• View in gallery

    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, and C), Cx. gelidus (D, E, and F) and Aedes vigilax (G, H, and I). ○ = intrathoracic inoculation; • = oral infection; ----- = intrathoracic inoculum titer line; –––– = oral inoculum titer line; – - – = limit of detection. PFU = plaque-forming units.

  • 1

    Tsai TF, 1994. Japanese encephalitis vaccines. Plotkin SA, Mortimer EA, eds. Vaccines. Second edition. Philadelphia: W.B. Saunders, 671–713.

  • 2

    Chambers TJ, Nestorowicz A, Mason PW, Rice CM, 1999. Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties. J Virol 73 :3095–3101.

    • Search Google Scholar
    • Export Citation
  • 3

    Monath TP, Guirakhoo F, Nichols R, Yoksan S, Schrader R, Murphy C, Blum P, Woodward S, McCarthy K, Mathis D, Johnson C, Bedford P, 2003. Chimeric live, attenuated vaccine against Japanese encephalitis (ChimeriVax™-JE): Phase II clinical trials for safety and immunogenicity, effect of vaccine dose and schedule, and memory response to challenge with inactivated Japanese encephalitis antigen. J Infect Dis 188 :1213–1230.

    • Search Google Scholar
    • Export Citation
  • 4

    Monath TP, McCarthy K, Bedford P, Johnson CT, Nichols R, Yoksan S, Marchesani R, Knauber M, Wells KH, Arroyo J, Guirakhoo F, 2002. Clinical proof of principle for Chimeri Vax™: recombinant live, attenuated vaccines against flavivirus infections. Vaccine 20 :1004–1018.

    • Search Google Scholar
    • Export Citation
  • 5

    Whitman L, 1939. Failure of Aedes aegypti to transmit yellow fever cultured virus (17D). Am J Trop Med 19 :19–26.

  • 6

    Bhatt TR, Crabtree MB, Guirakhoo F, Monath TP, Miller BR, 2000. Growth characteristics of the chimeric Japanese encephalitis virus vaccine candidate, ChimeriVax™-JE (YF/JE SA14-14-2), in Culex tritaeniorhynchus, Aedes albopictus and Aedes aegypti mosquitoes. Am J Trop Med Hyg 62 :480–484.

    • Search Google Scholar
    • Export Citation
  • 7

    Ritchie SA, Phillips D, Broom A, Mackenzie J, Poidinger M, van den Hurk A, 1997. Isolation of Japanese encephalitis virus from Culex annulirostris in Australia. Am J Trop Med Hyg 56 :80–84.

    • Search Google Scholar
    • Export Citation
  • 8

    Johansen CA, van den Hurk AF, Ritchie SA, Zborowski P, Nisbet DJ, Paru R, Bockarie MJ, Macdonald J, Drew AC, Khromykh TI, Mackenzie JS, 2000. Isolation of Japanese encephalitis virus from mosquitoes (Diptera: Culicidae) collected in the Western Province of Papua New Guinea, 1997–1998. Am J Trop Med Hyg 62 :631–638.

    • Search Google Scholar
    • Export Citation
  • 9

    Johansen CA, van den Hurk AF, Pyke AT, Zborowski P, Phillips DA, Mackenzie JS, Ritchie SA, 2001. Entomological investigation of an outbreak of Japanese encephalitis virus in the Torres Strait, Australia in 1998. J Med Entomol 38 :581–588.

    • Search Google Scholar
    • Export Citation
  • 10

    van den Hurk AF, Nisbet DJ, Hall RA, Kay BH, Mackenzie JS, Ritchie SA, 2003. Vector competence of Australian mosquitoes (Diptera: Culicidae) for Japanese encephalitis virus. J Med Entomol 40 :82–90.

    • Search Google Scholar
    • Export Citation
  • 11

    Karabatsos N, Buckley SM, 1967. Susceptibility of the baby-hamster kidney-cell line (BHK-21) to infection with Arboviruses. Am J Trop Med Hyg 16 :99–105.

    • Search Google Scholar
    • Export Citation
  • 12

    Rhim JS, Schell K, Creasy B, Case W, 1969. Biological characteristics and viral susceptibility of an African green monkey kidney cell line (Vero). Proc Soc Exp Biol Med 132 :670–678.

