INFECrION AND ImmuNrry, Dec. 1992, p. 4977-49830019-9567/92/124977-07$02.00/0Copyright © 1992, American Society for Microbiology

Vol. 60, No. 12

Immunogenicity of Conjugate Vaccines Consisting ofPneumococcal Capsular Polysaccharide Types 6B,

14, 19F, and 23F and a Meningococcal OuterMembrane Protein Complex

PHILIP P. VELLA,"* STEPHEN MARBURG,2 JOAN M. STAUB,1 PETER J. KNISKERN,1WILLIAM MILLER,1 A. HAGOPIAN,1 C. IP,1 RICHARD L. TOLMAN 2CYNTHIA M. RUSK,' L. S. CHUPAK,2 AND RONALD W. ELLIS1

Department of Cellular & Molecular Biology, Merck Sharp & Dohme Research Laboratories,West Point, Pennsylvania 19486,1 and Synthetic Chemistry Research, Merck Sharp &

Dohme Research Laboratories, Rahway, New Jersey 070652

Received 7 February 1992/Accepted 11 September 1992

In an effort to prepare pneumococcal (Pn) capsular polysaccharide (Ps) vaccines that would be immunogenicin infants, covalent conjugates were prepared for Pn types 6B, 14, 19F, and 23F. Each Ps type was covalentlybound to an outer membrane protein complex from Neisseria meningitidis serogroup B and evaluated forimmunogenicity in mice and infant monkeys. The conjugates induced specific anti-Ps antibody responses inmice and in infant rhesus and African green monkeys; a coniugate of 6B and outer membrane protein complexwas immunogenic at Ps doses as low as 20 ng. Although low levels of the Pn group-common cell wallpolysaccharide were present in all type-specific Ps preparations, anti-cell wall polysaccharide responsesinduced by covalent conjugates were <1% of the total anti-Ps response after two doses of vaccine. In contrast,the anti-cell wall polysaccharide response of a noncovalent conjugate represented 41% of the anti-Ps responseafter two doses. Relative T-cell dependence, a requirement for the human target population of infants less than18 months old, was demonstrated for all four Pn Ps con,jugates in an athymic mouse model. Therefore, thesePn Ps-outer membrane protein complex conjugate vaccines are excellent candidates for evaluation in humaninfants.

Invasive disease caused by Streptococcus pneumoniae ismost prevalent in children less than 2 years old; this includesgreater than 80% of pneumococcal (Pn) meningitis and Pnacute otitis media (AOM) cases and 70% of Pn bacteremia.S. pneumoniae is the leading cause of bacterial pneumoniarequiring hospitalization in infants and children and has thehighest case fatality ratio of the bacterial meningitides (19,25). Attack rates for AOM are difficult to estimate, butavailable case data indicate that at least three-fourths of allchildren will experience at least one episode by 6 years ofage (21). S. pneumoniae is responsible for approximately50% of all AOM, and Pn middle ear infections may exceed106 cases in the United States for children less than 2 yearsold (3). Types 6, 14, 19, and 23 account for >50% of initialand subsequent Pn AOM episodes as well as Pn meningitis,Pn bacteremia, and Pn pneumonia (18). There is evidencethat antibody induced in adults to the Pn capsular polysac-charide (Ps) is protective against Pn infection (5, 35, 38).

Antibiotic resistance of pneumococci appears to be on theincrease, and treatment ofAOM with antibiotics that are cellwall directed might contribute to further inflammation of themiddle ear through lysis of cell wall products in the middleear area (13). An immunoprophylactic approach to Pn inva-sive disease appears warranted, and children less than 2years old represent the prime target population for aneffective Pn Ps-based conjugate vaccine.A purified Pn Ps vaccine containing 14 Ps types first was

licensed in the United States in 1977 (20a). Elevated levels of

* Corresponding author.

postvaccination anti-Pn Ps antibodies persisted at least 2 and3.5 years in children and adults, respectively (42). Subse-quently 23-valent vaccines were licensed in 1983 and arerecommended for use in children 2 years old and older aswell as adults. T-cell-independent antigens, such as bacterialPs, are usually poorly or not immunogenic in children lessthan 2 years of age (6). Pn Ps of types 6A, 14, 18C, 19F, and23F induced poor antibody responses even in children 2 to 15years of age (9, 28, 32, 37).

