INFECTION AND IMMUNITY, Apr. 1983, p. 245-256 Vol. 40, No. 10019-9567/83/040245-12$02.00/0Copyright 0 1983, American Society for Microbiology

Further Studies on the Immunogenicity of Haemophilusinfluenzae Type b and Pneumococcal Type 6A Polysaccharide-

Protein ConjugatesCHIAYUNG CHU,t RACHEL SCHNEERSON,* JOHN B. ROBBINS, AND SURESH C. RASTOGIDivision ofBacterial Products, Office of Biologics, Food and Drug Administration, Bethesda, Maryland

20205

Received 13 September 1982/Accepted 10 December 1982

Conjugates were prepared by carbodiimide-mediated coupling of adipic acidhydrazide derivatives of Haemophilus influenzae type b (Hib), Escherichia coliK100, and pneumococcal 6A (Pn6A) polysaccharides with tetanus toxoid (TT), asan example of a "useful" carrier, and horseshoe crab hemocyanin (HCH), as anexample of a "nonsense" carrier. These conjugates were injected into NIH mice,and their serum antibody responses to the polysaccharides and proteins werecharacterized. As originally reported, Hib conjugates increased the immunogenic-ity of the capsular polysaccharide and elicited greater than the estimatedprotective levels of anti-Hib antibodies in most recipients after one injection andin all after the third injection (Schneerson et al., J. Exp. Med. 152:361-376, 1980).Both Hib conjugates induced similar anti-Hib responses. The K100-HCH conju-gate was more immunogenic than the K100-TT conjugate and elicited anti-Hibresponses similar to the Hib conjugates after the third injection. Simultaneousinjection of the K100 and the Hib conjugates did not enhance the anti-Hibresponse. The Pn6A-TT conjugate induced low levels of anti-Hib antibodies;when injected simultaneously with the Hib conjugates, the anti-Hib response wasenhanced, as all mice responded after the first injection and with higher levels ofanti-Hib than observed with the Hib conjugates alone (P < 0.05). The Pn6Aconjugates were not as immunogenic as the Hib conjugates. Pn6A-TT was moreeffective than was Pn6A-HCH; it elicited anti-Pn6A (>100 ng of antibody nitrogenper ml) in 6 of 10 mice after the third injection. The addition of the Hib-HCHconjugate to the Pn6A-TT conjugate increased the anti-Pn6A response with ahigher geometric mean antibody titer, and 9 of 10 mice responded after the thirdinjection. A preparation of diphtheria toxoid, TT, and pertussis vaccine increasedthe anti-Hib antibody levels after the first injection only in mice receiving Hib-TT,but not in mice receiving Hib-HCH, suggesting that additional carrier protein (TT)enhanced the anti-polysaccharide response. Simultaneous injection of Hib andPn6A conjugates with the same or different carriers resulted in an enhanced serumantibody response to each polysaccharide. The anti-tetanus toxin responsereached protective levels (>0.01 U/ml) in most mice after the first injection and inall mice after the second and third injections of Tf conjugates. A progressiveincrease in the anti-HCH response with each additional injection was noted inanimals receiving HCH conjugates. Animals receiving the diphtheria toxoid-TT-pertussis vaccine preparation responded with a greater increase in anti-carrierantibody than those receiving the conjugates alone. This method of synthesisprovided conjugates capable of inducing protective levels of antibodies to both thepolysaccharides and carrier proteins.

Prevention of invasive diseases, especially ity despite antibiotic and supportive therapy andmeningitis, due to Haemophilus influenzae type because of the increasing emergence of antibiot-b (Hib) has been advocated because of their ic-resistant organisms isolated from patients (55,frequency and continuing morbidity and mortal- 61). Serum antibodies to the capsular polysac-

charide of Hib have been shown to confer pro-

t Visiting Fellow, National Institutes of Health Fogarty tective immunity against invasive diseasesInternational Center, on leave from Institute of Biological caused by this organism (1, 3, 37, 39-41, 44, 45,Products, Shanghai, People's Republic of China. 48, 50, 54). The purified Hib polysaccharide has

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246 CHU ET AL.

limited usefulness as a vaccine because it doesnot induce a protective serum antibody responsein infants, the age group with the highest inci-dence of invasive Hib diseases (37, 38, 41, 45,58). In addition to this problem of age-relatedimmunogenicity, the Hib polysaccharide doesnot induce a booster response; a single injectionelicits the maximum response for each recipient(41, 58).The ability of polysaccharide-protein conju-

gates to increase the immunogenicity of a bacte-rial capsular polysaccharide, pneumococcustype 3, was first reported by Goebel and Averyin 1929 (17). Recently, we have reported asynthetic scheme for conjugates composed ofproteins and the capsular polysaccharide of Hib(47, 49, 51-53). The use of a spacer molecule,adipic acid dihydrazide (ADH), facilitated thecoupling reaction between the Hib polysaccha-ride and proteins providing high yields of conju-gates. In contrast to the slight or undetectableserum antibody response induced by the Hibpolysaccharide in laboratory mice, the conju-gates induced high levels of antibodies withbactericidal activity in almost all recipients. Theimmunogenicity of these conjugates was dosedependent within ranges that could be consid-ered for human infant use, was increased se-

quentially after two and three injections, andwas enhanced both by priming with the carrierprotein and by incorporation with Freund adju-vant. It was proposed that the conjugates bothincreased the immunogenicity of the Hib poly-saccharide and conferred upon it the propertiesof a "T-dependent antigen." In preliminary ex-periments, the Hib conjugates showed a similarincreased immunogenicity when injected intoprimates. This synthetic approach for preparingHib polysaccharide-protein conjugates was

found to be applicable for preparing similarcompounds with the capsular polysaccharides ofother human pathogens, pneumococcus 6A andEscherichia coli K13 (49, 51). Other workershave reported similar results with meningococ-cal and Hib polysaccharides by different syn-thetic schemes (2, 7, 24; E. C. Beuvery, V.Kanhai, P. C. van Putten, A. van der Kaader,and J. Haverkamp, Abstr. Conference on Patho-genic Neisseria, Montreal, 4 to 6 August 1982).