    • Search Google Scholar
    • Export Citation
  • 13

    Gorman BM, Leer JR, Filippich C, Goss PD, Doherty RL, 1975. Plaquing and neutralisation of arboviruses in the PS-EK line of cells. Aust J Med Tech 6 :65–71.

    • Search Google Scholar
    • Export Citation
  • 14

    Rosen L, Gubler D, 1974. The use of mosquitoes to detect and propagate dengue viruses. Am J Trop Med Hyg 23 :1153–1160.

  • 15

    Hardy JL, 1988. Susceptibility and resistance of vector mosquitoes. Monath TP, ed. The Arboviruses: Ecology and Epidemiology. Volume 1. Boca Raton, FL: CRC Press. 87–126.

  • 16

    Johnson BW, Chambers TV, Crabtree MB, Arroyo J, Monath TP, Miller BR, 2003. Growth characteristics of the veterinary vaccine candidate ChimeriVax™-West Nile (WN) virus in Aedes and Culex mosquitoes. Med Vet Entomol 17 :235–243.

    • Search Google Scholar
    • Export Citation
  • 17

    Johnson BW, Chambers TV, Crabtree MB, Bhatt TR, Guirakhoo F, Monath TP, Miller BR, 2002. Growth characteristics of ChimeriVax™-DEN2 vaccine virus in Aedes aegypti and Aedes albopictus mosquitoes. Am J Trop Med Hyg 67 :260–265.

    • Search Google Scholar
    • Export Citation
  • 18

    Johnson BW, Chambers TV, Crabtree MB, Guirakhoo F, Monath TP, Miller BR, 2004. Analysis of the replication kinetics of the ChimeriVax™-DEN 1,2,3,4 tetravalent virus mixture in Aedes aegypti by real-time reverse transcriptase-polymerase chain reaction. Am J Trop Med Hyg 70 :89–97.

    • Search Google Scholar
    • Export Citation
  • 19

    World Health Organization, 1998. Requirements for yellow fever vaccine (requirements for biological substances no. 3, revised 1995). World Health Organ Tech Rep Ser 872 :517–537.

    • Search Google Scholar
    • Export Citation

 

 

 

 

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

View More View Less
  • 1 Australian Army Malaria Institute, Brisbane, Queensland, Australia; School of Life Sciences, Queensland University of Technology, Brisbane, Queensland, Australia; Acambis Inc., Cambridge, Massachusetts

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 viremia = 4.8 PFU/mL, range = 0–30 PFU/mL of 0.9 days mean duration, range = 0–11 days). Some members of all three 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

Japanese encephalitis virus (JEV) is a member of the family Flaviviridae and is a leading cause of viral encephalitis in Asia. Case fatality rates of 29% have been reported with an additional 33–50% of survivors developing long-term neurologic sequalae.1 ChimeriVax™-JE is a live, attenuated, vaccine for JEV in which the premembrane (prM) and envelope (E) proteins genes of the yellow fever virus strain 17D (YF 17D) have been replaced with the vaccine strain JEV SA14-14-2 prM-E sequence.2 Persons inoculated subcutaneously with ChimeriVax™-JE develop a self-limiting, very low–titer viremia that poses a theoretical risk of secondary transmission by a mosquito vector. In a recent study (Acambis Inc., unpublished data), the viremia in humans after immunization with 3.0–5.0 log10 plaque-forming units (PFU) of ChimeriVax™-JE developed a mean peak viremia of 4.8 PFU/mL (range = 0–30 PFU/mL of 0.9 days mean duration, range = 0–11 days). The level of viremia was lower than reported for yellow fever 17D vaccine (YF-VAX®) with a mean peak viremia of 21.8 PFU/mL (range = 0–80 PFU/mL of mean duration 1.2 days, range 0–3 days).3,4 The low levels of viremia in humans after vaccination with ChimeriVax™-JE and YF 17D vaccines is a barrier to oral infection of hematophagous vectors. In addition, it has been known for many years that YF 17D vaccine virus has lost its ability to be transmitted by the yellow fever vector Aedes aegypti.5 In previous vector competence studies of ChimeriVax™-JE, Ae. aegypti, Ae. albopictus, and Culex tritaeniorhynchus mosquitoes did not become infected after oral feeding on blood-virus suspensions 6 containing 6.9 log10 PFU/mL.

