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Data suggest that the O-specific polysaccharide (O-SP) domain of the lipopolysaccharide (LPS) of Shigella species is both an essential virulence factor and a protective antigen and that a critical level of serum immunoglobulin G (IgG) to this antigen will confer immunity to shigellosis. Because covalent attachment of polysaccharides to proteins increases their immunogenicity, especially in infants and in young children, the O-SP of Shigella species were bound to medically useful proteins, and the safety and immunogenicity of the resultant conjugates were confirmed in adults and 4- to 7-year-old children. Succinylation of the carrier protein improved the immunogenicity of Shigella conjugates in mice and increased their yield. Based on these results, a clinical trial of O-SP conjugates of Shigella sonnei and Shigella flexneri 2a bound to succinylated mutant Pseudomonas aeruginosa exotoxin A (rEPAsucc) or native or succinylated Corynebacterium diphtheriae toxin mutant (CRM9 or CRM9succ) was conducted in healthy adults. The conjugates were safe and immunogenic. S. sonnei-CRM9, S. sonnei-CRM9succ, and S. sonnei-rEPAsucc elicited significant rises of geometric mean (GM) IgG anti-LPS within 1 week of injection (P < 0.001). At 26 weeks, the GM anti-LPS levels elicited by these three conjugates were similar and higher than their prevaccination levels (P < 0.0001). GM IgG anti-LPS levels elicited by S. flexneri 2a-rEPAsucc were significantly higher than those elicited by S. flexneri 2a-rCRM9succ at all intervals after injection. At 26 weeks, the levels of IgG anti-LPS in vaccinees were higher than their prevaccination levels (P < 0.0001). The serum antibody responses were specific, as there was no significant rise of anti-LPS to the heterologous O-SP in any vaccinee. Both conjugates elicited statistically significant rises of serum antibodies to the injected carrier protein. At 6 months, these five Shigella conjugates elicited higher fold rises than similar conjugates (D. N. Taylor et al., Infect. Immun. 61:3678–3687, 1993). Based on these data, we chose S. sonnei-CRM9 and S. flexneri 2a-rEPAsucc for evaluation in children.
Shigellosis remains a serious and common disease (16, 19, 23, 26, 28, 38, 46, 55, 56). In addition to causing watery diarrhea, shigellae are a major cause of dysentery (fever, cramps, and blood and/or mucus in the stool) (19, 46, 49, 55, 56). Not commonly appreciated is that dysentery, not watery diarrhea, retards growth in children (7, 9, 19, 30, 38, 46, 49, 55, 56).
Although Shigella dysenteriae type 1 was discovered as the cause of epidemic dysentery in Japan in 1898 (53), there is neither a licensed vaccine for it nor a consensus as to the mechanism(s) of host immunity to Shigella (11, 18, 31, 38, 46, 47). Vaccine development has been hampered by three factors: (i) the ineffectiveness of parenterally injected inactivated whole-cell vaccines which led to the belief that serum antibodies do not confer immunity (25, 32); (ii) the lack of a suitable animal model (46); and (iii) only indirect evidence of immune mechanism(s) in humans (11, 14, 38, 46–48).
The O-specific polysaccharide (O-SP) domain of lipopolysaccharide (LPS) is both an essential virulence factor and a protective antigen of Shigella (46). Convalescence from shigellosis confers LPS-specific immunity, although incomplete and of limited duration (6, 18, 31, 38, 46). The following data indicate serum immunoglobulin G (IgG) anti-O-SP confers immunity to shigellosis. (i) Correlation was found between the level of IgG LPS antibodies and resistance to shigellosis among Israeli solders (11, 14). (ii) There is an inverse relationship between the age incidence of shigellosis and the presence of IgG antibodies to the LPS of Shigella (41, 46). The peak incidence of shigellosis is in children and young adults; the disease is rare in infants and in older adults (14, 15, 20, 21, 23, 26, 28, 38, 46). Most newborns and adults have serum LPS antibodies that may be stimulated by cross-reacting bacteria (46–48). (iii) In a double-blind, vaccine-controlled randomized trial of our S. sonnei-rEPA (Pseudomonas aeruginosa recombinant mutant exoprotein A) conjugate (15), 1,447 Israel Defense Force (IDF) recruits from seven companies at separate field sites were vaccinated with S. sonnei-rEPA or one of two control vaccines. Shigellosis occurred in three units 2 to 3 months after vaccination; S. sonnei-rEPA induced an overall efficacy of 74% (P = 0.001). In one company, infection with S. sonnei occurred within 1 to 17 days of injection. Nevertheless, S. sonnei-rEPA conferred 43% (P = 0.04) protection, suggesting that our conjugates may be of value when administered during epidemics. A correlation was demonstrated only between the level of IgG anti-LPS and protection (15). Since serum antibodies are the main, if not the only, host mechanism induced by polysaccharide-protein conjugates, these data provide evidence that a critical level of IgG anti-LPS confers immunity to shigellosis (11, 46–48).
