Am. J. Trop. Med. Hyg., 68(2), 2003, pp. 218-221
Copyright © 2003 by The American Society of Tropical Medicine and Hygiene
LIMITED POTENTIAL FOR MOSQUITO TRANSMISSION OF GENETICALLY ENGINEERED, LIVE-ATTENUATED WESTERN EQUINE ENCEPHALITIS VIRUS VACCINE CANDIDATES
MICHAEL J. TURELL,
MONICA L. O’GUINN, AND
MICHAEL D. PARKER
Vector Assessment Branch, Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland
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ABSTRACT
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Specific mutations associated with attenuation of Venezuelan equine encephalitis (VEE) virus in rodent models were identified during efforts to develop an improved VEE vaccine. Analogous mutations were produced in full-length cDNA clones of the Cba 87 strain of western equine encephalitis (WEE) virus by site-directed mutagenesis in an attempt to develop an improved WEE vaccine. Isogenic viral strains with these mutations were recovered after transfection of baby hamster kidney cells with infectious RNA. We evaluated two of these strains (WE2102 and WE2130) for their ability to replicate in and be transmitted by Culex tarsalis, the principal natural vector of WEE virus in the United States. Each of the vaccine candidates contained a deletion of the PE2 furin cleavage site and a secondary mutation in the E1 or E2 glycoprotein. Both of these potential candidates replicated in mosquitoes significantly less efficiently than did either wild-type WEE (Cba 87) virus or the parental clone (WE2000). Likewise, after intrathoracic inoculation, mosquitoes transmitted the vaccine candidate strains significantly less efficiently than they transmitted either the wild-type or the parental clone. One-day-old chickens vaccinated with either of the two vaccine candidates did not become viremic when challenged with virulent WEE virus two weeks later. Mutations that result in less efficient replication in or transmission by mosquitoes should enhance vaccine safety and reduce the possibility of accidental introduction of the vaccine strain to unintentional hosts.
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INTRODUCTION
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The current investigational new drug (IND) vaccine for western equine encephalitis (WEE) virus is a formalin-inactivated vaccine derived from the CM-4884 strain of WEE virus. This vaccine requires three inoculations to achieve satisfactory immunity and can require additional booster doses. In an attempt to improve the current inactivated vaccine, specific mutations associated with attenuation of Venezuelan equine encephalitis (VEE) virus in rodent models were identified.1–3 Analogous mutations were inserted into full-length cDNA clones of the Cba 87 strain of WEE virus by site-directed mutagenesis, and isogenic virus strains with these mutations were recovered after transfection of baby hamster kidney (BHK) cells with infectious RNA.4
Live-attenuated vaccines offer many advantages over inactivated immunogens. These include administration of a single dose, more efficient induction of mucosal immunity, and generally longer duration of immunity. However, live arbovirus vaccines have the potential to be transmitted to secondary hosts and possibly revert to a more virulent phenotype. Reversion to virulence might occur in either the vertebrate or the arthropod host and might allow for the subsequent transmission of virulent virus. Thus, the potential for replication in and transmission by appropriate vectors should be a part of safety testing of newly developed arbovirus vaccines.
Therefore, we evaluated two attenuated strains of WEE virus (WE2102 and WE2130) for their ability to replicate in and be transmitted by Culex tarsalis, the principal natural vector of WEE virus in the United States.5–7 In addition, we evaluated these vaccine candidates for their ability to elicit protection of chickens from WEE virus challenge and the stability of the attenuated phenotype after intrathoracic inoculation of Cx. pipiens mosquitoes.
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MATERIALS AND METHODS
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Mosquitoes.
A laboratory strain of Cx. tarsalis (HP) provided by Dr. L. Kramer (University of California, Davis, CA) and a recently colonized strain of Cx. pipiens derived from specimens collected in New York and provided by Dr. J. Oliver (New York State Department of Health, Syracuse, NY) were used during these studies. Mosquitoes were held at 26°C with a 16:8 light:dark photoperiod. Larval mosquitoes were reared in pans containing dechlorinated tap water and provided ground catfish chow for nutrition.
Virus and virus assay.
We evaluated two attenuated strains of WEE virus, WE2102 and WE2130, produced by deletion of the furin cleavage site (RRPKR) at the junction of the WEE E3 and E2 glycoproteins from infectious clone pWE2000.4 These two viruses were rescued after transfection of BHK cells with in vitro transcripts from the mutated clone. In addition to the cleavage site deletion, strain WE2102 has a second site mutation, Glu
Lys, at amino acid 182 of the E2 glycoprotein. Strain WE2130 has a second site mutation, PheSer, at amino acid 257 of the E1 glycoprotein. We also used virus produced from a full-length infectious clone of wild-type virulent WEE (WE2000) and the virulent parent viral strain Cba 87.8
Plaque titers were determined on Vero cell monolayers grown in 6- or 12-well plastic cell culture plates.9 Serial 10-fold dilutions of each specimen were added to wells (0.1 mL/ well). After a one-hour absorption period, a nutrient overlay medium (Eagle’s basal medium with Earle’s salts, 7% heat-inactivated fetal bovine serum, 0.75% agarose, 100 units/mL of penicillin, 100 µg/mL of streptomycin, and 100 units/ml of nystatin) was added to each well and the plates incubated at 35°C for two days. Cells were then stained with 1 mL of this medium containing 0.017% neutral red without the fetal bovine serum and antibiotics. Plaques were enumerated the following day.