The objective of the present study was to assess the potential of mosquito species Cx. annulirostris, Cx. gelidus, and Ae. vigilax to become infected with ChimeriVax™-JE after ingesting a virus laden blood meal or after intrathoracic (IT) inoculation. Japanese encephalitis virus genotypes I or II have been isolated from these three mosquito species in Australia,79 and all three species of mosquito have been infected experimentally by membrane feeding with JEV isolates obtained from the Torres Strait of Australia.10

MATERIALS AND METHODS

Mosquitoes.

Culex gelidus larvae were collected from Ten-nant Creek (Northern Territory, Australia) by Peter Whelan (Northern Territory Health Services). Culex annulirostris eggs were provided by the Queensland Institute of Medical Research. Aedes vigilax eggs were supplied by Stephen Doggett (University of Sydney and Westmead Hospital). Colonies of each species were established at the Army Malaria Institute in an insectary maintained at 24–28°C and a relative humidity of 40–60%, with a 12:12 light:dark photoperiod. To maintain the colony, the larvae of each species were reared in dam water on a diet of commercial fish food (Kyorin, Himeji, Japan). For egg development, adults were offered a blood meal 3–5 times a week. All adult mosquitoes were maintained on a 20% (v/v) sucrose/water solution containing multivitamins (Myadec multivitamins and minerals; Nelson Laboratories, Warriewood, New South Wales, Australia) ad libitum. Mosquito colonies were established from larvae but to limit the possibility of adventitious infection of the colonies, 60 adults from each of the three colonies were screened for the presence of arboviruses by adding mosquito homogenate to 2-cm2 cultures of baby hamster kidney 21 (BHK-21) clone c15 cells11 and observing daily for 7–10 days for cytopathic effects (CPEs).

Viruses.

Yellow fever 17D vaccine (lot no. W6440-1 Stamaril®; Sanofi-Pasteur, Lyon, France) was passaged once in Vero (African green monkey) cells12 (European Type Culture Collection lot no. CB2617). Supernatant fluid was harvested when cell monolayers showed ≥ 75% CPE and stored at ≤ −60°C in 20% (v/v) heat-inactivated (56°C for 30 minutes) fetal bovine serum (FBS; Gibco-Invitrogen, Carlsbad, CA). Japanese encephalitis virus (Nakayama strain) was obtained from Queensland Health Scientific and Pathology Services (Townsville, Queensland, Australia) and was passaged in Vero cells in the same manner as YF 17D. ChimeriVax™-JE was supplied lyophilized (Acambis Inc., Cambridge MA) and was reconstituted in sterile 0.9% saline (AstraZeneca, Alderley Park, United Kingdom) before use. Titers of virus stocks were as follows: YF 17D = 7.3 log10 PFU/mL (in porcine stable-equine kidney cells [PS-EK]),13 JEV-Nakayama = 6.7 log10 PFU/mL (in BHK-21 c15 cells), and ChimeriVax™- JE = 6.1 log10 PFU/mL (in Vero cells).

