Elsevier

Vaccine

Volume 29, Issue 32, 18 July 2011, Pages 5114-5121
Vaccine

Response of MUTZ-3 dendritic cells to the different components of the Haemophilus influenzae type B conjugate vaccine: Towards an in vitro assay for vaccine immunogenicity

https://doi.org/10.1016/j.vaccine.2011.05.050Get rights and content

Abstract

Potency testing is mandatory for vaccine registration and batch release. Due to various limitations to in vivo potency testing, there is need for relevant in vitro alternatives. These alternative tests should preferably comprise cells from the target (human) species. The whole suite of immune responses to vaccination that occur in vivo in humans cannot be tested in vitro using a single cell type. Even so, dendritic cells (DC) form an important candidate cell type since they are pivotal in inducing and orchestrating immune responses. Cell lines are preferred over ex vivo cells for reasons of safety, accessibility, and reproducibility. In this first feasibility study we used the human cell line MUTZ-3, because it most closely resembles ex vivo human DC, and compared its response to monocyte-derived DC (moDC).

Haemophilus influenzae type B (HiB) vaccine was chosen because its components exert different effects in vivo: while the HiB antigen, polyribosyl ribitol phosphate (PRP) fails to induce sufficient protection in children below 2 years of age, conjugation of this polysaccharide antigen to outer membrane protein (OMP) of Neisseria meningitides, results in sufficient protection. Effects of PRP, OMP, conjugated PRP–OMP, and adjuvanted vaccine (PedVax HiB), on cytokine production and surface marker expression were established. PRP induced no effects on cytokine production and the effect on surface marker expression was limited to a minor decrease in CD209 (DC-SIGN). In both MUTZ-3 and moDC, OMP induced the strongest response both in cytokine production and surface marker expression. Compared to OMP alone conjugated PRP–OMP generally induced a weaker response in cytokine production and surface marker expression. The effects of PedVax HiB were comparable to conjugated PRP–OMP. While moDC showed a larger dynamic range than MUTZ-3 DC, these cells also showed considerable variability between donors, with MUTZ-3 DC showing a consistent response between the replicate assays. In our view, this makes MUTZ-3 DC the cells of choice. In conclusion, our results demonstrate that the MUTZ-3 DC assay allows discrimination between compounds with different immunogenicity. The potential of this cell line as (part of) an in vitro immunogenicity assay should be further explored.

Introduction

The efficacy of vaccines is dependent on their ability to induce an immune response of the appropriate type and of sufficient vigour, in order to induce lasting immunoprotection. For the purpose of vaccine marketing authorisation and batch release, testing its immunogenic potential in a relevant model is mandatory. Currently, most vaccine efficacy testing is performed in animal models with their inherent limitations. Firstly, animal models are only partly representative for the human immune response. Secondly, such in vivo tests generally show a high variability. Thirdly, in line with the 3R principle such as formulated by the EU (86/609/EEC) there is a desire to replace animal models with in vitro alternatives, especially in case of regulatory testing.

These issues can potentially be addressed by development of in vitro assays using human immune cells, either primary cells or immortalized cell lines. Using cells of human origin has the advantage of a lack of interspecies differences. Cell lines are preferred over ex vivo cells for reasons of safety, accessibility, and reproducibility. The whole suite of immune responses to vaccination that occur in vivo in humans cannot be tested in vitro using a single cell type. Even so, dendritic cells (DC) form an important candidate cell type since they are pivotal in inducing and orchestrating immune responses [1]. In recent years several dendritic cell lines have been developed to study their response to immunogens and their role in the immune system [2].

The myeloid leukemia cell line MUTZ-3 is the immortalized equivalent of CD34+ haematopoietic progenitors. To develop from their progenitors MUTZ-3 cells require the same cytokines (GM-CSF, IL-4, and TNF-α) as monocyte-derived DC (moDC). They can be induced to a phenotype consistent with interstitial or Langerhans DC, i.e. acquisition of CD14 and loss of CD1a, with interstitial DC being DC-SIGN+ and Langerhans DC being Langerin+ [3], [4]. Maturation resulted in increased expression of CD40, CD54, CD80, CD86, and HLA-DR. MHC class I, class II, and invariant (CD1) antigen processing and presentation is functional [3]. Comparison of various cell lines with moDC by gene profiling revealed that MUTZ-3 is the cell line that most closely resembles moDC [5]. Thus, DC derived from MUTZ-3 have several important characteristics in common with moDC, making the cell line a suitable alternative to DC obtained from blood donors. Furthermore, MUTZ-3 DC have already yielded promising results when explored as alternative model system to cord blood derived primary CD34+–DC for chemical contact allergens [6].

In our experimental set up we used the Haemophilus influenzae type B (HiB) vaccine, which does not require animal testing for batch release, because an HPLC assay for the polyribosyl ribitol phosphate (PRP) content has been accepted as potency test for this vaccine. However, HiB vaccine was chosen because it consists of 3 components each of which have a different contribution to the immune response. The capsular polysaccharide, PRP is the antigenic component of the vaccine to which the antibody response should be directed. However, a vaccine consisting only of PRP appeared only effective in adults and older children and failed to yield protection in children younger than 2 years, which is the group with the highest burden of disease [7], [8]. Therefore, a second-generation HiB vaccine was developed, in which PRP is conjugated to an immunogenic protein [9], [10]. Outer membrane protein from Neisseria meningitides (OMP) as immunogenic protein resulted in the most immunogenic conjugate vaccine tested in infants younger than 6 months of age [10]. In case of Liquid PedVax, similarly, PRP is conjugated to OMP. Furthermore, aluminium is present as adjuvant.

In the present study, we compared the effects of PRP, OMP, conjugated PRP–OMP and the adjuvanted vaccine on cytokine production and CD marker expression of MUTZ-3 DC and moDC.

Section snippets

MUTZ-3 cell line

The human CD34+ acute myeloid leukemia cell line MUTZ-3 (DSMZ, Braunschweig, Germany) was cultured in the presence of α-Minimum Essential Medium (Gibco), supplemented with 20% heat-inactivated FBS (Hyclone Laboratories), penicillin (100 U/ml)/streptomycin (100 μg/ml) (Gibco), 2 mM l-glutamine (Gibco), 50 μM 2-mercaptoethanol (Serva) and 27.5 U/ml GM-CSF (PeproTech).

The cells were maintained in 12-well tissue culture plates (Costar) at a concentration of 1–5 × 105/ml. The cells were passed twice a week.

General observations

Using moDC there was a considerable variability between donors in their response to treatment. Such variability was already observed in surface marker expression of the untreated moDC, probably reflecting the immune status of the donors (Table 1). The largest variability in surface marker expression was observed in HLA and DC-SIGN (Table 1). Donor 2 appeared to be the least responsive.

In MUTZ-3 DCs treatment effects on surface marker expression were much smaller than in case of moDC, leaving

Discussion

The in vitro DC-based cell assay shows different responses to the different components of the HiB vaccine. All combinations tested that contained OMP (OMP alone, PRP–OMP and PedVax) induced an increase in the co-stimulatory molecules CD80 and CD86 using both moDC and MUTZ-3 DC. PRP alone, on the other hand, failed to induce this response. The absence of a significant response to PRP is in agreement with the limited clinical immunogenicity found for PRP vaccines without conjugate. PRP is

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