The immunogenicity of polysaccharide-based vaccines, including conjugates, is age dependent (46–48). We improved the immunogenicity of Shigella conjugates as assayed in mice by introduction of another carrier protein, Corynebacterium diphtheriae CRM9 (a genetically derived nontoxic mutant of C. diphtheriae) (34) and by succinylation of the proteins prior to binding to the polysaccharide (27, 42, 43). Succinylation of a mutant Clostridium difficile toxin increased its solubility and its effectiveness for conjugates (43). Succinylation has been proposed for inactivating diphtheria and tetanus toxins and stabilizing the resultant toxoids (51).
We evaluated the safety and immunogenicity of S. sonnei and S. flexneri type 2a conjugates in adults prepared with these two carrier proteins, native or treated with succinic anhydride. These agents were approved for investigation by the National Institutes of Health (OH98-CH-N009), Food and Drug Administration (BB IND 7443), Office for Protection against Research Risks (SPA SF-5900-09), and Ministry of Health, Israel.
Healthy 18- to 40-year olds comprising workers in the clinics of the participating institutions, medical students, and some outsiders were recruited. Individuals were questioned about their health and whether they had been hospitalized or were receiving medication. Their vaccination histories were reviewed, and informed consent was obtained before admission to the study. Volunteers were excluded if they were or planned to be pregnant within 6 months of injection of the investigational vaccines, hospitalized for a chronic disease, had infection with human immunodeficiency virus type 1 (AIDS) or hepatitis, were taking medication on a continual basis, had multiple allergies, or had a febrile disease at the time of immunization or another infection that required medication. The oral temperature of each volunteer and a blood sample were taken before vaccination. Female volunteers underwent a urine pregnancy test.
Volunteers were randomized to receive one of five experimental vaccines. Each received a 0.5-ml injection, containing 25 μg of saccharide, into the left deltoid. Blood samples were also taken on day 3 and at 1, 4, and 26 weeks following vaccination. Liver function tests were assayed on days 0, 3, and 7. Serum antibodies to the O-SPs and carrier proteins were assayed at all intervals. The volunteers examined their injection sites in two dimensions and took their temperatures at 6, 24, and 48 h after the injection. They entered these data on a form for each of these intervals. Volunteers were asked to return to the clinic if they had a temperature higher than 38°C or induration (swelling) greater than 3.0 cm in diameter at the injection site. Each volunteer was called by a nurse or a physician at about 6 h and the next two mornings after the injection and questioned about their demeanor, temperature, and local reactions.
S. flexneri type 2a O-SP was assayed by the anthrone method (52). S. sonnei O-SP was assayed by the 2-phenylphenol assay for uronic acids (8). Protein was assayed by the Lowry method with bovine serum albumin as a standard (10), and nucleic acids were measured by A260. Derivatization with adipic acid dihydrazide was estimated with the trinitrobenzene sulfonic acid assay (10). ADP ribosyltransferase activity, with purified diphtheria and Shigella toxin as standards, was assayed as described elsewhere (40) by Allison O'Brien and Stephen Darnell, Uniformed Services University of the Health Sciences, Bethesda, Md., with Vero cells as the substrate. CRM9 had about 104, and S. sonnei-rEPA had 1.6 × 105, lower toxicity compared to diphtheria toxin.
