HYBRIDOMA Volume 11, Number 1, 1992 Mary Ann Liebert, Inc., Publishers

and Protective Activity of Murine Monoclonal Antibodies Directed Against the Capsular Polysaccharide of Type III Group B Streptococci




Facoltà di Medicina e Chirurgia, Università 1-98100 Messina, Italy

degli Studi di Messina,

ABSTRACT We have obtained 41 monoclonal antibodies directed against type III group B streptococci by immunizing Balb/c mice with formalin-killed bacteria. All of these antibodies reacted with purified type-specific carbohydrate by enzyme-linked immunosorbent assay and immunoprecipitation tests. The epitope recognized by all of these antibodies was associated with terminal sialic acid residues, as indicated by abrogation of immune reactions by treatment of the type-specific carbohydrate with neuraminidase. Two purified monoclonal antibodies (the IgM P9D8 and the lgG3 P4F12) were further characterized for their protective activity in a neonatal rat model of infection. P9D8 and P4F12 antibodies were significantly protective when administered in a dose of 0.5 and 2.5 mg/kg, respectively, at the same time as 3x105 colony forming units of type III streptococci. Protection was still observed when the antibodies were given up to 9h after challenge. No protection was afforded against infections with type la/c and II streptococci. Similarly, both antibodies effectively opsonized type III, but not la, lb or II bacteria, in an in vitro assay. These and similar, previously described, monoclonal antibodies may be useful, possibly after "humanization" by genetic engineering, for the therapy of neonatal group B streptococcal infections. INTRODUCTION

Group B streptococci (GBS) are a major cause of sepsis and meningitis in neonates. A significant percentage of GBS disease result in death or permanent disability despite appropriate antibiotic therapy (1). These bacteria have been classified into types la, lb, II, III and IV based on the reactivity of their capsular carbohydrate (CHO) with specific antisera. Type III strains are responsible for about two thirds of all neonatal GBS infections (1). It is generally agreed that the type-specific polysaccharide capsule is the main virulence factor of GBS. Heterologous type-specific antibodies are protective in experimental infections in rodents (2). Moreover low levels of maternal type-specific antibodies are associated with an


increased risk of neonatal infection (3). For these reasons type-specific antibody in the form of intravenous human immunoglobulin has been proposed as a possible therapeutic agent (4). In addition, active immunization of mothers with type-specific polysaccharide has been advocated as an effective preventive measure (5). Murine type-specific monoclonal antibodies (MAbs) have been produced by different laboratories and generally shown to be protective in mouse and neonatal rat models of GBS infections and in in vitro opsonic assays (6-8). Recently, a human group-specific MAb was reported to be protective in experimental infections with all of the clinical isolates tested that represented four capsule types (la, lb. II and III) (9,10). We describe here the properties and protective activities of murine MAbs that, as those previously characterized (7,8), react against the capsular polysaccharide of type III GBS and protect neonatal rats in experimental infections with type III GBS.


Reagents. Media and supplements for hybridoma production were from GIBCO (Mascia Brunelli, Milano, Italy).Anti-type III GBS polyclonal rabbit serum was a kind gift from Dr. Graziella Orefici, Istituto Superiore di Sanità, Roma, Italy. Anti-group B rabbit serum and Noble Agar were purchased from Difco (Direct International Distributors, Milano, Italy). All other chemicals were purchased from Sigma Chemical Co. (Mascia Brunelli). Bacterial Strains. The type III H738 strain, originally isolated from a septic neonate, was kindly provided by Dr. Bascom Anthony, National Institutes of Health, Bethesda, MD. All the other strains were obtained from Dr. Orefici and are listed in Table 1. All the clinical strains were typed in Dr. Orefici's laboratory, as previously described (11), and confirmed as GBS using anti-group B antisera. TABLE 1.