Conjugate vaccines have been developed to enhance theantibody responses of young children to Ps antigens. Con-jugation of Pn Ps and Haemophilus influenzae type b (Hib)capsular Ps to T-cell-dependent carriers has potentiated theantibody response to the Ps in otherwise poorly responsivehosts (2, 37, 43), although carrier priming may be necessaryfor conjugates employing diphtheria toxoid-derived conju-gates (33, 41). Hb conjugate vaccines are efficacious andhave been licensed for routine immunization of 2-month-oldinfants (4, 35). The use of the outer membrane protein(OMP) complex (OMPC) from Neisseria meningitidis sero-group B as an effective carrier has been shown to potentiateantibody levels to the Hib capsular Ps (polyribosyl ribitolphosphate [PRP]) in animals and humans (1, 8, 43). APRP-OMPC conjugate vaccine (PedvaxHIB; Merck Sharp &Dohme Research Laboratories) has been demonstrated to behighly effective in protecting high-risk Navajo infants vacci-nated at 2 months of age from Hib disease (35). Infantmonkeys have proven to be a useful model for the immuno-genicity of PRP-OMPC in human infants (41, 43, 44), in thatboth PRP and a noncovalent PRP-OMPC conjugate arenonimmunogenic and a covalent PRP-OMPC conjugate is

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immunogenic in both species after the first dose at 2 monthsof age with a booster effect after the second dose 1 to 2months later. In this paper we describe studies in which thePn Ps of types 6B, 14, 19F, and 23F were each covalentlylinked to OMPC and their immunogenicities in mice andinfant monkeys were demonstrated.

MATERIALS AND METHODS

Conjugate vaccines. Derivatization of Ps, OMPC, andpreparation and analysis of the covalent conjugate vaccineswere described previously (30). Current Ps/OMPC ratios arelisted in Table 2. Each type-specific Pn Ps is isolated fromfermentations of S. pneumoniae by alcohol precipitation. Toimprove solubility and handling during conjugation andsubsequent processing, the Pn Ps is subjected to molecularweight reduction by thermal or sonication treatment beforeconjugation (26); the sizes of the Pn Ps ranged from-350,000 to 600,000 kDa as determined on Sepharose 4Bcolumn chromatography. This is discussed in more detailelsewhere (30a). The OMPC is obtained from the outermembrane of N. meningitidis serogroup B strain B-il bydetergent extraction of intact cells after fermentation (20).Covalent conjugates are referred to as 6B-OMPC, 14-OMPC, etc. Physical mixtures of derivatized Pn Ps andOMPC are designated 6B+OMPC. Finally, conjugates pre-pared by a different method (39) where convalency could notbe demonstrated chemically (referred to as noncovalent) aredesignated 6B/OMP. The outer membrane protein used inthis conjugate was prepared by using an ammonium bicar-bonate extract of N. meningitidis serogroup B cells (39).Vaccines were tested as monovalent preparations adsorbedto aluminum hydroxide (0.45 mg of A13+ per ml) suspendedin 0.85% (wt/vol) saline solution containing 1:20,000 thimer-osal. Vaccine dosage levels were quantitated on the basis ofrate nephelometry assays specific for each Pn Ps type (34).Vaccine doses were selected based on infant monkey

studies performed previously with an Hib conjugate (PRP-OMPC) in which doses from 0.2 to 15.0 ,g induced compa-rable anti-PRP responses (41, 43). The polysaccharide/pro-tein ratios used (0.13 to 0.23) were selected because similarratios (0.06 to 0.26) for PRP-OMPC were observed to beimmunogenic in infant rhesus monkeys (44).Animals. Female, athymic (nu/nu; nude) mice 6 to 8 weeks

old and their congenic controls (nul+) were obtained fromHarlan Sprague Dawley, Indianapolis, Ind. Infant (2- to3-month-old) rhesus (Macaca mulata) and African green(Cercopithecus aethiops) monkeys of both sexes were pro-vided by New Iberia Research Center, Division of South-western Louisiana University, New Iberia, and CharlesRiver-Key Lois, Summerland Key, Fla., and were housedwith their mothers at these sites.

