Experimental Infection of Culex Annulirostris, Culex Gelidus, and Aedes Vigilax with a Yellow Fever/Japanese Encephalitis Virus Vaccine Chimera (Chimerivax™-Je)

Home , Culex annulirostris, Culex gelidus, Weary Dunlop

EXPERIMENTAL INFECTION OF CULEX ANNULIROSTRIS, CULEX GELIDUS, AND AEDES VIGILAX WITH A YELLOW FEVER/JAPANESE ENCEPHALITIS VIRUS VACCINE CHIMERA (CHIMERIVAX™-JE)

MARK REID,* DONNA MACKENZIE, ANDREW BARON, NATALIE LEHMANN, KYM LOWRY, JOHN AASKOV, FARSHAD GUIRAKHOO, AND THOMAS P. MONATH Australian Army Malaria Institute, Brisbane, Queensland, Australia; School of Life Sciences, Queensland University of Technology, Brisbane, Queensland, Australia; Acambis Inc., Cambridge, Massachusetts

Abstract. Australian mosquitoes from which Japanese encephalitis virus (JEV) has been recovered (Culex annulirostris, Culex gelidus, and Aedes vigilax) were assessed for their ability to be infected with the ChimeriVax™-JE vaccine, with yellow fever vaccine virus 17D (YF 17D) from which the backbone of ChimeriVax™-JE vaccine is derived

and with JEV-Nakayama. None of the mosquitoes became infected after being fed orally with 6.1 log10 plaque-forming units (PFU)/mL of ChimeriVax™-JE vaccine, which is greater than the peak viremia in vaccinees (mean peak vire- days). Some members of all three 11–0 ס PFU/mL of 0.9 days mean duration, range 30–0 ס PFU/mL, range 4.8 ס mia species of mosquito became infected when fed on JEV-Nakayama, but only Ae. vigilax was infected when fed on YF 17D. The results suggest that none of these three species of mosquito are likely to set up secondary cycles of transmission of ChimeriVax™-JE in Australia after feeding on a viremic vaccinee.

INTRODUCTION tralia,7–9 and all three species of mosquito have been infected experimentally by membrane feeding with JEV isolates ob- Japanese encephalitis virus (JEV) is a member of the fam- tained from the Torres Strait of Australia.10 ily Flaviviridae and is a leading cause of viral encephalitis in Asia. Case fatality rates of 29% have been reported with an MATERIALS AND METHODS additional 33–50% of survivors developing long-term neuro- logic sequalae.1 ChimeriVax™-JE is a live, attenuated, vac- Mosquitoes. Culex gelidus larvae were collected from Ten- cine for JEV in which the premembrane (prM) and envelope nant Creek (Northern Territory, Australia) by Peter Whelan (E) proteins genes of the yellow fever virus strain 17D (YF (Northern Territory Health Services). Culex annulirostris 17D) have been replaced with the vaccine strain JEV SA14- eggs were provided by the Queensland Institute of Medical 14-2 prM-E sequence.2 Persons inoculated subcutaneously Research. Aedes vigilax eggs were supplied by Stephen Dog- with ChimeriVax™-JE develop a self-limiting, very low–titer gett (University of Sydney and Westmead Hospital). Colonies viremia that poses a theoretical risk of secondary transmission of each species were established at the Army Malaria Insti- by a mosquito vector. In a recent study (Acambis Inc., un- tute in an insectary maintained at 24–28°C and a relative published data), the viremia in humans after immunization humidity of 40–60%, with a 12:12 light:dark photoperiod. To with 3.0–5.0 log10 plaque-forming units (PFU) of Chi- maintain the colony, the larvae of each species were reared in meriVax™-JE developed a mean peak viremia of 4.8 PFU/ dam water on a diet of commercial fish food (Kyorin, Himeji, PFU/mL of 0.9 days mean duration, range Japan). For egg development, adults were offered a blood 30–0 ס mL (range days). The level of viremia was lower than reported meal 3–5 times a week. All adult mosquitoes were maintained 11–0 ס for yellow fever 17D vaccine (YF-VAX௡) with a mean peak on a 20% (v/v) sucrose/water solution containing multivita- -PFU/mL of mean mins (Myadec multivitamins and minerals; Nelson Laborato 80–0 ס viremia of 21.8 PFU/mL (range duration 1.2 days, range 0–3 days).3,4 The low levels of vire- ries, Warriewood, New South Wales, Australia) ad libitum. mia in humans after vaccination with ChimeriVax™-JE and Mosquito colonies were established from larvae but to limit YF 17D vaccines is a barrier to oral infection of hematopha- the possibility of adventitious infection of the colonies, 60 gous vectors. In addition, it has been known for many years adults from each of the three colonies were screened for the that YF 17D vaccine virus has lost its ability to be transmitted presence of arboviruses by adding mosquito homogenate to by the yellow fever vector Aedes aegypti.5 In previous vector 2-cm2 cultures of baby hamster kidney 21 (BHK-21) clone c15 competence studies of ChimeriVax™-JE, Ae. aegypti, Ae. al- cells11 and observing daily for 7–10 days for cytopathic effects bopictus, and Culex tritaeniorhynchus mosquitoes did not be- (CPEs). come infected after oral feeding on blood-virus suspensions Viruses. Yellow fever 17D vaccine (lot no. W6440-1 Stama- 6 ௡ containing 6.9 log10 PFU/mL. ril ; Sanofi-Pasteur, Lyon, France) was passaged once in The objective of the present study was to assess the poten- Vero (African green monkey) cells12 (European Type Cul- tial of mosquito species Cx. annulirostris, Cx. gelidus, and Ae. ture Collection lot no. CB2617). Supernatant fluid was har- vigilax to become infected with ChimeriVax™-JE after in- vested when cell monolayers showed Ն 75% CPE and stored gesting a virus laden blood meal or after intrathoracic (IT) at Յ −60°C in 20% (v/v) heat-inactivated (56°C for 30 min- inoculation. Japanese encephalitis virus genotypes I or II utes) fetal bovine serum (FBS; Gibco-Invitrogen, Carlsbad, have been isolated from these three mosquito species in Aus- CA). Japanese encephalitis virus (Nakayama strain) was obtained from Queensland Health Scientific and Pathology Ser- vices (Townsville, Queensland, Australia) and was passaged * Address correspondence to Mark Reid, Australian Army Malaria in Vero cells in the same manner as YF 17D. ChimeriVax™- Institute, Weary Dunlop Drive, Gallipoli Barracks, Enoggera, JE was supplied lyophilized (Acambis Inc., Cambridge MA) Queensland 4051, Australia. E-mail: [email protected] and was reconstituted in sterile 0.9% saline (AstraZeneca, 659 660 REID AND OTHERS

