T Parkkali 1,HKäyhty 2, M Anttila 2, T Ruutu 1, T Wuorimaa 2, A Soininen 2, L Volin 1 and P Ruutu 2

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1 Bone Marrow Transplantation, (999) 24, Stockton Press All rights reserved /99 $ IgG subclasses and avidity of antibodies to polysaccharide antigens in allogeneic BMT recipients after with pneumococcal polysaccharide and Haemophilus influenzae type b conjugate vaccines T Parkkali,HKäyhty 2, M Anttila 2, T Ruutu, T Wuorimaa 2, A Soininen 2, L Volin and P Ruutu 2 Division of Haematology, Department of Medicine, Helsinki University Central Hospital; and 2 National Public Health Institute, Helsinki, Finland Summary: In a randomized study, 20 adult allogeneic BMT recipients were vaccinated at 6 months and 22 at 8 months after BMT with Haemophilus influenzae type b (Hib)- diphtheria toxoid conjugate vaccine (PRP-D), and 23 recipients at 8 months and 2 at 20 months with pneumococcal polysaccharide (Pnc PS) vaccine. IgG and IgG2 subclasses of Pnc PS and Hib antibodies and avidities of Pnc PS IgG antibodies were determined by EIA in sera from patients with at least a two-fold total antibody response to Pnc type 3, 6B, 9F or PRP-D. The Pnc PS vaccine induced predominantly IgG Pnc 3 antibody production. Anti-Pnc 6B and 9F responses were mainly IgG2. The time of the Pnc PS, at 8 or 20 months after BMT, did not influence the IgG subclass response pattern. The PRP-D vaccine induced predominantly IgG2 anti-hib production in the patients vaccinated at 6 months after BMT. The patients vaccinated at 8 months produced IgG and IgG2 antibodies more evenly. The same patient was able to produce predominantly IgG subclass antibodies to one antigen, Pnc 3, 6B, 9F or Hib, and IgG2 antibodies to another. The avidities of anti-pnc 6B and 9F month after were similar to those before, anti-pnc 3 avidity was lower than before but matured in 5 months. The IgG subclass distribution and avidity were similar in the patients with and without chronic GVHD. In conclusion, the IgG response to Pnc type 3 was predominantly IgG as in infants and IgG2 to PRP- D, Pnc 6B, and 9F as in adults. Early after BMT or the presence of chronic GVHD did not impair the quality of response to Pnc PS and PRP-D vaccines. Keywords: polysaccharide antibodies; IgG subclasses; avidity; BMT recipients; Streptococcus pneumoniae (Pnc) is the main cause of serious bacterial infections in allogeneic BMT recipients who survive 3 months after transplantation. 5 Haemophilus influenzae is also a significant cause of infections among Correspondence: Dr T Parkkali, Division of Haematology, Department of Medicine, Helsinki University Central Hospital, PO Box 340, Helsinki, FIN HYKS, Finland Received 30 November 998; accepted 25 April 999 allogeneic BMT recipients in the late post-transplant period. 6,7 BMT recipients without chronic GVHD are immunodeficient for to 2 years after allogeneic BMT and those with chronic GVHD as long as GVHD persists. 8,9 Serum IgM concentrations return to normal levels within 2 6 months, IgG within 3 8 months, and IgA within 3 36 months. Serum IgG and IgG3 recover earlier than IgG2 and IgG4. 7 Functional asplenia, low serum concentrations of antibodies to Pnc polysaccharides (PS), and impaired serum opsonic activity for Pnc may be the main mechanisms leading to increased susceptibility to pneumococcal infections. 2,,2 Allogeneic BMT recipients respond poorly to Pnc PS vaccines,,3 6 and the responses to one dose of Haemophilus influenzae type b (Hib)-conjugate vaccines have not been optimal. 4 7 The subclass distribution of IgG antibodies in the few adult BMT recipients studied after immunizing with Pnc PS has been variable. 8 The Hib tetanus toxoid-conjugated vaccine has elicited mainly IgG antibodies in the few BMT recipients studied. 7 Bactericidal activity of Hib antibodies has been shown to correlate with antibody avidity, 9 2 functional affinity of serum antibody to bind the antigen. 22 It is not clear whether IgG and IgG2 have differences in functional activity. The avidity of IgG2 subclass Pnc PS antibodies in healthy adults vaccinated with Pnc PS vaccine has been reported to be higher than that of IgG subclass antibodies. 23 On the other hand, IgG subclass Hib antibodies in healthy adults have similar or better functional activity than IgG2 antibodies. 24,25 Little is known about the IgG subclass distribution of specific antibodies in BMT recipients responding to s with Pnc or Hib vaccines and nothing is known about the avidities of these antibodies. The aim of the present study was to investigate the subclass distribution of Pnc PS and Hib IgG antibodies and the avidity of Pnc IgG antibodies in BMT recipients who, in our previous study, had shown at least a two-fold total antibody response to Pnc serotype 3, 6B, 9F or Hib after with Pnc PS and Hib-diphtheria toxoid conjugate vaccines. 6 Materials and methods Patients and immunizations Forty-five adult (age 6 years) BMT recipients who received a bone marrow transplant from an HLA-identical,