Immunization. Mice (8 per group) were immunized with0.5 ml of vaccine intraperitoneally on days 0, 7, and 28 andbled on day 35. Mice in the placebo group included with eachtest received 0.225 mg of aluminum hydroxide in saline. In aseparate test, some of the nu/+ mice (congenic controls)were immunized with a mixture of the four polysaccharidesand the OMPC. Since the nu/nu mice do not respond anybetter than the nul+ mice do, only the nul+ mice were usedas controls to test the Pn Ps mixed with the OMPC. Monkeyswere injected intramuscularly with vaccine on days 0 and 28,unless noted otherwise, with 0.25 ml of vaccine administeredinto the flank muscle of each leg (0.5 ml total; two sites wereselected rather than one to minimize the volume at any onesite because of the small muscle size). Monkeys were bled

just before injection and on day 42, unless noted otherwise.Doses were selected based on studies performed previouslywith an Hib conjugate vaccine (43). Injections and bleedingswere performed under the direction of W. E. Greer and thestaff at the New Iberia Research Center and P. W. Schillingand the staff at Charles River-Key Lois.Radioimmunoassay antibody determinations for Pn types.

Mouse and monkey sera were assayed for antibodies to PnPs types 6B, 14, 19, and 23F by a radioimmunoassay thatuses native, nonsized '4C-Pn capsular Ps (36). Standardhyperimmune anti-Pn Ps sera (rabbit) were purchased fromthe New York State Department of Health at Albany.Quantitative values for anti-Pn Ps were provided for eachtype from which linear standard curves were prepared foreach assay.ELISA antibody determinations for Ps, CPs, and OMPC.

Enzyme-linked immunosorbent assay (ELISA) procedureswere used to measure immunoglobulin G (IgG) against thenative, nonsized Pn capsular Ps, group-common C polysac-charide (CPs), OMPC, and IgM against the native, nonsizedPn capsular Ps. All ELISAs were conducted with sterile,optically certified microtiter plates (Corning no. 25801) withantigens solubilized in phosphate-buffered saline (PBS). In-cubations were performed on a rocking platform at 20 to25°C, and plates were washed three times with a PBSsolution on a ProWash system (Perkin-Elmer Cetus, Nor-walk, Conn.) with 0.5 ml of wash buffer per well and allowedto soak for 5 to 10 s. The wash buffer contained 0.05%(vol/vol) Tween 20 in PBS for anti-Pn Ps and anti-CPsdeterminations and 0.1% (vol/vol) Tween 20 for anti-OMPCassays. The CPs assay uses a sandwich ELISA techniquethat employs a polyclonal rabbit anti-CPs complex (lot h2,diluted 1:1,000 in PBS) and a CPs complex used at 1 ,ug/ml;both complexes were obtained from the Statens Seruminsti-tut, Copenhagen, Denmark. The serum diluent consisted ofPBS-0.05% Tween 20-1.0% (wt/vol) bovine serum albumin(BSA) for anti-Pn Ps and anti-CPs assays and 0.1% Tween20-0.5% BSA for anti-OMPC assays. Alkaline phosphataseconjugated to affinity-purified goat anti-human IgG or IgM(Kirkegaard and Perry Laboratories, Inc., Gaithersburg,Md.) was used at 0.25 ng/ml (IgG) or 1.0 ng/ml (IgM) foranti-Pn Ps determinations or at 1.0 ng/ml for anti-OMPCassays. The substrate, 0.1 ml of p-nitrophenyl phosphate(Sigma no. N-2765), was used at 1.0 mg/ml in 1 M diethanol-amine-0.001 M MgCl2 (pH 9.8) and warmed to 37°C for use.Sodium hydroxide (3 M) was added (50 pl per well) to stopall reactions after plates had incubated for 30 to 40 min at 20to 25°C. The A405 was read on a Biotek EL310 microplatereader. The specific absorbance of a standard was used in aweighted four-parameter logistics curve-fitting program byD. Robard and P. J. Munson (National Institute of ChildHealth and Human Development), as modified by D. M.Gross (Merck), to generate the standard curves for theanti-OMPC, anti-Pn Ps IgG, and anti-CPs assays. The stan-dard curves for the anti-Pn Ps IgM assays were generatedfrom a linear regression line-fitting program by T. Schofield(Merck).Anti-Pn Ps assay. For the IgG anti-Pn Ps assay, 0.1 ml of

