TAX and HBZ: hFc Ɣ 1 proteins as targets for passive immunotherapy

Abstract

Objectives: Human T leukemia virus type one (HTLV-1) causes two life-threatening diseases in around five percent of infected subjects, a T cell malignancy and a neurodegenerative disease. TAX and HBZ are the main virulence agents implicated in the manifestation of HTLV-1-associated diseases. Therefore, this study aims to produce these HTLV-1 factors as recombinant Fc fusion proteins to study the structures, their immunogenic properties as vaccines, and their capability to produce specific neutralization antibodies.

Materials and methods: TAX and HBZ sequences were chosen from the NCBI-nucleotide database, then designed as human Fc chimers and cloned into Pichia pastoris. Produced proteins were purified by HiTrap affinity chromatography and subcutaneously injected into rabbits. Rabbit Abs were purified by batch chromatography, and their neutralization activities for the HTLV-1-infected MT-2 cell line were assessed. Furthermore, the protective abilities of recombinant proteins were evaluated in Tax or HBZ immunized rabbits by MT-2 cell line inoculation and measurement of HTLV-1-proviral load.

Results: Specific Abs against Tax and HBZ can eliminate 2 million MT-2 cells in 1/1000 dilution in vitro. In challenging assays, the immunization of the animals using Tax or HBZ had no protective activity as HTLV-1 PVL was still positive.

Conclusion: The result suggests that recombinant TAX and HBZ: hFcγ1 proteins can produce a proper humoral immune response. Therefore, they could be considered a passive immunotherapy source for HTLV-1-associated diseases, while total TAX and HBZ proteins are unsuitable as HTLV-1 vaccine candidates.

Keywords: ATLL; HBZ; HTLV; Immunization passive; Pichia pastoris; Recombinant proteins; TAX.

Figure 1

Schematic illustration of the Protein constructs. A) Schematic illustration of the monomer genetic fusion of HTLV fusion proteins TAX, HBZ, and Fcү1 to create a TAX: Fcү1 and HBZ: Fcү1 fusion protein. B) Schematic illustration of the genetic fusion of the dimmer genetic fusion of HTLV fusion proteins TAX, HBZ, and Fcү1 to create a TAX: Fcү1 and HBZ: Fcү1 fusion protein. C) Schematic map of pPICZα: TAX/HBZ:hFcү1. The insert was cloned into the XhoIand NotI restriction enzyme sites of the pPICZα vector downstream to the AOX1 promoter. 5′ AOX1, alcohol oxidase 1 promoter; AOX1 TT, transcriptional terminator from Pichia pastoris AOX1 gene; TEF1 promoter, transcriptional elongation factor 1 promoter from Saccharomyces cerevisiae; EM7 promoter, synthetic prokaryotic promoter; Zeocin, Zeocin resistance gene; CYC1 TT, transcriptional terminator from Saccharomyces cerevisiae CYC1 gene; pUCori, pUC origin of replication

Figure 2

Protein modeling results for HBZ: hFcγ1. A) Solid ribbon views of the modeled protein, which include the dimeric form of Fc tag and HBZ. The red circle identifies the disulfide bond, and the amino acid residues were nominated. B) Both N and O glycosylation sites were displayed in yellow and green colors. The N glycosylation sites are located on Fc taq, while the O sites carry HBZ proteins. In dimeric form a total of 10 sites are available for glycosylation. C) The Ramachandran plot of the modeled protein displayed that 87.4% of whole recombinant proteins are located in the selected region. There are only 1.4% of residues located in the disallowed region

Figure 3

Protein modeling results for TAX: hFcγ1. A) Solid ribbon views of the modeled protein, which include the dimeric form of Fc tag and TAX. The red circle identifies the disulfide bond, and the amino acid residues were nominated. B) Both N and O glycosylation sites were displayed in yellow and green colors. The N glycosylation sites are located on Fc taq, while the O sites are carried on TAX proteins. In dimeric form a total of 13 sites are available for glycosylation. C) The Ramachandran plot of the modeled protein displayed that 79.9 % of whole recombinant proteins are located in the select region. There are only 2.5 % of residues located in the disallowed region

Figure 4

A Soft gel dot blotting for detection of TAX and HBZ: hFcγ1 optimum colonies. A) results suggest group number 3 (17, 18, 19) for Tax and B) (17, 19) for HBZ proteins have stronger transformed colonies

Figure 5

Dot blotting for detection of TAX and HBZ: hFcγ1 recombinant proteins and optimization of HRP antibody titration for the ELISA method. Our results demonstrate that titration of 1/3000 for HRP anti-human can have the best resolution

Figure 6

Analysis of the recombinant HBZ: hFcγ1 (a) and TAX: hFcγ1 (b) protein by 12 % SDS-PAGE stained with Coomassie Blue. A) Line 1 IVIG 1/100 and 2 HBZ: hFcγ1. B) Line 1, 2, and 3 TAX: hFcγ1, fractions of the eluted recombinant protein of approximately 51 and 65.5 kDa for HBZ and TAX: hFcγ1, respectively. Line Ladder: protein marker

Figure 7

Expression of recombinant HBZ: hFcγ1 and TAX: hFcγ1 protein was analyzed by immunoblot using anti-Human IgG-HRP. A) HBZ: hFcγ1 identification by NBT/BCIP substrate line 1: IVIG, 2: HBZ: hFcγ1. B) TAX: hFcγ1 fraction protein blotting by NBT/BCIP substrate line 1, 2, 3 TAX: hFcγ1. Ladder: Protein marker 10-180 kDa

Figure 8

Co-localization of FcγRI (CD64) on human PBMCs and TAX/HBZ: hFcγ1 recombinant fusion protein. Immunofluorescence staining of PBMCs showing TAX: hFcγ1 and HBZ: hFcγ1 recombinant fusion binds to FcγRI A and B, respectively. Red signal, PBMCs stained with PE anti-human CD64 (FcγRI) antibody; green signal, TAX: hFcγ1and HBZ: hFcγ1 recombinant fusion stained with goat anti-human IgG1-FITC antibody. C) Immunofluorescence staining of PBMCs with goat anti-human IgG1-FITC, PE anti-mouse CD64 (FcγRI) without Fc-fusion protein as a negative control (red signal, PBMCs stained with PE anti-human CD64 (FcγRI) antibody). Fcγ1, Fc fragment of human IgG1; FcγRI, Fcγ receptor I; PE, phycoerythrin; FITC, fluorescein isothiocyanate

Figure 9

A) Cell cytotoxicity assays (CCA) were done by Anti-TAX: hFcγ. B) CCA results for Anti-HBZ: hFcγ in 1/1000 titration. C) The negative control contained MT-2 cells plus normal saline. D) CCA demonstrates polyclonal specific antibody (1-1/1000 titration) against TAX:hFcγ1 and HBZ: hFcγ1 recombinant fusion protein to the evaluated killing ability of around 2 million HTLV malignant cell line. The CCA display 1/1000 titer of PCA for both TAX, and HBZ: hFcγ has the absolute ability to neutralize 2 million MT-2 cells

Figure 10

HTVL-I proviral examination by Real-Time PCR Tag-Man method. The human albumin is just presented for positive control, and none of the study groups were positive. The concentration of rabbit DNA was normalized around 60–70 ng/μl. The negative results for human albumin indicate the HTLV-1 genome detection results from inoculation, not the persistence of MT-2 cell lines