In our original report, the protein was treatedwith ADH and a water-soluble carbodiimide toform an adipic acid hydrazide (AH) derivative.These AH derivatives were then bound to cy-

anogen bromide-activated Hib polysaccharide.This synthetic scheme posed some problems. (i)the immune response to the native protein in-duced by the conjugates was slight; and (ii) theAH derivatives of tetanus toxoid (TT) andhorseshoe crab (Limulus polyphemus) hemocya-nin (HCH) were poorly soluble at pH 4.7, which

is considered optimal for the carbodiimide reac-tion (21).

Ideally, the conjugates would serve to induceprotective antibodies to the polysaccharide aswell as to the carrier protein. Accordingly, theconjugate should be constructed so as to pre-serve the immunogenicity of the native protein.In the present study, the conjugates were pre-pared by a modified scheme. The polysaccha-rides were first activated by cyanogen bromideand then reacted with ADH. The resultant AH-polysaccharide derivatives were bound to pro-tein carriers by the carbodiimide reaction. Theconjugates so prepared elicited both anti-poly-saccharide and antiprotein antibodies.The structures of the three polysaccharides

used are shown in Fig. 1; all three contain aribitol 5-phosphate in their repeat unit (12, 13,27, 42). The Hib and K100 polysaccharides havethe same composition and differ only in theirribose-ribitol bond; they have cross-reactiveantigenic and immunogenic properties. E. coliK100 was studied to see whether the anti-Hibresponse could be accelerated by the addition ofthis closely related polysaccharide. The K100-induced anti-Hib antibodies in adult volunteers(23, 48, 50). The Pn6A polysaccharide was cho-sen for study because that pneumococcal type isa common cause of diseases in infants andchildren, its immunogenicity is age dependentlike that of the Hib polysaccharide, it is acomparatively poor immunogen among the 14types in the polyvalent pneumococcal vaccine,and it was not cross-immunogenic with the Hibpolysaccharide in a clinical study (4, 5, 8, 9, 33,43, 56, 57). Two protein carriers were studied:TT as an example of a "useful" carrier andHCH as an example of a "nonsense" carrier.Experiments were designed to increase the yieldof bound polysaccharide in the conjugate. Theeffect of the additional injection of more thanone conjugate and of the diphtheria toxoid-TT-pertussis vaccine (DTP) vaccine to the Hibconjugates was also studied.

MATERIALS AND METHODSCarrier proteins. TT preparations were concentrat-

ed by vacuum dialysis (Union Carbide, Chicago, Ill.)

Pn6A--2)-oLD-Galp-(l1-3)-oL-D-Glcp-(1-3)ct-L-RhapD-(1 >3)-D-Ribitol-5-PO4

Hib--3)-D-Ribf-(l1 >1)-Ribitol-5-)PO4-

E. coli K100--.3)-D-Ribf-(l1 >2)-Ribitol-5--PO4-FIG. 1. Structure of capsular polysaccharides used

for preparation of the polysaccharide-protein conju-gates.

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H. INFLUENZAE AND PNEUMOCOCCUS CONJUGATES 247

and equilibrated against 0.2 M NaCl at 3 to 8°C to afinal concentration of about 40 mg of protein per ml.The concentrated TT preparations were centrifuged at20,000 x g for 20 min and passed through a 5 by 100-cm S-300 Sephacryl (Pharmacia Fine Chemicals, Pis-cataway, N.J.) column equilibrated in 0.2 M NaCI-0.01% NaN3 at 3 to 8°C. The peak corresponding to amolecular weight of 150,000 was pooled, concentratedby vacuum dialysis, and equilibrated against 0.2 MNaCl by exhaustive dialysis at 3 to 8°C. The finalmaterial was adjusted to 60 mg of protein per ml,centrifuged at 20,000 x g for 2 h at 4°C, passed througha 0.45-nm membrane (Nalge Co. Rochester, N.Y.),and stored at 3 to 8°C. The following TT preparationswere used: Shanghai Institute of Biological Products,Shanghai, People's Republic of China, lot no. 713;Institute Armand Frappier, lot no. AT3/4 7, 2433Lf/mg N protein, (courtesy of Jack Cameron); andWyeth Laboratories lot no. 4371, 2939 Lf/mgN, (cour-tesy of Alan Bernstein).The HCH was prepared from cell-free hemolymph

(donated by T.-Y. Liu, Food and Drug Administra-tion) (25). The hemolymph was centrifuged at 150,000x g at 10°C for S h. The supernatant was discarded,and the pellet was dissolved in 0.2 M NaCl andrecentrifuged at the same conditions. The blue pelletwas dissolved at approximately 100 mg/ml and passedthrough an S-300 Sephacryl column (see above), andthe peak corresponding to a molecular weight of65,000 was pooled, concentrated by vacuum dialysis,equilibrated against 0.2 M NaCl, and centrifuged at48,000 x g for 2 h at 100C, and the supernatant waspassed through a 0.45-nm membrane and stored at 3 to80C.

Polysaccharides. The Hib polysaccharide, preparedfrom strain 1482, contained less than 1% (wt/wt)protein and nucleic acid and less than 0.04% (wt/wt)lipopolysaccharide and had a Kd of 0.4 (molecularsize) (44, 47, 63, 64). The Pn6A polysaccharides weredonated by Allen Woodhour, Merck Sharp & DohmeResearch Laboratories, West Point, Pa., and FrankCano, Lederle Laboratories, Pearl River, N.Y. Bothpreparations passed the specifications for this type(10).

