various Salmonella vaccines for their ability to protect mice against Salmonella infection (1). The impetus behind these studies stems from a desire to develop an improved vaccine to protect human populations against typhoid fever. At present, various typhoid vaccines are in use around the world, but all consist of whole cells killed in different ways, such as heat killed, phenol preserved, acetone killed, or alcohol killed (18). Although many of these preparations have been shown in field trials to confer excellent protection of at least 7 years in duration (2), all of these vaccines are less than optimal, because they have considerable toxicity and require at least one booster dose. Since Salmonella typhi is exclusively a human pathogen, investigators have used infection of mice with the natural murine pathogen, Salmonella typhimurium, as a model for testing the efficacy of various vaccine preparations (15, 16, 23, 28). Over the years a rather vast literature has grown up using this mouse model of Salmonella infection, which has t Present address: State of Maryland Department of Health and Mental Hygiene, Eastern Shore Regional Laboratories, Salisbury, MD yielded information not only about the potency of various vaccines, but also about fundamental features of the host-parasite interactions in this infection. Studies in mice have led to the generally accepted conclusion that there are two different types of immune responses to Salmonella infection (23, 26, 36, 37). Living cells or killed bacteria administered in complete Freund adjuvant induce cellular and humoral immunity (7, 19, 23), whereas whole killed cells or lipopolysaccharide (LPS) give rise only to humoral immunity (14, 34). In all studies comparing the efficacy of various types of vaccines, live cells have been found to give the greatest degree of protection (1, 23, 31). This fact, together with several reports in the literature of very poor protection in the mouse model by killed whole vaccines (15, 23) (in spite of the excellent results in human field trials [18]), has led several investigators to favor the development of a live-cell vaccine for human use (6, 11, 13). There are, however, disadvantages to giving living organisms, including the danger of virulent revertants. Thus, when ribosome-rich extracts of various bacteria were found to be protective (41; T. K. Eisenstein, Ph.D. thesis, Bryn Mawr College, Bryn Mawr, Pa., 1969), there was considerable interest in the 435

2 436 ANGERMAN AND EISENSTEIN possibility that this type of subcellular preparation might be a superior type of nonliving vaccine. Ribosomal vaccines prepared from various Salmonella species have been tested in several different laboratories, including our own (8), and found to be highly protective. There is considerable controversy, however, as to the nature of their protective immunogens and their mechanism of action (9). Some investigators have suggested that these vaccines are unique among nonliving vaccines in eliciting cellular immunity (25, 42) and in providing levels of protection comparable to those of living cell vaccines (38, 40) Ṫo evaluate the potential of a ribosomal vaccine, we have systematically compared such a preparation in a number of parameters with acetone-killed cells, LPS, and live cells. The toxicity and protection studies have been reported already (1). It was found that when each vaccine was given at its optimal dose, and protection was tested 3 weeks postvaccination, live cells gave the best protection, ribosomes and acetone-killed cells gave high levels of protection only slightly less than those engendered by the live cells, and the LPS was markedly inferior. Of the nonliving vaccines, LPS was found to be the most toxic, and the acetone-killed cells and ribosomes were equal in their ratio of toxicity to efficacy (therapeutic index). In the present experiments, the humoral responses engendered by each of these vaccines were determined, and the vaccines were compared in terms of the duration of protection each induced over a 6- month period postvaccination. The results are discussed in terms of the efficacy of the various vaccines and the mechanisms of immunity to Salmonella infection. MATERIALS AND METHODS Animals. Female CD-1 mice, 19 to 21 g, were purchased from Charles River Mouse Farm, Wilmington, Mass. They were housed in plastic disposable cages with Absorb-Dri for bedding. Purina Mouse Chow was available ad libitum, and fresh water was provided in plastic sterilizable bottles. Mice of the same age were used in all experiments. Organism. S. typhimurium W118-2 was kindly provided by Samuel Formal of the Walter Reed Army Institute of Medical Research. This strain is strongly agglutinated with 0:1,4,5,12 and H:i;1,2 antisera (Difco) and exhibits typical biochemical reactions. The intraperitoneal 50% lethal dose (LD50) is 104 organisms for CD-1 mice, as determined by the method of Reed and Muench (30). Bacterial storage and culture. Stock cultures of strain W118-2 were stored lyophilized at -20'C. Organisms used for both immunization and challenge were prepared in the following manner. A lyophilized culture was rehydrated with brain heart infusion broth INFECT. IMMUN. and plated on blood agar. Several typical colonies were used to inoculate 10 ml of brain heart infusion broth, which was grown for 4 h at 360C to early log phase. A 5-ml portion of this broth was used to inoculate a 50- ml flask of brain heart infusion broth, which was incubated