Inoculation studies.
Two- to six-day-old female Cx. tarsalis were inoculated intrathoracically10 with 0.3 µL of a suspension containing approximately 10 6 plaque-forming units (PFU)/mL (102.5 PFU/mosquito) of one of the strains of WEE virus. Mosquitoes were placed in 0.5-liter cardboard containers with netting over the open end and held in an incubator maintained at 26°C with a 16:8 hour light:dark photoperiod. To determine the potential for replication of each of the WEE virus strains in mosquitoes, we removed five mosquitoes from each cage at selected times, triturated them individually in 1 mL of diluent (10% heat-inactivated fetal bovine serum in medium 199 with Earle’s salts, 100 units/mL of penicillin, 100 µg/mL of streptomycin, 100 units/ml of nystatin, and sodium bicarbonate), and froze them at -70°C until assayed for virus.
To determine the ability of mosquitoes to transmit virus by bite, mosquitoes inoculated seven or more days previously were allowed to feed individually on one-day-old leghorn chickens or 
3-day-old ICR mice. The chickens were bled the following day, and the blood was diluted 1:10 in diluent and tested for infectious virus by plaque assay. The mice were observed daily for mortality for 21 days.
To determine the potential for reversion to virulence after replication in mosquitoes, pools of 10 Cx. pipiens were inoculated as described earlier in this report. After incubation for six days at 26°C, they were triturated in diluent, clarified by centrifugation, titrated on Vero cell monolayers, diluted to 104.5 PFU/mL (103.5/chick), and inoculated subcutaneously (0.1 mL/chicken) into one-day-old leghorn chickens. These chickens were bled daily to monitor viremia.
Protection studies.
To determine the ability of these vaccine candidate strains to protect chickens from virulent WEE virus challenge, one-day-old leghorn chickens were inoculated subcutaneously with 0.1 mL of either the WE2102 or WE2130 strain of WEE virus (approximately 104 PFU/0.1 mL). These chickens were bled the next day to determine viremia levels. After 14 days, a serum sample was obtained from these chickens and age-matched negative control chickens. All birds were then challenged with 0.1 mL of either the virulent CBA 87 or WE2000 strain of WEE virus (approximately 105 PFU/0.1 mL). These chickens were again bled daily for two days to monitor viremia levels.
The maintenance and care of experimental animals complies with the National Institutes of Health guidelines for the humane use of laboratory animals. In conducting research using animals, the investigators adhered to the "Guide for the Care and Use of Laboratory Animals," as prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (NIH Publication No. 86-23, Revised 1996). The facilities are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International.
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RESULTS
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Mosquito inoculation studies.
All of the strains of WEE virus tested replicated in Cx. tarsalis (Table 1
). However, both of the vaccine candidate strains (WE 2102 and WE2130) grew to significantly lower titers (T 
6.4, degrees of freedom 7, P < 0.006) than did either the parent (WE2000) or the wild-type CBA 87 strains (Table 1 and Figure 1). In general, viral titers increased rapidly and reached their highest levels approximately 4–7 days after inoculation, and then gradually decreased with time (Figure 1).
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TABLE 1 Replication and transmission of selected strains of western equine encephalitis virus in Culex tarsalis after inoculation of approximately 102.5 plaque-forming units of virus*
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FIGURE 1. Replication of WE2000, Cba 87, and two live-attenuated vaccine candidate strains (WE2102 and WE2130) of western equine encephalitis virus in Culex tarsalis after intrathoracic inoculation of approximately 102.5 plaque-forming units (PFU) of virus. Five mosquitoes were sampled at each time point. Bars show the mean ± SE logarithm10 PFU per mosquito.
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Although both of the virulent strains were transmitted efficiently by bite after the mosquitoes had been infected by intrathoracic inoculation (13 of 14, 93%), mosquitoes inoculated with the WE2102 or WE2130 strains transmitted virus significantly less efficiently (2 of 14, 14%) (P 0.01, by Fisher’s exact test) (Table 1
). To evaluate the potential for reversion in an arthropod vector, we inoculated one-day-old chickens with either virus obtained from a pool of 10 Cx. pipiens inoculated with the various strains six days previously or with unpassed virus. Viremia profiles in chickens inoculated with the mosquito-passed viruses were similar to those in chickens inoculated with the unpassed strains (Figure 2). Thus, we found no evidence of phenotypic reversion after one mosquito passage.