Synthesis of the conjugates. The polysaccharideswere converted into sodium salt by passage throughDowex 50Wx8 (14). They were then activated withcyanogen bromide (Eastman Chemical Products, Inc.,Rochester, N.Y.) at pH 10.5 for 6 min and reacted with0.1 M ADH by tumbling overnight at 3 to 8°C. Thereaction mixture was dialyzed against 0.2 M NaCl withtwo changes for 24 h, followed by gel filtration throughG-100 Sephadex equilibrated in water, and freeze-dried. The resultant polysaccharides contained about2.0%o (wt/wt) adipic hydrazide (22).The AH polysaccharide derivatives and the proteins

were used at 20 ml/mg each, and coupling was donewith 0.1 M 1-ethyl-3-(3-dimethylamninopropyl)carbo-diimide-hydrochloride (Eastman) for 3 h at 4°C. Thereaction mixture was passed through a CL-4B Sepha-rose column, and the void volume peak was pooled,passed through a 0.45-nm filter, and stored at 4°C. Theconcentrations of proteins and polysaccharides wereassayed as described previously (26, 32, 47).

Serology. Serum anti-Hib antibodies and anti-Pn6Aantibodies were measured by radioimmunoassays (45,46). All samples were performed in duplicate, and the

results are expressed as the geometric mean (GM) forall animals in an experimental group and the number ofresponders (anti-Hib > 0.15 ,ug of antibody per ml;anti-Pn6A, >100 ng of antibody nitrogen (AbN) per ml(45, 46). Hyperimmune sera, used as references, weretaken from mice injected with 10 ,ug of TT or HCH incomplete Freund adjuvant (62). The antitoxin titerswere assayed as described by Barile et al. (6) withmale mice. Two instead of three mice were used foreach serum dilution. The mouse reference anti-TT wasassayed for its antitoxin activity by twofold dilutionsand found to contain 200 U of anti-toxin when com-pared with the U.S. reference tetanus antitoxin; othersera were titrated at 10-fold dilutions. An enzyme-linked immunosorbent assay (ELISA) was performedby the method of Engvall et al. (15). Samples of 4 ,ug ofTT or HCH per ml were coated to polyvinyl chlorideplates. Goat anti-mouse gamma globulin was conjugat-ed to alkaline phosphatase, and p-nitrophenyl phos-phate disodium (Sigma) was used as the substrate. Theconcentration of anti-HCH antibodies in the referenceserum was measured by the quantitative precipitinreaction.

Chemicals. The Hib and K100 polysaccharides weremeasured by the Bial reaction with a Hib polysaccha-ride, dried over P205, as a reference (26). The Pn6Apolysaccharide was measured by the orcinol reaction,and the protein was measured by the method of Lowryet al. (32). The hydrazide was measured by the methodof Inman (22).

Immunization. Six-week-old, female N:NIH(S)(NIH) mice were injected subcutaneously with salinesolutions of the conjugates containing 2.5 F.g of poly-saccharide chosen on the basis of previous experi-ments and because this amount of conjugate seemedcompatible with proposed dosage in human infants(37, 41, 44, 56, 58). When two or more conjugates wereinjected simultaneously they were mixed in a singlesyringe. When Hib and K100 conjugates were injectedsimultaneously, each was injected at 1.25 ,ug of poly-saccharide. When Hib or K100 (or both) was injectedwith Pn6A, 2.5 ,ug ofeach was used. Groups of 10 micewere injected three times at 2-week intervals with eachconjugate or combination of conjugates and were bled2 weeks after the first immunization and 1 week afterthe second and third immunizations. DTP (LederleLaboratories, Pearl River, N.Y.; lot. no. 661-506) wasinjected in 0.1-ml amounts at a different site in threeexperimental groups. Control animals, receiving onlypolysaccharide, were omitted in most experimentsbased upon an extensive experience in our laboratoryand others which has uniformly showed little or nodetectable antibody in most mice injected with a widedosage range of these polysaccharides (2, 47).

Statistics. The antibody levels were converted tologarithms to the base 10, and all statistical analyseswere performed on the log data. The term "mean"refers to the mean of log antibody levels. The antilogof a mean is the GM of a group of antibody levels.Group 5 (Pn6A-HCH) in Table 2 was excluded fromstatistical analyses, as there were no positive respons-es.Among groups receiving the same number of injec-

tions, means were compared by using the F test ofanalysis of variance. If means were significantly differ-ent, then they were arranged in increasing order anddivided into subgroups in which means are not signifi-

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248 CHU ET AL.

cantly different from each other (P < 0.05). TheNewman-Keuls method of the multiple comparisontest was used to create these subgroups which may beoverlapping, i.e., a mean may belong to more than onesubgroup (59). In Tables 2, 3, 5, and 6, subgroups areidentified by vertical and horizontal bars. For exam-ple, in Table 2 for the first injection, group 6 has thesmallest mean and group 11 has the largest. The firstsubgroup contains groups 6 and 4, the second sub-group contains groups 4 and 3, the third subgroupcontains groups 3, 8, 2, and 9, and the fourth subgroupcontains groups 8, 2, 9, 14, 13, 7, 1, 10, 12, and 11. Thisshows that groups 6 and 4 are not different and groups4 and 3 are not different but groups 6 and 3 arestatistically different, etc.

