Eur. J . Immunol. 1988.18: 601-606 Human monoclonal antibodies against Haemophilus influenme type b 601

Denis Martin’, Yolande Larose, Josee Hamel, Jacqueline Lagace and Bernard R. Brodeur

Hybridoma Section, Bureau of Microbiology, Laboratory Centre for Disease Control, Ottawa

Heterohybridomas secreting human monoclonal antibodies against Haemophilus influenzae type b

Seven human monoclonal antibodies (HmAb) directed against outer membrane anti- gens of Haemophilus influenzae type b (Hib) were produced by fusing Sp2/HPT heteromyeloma cells with human tonsillar lymphocytes sensitized in vitro for 6 days. The heterohybridomas were maintained in culture for at least one year and secreted, when cultured in Dulbecco’s modified Eagle’s medium without fetal calf serum, between 1 and 15 pg/106 cells/m1/24 h. All of the HmAb were IgGs except HiH-12 which is an IgM. Antibodies directed against the lipopolysaccharide and proteins of apparent molecular masses of 43, 37 and 27 kDa were identified by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of outer mem- brane. Binding radioimmunoassay with live bacteria showed that five out of seven HmAb adsorbed to cell surface-exposed antigenic determinants. HmAb HiH-6, HiH- 7 and HiH-10 reacted with a surface-accessible determinant on the 43-kDa outer membrane protein. In a dot enzyme immunoassay, these HmAb recognized 103 out of 111 Hib strains isolated worldwide. The strains were selected to represent the most common genotypic variations among Hib. None of these HmAb reacted with other bacterial species tested. These HmAb may serve to study the bacterial surface anti- gens implicated in the human humoral response and protection to Hib infections.

1 Introduction

Haemophilus influenzae type b (Hib) is the most common cause of bacterial meningitis in children. Antibodies directed against the capsular polysaccharide contribute to protection against Hib diseases [l, 21. However, purified Hib capsular antigen failed to induce the synthesis of protective antibodies in infants less than 18 months [3-51 and in individuals with certain genetic immunodeficiencies [6]. The capsular polysac- charide has been coupled to a protein carrier such as diph- theria toxoid to improve its immunogenicity in young children [7, 81, Infants less than six months of age do not respond to this conjugated vaccine and it is not known whether or not the responses to this vaccine will be subject to genetic regulation ~ ~ 9 1 .

Passive immunization with human immunoglobulins (Ig) has been used to provide protection against Hib infections in infants less than six months of age [lo]. Human monoclonal antibodies (HmAb) represent an interesting alternative for the passive immunization of this high-risk population. In compari- son to human polyvalent sera, large quantities of homo- geneous and well characterized protective HmAb can be pro-

[I 65681

Supported by a post-doctoral fellowship from the Natural Sciences and Engineering Research Council of Canada.

Correspondence: Bernard R. Brodeur, Head, Hybridoma Section, Bureau of Microbiology, Laboratory Centre for Disease Control, Tunney’s Pasture, Ottawa K1A OL2, Canada

Abbreviations: (H)mAb: (Human) monoclonal antibody(ies) Hib: Haemophilus influenzae type b SDS-PAGE: Sodium dodecyl sulfate- polyacrylamide gel electrophoresis LPS: Lipopolysaccharide O M Outer membrane ELISA: Enzyme-linked immunosorbent assay DMEM: Dulbecco’s modified Eagle’s medium HGPRT: Hypoxan- thine-guanine phosphoribosyltransferase PEG: Polyethylene gly- col BSA: Bovine serum albumin HAT: Hypoxanthine amino- pterin, thymidine CFU: Colony-forming units ET: Electrophoretic type PBS: Phosphate-buffered saline

duced in cell culture [ll-131. There have been several reports on the development of murine mAb specific to Hib capsular polysaccharide [ 141, and other outer membrane antigens [15-171. It has also been reported that mouse mAb specific to the 98-kDa outer membrane (OM) protein [lS] and the lipo- polysaccharide (LPS) [ 163 were protective in experimental models of Hib infections. However, mouse mAb appear to be of limited use in the treatment of human infection, mainly because of the risk of developing antibodies to mouse Ig lead- ing to sensitization and allergic responses [ll, 181. Hunter et al. [19] and Gigliotti et al. [20] have reported the production of HmAb against Hib capsular polysaccharide and that these HmAb provided significant protection in an infant rat model of Hib infection.