Virus titrations

Mosquitoes were killed with CO2 gas and triturated using plastic pestles in sterile micro-centrifuge tubes in 1 mL volumes of RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) containing 0.3g/L of sodium bicarbonate and supplemented with 20% (v/v) heat-inactivated FBS (HI-FBS), 100μg/mL of streptomycin, 100 U/mL of penicillin, 2 mM l-glutamine (SPG) (Sigma-Aldrich), and 2.5 μg/mL of amphotericin B (Gibco-Invitrogen). The homogenate was clarified by centrifugation (10,377 × g for 2 minutes) and the supernatant was stored at ≤ −60°C until assayed. Virus titers were assayed using a modified plaque titration method.13 Briefly, supernatants from the mosquito homogenates were diluted 10-fold in duplicate 2% (v/v) HI-FBS RPMI 1640 medium/SPG and 100 μL was added to 2-cm2 cell monolayers. Japanese encephalitis virus (Nakayama) and ChimeriVax™-JE were titrated on BHK-21 c15 cells and YF 17D on PS-EK cells (because YF 17D did not plaque consistently on BHK-21 c15 cells). After 1 hour ± 10 minutes, 1.5% (w/v) carboxymethylcellulose (Sigma-Aldrich) with a final concentration of 2% (v/v) HI-FBS RPMI 1640 medium/SPG was added to each culture and incubated at 37 ± 1°C for 3–5 days in an atmosphere of 5% (v/v) CO2 in air. Monolayers were fixed and stained with 0.5% (w/v) CI basic violet in 5% (v/v) formalin:phosphate buffered saline (Sigma-Aldrich) before plaques were counted. The limit of sensitivity of the assay was ≥ 1.7 log10 PFU/mosquito. To enable log transformations of average plaque counts, cell monolayers with no detectable plaques were given the nominal value of half the limit of detection (0.85 log10 PFU).

Intra-thoracic inoculation of mosquitoes

Freshly thawed stocks of virus were maintained on ice for 2 hours ± 20 minutes. Female mosquitoes of the three species 1–4-days post-emergence, were immobilized with CO2 gas and manipulated on a laboratory cold plate (2 ± 1°C) (Thermoline Scientific, Sydney, New South Wales, Australia). Mosquitoes were inoculated IT with 0.15 ± 0.08 μL of virus stock using heat-drawn, 1-mm capillary tubes (Harvard Apparatus, Eden-bridge, United Kingdom) as previously described (Narishige, Tokyo, Japan).14 After inoculation, mosquitoes were transferred in batches of 20 to primary mesh-covered plastic cups and maintained at 27 ± 1°C, at a relative humidity of 80 ± 5%, with a 12:12 light:dark photo phase for up to 18 days. Five mosquitoes were sampled every 24 hours for 8 days and again at days 10, 12, 15, and 18.

Oral infection of mosquitoes

Defibrinated sheep’s blood (Institute of Medical and Veterinary Science, Adelaide, Queensland, Australia) was washed twice in serum free RPMI 1640 medium and the erythrocyte pellet was reconstituted to its original volume using freshly thawed virus stocks. Batches of 50, 1–4-day-old female mosquitoes of each species were starved for 24 ± 6 hours prior to feeding on the virus/erythrocyte suspension under a commercial sausage skin membrane warmed to 37 ± 1°C in a water-jacketed membrane feeder (University of Queensland, Brisbane, Queens-land, Australia). Culex gelidus would not feed from membrane feeders and therefore were offered cotton pledgets soaked in the virus/erythrocyte suspensions warmed to 37 ± 1°C immediately prior to feeding. All mosquitoes were fed over a time interval of 2 hours ± 20 minutes and fully engorged mosquitoes maintained for up to 18 days as described for mosquitoes infected IT. The average blood meal for each species was estimated by weighing a sample of 20 mosquitoes pre-feeding and post-feeding (Cx. annulirostris = 1.3 ± 0.9 μL, Cx. gelidus = 1.9 ± 0.6 μL, and Ae. vigilax = 1.7 ± 1.5 μL).

Stability of virus stocks

Experiments were conducted to quantify the reduction in virus titer for the virus/erythrocyte suspensions and IT inocula for each virus at 37 ± 1°C or 0°C after a 2 hour ± 20 minute time interval. Five replicates were assayed without freeze-thawing before and after the time interval and the average titer was compared by a paired, one-tailed Student’s t-test (α = 0.05) to determine whether significant decreases in virus titer had occurred.