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FIGURE 2. Replication of WE2000 and two live-attenuated vaccine candidate strains (WE2102 and WE2130) of western equine encephalitis virus in one-day-old leghorn chickens after subcutaneous inoculation of approximately 104 plaque-forming units of virus. Four chickens were inoculated with each virus strain. Open symbols represent mean titers after passage in Culex pipiens by intrathoracic inoculation, while solid symbols represent mean titers of the unpassed viral stocks.
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Protection studies.
Both the WE2102 and WE2130 strains of WEE virus induced low-level viremias when inoculated into one-day-old chickens (Table 2). When these chickens were challenged two weeks later with either the WE2000 or CBA 87 strains of virulent WEE virus, none of these chickens developed a detectable viremia, although all nine chickens not previously infected with WE2102 or WE2130 virus developed a moderate viremia (Table 2). Thus, infection with either WE2102 or WE2130 protected the chickens from infection with a virulent strain of WEE virus.
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TABLE 2 Replication of selected strains of western equine encephalitis virus in one-day-old chickens after inoculation of approximately 104 plaque-forming units of virus and protection from challenge with wild-type virus*
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DISCUSSION
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Live-attenuated virus vaccines offer many advantages over the use of inactivated immunogens. However, a major concern with the use of live-attenuated virus vaccines is the potential for spread of either the vaccine or a pathogenic revertant to susceptible hosts. This was documented by the isolation of TC-83 virus, the current IND live-attenuated VEE vaccine, from field-collected mosquitoes after TC-83 was used to vaccinate equines during an outbreak of VEE in the early 1970s.11
Thus, one needs to consider the risk of introducing a virulent pathogen when using a live-attenuated virus vaccine. This risk is reduced if the vaccine strain produces a low to undetectable viremia unlikely to infect a mosquito. Various studies have shown that when mosquitoes are infected after ingesting very low doses of virus, the virus is less likely to disseminate to the hemocoel and thus be capable of transmission.12 The risk is further reduced if the vaccine candidate is less efficiently transmitted than the parent strain. In addition, evidence that the vaccine strain retains its vaccine-like phenotype after mosquito passage indicates that in the rare occasion in which the virus might be transmitted by a mosquito, the transmitted virus would not be likely to initiate an infection with virulent virus.
Culex tarsalis was selected for these studies because it is an incriminated vector of WEE virus.5–7 In addition to being a competent laboratory vector, it is a common mosquito that readily feeds on birds and mammals throughout its range.13 Although the potential vaccine candidates replicated in female Cx. tarsalis after intrathoracic inoculation, viral titers were significantly lower than those attained in mosquitoes inoculated with parental viruses. Similarly, only two (14%) of 14 mosquitoes with a disseminated infection with the vaccine candidates transmitted virus by bite, while 13 (93%) of 14 mosquitoes transmitted the two wild-type strains. This is consistent with results from a study with V3526, a furin cleavage site deletion mutant of VEE virus in which only 18% of female Ochlerotatus taeniorhynchus inoculated with this vaccine candidate strain transmitted virus, while 81% of those inoculated with the virulent parent virus transmitted VEE virus by bite.14 In addition, even after replication in a mosquito, the vaccine strains retained their attenuated characteristics based on absence of mortality and relatively low viremia levels in chickens.
The WE2102 and WE2130 strains contain a deletion of the furin cleavage site in PE2, as well as a second site mutation in E1 or E2. As would be expected, the deletion mutants are genetically stable, and showed no apparent phenotypic reversion on passage in mosquitoes. Based on the low viremias in chickens in this study, reduced ability to replicate in and be transmitted by a known competent vector of WEE virus, and evidence that WE2102 and WE2130 do not revert to virulence after mosquito passage, it is unlikely that mosquito transmission of these viruses would occur.
Received November 15, 2001.
Accepted for publication October 30, 2002.
Acknowledgments: We thank L. Kramer (University of California, Davis, CA) for providing Cx. tarsalis mosquitoes and J. Oliver (Arthropod-Borne Disease Program, New York State Department of Health, Syracuse, NY) for providing Cx. pipiens mosquitoes. We also thank D. Dohm, J. Blow, and K. Kenyon for critically reading the manuscript.
Disclaimer: The views of the authors do not purport to reflect the positions of the Department of the Army or the Department of Defense.
Authors’ address: Michael J. Turell, Telephone: 301-619-4921, Monica L. O’Guinn, Telephone: 301-619-4689, and Michael D. Parker, Telephone: 301-619-4916, Vector Assessment Branch, Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Frederick, MD 21702-5011.
Reprint requests: Michael J. Turell, Vector Assessment Branch, Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Frederick, MD 21702-5011, Fax: 301-619-2290, e-mail: michael.turell{at}det.amedd.army.mil
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ABSTRACT
INTRODUCTION