at 360C on a rotary shaker until turbid (about 5 x 108 cells per ml). The concentration of cells in the culture was estimated by direct microscopic counts. The flask was put on ice, and a 1-ml portion was mixed with 4 ml of a 5% paraformaldehyde solution to stop motility. The bacteria were then counted in a Petroff-Hausser chamber, and the culture was diluted in saline to the desired concentration. The actual number of viable bacteria in the culture was determined by duplicate plate counts. Preparation of vaccine. Vaccine preparations were exactly the same as those used in the previous study (1). Briefly, LPS was obtained by phenol-water extraction as described by Nowotny (27), acetonekilled cells were prepared by following the procedure of Landy (21), and ribosomal vaccine was extracted by using the Fogel and Sypherd method (12). Immunization. Mice were immunized with one intraperitoneal (i.p.) injection of vaccine in a volume of 0.5 ml. When appropriate, vaccines were suspended or diluted with sterile, nonpyrogenic saline (Abbott Laboratories). Controls received saline. For immunization with live cells, organisms were grown to log phase as described above, counted, and diluted to the desired concentration. The number of cells injected was confirmed by duplicate plate counts. In the previous paper in this series (1), the use of optimal dosages for each vaccine, rather than equal microgram dosages of different preparations, was extensively justified. The rationale behind this concept lies with the desire to test which vaccine is the best, under conditions in which each vaccine is performing optimally. Studies to compare the specific activity of various vaccines (i.e., protective capacity per microgram of vaccine) were carried out (1) but are not relevant to the design of the experiments to be presented here. Challenge. At the desired time interval after immunization, mice were challenged i.p. with the desired dosage of live strain W118-2 in 0.5 ml of saline. Cells were grown, and the inoculum was standardized and verified as described above. Protection was assessed by 30-day survival. Collection of serum. Groups of six mice were immunized i.p. and, at the desired time interval postvaccination, were anesthetized with chloroform and bled from the heart. Individual serum samples were clotted at room temperature for 1 h and then kept overnight at 4 C. Equal amounts of serum were collected from each of the six blood samples and were pooled. A portion of the pooled serum to be used for hemagglutination (HA) was absorbed twice for 30 min at 36 C with an equal volume of washed human erythrocytes to remove nonspecific agglutinins. The serum was either used immediately or stored in small volumes at -20 C. For the serology on mice immunized at staggered intervals of 3 weeks to 6 months, all animals were bled on the same day. Sera were stored in the refrigerator, and all samples were tested the next day against the same batch of coated and uncoated erythrocytes.

3 VOL. 27, 1980 HA titers to W118-2 LPS. The same LPS used for vaccination was also used for the passive HA assays. The LPS was attached to human, Rh-negative, group 0 erythrocytes according to the method of Neter as described by Nowotny (27). Ten milligrams of LPS was dissolved in 5 ml of sterile saline and boiled for 2 h, after which the volume was measured and solid NaCi was added to a final concentration of 0.85%. Washed, packed fresh human erythrocytes in a volume of 0.5 ml were added to 2.5 ml of the boiled LPS solution, and the mixture was incubated at 360C for 1 h. The coated cells were washed three times with saline and suspended to a final concentration of 1%. The same concentration of uncoated erythrocytes was used as a control. Serum (0.025 ml) was serially diluted with an equal volume of saline containing 1% bovine serum albumin in microtiter plates with tapered walls (Microbiological Associates). A ml volume of the 1% suspension of erythrocytes was added to each well. The plates were incubated at room temperature for 2 h, and the wells were scored from 4+ to 1+. The wells were scored again after being held overnight at 40C. The titer was taken as the highest dilution giving a 3+ reaction. Typing serum (0:1,4,5,12; Difco) and a standard, positive serum sample raised against acetonekilled cells were used as positive controls. Serum from control mice injected with saline served as the negative control. Whole-cell agglutination (WCA). The same serum samples used for determination of HA titers were also tested for whole-cell agglutinin levels. Serum (0.5 ml) was added to the first of a series of 10 tubes (16 by 100 mm) containing an equal volume of saline and then was serially diluted to give a final volume of 0.5 ml per tube. Then, 0.5 ml of a 5 x 108 suspension of acetone-killed cells was added to each tube. The tubes were incubated at 360C for 1 to 2 h and then held overnight at 40C. Tubes were scored from 4+ to 1+, and the titer was determined as the highest dilution giving a 3+ reaction. As positive controls, typing serum (Difco) and a positive serum raised against killed cells were used. As a negative control, serum was obtained from mice injected with saline. Statistics. The levels of significance for the observed frequencies (Pi) were determined by Fisher's exact test for 2 x 2 tables (5). IMMUNITY TO SALMONELLA INFECTION 437 RESULTS Dose response: anti-lps titers. Groups of six mice were immunized i.p. with graded doses of LPS, acetone-killed