Nucleotide sequencing

Culex annulirostris mosquito homogenates 876, 877, and 878 obtained 12 days after IT inoculation with ChimeriVax™-JE were passaged once in Vero cells and titrated in BHK-21 c15 cells as previously described. Viral RNA was extracted from the supernate of infected cultures using the High Pure Viral RNA kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Viral RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) using random hexamers (Roche Diagnostics). The region of the genome coding for capsid (C) and prM proteins was amplified by polymerase chain reaction (PCR) using Taq polymerase (Roche diagnostics) and primers previously described,7 as well as with JEV E protein–specific primers (JEV-19F: 5′-GGCAATCGTGACTTCATAGAAG-3′ and JE-591R: 5′-TCCACTCCTTGGCTCACAGTC-3′) on a thermocycler (Eppendorf, Hamburg, Germany). Cycling conditions were 94°C for 2 minutes followed by 35 cycles at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds. The PCR products were stained with ethidium bromide after electrophoresis on a 2% (w/v) agarose Tris-borate-EDTA gel (Sigma-Aldrich) and visualized with ultraviolet light. The 380-base pair C/prM and 572-base pair envelope gene bands were sequenced in both directions by dideoxynucleotide dye termination (BigDye terminator cycle sequencing kits; Applied Biosystems, Foster City, CA) ) using the oligonucleotide primers previously mentioned in this report and an automated sequencer (Applied Biosystems).

Statistical analysis

The Kruskal-Wallis test was used to compare the virus titer between the mosquito groups, viruses, and inoculation methods by ranking each data point and comparing mean ranks between the virus groups. Analysis was undertaken using SPSS version 7.0 software (SPSS Inc., Chicago, IL).

RESULTS

The titer of YF 17D virus in a suspension of erythrocytes decreased from 7.3 to 6.7 log10 PFU/mL (P = 0.023) over 2 hours ± 20 minutes at 37 ± 1°C. The oral feeding dose for YF 17D was therefore adjusted accordingly. The titer of virus in other IT inocula or in virus/erythrocyte suspensions did not decrease significantly.

ChimeriVax™-JE was not observed in Cx. annulirostris mosquitoes at detectable levels after oral or IT infection (Figure 1A). In contrast, wild-type JEV-Nakayama titers in-creased to approximately 6.0 log10 PFU/mosquito in Cx. annulirostris infected IT and several mosquitoes (7%, 4 of 60) infected orally developed titers of approximately 5.0 log10 PFU/mosquito (Figure 1B). No multiplication of YF 17D was detected in this species of mosquito after oral infection and titers in mosquitoes inoculated IT (22%, 13 of 60) did not increase above that of the estimated inoculum (3.5 log10 PFU/mosquito) (Figure 1C).

There was no evidence of ChimeriVax™-JE virus in Cx. gelidus after oral infection. Moreover, titers of Chimeri Vax™-JE in this species inoculated IT (8%, 5 of 60) rarely increased above the titer of the estimated inoculum (2.3 log10 PFU/mosquito) (Figure 1D). In contrast, JEV-Nakayama virus was observed at high titer after IT inoculation in almost all (92%, 55 of 60) mosquitoes. Moreover, 18% (11 of 60) of Cx. gelidus mosquitoes infected orally contained JEV-Nakayama at an approximate titer of 5.5 log10 PFU/mosquito (Figure 1E). The titers of YF 17D in Cx. gelidus were similar to those of ChimeriVax™-JE with only 2% (1 of 60) of mosquitoes infected after feeding and 20% (12 of 60) after IT inoculation (Figure 1F).

ChimeriVax™-JE was not detected in Ae. vigilax infected orally, but titers were detected in 70% (42 of 60) of mosquitoes after IT inoculation but at a low mean titer of 2.4 log10 PFU/mosquito (estimated inoculum = 2.3 log10 PFU/mosquito) (Figure 1G). Japanese encephalitis virus (Nakayama) was detected in 88% (53 of 60) of Ae. vigilax inoculated IT reaching titers as high as 6.2 log10 PFU/mosquito. After oral infection, JEV-Nakayama was detected in 12% (7 of 60) of the Ae. vigilax assayed (Figure 1H). Yellow fever virus 17D was detected in 10% (6 of 60) of Ae. vigilax fed orally on this virus but titers were all less than the titers of the estimated inoculum (3.9 log10 PFU/mosquito). Yellow fever virus 17D was detected in 100% (60 of 60) mosquitoes inoculated IT but the mean titer (3.5 log10 PFU/mosquito) was also similar to that of the estimated inoculum (3.5 log10 PFU/mosquito) (Figure 1I).