RESULTSEfficiency of coupling reaction. The effect of

the concentration of the protein and the Hibpolysaccharide during the coupling reactionupon the yield of conjugates was studied. Inpreliminary studies, it was found that the yieldof conjugates, based upon the amount of poly-saccharide bound, was variable; the concentra-tion of the reactants in the 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide - hydrochloride -

mediated coupling reaction seemed to be impor-tant in determining this variation. Figure 2shows a representative experiment with TT tostudy this problem. The concentrations of boththe AH-Hib polysaccharide and TT were keptequal and increased from 5.0 to 30.0 mg/ml. Theconcentration of 1-ethyl-3-(3-dimethylaminopro-pyl)carbodiimide-hydrochloride was kept con-stant. After incubation for 3 h at 4°C and pH 4.9± 0.1, the reaction mixture was dialyzed againstphosphate-buffered saline overnight at 3 to 80Cand centrifuged at 20,000 x g at 4°C for 20 min,and the supernatant was passed through a 4BSepharose column. There was a progressiveincrease in the efficiency of the coupling reac-tion as the concentration of the reactants in-creased; there was 8% recovery when the reac-tants were 5 mg/ml and a 62% recovery when thereactants were 30 mg/ml. In addition, the pro-tein/polysaccharide ratio decreased from about3.0/1 at 5.0 mg/ml to 1.4/1 to 1.6/1 at 30 mg/ml.On the basis of these data, and because of thedifficulty using higher concentrations of proteinswith limited amounts of tetanus toxoid, theprotein and polysaccharide were used at 20mg/ml for the coupling reactions.Not shown are experiments in which the ratio

of protein to polysaccharide was varied from 1/3to 3/1. No increased efficiency of binding wasobserved throughout this range of reactants.

Characterization of the conijugates. The yieldand composition of the conjugates used in theseexperiments are shown in Table 1. The protein/polysaccharide ratio varied from 2.8 for Pn6A-HCH to 1.4 for Hib-TT. The yields ranged from

0.3

0.2

0.1

c6

0.3

0.2

0.1

10 20 30 40 50 60 10 2D 30 40 50 60

VOWME EFFWENT (ml) POLYSACCHARIDE--PROTEINFIG. 2. Effect of concentration of reactants upon

the composition and yield of Hib-TT conjugates. Thesolid line depicts the concentration of the polysaccha-ride, and the broken line shows the protein concentra-tion of the effluent. The concentration of the AHderivative of the Hib polysaccharide and TT wasvaried; the concentration of 1-ethyl-3-(3-dimethyla-minopropyl)carbodiimide-hydrochloride was keptconstant. After the 3-h coupling reaction at 4°C and atpH 6.0, the mixture was dialyzed against saline at 2 to8°C for about 18 h, centrifuged at 12,000 x g at 4°C for20 min, and passed through a 4B Sepharose column.Increasing the concentration of the Hib polysaccha-ride and TT increased both the yield of materialpassing through the void volume of the column (V0)and the ratio of polysaccharide to protein in the finalconjugate.

a low of 2.2% (K100-HCH) to 52% (Pn6A-HCH).

Anti-Hib antibody response. Table 2 shows theserum anti-Hib responses induced by the sixconjugates. The GM of anti-Hib antibodies in-duced by the two Hib conjugates after the firstimmunization exceeded the estimated protectivelevel (0.15 ,ug of antibody per ml). The GM andpercentage of responders increased after thesecond and third immunizations with these twoconjugates. After the third immunization, allanimals responded with greater than 0.15 ,ug ofantibody per ml. There were no differencesbetween the anti-Hib responses to these twoconjugates after any of the three immunizations.The K100 conjugates elicited a lesser anti-Hib

response than that of the Hib conjugates afterthe first immunization. Thereafter, the K100-HCH conjugate elicited both a higher GM and a

5 mg/ml 4 15 mg/ml 48% 28%

I X iiNI l I F |I

* 20mg/mi + 30 mg/ml 4A 3462%

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H. INFLUENZAE AND PNEUMOCOCCUS CONJUGATES 249

TABLE 1. Characterization of polysaccharide-protein conjugatesa

Prt/Conjugate Protein Polysaccharide psb Yield

Hib-TT 407 252 1.6 33Hib-Trr 490 342 1.4 45Hib-HCH 565 268 2.1 25K100-TT 544 236 2.3 25K100-HCH 78 39 2.0 2.2Pn6A-TT 90 53 1.7 50Pn6A-HCH 504 180 2.8 52

a The polysaccharide was measured by the Bialreaction (26), and the protein was measured by themethod of Lowry et al. (32). The yield was determinedby calculating the amount of polysaccharide in thevoid volume of a calibrated 4B Sepharose columncompared with the amount of starting material.

b Prt/Ps, Protein/polysaccharide.cIThe second preparation of Hib-Tr conjugate was

not used for immunization in this experiment.

higher proportion of responders than the K100-TT conjugate (P < 0.05). After the second andthird immunization, the K100-HCH conjugateinduced anti-Hib antibodies similar to those elic-ited by the two Hib conjugates.The Pn6A-TT conjugate elicited a slight anti-

Hib response after the second and third immuni-zations; this response was less than that inducedby the two Hib conjugates (in Pn6A-TT versus

Hib-HCH, P < 0.0025 after the second immuni-zation and P < 0.001 after the third immuniza-tion; in Pn6A-TT versus Hib-TT, P < 0.001 forthe second and third immunizations) and theK100-HCH conjugate (P < 0.01). The Pn6A-HCH conjugate did not elicit anti-Hib antibodiesafter any of the three injections.The effect of injecting both Hib conjugates

was similar to that observed with the monova-lent preparations. The total Hib polysaccharidedose was 2.5 pLg in the mice receiving either themonovalent or the bivalent preparations. Therewere no differences between the anti-Hib anti-bodies in the groups that received both Hibconjugates after any of the three immunizationsby using the criteria of either the GM or thepercentage of responders. Similarly, there wasno enhancement of anti-Hib when Hib conju-gates were injected simultaneously with either ofthe K100 conjugates, although the GM of anti-Hib antibodies induced by the K100-HCH andthe Hib conjugates were higher than those elicit-ed by the K100-TT along with the Hib conju-gates (P < 0.05).The GM and percentage of responders was

greater after the first injection in mice receivingHib-HCH and either of the Pn6A conjugates (P< 0.05). There was a 100% response, and theGMs were 1.79 for Pn6-HCH and 1.86 for Pn6A-TT when combined with the Hib-HCH as com-pared with 0.42 for Hib-HCH alone. After the

