The thymus-dependent nature of the murine antibody response to a monoclonal anti-idiotypic antibody to the Neisseria meningitidis serogroup C capsular polysaccharide

Microbial Pathogenesis 1990; 8 : 411-419

The thymus-dependent nature of the murine antibody response to a monoclonal anti-idiotypic antibody to the Neisseria meningitidis serogroup C capsular polysaccharide M . A . Julie Westerink,'* Peter C . Giardina,' Anthony A . Campagnari 2 and Michael A . Apicella' 2 'Department of Medicine and DDepartment of Microbiology, State University of New York at Buffalo, Buffalo, New York, U.S.A . (Received March 15, 1990; accepted March 16, 1990)

Westerink, M . A . J . (Dept of Medicine, State University of New York at Buffalo, Buffalo, New York, U .S .A .), P . C . Giardina, A . A . Campagnari and M . A . Apicella . The thymus-dependent nature of the murine antibody response to a monoclonal anti-idiotypic antibody to the Neisseria meningitidis serogroup C capsular polysaccharide . Microbial Pathogenesis 1990; 8 : 411-419 . Idiotype vaccines are proteins which may offer an alternative strategy for the conversion of a thymus-independent antigen into a thymus-dependent immunogen . To examine this question, we have studied the nature of the immune response to a monoclonal anti-idiotypic antibody, designated 6F9, which acts as a surrogate of Neisseria meningitidis serogroup C capsular polysaccharide, and compared this response to the nominal antigen, the meningococcal Cpolysaccharide (MCP) . BALB/c mice immunized with an optimal dose (100 µg) of 6F9 generate a specific anti-MCP IgG response which is maximal after 4 weeks . Secondary immunization with 6F9 results in a three- to five-fold increase in the specific IgG response . Mice given an optimal immunizing dose of MCP (5 ug) failed to generate an anti-MCP IgG response . No secondary response is detectable in mice immunized with MCP . Animals immunized with 6F9 and subsequently challenged with live meningococci group C show a significant anti-MCP IgG response . BALB/c nu/nu mice fail to generate an anti-MCP IgG antibody response to 6F9, while the nu/+ controls generate an anti-MCP IgG antibody titer 100 times that of the MCPimmunized mice . Neonatal mice that failed to respond to MCP developed early IgM and a subsequent IgG anti-MCP response after immunization with 6F9 . These data demonstrate that the anti-idiotype 6F9, the combining site of which contains a surrogate image of the meningococcal group C capsular polysaccharide, evokes the responses expected of a Tdependent antigen . Key words : anti-idiotypic antibody ; Neisseria meningitidis serogroup C ; capsular polysaccharide .

Introduction Nisonoff and Lamoyi, and Roitt and co-workers"' suggested in 1981 that internal image antibodies might be exploited as a new type of vaccine . It has been well established that anti-idiotypic antibodies (anti-Id) can induce the secretion of antigenspecific antibodies in the absence of antigen, and thus act as a so-called internal image or surrogate antigens . Several authors have employed this technique successfully *Author to whom correspondence should be addressed : M . A . J . Westerink, SUNY at Buffalo, Department of Medicine, Division of Infectious Diseases, 462 Grider Street, Buffalo, NY 14215, U .S .A . 0882-4010/90/060411 +09 $03 .00/0