Sequence analysis of the C, prM, and E protein genes of virus from mosquitoes 876, 877, and 878, the three Cx. annulirostris mosquitoes showing high virus titers after IT inoculation of ChimeriVax™-JE, indicated infection with JEV-Nakayama virus rather than ChimeriVax™-JE. A review of work logs suggests these three mosquitoes were misidentified during collection. The data points corresponding to these mosquitoes were removed from the Cx. annulirostris Chimeri Vax™-JE IT analysis, giving a total of 57 mosquitoes for the analysis (Figure 1A).

Virus titers of ChimeriVax™-JE in all mosquito species were less than those for JEV-Nakayama inoculated IT or orally (P < 0.001). Titers of ChimeriVax™-JE were also less than those for YF 17D in all mosquito species inoculated IT (P = 0.002) and orally (P = 0.008).

DISCUSSION

Neither Cx. annulirostris, Cx. gelidus, nor Ae. vigilax mosquitoes became infected with ChimeriVax™-JE vaccine virus when fed with a blood meal containing doses of virus that exceeded the maximum viremia observed in humans inoculated with ChimeriVax™-JE vaccine. Furthermore, when the midgut infection barrier15 was circumvented by IT inoculation, titers of ChimeriVax™-JE were significantly less than those of either control viruses: JEV-Nakayama or YF 17D (P < 0.001).

This study has identified minor differences in the susceptibility of Culex and Aedes species to infection with ChimeriVax™-JE and YF 17D. Bhatt and others6 compared the differences in the susceptibility of Cx. tritaeniorhynchus, Ae. aegypti, and Ae. albopictus to infection with ChimeriVax™-JE, JEV SA14-14-2, JEV SA14, and YF 17D. In contrast, Cx. tritaeniorhynchus was a highly efficient vector for both the live, attenuated JEV SA14-14-2 vaccine (currently being used in China, South Korea, and Vietnam) and wild-type JEV (SA14). Culex tritaeniorhynchus, which was the only Culex species examined, did not become orally infected with either ChimeriVax™-JE or YF 17D viruses inoculated IT with approximately 5.5 log10 PFU/mosquito. In the present study, both Cx. gelidus and Cx. annulirostris demonstrated an ability 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 infected after feeding with YF 17D.6 In contrast, YF 17D was detected in Ae. vigilax 12 and 18 days after feeding. Yellow fever virus 17D was also detected in one Cx. gelidus mosquito 24 hours after feeding. This may have been the inoculated virus that had survived digestion rather than indicating multiplication of the virus.

Although both JEV Nakayama and JEV SA-14-14-2 are JEV genotype III, this study would have benefited from the use of JEV SA-14-14-2 in place of JEV-Nakayama as the JEV control. At the time of this study, JEV SA-14-14-2 was not permitted to be used in our facilities.

ChimeriVax™ vaccines for JE, dengue and West Nile have now been assessed in the arbovirus vectors Cx. tritaeniorhynchus, Cx. quinquefasciatus, Cx. nigripalpus, Ae. aegypti, and Ae. albopictus.6,1618 These studies and the study presented here suggest that ChimeriVax™ viruses are phenotypically similar to YF 17D for mosquito attenuation5 (first established in 1939), irrespective of the flavivirus prM or E gene presented in the chimera. Yellow fever vaccines (and genetically modified derivatives) have been and continue to be manufactured from certified sub-strains of YF 17D under a seed lot system19 to maintain the attenuated vaccine phenotype. The inability of these vaccines to cause disseminated infection after oral ingestion of high viral doses, together with the very low viremia levels in humans after vaccination, suggest that mosquitoes pose limited risk for establishing secondary cycles of the ChimeriVax™-JE vaccine in Australia after feeding on a viremic vaccinee.