cells, or ribosomes. At 3 weeks postimmunization, sera were collected from the mice, pooled in equal quantities, and tested for antibody to LPS by passive HA using LPS-coated human erythrocytes. Figure 1 shows the antibody response as a function of increasing dosage of vaccine. Low doses (1 to 100,Ig) of each vaccine were capable of substantially raising the titer, and there was a rapid rise with increasing doses in the low dose range. The titers reached a maximum at about 100lg of antigen. In a previous study, in which a systematic comparison had been made of the protective capacity and toxicity of these four vaccines, an optimal dose had been determined for each preparation based on its ability to give maximal protection with minimal toxicity (1). The optimal doses were 100 lag for LPS, 250,tg for acetone-killed cells, 1,000 jg for ribosomes, and 6.5 x 103 live cells. As shown in Fig. 1, the optimal immunizing dose of each vaccine gave a maximal antibody response in the plateau range of its respective curve. Note that mice immunized with a neartoxic dose of killed cells showed a decrease in antibody titer. Comparison of WCA titers and LPS titers with survival. Groups of 10 mice each were immunized with the optimal doses of each vaccine and 3 weeks postimmunization were challenged with 1,000 LD50 of live W Additional groups of six animals each were used to collect immune sera 21 days postimmunization. WCA titers and anti-lps titers were determined for each group. In Fig. 2, the serological responses are compared with the survival data. The major conclusion to be drawn from these data is that a high titer in the passive HA test is Mg RIB FIG. 1. Effect of vaccine dosage on the anti-lps HA titers. Three weeks postimmunization with the designated antigen, pooled sera of six mice were collected and titrated by passive HA using W118-2 LPS-coated erythrocytes. AKC, Acetone-killed cells; RIB, ribosomal vaccine.

4 438 ANGERMAN AND EISENSTEIN cc cl 0 co z mg 250 jig 1000 m9g 01 LDso FIG. 2. Correlation of antibody titers with protection at 3 weeks postimmunization. Mice were challenged with 107 live cells of W (a) Anti-LPS HA titer; (U) WCA; (E) protection against 1,000 LDrko. not sufficient to insure high levels of protection. The magnitude of the anti-lps response in mice immunized with the three different nonviable vaccines was similar, but the protection elicited by LPS was only 10% of that provided by killed cells and ribosomes. Thus, immunization with LPS gives high anti-lps titers, but low levels of protection. In contrast, live-cell immunization resulted in negligible anti-lps titers, but in a substantial level of protection. Comparison of the titers of the LPS- and the live-cell-vaccinated animals shows that the two groups of mice had identical WCA titers (1:16), but widely different HA titers (1:256 versus 1:2). These results suggest that a positive agglutination reaction is dependent on factors other than the anti-o response, which is detected by passive HA Ṅote that both acetone-killed cells and the ribosomal vaccine induce high levels of protection, as well as substantial antibody titers, as measured in both assays. Duration of protection. The foregoing studies used a 3-week interval between immunization and challenge. To determine which vaccine affords the greatest protection over longer periods of time, groups of 10 mice were immunized with the optimal dose of each vaccine at 6 months, 4 months, 2 months, or 3 weeks before challenge with a high or low dose of live organisms. At each time period, there were two control groups of six mice each which were injected with saline and challenged with the low dose. Mice were all received in the same shipment. The time of immunization was staggered so that all animals were of the same age at the time of challenge, and all groups received exactly the same high or low challenge dose. Figure 3 shows the survival data for mice receiving the low challenge dose cr U0 (400 LD5o), and Fig. 4 shows the results for the high challenge dose (710 LD50). All control mice succumbed. At 3 weeks postimmunization, all vaccines appeared equal in giving excellent protection against either the high or the low challenge dose. This result is in agreement with our previous studies (1). At 2 months postimmunization, the live-cell vaccine, the acetone-killed cells, and the ribosomal vaccine showed a drop in potency, although the differences between the 3-week and the 2-month survival values were not statistically significant. In contrast, the LPS afforded no protection at 2 months. At 4 months 100 B0.;60 ~: IS0 Days Post-I'miunization FIG. 3. Duration ofprotection by various vaccines. Groups of 10 mice each were all received on the same date and were immunized at staggered intervals with the optimal dose of the designated vaccine. (0) LPS, 100 jg of LPS; (M) killed cells, 250 jig of acetonekilled cells; (-) ribosomal preparation, 1,000 jig; (F) live cells, 103 cells. All animals were challenged on the same day with 4 x 106 live cells of W INFECT. IMMUN Days Post-Immunization FIG. 4. Duration ofprotection by various vaccines. Groups of 10 mice each were all received on the same date and were immunized at staggered intervals with the optimal dose of the designated vaccine. (E) LPS, 100 pg of LPS; (0) killed cells, 250 ug of acetonekilled cells; (U) ribosomal preparation, 1,000 pg; (F) live cells, 103 cells. All animals were challenged on the same day with 7.1 x 106 live cells of W118-2.

