Antibacterial polysaccharide antibody deficiency after allogeneic bone marrow transplantation

Journal of Clinical Immunology, Vol. 10, No. 3, 1990

Antibacterial Polysaccharide Antibody Deficiency After Allogeneic Bone Marrow Transplantation I. QUINTI, 1'5 A. VELARDI, 2 S. LE MOLI, 3 E. GUERRA, 1 R. D'AMELIO, 3 P. MASTRANTONIO, 4 M. F. MARTELLI, 2 and F. AIUTI 1

Accepted: March 8, 1990

major pathogens of subjects with humoral immunodeficiencies and during childhood (2-5). However, the mechanism of immune response against polysaccharide antigens is not completely understood, and problems such as unresponsiveness under 2 years of age as well as the heterogeneity of the repertoire of human antibodies are not well defined. Since the combined cellular and humoral immunodeficiency of marrow graft survivors recover slowly and infections may be a serious problem in the months following engraftment (6-8), we analyzed the repertoire of antipolysaccharide antibodies in a population of allogeneic bone marrow-engrafted adults immunized with meningococcal groups A and C and pneumococcal antigens in comparison with a group of immunized adult healthy subjects. The patients showed low antibody titers of the IgM class and no bactericidal activity in vitro; the analytical isoelectrofocusing showed the appearance of a restricted pattern of clonotypes only in a few subjects.

Allogeneic bone marrow-engrafted adults immunized with meningococcal types A and C and pneumococcal type 14 polysaccharide antigens showed only low antibody titers of the IgM class, no antibody titers of the IgG or IgA classes, and no bactericidial activity in vitro. The analytical isoelectrofocusing showed the appearance of a restricted pattern of clonotypes in a minority of subjects. These observations are consistent with the hypothesis that B cells in bone marrow transplant patients express some characteristics of neonatal B cells and suggest that polysaccharide-protein conjugates, rather than isolated polysaccharide, might be utilized in the setting of bone marrow transplantation. KEY WORDS: Polysaccharide antigens; antibody deficiency; bone marrow transplantation; analytical isoelectrofocusing.

INTRODUCTION Long-term survivors after allogeneic bone marrow transplantation (BMT) may experience recurrent bacterial infections in the first months following BMT. We recently demonstrated that the humoral immune system of adults undergoing successful marrow engraftment reproduces some of the maturational steps that occur in normal B-cell ontogeny during the first year of life (1). In particular, we found a marked IgG2 deficiency. This defect is often associated with an impaired response to polysaccharide antigens from bacteria that represent the

METHODS Patients. Thirteen patients with hematologic malignancies underwent allogeneic BMT. All patients were transplanted in remission and prepared for transplantation by cytoreduction therapy with antithymocyte horse Ig (Merieux, Lyon, France) and procarbazine (12 mg/kg/day) followed by hyperfractionated total-body irradiation (1400 rads) and cyclophosphamide (60 mg/kg/day). The marrow donors were HLA-identical, mixed lymphocyte culture-compatible siblings (age >20 years). To prevent GvHD, donor marrow was depleted of T cells by sequential soybean agglutination and sheep red blood-cell rosetting, by the procedure described

1Department of Allergy and Clinical Immunology, University of Rome, Rome, Italy. ZDivision of Hematology, University of Perugia, Perugia, Italy. 3DASRS, Laboratory of Hygiene and Immunology, Practica di Mare, Rome, Italy. 4Istituto Superiore di SanitY, Rome, Italy. 5To whom correspondence should be addressed at Department of Allergy and Clinical Immunology, University of Rome "La Sapienza," Viale dell'Universit~ 37, 00185 Rome, Italy.