TABLE 2. Serum anti-Hib response of mice injected with HiB, K100, Pn6A polysacchari4es conjugated toHCH or TT alone or with DTP adsorbeda

GM F.g of 4pti-Hib antibody per ml (no. of responders/total) afterGroup Conjugates injection

1 2 3

1 Hib-HCH 0.42 (8/10) 0.74 (8/10) 7.49 (11/11)2 Hib-TT 0.20 (6/9) 2.32 (10/10) 5.17 (9/9)3 K100-HCH 0.12 (4/10) 1.43 (10/10) 7.72 (10/10)4 K100-TT 0.06 (2/10) 0.27 (5/10) 0.69 (8/8)5 Pn6A-HCH 0.03 (0/10) 0.06 (1/10) 0.04 (0/10)6 Pn6A-TT 0.04 (1/10) 0.13 (4/10) 0.45 (7/10)7 Hib-HCH + K100-HCH 0.40 (8/10) 4.64 (10/10) 8.47 (10/10)8 Hib-HCH + K100-TT 0.19 (7/10) 1.66 (9/9) 3.93 (11/11)9 Hib-HCH + Hib-TT 0.24 (6/10) 4.53 (10/10) 12.01 (9/9)10 Hib-HCH + Pn6A-HCH 1.70 (10/10) 4.09 (9/9) 5.31 (10/10)11 Hib-HCH + Pn6A-TT 1.86 (10/10) 3.83 (10/10) 8.79 (10/10)12 Hib-TT + DTP 1.74 (10/10) 9.36 (10/10) 18.10 (10/10)13 Hib-HCH + DTP 0.39 (7/9) 7.62 (10/10) 10.37 (10/10)14 Hib-HCH + K100-HCH + 0.36 (10/10) 4.72 (10/10) Not done

Pn6A-HCH + DTPa GMs are ranked in increasing order and grouped by the multiple comparison method (59).First injection: 6, 4, 3, 8, 2, 9, 14, 13, 7, 1, 10, 12, 11.

Second injection: 6, 4,1,3,8,2,11,10,9,7,14,13 12 Third injection: 6, 4, 8, 2, 10, 1, 3, 7, 11, 13, 9, 12.

Anti-Hib antibody of 6-week-old female NIH mice injected subcutaneously with Hib, K100, and Pn6Aconjugates 1, 2, and 3 times is shown.

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250 CHU ET AL.

second injection, the GM was higher in the micereceiving the Pn6A conjugates along with theHib-HCH than in mice receiving monovalentHib-HCH (P < 0.05); this difference was nolonger detectable after the third immunization.The combination of the Pn6A conjugate withHib-HCH elicited a 100% response after thesecond and third injections. In these groups, thedose of Hib polysaccharide was 2.5 p.g as com-pared with 1.25 ,ug of Hib polysaccharide in thegroups receiving the combination of Hib andK100 polysaccharides.Anti-Pn6A antibody response. In general, the

Pn6A polysaccharide conjugates were less im-munogenic than the Hib conjugates (Table 3).The more immunogenic of the two Pn6A conju-gates, Pn6A-TT, induced only 2 of 10 respondersafter the first injection (GM, 13.5 ng of AbN perml). This progressively increased to 6 of 10responders (GM, 159.3 ng of AbN per ml) afterthe third immunization. The Pn6A-HCH conju-gate induced only 2 of 10 responders after thethird immunization with aGM of 21.0 ng ofAbNper ml.When Hib-HCH and Pn6A-HCH were inject-

ed simultaneously, they induced an anti-Pn6Aresponse only after the second and third injec-tions. This combination elicited anti-Pn6A anti-bodies in 4 of 10 mice with a GM of 54.4 ng ofAbN per ml. The combination of Pn6A-TT andHib-HCH elicited 9 responders out of 10 miceafter the third injection with a GM of 418 ng ofAbN per ml.

Anti-tetanus toxin response. The anti-Tf titersare expressed as units of antitoxin with the U.S.standard anti-tetanus toxin as the reference.These units were derived from the ELISA as-

says of the individual sera compared with themouse reference serum (200 U/ml).

Table 4 shows the relation between theamount of antitoxin and anti-Tl as obtained byELISA for four representative mouse sera.

There is only a fair correlation, as two of thevalues for anti-lT and ELISA are compatible.Accordingly, the protective level of anti-TT(0.01 U/ml) in sera assayed by ELISA can onlybe estimated. Table 5 shows the anti-lT inducedby the conjugates alone or in combination withDTP. There was no anti-lT induced by the Hiband K100 conjugates prepared with HCH. Allthree conjugates prepared with 1T induced an

antitoxin response. All animals that receivedany conjugate or combination of conjugates pre-pared with TT responded with protective levels('0.01 U/ml) of anti-TT after the second andthird immunizations. There was no difference inthe antitoxin response induced by the three TTconjugates after any of the three immunizations.As would be expected, the addition of DTPyielded the highest anti-TT of all the groups.After the third immunization, the mice receivingHib-TT conjugate responded with the highestanti-TT (108.9 U/ml) as compared to those re-ceiving DTP and conjugates prepared with HCH(P < 0.05).Anti-HCH response. Serum anti-HCH anti-

bodies were detected in all groups injected withan HCH conjugate alone or in combination of anHCH with a TT conjugate. As expected, no anti-HCH antibodies were detected in groups inject-ed with TT conjugates (Table 6). We did not listthe number of responders since there is no

known protective activity for anti-HCH antibod-ies.The level of anti-HCH antibodies increased

with the number of injections in all groups. TheK100-HCH and Pn6A-HCH conjugates inducedsimilar levels of anti-HCH antibodies after each