Pn 6B Ps (5.0 ,g/ml) or 23F Ps (20.0 ,ug/ml) was added toeach microtiter plate well and incubated for 1 h; for the IgMassay, 0.1 ml of Pn 6B Ps (5.0 ,ug/ml) was added to each well,and the plates were incubated for 16 to 20 h. The plates werewashed, 0.1 ml of diluted serum was added to each well, andthe plates were incubated for 2 h and then washed. After PnPs was added, the IgM anti-Pn Ps assay plates were blockedwith 2.0% BSA in PBS (0.15 ml per well) before the test sera

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were added. An alkaline phosphatase conjugate (0.1 ml) wasadded to each well, and the plates were incubated for 2 h andthen washed. A substrate (0.1 ml) was added to each well,the plates were incubated, and then the reactions were

stopped. The A405 was read. A pool of human serum fromadults vaccinated with a 14-valent Pn Ps vaccine was pur-

chased from D. Granoff, Washington University, St. Louis,Mo. IgG was purified on protein G and Sepharose 6Bcolumns (Pharmacia), and anti-Pn Ps values were estab-lished by quantitative precipitation. Two other human serum

pools from the same source serve as controls in each assay.Unfortunately, there is no general reference standard cur-rently in use among the various laboratories involved inpneumococcal vaccine research. An IgM standard was pre-

pared from the human serum pool described above bypassing the pooled sera through a Sepharose S300 HRcolumn (Pharmacia); passes of the eluate were made until>95% IgM purity was achieved. Two other human serum

pools served as controls for each assay.Anti-Pn CPs assay. Samples of 50 ,ul of rabbit anti-CPs

polyclonal serum were added to microtiter plate wells andincubated for 18 to 20 h at 20 to 25°C, and then the plateswere washed. CPs complex was added (50 p,l per well, 1p,g/ml), and the plates were incubated for 1 h and thenwashed; then 0.1 ml of blocking buffer solution (PBS with0.05% Tween 20 and 2.0% BSA) was added to each well, andthe plates were incubated for 1 h and then washed. Dilutedserum (50 ,ul per well) was added, and the plates were

incubated for 2 h and then washed; then 50 ,ul of alkalinephosphatase conjugate was added, and the plates were

incubated for 2 h and then washed. The addition of warmedsubstrate (0.1 ml per well) was followed by a 30- to 40-minincubation, after which the reaction was stopped with so-

dium hydroxide. The A405 was read. An anti-Pn CPs IgGstandard was prepared in a manner similar to that describedabove for the anti-Pn Ps ELISA. Pooled sera from humansvaccinated with Pn Ps served as controls for each assay.Anti-OMPC assay. OMPC (4.0 p.g/ml) in 0.05 M NaHCO3

(pH 9.5) was added (0.1 ml per well), and the plates wereincubated for 18 to 20 h and then washed. A blocking buffer(PBS-0.1% Tween 20-2.0% BSA) was added to each well toprevent nonspecific binding. The plates were incubated for 1h and then washed. Diluted sera (0.1 ml per well) were

added, and the plates were incubated for 2 h and thenwashed; 0.1 ml of alkaline phosphatase conjugate per wellwas added, and the plates were incubated for 1 h and thenwashed four times. Warmed substrate (0.1 ml per well) wasadded, the plates were incubated for 30 to 40 min, and thenthe reaction was stopped with sodium hydroxide. The A405was read. The standard serum used was prepared from a

human adult that had been vaccinated with a PRP-OMPCconjugate. The serum was purified through a protein Aaffinity column (Pharmacia), dialyzed, purified through an

OMPC-cyanogen bromide-activated Sepharose 4B column,dialyzed, concentrated by ultrafiltration, and quantitated byIgG radial immunodiffusion. The purified IgG was used toprepare standard curves for each assay; the unknown testserum values were calculated from these standard curves.

Statistics. The statistical analysis for the athymic mouse

studies was performed by a two-sided, two-sample t test onlog titers.

