This study presents the most detailed analysis to date of in vivo humoral immune responses in subjects after rituximab treatment, including responses to both recall antigens and neoantigens, particularly during the period of maximal B-lymphocyte depletion. Of significance, because the subjects had not received prior or concurrent immunomodulatory drugs that might affect general immune responses or B-lymphocyte function, the data represent an uncontaminated test of rituximab's effect. Such data provide important safety information and needed guidance for immunizations in patients receiving treatment with rituximab. Our study confirms the maintenance of protective antibody titers and the successful (albeit slightly depressed) recall and de novo responses after B-lymphocyte recovery and, more importantly, that antigen exposure during B-lymphocyte depletion did not preclude subsequent responses.
As others and we have reported previously, B lymphocytes recover over a period of 1 year after rituximab treatment.24-26
The recovery is not uniform because naive B lymphocytes completely recover by 1 year, whereas memory B lymphocytes remain depleted.25,26
In the current study the memory B-lymphocyte population was initially depleted to the same degree as total CD19 B lymphocytes and naive B lymphocytes. However, memory B lymphocytes were much slower to recover. Moreover, we did not observe an increase in the memory B-lymphocyte population 1 month after the immunizations given at month 12.
Rituximab has little effect on circulating antibody titers to previous immunizations in either adults5,27
Although rituximab results in a reduction in IgM levels, it does not affect serum IgG levels.5,26,29
IgG antibodies are made by long-lived plasma cells that do not express CD20 on their surface and thus are not eliminated with rituximab. In healthy adults the half-life of anti-MMR titers after live virus vaccine have been estimated to be greater than the life of a person,30
and we saw no change in titers in our study.
Several studies have evaluated the responses to various antigens after treatment with rituximab. Recall responses to tetanus are substantially reduced when immunization is given early (4 weeks)6
or late (6 and 9 months)31
after completing rituximab. Recall responses to diphtheria immunization after rituximab treatment have not been reported previously. In our study the response to Td after rituximab was diminished compared with that seen in the placebo group, but adequate protection was achieved. Although it has been reported in patients with long-standing T1D that responses to diphtheria toxoid are reduced, we saw no such effect (the anti-diphtheria geometric mean titer after booster vaccination of 4.84 IU/mL [95% CI, 3.53-6.63 IU/mL; range, 0.52-11.72 IU/mL] was in the normal range).32
Although response to Pneumococcus species is often used in evaluation of immunodeficiency, we did not include it because polysaccharide vaccines are associated with hyporesponsiveness, and their use could potentially jeopardize the recall response of the recipients when re-exposed to the same antigen.
The de novo
response to hepatitis A immunization has been studied in patients treated with rituximab for malignant diseases, but the results were ambiguous.6
Patients with T1D have been reported to have an attenuated anti–hepatitis A response (ELISA titer: 53 IU/L in patients with T1D vs 212 IU/L in healthy control subjects, P
In our placebo group the anti–hepatitis A response was 57 mIU/mL (95% CI, 37-88 mIU/mL; range, 7-269 mIU/mL); 14 of 18 achieved a protective titer of greater than 20 mIU/mL. We saw a significant increase in hepatitis A responses after recovery from rituximab, although to a lower degree than in the control group.
In healthy subjects phiX174 circulates for 3 to 4 days after primary intravenous immunization until the induced IgM antibody response neutralizes and eliminates the phage before day 7. Because no phiX174 was detected at 7 days after initial immunization, even the very low amount of IgM produced by the rituximab-treated subjects was sufficient to neutralize and remove this antigen from the circulation. Thus the rituximab-induced B-lymphocyte defect is not as profound as in patients with X-linked agammaglobulinemia because of mutation of Bruton tyrosine kinase or in those with T-B severe combined immunodeficiency in whom clearance of phage can be delayed for weeks9,10
and resembles that seen in patients with X-linked agammaglobulinemia with some residual function of Bruton tyrosine kinase, children with adenosine deaminase deficiency, patients with Wiskott-Aldrich syndrome,13
or a subset of patients with common variable immune deficiency.9-12
A possible source of this IgM could be marginal zone B lymphocytes that, despite expressing CD20, have been suggested to be resistant to rituximab depletion.33
Germinal center B lymphocytes, the site of isotype switching, are sensitive to rituximab and thus could explain the lack of an IgG response while rituximab is present in the circulation.33
An important aspect of our study is the analysis of a recall antibody response to an antigen first seen during the period of B-lymphocyte depletion and again when B lymphocytes have recovered. It was hypothesized that such immunization precludes a subsequent response to the antigen at the time of subsequent immunizations similar to what has been seen in patients with HIV infection.18,19
However, the response of rituximab-treated subjects to a tertiary and quaternary immunization with phiX174 recapitulates the pattern seen in the placebo-treated subjects and healthy control subjects after primary and secondary immunization. This indicates that in the presence of rituximab, a memory B-lymphocyte response was not generated. Rituximab has been shown to persist in the blood up to 9 months after the first dose of rituximab.5
memory B lymphocytes express CD20, they are expected to be lysed as soon as they begin to appear.
DiLillo et al34
reported an extensive analysis of immunizations in mice that were depleted with an anti-murine CD20 antibody. Although their results are similar to ours in some ways, there are some differences as well, perhaps related to differences in physiology between murine and human responses.
Heterologous (ie, bystander) immune responses have been postulated as a mechanism of antibody persistence.35
Bernasconi et al35
hypothesized “that during an antigen-specific response, the increased availability of activated T cells will lead to increased production of plasma cells of unrelated specificities.” They reported that tetanus immunization increased both tetanus-specific and antigen-nonspecific circulating plasma cells. A corollary to this finding would be an increase in detectable levels of nonspecific antibody, but they did not report data for nonspecific antibodies. Di Genova et al36
found that an anti-tetanus antibody response was not associated with changes in antibodies to Candida
species or purified protein derivative. Amanna et al30
saw no increase in antibody titers to 7 independent antigens in healthy subjects followed for 60 days after a vaccinia virus booster immunization. We, too, did not see any change in nonspecific antibody titers to MMR 1 month after multiple immunizations with Td, hepatitis A, and phiX174. These 3 independent assessments bring into question the bystander hypothesis for antibody persistence long after antigen exposure.
In summary, treatment with rituximab has a profound effect on immunization given during the time of B-lymphocyte depletion. With recovery, the immune response returns toward normal. Immunization during the time of B-lymphocyte depletion, although ineffective, does not preclude a subsequent response to the antigen.