In this report we describe the production and characterization of seven HmAb directed against Hib LPS and OM proteins. These stable heterohybridomas have secreted HmAb in cul- ture for more than twelve months. These HmAb may help us to understand further the human immune response against antigens present on the bacterial surface, and could eventually be used in humans for therapeutic purposes.

2 Materials and methods

2.1 Bacterial strains and culture conditions

A total of 111 Hib isolates were kindly provided by Dr. James M. Musser, University of Rochester, NY. Electrophoretic typ- ing (ET), performed by Dr. Musser’s laboratory, indicated that the major genetically distinct groups of Hib were repre- sented in this collection [21]. These strains were isolated from patients living in Africa, Europe, Malaysia, New Guinea and the United States. Six strains of other serotypes, three untyp- able strains and the 3068 type b isolate were obtained from Sainte-Justine Hospital, Montreal, QuCbec, Canada. Other bacterial species such as E. coli, N . gonorrhoeae, N . meningiti- dis, N . mucosa, protein A-negative S . aureus, S . epidermidis and S . pneumoniae were obtained from our own collection.

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1988 0014-2980/88/0404-0601$02.50/0

602

The H. influenzue strains were grown overnight on chocolate agar plates (I. A. F. Production Inc., Laval, QuCbec) at 37 "C in an atmosphere containing 5% COz. Cultures were stored at -70°C in brain heart infusion broth containing 20% (v/v) gly- cerol or kept lyophilized. Identification of type b serotype strains was determined by a slide agglutination test using a commercially available sera (Difco Laboratories, Detroit, MI) and by dot immunoassay using a serotype-specific mouse mAb

D. Martin, Y. Larose, J. Hamel et. al.

Hb-2 [22].

Eur. J. Immunol. 1988.18: 601-606

2.2 OM and LPS preparations

The lithium chloride extraction of OM from Hib strain 3068 was performed as described by Hamel et al. [17]. The LPS was isolated from Hib strain by a rapid phenol-water extraction procedure described by Izana [23].

2.3 In varo immunization of human tonsil lymphocytes

Fresh human tonsillar tissue was obtained from the Children's Hospital of Eastern Ontario, Ottawa. The tonsils were rinsed with 95% ethanol, washed in Dulbecco's modified Eagle's medium (DMEM; Gibco Laboratories, Grand Island, NY) supplemented with 50 yg/ml gentamycin and then cut into small pieces. The cells were dispersed and suspended in 20 ml of medium. The cell suspension was layered over a Ficoll- Paque gradient (Pharmacia, Piscataway, NJ) and centrifuged at 400 x g for 30 min at room temperature. The cells were recovered from the interface and washed twice in medium.

The in vifro immunization procedure used was described pre- viously [24]. Briefly, human tonsil lymphocyte suspensions were depleted of T cells as follows: 800-pl of 1 x lo7 lympho- cytes in DMEM with gentamycin and 5% fetal calf serum were incubated with 100 p1 of OKT8 antibodies (Ortho Phar- maceutical Co., Raritan, NJ) at a 1 : 8 dilution for 20 min at 37°C. One hundred yl of rabbit complement was then added and the cells were incubated for 45 min at 37 "C with shaking every 15 min. After washing in DMEM, cells were layered over a Ficoll-Paque gradient, centrifuged and the viable cells were recovered from the interface. After lysis, less than 2% of residual cells were OKT8' as judged by immunofluorescence. The cells were cultured for 6 days in flat-bottom 24-well plates (Nunc, Roskilde, Denmark) at 4 x lo6 cellsfwell at 37 "C in a humidified 10% C 0 2 atmosphere. The basic culture medium used for the in vitro immunization consisted of 2 ml of DMEM per well, supplemented with 15% (v/v) human T cell growth factors (Lymphocult, Biotest Diagnostics, Frankfurt, FRG), 15% (vfv) fetal calf serum, 50 pg/ml of gentamycin, 2 mM L- glutamine, 1% (v/v) of MEM essential amino acids (Gibco), 0.05 mM sodium pyruvate (Microbiological Associates, Bethesda, MD), 5 X M of 2-mercaptoethanol (Bio-Rad Laboratories, Richmond, CA), and Hib OM preparations, ranging from 0.001 to 0.1 pg of proteidml.