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and raised protective antibodies in animals against various pathogens, including Escherichia coli K13, 3 hepatitis B,4 Streptococcus pneumoniae' and a number of parasitic and viral systems .' Despite the development of effective antibiotics and polysaccharide vaccines that appear efficacious in adults and older children,` the polysaccharide encapsulated bacteria, including Haemophilus influenzae, E. coli and Neisseria meningitidis remain the most common cause of serious infections in infants and young children ." , The immune response to polysaccharide antigens has several characteristics which distinguish it from the antibody response to protein antigens . These characteristics include thymus-independence, 12 failure to stimulate a memory response and to undergo affinity maturation (i .e . lack of booster response), 13 and late development in .14 ontogeny .1s Anti-Id-mediated B-cell activation may be of particular interest in systems which rely on an adequate anti-capsular polysaccharide response for protective immunity . It has been demonstrated 15 that the development of antibodies to meningococcal polysaccharides during ontogeny in BALB/c mice parallels the immunity to these capsular polysaccharides in man ." Neonatal and adult BALB/c mice would be suitable models for studying the immune response in an immature and a mature immune system to an anti-idiotypic antibody which acts as a surrogate antigen of meningococcal Cpolysaccharide (MCP) . Rubenstein et al. 17 18 and Stein 3 have demonstrated that anti-idiotypic antibodies can prime neonatal mice. However, evidence that anti-idiotypic antibodies that are surrogates of polysaccharide antigens can stimulate a T-dependent antibody response in immunologically immature animals has not been well established . We have developed a monoclonal anti-idiotypic antibody, 6F9, which contains the surrogate image on the combining antibody site which mimics MCP .` In this report, we will characterize the nature of the immune response elicited by this anti-idiotypic antibody in neonatal and adult BALB/c mice . Our results indicate that the immune response to the anti-idiotype 6F9 is capable of evoking a T-dependent antibody response to the MCP in adult and neonatal animals . Results Dose response curve To determine the optimal immunizing dose of MCP, BALB/c mice (n = 5 per group) were immunized intraperitoneally (i .p .) with 0 .1, 1, 5, 10 or 50 pg of MCP . The mice were bled on day 5 and the anti-MCP IgM antibody titer was determined by ELISA . The optimal immunizing dose of MCP was determined to be 5 pg . Larger doses of MCP resulted in a decrease in specific antibody . Using a similar method, a dose response curve was generated by immunizing mice i .p . with the anti-idiotype 6F9 . The specific anti-meningococcal C IgM antibody response increased as the dose increased from 1 to 100 pg . A larger dose resulted in a decreased response . The optimal immunogenic dose was considered to be 100 pg of 6F9 (Fig . 1) . Kinetics of the antibody response To study the kinetics of the antibody response, BALB/c mice were immunized with an optimal immunizing dose of MCP (5 pg) or 6F9 (100 pg) . Five animals from each group were killed on a weekly basis and the specific anti-MCP IgM and IgG response was determined . Mice immunized with MCP generated a group C-specific IgM response which was maximal by day 7 . This level of response remained constant over the four-week period of study . Mice immunized with 6F9 also generated a group C-



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Fig . 1 . ELISA analysis of mouse sera for anti-MCP IgM antibody response . BALB/c mice (n = 5 per group) were immunized i .p. with 0 .01, 0 .1, 1, 5, 50, 100 or 200,ug of MCP (o) or 6F9 (o) . Mice injected with PBS (o) served as negative controls . The anti-MCP IgM antibody response was measured after 5 days . The dose response curves were generated by regression analysis of data from separate analyses at each point .

specific IgM response which was maximal at day 7, but then decreased significantly (P < 0 .01) . As can be seen in Fig . 2, an IgG antibody response was not detectable in the sera of mice immunized with MCP . In contrast, mice immunized with 6F9 showed a significant IgG response which was approximately 100-fold greater than the background titer . This IgG response was first detectable at day 14 and continued to rise to day 28 .

Secondary antibody response To study the secondary immune response, mice (n = 5 per group) were immunized i .p . with an optimal dose of MCP or 6F9 on day 0 and received a second optimal dose of immunogen on day 21 . The anti-MCP IgG response was determined on day 26

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Fig . 3 . ELISA analysis of the secondary immune response to MCP and 6F9 . BALB/c mice (n = 5 per group) were immunized i .p . with 5 pg of MCP (o) or 100 pg of 6F9 (o) on day 0 and day 21 . The control group of mice received a single injection of MCP (A) or 6F9 (o) on day 0 . The anti-MCP IgG response was measured on day 26 in microdilution wells coated with 10 µg MCP per ml . Curves were generated by regression analysis of data from separate analyses at each point .

(Fig . 3) in both groups . As expected, mice immunized with MCP failed to demonstrate an IgG response . In contrast, mice immunized with 6F9 demonstrated the development of immunologic memory . The IgG antibody response of mice that received a booster dose of 6F9 was five-fold higher than in the mice that received a single dose of 6F9 . Similar secondary responses were observed in animals immunized with an optimal dose of MCP or 6F9 on day 0, who then received 10 3 cfu of live group C meningococci on day 21 (Fig . 4) . In the mice immunized with 6F9 we observed a three- to five-fold increase in the specific IgG response after they received a dose of live meningococci . Parallel experiments on mice immunized with MCP and live meningococci demonstrate that these animals did not develop a secondary anti-MCP IgG response .