GBS Strains Used in the Present

Serotype la lb la/c II III

'Isolated from the blood



Clinical isolates

MEDT.069 T97.423

258*,247,239,229 064,05,063,214,89* 363*,255*,259*,104,251, 252,237,242,90*,H738*

immunizing strains 090 H3B6 A909 18RS21 D136C

orcerebrospinal fluid of septic neonates

Purification of GBS CHO Antigens. The group and type-specific CHOs were purified from the culture supernatants of the H738 strain (type III) by ion-exchange chromatography and gel filtration as described (12,13) with minor modifications. Briefly, Todd Hewitt broth supplemented with yeast estract (3g/l) was dialyzed against H2O, autoclaved, and supplemented with glucose and disodium phosphate to final concentrations of 90 and 45 mM, respectively, before

inoculation with strain H738. The supernatants from early stationary cultures were concentrated by filtration, neutralized to a pH of 7.0, heat inactivated (56° C for 30 min) and dialyzed against 0.01 M Tris buffer (pH 7.2). This material was mixed with DEAE Sephacel (Pharmacia


S.p.A.,Cologno Monzese, Italy) and eluted in a column with a linear NaCI gradient (0 to 0.75M) in Tris buffer. Fractions reacting with group or type-specific serum by immunodiffusion were pooled separately, dialyzed against water, lyophilized and further purified by gel filtration on a Sepharose6B column (Pharmacia) eluted with Tris buffered saline (TBS; 0.01 M Tris, 0.15M NaCI, pH 7.2). The purified type III and group CHO preparations had both a protein content of < 0.2% by weight as determined by the Bio-Rad protein assay (Bio-Rad Laboratories SRL, Segrate, Italy). Sialic acid (14) and rhamnose (15), accounted for 24% and 0.5%, respectively, of the weight of the type-specific CHO and for 2,048) received the fist of two intraspenic injections separated by a 2-week interval and consisting of 1X107 bacteria in PBS (50ul). Three days afterthe last intrasplenic injection, spleen cells were fused


SP2/0-Ag14 myeloma using 50% polyethylene glycol 1500 (BDH, Pole, England) according to a published procedure (16). Hybridomas were selected in HAT medium (RPMI 1640, 15% Myoclone fetal bovine serum, 100 uM hypoxantine, 16 u.M thymidine, 0.4 u.M aminopterin, 50 u.g/ml streptomycin sulphate, 50 Ul/ml benzylpenicillin, 10uM HEPES) in 96-well tissue culture plates. Wells showing growth were screened by agglutination and ELISA. Positive wells were expanded in HT medium (same composition as the HAT medium but without aminopterin),supplemented with 1x105/ml Balb/c splenocytes as feeder cells. After the hybridomas were adapted to grow in regular RPMI medium with 10% FBS in 20 ml volumes, the cells were stored frozen and the spent supernatants were concentrated by precipitation with 33% saturated ammonium sulfate. The pellets were dialyzed in Hank's balanced salt solution (GIBCO),adjusted to a protein concentration of 1 mg/ml and used for immunodiffusion tests against CHO antigens and monospecific rabbit sera (Sigma) for isotype determination. Selected hybridomas were cloned twice by limiting dilution and injected into mice for ascites production. IgM and lgG3 MAbs were purified from ascites fluid by ammonium sulfphate precipitation followed by gel filtration on Sephacryl S-300 (Pharmacia) oraffinity chromatography on Protein A-Sepharose 4-B, respectively (17). The purity of these preparations was checked by silver staining of polyacrylamide gels electrophoresed in a PhastSystem (Pharmacia) according to manufacturer's instructions. with the

Protection Tests. These were done in neonatal (0.7) with the type III, but not the group, CHO (Table 2). The hybridomas were expanded



III- and

Group-Specific Polysaccharides from GBS Reactivity against Purified Type of Hybridoma Supernatants Clone ELISA (A405) Isotype type CHO P4F6 P2H10 P10A7 P10G10 P3H10 P5D8 P2D4 P6C7 P5D2 P9D8 P2F2 P4D8 P9B7 P8H11 P4D9 P2F7 P2F3 P1C4 P9E10 P10A2 P2H12 P10A9 P4H9 P1A11 P6D12 P8B6 P10F11 P4F7 P4A3 P4F12 P2G6 P10A11 P10D9 P6A3 P7F7 P7E1 P3F8 P3D9 P3C7 P3H3 P3B6

group CHO


0.11 0.06 0.09 0.08 0.07 0.10 0.15 0.08 0.18 0.06 0.18 0.04 0.06 0.10 0.09 0.12 0.07 0.10 0.09 0.08 0.14 0.12 0.18 0.06 0.08 0.07 0.04 0.08 0.10 0.15 0.10 0.11 0.15 0.11 0.04 0.16 0.10 0.21 0.12 0.09 0.10

