Production of antigen-specific human IgGs by in vitro immunization

Abstract

Background: We previously developed in vitro immunization based on a fusion protein containing the transcriptional transactivator (Tat) of human immunodeficiency virus and a double domain, called ZZ, derived from protein A of Staphylococcus aureus. In this approach, naïve human peripheral blood mononuclear cells (PBMCs) trigger a specific IgM antibody (Ab) response in the presence of ZZTat. In the present study, we attempted to raise a specific IgG Ab response.

Results: We found that PBMCs incubated with ZZTat and a mixture containing anti-CD40, IL4 and IL21 secrete anti-Tat IgG Abs in their supernatants, indicating that the cytokine cocktail provides an isotypic switch. Then, we deciphered the Tat determinant involved in the phenomenon and found that it is located in the region 22-57 and that, within this region, the cysteine-rich domain and the basic residues play a crucial role. Finally, we prepared a fusion protein containing a fragment derived from the NY-ESO-1 cancer/testis antigen (Ag) and showed that PBMCs incubated with ZZfNY-ESO-1Tat trigger a specific anti-fNY-ESO-1 IgG Ab response, which demonstrates the possibility of transferring immunizing ability to an Ag unrelated to Tat.

Conclusion: Our ZZTat-based in vitro immunization approach that offers the possibility to raise an IgG Ab response against NY-ESO-1 might represent a valuable first stage for the generation of fully human IgG specific Abs.

Fig. 1

a An anti-Tat IgG Ab response is raised in vitro when PBMCs are incubated with ZZTat101 and a cytokine/activator cocktail. PBMCs (5 × 10⁵ cells per well) were incubated with or without CD40L/IL-4/IL-21, anti-CD40/IL-4/IL-21, ZZTat101, ZZTat101 + CD40L/IL-4/IL-21, ZZTat101 + anti-CD40/IL-4/IL-21, respectively. Then, supernatants were collected and added to Tat101-coated plates. After overnight incubation, a peroxidase-conjugated anti-human IgG was added and enzymatic activity was determined using ABTS as substrate. b The anti-Tat IgG Ab response is specific to Tat101. Supernatants from PBMCs incubated with ZZTat101 + anti-CD40/IL-4/IL-21 were mixed with either a dilution buffer or a fixed amount (10 μg/mL) of three different Ags (Tat101, ovalbumin, lysozyme) and incubated in Tat101-coated plates. Presence of anti-Tat IgG was assessed as in A (**: p < 0.01; ***: p < 0.001)

Fig. 2

a The covalent coupling of Tat101 to ZZ is absolutely required to trigger an anti-Tat IgG Ab response. PBMCs were incubated with or without anti-CD40/IL-4/IL-21, ZZTat101, ZZTat101 + anti-CD40/IL-4/IL-21, ZZ + anti-CD40/IL-4/IL-21, ZZ + Tat101 + anti-CD40/IL-4/IL-21, Tat101 + anti-CD40/IL-4/IL-21, respectively. Then, supernatants were collected and added to Tat101-coated plates. Presence of anti-Tat IgG was assessed as in Fig. 1a. b The anti-Tat IgG response requires preservation of the Tat lysines. PBMCs were incubated with or without anti-CD40/IL-4/IL-21, ZZTat101 + anti-CD40/IL-4/IL-21, ZZTat101ϕ + anti-CD40/IL-4/IL-21, respectively. Presence of anti-Tat IgG was assessed in supernatants as in Fig. 1a (*: p < 0.05)

Fig. 3

The 22–57 region of Tat suffices to raise an IgG response and preservation of the cysteine residues is absolutely required for the phenomenon. PBMCs were incubated for seven days with or without anti-CD40/IL-4/IL-21, ZZTat101 + anti-CD40/IL-4/IL-21, ZZTat22-57 + anti-CD40/IL-4/IL-21, ZZTat22-57C(22–37)S + anti-CD40/IL-4/IL-21, respectively. Then, supernatants were collected and added to Tat101-coated plates. Presence of anti-Tat IgG was subsequently assessed as described in Fig. 1a

Fig. 4

An optimal anti-Tat specific B-lymphocyte response was reached after 11 days of in vitro immunization. PBMCs were incubated for eight, eleven or thirteen days in the presence or absence of anti-CD40/IL-4/IL-21, ZZTat101 + anti-CD40/IL-4/IL-21, respectively. Then, PBMCs were transferred from culture plates to ELISPOT plates previously coated with Tat101. After an additional 24-h incubation at 37 °C, a biotinylated anti-human IgG was added. Spot-forming cells were then detected using a streptavidin-alkaline phosphatase conjugate and NBT/BCIP as substrate. Spot-forming cells were quantified with an automated ELISPOT reader

Fig. 5

ZZTat101 is able to confer on fNY-ESO-1 the ability to induce B-lymphocytes to secrete anti-fNY-ESO-1 IgG antibodies. a PBMCs were incubated for eleven days without anti-CD40/IL-4/IL-21, with ZZTat22-57 + anti-CD40/IL-4/IL-21 or ZZfNY-ESO-1Tat22-57 + anti-CD40/IL-4/IL-21, respectively. Then, supernatants were collected and added to anti-human IgG-coated plates. After an incubation of 2 h, biotinylated f-NY-ESO-1 was added to each well. After overnight incubation, an acetylcholinesterase-labeled streptavidin conjugate was added and enzymatic activity was determined using Ellman's reagent. b PBMCs were incubated for eleven days with or without anti-CD40/IL-4/IL-21, ZZfNY-ESO-1Tat22-57 + anti-CD40/IL-4/IL-21, respectively. PBMCs were transferred from culture plates to ELISPOT plates previously coated with fNY-ESO-1. After an additional 24-h incubation at 37 °C, a biotinylated anti-human IgG was added. Spot-forming cells were then detected using a streptavidin-alkaline phosphatase conjugate and NBT/BCIP as substrate. Spot-forming cells were quantified with an automated ELISPOT reader