Porcine endogenous retrovirus transmission characteristics of galactose alpha1-3 galactose-deficient pig cells

Abstract

Galactose alpha1-3 galactose (Gal) trisaccharides are present on the surface of wild-type pig cells, as well as on viruses particles produced from such cells. The recognition of Gal sugars by natural anti-Gal antibodies (NAb) in human and Old World primate serum can cause the lysis of the particles via complement-dependent mechanisms and has therefore been proposed as an important antiviral mechanism. Recently, pigs have been generated that possess disrupted galactosyl-transferase (GGTA1) genes. The cells of these pigs do not express Gal sugars on their surface, i.e., are Gal null. Concerns have been raised that the risk of virus transmission from such pigs may be increased due to the absence of the Gal sugars. We investigated the sensitivity of porcine endogenous retrovirus (PERV) produced from Gal-null and Gal-positive pig cells to inactivation by purified NAb and human serum. PERV produced in Gal-null pig cells was resistant to inactivation by either NAb or human serum. In contrast, although Gal-positive PERV particles were sensitive to inactivation by NAb and human serum, they required markedly higher concentrations of NAb for inactivation compared to the Gal-positive cells from which they were produced. Complete inactivation of Gal-positive PERV particles was not achievable despite the use of high levels of NAb, indicating that NAb-mediated inactivation of cell-free PERV particles is an inefficient process.

FIG. 1.

Schematic of the galactosyltransferase gene GGTA1 and replacement vector pGalGTΔS-loxNeo. The genomic organization of the region of the genomic GTTA1 allele targeted by the GTTA1 replacement vector pGalLoxNeo is shown. Primer locations used for assessment of the 5′ and 3′ targeting events as well as the diagnostic restriction sites are presented. Note that integration-specific PCRs are achieved by amplification across the genomic DNA-to-vector boundaries.

FIG. 2.

Production of Gal-null porcine cells. (A) Targeting of the GGTA1 allele of PED cells using the pGalGTΔS-loxNeo vector. Arrows indicate PCR bands derived from targeted (T) and wild-type (WT) amplicons for the 5′ (primers F238 and R823) and 3′ (F527 and GR2520) genomic-DNA targeting assays. Similar results were obtained with single cell clones of Gal-null ST-IOWA cells. (B) FACS analysis of GGTA1-targeted ST-IOWA cells. Following targeting of the first allele, Gal-null cells were selected using three rounds of baboon NAb (50 μg/ml) and rabbit complement selection. The Gal status was analyzed by FACS analysis with IB4 lectin. Dark profile, unstained cells; light profile, IB4-stained cells. Similar results were obtained with PED cells. (C) Gal-null ST-IOWA cells are resistant to NAb-mediated lysis. ST-IOWA cells were exposed to purified NAb in the presence of supplementary complement, and cell lysis was determined by cell counting. ▪, Gal-positive ST-IOWA cells; □, Gal-null ST-IOWA cells.

FIG. 3.

Gal-positive PERV is sensitive to inactivation by purified NAb (A) and human serum (B). The sensitivities of Gal-positive (filled symbols) and Gal-null (open symbols) PERV were assessed using cell-free LacZ pseudotype infection of Gal-null ST-IOWA^−/− cells. (C) The infectivity of Gal-positive PERV-A is reduced, but not eliminated, following exposure to NAb. Replication-competent PERV-A was produced in either wild-type ST-IOWA^+/+ cells (○, •), Gal-null ST-IOWA^−/− cells (▪, □), or 293 cells (▴, ▵) and treated with either NAb at 50 μg/ml (open symbols) or fetal bovine serum (filled symbols). The treated supernatant was used to infect 293 cells, and infection was monitored by RT assay.

FIG. 4.

Replication competence of PERV released from engineered miniature swine PED cells. The replication competence of the ecotropic virus produced by miniature swine PED cells was assessed by using PERV infectivity assays in Gal-positive wild-type ST-IOWA^+/+ (A) and Gal-null ST-IOWA^−/− (B) target cells. The inoculating virus was produced from either PED^+/+ (▵), PED^+/− (○), or PED^−/− (□) cells, and infection was monitored by RT assay.