Evaluation of Rabies Biologics against Irkut Virus Isolated in China

Ye Liu, Qi Chen, Fei Zhang, Shoufeng Zhang, Nan Li, Hai Lian, Ying Wang, Jinxia Zhang, Rongliang Hu

Laboratory of Epidemiology and Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Military Veterinary Research Institute, Academy of Military MedicalSciences, Changchun, China

An Irkut virus (IRKV) was recently isolated from a bat in China. The protective ability of rabies biologics available in the Chi-nese market and experimental biologics against the rabies virus (RABV) and IRKV were assessed in a hamster model via preex-posure prophylaxis (PrEP) and postexposure prophylaxis (PEP) experiments. The results demonstrated that a single dose of ra-bies vaccine did not induce adequate protection against IRKV infection. However, routine PrEP with three doses of vaccineinduced complete protection against IRKV infection. Higher doses of RABV immunoglobulins and alpha interferon were re-quired during PEP to protect hamsters against IRKV versus RABV infection. Experimental recombinant vaccines containingIRKV glycoproteins induced more-reliable protection against IRKV than against RABV infection. Those findings may be ex-plained by limited cross-neutralization of these viruses (confirmed via in vitro tests) in conjunction with antigenic distances be-tween RABV and IRKV. These results indicate that the development and evaluation of new biologics for PrEP and PEP are re-quired to ensure sufficient protection against IRKV infection in China and other territories where this virus is present.

Rabies is an acute progressive encephalomyelitis caused by neg-ative-sense single-stranded RNA viruses from the genus Lys-

savirus, family Rhabdoviridae. At present, the genus includes 12established viral species and 3 viruses awaiting taxonomic assess-ment (1–4). Of these, classic rabies virus (RABV) is the mostbroadly distributed, causing �99% of human lyssavirus casesworldwide (1). In China, RABV causes 2,000 to 3,000 reportedhuman deaths per year. Almost all Chinese lyssavirus isolates be-long to RABV species originating from carnivores (5). However,several human rabies cases of bat origin, in which the viruses werenot identified, were reported (6). In 2012, for the first time, alyssavirus was isolated from a greater tube-nosed bat (Murina leu-cogaster) in the Jilin Province of China. It was identified as Irkutvirus (IRKV) (isolate IRKV-THChina12) (6). Phylogenetic anal-ysis demonstrated that structural proteins of this virus shared highsequence identity (�98%) with IRKV isolate Ozernoe, from ahuman case of rabies following a bat bite in Russia in 2007 (7).Several studies have investigated the serological cross-reactivity oflyssaviruses and the protective ability of commercially availableRABV biologics against non-RABV lyssaviruses. In general, it hasbeen shown that rabies biologics provide sufficient protectionagainst lyssaviruses from phylogroup I (8). However, a more re-cent study demonstrated that rabies biologics available in theUnited States elicited only partial protection against several non-RABV lyssaviruses, including IRKV (9). The need existed, there-fore, to further assess the serological cross-reactivity betweenRABV and IRKV and to evaluate whether rabies biologics avail-able in China and elsewhere provide reliable protection againstIRKV infection. In this study, we conducted a series of preexpo-sure prophylaxis (PrEP) and postexposure prophylaxis (PEP) ex-periments in an animal model, to evaluate the protective effects ofseveral commercially available and experimental biologics againstIRKV infection.

MATERIALS AND METHODSSequence comparisons of viral glycoproteins. Multiple alignments oflyssavirus glycoprotein sequences were performed using the DNASTARLasergene program (DNASTAR, Madison, WI) and the BioEdit program(10).