Fig . 4 . ELISA analysis of mouse sera for anti-MCP IgG antibody response . BALB/c mice (n = 5 per group) were immunized with 5 pg of MCP (o) or 100 pg of 6F9 (o) on day 0 and received a booster injection with 103 cfu of live group C meningococci i .p . on day 21 . The control group of mice consisted of mice immunized with 5 pg MCP (A) or 100 pg 6F9 (o) on day 0. The anti-MCP IgG antibody response was measured on day 26 . Curves were generated by regression analysis of data from separate analyses at each point.



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Fig . 5 . ELISA analysis of nu/nu mouse sera for anti-MCP antibody response . Athymic BALB/c nu/nu mice (n = 5 per group) were immunized with 5 ,ug of MCP (o) or 100 pg of 6F9 (o) . BALB/c nu/+ mice served as controls and also received 5 pg MCP (o) or 100 pg 6F9 (o) on day 0 . The anti-MCP IgM antibody response was determined on day 5 (a) . The anti-MCP IgG antibody response was determined on day 28 (b) . Curves were generated by regression analysis of data from separate analyses at each point .

Antibody response in nu/nu mice Athymic BALB/c nu/nu mice and a control group of nu/+ mice (n = 5 per group) were immunized i .p . with an optimal dose of MCP or 6F9 on day 0 . The mice were bled on day 28 and the sera tested for an anti-MCP IgM and IgG antibody response . The results of this study are shown in Fig . 5(a) and (b) . Athymic nu/nu mice, which are incapable of responding to T-dependent antigens, failed to show an anti-MCP response when immunized with 6F9, while the nu/+ control mice showed a response similar to normal BALB/c mice . Since the nu/nu mice are capable of responding to Tindependent antigens, an antibody response of similar magnitude to normal BALB/c mice was seen in mice immunized with MCP . Primary and secondary immune response in neonatal mice To determine if 6F9 could generate a similar response in neonatal mice the following studies were undertaken . Dose response curves were generated for neonatal mice (810 days old) by immunizing neonates i .p . with 1, 2 or 5 ug MCP or 5, 25, 50 or 100 pg 6F9 . The specific anti-MCP IgM antibody response was measured after 5 days . The optimal immunogenic dose for MCP was 1 µg and for 6F9 was 25 ug . The antibody response to both MCP and 6F9 was relatively poor (five- to eight-fold less) in comparison to response in adult mice (data not shown) .



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Fig . 6 . Results of the ELISA analysis of neonatal mouse sera for anti-MCP IgG antibody response . Neonatal BALB/c mice (n = 10 per group) 8-10 days of age were immunized with 1 pg MCP (A) or 25 µg of 6F9 (o) on day 0 . Five mice in each group were boosted on day 21 with 5 pg MCP (o) or 100 pg 6F9 (o) . The anti-MCP IgG antibody response was determined on day 26 . Curves were generated by regression analysis of data from separate analyses at each point .

Neonatal mice (n = 5 per group) were then immunized with 1 µg of MCP or 25 pg 6F9 on day 0 and boosted on day 21 with 5 pg of MCP or 100 ug 6F9 . The specific anti-MCP IgG response was determined five days later on day 26 (Fig . 6) . Mice immunized with MCP did not have a detectable anti-MCP IgG response, while mice immunized as neonates with 6F9 had a significant anti-MCP IgG response which was comparable to the IgG response seen after 4 weeks in adult mice immunized with 6F9 . These experiments indicate that the anti-Id 6F9 is capable of priming neonatal mice in contrast to MCP, the nominal antigen . Discussion Jerne first published the network hypothesis in 1974, defining the immune system as a web of interacting idiotopes . 23 The normal immune system features an interlocking network of antibodies directed at one another's idiotopes . Inherent in the network theory is the idea that at least some of these anti-idiotype antibodies are internal images of external antigens . Data collected in recent years show that anti-idiotypic antibodies directed at the hypervariable site of antimicrobial antibodies can be used to induce neutralizing antibodies in adult anima ls4,5,24 .25 or prime immunologically immature animals .' Collectively, these data strongly suggest that B-cell defined anti-idiotypes which mimic antigen may be potentially useful as antimicrobial vaccines . Anti-Id based vaccines may represent viable vaccine candidates in situations where it is difficult to obtain adequate amounts of purified antigen or when a conventional attenuated vaccine has a high propensity for reverting to a virulent form . In addition, anti-Id based vaccines may be alternative vaccines in immunologically incompetent hosts, such as neonates, which are incapable of responding to currently licensed capsular polysaccharide vaccines . Idiotype vaccines are proteins and therefore may act as T-dependent antigens . However only two groups" ," have shown that an anti-Id vaccine is able to induce a B- and T-cell-mediated response . An anti-Id which mimicked a T-dependent antigen was used in both systems . McNamara etal. and Stein and Soderstrom 3.5 have previously shown the usefulness of