O-SPs from Plesiomonas shigelloides O7 (possessing a structure identical to that of the O-SP of S. sonnei) and S. flexneri type 2a were purified as described elsewhere (57). Both lots, containing less than 1% protein and nucleic acids, were passed through a 2.5- by 100- cm column in 0.2 M NaCl. The void-volume fractions were dialyzed extensively against pyrogen-free water and freeze-dried.
For production of CRM9, strain C7 (β)(tox-201, tox-9) was obtained from Randall Holmes, Uniform Services University of the Health Sciences, and David M. Neville, Jr., National Institute of Mental Health, National Institutes of Health. C7 (β)(tox-201, tox-9) was cultivated in modified CY medium without deferration and purified as described with the additional step of gel filtration through Superdex 200 (42, 54). Fractions containing CRM9, located by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), were pooled, sterile filtered, concentrated to ~30 mg/ml, and stored at −70°C (54). rEPA was prepared as described elsewhere (22, 57).
Succinic anhydride was added to the protein at ratios of 1:10 for CRM9 and 1:5 for rEPA (42). Residual lysine was used as an indirect measure of succinylation; CRM9 had 15.65 lysine units versus 2.65 for its succinylated derivative (CRM9succ) and rEPA had 25.4 lysine units versus 17.3 for its succinylated derivative (rEPA suec) (24). Double immunodiffusion against the corresponding antisera showed a line of identity between the succinylated and native proteins (not shown). SDS-PAGE showed a slight decrease in migration of both succinylated proteins compared to the native proteins (not shown). By Limulus amoebocyte lysate (LAL) assay, both proteins contained <0.04 endotoxin units of (EU) protein μg.
S. sonnei O-SP was prepared as described elsewhere (57). The final product (S. sonnei-AH) contained 5.6% adipic acid hydrazide, and LAL assay showed 0.125 EU of polysaccharide μg. Double diffusion in agar showed an identity reaction with the O-SP (not shown).
S. flexneri 2a O-SP was prepared as described elsewhere (57). The final product had 4.4% adipic acid hydrazide, and LAL assay showed 0.006 EU per μg of polysaccharide. Double diffusion in agar showed an identity reaction with the O-SP (not shown).
S. sonnei-AH was added to either CRM9, CRM9succ, or rEPAsucc, and the mixture was stirred at room temperature in a pH stat. EDC was added to 0.1 M, and the pH was maintained at 5.8 (33). The reaction mixture was dialyzed against 0.2 M NaCl–0.25 mM sodium phosphate–0.01% thimerosal (pH 7.0) at 4°C for 3 days and passed through Sepharose CL-4B; the void-volume peak was diluted with pyrogen-free saline to about 40 μg of saccharide/ml.
Conjugation was performed as described previously (42). The conjugate was diluted with pyrogen-free saline to about 40 μg of saccharide/ml/.
Sera from the volunteers were separated immediately and stored at −70°C until assayed. Serum IgG, IgA, and IgM to the LPS of S. sonnei and of S. flexneri 2a were measured by enzyme-linked immunosorbent assay (ELISA) (57). Monoclonal antibodies HP6043 (anti-human IgG), HP6084 (anti-human IgM), and HP6107 (anti-human IgA), from George Carlone, Centers for Disease Control and Prevention, were used to assay both LPS and carrier protein antibodies. Antibody levels were calculated by parallel line comparison to a standard serum (included on each plate) assigned a value of 100 U (44). Serum IgG to P. aeruginosa exotoxin A and diphtheria toxin were also measured by ELISA (4, 57). A positive antibody response was defined as a fourfold or greater rise above the prevaccination level.
Serum antibodies elicited in mice by the five conjugates have been reported elsewhere (42). Briefly, saline solutions containing 2.5 of μg O-SP/100 μl were injected subcutaneously into 5-week-old outbred female mice; IgG antibodies were detected in all mice after the third injection. CRM9 served as a more immunogenic carrier for serum anti-LPS than rEPA. Conjugates composed of succinylated proteins elicited higher levels of IgG anti-LPS than did those prepared from native components. Conjugates prepared with native proteins elicited higher levels of IgG protein antibodies than did conjugates composed of succinylated proteins.