(PEG 1450; Eastman Kodak, Rochester, NY) in DMEM medium. The PEG solution was adjusted to pH 6.8 and filter- sterilized just before use. The lymphocytes and the myeloma cells were adjusted to 1 X lo7 cells/ml, washed once in serum- free DMEM, and fused in a 50 ml conical tube at room tem- perature using 0.5 ml PEG. The PEG solution was added slowly over a 60-s period. The tube was rotated gently for another 90 s followed by the addition of 10 ml of DMEM over a 10-min period. Fused cells were incubated at 37°C in a humidified 10% COz atmosphere for 20 min in DMEM, washed, and suspended at 1 x lo6 cells/ml in DMEM sup- plemented with 20% fetal calf serum, 2 mM L-glutamine and 50 pg/ml gentamycin. One hundred-yl aliquots of the cell sus- pension were dispended in 96-well tissue culture plates (Nunc) which contained a feeder layer of 3 X lo3 murine mac- rophages. Twenty-four hours after fusion the medium was aspirated and replaced by DMEM containing hypoxanthine, aminopterin and thymidine (HAT; Sigma, St. Louis, MO). The medium was changed every 48 h.

2.5 Determination of Ig class and enzyme-linked immunosorbent assay (ELISA) procedure

The class, subclass and light chain specificity of the human Ig were determined by ELISA using commercially available rea- gents (ICN Immunobiologicals, Lisle, IL; Fisher Biotech, Ottawa, Ontario, Canada). Supernatants of wells containing growing clones were screened by ELISA for the presence of antibodies to Hib as previously described by Hamel et al. [17] using alkaline phosphatase-conjugated goat anti-human Ig (Copper Biomedical Inc., Malvern, PA).

2.6 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

OM proteins were resolved by electrophoresis using the Bio- Rad Mini Protean system according to the method of Laemmli [26]. The stacking gel and the resolving gel contained 4% (w/v) and 10% (wfv) acrylamide (Bio-Rad), respectively. The sam- ples were prepared by mixing 1 part of OM antigens, adjusted to 1 mg of proteidml, with 4 parts of the sample buffer [62.5 mM Tris-HC1, pH 6.8, 1% (v/v) glycerol, 2% (w/v) SDS, 0.5% (v/v) 2-mercaptoethanol and 0.5% (w/v) bromophenol blue] and heated for 5 min at lOO"C, or 30 min at 37°C. Elec- trophoresis was performed at a constant voltage of 100 V until the bromophenol blue dye entered the separating gel at which time the voltage was increased to 200 V. The gels were stained with Coomassie blue and then destained as described by Weber and Osborn [27]. The following proteins (Pharmacia) were used as molecular mass standards: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), a-lactalbumin (14.4 kDa).

2.7 Immunoblotting procedure 2.4 Fusion procedure

The fusion procedure was performed as described by Brodeur et al. [25] with the following modifications. Tonsil lympho- cytes immunized in v i m for 6 days were fused in a 1 : 1 ratio with the nonsecreting HGPRT-deficient heteromyeloma Sp2/ HPT in a solution containing 50% (w/v) polyethylene glycol

After SDS-PAGE, the proteins were transferred elec- trophoretically from the gel to nitrocellulose paper (Bio-Rad) by the method of Towbin et al. [28]. A gradual potential from 5 to 40 V was applied to the gel-nitrocellulose paper sandwich for 1 h in an electroblot buffer consisting of 25 mM Tris-HC1, 192 mM glycine and 20% (v/v) methanol, pH 8.3. The nitrocel-

Eur. J. Immunol. 1988.18: 601-606 Human monoclonal antibodies against Haemophilus influenzae type b 603

lulose paper was soaked for 30 min at room temperature in a solution of either 0.03% (vh) Tween-PBS or 0.25% (wh) BSA-PBS (Sigma). The blots were incubated overnight at 4°C with concentrated culture supernatants. Mouse hyperimmune sera diluted in 5% (w/v) skimmed milk (Carnation) were used as control. The papers were washed for 10 min in PBS fol- lowed by two more washes in 0.02% (v/v) Tween-PBS and rinsed for 10 min in PBS. The blots were then soaked for 60 rnin at 37°C in 5% (wh) skimmed milk containing "'I- labeled sheep anti-human Ig (Amersham, Arlington Heights, IL) 0.25 pCi = 9.25 kBq. After four washes as described above, the paper strips were exposed for 48 h at -70°C to Dupon Cronex film (Wilmington, DE) in a Kodak X-omatic cassette fitted with intensifying screens.