RESULTS

Immunogenicity in mice. The nude mouse (nu/nu), whichlacks T cells, and its congenic counterpart (nul+) are a

TABLE 1. Anti-Pn Ps responses to Pn-OMPC conjugates inathymic mice and their congenic littermates

Dose (Ig) GMT, Fg of anti-Pn Ps/ml (95%confidence intervals) in:

VaccinePn Ps OMPC nu/nu (athymic) nu/+ miceamice

6B-OMPC 0.5 3.4 4.8 (2.4, 9.7) 523.3 (443.1, 617.9)14-OMPC 0.5 2.3 11.7 (7.5, 18.4) 338.4 (273.8, 418.4)19F-OMPC 0.5 4.0 2.1 (1.1, 4.2) 96.8 (67.1, 139.5)23F-OMPC 0.5 3.6 0.2 (0.1, 0.3) 4.8 (0.9, 24.0)

a P < 0.05 compared with the nu/nu response (two-sided two-sample t teston log titer).

useful model for testing the relative T-cell dependence of theimmune response to a particular antigen. All four Pn Ps-OMPC conjugates were immunogenic in the nul+ mice, andthe responses for each Pn type were significantly higher(>10-fold) than the anti-Pn Ps response of the nulnu mice(Table 1). Geometric mean titers (GMTs) ranged from 4.8 to523.3 F±g/ml for the four Pn types. When an aluminumhydroxide placebo was injected into nul+ mice, anti-Pn Pscapsular responses were not observed (<0.2 ,ug/ml; data notshown). A physical mixture of Pn Ps type 6B, 14, 19F, or 23Fwith OMPC induced marginal anti-Pn capsular Ps responsesin nul+ mice (GMTs, 0.2 to 0.6 ,ug/ml; data not shown).Immunogenicity in infant monkeys. Given the utility of

infant monkeys as a model for the immunogenicity of PRP-OMPC in human infants, we tested Pn Ps-OMPC conjugatevaccines in infant monkeys. Both African green and rhesusmonkeys were used, given the constraints on the periodicavailability of both species over time.

All four Pn Ps-OMPC conjugate vaccines were immuno-genic (in the radioimmunoassay) after one or two doses inboth species of monkey. The anti-Pn Ps GMTs in infantAfrican green monkeys were >1.0 ,ug/ml for all conjugatesafter one dose and >30.0 ,ug/ml after two doses (Table 2).Responses after two doses increased at least 2.5-fold overthose after one dose for all types. Doses of 0.5 to 5.0 ,uginduced comparable anti-Pn Ps GMTs after one or two dosesof 6B- or 23F-OMPC (Table 2). Furthermore, a 1.0-,ug doseof a physical mixture of 23F Ps with OMPC or 23F Ps alonedid not elicit any detectable anti-23F antibody response(<0.2 ,ug/ml).-Anti-Pn 6B IgG responses of infant African green mon-

keys were determined after one (106 and 170 ,ug/ml) and two(381 and 1,026 ,ug/ml) doses of 0.5 and 5.0 p.g, respectively,whereas infant rhesus monkeys had responses of 201 and 47I±g/ml after two doses of 1.0 and 10.0 ,ug, respectively (datanot shown). Anti-Pn 6B IgM responses after one and twodoses of 5.0 ,g were 11 and 14 ,ug/ml, respectively (data notshown). Anti-Pn 23F Ps IgG responses in infant rhesusmonkeys were 1,358 and 2,731 ,ug/ml after two doses of 0.5and 5.0 ,ug, respectively.The 6B-OMPC conjugate was immunogenic in infant

rhesus monkeys, although anti-Pn Ps responses were ca. 2-to 10-fold lower than those observed in infant African greenmonkeys (Table 3). As in infant African green monkeys, aphysical mixture of Pn 6B Ps and OMPC did not elicitanti-6B responses (<0.2 1Lg/ml in infant rhesus monkeys),and a 25-,g dose of noncovalent 6B/OMP conjugate eliciteda minimal anti-6B response (GMT, 4.4 ,ug/ml after twodoses, data not shown).