TABLE 3. Serum anti-Pn6A antibody response of mice injected with HiB or Pn6A polysaccharidesconjugated to HCH or TT alone or with DTP adsorbeda

GM ng of AbN per ml of anti-Pn6A (no. of responders/total)Group Conjugates after injection

1 2 3

5 Pn6A-HCH 13.7 (0/10) 7.1 (1/9) 21.0 (2/10)6 Pn6A-TT 13.5 (2/10) 3.1 (3/10) 159.0 (6/10)10 Hib-HCH + Pn6A-HCH 4.2 (0/10) 52.9 (3/9) 54.4 (4/10)11 Hib-HCH + Pn6A-TT 14.6 (0/10) 59.1 (4/10) 418.0 (9/10)12 Hib-TT + DTP 5.9 (0/10) 1.2 (0/10) 2.5 (0/10)13 Hib-HCH + DTP Not done Not done 53.0 (3/9)

a The GMs are ranked in increasing order and grouped by the multiple comparison method (59).First injection: 10, 12, 6, 5, 11.

Second injection: 12, 6, 5, 10, 11.,j IThird injection: 12, 5, 13, 10, 69 11.

Anti-Pn6A antibody of 6-week-old female NIH mice injected subcutaneously with conjugates containing 2.5,ug of polysaccharides one, two, and three times is shown.

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TABLE 4. Serum anti-tetanus toxin antibodies,measured by neutralization and ELISA, in mice

immunized with Hib-TT conjugates with and withoutDTPa

Anti-rTT (U/ml)Conjugate No. of

injections Neutral- ELISAization

Hib-TT 2 <0.02 0.025Hib-TT 3 >0.2-<2.0 0.144Hib-T + DTP 2 >0.2-<2.0 22.69Hib-lT + DTP 3 >20-<200 168.0

a Individual sera taken from mice injected two orthree times with Hib-TT conjugates with and withoutsimultaneous injection of 0.1 ml of DTP at a differentsite, were assayed for their anti-TT antibody by bothELISA and toxin neutralization.

injection. The addition of DTP elicited higherlevels of anti-HCH antibodies than the Hib-HCH conjugate alone (P < 0.01). The anti-HCHantibodies elicited by the combination of Hib-HCH plus K100-HCH plus Pn6A-HCH plusDTP (group 14), containing approximately 8.0,ug of HCH for each injection, were the highest(P < 0.05).

Cross-reaction between Hib and Pn6A polysac-charides. Hib-TT and Hib-HCH injected alonedid not induce anti Pn6A antibodies. The anti-Pn6A response elicited by the Pn6A-TT conju-gates after the third immunization was enhancedby the addition of the Hib-HCH after the thirdinjection (Table 3). Injection of the combinationresulted in a higher GM and number of respond-

ers than did the injection ofPn6A-TT alone (GMof 418 versus 159 and 9 versus 6 responders,respectively, out of 10 mice). Unexpectedly, themore immunogenic of the two Pn6A conjugates,Pn6A-TT, induced anti-Hib especially after thethird injection (GM of anti-Hib, 0.45; 7 respond-ers out of 10 mice) (Table 2). This same conju-gate, Pn6A-TT, given in combination with Hib-HCH enhanced the anti-Hib antibodies after thefirst injection as compared with Hib-HCH alone(GM of 1.8 and 10 responders out of 10 miceversus GM of 0.42 and 7 responders out of 10mice; P < 0.05).Pn6A-HCH was less immunogenic than Pn6A-

TT, yet both Pn6A conjugates gave a similarenhancement of response to anti-Hib when giv-en with Hib-HCH after the first injection ascompared with Hib-HCH alone. There was nodemonstrable effect of these Pn6A conjugatesadded to Hib-HCH on the anti-Hib antibodiesafter the second and third injections; all miceresponded in these groups with high levels ofanti-Hib antibodies.

Effect on anti-Hib response of simultaneousiDjection of conjugates with DTP adsorbed. DTP(0.1 ml) was injected simultaneously at anothersite with Hib-TT or Hib-HCH to study the effectof this common pediatric vaccine on the anti-Hibresponse to the conjugates (Table 2). DTP en-hanced the anti-Hib response to the Hib-TT, butnot to the Hib-HCH, after the first injection; thenumber of responders was increased from 6 to10 responders out of 10 mice, and the GMincreased from 0.20 to 1.74 ,ug/ml (P < 0.05).

TABLE 5. Serum anti-TT responses in mice immunized with Hib, K100 or Pn6A conjugated to TT or HCHalone or in combination with DTP

GM anti-TT U/ml (no. of responders/total) after injectionGroup Conjugates

1 2 3

2 Hib-TT 0.018 (6/6) 0.049 (6/6) 0.172 (4/4)3 K100-HCH 0 0 04 K100-TT 0.034 (4/8) 0.201 (10/10) 0.290 (9/9)6 Pn6A-TT 0.020 (7/8) 0.093 (9/9) 0.53 (9/9)8 Hib-HCH + K100-TT 0.023 (10/10) 0.177 (9/9) 0.064 (11/11)9 Hib-HCH + Hib-TT 0.009 (6/10) 0.027 (9/10) 0.099 (7/)11 Hib-HCH + Pn6A-TT 0.051 (10/10) 0.128 (9/9) 0.407 (9/9)12 Hib-TT + DTP 0.331 (10/10) 6.92 (10/10) 108.9 (10/10)13 Hib-HCH + DTP 0.623 (9/9) 4.85 (10/10) 36.5 (10/10)14 Hib-HCH + K100-HCH + 0.485 (10/10) 2.98 (8/8) Not done

Pn6A-HCH + DTPa The GMs are ranked in increasing order and grouped by the multiple comparison method (59).First injection: 9, 2, 6, 8, 4, 11, 12, 14, 13.