Viruses and cells. IRKV isolate IRKV-THChina12, which was ob-tained in 2012 from a Murina leucogaster bat in Tonghua county, JilinProvince (6), was amplified in a single intracerebral mouse passage. RABVisolate BD06, which was obtained in 2006 from a rabid dog in China, wasmaintained in dog brains via serial passages (11). The titers of IRKV-THChina12 and BD06 suspensions used in the study in the Syrian ham-ster model were 103.0 to 103.5 times the intramuscular (i.m.) 50% lethaldose (LD50)/ml. One hundred times the hamster LD50 (the lowest intra-muscular 100% lethal dose) of IRKV-THChina12 and BD06 was used forchallenge. RABV strain CVS-11 was grown in BHK-21 cells and used inthe virus neutralization tests described below. BHK-21 and HEK-293 cellswere maintained at 37°C in Dulbecco’s minimum essential medium(DMEM) supplemented with 2% newborn calf serum, 100 U/ml penicil-lin G, and 100 �g/ml streptomycin sulfate, in a 5% CO2 humidified incu-bator.

Animals. Two-month-old female adult Syrian hamsters weighing ap-proximately 100 g were obtained from the Changchun Institute of Biolog-ical Products (China) and were randomly divided into 36 groups of 10hamsters (groups 1 to 36) (Table 1). Following challenge, the hamsterswere observed for 28 days. Animals that showed clinical signs of rabies andanimals that survived 28 days of observation were euthanized by CO2

intoxication. Brains were removed and tested by direct fluorescent anti-body (DFA) testing for the presence of RABV or IRKV antigens (12). Allanimal experiments described in this paper were conducted according tothe Guideline on Humane Treatment of Laboratory Animals, stipulatedby the Ministry of Science and Technology (MOST) of the People’s Re-public of China (13), and were approved by the Animal Welfare Commit-tee of the Military Veterinary Research Institute (Changchun, China).

Rabies biologics. The following commercial rabies biologics wereused in this study: a locally produced Vero cell vaccine for human use(strain PV2061, Chengda Suda, 0.5 ml/dose, lot 201209276; LiaoningChengda Biotechnology Co.), an imported veterinary vaccine (strain Pas-teur RIV, Nobivac Rabies, 1 ml/dose, lot A154A01; Intervet Interna-

Received 19 June 2013 Returned for modification 30 July 2013Accepted 7 August 2013

Published ahead of print 14 August 2013

Address correspondence to Rongliang Hu, ronglianghu@hotmail.com.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JCM.01565-13

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tional), a commercially available human RABV immunoglobulin (HRIG)(Wusheng, 20 IU/kg, lot 20111101; Wuhan Institute of Biological Prod-ucts Co.), and recombinant human alpha interferon 2a (IFN-�2a)(5,000,000 IU/ml, lot 20110443; Changchun Institute of Biological Prod-ucts Co.).

Construction of recombinant human adenovirus type 5 expressingRABV and IRKV glycoproteins. To compare the cross-immunogenicityof the viral glycoproteins of IRKV and RABV, the recombinant productsrecombinant human adenovirus type 5 (rHAd5)-THChina12-G andrHAd5-BD06-G were generated using an E1- and E3-deleted cytomega-lovirus (CMV) adenoviral expression system (RAPAd; Cell Biolabs, Inc.,CA) (14). In a typical procedure, BD06 and IRKV-THChina12 glycopro-tein genes were inserted into multiple cloning sites of the shuttle vectorpacCMVK-NpA. Then, HEK-293 cells were cotransfected with HAd5backbone vector pacAd5 9.2–100 (devoid of the left inverted terminalrepeat [ITR] and the packaging signal) using Lipofectamine (Invitrogen,CA) and were passaged until a typical cytopathic effect was observed(without the need to perform multiple plaque isolations). Expression of

the lyssavirus glycoproteins in the transfected HEK-293 cells was deter-mined using Light Diagnostics rabies FITC-globulin conjugate (EMDMillipore Corp., MA) (6).