All preparations of type-specific Pn Ps contain the group-

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TABLE 2. Anti-Pn Ps and anti-OMPC responses of infant African green monkeys to Pn Ps-OMPC conjugatesadsorbed to aluminum hydroxide

GMT of anti-Pn Ps GMT of anti-OMPCVaccine Dose (pg) No. of S-CMHCO/ (tg/ml) on day: (,g/ml) on day:

monkeys lysinePs OMPC 0 28 42 0 28 42

Pn 6B-OMPC 5.0 33.5 4 0.077 <0.2 81.6 212.9 <0.7 85.1 787.80.5 3.3 4 0.077 <0.2 62.4 164.9 <0.7 53.6 183.4

Pn 14-OMPC 0.5 2.2 4 0.098 <0.2 1.4 30.0 <0.7 44.7 216.2Pn 19F-OMPC 0.5 3.9 3 0.053 <0.2 5.4 53.2 <0.7 130.1 210.3Pn 23F-OMPC 5.0 35.0 3 0.031 <0.2 71.4 485.9 <0.7 338.5 1,136.5

0.5 3.5 4 0.031 <0.2 23.9 357.6 <0.7 177.4 590.6Pn 23F+OMPC 0.5 3.5 2 NAb <0.2 <0.2 <0.2 <0.7 115.4 8,110.2Pn 23F Ps 0.5 0.0 3 NA <0.2 <0.2 <0.2 <0.7 <0.7 <0.7

a S-CMHC, S-carboxymethylhomocysteine.NA, not applicable.

common CPs, which might conjugate to OMPC and therebypotentiate its immunogenicity. Therefore, the anti-CPs re-sponses to the Pn Ps-OMPC conjugate vaccines were eval-uated. The anti-CPs IgG responses of infant African greenand infant rhesus monkeys were <1.0 ,ug/ml after two 0.5- to10-pg doses of 6B- or 23F-OMPC covalent conjugates. Theanti-CPs response induced by the covalent conjugates aftertwo doses represented c0.5% of the total IgG anti-Pn Psresponses observed for any of the 6B- or 23F-OMPC vac-cines tested (data not shown). The highest anti-CPs response(GMT, 1.8 ,ug/ml) observed after two doses of vaccine wasfor a noncovalent 6B/OMPc conjugate in infant rhesusmonkeys, which likewise was the highest response (41% ofthe total IgG) when compared with the anti-Pn Ps responseafter two doses; the anti-Pn 6B IgG response (GMT) was 4.4p,g/ml (data not shown).Anti-OMPC responses were measured to determine

whether the carrier was immunogenic after conjugation.Anti-OMPC responses were observed for Pn Ps-OMPCconjugates of 6B, 14, 19F, and 23F in infant African greenmonkeys (Table 2). The anti-OMPC responses induced byeach of the four conjugates range from 44.7 to 338.5 jig/mlafter one dose to 183.4 to 1,136.5 ,ug/ml after two doses. Theanti-OMPC response observed for a physical mixture of Pn23F and OMPC was 115.4 ,g/ml after one dose and 8,110.2,g/ml after two doses.

DISCUSSIONOf the 83 types of pneumococci, only a few are responsi-

ble for most pediatric disease. Types 6B, 14, 19F, and 23Fare the most common and account for approximately half ofinvasive Pn disease in children. An effective vaccine againstthese types would provide an excellent basis for a pediatric

TABLE 3. Anti-Pn Ps responses of infant rhesus monkeys toPn 6B Ps-OMPC conjugate adsorbed to aluminum hydroxide

GMT of anti-Pn PsVaccine Dose No. of (1Lg/ml) on day:(>g of Ps) monkeys

0 28 42

Pn 6B-OMPC 10.0 3 <0.2 4.4 17.91.0 3 <0.2 6.5 67.20.1 3 0.3 7.0 31.40.02 3 <0.2 2.5 10.7

Pn 6B+OMPC 10.0 3 <0.2 <0.2 <0.2

pneumococcal vaccine. A similarly prepared Hib conjugatevaccine (PRP-OMPC) was evaluated in the infant monkeymodel, which is a reliable predictor of immunogenicity inhuman infant clinical trials (43). Pn Ps types 6B, 14, 19F, and23F covalently conjugated to the T-cell-dependent OMPCcarrier elicit antibody responses in mice and infant monkeys.