2 672 MLC-negative sibling donor between January 985 and November 989 at Helsinki University Central Hospital were randomized into two different schedules, beginning either at 6 (early group) or 8 (late group) months after BMT as described earlier. 6 Briefly, 20 of the 23 patients in the early group were vaccinated at 6 months (delay in vaccine availability in three cases) and the 22 patients in the late group at 8 months after BMT with Haemophilus influenzae type b (Hib)-diphtheria toxoid conjugate vaccine (PRP-D, ProHIBIT; Connaught Laboratories, Swiftwater, PA, USA) containing 20 g of Hib capsular polysaccharide linked covalently with 20 g of diphtheria toxoid. All 23 early group patients were vaccinated at 8 months and 2 of the 22 late group patients at 20 months after transplantation with pneumococcal polysaccharide (Pnc PS) vaccine (Pneumovax; Merck, Sharp and Dohme, West Point, PA, USA) containing 25 g capsular polysaccharide from each of the 23 Pnc serotypes. Both vaccines were administered intramuscularly. The patients with at least a two-fold total antibody response to Pnc serotype 3, 6B, 9F or PRP-D at month after 6 were used in this study for measuring the IgG subclass responses to Pnc types 3, 6B, 9F or Hib, respectively, and the avidity of Pnc PS IgG antibodies. Of the 20 patients with a two-fold increase of antibodies to at least one of the tested antigens (Pnc type 3, 6B, 9F or Hib), nine were in the early group and in the late group. The median age of the patients was 26.5 years (range 8 49 years). Nine patients had AML, five ALL, four CML, one multiple myeloma and one Burkitt s lymphoma. The conditioning treatment was CY + TBI in 5 cases and Bu + CY in five patients. 6 Four patients received CsA, 4 patients CsA + a short course of MTX, and two patients corticosteroids and MTX as GVHD prophylaxis. Eight patients (four in each group) suffered from acute GVHD. Six patients (four in the early, two in the late group) had clinical manifestations of chronic GVHD within 2 months prior to or at the time of the Hib or Pnc s. Chronic GVHD was graded as extensive in two cases. Chronic GVHD was treated with methylprednisolone alone or in combination with azathioprine. Serum samples The IgG subclasses of antibodies to Pnc serotypes 3, 6B, 9F and Hib were measured from serum samples drawn from the donors and patients before transplantation and from the patients prior to and month after. The avidities of Pnc IgG antibodies were determined in sera collected from the patients before transplantation, before, and and 6 months after. Control samples for avidity assays For comparison of avidities of Pnc PS IgG antibodies we used serum samples from healthy adults who had been vaccinated with Pnc PS vaccine. 26 EIA for Pnc PS IgG and IgG2 antibodies An EIA specific for IgG and IgG2 anti-pnc PS as described by Soininen et al 27 was used for measurement of the IgG subclasses of Pnc PS antibodies. Microtitre plates (MaxiSorp; Nunc, Roskilde, Denmark) were coated with Pnc PS 3 (5 g/ml together with methylated human serum albumin), 6B ( g/ml), and 9F ( g/ml) (American Type Culture Collection, Rockville, MD, USA) in pyrogenfree phosphate-buffered saline (PBS) incubating at 37 C for 5 h and then overnight at 4 C. After washing the plates were blocked with % foetal calf serum in PBS (F-PBS) for h at 37 C. The first dilution of the serum samples was made in F- PBS containing or 20 g/ml pneumococcal C polysaccharide absorbent (Statens Seruminstitut, Copenhagen, Denmark). Further dilutions were made in F-PBS. After incubation and washings monoclonal mouse anti-human IgG and IgG2 were added, (clone HP 6070 and clone HP 6002, respectively; Zymed Laboratories, San Francisco, CA, USA) followed by alkaline phosphatase-conjugated rabbit anti-mouse IgG antibody (Jackson Immunoresearch Laboratories, West Grove, PA, USA). The substrate was added and the absorbance was measured on an EIA reader (Labsystem Multiscan, Helsinki, Finland) at a wave length of 405 nm. The IgG and IgG2 concentrations of antibodies to Pnc PSs in the pneumococcal reference serum lot 89-SF (Food and Drug Administration, Bethesda, MD, USA) 28 with a known total IgG antibody concentration have been assigned previously. 