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. 2008 Apr;57(4):493-505.
doi: 10.1007/s00262-007-0388-y. Epub 2007 Sep 18.

Listeriolysin O is an improved protein carrier for lymphoma immunoglobulin idiotype and provides systemic protection against 38C13 lymphoma

Paul Neeson  1 , Zhen Kun Pan, Yvonne Paterson
Affiliations

Listeriolysin O is an improved protein carrier for lymphoma immunoglobulin idiotype and provides systemic protection against 38C13 lymphoma

Paul Neeson et al. Cancer Immunol Immunother. 2008 Apr.

Abstract

Follicular lymphoma (FL) is a disease that responds to current treatment regimens; however, patients in general relapse with increasingly refractory disease. Idiotype-based vaccines are currently under trial for the treatment of FL. These vaccines comprise the patient's BCR idiotype (Id) as the tumor antigen conjugated to the protein carrier Keyhole Limpet Hemocyanin (KLH); however, other protein carriers may enhance the immune response to the lymphoma Id. In this study we investigated whether an alternate carrier, Listeriolysin O (LLO), would amplify the immune response to Id protein and provide better protection against challenge by 38C13 murine lymphoma. The Id-LLO vaccine compared favorably against Id-KLH in tumor-protection studies and both vaccines provided systemic immunity against 38C13 lymphoma. However, the immune response to the two conjugates was different in that Id-LLO induced a more powerful Th1 response characterized by high titer IgG2a anti-Id antibodies after one immunization and the presence of CD4 cells secreting IFN-gamma. In vivo studies demonstrated that immune serum contributed to the anti-lymphoma efficacy seen following Id-LLO immunization. Interestingly, Id-LLO immunized mice, when challenged twice with 38C13 lymphoma provided better protection against challenge by the BCR loss variant 38C13-V2, suggesting that Id-LLO immunized mice have more potential to develop epitope spreading than Id-KLH. In conclusion, Id-LLO compared favorably against Id-KLH in its anti-lymphoma efficacy. Furthermore, Id-LLO induced a more potent humoral and cell-mediated immune response and promoted epitope spreading after lymphoma challenge. Thus, anti-Id vaccines incorporating LLO may be a better therapeutic option for treatment of B-cell lymphoma.

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Figures

Fig. 1
Fig. 1
Id-LLO immunization protects C3H/HeN mice from 38C13 lymphoma challenge. C3H/HeN mice were immunized with 38Id or 38Id conjugates and subsequently challenged with 38C13 lymphoma, depicted in (a). The development of s.c. lymphoma was monitored for each mouse over the next 60 days (b), and the results presented as the frequency of each vaccine group “tumor free”. Statistical analysis of the incidence of tumor-free mice for each vaccine group was performed for the course of the study (non-parametric Kaplan–Meier, Log Rank Mantel-Cox test) using SPSS software. An asterisk represents where a test vaccine result is statistically different (P < 0.05) compared to the control groups. These results are representative of two separate experiments
Fig. 2
Fig. 2
Idiotype-LLO immunization induces high-titer anti-idiotype antibodies with a higher IgG2a:IgG1 ratio than those induced by Id-KLH. The presence of 38C13 anti-idiotype antibodies was determined by a sandwich ELISA on sera taken from C3H/HeN mice pre-immunization, 12 days post first or second immunization. Data are plotted as the mean titer ± standard error of eight mice per vaccine group (a). The sera with positive titers were further characterized by an IgG-isotyping assay. This was performed by ELISA, results are presented for sera collected post first or second immunization with either Id-LLO or Id-KLH (b). An asterisk depicts where a significant difference (Student’s t-test, P < 0.05) was detected between the two vaccine groups post immunization
Fig. 3
Fig. 3
Id-LLO immunization induces a Th1 CD4 T cell response. DLNs were collected 14 days after s.c. immunization and cells harvested. CFSE-stained DLN cells were then incubated with purified proteins for 5 days before being re-stimulated with PMA/Ionomycin for 5 h in the presence of monensin. Cells were then stained for surface CD4, fixed and stained for intracellular cytokine. Data were acquired on a FACSCalibur and analyzed by FlowJo software. The results are presented as the percentage (mean ± SD) of gated cells secreting cytokines, a CD4 T cell IFN-γ secretion for each vaccine group, b CD4 T cell IL-4 secretion for each vaccine group, c CD4 proliferation results for each vaccine group. Student t-test was used to analyze the data, the symbol asterisk indicates where in vitro protein stimulation induced a significantly different result (P = <0.05) compared to media only in that vaccine group
Fig. 4
Fig. 4
Transfer of serum after Id-LLO immunization protects from in vivo challenge with 38C13 lymphoma. A modified Winn assay was performed to assess the role of serum effectors in the anti-lymphoma effect in vivo. The experimental design is depicted in a, mice were immunized with two rounds of Id-LLO + mGMCSF, the control group was naïve mice. Fourteen days after the second round of immunization, peripheral blood was collected and a pool of serum prepared. The pooled serum was then co-inoculated s.c. with 38C13 cells on the left flank into recipient mice (8 per group). The mice were then monitored for 60 days to assess the frequency of lymphoma development in each test or control group. The results for serum transfer are shown in b. Statistical analysis of the incidence of tumor-free mice for each vaccine group was performed for the course of the study (non-parametric Kaplan–Meier, Log Rank Mantel-Cox test) using SPSS software. An asterisk represents where a statistical difference (P < 0.05) in anti tumor efficacy was detected between effectors from immunized and naïve mice
Fig. 5
Fig. 5
Mice immunized with 38Id-KLH or 38Id-LLO have systemic immunity and are protected from 38C13 challenge on the opposite flank to the initial immunization and challenge. Immunized C3H/HeN mice that survived the first tumor challenge (Fig. 1b) were subsequently challenged on the opposite flank (right side) with 1 × 103 38C13 cells and monitored for tumor formation over the next 60 days. Results are depicted as the percentage mice in each vaccine that are tumor free for each time point. Naïve age-matched C3H/HeN mice (n = 4) served as negative controls for this study. The data for each vaccine group (or the naïve control group) were analyzed by non-parametric Kaplan–Meier, Log Rank Mantel-Cox test using SPSS software. An asterisk represents where a statistical difference (P < 0.05) in anti-tumor efficacy was detected between immunized and naïve mice
Fig. 6
Fig. 6
LLO conjugated to the BCR idiotype induces more effective epitope spreading than KLH. Immunized mice that survived two previous challenges with the parental 38C13 lymphoma were subsequently challenged s.c. with the BCR-loss variant 38C13-V2 cells. The mice were then monitored for 60 days for the formation of s.c. lymphoma. The data for each vaccine group (or the naïve control group) were then analyzed by non-parametric Kaplan–Meier, Log Rank Mantel-Cox test using SPSS software. An asterisk represents where a statistical difference (P < 0.05) in anti tumor efficacy was detected between immunized and naïve mice. Results are representative of two separate experiments
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