0.92 1.02 0.95 0.88 1.25 1.15 0.98 1.08 1.69 1.04 0.96 1.16 1.21 1.35 0.90 1.14 0.83 0.75 0.71 1.03 0.95 1 .26 0.85 1.10 1 .48 0.72 0.96 0.70 0.94 0.82 0.96 0.88 0.96 0.81 0.75 0.90 0.75 0.80 0.74 0.84

to 20-40 ml cultures and the



IgM IgM IgM igG3 IgM IgM igA IgM IgM IgM IgM IgM IgM IgM lgG2b lgG3

lgG3 IgM IgM IgM IgM IgM IgM IgM IgM IgM IgM IgM

spent supernatants were concentrated by ammonium sulphate precipitation followed by dialysis. Isotype determination, performed on these materials, revealed 36 IgM, 3 lgG3, 1 lgG2b and 1 IgA (Table 2). Specificity for the type CHO was then confirmed using a panel of GBS strains (Table 1 ). As expected, all the supernatants agglutinated 100% of the type III GBS, but none of the strains belonging to other serotypes. Agglutination of the type III GBS was totally inhibited by 1 u.g/ml


of purified type III, but not group, CHO. In addition, all the concentrated supernatants precipitation reactions in double diffusion assays against purified type III CHO.


Lack of Reactivity with Non-Sialated Type III CHO. Type III CHO is known to occur in two forms. One contains intact sialic acid residues while the other is non-sialated (7).Thus it was important to determine which form was recognized by our antibodies. Treatment of bacteria with neuraminidase completely prevented the agglutination produced by all supernatants but not by polyclonal type- and group-specific sera. Similarly, neuraminidase or strong acid treatment of the purified type CHO resulted in abrogation of precipitation reactions in double diffusion tests against all of the concentrated supernatants. In order to further confirm specificity for epitopes associated with sialic acid, MAbs P9D8 and P4F12 were cloned twice, purified from ascites fluid and tested by IEP against native and neuraminidase-treated type III CHO (Figure 1). The native CHO migrated towards the anode and reacted strongly with both MAb P9D8 and polyclonal type-specific rabbit serum. In contrast, neuraminidase-treated type CHO migrated towards the cathode and reacted faintly with the serum only. Similar results were obtained with P4F12 (not shown). These data indicate that loss of sialic acid residues from the type CHO resulted, as expected, in a loss of both negative charges and reactivity with MAbs, but not with polyclonal sera.


+ MAb P9D8 (anti-type III) -Native type III carbohydrate Polyclonal anti-type III serum Neuraminidase-treated type III carbohydrate MAb P9D8 (anti-type III) _


-Group carbohydrate Polyclonal anti-group serum —



FIGURE 1. Grabar-Williams Immunoelectrophoresis Analysis of NeuraminidaseTreated and Native Type III Polysaccharide from GBS. MAb P9D8 reacted with a negatively charged, native form of type III polysaccharide, but not with the neuraminidase-treated, positively charged antigen. Group-specific carbohydrate and anti-group serum were included for comparison.

Protective and


Figure 2. shows that the purified MAbs P9D8 (IgM) and (lgG3) mg/Kg, respectively, protected neonatal rats infection a control MAb (P2E8, also an IgM) did not with strain H738 while against (type III), afford any protection. MAbs P9D8 and P4F12 at doses of 1 and 5mg/kg, respectively, were able to almost completely prevent deaths of the infected pups even when administered 4 h after challenge. Partial protection was observed when the MAbs were injected 9h after challenge. P4F12


at doses as low as 0.5 and 2.5


> oc


ü or UJ





FIGURE 2. Effect of MAbs P9D8 (IgM ) and P4F12 (lgG3) on Survival of Neonatal Rats Infected with the Type III Strain H738. MAbs were given immediately (panels A. and D.), 4h (panels B.and E.), or9h (panelsC.andF.) afterchallenge. Legends:« =P9D8 1 mg/kg; A=P9D8 0.5mg/kg; O=control MAbP2E85mg/kg; A =P4F12 5mg/ kg;D=P4F12 2.5mg/kg; • =Significantly ( rr Z)





Ü rr UJ










FIGURE 3.Effect of MABs P9D8 ( ;1 mg/kg), P4F12 (A;5mg/kg) and Control MAb P2E8 (O; 5mg/kg) on Survival of Neonatal Rats Infected with Clinical Isolates of Group B Streptococci. * =Significantly (

Specificity and protective activity of murine monoclonal antibodies directed against the capsular polysaccharide of type III group B streptococci.

We have obtained 41 monoclonal antibodies directed against type III group B streptococci by immunizing Balb/c mice with formalin-killed bacteria. All ...
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