Alderley Park, United Kingdom) before use. Titers of virus 1°C immediately prior to feeding. All mosquitoes were fed ס stocks were as follows: YF 17D 7.3 log10 PFU/mL (in por- over a time interval of 2 hours ± 20 minutes and fully en- gorged mosquitoes maintained for up to 18 days as described ס cine stable-equine kidney cells [PS-EK]),13 JEV-Nakayama

6.7 log10 PFU/mL (in BHK-21 c15 cells), and ChimeriVax™- for mosquitoes infected IT. The average blood meal for ס JE 6.1 log10 PFU/mL (in Vero cells). each species was estimated by weighing a sample of 20 mo- ס Virus titrations. Mosquitoes were killed with CO2 gas and squitoes pre-feeding and post-feeding (Cx. annulirostris 1.3 ± 1.7 ס ␮L, and Ae. vigilax 0.6 ± 1.9 ס triturated using plastic pestles in sterile micro-centrifuge ± 0.9 ␮L, Cx. gelidus tubes in 1 mL volumes of RPMI 1640 medium (Sigma- 1.5 ␮L). Aldrich, St. Louis, MO) containing 0.3g/L of sodium bicar- Stability of virus stocks. Experiments were conducted to bonate and supplemented with 20% (v/v) heat-inactivated quantify the reduction in virus titer for the virus/erythrocyte FBS (HI-FBS), 100␮g/mL of streptomycin, 100 U/mL of peni- suspensions and IT inocula for each virus at 37 ± 1°Cor0°C cillin, 2 mM L-glutamine (SPG) (Sigma-Aldrich), and 2.5 ␮g/ after a 2 hour ± 20 minute time interval. Five replicates were mL of amphotericin B (Gibco-Invitrogen). The homogenate assayed without freeze-thawing before and after the time in- was clarified by centrifugation (10,377 × g for 2 minutes) terval and the average titer was compared by a paired, one- -to determine whether sig (0.05 ס ␣) and the supernatant was stored at Յ −60°C until assayed. tailed Student’s t-test Virus titers were assayed using a modified plaque titra- nificant decreases in virus titer had occurred. tion method.13 Briefly, supernatants from the mosquito ho- Nucleotide sequencing. Culex annulirostris mosquito ho- mogenates were diluted 10-fold in duplicate 2% (v/v) HI-FBS mogenates 876, 877, and 878 obtained 12 days after IT inocu- RPMI 1640 medium/SPG and 100 ␮L was added to 2-cm2 cell lation with ChimeriVax™-JE were passaged once in Vero monolayers. Japanese encephalitis virus (Nakayama) and cells and titrated in BHK-21 c15 cells as previously described. ChimeriVax™-JE were titrated on BHK-21 c15 cells and YF Viral RNA was extracted from the supernate of infected cul- 17D on PS-EK cells (because YF 17D did not plaque consis- tures using the High Pure Viral RNA kit (Roche Diagnostics, tently on BHK-21 c15 cells). After 1 hour ± 10 minutes, 1.5% Mannheim, Germany) according to the manufacturer’s in- (w/v) carboxymethylcellulose (Sigma-Aldrich) with a final structions. Viral RNA was reverse transcribed with Moloney concentration of 2% (v/v) HI-FBS RPMI 1640 medium/SPG murine leukemia virus reverse transcriptase (Promega, Madi- was added to each culture and incubated at 37 ± 1°C for 3–5 son, WI) using random hexamers (Roche Diagnostics). The days in an atmosphere of 5% (v/v) CO2 in air. Monolayers region of the genome coding for capsid (C) and prM proteins were fixed and stained with 0.5% (w/v) CI basic violet in 5% was amplified by polymerase chain reaction (PCR) using Taq (v/v) formalin:phosphate buffered saline (Sigma-Aldrich) be- polymerase (Roche diagnostics) and primers previously de- fore plaques were counted. The limit of sensitivity of the assay scribed,7 as well as with JEV E