2.8 Dot enzyme immunoassay

A dot enzyme immunoassay was used for rapid screening of the HmAb against a large number of bacterial strains. The bacteria were grown overnight on chocolate agar plates. The microbial lawns were removed from the surface of the agar plate with dacron swabs and suspended in PBS, pH 7.2. For each strain the cell suspension was adjusted spectrophotomet- rically to 5 x lo6 colony-forming units (CFU)/ml. The Bio-Dot microfiltration apparatus (Bio-Rad) was used to perform the immunoassay. Fifty p1 per well of each bacterial suspension was filtered through the nitrocellulose by gravity. Each well was washed twice with 200 pl of 0.02% (vh) Tween-PBS and 200 pl of 0.5% (w/v) gelatin-PBS (Fisher, Ottawa, Canada). The sheet was removed from the apparatus, washed twice with Tween-PBS and soaked in a PBS solution containing 1% (wh) gelatin for 30 rnin at room temperature. The nitrocellulose sheet was incubated 1 h at 37°C with undiluted or concen- trated cultured supernatants. The sheet was washed twice in PBS and incubated with peroxidase-conjugated goat anti- human Ig (Cooper Biomedical). After incubation for 1 h at 37 "C and three washes, the blots were soaked in a solution of o-dianisidine prepared as described by Towbin et al. [28].

2.9 Adsorption of HmAb

One ml of hybridoma culture supernatant was mixed with lo9 live intact Hib, untypable, or N . gonorrhoeae cells, and was incubated with constant shaking for 2 h at 4°C. After three washes in PBS, the bacterial cells were suspended in 1% (w/v) BSA/PBS solution containing '251-labeled sheep anti-human Ig (Amersham) 0.25 pCi/ml for 1 h at room temperature with constant a itation. The bacteria were washed twice in PBS, and the l2 I counts per minute were detected using a 1282 Compugamma (LKB Instruments Inc., Rockville, MD). Bacterial counts before and after the adsorption with HmAb reflected no loss of viability.

!

2.10 Enzymatic treatment of OM

For protein digestion, 25 pg of proteinase K (Boehringer, Mannheim, FRG) or 150 pg of protease from Streptomyces griseus type VI (Sigma) in PBS, pH 7.2, was added to 15 pl (protein 1 mglml) of Hib OM preparation. After incubation for 15 min at 37"C, the OM protein concentration was

tion plate (Flow Laboratories, Mississauga, Ontario, Canada). The supernatant of each clone was reacted with enzymatically treated and control OM preparations.

3 Results

3.1 Generation of heterohybridomas producing HmAb against Hib

Growth of human tonsil lymphocytes in the presence of 15% human T cell growth factors and 15% fetal calf serum was maintained over the 6-day sensitization period. The OM pro- tein concentration necessary for optimal in vitro antibody pro- duction varied among donors, with the most effective dose of antigen ranging from 1 and 100 nglml.

The nonsecreting heteromyeloma Sp2/HPT cell line used in this study was obtained after three successive fusions of the Sp2/0 mouse myeloma with human peripheral blood and tonsil lym hocytes. The heteromyeloma Sp2/HPT is sensitive to

2 mM ouabain. Karyotype analysis has demonstrated the pres- ence of 4 to 6 human chromosomes integrated into the mouse genome.

This heteromyeloma cell line was fused with human tonsil lym- phocytes that were previously sensitized in vitro against Hib antigens. Seven stable heterohybrids secreting specific HmAb against Hib have been cloned and maintained in culture for at least 12 months. These hybrids are maintained in DMEM sup- plemented with 10 to 15% fetal calf serum, and have a doubl- ing time of between 20 to 30 h (Table 1). The level of Ig secretion when cultured in DMEM without serum ranged from 1 to 15 pg/ml/106 cells124 h. The Ig class and subclass were determined by ELISA. One of the HmAb, HiH-12 is an IgM, whereas all others are IgGs.