Figure 1.
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, and C), Cx. gelidus (D, E, and F) and Aedes vigilax (G, H, and I). ○ = intrathoracic inoculation; • = oral infection; ----- = intrathoracic inoculum titer line; –––– = oral inoculum titer line; – - – = limit of detection. PFU = plaque-forming units.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 4; 10.4269/ajtmh.2006.75.659

*

Address correspondence to Mark Reid, Australian Army Malaria Institute, Weary Dunlop Drive, Gallipoli Barracks, Enoggera, Queensland 4051, Australia. E-mail: markreid2@optusnet.com.au

Authors’ addresses: Mark Reid, Donna MacKenzie, Andrew Baron, and Natalie Lehmann, Australian Army Malaria Institute, Weary Dunlop Drive, Gallipoli Barracks, Enoggera, Queensland, 4051 Australia. Kym Lowry and John Aaskov, Australian Army Malaria Institute, Weary Dunlop Drive, Gallipoli Barracks, Enoggera, Queensland, 4051 Australia and School of Life Sciences, Queensland University of Technology, GPO Box 2434, Brisbane, Queensland, 4001 Australia. Farshad Guirakhoo, Acambis Inc., 38 Sidney Street, Cambridge MA 02139. Thomas P. Monath, Kleiner Perkins Caulfield & Byers, 21 Finn Rd., Harvard, MA 01451.

Acknowledgments: We thank Cassie Jansen and Dr. Andrew van den Hurk (Queensland Health Pathology and Scientific Services, Brisbane, Queensland, Australia), and Corporal Raethea Huggins, Lieutenant Robert Marlow, Major Steven Frances, and Lieutenant Colonel Robert Cooper (Australian Army) for entomologic assistance and technical advice. We also thank Helen Gramatonev (statistical consultant) for statistical support.

Financial support: This study was supported by Acambis Inc. (Cambridge, MA) and the Joint Health Support Agency, Defence Health Services (Canberra, Australia).

Disclosure: Mark Reid has acted as a paid consultant to Acambis Inc. in relation to JE vaccine trials. Farshad Guirakhoo and Thomas P. Monath are current and former employees of Acambis Inc. These statements are made in the interest of full disclosure and not because the authors consider this to be a conflict of interest.

Disclaimer: All investigations were approved by the Office of the Gene Technology Regulator and the Australian Quarantine Inspection Service (Canberra, Australia). The use of mice (for mosquito colony maintenance) was approved by the Animal Experimentation and Ethics Committee of the Australian Army Malaria Institute in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, Version 7. The opinions expressed herein are those of the authors and do not necessarily reflect those of the Australian Defence Health Services or any extant policy.

REFERENCES

  • 1

    Tsai TF, 1994. Japanese encephalitis vaccines. Plotkin SA, Mortimer EA, eds. Vaccines. Second edition. Philadelphia: W.B. Saunders, 671–713.

  • 2

    Chambers TJ, Nestorowicz A, Mason PW, Rice CM, 1999. Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties. J Virol 73 :3095–3101.

    • Search Google Scholar
    • Export Citation
  • 3

    Monath TP, Guirakhoo F, Nichols R, Yoksan S, Schrader R, Murphy C, Blum P, Woodward S, McCarthy K, Mathis D, Johnson C, Bedford P, 2003. Chimeric live, attenuated vaccine against Japanese encephalitis (ChimeriVax™-JE): Phase II clinical trials for safety and immunogenicity, effect of vaccine dose and schedule, and memory response to challenge with inactivated Japanese encephalitis antigen. J Infect Dis 188 :1213–1230.

    • Search Google Scholar
    • Export Citation
  • 4

    Monath TP, McCarthy K, Bedford P, Johnson CT, Nichols R, Yoksan S, Marchesani R, Knauber M, Wells KH, Arroyo J, Guirakhoo F, 2002. Clinical proof of principle for Chimeri Vax™: recombinant live, attenuated vaccines against flavivirus infections. Vaccine 20 :1004–1018.

    • Search Google Scholar
    • Export Citation
  • 5

    Whitman L, 1939. Failure of Aedes aegypti to transmit yellow fever cultured virus (17D). Am J Trop Med 19 :19–26.

  • 6

    Bhatt TR, Crabtree MB, Guirakhoo F, Monath TP, Miller BR, 2000. Growth characteristics of the chimeric Japanese encephalitis virus vaccine candidate, ChimeriVax™-JE (YF/JE SA14-14-2), in Culex tritaeniorhynchus, Aedes albopictus and Aedes aegypti mosquitoes. Am J Trop Med Hyg 62 :480–484.