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anti-Id vaccines by inducing and priming for protective antibodies against encapsulated organisms . However these investigators did not report the characteristics of the immune response . In these studies it was necessary to either couple the anti-Id to a protein carrier or use an adjuvant to induce these protective antibodies . The use of adjuvants and/or carrier molecules seemed to be a prerequisite in many instances for optimal 28,29

antibody production by anti-idiotypes . The data regarding the immune response to T-independent antigens is primarily based on the studies with type III pneumococcal polysaccharide, 30 H . influenzae type 5.32,33 B polysaccharide, 31 group A and C meningococcal polysaccharide' and dextran ." Using a similar murine model, we have investigated the nature of the immune response evoked by MCP and by an anti-idiotypic antibody which mimics the group C capsular polysaccharide . The results of our studies demonstrate that the anti-Id 6F9 is capable of eliciting not only an anti MCP IgM response, but also a significant anti-MCP IgG response in contrast to the nominal antigen . These results suggest that 6F9, the surrogate image of a T-independent antigen acts as a T-dependent antigen . Athymic nu/nu mice, incapable of responding to Tdependent antigens, were used to confirm these findings . The results of these studies showed that nu/nu mice did not respond to 6F9 while the nu/+ controls showed a normal anti-MCP antibody response . The nu/nu mice immunized with MCP were capable of responding to this T-independent antigen as were the nu/+ controls . A significant secondary response or booster effect was seen in mice who received a second immunization with 6F9 . This booster response was not detected in the mice immunized with MCP . Furthermore, mice immunized with 6F9 and subsequently boosted with live group C meningococci showed a similar booster response . These data suggest that the subset of T memory cells stimulated by 6F9 are the T cells involved in the MCP response when confronted with live group C meningococci . Finally, data obtained from neonatal mice showed that these mice can be primed effectively with the anti-Id 6F9 and show a significant anti-MCP IgG response upon reimmunization . In contrast to our previous work 19 we did not find it necessary to use a protein carrier molecule or adjuvant in this study . In our preliminary experiments the animals received a suboptimal dose of antigen which may account for the necessity to use a protein carrier and/or adjuvant . This may be explained by the dose response experiments carried out in the present study which shows the optimal dose of 6F9 to be 100 µg per mouse . Other investigators 28' 29 have found the use of carrier molecules and/or adjuvants necessary . In some of these cases a suboptimal dose of anti-Id may also have been used . In addition, we speculate that a number of anti-Id molecules may require a carrier molecule and/or adjuvant for correct or effective antigen presentation and processing . Finally, the location of the surrogate image on the immunoglobulin molecule (variable heavy versus variable light chain, or a combination of both) may play a crucial role in this phenomenon . Recent advances in immunoglobulin and mRNA sequencing techniques will enable us to clarify these issues . In conclusion, we have demonstrated that the anti-Id 6F9 whose combining site contains a surrogate image of a T-independent antigen, group C meningococcal polysaccharide, and acts as a T-dependent antigen .