Antibody levels, expressed as geometric means (GM), were calculated by using log transformation data and compared by paired and unpaired t test or Wilcoxon rank sum test; levels of less than the sensitivity of the ELISA were assigned one-half of that level. We used the chi-square test for contingency tables (e.g., proportion of adverse effects) and the Wilcoxon rank sum test for continuous variables. P values of <0.05 were considered statistically significant.
None of the 152 volunteers had serious local reactions or a temperature above 37.6°C up to 48 h after injection. Four volunteers, each injected with a different conjugate, had mild and uncomplicated diarrhea starting 1 to 2 days after injection, likely due to an intercurrent infection.
One volunteer, a medical student who received S. sonnei-CRM9, had acute onset of urticaria and shortness of breath 2 days after injection. These symptoms abated shortly after corticosteroid treatment, and she has remained well to date. She had no history of allergies, and no cause for this episode was established.
One week after injection, three volunteers had palpable lymph nodes and five had localized redness and swelling of 0.1 to 1.9 cm. A recipient of S. flexneri-CRM9succ had local swelling of 5 cm.
A pediatrician who received S. sonnei-rEPAsucc had normal creatine and liver enzymes prior to vaccination. Three days after vaccination, his alkaline phosphatase, serum glutamic oxaloacetic transaminase, and serum glutamic pyruvic transaminase levels were 87, 112, and 240 U/liter; respectively. At 1 week, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, and lactate dehydrogenase levels were 174, 339, and 494 U/liter, respectively. The vaccinee was asymptomatic, tested negative for hepatitis A, B, and C and Epstein-Barr virus but tested positive for cytomegalovirus (CMV) both IgG and IgM anti-CMV titers were elevated: prevaccination, 1.21 (IgG) and 3.54 (IgM); 3 days postvaccination, 2.11 and 4.71; 1 month postvaccination, 3.16 and 1.86. He remained asymptomatic, and his enzyme levels returned to normal within 2 weeks. All other volunteers had normal levels of liver enzymes.
The ratios of protein to polysaccharide of the five conjugates were similar, ranging from 1.35 for S. sonnei-CRM9 to 2.58 for S. flexneri 2a-rEPAsucc. The pyrogen contents of all five conjugates were low: all passed the rabbit thermal induction assay undiluted (58).
All volunteers had preexisting anti-LPS of the three Ig classes to both Shigella O-SPs, and there were no significant differences in these levels between the conjugate groups. None of the conjugates elicited a significant GM, antibody rise at 3 days following injection.
All three conjugates elicited a significant rise of anti-LPS of the three Ig classes at 1 and 4 weeks after injection (P < 0.0001).
All three conjugates elicited a statistically significant rise of the GM anti-LPS 1 week after immunization (P = 0.0001): 66 of 81 volunteers (82%) had a ≥4-fold rise over their prevaccination levels. The GM levels at 4 weeks were higher than those at 1 week (P = 0.01). The peak level of IgG anti-LPS was at the 4-week interval for all three conjugates: 81 of 82 volunteers (99%) had a ≥4-fold rise over the preimmunization levels. At 26 weeks, these levels declined about twofold and were not significantly different from each other: 70 of 77 volunteers (91%) had anti-LPS levels ≥4-fold higher than before vaccination (P = 0.0001).
Except for the IgG levels at 1 week (S. sonnei-CRM9, 42.5 EU > S. sonnei-rEPAsucc, 15.4; P = 0.03), the GM IgG levels at each interval were not statistically different.
The peak GM levels of IgM and IgA occurred at the first week with the exception of IgA anti-LPS elicited by S. sonnei-rEPAsucc, which was at 4 weeks. The level elicited by S. sonnei-CRM9 was higher than those elicited by the other two conjugates (56.7 versus 16.7, P = 0.02). At the 26-week interval, IgM levels declined to the prevaccination level. IgA anti-LPS at 26 weeks declined at least twofold from their peak levels but remained higher than the preinjection levels (P = 0.0001).
Both conjugates elicited a rise of anti-LPS of the three Ig isotypes 1 week following injection (P = 0.0001).