.- 11 1JSecond injection: 9, 2, 6, 11, 8, 4, 14, 13, 12

Third injection: 8, 9, 2, 4, 11, 6, 13, 12.L|'J'.,.JI LJ

Groups of 10 mice were injected with conjugates containing about 3.0 to 4.0 ,ug of either TT or HCH. Theirsera were assayed for anti-TT by ELISA. These results were converted to anti-TT units with the use of areference antiserum.

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TABLE 6. Serum anti-HCH antibodies in mice immunized with Hib, K100, or polysaccharides conjugated toHCH or TT with and without DTP adsorbeda

GM anti-HCH (,ug of antibody per ml of serum) after

Group Conjugates injection1 2 3

1 Hib-HCH 0.08 0.19 0.622 Hib-TT Not done 0 Not done3 K100-HCH 0.12 0.58 0.904 K100-TT Not done 0 Not done5 Pn6A-HCH 0.09 0.29 0.997 Hib-HCH + K100-HCH 0.10 0.33 0.788 Hib-HCH + K100-TT 0.09 0.36 0.699 Hib-HCH + Hib-TT 0.07 0.18 0.4510 Hib-HCH + Pn6A-HCH 0.13 0.36 0.9911 Hib-HCH + Pn6A-TT 0.11 0.16 0.4313 Hib-HCH + DTP 0.11 0.46 1.514 Hib-HCH + K100-HCH + 0.17 1.05 Not done

Pn6A-HCH + DTP

a The GMs are ranked in increasing order and grouped by the multiple comparison method (59).First immunization: 14, 10, 3, 13, 11, 7, 8, 5, 1 9.

Second immunization: 14, 3, 13, 10, 8, 7, 5, 1, 9, 11.L

Third immunization: 13, 10, 5, 3, 7, 8, 1, 9, 11.I,Groups of 10 mice were injected with conjugates containing about 3.0 to 4.0 ,ug of either HCH or TT. The

animals were bled as described in the text, and their sera were assayed for anti-HCH by ELISA.

After the second and third immunizations, allmice in both groups responded with higher lev-els of anti-Hib antibodies, but there was no

statistically significant difference between theHib-TT groups with and without DTP. In theHib-HCH groups, DTP enhanced the anti-Hibresponse from 8 out of 10 mice and a GM of 0.74,ug/ml to 10 out of 10 and a GM of 7.62 (P <

0.05). After the third immunization, all miceresponded with high levels of anti-Hib antibod-ies, and there was no significant differencecaused by the addition of DTP.DTP was also injected simultaneously with

three conjugates: Hib-HCH, K100-HCH, andPn6A-HCH. All animals responded after thefirst and second injection, although the anti-Hiblevel was similar to that elicited by the Hibconjugates alone.

Effect of adsorption of conjugates on alum. Notshown are the anti-Hib antibody responses ofmice that received the Hib-TT conjugate ad-sorbed on alum. There was no difference in themice receiving the alum-adsorbed conjugatecompared to the response elicited by the conju-gate in saline.

DISCUSSIONThe serum anti-Hib antibody response, elicit-

ed by the conjugates prepared by derivatizationof the Hib polysaccharide rather than the proteincarrier, was similar to those reported for the

original method (47, 53). The attachment of thespacer (ADH) to the polysaccharide did notadversely affect its solubility or reaction withtyping antiserum and eliminated the problems ofsolubility encountered with some of the AHprotein derivatives. There was no differencebetween the anti-Hib antibody response inducedby conjugates prepared with the "useful" carri-er, TT, over that prepared with a " nonsense"carrier, HCH. Therefore, to avoid injection ofadventitious antigens into infants, we suggestthat conjugates should be prepared with "usefulcarriers." This method of synthesis seems to bea general one as AH polysaccharides have beencoupled to several "useful" carrier proteins (R.Schneerson, C. Chu, M. C. Hardegree, J. andB. Robbins, manuscript in preparation). Beu-very et al. have reported on the antibody re-sponse elicited by conjugates composed ofgroup A meningococcal polysaccharide and TTprepared without a spacer and with spacerscontaining two- and six-carbon chains (Beuveryet al., Abstr. Conference on Pathogenic Neisser-ia). These three conjugates elicited a similarprimary anti-group A response. However, therewas a graded anti-group A secondary responserelated to the length of the carbon spacer; thehighest response was elicited by the conjugatewith the six-carbon spacer. The conjugate pre-pared with the six-carbon spacer also elicited thehighest serum antibodies to the carrier protein(TT) after both primary and secondary immuni-

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H. INFLUENZAE AND PNEUMOCOCCUS CONJUGATES 253

zations. They postulated that the six-carbonspacer allowed more interaction of the two mac-romolecules comprising the conjugate with lym-phoid cells and thus a greater immunogenicitythan the conjugates prepared with no spacer or atwo-carbon spacer.The combination of the Hib-TT conjugate and

