In 2000, the FDA licensed the 7-valent protein-conjugated pneumococcal vaccine (Prevnar (PNCRM7®)) containing 7 serotypes (4, 6B, 9V, 14, 18C, 19F, 23F) that cause 70–80% of invasive pneumococcal infections in North America (12,13). Five of the serotypes (6B, 9V, 14, 19F, 23F) are responsible for the majority of antibiotic resistant pneumococcal infections worldwide (12–14). Conjugation of pneumococcal saccharides to a carrier protein (CRM₁₉₇, a non-toxic variant of diphtheria toxin) results in T cell-dependent B cell generation of memory B cells that respond to the attached sugars (12,13). In contrast, PPV23, elicits a T cell independent B cell response and fails to induce antigenic memory, precluding the development of an anamnestic response following antigenic re-exposure (14). PNCRM7 vaccination has dramatically decreased the incidence of pneumococcal disease in healthy infants and children (15), populations who possess circulating B cells that are phenotypically (CD20+CD5+CD38+CD1c+) and functionally similar to those of HCT recipients (16).

Serotype-specific pneumococcal capsular polysaccharide IgG was measured by enzyme-linked immunsorbant assay (ELISA). The FDA standard reference serum 89-S was used as the calibration standard. Titers against pneumococcal serotypes 1,3,14,19F, 23F, and 7F were measured in order to distinguish responses specific to PPV23 (1,3, 7F) from responses common to both vaccines (14, 19F, 23F). Response to PNCRM7 or PPV23 was defined as seroconversion or >3 fold rise in titer against serotypes 14, 19F, and 23F, three significant causes of antibiotic resistant pneumococcal infections in HCT recipients (2, 5). Initial antibody response was measured at a median of 3.0 months (range: 2–12 months) after completion of PNCRM7 and HIB immunization. PPV23 response was measured at a median of 3.2 months (range 1.5–12 months) following immunization. HIB response, measured by ELISA, was defined as seroconversion (> 1 ug/ml) or >3 fold increase in antibody titer. The lower limits of quantification for pneumococcal and H flu antibody levels were 0.5 ug/ml and 0.15 ug/ml, respectively.

Immunofluorescence studies. Two color immunofluorescence was performed on the circulating lymphocytes all patients within three months of vaccine initiation. Directly fluoresceinated (FITC) antibodies, including CD45 (common leukocyte antigen, positive control), MsIgG, CD3 (pan T-cell), CD20 (pan-B cell), CD14 (monocyte marker), and phycoerythrin-conjugated (PE) antibodies MsIgG, CD8 (cytotoxic or suppressor T cell marker), CD4 (helper T cell), CD45RA (naïve T cell subset), and CD56 (natural killer cells) were purchased from Becton Dickinson (Mountain View, CA). Immunofluorescence was performed on whole blood as previously described (19). Immunofluorescence samples were analyzed on a FACScan (Becton Dickinson). The lymphoid populations to be analyzed were gated on using log 90 degree and forward angle scatter characteristics. The leukocyte-specific monoclonal antibody CD45 was used to gate out any residual red cells. Monocyte contamination was ruled out by lack of reactivity (<1%) of the gated lymphoid cells with the monocyte specific marker CD14.

Sixty-two percent (79/127) of patients responded to PNCRM7 (Table 2a, ​,2b).2b). A dichotomy existed between the response observed in children (median age: 9 years (0.1–17.9) and adults (median age: 41 years(18–64). Eighty-eight percent of children responded to the heptavalent pneumococcal conjugate vaccine compared to 44% of adults (p<0.001). In contrast only 1 of 18 patients, median age: 42.5 years, immunized with PPV23 responded to serotypes 14, 19F, and 23F. Of the 16 adult recipients of PPV23, only 1 of 16 responded compared to 34 of 76 adults given the conjugated vaccine (p=0.003). PNCRM7 response in children and adults was not affected by the use of T cell depletion or donor type (Table 2b). Five of 12 patients who received rituximab responded to PNCRM7, including 2 of 4 children and 3 of 8 adults. For PNCRM7 vaccination, results from multivariate logistic regression model indicated that age at transplant significantly predicted response (p=0.0002). DLI and disease diagnosis were associated with response, but did not significantly predict response after adjusting for age at transplant (Table 2a).

HIB response was evaluable in 115 of 127 recipients of PNCRM7 and 13 of 18 recipients of PPV23. Overall, 86% of PNCRM7 recipients responded to HIB, including 96% of children (48/50) and 79% (51/65) of adults (p=0.006). Response to the Hib conjugate vaccine was observed in 77% and 100% of patients who failed PNCRM7 or PPV23, respectively. Since age was the only prognostic factor associated with response to the HIB conjugate vaccine, a single variable logistic regression model was used to predict the probability of response based on age at transplant (Table 3). A graph depicting the probability of response to HIB and PNCRM7 based on the age at transplant is shown in Figure 1.

Correlation of PNCRM7 response with in vitro parameters of immune reconstitution including the PHA response and circulating numbers of CD3, CD4, CD4+CD45RA naïve T cells, CD8, and CD19 cells/ul was evaluated. In patients >50 years of age, 11 of 19 patients vaccinated after reaching minimal milestones of immune reconstitution (CD4 >200/ul, IgG >500 mg/dl, PHA within 60% lower limit of normal) responded to PNCRM7, compared with 0 of 8 vaccinated prior to reaching these milestones (p=0.006). Among patients vaccinated after achieving minimal milestones of immune reconstitution, there was no correlation between response and the absolute level of CD3 T cells, CD8+ T cells, CD4, or CD19+ B cells (data not shown). In contrast, the level of circulating CD4+CD45RA+ cells/ul, p<0.01, correlated with response (figure 2).