Conjugation of a variety of bacterial Ps to different proteincarriers has resulted in the induction of enhanced T-cell-dependent anti-Ps responses (23, 31, 43). Mice and nonhu-man primates have responded poorly or not at all to theseand several other nonconjugated bacterial Ps and to conju-gates, such as PRP oligosaccharide linked to nontoxic diph-theria toxoid from mutant strain Corynebacterium diphthe-riae CRM197, PRP-diphtheria toxoid, or Pn 6A-tetanustoxoid, that appear to be dependent on some type of coprim-ing with diphtheria toxoid or tetanus toxoid (7, 10, 37, 41). Inthe current study, Pn Ps conjugated to OMPC inducedanti-Pn Ps responses in normal mice (nu/+) that weresignificantly higher than those in the respective groups ofathymic mice, thus indicating relative T-cell dependence.However, the level of anti-Pn capsular Ps response observedin the nu/nu mice suggests that the conjugate also has aT-cell-independent characteristic that may be attributable tothe manner of presentation by the OMPC. The Pn Ps whenmixed physically with OMPC are poorly immunogenic innu/+ mice in our studies. A noncovalent conjugate of Pn6B/OMP (39) was poorly immunogenic in infant rhesusmonkeys with a coupling procedure described previously(40). This would suggest that the conjugation methodologyand configuration of the conjugate are key factors in elicitinganti-Pn Ps potentiation and T-cell dependency.Schneerson et al. demonstrated that a Pn 6A-tetanus

toxoid conjugate administered in 5- or 50-,ug doses waspoorly immunogenic in both juvenile and infant rhesusmonkeys (37). The coadministration of PRP-tetanus toxoidwith Pn 6A-tetanus toxoid did not improve the anti-Pn 6Aresponse for either dose. This may reflect inefficient antigenpresentation or a carrier that is not sufficiently immunogenic;covalent conjugation may not be the sole determinant ofimmunogenicity in an immunologically immature host.

In contrast to the previously reported studies in animals,covalent Pn Ps-OMPC conjugates have induced anti-Pn Psresponses of 11 to 486 Lg/ml in infant rhesus and Africangreen monkeys receiving two vaccine doses of 0.02 to 10.0,ug. The anti-Pn 6B IgG responses after one and two doses(5.0 ,ug each) represent 93.5 and 98.7%, respectively, of theanti-Pn 6B IgM responses. A similar pattern was observedpreviously for anti-PRP responses to an Hib conjugate (43).

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The OMPC and conjugation chemistry (30) used here havebeen demonstrated to potentiate the immunogenicity of HibPRP (1, 43). The components detected in the OMPC areproteins of classes P1.2, P2a, and P4 (43) with approximately3 to 5% lipopolysaccharide. All three proteins participate inthe Hib conjugation reaction as described previously (43),and it is likely that this situation is occurring with Pnconjugates since the same carrier, Ps derivatization, andOMPC derivatization methods are being employed. Theconfiguration of the OMPC does not appear to have beenaltered during conjugation, since IgG antibody induced toconjugated OMPC is being measured in an ELISA withnonderivatized OMPC. Also, conjugated OMPC in PRP-OMPC vaccines induces anti-OMPC serum bactericidal an-tibody as measured in a complement-dependent serum bac-tericidal assay with the homologous N. meningitidisserogroup B strain (source of OMPC) as the challengeorganism (43). Serum bactericidal antibody responses to theOMPC in the Pn conjugates have not yet been determined. Aconjugate of 6B is immunogenic at doses as low as 0.02 ,g ininfant rhesus monkeys; excellent primary and secondaryresponses are observed. It may suggest that extensiveT-helper cell activity is being induced in these monkeys,given the high levels of anti-Ps that are measured.Although CPs is present in the native Pn Ps (5 to 15%) and

in the sized polysaccharides (1 to 3%) used in these studies(22), covalent conjugates of 6B- and 23F-OMPC induceanti-CPs responses of <1.0 p,g/ml, which represents <1.0%of the specific anti-Pn Ps response after two doses in infantrhesus and African green monkeys. In contrast, anti-CPslevels elicited by a noncovalent 6B/OMP conjugate were41% of the anti-6B response. This may be attributable to thehigher CPs content of the native Pn Ps used in the noncova-lent conjugate and or the different coupling chemistry thatwas utilized. Koskela demonstrated that a 14-valent Pn Psvaccine induced IgG, IgA, and IgM anti-CPs, but the in-creased response was less than twofold and did not contrib-ute substantially to the type-specific antibody response (27).Most individuals appear to harbor CPs antibodies as a resultof natural exposure to pneumococci, but the relevance ofboosted anti-CPs, if they were induced, is unknown.