protein–specific primers Ն Ј Ј was 1.7 log10 PFU/mosquito. To enable log transformations (JEV-19F: 5 -GGCAATCGTGACTTCATAGAAG-3 and of average plaque counts, cell monolayers with no detectable JE-591R: 5Ј-TCCACTCCTTGGCTCACAGTC-3Ј)ona plaques were given the nominal value of half the limit of thermocycler (Eppendorf, Hamburg, Germany). Cycling con- detection (0.85 log10 PFU). ditions were 94°C for 2 minutes followed by 35 cycles at 94°C Intra-thoracic inoculation of mosquitoes. Freshly thawed for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds. stocks of virus were maintained on ice for 2 hours ± 20 min- The PCR products were stained with ethidium bromide after utes. Female mosquitoes of the three species 1–4-days post- electrophoresis on a 2% (w/v) agarose Tris-borate-EDTA gel emergence, were immobilized with CO2 gas and manipulated (Sigma-Aldrich) and visualized with ultraviolet light. The on a laboratory cold plate (2 ± 1°C) (Thermoline Scientific, 380-base pair C/prM and 572-base pair envelope gene bands Sydney, New South Wales, Australia). Mosquitoes were in- were sequenced in both directions by dideoxynucleotide dye oculated IT with 0.15 ± 0.08 ␮L of virus stock using heat- termination (BigDye terminator cycle sequencing kits; Ap- drawn, 1-mm capillary tubes (Harvard Apparatus, Eden- plied Biosystems, Foster City, CA) ) using the oligonucleotide bridge, United Kingdom) as previously described (Narishige, primers previously mentioned in this report and an auto- Tokyo, Japan).14 After inoculation, mosquitoes were trans- mated sequencer (Applied Biosystems). ferred in batches of 20 to primary mesh-covered plastic cups Statistical analysis. The Kruskal-Wallis test was used to and maintained at 27 ± 1°C, at a relative humidity of 80 ± 5%, compare the virus titer between the mosquito groups, viruses, with a 12:12 light:dark photo phase for up to 18 days. Five and inoculation methods by ranking each data point and com- mosquitoes were sampled every 24 hours for 8 days and again paring mean ranks between the virus groups. Analysis was at days 10, 12, 15, and 18. undertaken using SPSS version 7.0 software (SPSS Inc., Chi- Oral infection of mosquitoes. Defibrinated sheep’s blood cago, IL). (Institute of Medical and Veterinary Science, Adelaide, Queensland, Australia) was washed twice in serum free RPMI 1640 medium and the erythrocyte pellet was reconsti- RESULTS tuted to its original volume using freshly thawed virus stocks. Batches of 50, 1–4-day-old female mosquitoes of each species The titer of YF 17D virus in a suspension of erythrocytes ס were starved for 24 ± 6 hours prior to feeding on the virus/ decreased from 7.3 to 6.7 log10 PFU/mL (P 0.023) over 2 erythrocyte suspension under a commercial sausage skin hours ± 20 minutes at 37 ± 1°C. The oral feeding dose for YF membrane warmed to 37 ± 1°C in a water-jacketed mem- 17D was therefore adjusted accordingly. The titer of virus in brane feeder (University of Queensland, Brisbane, Queens- other IT inocula or in virus/erythrocyte suspensions did not land, Australia). Culex gelidus would not feed from mem- decrease significantly. brane feeders and therefore were offered cotton pledgets ChimeriVax™-JE was not observed in Cx. annulirostris soaked in the virus/erythrocyte suspensions warmed to 37 ± mosquitoes at detectable levels after oral or IT infection INFECTION OF AUSTRALIAN MOSQUITOES WITH CHIMERIVAX™-JE 661