10- iT M aminopterin, 2.5 pg/ml azaserine, and resistant to

3.2 HmAb reactivity with proteolytic enzyme-treated OM

Enzymatically treated and untreated OM preparations of Hib strain 3068 were used as coating antigens for ELISA and dot assay. The enzymatic treatments altered the OM protein struc- tures such that the mAb reactivities could not be detected in ELISA assay (Table 2). HmAb tested against enzyme-treated OM were retested against untreated OM to establish that the reactivity was not impaired by residual enzymatic activity. A

Table 1. Characterization of HmAb against Hib

Hybridoma Class Stability in culture Growth"' Level of of Ig (months) rate secretionb'

WH- 1 I g G d 12 30 1 .o HiH-2 IgGz,). 15 28 8.0 WH-3 IgG2,x 12 26 3.0 HiH-6 IgG,k 12 26 6.0 HiH-7 IgG ,A 12 20 6.0 WH-10 IgG3,h 12 27 15.0 WH-12 JgM 12 20 1.2

adjusted to 5 pglml with coating buffer, pH 9.6, and 100 of that suspension was dispensed into a Linbro E.I.A. microtitra-

a) Growth rate: doubling time calculated in hours. b) Secretion: pg/mV106 cells/24 h in DMEM without serum.

604

dot enzyme immunoassay was also used to determine reactiv- ity of the HmAb against LPS purified from Hib strain 3068. HmAb HiH-2 and HiH-3 reacted with the purified LPS and enzymatically treated OM preparations. The other HmAb did not recognize the purified LPS and their corresponding epitopes on the enzymatically treated OM preparations.

D. Martin, Y. Larose, J . Hamel et. al. Eur. J. Immunol. 1988.18: 601-606

3.3 Localization of antibody-specific epitopes

The Western immunoblotting technique was used to deter- mine the specific OM antigens to which the HmAb bound. Mouse hyperimmune serum against OM preparations of Hib strain 3068 was used as a positive control (Fig. 1). Five HmAb reacted with OM proteins, while the two others, HiH-2 and HiH-3, recognized epitopes present on the LpS. The 43-kDa

Figure 1. Autoradiogram of the immunoblots of Hib 3068 strain OM Preparations after probing with HmAb HiH-1 (A), HiH-2 (B), HiH-3 (C), HiH-6 (D), HiH-7 (E), HiH-10 (F), HiH-12 (G), and mouse hyperimmune sera (H). Standard proteins are identified with their kDa values on the left.

Table 2. HmAb reactivity with enzymatically treated OM and purified LPS from Hib strain 3068

Hybridorna ELISA valuesaJ Proteinasc LPS" Untreated Protease-treated K-treated

OM OMb' Ohlb'

HiH- 1 0.103 0.001 0.004 - HM-2 1.377 0.512 0.598 + HiH-3 0.535 0.120 0 394 + HiH-6 0.326 0.017 0.024 - HiH-7 0.351 0.015 0.018 - w-lo 0.442 0.018 0.019 - HiH-12 0 118 0 012 0.009 - cmam

Figure 2. Autoradiogram showing the effect of the temperature of soiubiliziation of the OM of Hib 3668 strain on the reactiviiy of HiH-1. OM were heated in electrophoresis sample buffer containing 2% SDS and 0.5% of 2-mercaptoethanol for 30 min at 3 7 ~ ~ (a), and min at 1000~ (b). Standard proteins are identified with their kDa values on

a) Absorbance at 410 nm after 1 h at room temperature. b) After an incubation of 15 min at 37°C with the enzymes, the OM

protein concentration was adjusted to 5 pg/ml with coating buffer, and 100 ul was disDensed into each well. *LA 1-2*

LllG 1Gll. c) The hybndoma supernatants were reacted with purified LPS from

Hib in a dot enzyme immunoassay.

Table 3. Binding properties of HmAb to intact bacterial cells

Hybridoma lrI-labeled anti-human Ig (cpm) bound Haemophilus influenme N. gonorrhoeae T w b Untypahle

HiH-la) m-2 HiH-3 i-m-6 HiH-7 HiH.10 HIX-!~ W)

#1b' 51 191 33 054 48 HI1 43 I 8 4 40 630

1445 526

71 1 409

1391 494 715 831

2910 f 202

316 750 673 39 1 503 735 506 734

a) Hybridoma culture supernatants were incubated with live intact bacterial cells. The bacteria were then incubated with 1251-labeled sheep anti-human Ig and pelleted. The bacterial cell-bound lz5I was counted. Background without bacteria was lower than 400 cpmlmin.

b) Mean cpm of duplicate experiments. c) HmAb specific against cytomegalovirus.

major OM protein was identified by HiH-6, HiH-7 and HiH- 10. HiH-1 and HiH-12 reacted against two protein bands with apparent molecular masses of 27 and 37 kDa. The reactivity of HiH-1 was also tested against OM preparations treated at 100°C for 5 min and at 37°C for 30 min (Fig. 2). When the OM preparation was heated at 37°C for 30 min, HiH-1 reacted only with one protein band with apparent molecular mass of 27 kDa, instead of the two bands normally observed on the autoradiogram.