Evaluation of the efficacy of rabies biologics against RABV andIRKV infection in animal models. For preexposure prophylaxis (PrEP)experiments (http://www.who.int/rabies/human/WHO_strategy_prepost_exposure/en/index.html), 14 groups (groups 1 to 14) received intramuscu-lar injections (in the gastrocnemius muscle) of 50 �l of commercial human orveterinary vaccines or experimental recombinant vaccines (rHAd5-TH12-Gand rHAd5-BD06-G) (Table 1). Negative controls (groups 15 and 16) re-ceived only phosphate-buffered saline (PBS) (Table 1). Four weeks after thelast vaccination, the hamsters were challenged i.m. with BD06 or IRKV-THChina12 (100 times the hamster i.m. LD50) (Table 1). Each hamster wasimmobilized and a blood sample was taken from the retro-orbital plexusbefore challenge. Serum samples were collected by centrifugation at 5,000 � gfor 10 min and were subjected to fluorescent antibody virus neutralization(FAVN) testing based on RABV strain CVS-11 (15).

In the postexposure prophylaxis (PEP) experiments, 20 groups

TABLE 1 Design of experiments on preexposure and postexposure prophylaxis in a hamster model

Treatment andgroup no. Rabies biologic(s) Inoculation amount/dose Challenge virus

PrEP1 Human vaccine 1 dose BD062 Human vaccine 1 dose IRKV-THChina123 Human vaccine 3 dosesa BD064 Human vaccine 3 dosesa IRKV-THChina125 Veterinary vaccine 1 dose BD066 Veterinary vaccine 1 dose IRKV-THChina127 rHAd5-TH12-G 1 dose BD068 rHAd5-TH12-G 1 dose IRKV-THChina129 rHAd5-TH12-G 3 dosesa BD0610 rHAd5-TH12-G 3 dosesa IRKV-THChina1211 rHAd5-BD06-G 1 dose BD0612 rHAd5-BD06-G 1 dose IRKV-THChina1213 rHAd5-BD06-G 3 dosesa BD0614 rHAd5-BD06-G 3 dosesa IRKV-THChina1215 Negative control (PBS) 1 dose BD0616 Negative control (PBS) 1 dose IRKV-THChina12

PEP17 HRIG 20 IU/kg BD0618 HRIG 20 IU/kg IRKV-THChina1219 HRIG 200 IU/kg BD0620 HRIG 200 IU/kg IRKV-THChina1221 Human vaccine 4 dosesb BD0622 Human vaccine 4 dosesb IRKV-THChina1223 HRIG � human vaccine 200 IU/kg � 4 dosesb BD0624 HRIG � human vaccine 200 IU/kg � 4 dosesb IRKV-THChina1225 HRIG � rHAd5-TH12-G 200 IU/kg � 4 dosesb BD0626 HRIG � rHAd5-TH12-G 200 IU/kg � 4 dosesb IRKV-THChina1227 HRIG � rHAd5-BD06-G 200 IU/kg � 4 dosesb BD0628 HRIG � rHAd5-BD06-G 200 IU/kg � 4 dosesb IRKV-THChina1229 IFN-�2a 3 dosesc BD0630 IFN-�2a 3 dosesc IRKV-THChina1231 HRIG � IFN-�2a 200 IU/kg � 3 dosesc BD0632 HRIG � IFN-�2a 200 IU/kg � 3 dosesc IRKV-THChina1233 Human vaccine � IFN-�2a 4 dosesb � 3 dosesc BD0634 Human vaccine � IFN-�2a 4 dosesb � 3 dosesc IRKV-THChina1235 Negative control (PBS) 1 dose BD0636 Negative control (PBS) 1 dose IRKV-THChina12

a Hamsters were inoculated with a priming dose, followed by boosters 7 and 28 days later (50 �l/hamster), before exposure.b Hamsters were inoculated with vaccine on days 0 (100 �l/hamster), 7 (50 �l/hamster), and 21 (50 �l/hamster) after exposure.c Hamsters were injected with IFN-�2a on days 0, 1, and 2 (100,000 IU/hamster) after exposure.