160 0271-9142/90/0500-0160506.0(I/0© 1990PlenumPublishingCorporation

ANTIBODY DEFICIENCY IN BMT PATIENTS

by Reisner et al. (9). Residual T cells in the marrow inoculum never exceeded 0.2%. This was evaluated by direct immunofluorescence utilizing fluorescein isothiocyanate (FITC)-conjugated anti-Leu4 antibody (Beckton-Dickinson, Oxnard, CA). Marrow graft recipients received no immunosuppressive therapy for GvHD prophylaxis in the posttransplant period. Only one patient developed grade I acute GvHD and none had chronic GvHD. Engraftment of donor marrow was documented by blood group change or by disappearance of the Phl chromosome. During the first 6 months after BMT patients were treated with human gamma-globulins for intravenous use. The immunization with polysaccharide antigens was done after a period ranging from 7 to 15 months post-BMT. Control subjects were 70 healthy military recruits age-matched with the donors, Vaccination. All patients were in remission at the time of immunization. Each subject received a single injection of a 0.5-ml dose of vaccine containing 50 ~g of each polysaccharide in the left tricep muscle. The vaccine consisted of a mixture of purified polysaccharides from 14 pneumococcal typesmU.S. 1, 2, 3, 4, 5, 51, 8, 9, 12, 14, 56, 19, 23, and 25--and from meningococcus A and C (BioMerieux, Lyon, France). The contemporary administration of pneumococcal and meningococcal vaccines was well tolerated in all subjects. Serum samples were obtained from each subject before and after 18 and 90 days from vaccination. Antigen-Specific Ig Assay. Antibody quantity determination was carried out using an enzyme-linked immunosorbent assay (ELISA) for IgG and IgM classes. Flat-bottom polystyrene microtest plates (Pro-bind, Falcon) were coated with meningococcal polysaccharide A or C and with pneumococcal polysaccharide type 14 (Merieux, Lyon France) at a concentration of 10 txg/mt in carbonate-bicarbonate buffer (0.005 M, pH 9.6) for 3 hr at 37°C and overnight at 4°C. To minimize nonspecific absorbtion of serum proteins to the plastic, the wells were incubated with blocking solution of 0.1% gelatin in phosphate-buffered-saline (PBS) for 2 hr at room temperature. After four washings with PBS supplemented with 0.05% of Tween 20 (PBS-T), serum samples were added at dilutions of 1:100 in PBS with 5% goat serum and incubated for 2 hr at room temperature. After four washings with PBS-Tween, a goat F(ab) 2 anti-human IgG or IgM or IgA alkaline phosphatase-conjugated antiserum (Zymed, San Francisco, CA) diluted 1:1000 was added. After an

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incubation of 2 hr at 37°C, plates were washed three times with PBS-Tween and once with Tris-HC1, 0.1 M, pH 8.6), and 100 ixl of p-nitrophenyi phosphate (Sigma, St. Louis, MO) at 1 txg/ml in Tris-HCl (1 M, pH 8.6) was added to each well. After 20 min the OD of the reaction was read at 405 nm by an automated photometer (Titertek, Multiskan). Controis for each plate included standard antisera with a defined antibody content, kindly provided by Institut Merieux, Lyon. Labeling of Polysaccharide. Tyramine groups were coupled directly to meningococcal polysaccharide A (PsA) by cyanogen bromide activation (I0). Tyraminated PsA was then radioiodinated by the chloramine-T method (11). Isoelectrofocusing (IEF). Antibodies to meningococcal polysaccharide A were separated by is0electrofocusing in agarose gel and visualized by autoradiography after labeling with ~25I-PsA. Thin agarose gels (0.5 ram) with 1% agarose IEF (Pharmacia, Uppsala, Sweden), 10% sorbitol, and 3% Ampholyte (Pharmacia), pH 3.5-9.5, were formed by pouting agarose solution at 65°C into a preheated casting assembly wherein the gel solidified and bound firmly to Gel Bond (Pharmacia) film-bubbled glass plates. Focusing was performed in horizontal slabgels on a Bio-Rad apparatus, cooled at 10°C by a Multitemp apparatus (Pharmacia). The electrode strips contained 0.5 N sodium hydroxide (catholyte) or 0.5 M acetic acid (anolyte). Twenty micrograms of serum, diluted 1/4 in 0.9% NaCI, was applied near the anode on paper sample strips. Proteins were allowed to migrate and focus for about 90 rain at a constant power of 600 W. The pH gradient was measured by cutting pieces of gel every centimeter and placing them in 0.5 ml of HzO. After focusing, the gels were then treated according to Insel et al. (12), by incubation for 1 hr in 21% Na2SO4, followed by two 1-hr incubations in 18% Na2SO4, and then in 18% NazSO 4 with 1% bovine serum albumin (BSA) and 0.01 M phosphate buffer (PB), pH 7. The gels were then overlaid with 125I-PsA (2 x 107 cpm) in 50 ml 18% NaaSO 4 with BSA-PB overnight. Gels were repeatedly washed with 18% NazSO 4 for 24 hr, then fixed with 0.1% glutaraldehyde in 18% NazSO4 for 1 hr at room temperature. After further washings with PBS, followed by two 1-hr washings in water, the gels were dehydrated by two 1-hr incubations with 40% ethanol, air-dried, and autoradiographed (48 hr with Kodak X-Omat or AR films), Bactericidal Activity. A microbactericidal assay based on the method described by Gold and Wild