3.4 Binding properties of the HmAb

To determine if the HmAb were directed against cell surface- exposed epitopes, hybridoma culture supernatants were incu- bated with intact Hib cells. As controls, untypable H. influen- tae and N. gonorrhoeae cells were also incubated with the HmAb. HiH-1 and HiH-12 did not adsorb on the surface of intact bacterial cells. The other HmAb were able to recognize their corresponding epitopes on the surface of Hib cells, but not on the surface of the untypable or N . gonorrhoeae cells (Table 3 ) . A HmAb specific to cytomegalovirus was used as control, and did not adsorb on the surface of either bacterial cells tested.

Eur. J. Immunol. 1988.18: 601-606 Human

Table 4. Specificity of HmAb

Hybridorna Haemophilicr influenzae Type b Untypable Other Other bacterial

scrotypes"' specicP

HiH-I IllilllC' 3 0 616 Oil HiH-2 90'1 1 1 O(3 Oi6 0.7 HiH-3 Wlll 013 016 0!7 HiH-6 10311 11 113 0th O f l HM-7 10311 1 1 1 /3 016 on HiH-10 103/111 213 016 on HiH-12 61;111 3, '3 0/6 0:l

a) H . influenzae strains of serotype other than b. b) E . coli, N . gonorrhoeae, N . meningitidis, N. mucosa, S. epider-

midis, S. pneumoniae and S. aureus. c ) Number of positivehumber of strains tested by a dot enzyme im-

munoassay.

3.5 Specificity of the HmAb

A dot enzyme immunoassay was used for rapid screening of the reactivity of the HmAb against a large number of bacterial strains (Table 4). The seven HmAb did not recognize any of the other bacterial species tested, HiH-2, HiH-3 and HiH-12 which reacted with LPS or the 27-37-kDa OM protein have identified, respectively, 81%, 59% and 54% of the Hib strains tested. One hundred and three out of 111 (92%) Hib strains were recognized by HiH-6, HiH-7 and HiH-10. These three HmAb specifically reacted with the 43-kDa OM protein (Fig. 1). HmAb HiH-1 identified all Hib strains, untypable, and other serotypes tested.

4 Discussion

In this study, we report the production of HmAb against the LPS and proteins present in the OM of Hib strains. The major problems encountered with currently available human myeloma cell lines are their low hybridization frequency, slow growth rate, hybrid instability, and the secretion of antibody by several parental myelomas [12]. To overcome these prob- lems, mouse-human hybrid myeloma cell lines were developed and used as fusion partners to produce heterohybridomas se- creting HmAb [ll, 291. In previous reports [25, 301, we have described the development of two heteromyelomas. To obtain these HmAb, human tonsillar lymphocytes were fused with the mouse-human heteromyeloma Sp2/HPT. The HAT-sensi- tive, nonsecreting Sp2/HPT fusion partner was obtained fol- lowing several fusions between the Sp2/0 mouse myeloma and human tonsil and peripheral blood lymphocytes. It contains 4 to 6 human chromosomes incorporated in the mouse genome. The presence of these human chromosomes may be responsi- ble for the stability of additional human chromosomes incor- porated during a cell fusion [31]. Seven stable, well-growing and secreting specific human Ig clones have been generated and maintained in culture for at least one year.

In a previous report [24], we observed that in addition to a satisfactory fusion partner, the source of the lymphocytes and the in vitro sensitization of the lymphocytes were critical for the generation of HmAb. For these reasons, human tonsillar lymphocytes were used instead of peripheral blood lympho- cytes. They were primed in vitro for 6 days before a fusion in

monoclonal antibodies against Haemophilus influenrue type b 605

order to increase the number of specific secreting B cells. The antigen concentration necessary to induce a strong specific response was between 1 and 100 ng of OM proteindml. It was demonstrated that the optimum antigen concentration for in vitro immunization varied among human donors [24,30].