27 The IgG subclass concentrations in the sera of the donors and patients were calculated by comparing the titres of the donor and patient sera to the titres of the pneumococcal reference serum 89-SF. The results are given as g/ml. The lower detection limits were 0.04, 0.03 and 0. g/ml for IgG and 0.07, 0.04 and 0. g/ml for IgG2 in type 3, 6B and 9F assay, respectively. Undetectable concentrations of antibodies were assigned the value of half of the lower limit of detection. EIA for Hib IgG and IgG2 antibodies An IgG/IgG2-specific EIA for Hib PS was a modification of the assay described before. 29 In short, the microtitre plates (Costar 359; Cambridge, MA, USA) were coated with Haemophilus influenzae type b oligosaccharide-human serum albumin conjugate received from Dr Porter Anderson (University of Rochester, Rochester, NY, USA) 30 and blocked as above, except that the overnight incubation occurred at 37 C. The results of the Hib antibody subclass determinations are given as titres. For calculations, titres 25 are given an arbitrary value of 2.5. EIA for measuring avidity of Pnc PS IgG antibodies An EIA method described earlier by Anttila et al 3 was used to determine the relative avidity of Pnc PS IgG antibodies. Microtitre plates (MaxiSorp) were coated, as above, with Pnc PSs. To dissociate formed antigen antibody complexes, 0.5 m sodiumthiocyanate (NaSCN) diluted in F-PBS was used. Dilution series with inhibitor (NaSCN) and control (F-PBS) were made from each sample. The plates were incubated at 22 C for 5 min and washed. After that alkaline phosphatase-conjugated anti-human IgG (Sigma, St Louis, MO, USA) diluted in F-PBS was added followed by the substrate, p-nitrophenyl phosphate (Sigma). The results

3 are expressed as relative avidity indexes (AI), assigned as the percentage of antibodies that remained bound to the antigens after NaSCN treatment. AIs were calculated by comparing the titres of the inhibited and non-inhibited series of the sample: AI = titre (NaSCN+ ) titre (NaSCN ) 0. Results IgG subclass responses to pneumococcal polysaccharide and Haemophilus influenzae type b conjugate vaccines Of the 5 patients with a two-fold total antibody rise for one or more of the Pnc serotypes investigated, 2, five and five patients showed this response to types 3, 6B and 9F, respectively. Both the donors and patients prior to transplantation as well as the patients before and after showed an IgG2 subclass predominance in antibodies to Pnc PSs (Table ). The specific IgG and IgG2 responses are shown in Figure. The two early-group patients did not differ from the late-group patients in the ability to produce IgG or IgG2 subclass anti-pnc 3 after. The mean fold increases in IgG and IgG2 concentrations after were 8.3 and 4.2 to Pnc serotype 3, 8.9 and 99. to Pnc 6B, and 3.3 and 7.8 to Pnc 9F, respectively (Table 2). Two early-group patients showed no IgG response to Pnc 6B, and one early-group patient showed no IgG response to Pnc 9F. The Hib antibody results are expressed as titres, and thus the geometric means (GMs) of IgG and IgG2 cannot be compared with each other. Two of the 7 patients with a two-fold response in the total antibody to PRP-D were excluded from the analysis, as neither the IgG nor the IgG2 titre increased after suggesting that they had had an IgM and/or IgA response. The GM titres of IgG and IgG2 anti-hib were similar in donors and patients prior to BMT (Table ). The changes in specific IgG and IgG2 titres after BMT are shown in Figure. The GM titres of both IgG and IgG2 Hib antibodies were higher in the early-group patients at 6 months after BMT than in the lategroup patients at 8 months after BMT. Five early-group and one late-group patient had no IgG response, and one late-group patient showed no IgG2 response. The mean fold responses to in the IgG and IgG2 titres were 3.7 and 8.5 in the early and 8.3 and 5.2 in the late group, respectively (Table 