    • Search Google Scholar
    • Export Citation
  • 7

    Ritchie SA, Phillips D, Broom A, Mackenzie J, Poidinger M, van den Hurk A, 1997. Isolation of Japanese encephalitis virus from Culex annulirostris in Australia. Am J Trop Med Hyg 56 :80–84.

    • Search Google Scholar
    • Export Citation
  • 8

    Johansen CA, van den Hurk AF, Ritchie SA, Zborowski P, Nisbet DJ, Paru R, Bockarie MJ, Macdonald J, Drew AC, Khromykh TI, Mackenzie JS, 2000. Isolation of Japanese encephalitis virus from mosquitoes (Diptera: Culicidae) collected in the Western Province of Papua New Guinea, 1997–1998. Am J Trop Med Hyg 62 :631–638.

    • Search Google Scholar
    • Export Citation
  • 9

    Johansen CA, van den Hurk AF, Pyke AT, Zborowski P, Phillips DA, Mackenzie JS, Ritchie SA, 2001. Entomological investigation of an outbreak of Japanese encephalitis virus in the Torres Strait, Australia in 1998. J Med Entomol 38 :581–588.

    • Search Google Scholar
    • Export Citation
  • 10

    van den Hurk AF, Nisbet DJ, Hall RA, Kay BH, Mackenzie JS, Ritchie SA, 2003. Vector competence of Australian mosquitoes (Diptera: Culicidae) for Japanese encephalitis virus. J Med Entomol 40 :82–90.

    • Search Google Scholar
    • Export Citation
  • 11

    Karabatsos N, Buckley SM, 1967. Susceptibility of the baby-hamster kidney-cell line (BHK-21) to infection with Arboviruses. Am J Trop Med Hyg 16 :99–105.

    • Search Google Scholar
    • Export Citation
  • 12

    Rhim JS, Schell K, Creasy B, Case W, 1969. Biological characteristics and viral susceptibility of an African green monkey kidney cell line (Vero). Proc Soc Exp Biol Med 132 :670–678.

    • Search Google Scholar
    • Export Citation
  • 13

    Gorman BM, Leer JR, Filippich C, Goss PD, Doherty RL, 1975. Plaquing and neutralisation of arboviruses in the PS-EK line of cells. Aust J Med Tech 6 :65–71.

    • Search Google Scholar
    • Export Citation
  • 14

    Rosen L, Gubler D, 1974. The use of mosquitoes to detect and propagate dengue viruses. Am J Trop Med Hyg 23 :1153–1160.

  • 15

    Hardy JL, 1988. Susceptibility and resistance of vector mosquitoes. Monath TP, ed. The Arboviruses: Ecology and Epidemiology. Volume 1. Boca Raton, FL: CRC Press. 87–126.

  • 16

    Johnson BW, Chambers TV, Crabtree MB, Arroyo J, Monath TP, Miller BR, 2003. Growth characteristics of the veterinary vaccine candidate ChimeriVax™-West Nile (WN) virus in Aedes and Culex mosquitoes. Med Vet Entomol 17 :235–243.

    • Search Google Scholar
    • Export Citation
  • 17

    Johnson BW, Chambers TV, Crabtree MB, Bhatt TR, Guirakhoo F, Monath TP, Miller BR, 2002. Growth characteristics of ChimeriVax™-DEN2 vaccine virus in Aedes aegypti and Aedes albopictus mosquitoes. Am J Trop Med Hyg 67 :260–265.

    • Search Google Scholar
    • Export Citation
  • 18

    Johnson BW, Chambers TV, Crabtree MB, Guirakhoo F, Monath TP, Miller BR, 2004. Analysis of the replication kinetics of the ChimeriVax™-DEN 1,2,3,4 tetravalent virus mixture in Aedes aegypti by real-time reverse transcriptase-polymerase chain reaction. Am J Trop Med Hyg 70 :89–97.

    • Search Google Scholar
    • Export Citation
  • 19

    World Health Organization, 1998. Requirements for yellow fever vaccine (requirements for biological substances no. 3, revised 1995). World Health Organ Tech Rep Ser 872 :517–537.

    • Search Google Scholar
    • Export Citation
Save