To date, this is the largest study evaluating PNCRM7 responses following allogeneic HCT and the first to evaluate PNCRM7 responses in adult recipients of an unmodified or T cell depleted unrelated HCT or adults following a T cell depleted HCT from any donor type. Although retrospective, it includes 80% of all patients transplanted during a defined period (2002–2005) who survived disease-free >1 year. Molrine et al. evaluated PNCRM7 response in 65 unmodified HLA-matched related HCT recipients (median age: 40 years) immunized with PNCRM7 at 3, 6, and 12 months, 30 of whom received grafts from donors immunized before bone marrow harvest (32). After the first immunization, antibody responses in the immunized donor group were significantly higher for 5 of the 7 vaccine serotypes. Although response after the 3^rd PNCRM7 immunization was not evaluable in approximately 30% of the starting population due to relapse (n=8), death from other causes (n=5), thrombocytopenia (n=3), missed vaccines (n=2), or DLI (n=1), 60% of the remaining patients in both groups demonstrated significant pneumococcal titers to all 7 serotypes. In a prospective, randomized double blind trial in adult recipients of an HLA-matched related HCT, Kumar and colleagues compared the effect of vaccinating donor-recipient pairs with a single PPV23 or PNCRM7 (33). Donors were vaccinated at least two weeks prior to stem cell harvest; patients at 6 months post HCT. At 12 months post HCT, the mean number of serotypes for which a response was observed was 1.8 in the PPV23 group and 3.1 in the PNCRM7 group. Similar to the study of Storek et al., no advantage to pre-HCT immunization of the donor and/ or host with PPV23 was observed (34). Meisel et al. studied the response of 43 patients < 17 years of age after a related (45%) or unrelated (55%) HCT. demonstrating that 74% of 43 patients responded to all 7 vaccine serotypes following the third immunization (35). In a study of 30 children vaccinated according to the Royal College of Paediatrics and Child Health guidelines, Patel and colleagues demonstrated that 80% responded to each of the serotypes contained in PNCRM7 when given two monthly doses initiated at 15 months or 21 months following an HLA-matched sibling (n=10) or unrelated HCT (n=20), respectively (36, 37). Considering the poor response of transplant recipients to PPV23 (9–11, 31, 33, 34) and its failure to reduce the incidence of pneumococcal pneumonia even in healthy middle-aged and elderly adults (38), this and the above studies (33,35) support further trials of PNCRM7 in adults and children following HCT.

The EBMT (4) and CDC guidelines (2) advocate revaccination at fixed intervals following transplant, irrespective of patient age, donor or hematopoietic cell type, intensity of conditioning, presence of chronic graft versus host disease, or use of post transplant rituximab. Nevertheless, many of these variables affect the kinetics of T and B cell reconstitution following HCT (19, 39), and likely the ability of patients to respond and maintain protective titers following vaccination (40). Only the Royal College of Paediatrics and Child Health guidelines stratify the timing of vaccination on the basis of donor type (autologous and HLA matched sibling HCT versus all other donors) due to the delayed recovery of immune competence often associated with receipt of an unrelated or HLA mis-matched donor transplant (19, 39). Our study demonstrates that in older patients response to PNCRM7 is significantly better in those vaccinated after acquisition of minimal milestones of immune reconstitution, milestones reached at different time points post HCT, depending on age and presence of chronic GVHD (19, 39). It also shows that even within a population of patients who have achieved minimal milestones of immune reconstitution, not all patients respond suggesting that qualitative and/or quantitative differences in effector and memory helper T and B cells are likely to differentiate responders and non-responders. It is possible that certain vaccines such as tetanus and polio may elicit responses in the majority of patients early post HCT due to expansion of memory T and B cells transferred with the graft. In contrast, vaccines such as recombinant Hepatitis B (41) and pneumococcus may constitute neoantigens requiring generation of T and B cells from donor lymphoid progenitors developing within the host thymus and other tissues of the lymphoid system. Our study demonstrated a relationship between higher numbers of circulating CD4+CD45RA+ T cell numbers and improved PNCRM7 response suggesting that responders had achieved a significant level of reconstitution from precursor populations developing within the host. The correlation; however, was not absolute. Thus, impaired reconstitution of B cell precursors or antigen presenting cells may persist in some patients despite de novo T cell reconstitution.

Although invasive pneumococcal infections occur more frequently in patients with chronic GVHD, deaths from these infections occur in patients with and without chronic GVHD (1–5) as well as in autologous transplant recipients (5). This study, as well as the majority of studies evaluating PNCRM7 response to date, have not included large numbers of patients with chronic GHVD, either by design or early death from chronic GVHD in enrolled patients (32, 33,35, 36). Clearly, there is a need to design multi-center prospective trials which do include patients with limited and potentially extensive chronic GVHD in order to identify the most effective vaccination strategy to elicit protection against pneumococcal disease in the highest proportion of patients at the earliest time possible. Inclusion in such trials of correlative studies analyzing in vitro immune reconstitution should identify immune characteristics most predictive of early and durable immune responses.