2). The proportions of the IgG and IgG2 subclasses in the IgG response to the same antigen varied greatly between individuals (Table 2). On the other hand, in several cases a patient responding to several antigens tested produced predominantly IgG to one antigen and IgG2 to another. The few patients with chronic GVHD did not differ from those without. Two patients (Nos 9 and 9) had extensive chronic GVHD at the time of s and thereafter. Nevertheless, they responded to two to three antigens with an IgG2 predominance. Avidity of Pnc antibodies The AI of Pnc 3 IgG antibodies varied greatly between individual patients before transplantation and either increased or decreased after transplantation (Figure 2). All patients except one early-group patient had a decrease in the AI after. However, during the next 5 months the AIs of Pnc 3 antibodies increased. The mean AI of anti-pnc 3 was 52 before BMT, 56 before, and 23 month after. By 6 months after the mean AI increased to 49. The mean AI of anti-pnc 3 in eight healthy adults did not differ from the AIs of the BMT recipients: 50 (range 3 84) before 673 Table Serum IgG and IgG2 subclass anti-pnc PS 3, 6B and 9F concentrations (geometric mean, g/ml) and anti-hib titres (geometric mean) prior to transplantation, and before and after in patients responding to Pnc PS and Hib conjugate vaccines and in their donors Donor Patient prior to Patient prior Patient month after transplantation to IgG IgG2 IgG IgG2 IgG IgG2 IgG IgG2 Pnc 3 (n) early group (2) late group () both groups Pnc 6B (n) early group (4) late group () both groups Pnc 9F (n) early group (3) late group (2) both groups Hib (n) early group (9) late group (6) both groups

4 674 0 Pnc 3 lgg 0 Pnc 3 lgg n = 5 n = 4 Pre-BMT Pnc 6B lgg 0.0 Pre-BMT Pnc 6B lgg n = 4 n = 4 Pre-BMT Pre-BMT Pnc 9F lgg Pnc 9F lgg n = Pre-BMT Hib lgg 0.0 Pre-BMT Hib lgg Titre Titre 0 0 n = 7 n = 6 n = 4 n = 6 n = 3 n = 4 n = 5 Pre-BMT Pre-BMT Figure IgG and IgG2 antibody levels to pneumococcal serotypes 3, 6B and 9F, and to Haemophilus influenzae type b (Hib) in BMT recipients before BMT and, and month after with pneumococcal polysaccharide and Hib-diphtheria toxoid conjugate vaccines. ( ) Early group; ( ) late group; ( ) represents patients from both groups below detection limit, and n refers to the number of patients (when more than one observation is indicated by one symbol).

5 Table 2 IgG subclass fold responses to Haemophilus influenzae type b-diphtheria toxoid conjugate and pneumococcal polysaccharide vaccines in BMT recipients 675 Patient Group cgvhd Hib Pnc 3 Pnc 6B Pnc 9F IgG IgG2 IgG IgG2 IgG IgG2 IgG IgG2 25 early a early + b early early early early early early early ++ c late late late late late late late late late late late a No chronic GVHD. b Limited chronic GVHD. c Extensive chronic GVHD. and 27 (range 2 6) month after. None of the healthy adults showed an increase in the AI after. The AI of anti-pnc 6B decreased in all except one earlygroup patient between BMT and (Figure 2). After the AI of anti-pnc 6B clearly increased in two patients. In only one patient did the AI show a slight increase between and 6 months after. The AIs of anti-pnc 6B of the healthy adults ranged from 3 to 75 (mean 52) before and from 2 to 54 (mean 37) month after. One of the healthy adults had a slight increase in the AI after. The AI of anti-pnc 9F decreased in four of the five patients between BMT and. After the AI increased in three patients. One patient showed a clear increase in the AI between and 6 months after. The AI of anti-pnc 9F in the healthy adults ranged from 8 to 94 (mean 50) prior to and from 7 to 7 (mean 39) month after. One of the healthy adults had an increase in the AI after. In the five patients with chronic GVHD the AIs were not lower than in those without. The two patients with extensive chronic GVHD produced antibodies of relatively high avidity to Pnc 9F (AIs 85 and 52) and 6 months (68 and 84) after, and the AI of anti-pnc 3 matured from 48 to 92 between and 6 months after in one of these two patients. Discussion We studied the IgG subclass distribution of Pnc and Hib PS antibodies and the avidity of Pnc PS IgG antibodies in HLA-matched sibling adult BMT recipients who had shown a two-fold total antibody response to Pnc PS or Hib conjugate vaccines. 