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(groups 17 to 36) were challenged in the gastrocnemius muscle in the lefthind leg with BD06 or IRKV-THChina12 (100 times the hamster LD50)(Table 1). Four hours after challenge, HRIG (20 or 200 IU/kg) was admin-istered i.m. at the site of virus challenge (Table 1). The human vaccine wasadministered in the gastrocnemius muscle in the right hind leg, via the2-1-1 regimen, on days 0 (4 hours after challenge, in the triceps brachiimuscle in the right foreleg and the gastrocnemius muscle), 7, and 21(Table 1) (http://www.who.int/rabies/human/WHO_strategy_prepost_exposure/en/index.html). IFN-�2a was administered in the tricepsbrachii muscle in the left foreleg on days 0 (4 hours after challenge), 1, and2 (Table 1). Negative controls (groups 35 and 36) received PBS postchal-lenge (Table 1). On day 3 after challenge, a serum sample was taken fromeach hamster using the same method as described above, for FAVN testingbased on RABV strain CVS-11 (15).

Statistical analyses. The 95% confidence intervals for virus titers in-dicated in the text were calculated by the Neoprobit method (16). Fisher’sexact test was used to compare the survival rates for animals in the groupschallenged with different viruses and subjected to different prophylactictreatments. Variance analyses were performed to determine statisticallysignificant differences in antibody titers by one-way analysis of variance(ANOVA). The analyses were performed using the SPSS 16.0 package(SPSS Inc., Chicago, IL). The results of the comparisons between groupswere considered significant at P � 0.05.

RESULTSGlycoprotein sequence comparisons. Comparison of the de-duced amino acid (aa) sequences revealed that the ectodomain ofRABV contains N-linked glycosylation sites at positions 37, 158(PV2061 and Flury-LEP), 247, and 319, with two at positions 247and 319 being present in IRKV (Fig. 1). Comparison of majorantigenic sites (17–19) demonstrated that antigenic sites I (aa 226to 231), II (aa 34 to 42 and 198 to 200), III (aa 330 to 338), and G1(aa 242 to 243) in the ectodomain were different in IRKV than inRABV; however, antigenic sites IV (aa 251), G5 (aa 261 to 264),and VI (aa 264) and a B-cell epitope (aa 14 to 19) were fullyconserved (Fig. 1).

Protective effects of rabies biologics against IRKV infection.In the experiment with one-dose vaccination, all hamsters (excepttwo immunized with rHAd5-TH12-G) survived the challengewith RABV; however, only 80% of the hamsters survived the chal-lenge with IRKV (Table 2). In the conventional PrEP experiment,all hamsters immunized with 3 doses of human rabies vaccinessurvived challenges with RABV and IRKV (Table 2). All hamstersdeveloped significant titers of virus-neutralizing antibodies(VNAs) in the serum, and the VNA titers of serum samples fromhamsters immunized with 3 doses of vaccine were greater thanthose of hamsters immunized with one dose of vaccine.

The recombinant vaccines rHAd5-TH12-G and rHAd5-BD06-G were less efficient. No survival rates of 100% wereachieved with one-dose vaccination, although these vaccines pro-tected animals better against infection from a homologous virus(80%) than against a heterologous virus (20%) (P � 0.05). How-ever, all hamsters immunized with 3 doses of recombinant vac-cines survived challenges with RABV and IRKV (Table 2). InFAVN testing, virus neutralization titers of serum samples fromhamsters immunized with 3 doses of rHAd5-BD06-G were about27 times higher than those of hamsters immunized with 3 doses ofrHAd5-TH12-G (Table 2).

In the PEP experiment (Table 3), 20 IU/kg of HRIG injected atthe challenge site did not protect hamsters against IRKV infection,whereas 80% of hamsters survived RABV challenge. After treat-ment with 200 IU/kg of HRIG, the protection against IRKV infec-

tion increased to 60% (P � 0.05). The difference in VNA titersbetween groups that received 20 IU/kg and 200 IU/kg of HRIG(0.45 � 0.21 and 3.50 � 0.95 IU/ml, respectively) was statisticallysignificant (P � 0.05). Furthermore, the combination of HRIGand IFN-�2a improved the survival rate (P � 0.05). The additionof human vaccines to prophylactic regimens decreased VNA titersand survival rates for animals in the groups challenged with IRKV(P � 0.05); however, the addition of recombinant vaccines did notsignificantly decrease VNA titers or survival rates. Incubation pe-riods for hamsters challenged with RABV were 8 to 13 days andthose for hamsters challenged with IRKV were 6 to 13 days. Fol-lowing the incubation periods, rabies-like symptoms, such as leth-argy, muscle weakness, and progressive paralysis of one or bothhind limbs, were observed.