Using B cells primed in vitro, several clones secreting specific antibodies against Hib LPS and OM proteins were obtained. The LPS nature of the antigen recognized by HiH-2 and HiH-3 clones was suggested by their conserved reactivity with enzy- matically treated OM and purified LPS. The same experi- ments showed that the five remaining HmAb were directed against protein antigenic determinants. Furthermore, Western immunoblotting followed by autoradiography revealed that these HmAb reacted with protein bands with apparent molecular masses of 27,37 and 43 kDa. The OM protein with an apparent molecular mass of 27 kDa is heat modifiable and the 37-kDa band results from the modification of this protein [32]. Both the non-heat-modified form and the heat-modified form proteins were immunologically reactive with the HmAb HiH-1.

Binding assays demonstrated that the HmAb specific to the 43-kDa OM protein and to the LPS recognized accessible epitopes on the surface of intact bacterial cells. Granoff and Munson [6] also observed that epitopes on the 43-kDa OM protein are accessible to antibodies on the cell surface. Gulig and Hansen [16] described a murine mAb specific to Hib LPS which has the ability to erradicate systemic Hib disease in the infant rat model. These results indicate that the protective mAb specific to LPS adsorbed in v ivo on the surface of intact cells. HmAb tonsil HiH-1 and HiH-12 did not react with intact bacteria. Munson and Granoff [6] were not able to demon- strate that a portion of the 37-kDa OM protein was exposed on the surface of the bacteria. The HmAb which adsorbed on the surface of intact bacterial cells are interesting candidates for the in vitro and in v ivo evaluation of their biological activity.

To verify the specificity of the HmAb, we used a highly diver- sified collection of H. influenzae strains. One hundred and eleven Hib isolates were selected from a collection of strains isolated worldwide to include the most common genotypes. We observed that HiH-6, HiH-7 and HiH-10, which reacted with the 43-kDa OM protein, recognized all the ET1 and ET12 strains tested. Musser et al. [21] reported that 65% of the strains responsible for meningitis belong to these two groups. These two ET profiles found in H . influenzae strains seem to have a very wide geographical distribution. Furthermore, of the eight strains missed by these three HmAb, seven were classified in the ET25. It is of interest that this ET profile is generally rare in North America [21]. In contrast to HmAb HiH-6, HiH-7 and HiH-10, no correlation was observed between the reactivity to Hib isolates of LPS-specific HiH-2 and HiH-3 and the electrophoretic types. Serotype b, other serotypes and untypable H. influenzae strains tested were all identified by HiH-1. The epitope recognized by this HmAb is not accessible on the surface of intact bacteria.

This study demonstrates that in vitro immunization of human tonsillar lymphocytes combined with the use of a heteromye- loma fusion partner can lead to the generation of stable heterohybrids secreting HmAb. The biological activities of the HmAb are currently under investigation in in vitro assays and in animal Hib infections [34].

Received January 4, 1988; in revised form January 26, 1988.

606 D. Martin, Y . Larose, J. Hamel et. al. Eur. J. Immunol. 1988.18: 601-606

5 References

1 Schneerson, R., Rodrigues, L. P., Parke, J . C., Jr. and Robbins, J. B., J. Immunol. 1971. 107: 1081.

2 Anderson, P., Johnston, R. B., Jr. and Smith, D. H., J. Clin. Invest. 1972. 51: 31.

3 Makela, P. H., Peltola, H., Kayhty, H., Jousimies, H., Pettay, O., Ruoslahti, E., Sivonen, A. and Renkonen, 0. V., J. Infect. Dis. 1977. 136: (Suppl.) 43.

4 Smith, D. H., Peter, G., Ingram, D. L., Harding, A. L. and Anderson, P., Pediatrics 1973. 52: 637.

5 Peltola, H., Kayhty, H., Sivonen, A. and Makela, P. H., Pediat- rics 1977. 60: 730.

6 Granoff, D. M. and Munson, R. S . , Jr., J . Infect. Dis. 1986.153: 448.

7 Eskola, J., Kayhty, H., Peltola, H., Karamko, V., Makela, P. H., Samuelson, J. and Gordon, L. K., Lancet 1985. i: 1184.

8 Lepow, M. L., Barkin, R. M., Berkowitz, C. D., Brunell, P.A., James, D., Meier, K., Ward, J., Zahradnik, J. M., Samuelson, J., McVerry, P. H. and Gordon, L. K., J. Infect. Dir. 1987.156: 591.