6 Of the BMT recipients 50% responded to one or more of the four PS antigens studied, in contrast to 84 94% of healthy adults with a two-fold total antibody response to Hib and Pnc PS types 3 and 9F after, and only 58% to Pnc type 6B. 6 Results in the present study show that the patients were able to produce both IgG and IgG2 subclass polysaccharide antibodies. The relative avidity of anti-pnc 3 was lower month after than before but increased to the preimmunization level over 5 months. The AIs of anti- Pnc 6B and 9F were mainly at a relatively high level soon after. The IgG subclass distribution and relative avidity of the polysaccharide antibodies varied greatly between individual BMT recipients. The BMT recipients in the present study were adults. Age of the BMT recipient may influence the IgG subclass distribution of antibodies to capsular polysaccharides. After intensive chemotherapy or allogeneic BMT, immune reconstitution occurs earlier in children than in older patients, as the thymus of a younger patient has a greater capacity to generate new T lymphocytes. 32,33 Antibodies to pneumococcal capsular polysaccharides are mainly of the IgG subclass in young children, 34 but the Pnc PS vaccine induces predominantly IgG2 subclass antibody production in adults. 35,36 The IgG2 response to the Pnc PS vaccine in paediatric BMT recipients has been impaired compared to that of healthy age-matched controls. 37 In previous studies including both adult and paediatric BMT recipients, the Hib conjugate vaccine has induced predominantly IgG subclass Hib antibodies and plain Hib PS and Pnc PS vaccines have alternatively induced predominantly IgG or IgG2

6 676 AI AI AI Pnc 3 Pre-BMT Pnc 6B Pre-BMT Pnc 9F Pre-BMT Figure 2 Avidity indices (%) of IgG antibodies to pneumococcal serotypes 3, 6B, and 9F in BMT recipients before BMT, before, and and 6 months after with pneumococcal polysaccharide vaccine. ( ) Early group; ( ) late group. subclass antibodies. 7,8 The same Hib conjugate vaccine, which we used, has induced IgG-rich anti-hib responses more often in healthy children than in adults. 38 IgG3 and IgG4 subclass antibody production against capsular polysaccharides is scanty after, and therefore only the IgG and IgG2 subclasses of anti-pnc PSs and anti-hib were measured in the present study. IgG2 subclass pneumococcal antibodies predominated in the sera of the present patients before and after. BMT recipients have low serum IgG2 concentrations for months to years after transplantation, 7,4 resembling, in this respect, young children. Although the magnitude of the total antibody response to the Pnc PS vaccine in adult BMT recipients is poor as in young children, the antibodies to capsular polysaccharides are predominantly of the IgG2 subclass as in healthy adults. The present BMT recipients produced IgG rather than IgG2 subclass antibodies to Pnc serotype 3 more effectively, but the few recipients with a two-fold total antibody response to Pnc types 6B and 9F produced predominantly IgG2 antibodies. The IgG predominance in anti-pnc 3 production was unexpected, as the typical anti-pnc 3 response shows IgG2 predominance in adults. 35 Pnc type 3 is a good, type 9F an intermediate, and type 6B a poor immunogen. 42 Possibly Pnc type 3 induced response in immunologically more immature recipients than did types 6B and 9F, and the IgG subclass pattern in the anti-pnc 3 response resembled that of infants with IgG predominance. Only Hammarström et al 8 have previously studied the IgG subclass response to the Pnc PS vaccine in a few adult BMT recipients. Some of their patients showed no response, some produced predominantly IgG, and others IgG2 to Pnc type 3 as well as to Pnc types 6A and 23F. In paediatric BMT recipients the IgG and IgG2 responses have been of the same degree and have correlated with each other. 43 The PRP-D conjugate vaccine tended to induce a higher mean fold increase in the IgG2 than in IgG subclass in the early-group recipients. The late-group patients produced slightly more IgG than IgG2 subclass antibodies. In previous studies the PRP-D vaccine has induced predominantly IgG2 subclass Hib antibodies in the majority of healthy adults and IgG antibodies in some individuals. 38,39 In children the response to PRP-D has been predominantly IgG. 38 Thus, the present early-group patients responded as adults and the late-group patients more as children. The tetanus toxoid-conjugated Hib capsular polysaccharide vaccine given 4 months after BMT induced predominantly IgG subclass antibodies in BMT recipients. 