S. flexneri 2a-rEPAsucc elicited higher GM levels than S. flexneri type 2a-CRM9succ at all intervals. Both conjugates elicited a statistically significant rise of the GM anti-LPS 1 week after immunization. For S. flexneri 2a-rEPAsucc, the GM was 77.5 versus 14.0 (P = 0.0001), and 14 of 29 volunteers (48%) had a ≥4-fold rise above the prevaccination level. For S. flexneri 2a-CRM9succ, the GM was 50.3 versus 10.6 (P = 0.0001), and 14 of 30 volunteers (47%) had a ≥4-fold rise over their prevaccination levels.
A statistically significant rise over the level at the 1-week interval was elicited by both conjugates at 4 weeks. For S. flexneri 2a-rEPAsucc, the GM was 420 versus 77.5 (P = 0.0001), with 28 of 30 volunteers (93%) having ≥4-fold rise above their preimmunization levels. For S. flexneri 2a-CRM9succ, 1 the GM was 221 versus 50.3 (P = 0.0001), and 28 of 32 volunteers (88%) had ≥4-fold or higher levels compared to those before immunization.
At the 26-week interval, the GM levels in both groups had declined about 60% from their maximal levels: the GM of the S. flexneri 2a-rEPAsucc group was higher than that of the S. flexneri 2a-CRM9succ group (179 versus 94.6, P = 0.045). For S. flexneri 2a-rEPAsucc, 22 of 28 volunteers (75%) had a ≥4-fold rise over their preimmunization levels; 20 of 29 volunteers (69%) had a ≥4-fold rise for S. flexneri 2a-CRM9succ. The GM levels of the two conjugate vaccine groups were higher than those prior to injection (P = 0.0001).
Both conjugates elicited comparatively low but statistically significant rises of anti-LPS at weeks 1 and 4 after immunization (P = 0.0001). The peak levels of IgM anti-LPS were at week 4; these levels declined to about 2.5 times those prevaccination but at 26 weeks were significantly higher than those prior to injection (P = 0.0001).
Both conjugates elicited a statistically significant rise of IgA anti-LPS 1 week after vaccination. Only S. flexneri 2a-rEPAsucc elicited an additional rise from week 1 to week 4 (31.9 versus 16.4, P = 0.02). At the 26-week interval, the GM levels declined about 60% for both groups: the GM elicited by S. flexneri 2a-rEPAsucc was higher than that of elicited by S. flexneri 2a-CRM9 (12.1 versus 3.96, P = 0.005).
None of the conjugates elicited statistically significant GM rises of any isotype at any interval to the heterologous O-SP (not shown).
The GM serum levels and increases of IgG anti-LPS after 26 weeks following immunization of volunteers in this study were compared to those elicited by similar conjugates in IDF recruits in a previous study (57). All three S. sonnei conjugates in this study elicited similar levels of anti-LPS at 6 and 26 weeks after immunization as S. sonnei-rEPA (IDF). The three conjugates in this study also elicited similar fold rises (23.4, 16.9, and 26.6) at 26 weeks, all of which were higher than values for the S. sonnei-rEPA used in the previous study (9.2 EU) (57).
In this study, S. flexneri 2a-rEPAsucc was more immunogenic than S. flexneri 2a-CRM9succ (Table (Table3)3) and S. flexneri-rEPA (IDF), as shown by the higher anti-LPS levels at both 6 (420 EU versus 126) and 26 (179 EU versus 93.7) weeks and by the fold rise at 26 weeks (12.8 versus 3.7).
All vaccinees had preexisting levels of antibodies to the two toxins, and none of the conjugates elicited a significant antibody response to the homologous carrier protein at the 3-day interval following injection.