Opsonophagocidal antibody is induced in infant rhesusmonkeys by 6B-, 23F-, 19F-, and 14-OMPC conjugates (16)when a method described previously for Hib (17) is used.Antibody to Pn facilitates phagocytosis (11, 15), and onewould expect opsonic activity to play a major role inpreventing Pn infection. Additional functional activity forPn-OMPC conjugates has been demonstrated in an AOMmodel in chinchillas (14), where 6B- and 19F-OMPC wereimmunogenic and effective in preventing purulentAOM (12).Studies with additional serotypes are ongoing.The use of aluminum hydroxide as an adjuvant extends

from preclinical studies with PRP-OMPC, in which furtherpotentiation of the immune response was observed in infantrhesus monkeys (43). In humans, PRP-OMPC adsorbed toaluminum hydroxide was associated with superior tolerabil-ity compared with an aqueous saline conjugate formulation(29).Anti-OMPC responses were induced by all Pn Ps-OMPC

conjugates in infant African green monkeys. The immuno-genicity of OMPC appears to be substantial after one andtwo doses with a definite booster response observed after thesecond dose; doses of <5 ,ug of OMPC induced anti-OMPCresponses in excess of 175 ,ug/ml, indicating that OMPC is anexcellent immunogen. The anti-OMPC response of a physi-cal mixture of Pn 23F and OMPC was similar to the covalent

conjugate of 23F after one dose but exceeded 8,100 ,g/mlafter two doses. The high anti-OMPC response (> 10-fold) tothe physical mixture relative to the response to Pn 23F-OMPC (0.5-Rg dose) is not surprising, since the covalentlybound Pn Ps would be expected to cover a portion of thespecific antigenic determinant sites of the OMPC. Thisobviously does prevent excellent potentiation of anti-Pn Psin the covalent conjugates.The immunogenicity data in infant monkeys suggest

T-cell-dependent properties and appear to predict immuno-genicity in children less than 2 years of age. Preliminaryimmunogenicity data in children and infants less than 6months old indicate that a Pn 6B-OMPC covalent conjugateis immunogenic (24); larger numbers of vaccinees will accu-mulate as the study continues. Sera from this study indicatethat opsonins are induced by the conjugate in infants (16).The presence of a covalent bond, conjugate configuration,

mode of antigen presentation, aluminum hydroxide adju-vant, and immunogenic properties of the OMPC as a carriermay all contribute to its effectiveness in potentiating anti-PnPs responses in mice and monkeys. Covalent conjugatesprepared with OMPC for Hib conjugates were T cell depen-dent and immunogenic in infant monkey species, whereasnoncovalent Hib conjugates with similar PRP and OMPCwere not T cell dependent or immunogenic. Covalency alonemay not be enough if the carrier is not sufficiently immuno-genic. Seid demonstrated covalency for HbOC, but theconjugate is not immunogenic in diphtheria toxoid-naiveanimals (41) and humans (33). The infant monkey model hasbeen shown to be a reliable indicator of the immunogenicityof PRP-OMPC in young children and infants (1, 43). Cur-rently, various doses of multivalent conjugate formulationsin infant African green monkeys are being studied forimmunogenicity and antibody persistance; this will be dis-cussed in another manuscript (unpublished data). Clinicalstudies with the other conjugate vaccines are in progress todetermine the reliability of the infant monkey model inpredicting the immunogenicities of the various Pn Ps-OMPCconjugates in humans.

ACKNOWLEDGMENTSWe thank J. Armstrong, P. Burke, K. Dolan, R. Hepler, W.

Hurni, and S. Szymanski for technical assistance, T. Schofield forstatistical analysis, and W. E. Greer and the staff at New IberiaResearch Center, Southwestern Louisiana University, and P. W.Schilling and the staff at Charles River-Key Lois at SommerlandKey, Fla., for performance of immunogenicity tests in infant mon-keys.

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