DISCUSSION

For the first time, an IRKV was recently isolated from a bat inChina through the active rabies surveillance program. AlthoughChinese IRKV belongs to the same phylogroup, 1, as the widelydistributed RABV, it is more closely related phylogenetically toEuropean bat lyssavirus type 1 and Duvenhage virus than to RABV(6). Phylogenetic relationships and genetic distances between lys-saviruses partially reflect the extent of serological cross-reactivity.For example, it was suggested that 72 to 74% amino acid sequenceidentity within glycoprotein ectodomains provides sufficientcross-neutralization between lyssaviruses (8). The same can beinferred for the protection elicited by rabies biologics (which allare based on several well-characterized RABV strains) againstnon-RABV lyssaviruses. Commercial rabies biologics have beenfully efficacious against a phylogenetically related Australian batlyssavirus (20). However, the protection elicited by several rabiesbiologics against IRKV in animal models was limited (9).

Additional attention should be paid to conservation of anti-genic sites on lyssavirus glycoproteins. This is particularly impor-tant as several virus-neutralizing monoclonal antibodies (MAbs)were offered for replacement of conventional HRIG in humanrabies PEP (21–28). For example, a linear epitope with the keyresidues LCGV within antigenic site I serves for binding of MAbsCR57 and 62071-3 (22, 23). Similarly, conserved conformationalantigenic sites II and III contain binding epitopes for several otherMAbs (24–28). Amino acid differences in these epitopes may alterMAb binding and thus abolish neutralization of non-RABV lyssa-viruses, including IRKV. Therefore, MAbs selected for antibodycocktails for use in PEP must be scrutinized for their ability toneutralize non-RABV lyssaviruses.

In this study, the efficacy of rabies biologics available in Chinaagainst IRKV was determined in routine and modified PrEP andPEP experiments, as described previously (9). Although a singledose of rabies vaccine did not induce adequate protection againstIRKV infection, routine PrEP (three vaccine doses, on days 0, 7,and 28) induced strong protection against RABV and IRKV infec-tion.

In the PEP experiments, however, only very high doses ofRABV immunoglobulins conferred partial protection of animalsagainst IRKV infection, whereas the routine PEP regimen (20IU/kg body weight of HRIG injected only once, followed by the2-1-1 vaccine regimen) did not protect animals against IRKV in-fection at all. Moreover, combination of HRIG with vaccines de-creased the VNA titers and survival rates of hamsters, comparedwith the groups that were given HRIG only, likely because of in-

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FIG 1 Comparative amino acid sequence alignment of glycoprotein ectodomains of IRKV and RABV. Numbers, amino acid positions. Boxes, antigenic sites, i.e.,site I (aa 226 to 231), site II (aa 34 to 42 and 198 to 200), site III (aa 330 to 338), site IV (aa 251), site G1 (aa 242 to 243), site G5 (aa 261 to 264), and site VI (aa264) and a novel B-cell epitope (aa 14 to 19). Arrows, four putative N-glycosylation sites.

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terference between the vaccines and the immunoglobulins. Shortincubation periods in a hamster model may not provide sufficienttime for the development of active immune responses, with pro-tection against infection being based solely on antibodies deliv-ered passively. These antibodies may fail to protect animalsagainst IRKV infection based on limited cross-neutralization ac-tivity (Tables 2 and 3). It is possible that PEP models with longerincubation periods or situations involving real-life exposureswould allow sufficient time for the development of active immuneresponses to vaccination and would protect animals and humansagainst IRKV infection, as may be inferred from our PrEP exper-iments. However, short incubation periods may occur after real-life exposures as well (29), and this increases the demand for thedevelopment of novel immunoglobulin preparations (includingMAbs) for protection against IRKV and other non-RABV lyssavi-ruses.