5 VOL. 27, 1980 IMMUNITY TO SALMONELLA INFECTION 439 postvaccination the protection engendered by vaccines. For the next 4 months the titer remained at the same high level found in the acetone-killed cells had decayed to 50% of its level at 3 weeks postvaccination. The reduction killed-cell- and ribosome-immunized mice. is significant for both high and low challenge These same serum samples were also used for doses, and the protective capacity continued to determination of WCA titers. The results (Fig. diminish to even lower levels by 6 months postvaccination. Acetone-killed cells afforded significant protection above controls at 4 months (120 days) but not 6 months (180 days) postvaccination. At 4 or 6 months postvaccination, LPS still afforded the least protection when compared with the other vaccines, but interestingly, and surprisingly, there were more LPS-vaccinated survivors at these time periods than at 2 months (60 days). The live-cell vaccine and the ribosomal vaccine showed a somewhat slower loss of their protective capacities compared with acetone-killed cells, but the differences were not statistically significant. When the low challenge dose was used, the ribosomes, the live cells, and the acetone-killed cells afforded significant levels of protection at 120 days (4 months) postimmunization, whereas the LPS did not. At 6 months postvaccination only the live cells and ribosomal vaccine afforded significant protection against the low challenge dose. When the animals were subjected to the high challenge dose, only the ribosomal vaccine and the live cells gave significant protection 60 and 120 days postvaccination, and none of the vaccines gave significant protection at 180 days. Thus, the ribosomal vaccines induced an immunity comparable in duration to that of live cells, and somewhat higher than that resulting from vaccination with whole killed cells or LPS. The results suggest that ribosomal vaccines may afford a somewhat greater duration of protection than the other nonliving vaccines tested. Duration of the antibody response. Additional groups of six mice each were immunized with the optimal doses of each vaccine at 6 months, 4 months, 2 months, and 3 weeks postvaccination. Sera were collected and pooled, and the anti-lps and WCA titers for each group were determined. This experiment was conducted simultaneously with the duration-of-protection experiment. The duration of the anti- LPS response is shown in Fig. 5. The HA titers induced by each of the three nonviable vaccines were similar, showing a rapid rise by 3 weeks postimmunization. This high level of antibody was maintained over the next 5 months in ribosome- and killed-cell-vaccinated mice, but the titer in LPS-vaccinated mice decreased rapidly at 2 months and then remained at a low level for the remaining 4 months. Mice immunized with live cells showed no anti-lps response until 6 weeks postimmunization, but by 2 months the titer was equal to that elicited by the nonviable 6) show a different response from that seen when anti-lps titers were measured. Killed cells and ribosomes gave high agglutination titers at 3 weeks postimmunization, and they remained constant for the next 5 months. Immunization with LPS resulted in an agglutination titer that was only half of that given by the other nonviable vaccines, and this titer decreased to a negligible level by 4 months postvaccination. Although immunization with live cells gave no anti-lps titer at 3 weeks, a substantial agglutination titer, equal to that seen in LPS-immu- U, I- z 0 c: MONTHS POST-IMMUNIZATION FIG. 5. Duration of the anti-lps passive HA antibody response. Groups of six mice were immunized i.p. with the optimal doses of each vaccine. At the indicated timespostvaccination, serum was collected, pooled, and titrated. AKC, Acetone-killed cells; RIB, ribosomal vaccine; Live, live-cell vaccine. z ' 2 10 ~~~AKC RIB 0IV MONTHS POST- IMMUNIZATION FIG. 6. Duration of the WCA antibody response. Groups of six mice were immunized i.p. with the optimal doses of each vaccine. At the indicated times postvaccination, serum was collected, pooled, and titrated. AKC, Acetone-killed cells; RIB, ribosomal vaccine; Live, live-cell vaccine.