Materials and methods Animals. BALB/c mice were obtained from West Seneca Laboratories (West Seneca, New York) . The animals were bred at the animal facility of the Clinical Center of the State University of New York at Buffalo . Adult mice were immunized at 6-8 weeks of age . Neonatal mice 8-10



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days of age were used . Athymic nu/nu BALB/c mice and the nu/+ controls between the age of 6-8 weeks of age were obtained from Jackson Laboratories (Bar Harbor, Maine) . Bacterial strains. N. meningitidis serogroup C strain MP13 was obtained from our own collection . It was stored frozen at -70°C in glycerinated Muller-Hinton broth . It was reconstituted and grown on supplemented GC agar (Difco Laboratories, Detroit, Michigan) in 5% CO 2 at 37°C . This strain produces an 0-acetyl negative capsular polysaccharide . 20 Meningococcal C-polysaccharide . Vaccine quality group C meningococcal polysaccharide (MCP) was a gift from Dr Philip Vella, Merck, Sharp and Dohme Research Laboratories (West Point, New Jersey) . Monoclonal anti-idiotype antibodies . The development of the monoclonal anti-idiotype antibody 6F9 has been described previously ." Briefly, pristane treated BALB/c mice were immunized with hybridoma cells secreting 1 E4 (AB1 or idiotype) . After a 3-week incubation period the splenocytes were removed and fused with SP2/0 Ag14 plasmacytoma cells according to standard procedure . 21 Hybridoma supernatants were screened for recognition of AB1 by dot assay, inhibition ELISA and indirect immunofluorescence . Monoclonal anti-idiotype 6F9, isotype IgG2b, was selected for further experiments . Large quantities of AB2, free of other contaminating proteins were obtained by growing the hybridoma cells in dialysis membranes . 22 The antibodies were purified using P300 molecular sieve chromatography (Bio-Rad Laboratories, Richmond, California) . EL/SA analysis. Purified capsular polysaccharide was diluted in carbonate buffer, pH 9 .6, to a final concentration of 10 pg/ml . The wells of 96-well polyvinyl plates (Linbro/Titertek, Flow Laboratories, McLean, Virginia) were coated with 100 yl polysaccharide buffer and incubated for 1 h at 37°C and then overnight at 4°C . Plates were washed three times with 0 .01 M PBS0 .05% Tween 20 . The plates were blocked for 1 h with 3% gelatin at 37°C and washed . Serial dilutions of serum starting at 1 : 10 to 1 : 360 were prepared, and 100 µl was added per well . Plates were incubated for 1 h at 37°C and washed three times . The plates were incubated with anti-mouse IgG or IgM peroxidase-labeled conjugate (Kirkengaard & Perry Laboratories, Gaithersberg, Maryland), washed three times and developed with 100 µl of substrate buffer containing 0 .02% o-phenylene diamine (Sigma Chemical Company, St Louis, Missouri) . The absorbance was read with the EIA reader (Bio-Tek Instruments, Burlington, Vermont) . Statistics . Statistical analysis was obtained using an Apple Macintosh SE computer with a Stat View 512+ program . Analysis of variance (ANOVA) was used to determine differences between groups . The Fisher PLSD was used for post-hoc ANOVA comparisons with a P value of <0 .01 considered significant . This work was supported in part by the Ralph Hochstetter Medical Research Fund in honor of Dr Henry C . and Bertha H . Buswell, by a grant from the World Health Organization Steering Committee on Encapsulated Bacteria, and by Public Health Service grants A126279 and AI18384 from the National Institute of Allergy and Infectious Diseases . This work is dedicated to Leendert G . Westerink, Ph .D . (1913-1990) .

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Rubenstein LJ, Goldberg B, Hiernaux J, Stein KE, Bona CA. Idiotype-antiidiotype regulation V . The requirement for immunization with antigen or monoclonal antiidiotypic antibodies for the activation of b,6 and b2,1 polyfructosan-reactive clones in BALB/c mice treated at birth with minute amounts of anti-A48 idiotype antibodies. J Exp Med 1983 ; 158 : 1129-44, 19 . Westerink MAJ, Campagnari AA, Wirth MA, Apicella MA . Development and characterization of an ani-idiotype antibody to the capsular polysaccharide of Neisseria meningitidis serogroup C . Infect Immun 1988 ; 56 :1120-7 . 20, Apicella MA . Identification of a subgroup antigen on the Neisseria meningitidis group C capsular polysaccharide . J Infect Dis 1974 ; 129 : 147-53 . 21 . Kennett R . Cell fusion . Methods Enzymol 1979 ; 58 : 345-59 . 22 . Sjogren-Jansson E, Jansson S . Large-scale production of monoclonal antibodies in dialysis tubing . J Immunol Methods 1985 ; 84 : 359-64 . 23 . Jerne NK . 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