7 The PRP-D vaccine may have weaker T cell dependence, showing lower immunogenicity and immunological priming effect in infants than Hib PS conjugated to tetanus toxoid. 44,45 IgG is the predominant subclass in T cell-dependent responses. 46 The grade of T cell dependence of the Hib conjugate vaccines may partly explain the differences in the IgG subclass responses among different BMT recipient populations. It is also possible that in the present study only immunologically relatively mature recipients showed a two-fold total antibody response to the PRP-D vaccine and produced predominantly IgG2 subclass antibodies as healthy adults. On the whole, the results of different IgG/IgG2 studies are difficult to compare with each other, as the methodologies in the antibody determinations, T cell dependence of vaccines, and schedules vary between the studies. The early of the present recipients did not prevent IgG2 subclass response but rather favoured it. The recipients vaccinated at 6 and 8 months after BMT were probably more immunodeficient at the time of than the recipients vaccinated at 8 and 20 months. In spite of this, the early-group recipients produced predominantly IgG2 subclass antibodies more often than the late-group recipients. However, the number of patients was very small and unevenly distributed in the two groups. Among the recipients responding to at least two antigens, the same recipient was able to produce predominantly IgG to one antigen and IgG2 to another. To our knowledge, the avidity of antibodies induced by

7 the Pnc PS vaccine has not been studied in BMT recipients. Avidity is a measure of the functional activity of antibodies. 9,2,22 Low-avidity polysaccharide antibodies may show impaired bactericidal and opsonic activity, but the precise clinical implication of antibody avidity, in terms of protective efficacy, is unknown. It is likely that antibody avidity becomes biologically important especially in relatively low serum antibody concentrations. 47 We measured the relative avidity of IgG antibodies against pneumococcal capsular polysaccharides by dissociating previously formed antibody antigen complexes by sodium thiocyanate. The avidities of anti-pnc 7F and 4 in elderly persons have been shown to be similar and comparable to those in younger adults, prior to, 4 weeks, and 5 years after with a 23-valent Pnc PS vaccine. 48 The present recipients responding to the Pnc PS vaccine produced antibodies of the same avidity as before, except to Pnc type 3. The avidity of anti-pnc 3 matured later. It may be that a good immunogen, Pnc 3, stimulates B cell clones of both high and low avidity antibodies. With time the low avidity clones are probably selected away, and the relative avidity increases. The quality of the polysaccharide antibodies in BMT recipients may be important in regard to protection, as the antibody concentrations achieved by with polysaccharide vaccines remain relatively low.,3,6,37 IgG2 subclass antibodies induced by a plain polysaccharide vaccine show higher avidity than IgG antibodies to Pnc PS type After the AIs of anti-pnc 3 decreased, probably due to the predominance of IgG in the response. However, in all individual patients the relative avidity of antibodies to the Pnc serotypes investigated did not follow the IgG/IgG2 distribution in the response. The six patients with chronic GVHD did not differ from the others in their ability to produce IgG or IgG2 subclass antibodies. Information concerning the influence of chronic GVHD on the total serum IgG2 concentration and specific IgG2 antibody response to against encapsulated bacteria is scanty and somewhat controversial. 7,8,37,43 The correlation between specific IgG2 response to Pnc PS vaccine and total serum IgG2 concentration in BMT recipients is uncertain. 8,37 We did not measure total IgG2 concentrations in the sera of our patients. In the present study the patients with chronic GVHD had the highest fold increases in IgG2 anti-pnc 3, -Pnc 9F and -Hib among all responding recipients. In conclusion, adult allogeneic BMT recipients, who respond to Pnc PS and Hib conjugate vaccines, are able to produce both IgG and IgG2 subclass antibodies. The relative avidities of Pnc PS antibodies are comparable to those of healthy adults. 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