6 440 ANGERMAN AND EISENSTEIN nized mice, was demonstrable at that time. Correlation of the duration of the antibody response with survival. In Fig. 7, the LPS and agglutination titers were plotted together with the survival data for each of the vaccines. For the nonviable vaccines, the curves for LPS and WCA antibody seem to parallel each other. In general, the antibody curves also follow the survival data; that is, when there is a decrease in survival, there is also a decrease in antibody level, although usually not of the same magnitude. As expected, this generalization did not apply to live-cell immunization. When the profiles of mice vaccinated with acetone-killed cells or ribosomes are compared, it can be seen that the antibody titers in both groups of animals are similar; yet at 6 months postimmunization, the ribosomes gave significant protection against the low dose challenge, but acetonekilled cells did not. DISCUSSION The Salmonella literature is replete with controversy concerning many aspects of the fundamental mechanisms of host defense to this organism, and as a consequence what kind of vaccine should be used to engender an optimal immune response. One area of controversy has centered around the importance of anti-o anti- R 10o MONTHS POST-IMMUNIZATION.100 WCA c lo1 10 s%45 I4 INFECT. IMMUN. body in protection. As shown in these results, it is possible to develop a strong immunity with live cells at 3 weeks postimmunization in the absence of any anti-o antibody as determined by passive HA. This observation has led some investigators to the conclusion that anti-o antibody is not important in protection (15, 23). The results presented in this paper show that highly purified phenol-water-extracted LPS will protect mice at 3 weeks postvaccination against a challenge dose as high as 400 or 710 LD50, although not against 1,000 LD50. Since, as far as is known, the 0 antigens are the only major immunodeterminants on this molecule, it is probable that the protection engendered by this vaccine is dependent on the anti-o response. This interpretation is supported by the work of Lindberg et al. (22) and Svenson and Lindberg (34), showing that synthetic 0 antigens, as well as highly purified 0 antigenic determinants coupled to protein carriers, protect mice against Salmonella infection. However, the results presented here also show that a high anti-o titer, as measured by a high HA, is not sufficient to assure high levels of protection, since the HA titers of LPS-immunized mice and mice receiving acetone-killed cells or ribosomes did not differ, but the acetone-killed cells and ribosomal vaccine were superior to LPS in both their mag A- no0~ Q I 4 P. LPS.41D L ~~~~~~4005LDIO 4 f IMMUNIZED WITH LIVE 2 4 MONTHS POST-IMMUNIZATION A _ I en *~~~~~~~~~~~~ loo ois - MU 710LDo I P.~~~~~~~~~4b hi400 1_13, I j610 to WA.41. 2X 710 LDy, Qs u IMMUNIZED WITH AKC 4 0.e 1u 4 To MONTHS M POST-IMMUNIZATION 0* MONTHS POST-IMMUNIZATION FIG. 7. Correlation of duration ofprotection with antibody responses. (A) Mice immunized with LPS; (B) mice immunized with live cells; (C) mice immunized with ribosomal vaccine (RIB); (D) mice immunized with acetone-killed cells (AKC). (E) Percent survival with 400-LD50 challenge. (O--- -0) Percent survival with 710- LD50 challenge. (V) Anti-LPS HA titer; (0-x) anti- WCA titer.