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Table I. Geometric Mean ± SD of Specific IgG and IgM (OD) Before (Day 0) and After (Days 30 and 90) Immunization Day Anti-meningococcal PsA IgG

IgM

Anti-meningococcal PsC IgG

IgM

Anti-pneumococcus type 14 IgG

IgM

Transplant group

Controls

0 30 90 0 30 90

250.7 287.9 308.9 198.6 367.8 767.9

- 33,1 - 100.5 -+ 234.1 - 66.6 - 100.9 - 412.8

306.1 --- 265.3 813.3 + 417.2 m 205.7 ± 185.7 472.4 - 330.1 m

0 30 90 0 30 90

242.3 282.3 218.9 184.6 343.8 863.7

± 27.7 ± 47.9 - 224.5 + 55,9 -+ 120.4 ± 355.7

242.7 --- 320.6 795.2 - 488.3 -184.8 --- i37.6 433,7 + 280.1 --

0 30 90 0 30 90

281.1 280.4 316.2 650.8 763.3 1415.1

± 69.6 ± 63.9 -----121.1 -+ 105.6 ± 222.1 --- 438.4

ma

--

aNot done.

was used (13). Briefly, H. meningitidis groups A and C were grown in Mueller-Hinton broth for 24 hr, cultured for 18 hr at 37°C in blood-agar plates, and transferred in PBS at a concentration of 3 z 54 bacteria/ml. Twenty-five microliters of heatinactivated serum, serially diluted in a microtitration plate, was incubated at 37°C for 30 min with 25 ~I of PBS + 0.001% phenol red + 0.01% BSA and 0.25 Ixl of bacterial suspension + 0.25 txl of rabbit complement. Then the number of viable bacteria was determined as colony-forming units by transfering 50 ixl from each microtiter well to MuellerHinton agar plates. Serum antibody titer was the greatest dilution in which at least a 50% decrease in number of colonies was observed in comparison to the control (0.25 ~1 of bacteria +0.25 ~1 of PBS + BSA + phenol red). RESULTS

months post-BMT are shown in Table I: there were no significant changes in the mean concentrations of specific IgG before and 30 and 90 days after immunization. The control group showed statistically higher concentrations of specific IgG and IgM after immunization in comparison to the preimmunization values (P < 0.0001 for IgG and IgM) and to the transplant group postimmunization values (P < 0.0001 for IgG and P < 0.05 for IgM); preimmunization titers to all antigens in the control group were similar to those in the transplant group, with a wider distribution of values. In the BMT group, in fact, B-lymphocyte activation in vivo, in response to antigens, results mainly in IgM production, with a significant increase in specific IgM to meningococcal antigens on day 30 (P < 0.0001) and on day 90 (P < 0.0001) and in specific IgM to pneumococcus type 14 antigen on day 90 (P < 0.0001). No specific IgA were found in pre- and postimmunization sera (data not shown).

Human Meningococcat and Pneumococcal Polysaccharide Antibodies

Bactericidal Antibody Activity

The geometric mean serum concentrations of antibody to the pneumococcal type 14 antigen and meningococcal types A and C antigens in a group of 13 graft marrow recipients immunized with polysaccharide antigens after a period ranging from 7 to 15

Bactericidal activity before and after meningococcal polysaccharides A and C immunization are shown in Table II. Only 1 of 10 patients showed an increase in the serum titer with bactericidal activity in vitro to meningococci A and C, while one patient

Journal of Clinical Immunology, Vol. 10, No. 3, 1990

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Table II. Bactericidal Activity Before (a) and After (b) Meningococcal Polysaccharide A and C Immunization in 10 Bone Marrow-Engrafted Adults Patient No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

a b a b a b a b a b a b a b a b a b a b

Meningococcus A

Meningococcus C