Interferon can inhibit rabies virus activity in vivo and does notinterfere with circulating VNAs (30). This was confirmed in ourPEP experiment. Perhaps addition of interferon to PEP regimenswould increase the protection of humans against non-RABV lys-saviruses, although this must be further evaluated in higher-mam-malian models and clinical trials.

A replication-deficient vector derived from human adenovirustype 5 was chosen for the cross-immunogenicity experiments,since it has been well studied for expression of RABV proteins andhas been evaluated in animal models (31). In our experiments, arecombinant vaccine expressing IRKV glycoproteins providedsignificantly better results in animals challenged with IRKV thandid a recombinant vaccine expressing RABV glycoproteins. How-ever, it did not provide reliable protection against RABV chal-lenge. Chimeric lyssavirus glycoproteins from partial RABV andIRKV genes should be considered, to achieve protection againstthese two viruses (32, 33).

Surveillance for non-RABV lyssaviruses in China is very lim-ited, as is diagnostic capacity across the country. The threat ofIRKV to animals and humans is difficult to estimate. Although atleast two bat-associated human rabies cases in China have beenreported (6), IRKV was not identified in those cases, as the diag-noses were based on clinical signs and exposure history only.

In conclusion, commercially available rabies biologics do notprovide protection against IRKV infection as reliable as that

against RABV infection. Before the development of new biologics(e.g., chimeric vaccines and MAbs), the combination of higherdoses of RABV immunoglobulins with interferon may be effectivetreatment for exposure to IRKV and other non-RABV lyssavi-ruses. In addition, active field surveys should be performed toinvestigate more thoroughly the prevalence and circulation pat-terns of IRKV in China, to assess properly the public health andveterinary implications.

ACKNOWLEDGMENTS

This project was funded by the Key Project (grant 30630049) and theYouth Program (grants 30900058 and 31001070) of the National NaturalScience Foundation of China and the National 973 Project (grant2011CB500705).

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TABLE 2 Protection against RABV and IRKV elicited in hamsters byvaccines via preexposure prophylaxis

Groups

Prechallenge VNAtiter (mean � SD)(IU/ml)a

No. of surviving hamsters/no.challenged with:

RABV BD06 IRKV-THChina12

1 and 2 7.47 � 2.06 10/10b 8/10b

3 and 4 94.07 � 21.06 10/10 10/105 and 6 6.02 � 3.63 10/10 8/107 and 8 0 2/10c 8/10c

9 and 10 1.17 � 0.32 10/10 10/1011 and 12 2.51 � 2.43 8/10 2/1013 and 14 27.22 � 11.84 10/10 10/1015 and 16 0 0/10 0/10a VNA titers were determined in FAVN tests against the CVS-11 RABV strain on theday of challenge.b P � 0.05.c P � 0.05.

TABLE 3 Protection against RABV and IRKV elicited in hamsters inpostexposure prophylaxis experiments

Groups

VNA titer(mean � SD)(IU/ml)a

No. of surviving hamsters/no.challenged with:

RABV BD06 IRKV-THChina12

17 and 18 0.45 � 0.21 8/10b 0/10b

19 and 20 3.50 � 0.95 10/10c 6/10c

21 and 22 0 0/10 0/1023 and 24 1.10 � 0.42 10/10 2/1025 and 26 2.39 � 1.15 10/10 6/1027 and 28 1.90 � 1.32 10/10b 4/10b

29 and 30 0 4/10 4/1031 and 32 3.23 � 1.09 10/10 8/1033 and 34 0 3/10 3/1035 and 36 0 0/10 0/10a Antibody titers were determined in FAVN tests against the CVS-11 RABV strain onday 3 after challenge with the viruses.b P � 0.05.c P � 0.05.

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