All volunteers had >0.01 U of antitoxin prior to injection, and there was no statistically significant difference between the three groups. All three conjugates containing CRM9 elicited a significant rise of diphtheria toxin antibodies 1 week after injection, with peak levels at 4 weeks. S. sonnei-CRM9, the conjugate with the native protein carrier, elicited the highest level (8.89 EU), but this was not significantly different from the response elicited by conjugates with the succinylated protein (6.08 for S. sonnei-CRM9succ and 4.28 for S. flexneri-CRM9succ). Both S. sonnei conjugates with CRM9 as the carrier elicited higher levels than S. flexneri-CRM9succ at 1 week (6.37 and 5.03 versus 2.54, P = <0.05). At 26 weeks, although the level was highest in the recipients of S. sonnei-CRM9 (2.34), there was no significant difference between the GM elicited by the CRM9-containing conjugates, and all had about twice the preinjection levels.
Both the S. sonnei and S. flexneri type 2a conjugates contained rEPAsucc as the carrier, and both elicited a lesser response (about 3.7-fold rise at the 4-week interval) to the ETA compared to the fold rise to the carrier. The GM level of anti-rEPA declined at 26 weeks to about twice the preinjection level; there was no difference in the fold rises between the two conjugates.
None of the conjugates elicited a significant GM rise in antibodies to the heterologous carrier protein (not shown).
None of the vaccinees developed fever within 48 h of injection, and local reactions were minor and infrequent; no volunteer missed a day of work or of school because of the vaccination.
We cannot explain the episode of shortness of breath and urticaria in one vaccinee 2 days after injection of S. sonnei-CRM9; she responded quickly to an injection of corticosteroid. The vaccinee had not experienced such a reaction following other vaccinations and has remained well since that episode. Her pre and postimmunization antibody levels were not unique. A serologically documented asymptomatic CMV infection provides an explanation for the transient rise in liver enzymes in a recipient of S. sonnei-rEPAsucc.
The safety and immunogenicity of our Shigella conjugates in U.S. Army and IDF recruits and in 4- to 7-year-old Israelis have been reported elsewhere (4, 12, 15, 57). The safety of our O-SP conjugates for Salmonella paratyphi A, Vibrio cholerae O1, and Escherichia coli O157 has been documented elsewhere (29, 38, 39).
Reinjection of S. flexneri 2a O-SP conjugates induced a booster response in adults and in 4- to 7-year-old children not observed with other polysaccharide conjugates (4, 48, 57). Although it is not possible to compare the levels of the IgG anti-LPS in mass units, the differences in fold rises suggest that the O-SP of S. sonnei is more immunogenic than that of S. flexneri 2a. Another difference is that the IgG responses elicited by our O-SP conjugates persisted for at least 2 years, considerably longer than after disease (4, 12, 13, 20, 21, 37).
The immunogenicity of polysaccharide conjugates in mice or in human adults may not always predict their activity in infants or young children (1, 2). Our choice for the five conjugates was based on their immunogenicity in young outbred mice (41). The three S. sonnei O-SP conjugates, S. sonnei-CRM9, S. sonnei-CRM9succ, and S. sonnei-rEPAsucc, induced similar IgG anti-LPS responses in adults. We chose S. sonnei-CRM9 to evaluate in young children because it is the simplest of the three conjugates to synthesize. S. flexneri 2a containing rEPAsucc was the more immunogenic of the two conjugates and was chosen for study in young children.
Shigellosis induced a rise of anti-LPS of all three Ig isotypes; the increase in IgG anti-LPS is the highest and has the longest duration (11, 13, 20, 21, 37, 57). The magnitude of the anti-LPS response is related to the severity of symptoms, and anti-LPS levels of the three isotypes decline to those in acute-phase sera in less than 1 year (13, 39). This comparatively rapid decline of serum anti-LPS provides an explanation for the limited duration of disease-induced immunity (6, 18). The higher and longer-lasting levels of IgG anti-LPS suggest that our conjugates will induce a more complete and long-lasting immunity than shigellosis.
Finally, we reported preparation of highly immunogenic conjugates with synthetic saccharides corresponding to the O-SP of S. dysenteriae type 1 (45). Both the length and the density of the saccharide were related to the immunogenicity of the conjugate in mice (1, 2). The use of synthetic saccharides may permit the development of more immunogenic polysaccharide conjugates (48).
We gratefully acknowledge Sylvia Koo of the Immunology Service, Department of Laboratory Medicine, National Institutes of Health, for assay of CMV antibodies.