DTP, which elicited the highest levels of anti-Hib, did not induce anti-Pn6A antibodies afterthree injections. The combination of Hib-HCHand DTP induced a slight anti-Pn6A responseafter the third injection. Similarly, Pn6A-TTinduced a slight anti-Hib response, which in-creased with the number of injections. WhenHib-HCH was injected with either Pn6A-HCHor Pn6A-TT, both the anti-Hib and anti-Pn6Aresponses were increased over that induced byeither conjugate alone. Therefore, the injectionof two conjugates did not exert a negative effect,and it was encouraging to see that the combina-tion of Hib-HCH and Pn6A conjugates induced10 of 10 responders after only one injection. Oneexplanation for this enhanced anti-Hib and anti-Pn6A response was that the total amount of thecross-reacting polysaccharides was two times(5.0 ,ug1dose) than that injected with either con-jugate alone. The steep dose-response curveobserved for the Hib conjugates makes this apossibility (2, 47, 52). Another explanation forthe enhancement in the antibody responses toboth polysaccharides elicited by their simulta-neous administration as conjugates may be dueto the cross-immunogenicity between the Hiband Pn6A polysaccharides reported in hyperim-mune animal sera (36), but hitherto unreportedin humans. The structural basis for this cross-reactivity is that both the Hib and Pn6A containribitol 5-phosphate in their repeat unit (12, 42).The cross-reactivity between the Hib and Pn6Apolysaccharides in mice, elicited by the singleconjugate, was only observed after two andthree injections; in the aforementioned clinicalstudy, the adult volunteers received only oneinjection of the Pn6A polysaccharides (43, 55). Asimilar enhancement of both anti-Hib and anti-Pn6A antibodies has been observed in primatesinjected with these Hib and Pn6A conjugates(Chu, Schneerson, Robbins, and Hardegree, un-published data).No advantage upon anti-Hib antibody forma-

tion was achieved by using the K100 conjugatesalone or in combination with Hib-HCH. Howev-er, in contrast to the studies with combinationsof Pn6A and Hib conjugates, the total amount ofpolysaccharide in the experiments when K100and Hib conjugates were combined was thesame (2.5 ,ug per dose) as that administered withthe monovalent preparations. Further experi-ments are planned to study the effect of variousdoses and combinations of Hib, Pn6A, and K100

conjugates upon the enhancement and accelera-tion of the anti-polysaccharide response.DTP-adsorbed vaccines contain two compo-

nents, Bordetella pertussis cells and aluminiumsalts, known to modify the human serum anti-body response (18). Moxon et al. reported thatsimultaneous injection of purified Hib polysac-charide and DTP, mixed together, into 2- to 5-year-old children depressed the anti-Hib re-sponse (35). King et al. reported that theimmunogenicity of Hib polysaccharide, previ-ously incubated wtih B. pertussis cells, wasincreased in infants (28, 55). The nature of theassociation between the Hib polysaccharide andthe B. pertussis was not reported. The effect ofB. pertussis upon the response to another capsu-lar polysaccharide, pneumococcal type 3, inmice has been studied. When B. pertussis wasinjected intraperitoneally, there was a depres-sant effect upon the anti-type 3 response (29).However, B. pertussis exerted an enhanced anti-type 3 response when it was injected subcutane-ously (60). The simultaneous administration ofDTP with the Hib conjugates appeared to havetwo effects. The first was an accelerated anti-Hib response after the first injection of Hib-TTwith DTP; both the GM and number of respond-ers were greater than those elicited by Hib-TTalone. This accelerated anti-Hib response wasnot noted when DTP was added to the Hib-HCHconjugate, despite the fact that a similar re-sponse was elicited by these two Hib conjugatesinjected singly. The enhancement may havebeen due to the additional tetanus component inthe DTP exerting a "carrier" effect. According-ly, further experiments are planned to study theeffect of simultaneously injecting diphtheria tox-oid and TT along with polysaccharide conju-gates prepared with these two pediatric vaccinecomponents. The second effect of DTP was anenhancement of both the anti-polysaccharideand anti-carrier antibody levels after the secondand third injections. This enhancement mayhave been due to the adjuvant effect of thelymphocytosis-promoting factor from the B. per-tussis cells or the aluminium salts (or both) (16,29, 31). This adjuvant effect, described for thelymphocytosis-promoting factor and similar tothat reported for cholera toxin (11), may havebeen responsible for the enhancement of theanti-Hib response reported by King et al. (28). Itwas not possible to decide whether the DTPexerted an adjuvant effect upon the anti-TTantibodies in the groups receiving TT conju-gates. We failed to detect an adjuvant effectupon anti-Hib levels in animals injected with anHib conjugate adsorbed with aluminium salts.

Mitani et al. reported that conjugates, pre-pared with pullalan (linear copolymer of malto-triose) and Ti elicited considerably lower levels

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254 CHU ET AL.

of immunoglobulin E (IgE) anti-TT antibodiesthan a similar dose of Formalin-treated, alumin-ium-precipitated TT (34). Both the conjugateand the formalinized, precipitated TT elicitedsimilar amounts ofIgM and IgG anti-TT antibod-ies. The conjugation procedure reduced the tox-icity of the TT to satisfactory levels. The pulla-lan-TT conjugate also elicited less IgE anti-tetanus antibodies than the formalinized,aluminium-precipitated TT (20, 31). This pro-vides supportive information that conjugatessuch as those described in this paper may beconsidered for clinical trials.Our experiments have shown that a "useful"

carrier is as effective as a "nonsense" carrier inmice. Therefore, it would seem that a "useful"carrier would be preferred for human use pro-vided it does not interfere with established im-munization programs or the acquisition of "nat-ural" immunity. Additional possible candidatesfor carrier proteins would be outer membraneproteins of Hib that induce bactericidal antibod-ies (19, 22, 31a).As yet we have no explanation for the lesser

immunogenicity of K100-TT as compared toK100-HCH or Pn6A-HCH as compared withPn6A-TT. There was no demonstrable differ-ence in the molecular size characteristics orchemical composition of these less immunogenicconjugates. This is an important problem, as itwill be necessary to accurately predict the im-munogenicity of these conjugates if they are tobe considered for clinical studies.

ACKNOWLEDGMENTS

We are grateful to Carolyn Hardegree for assay of the anti-toxin titers of the sera and for her review of this manuscript.We are also grateful to Gerald Schiffman, State University ofNew York, Downstate Medical Center, Brooklyn, N.Y., forhis determinations of the anti-Pn6A antibodies.

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