9 Eskola, J . , Peltola, H., Takala, A. K., Kayhty, H., Hakulinen, M., Karanko, V., Kela, E., Rekola, P., Ronnberg, P.-R., Samuel- son, J. S., Gordon, L. K. and Makela, P. H., N. Engl. J. Med. 1987. 317: 717.

10 Santosham, M., Reid, R., Ambrosino, D. M., Wolff, M. C., Almeido-Hill, J., Priehs, C., Aspery, K. M., Garrett, S. , Croll, L., Foster, S., Burge, G., Page, P., Zacher, B., Moxon, R. and Siber, G. R., N. Engl. J . Med. 1987. 317: 923.

11 James, K. and Bell, G. T., J . lmmunol. Methods 1987. 100: 5. 12 Kozbor, D. and Roder, J. C., lmmunol. Today 1983. 4: 72. 13 Shay, J. W., in Engleman, E. G., Foung, S. K. H., Larrick, J. and

Raubitschek, A. (Eds.), Human hybridomas and monoclonal anti- bodies, Plenum Press, New York 1985, p. 5.

14 Gigliotti, F. and Insel, R. A., J . Infect. Db. 1982. 146: 249. 15 Kimura, A., Gulig, P. A., McCracken, G. H., Jr., Loftus, T. A.

16 Gulig, P. A., and Hansen, E. J., Infect. lmmun. 1985. 49: 819. and Hansen, E. J., Infect. Immun. 1985. 47: 253.

17 Hamel, J., Brodeur, B. R.,Larose, Y., Tsang, P. S., Belmaaza, A. and Montplaisir, S . , J. Med. Microbiol. 1987. 23: 163.

18 Abrams, P. G., Rossio, J. L., Stenvenson, H. C. and Foon, K. A., Methods Enyzmol. 1986. 121: 107.

19 Hunter, K. W., Jr., Fischer, G. W., Hemming, V. G., Wilson, S. R., Hartzman, R. J. and Woody, J. N., Lancet 1982. ii: 798.

20 Gigliotti, F., Smith, L. and Insel, R. A., J . Infect. Dis. 1984. 149: 43.

21 Musser, J. M., Granoff, D. M., Pattison, P. E. and Selander, R. K., Proc. Natl. Acad. Sci. USA 1985. 82: 5078.

22 Hamel, J., Brodeur, B. R., Belmaaza, A., Montplaisir, S. , Mus- ser, J. M. and Selander, R. K., J. Clin. Microbiol. 1987.25: 2434.

23 Inzana, T J., J . Infect. Dis. 1983. 148: 492. 24 Lagad, J. and Brodeur, B. R., J. Immunol. Methods 1985. 85:

127. 25 Brodeur, B. R., LagacC, J., Larose, Y., Martin, M. L., Joly, J. R.

and PagC, M. in Schook, L. B. (Ed.), Monoclonal antibody pro- duction techniques and applications, Marcel Dekker Inc., New York 1987, p. 51.

26 Laemmli, U. K., Nature 1970.227: 680. 27 Weber, K. and Osborn, M., J. Biol. Chem. 1969.244: 4406. 28 Towbin, H., Staehelin, T. and Gordon, J., Proc. Natl. Acad. Sci.

USA 1979. 76: 4350. 29 Kozbor, D., Roder, J. C., Sierzega, M. E., Cole, S. P. C. and

Croce, C. M., Methods Enzymol. 1986. 121: 120. 30 Martin, D., Brodeur, B. R., Larose, Y., Faucher, S. and Hamel,

J . , in Borrebaeck, C. (Ed.), Proc. Znt. Symp. on in vitro immuni- zation in Hybridoma Technology, Publ. Elsevier Sc. 1988, in press.

31 Teng, N. N. H., Lam, K. S., Riera, F. C. and Kaplan, H. S . , Proc. Natl. Acad. Sci. USA 1983. 80: 7308.

32 Coulton, J. W. and Wan, D. T. F., Can. J . Microbiol. 1983. 29: 280.

33 Munson, R. S. , Jr. and Granoff, D. M., Infect. lmmun. 1985. 49: 544.

34 Brodeur, B. R., Tsang, P. S., Hamel, J., Larose, Y. and Mont- plaisir, S . , Can. J . Microbiol. 1986. 32: 33.