Reduced sensitivity to human serum inactivation of enveloped viruses produced by pig cells transgenic for human CD55 or deficient for the galactosyl-alpha(1-3) galactosyl epitope

Abstract

Complement activation mediated by the major xenogeneic epitope in the pig, galactosyl-alpha(1-3) galactosyl sugar structure (alpha-Gal), and human natural antibodies could cause hyperacute rejection (HAR) in pig-to-human xenotransplantation. The same reaction on viruses bearing alpha-Gal may serve as a barrier to zoonotic infection. Expressing human complement regulatory proteins or knocking out alpha-Gal epitopes in pig in order to overcome HAR may therefore pose an increased risk in xenotransplantation with regard to zoonosis. We investigated whether amphotropic murine leukemia virus, porcine endogenous retrovirus, and vesicular stomatitis virus (VSV) budding from primary transgenic pig aortic endothelial (TgPAE) cells expressing human CD55 (hCD55 or hDAF) was protected from human-complement-mediated inactivation. VSV propagated through the ST-IOWA pig cell line, in which alpha-galactosyl-transferase genes were disrupted (Gal null), was also tested for sensitivity to human complement. The TgPAE cells were positive for hCD55, and all pig cells except the Gal-null ST-IOWA expressed alpha-Gal epitopes. Through antibody binding, we were able to demonstrate the incorporation of hCD55 onto VSV particles. Viruses harvested from TgPAE cells were relatively resistant to complement-mediated inactivation by the three sources of human sera tested. Additionally, VSV from Gal-null pig cells was resistant to human complement inactivation. Such protection of enveloped viruses may increase the risk of zoonosis from pigs genetically modified for pig-to-human xenotransplantation.

FIG. 1.

hCD55 and α-Gal expression on porcine cells. (a) Expression of CD55 was analyzed by FACs on both transgenic (TgPAE) and nontransgenic (PAE) pig cells. Staining by anti-hCD55 (shaded histogram), no primary antibody (negative control; solid line), or anti-hCD46 (dotted line, similar profile to that of the no-antibody control) followed by FITC-labeled secondary anti-mouse immunoglobulin antibodies (upper panels) is shown. Staining for α-Gal was a one-step incubation; hence, −lectin on the histogram represents cells only, and +lectin represents cells incubated with IB-4 lectin conjugated to FITC (lower panels). (b) α-Gal expression on ST-IOWA wild-type and ST-IOWA Gal-null (−/−) cells. Expression of the α-Gal antigen was analyzed by FACs, using the IB-4 lectin. A rightward shift of the histogram in the presence of lectin (+lectin) compared to the histogram generated in the absence of lectin (−lectin) indicates expression of the α-Gal antigen. The level of expression was determined using the MFI shift, generated by the Cell Quest FACScan software. The MFI shift was calculated by determining test MFI and subtracting that of negative controls (no primary antibodies for hCD55 and hCD46 expression and no lectin for α-Gal expression), and all the mean shifts are shown in Table 1.

FIG. 2.

Demonstration of hCD55 incorporation on VSV particles by a viral pull-down assay. VSV harvested through HeLa, TgPAE A, and PAE E cells in the presence of antibody was incubated with protein G-expressing bacterial cells (OMNISORB). Antibodies used were three anti-human CD55 antibodies, BRIC 216, BRIC 471, and anti-DAF; anti-human CD46 J4-48 (hCD46); and anti-CD59 BRA-10G (hCD59). Five micrograms of anti-DAF and 1 μg of the other antibodies were used for incubation with virus and protein G cells. Titers of VSV for pulled-down and input particles were determined by TCID₅₀ assay. The ratio of these titers is shown as percent binding.

FIG. 3.

MLV-A sensitivity to human complement. (A) LacZ(MLV-A) harvested from three transgenic (TgPAE A, B, and C) and three nontransgenic (PAE D, E, and F) primary pig cells was assayed for its sensitivities to human complement by an infection assay. Virus harvested from HeLa cells is inherently resistant to human complement and was used as a control in two different experiments. Mean percentage titers from duplicated experiments relative to those achieved when using heat-inactivated human serum are shown. (B) Three different human sera, serum 1 (straight line), serum 2 (dotted line), and serum 3 (dashed line), were tested for their ability to inactivate MLV-A harvested from TgPAE A or PAE D cells. Virus harvested from HeLa cells was used as a control. Titers are expressed as a mean percentage relative to titer achieved using heat-inactivated serum from duplicated experiments.

FIG. 4.

PERV sensitivity to human complement. PERV-A 14/220 harvested from two transgenic pig cell lines, TgPAE A and TgPAE B, and one nontransgenic pig cell line, PAE D, were tested for their sensitivities to human sera 1 and 3. Virus harvested from HeLa cells was used as a control for resistant virus. Titers are expressed as a mean percentage relative to titers achieved using heat-inactivated serum of duplicated experiments.

FIG. 5.

Sensitivity of VSV to human complement. (A) VSV harvested from two transgenic pig cells, TgPAE A and TgPAE C, and two nontransgenic pig cells, PAE D and PAE F, were tested for their sensitivities to human serum 1. Virus harvested from HeLa cells was used as a positive control. (B) VSV harvested from ST-IOWA Gal^+/+ (IOWA-Gal+/VSV) or ST-IOWA Gal^−/− (IOWA/Gal-/VSV) cells was tested for sensitivity to human complement using serum 1. HeLa/VSV virus was used as a positive control. TCID₅₀ titers are expressed as a mean percentage relative to titers achieved using heat-inactivated serum from duplicated experiments.