7 VOL. 27, 198 nitude and duration of protection. Thus, the more complex vaccines must contain other factors which influence the immune response. It was found that both of these latter vaccines result in high WCA titers as well as high HA titers, whereas LPS gave a high HA titer but a low WCA titer. These results can be explained by one of two hypotheses. If both tests depend on anti-o antibody, then there must be differences in the classes of antibody that initiate the two reactions. Alternatively, one must postulate that the agglutination reaction as we perform it is partly dependent on antibodies to another antigen or to a slightly different conformation of 0-antigens that may be present in the more complex vaccines. In this regard, it might be noted that we used acetone-killed cells for the agglutination test, whereas the classic Widal test uses heat-killed cells to remove the inhibitory Vi antigen of S. typhi. It is possible that acetone killing preserves some heat-labile antigen. Since there is mounting evidence that Salmonella has protein antigens that are protective (17, 29, 35), it is possible that the development of a visible agglutination reaction in our system was partly dependent on antibodies to a protein determinant. In general, the results for the nonviable vaccines show a correlation between agglutination titer and protection, but not between HA titer and protection. When the various vaccines are compared for the duration of the protection they induce, the results show that the LPS is the least effective in providing long-lasting immunity. The observation that the protection rate of the LPS vaccine was high at 3 weeks, diminished to 0% at 2 months postimmunization, and then rose to detectable levels again at 4 and 6 months is difficult to interpret, but it may be due to possible cyclical immune responses to LPS, which others have observed (3, 4). Among the more interesting results from these studies was the observation that the live-cell vaccine and the ribosomal preparation were somewhat more effective than acetone-killed cells or LPS in giving a significant level of protection over a longer period of time. The reason why live cells give a greater duration of immunity could be related to several factors. First, live cells are known to induce a cellular immune response (23). As shown in the present results, with time, live cells also induced a high and sustained HA response. Although as already noted, the HA response does not insure high levels of protection, it could, in conjunction with cellular immunity, contribute to the overall level of host resistance. The constant antigenic stimulus provided by viable, persistent salmonellae could also account for the prolonged protection IMMUNITY TO SALMONELLA INFECTION 441 engendered by live cells. The reason why the ribosomal vaccine behaves like the live cells is not readily apparent. Unlike the live cells, the ribosomal vaccine shows a pattern of humoral responses that are similar to those of acetone-killed cells in kinetics, magnitude, and duration. Thus, ribosomal vaccines do not act as adjuvants to enhance the HA or WCA titers. It is possible that the ribosomal vaccines contain additional antigens that are not found in the acetone-killed cells and are not present in LPS. It is also possible that the ribosomal vaccines elicit antibody formation of different classes, slightly different specificity, affinity, or avidity, than killed cells or LPS, and that the antibodies induced by the ribosomal preparation may be slightly more effective in protection, or may be of longer half-life. For example, it is possible that the kinetics of the immunoglobulin G to immunoglobulin M ratios of the antibody response may vary between mice immunized with the different vaccines, especially over long periods of time. It is noteworthy that the ribosomal vaccine has been shown to behave differently from the purified LPS when tested in C3H/HeJ mice. In these animals, LPS induces no anti-o response and it is not protective. The ribosomal extract, however, gives an anti-o response and is protective (10). A 19S cytophylic immunoglobulin also has been implicated in protection against Salmonella infection, although little is known about its properties (20, 24, 25, 32). Only live organisms and ribosomal vaccine have been reported to induce this type of response. It could also be postulated that, since the ribosomal extraction method is such a gentle procedure, it preserves antigens in a more active state. If, however, the enhanced long-term protection of the ribosomal vaccine is due to differences in the specificity or quality of the antibodies produced, one would have to postulate that these differences are not detectable by the antibody assays used. An alternative explanation for the longer duration of protection by ribosomal vaccines is that they persist longer in the tissues than do killed cells or LPS, and thus exert an antigenic stimulus over a longer period of time. It has been unique proposed that ribosomal vaccines are among nonviable vaccines in stimulating cellular immunity (25, 33). If this is true, it could account for the greater duration of protection they induce. At present, it remains to be determined whether the longer-term protection by ribosomal vaccines observed in these studies relates to their ability to induce cell-mediated immunity, or to their ability to induce a spectrum of humoral responses different from those of the other nonliving vaccines.

8 442 ANGERMAN AND EISENSTEIN These results on duration of protection, taken together with the previous studies on the toxicity and the magnitude of the short-term protection of these various vaccines, show that ribosomal vaccines are equal in all parameters to acetonekilled cells, and have the advantage of providing longer-lasting immunity. However, these results, as well as those of our previous study (1), also show that sublethal doses of living virulent organisms are slightly more effective than acetonekilled cells or ribosomal vaccine, in terms of providing protection against high challenge doses and in giving long-lasting immunity. ACKNOWLEDGMENTS We thank Morton Klein for his encouragement and his helpful discussions on experimental design. The excellent technical assistance of Susan O'Donnell is appreciated. This work was supported by Public Health Service grant Al from the National Institute for Allergy and Infectious Diseases. LITERATURE CITED 1. Angerman, C. R., and T. K. 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