Anti-EBOV GP IgGs Lacking α1-3-Galactose and Neu5Gc Prolong Survival and Decrease Blood Viral Load in EBOV-Infected Guinea Pigs

Abstract

Polyclonal xenogenic IgGs, although having been used in the prevention and cure of severe infectious diseases, are highly immunogenic, which may restrict their usage in new applications such as Ebola hemorrhagic fever. IgG glycans display powerful xenogeneic antigens in humans, for example α1-3 Galactose and the glycolyl form of neuraminic acid Neu5Gc, and IgGs deprived of these key sugar epitopes may represent an advantage for passive immunotherapy. In this paper, we explored whether low immunogenicity IgGs had a protective effect on a guinea pig model of Ebola virus (EBOV) infection. For this purpose, a double knock-out pig lacking α1-3 Galactose and Neu5Gc was immunized against virus-like particles displaying surface EBOV glycoprotein GP. Following purification from serum, hyper-immune polyclonal IgGs were obtained, exhibiting an anti-EBOV GP titer of 1:100,000 and a virus neutralizing titer of 1:100. Guinea pigs were injected intramuscularly with purified IgGs on day 0 and day 3 post-EBOV infection. Compared to control animals treated with IgGs from non-immunized double KO pigs, the anti-EBOV IgGs-treated animals exhibited a significantly prolonged survival and a decreased virus load in blood on day 3. The data obtained indicated that IgGs lacking α1-3 Galactose and Neu5Gc, two highly immunogenic epitopes in humans, have a protective effect upon EBOV infection.

Fig 1. Effect of the immunization of double KO pig with EBOV VLPs.

Serum samples obtained from an immunized double KO pig before (day 0), during (day 15, 30 and 57) or after immunizations (day 83) were analyzed using ELISA for specific anti-GP antibodies. The mean OD at each time point for each dilution is represented. Anti-EBOV GP titers of the latest collected pig sera was about 1:100,000.

Fig 2. Monitoring of the weight of guinea pigs treated with anti-EBOV IgGs.

Each curve represents one group of 5 guinea pigs. The standard deviation is represented for each group at each time point. * p<0.05 when comparing all guinea pigs having received anti-EBOV IgGs to animals having received IgGs from a non-immunized pig, using a repeated measures two-way ANOVA test.

Fig 3. Kaplan Meier survival curves of guinea pigs after EBOV infection, according to treatment with anti-EBOV IgGs.

A: Survival of guinea pigs having received the anti-EBOV IgGs (n = 10) compared to guinea pigs receiving the non-immune DKO IgGs (n = 5, *p = 0.0424, using a Log Rank test). B: Survival of guinea pigs having received one (DO) or two (D0 and D3) doses of polyclonal anti-EBOV IgGs.

Fig 4. Viral loads in serum at day 3 post-EBOV infection.

A: Levels of circulating EBOV transcripts at day 3 post infection in the serum of each animal was evaluated by RT-qPCR using primers targeting EBOV polymerase gene. The horizontal bars represent the median values. The median D3 virus load was 839,333 following the injection of non-immune IgGs from a DKO pig and 259 following the injection of anti-EBOV antibodies (p = 0.055, using a Mann-Whitney test). The limit of detection of the test was of 180 relative genome copies/ml, and all values under this threshold were considered as negative data (as represented on the graph). B: Correlation between viral load on day 3 and guinea pig survival. Kendall's rank correlation coefficient showed a significant negative correlation (p = 0.0003) between EBOV viral load at day 3 and survival following infection, when considering all pooled data. Non-treated animal’s values are displayed as circles whereas treated animal’s values are displayed as squares.