ס (Figure 1A). In contrast, wild-type JEV-Nakayama titers in- PFU/mosquito (estimated inoculum 2.3 log10 PFU/ creased to approximately 6.0 log10 PFU/mosquito in Cx. an- mosquito) (Figure 1G). Japanese encephalitis virus (Na- nulirostris infected IT and several mosquitoes (7%, 4 of 60) kayama) was detected in 88% (53 of 60) of Ae. vigilax inocu- infected orally developed titers of approximately 5.0 log10 lated IT reaching titers as high as 6.2 log10 PFU/mosquito. PFU/mosquito (Figure 1B). No multiplication of YF 17D was After oral infection, JEV-Nakayama was detected in 12% (7 detected in this species of mosquito after oral infection and of 60) of the Ae. vigilax assayed (Figure 1H). Yellow fever titers in mosquitoes inoculated IT (22%, 13 of 60) did not virus 17D was detected in 10% (6 of 60) of Ae. vigilax fed increase above that of the estimated inoculum (3.5 log10 PFU/ orally on this virus but titers were all less than the titers of the mosquito) (Figure 1C). estimated inoculum (3.9 log10 PFU/mosquito). Yellow fever There was no evidence of ChimeriVax™-JE virus in Cx. virus 17D was detected in 100% (60 of 60) mosquitoes inocu- gelidus after oral infection. Moreover, titers of Chimeri lated IT but the mean titer (3.5 log10 PFU/mosquito) was also

Vax™-JE in this species inoculated IT (8%, 5 of 60) rarely similar to that of the estimated inoculum (3.5 log10 PFU/ increased above the titer of the estimated inoculum (2.3 log10 mosquito) (Figure 1I). PFU/mosquito) (Figure 1D). In contrast, JEV-Nakayama vi- Sequence analysis of the C, prM, and E protein genes of rus was observed at high titer after IT inoculation in almost all virus from mosquitoes 876, 877, and 878, the three Cx. annu- (92%, 55 of 60) mosquitoes. Moreover, 18% (11 of 60) of Cx. lirostris mosquitoes showing high virus titers after IT inocu- gelidus mosquitoes infected orally contained JEV-Nakayama lation of ChimeriVax™-JE, indicated infection with JEV- at an approximate titer of 5.5 log10 PFU/mosquito (Figure Nakayama virus rather than ChimeriVax™-JE. A review of 1E). The titers of YF 17D in Cx. gelidus were similar to those work logs suggests these three mosquitoes were misidentified of ChimeriVax™-JE with only 2% (1 of 60) of mosquitoes during collection. The data points corresponding to these infected after feeding and 20% (12 of 60) after IT inoculation mosquitoes were removed from the Cx. annulirostris Chimeri (Figure 1F). Vax™-JE IT analysis, giving a total of 57 mosquitoes for the ChimeriVax™-JE was not detected in Ae. vigilax infected analysis (Figure 1A). orally, but titers were detected in 70% (42 of 60) of mosqui- Virus titers of ChimeriVax™-JE in all mosquito species toes after IT inoculation but at a low mean titer of 2.4 log10 were less than those for JEV-Nakayama inoculated IT or

