IgY antibodies against Ebola virus possess post-exposure protection and excellent thermostability

Ebola virus (EBOV) is the most virulent pathogens that cause hemorrhagic fever with high mortality rates in humans and nonhuman primates. The postexposure antibody therapies to prevent EBOV infection are considered efficient. However, due to the poor thermal stability of mammalian antibody, their application in the tropics has been limited. Here, we developed a thermostable therapeutic antibody against EBOV based on chicken immunoglobulin Y (IgY). The IgY antibodies demonstrated excellent thermal stability, which retained their neutralizing activity at 25°C for one year, in contrast to conventional polyclonal or monoclonal antibodies (MAbs). We immunized laying hens with a variety of EBOV vaccine candidates and confirmed that VSV Δ G/EBOVGP encoding the EBOV glycoprotein could induce high titer neutralizing antibodies against EBOV. The therapeutic efficacy of immune IgY antibodies in vivo was evaluated in the newborn Balb/c mice model. Lethal dose of virus challenged mice were treated 2 or 24 h post-infection with different doses of anti-EBOV IgY. The group receiving a high dose of 106 NAU/kg (neutralizing antibody units/kilogram) achieved complete protection with no signs of disease, while the low-dose group was only partially protected. In contrast, all mice receiving naïve IgY died within 10 days. In conclusion, the anti-EBOV IgY exhibits excellent thermostability and protective efficacy, and it is very promising to be developed as alternative therapeutic entities. Author Summary Although several Ebola virus therapeutic antibodies have been reported in recent years, however, due to the poor thermal stability of mammalian antibody, their application in tropical endemic areas has been limited. We developed a highly thermostable therapeutic antibody against EBOV based on chicken immunoglobulin Y (IgY). The IgY antibodies demonstrated excellent thermal stability, which retained their neutralizing activity at 25°C for one year. The newborn mice receiving passive transfer of IgY achieved complete protection against a lethal dose of virus challenge indicating that the anti-EBOV IgY provides a promising countermeasure to solve the current clinical application problems of Ebola antibody-based treatments in Africa.


Introduction 55
Ebola virus (EBOV) belongs to the Filoviridae family and the known cause of severe 56 hemorrhagic fever in humans and nonhuman primates (NHPs). Since the epidemic of Zaire

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The ongoing outbreak in the DRC is the second-largest Ebola epidemic on record, with 2 279 62 lives lost and 3 462 confirmed infections since August 2018, which prompted WHO to declare 63 this epidemic a public health emergency of international concern. Pandemic potential, high 64 mortality, high infectivity, and lack of preventive and therapeutic approaches make EBOV a 65 Class A pathogen that seriously threatens public health.

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The intermittent and continuous outbreak of Ebola disease (EVD) poses a challenge for lethal challenge in newborn mice. Our results suggest that the potent IgY warrant further 110 development as prophylactic and therapeutic reagents for EVD.

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Preparation of immunogens 113 Vaccine-elicited neutralizing antibodies (NAbs) are associated with protection against 114 Filoviridae family mediated disease. In order to obtain the most potent anti-EBOV antibody,

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Western blot confirmed that these immunogens could express or contain EBOV GP that can 119 induce NAbs in animals (Fig 1). Due to the differences in humoral immune responses induced 120 by different vaccines, we need to screen for the most suitable immunogen for IgY antibody 121 production.

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Thirty-five laying hens were randomly divided into seven groups, which were immunized four 127 times with each immunogen or PBS control intramuscularly (i.m.) at a 14-day interval. Eggs

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were collected at 0, 2, 4, 6, 8 weeks, and IgY antibodies were purified from egg yolk for ELISA 129 and NAbs test. Both titers in all groups were gradually increased after the first immunization.

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Passive transfer of IgY protect newborn BALB/c mice from lethal challenge 153 To determine whether the anti-EBOV IgY antibodies are protective against EBOV, passive 154 protection experiment was performed in newborn BALB/c mice (within the first 3 days of life).

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Forty newborn BALB/c mice were divided into eight groups, which were challenged 156 subcutaneously (s.c.) with 10 4 TCID50 VSVΔG/EBOVGP. Two hours or 1 day post-infection 157 (dpi), each mouse was adoptively transferred with IgY twice daily for 3 days, and control group 158 mice treated with naive IgY (Fig 4a). To determine the correlation between the transferred IgY 159 dosage and therapeutic efficacy, three different dosages with 10 4 , 10 5 , or 10 6 NAU/kg (NAb  year. However, the antibody titer stored at 37℃ gradually decreased from the second month,

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and only about 20% of the antibody activity remained by the end. In contrast, the activity of the 195 IgY stored at 45℃ is lost faster, and the NAbs titer cannot be measured by the third month.

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These results proved that the anti-EBOV IgY has excellent thermal stability, can be stored at 197 room temperature (RT) for up to one year, and can maintain one month of activity at 37℃ 198 without significant changes. Even at a high temperature of 45℃, it still can short-term retention 199 of activity (Fig 6). It is suggested that this anti-EBOV IgY can be used as an emergency

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Purification of yolk IgY antibody 367 IgY was isolated from the egg yolk using the water dilution method, a rapid and simple method 368 was used to separate IgY from the yolk. The separation method is improved based on previous

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For Western blot analysis, the proteins were electrically transferred onto a polyvinylidene 389 difluoride (PVDF) membrane using a semi-dry blotting apparatus (15V, 40 min, RT), then 390 blocked with Tris-buffered saline containing 0.05% Tween 20 (TBS-T) and 5% non-fat dry 391 milk for 1 h at RT and was incubated overnight at 4 °C with a 1:5000 dilution of mouse anti-