FIGURE 1. Titer of ChimeriVaxTM-JE (A, D, and G), JEV-Nakayama (B, E, and H) and YFV 17D (C, F, and I) in Culex annulirostris (A, B, intrathoracic inoculum ס ----- ;oral infection ס intrathoracic inoculation; ᭹ ס and C), Cx. gelidus (D, E, and F) and Aedes vigilax (G, H, and I). ᭺ .plaque-forming units ס limit of detection. PFU ס – - – ;oral inoculum titer line ס –––– ;titer line 662 REID AND OTHERS orally (P < 0.001). Titers of ChimeriVax™-JE were also less Received March 27, 2006. Accepted for publication June 4, 2006. than those for YF 17D in all mosquito species inoculated IT Acknowledgments: We thank Cassie Jansen and Dr. Andrew van ,den Hurk (Queensland Health Pathology and Scientific Services .(0.008 ס and orally (P (0.002 ס P) Brisbane, Queensland, Australia), and Corporal Raethea Huggins, Lieutenant Robert Marlow, Major Steven Frances, and Lieutenant Colonel Robert Cooper (Australian Army) for entomologic assis- DISCUSSION tance and technical advice. We also thank Helen Gramatonev (statistical consultant) for statistical support. Neither Cx. annulirostris, Cx. gelidus, nor Ae. vigilax mos- Financial support: This study was supported by Acambis Inc. (Cam- quitoes became infected with ChimeriVax™-JE vaccine virus bridge, MA) and the Joint Health Support Agency, Defence Health Services (Canberra, Australia). when fed with a blood meal containing doses of virus that exceeded the maximum viremia observed in humans inocu- Disclosure: Mark Reid has acted as a paid consultant to Acambis Inc. in relation to JE vaccine trials. Farshad Guirakhoo and Thomas P. lated with ChimeriVax™-JE vaccine. Furthermore, when the Monath are current and former employees of Acambis Inc. These 15 midgut infection barrier was circumvented by IT inocula- statements are made in the interest of full disclosure and not because tion, titers of ChimeriVax™-JE were significantly less than the authors consider this to be a conflict of interest. those of either control viruses: JEV-Nakayama or YF 17D (P Disclaimer: All investigations were approved by the Office of the < 0.001). Gene Technology Regulator and the Australian Quarantine Inspec- This study has identified minor differences in the suscepti- tion Service (Canberra, Australia). The use of mice (for mosquito colony maintenance) was approved by the Animal Experimentation bility of Culex and Aedes species to infection with Chi- and Ethics Committee of the Australian Army Malaria Institute in 6 meriVax™-JE and YF 17D. Bhatt and others compared the accordance with the Australian Code of Practice for the Care and Use differences in the susceptibility of Cx. tritaeniorhynchus, Ae. of Animals for Scientific Purposes, Version 7. The opinions expressed aegypti, and Ae. albopictus to infection with ChimeriVax™- herein are those of the authors and do not necessarily reflect those of the Australian Defence Health Services or any extant policy. JE, JEV SA14-14-2, JEV SA14, and YF 17D. In contrast, Cx. tritaeniorhynchus was a highly efficient vector for both the Authors’ addresses: Mark Reid, Donna MacKenzie, Andrew Baron, live, attenuated JEV SA14-14-2 vaccine (currently being used and Natalie Lehmann, Australian Army Malaria Institute, Weary Dunlop Drive, Gallipoli Barracks, Enoggera, Queensland, 4051 Aus- in China, South Korea, and Vietnam) and wild-type JEV tralia. Kym Lowry and John Aaskov, Australian Army Malaria In- (SA14). Culex tritaeniorhynchus, which was the only Culex stitute, Weary Dunlop Drive, Gallipoli Barracks, Enoggera, Queens- species examined, did not become orally infected with either land, 4051 Australia and School of Life Sciences, Queensland Uni- ChimeriVax™-JE or YF 17D viruses inoculated IT with ap- versity of Technology, GPO Box 2434, Brisbane, Queensland, 4001 Australia. Farshad Guirakhoo, Acambis Inc., 38 Sidney Street, Cam- proximately 5.5 log10 PFU/mosquito. In the present study, bridge MA 02139. Thomas P. Monath, Kleiner Perkins Caulfield & both Cx. gelidus and Cx. annulirostris demonstrated an ability Byers, 21 Finn Rd., Harvard, MA 01451. to maintain low titer infections with ChimeriVax™-JE and YF 17D after IT inoculation. Bhatt and others demonstrated that neither Ae. aegypti or Ae. albopictus species became in- REFERENCES fected after feeding with YF 17D.6 In contrast, YF 17D was detected in Ae. vigilax 12 and 18 days after feeding. Yellow 1. Tsai TF, 1994. 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