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Am. J. Trop. Med. Hyg., 60(6), 1999, pp. 1041–1044 Copyright q 1999 by The American Society of Tropical Medicine and Hygiene LIMITED POTENTIAL FOR MOSQUITO TRANSMISSION OF GENETICALLY ENGINEERED, LIVE-ATTENUATED VENEZUELAN EQUINE ENCEPHALITIS VIRUS VACCINE CANDIDATES MICHAEL J. TURELL, GEORGE V. LUDWIG, JOHN KONDIG, AND JONATHAN F. SMITH Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland Abstract. In an attempt to improve the current live-attenuated vaccine (TC-83) for Venezuelan equine encephalitis (VEE), specific mutations associated with attenuation of VEE virus in rodent models were identified. These mutations were inserted into full-length cDNA clones of the Trinidad donkey strain of VEE virus by site-directed mutagenesis, and isogenic virus strains with these mutations were recovered after transfection of baby hamster kidney cells with infectious RNA. We evaluated 10 of these strains for their ability to replicate in and be transmitted by Aedes taeniorhynchus, a natural vector of epizootic VEE virus. Two vaccine candidates, one containing a deletion of the PE2 furin cleavage site, the other a combination of three separate point mutations in the E2 glycoprotein, replicated in mosquitoes and were transmitted to hamsters significantly less efficiently than was either parental (wild type) VEE virus or TC-83 virus. Although the attenuated strains were transmitted to hamsters by mosquitoes, after intrathoracic inoculation, there was no evidence of reversion to a virulent phenotype. The mutations that resulted in less efficient replication in, or transmission by, mosquitoes should enhance vaccine safety and reduce the possibility of environmental spread to unintentional hosts. The current investigational new drug (IND) vaccine (TC83) for Venezuelan equine encephalitis (VEE) strain IAB virus is a live-attenuated virus that causes reactogenicity in 20% of recipients and fails to elicit a positive seroresponse in 20% of recipients.1 Current efforts to develop an improved live-attenuated vaccine for VEE identified specific mutations associated with attenuation of VEE virus in rodent models.2– 4 These attenuating mutations have been inserted into a fulllength cDNA clone of wild-type VEE virus (IAB) to produce selected isogenic strains containing one or more attenuating mutations. These attenuated strains are currently being evaluated for their potential as a live-attenuated VEE virus vaccine. Live-attenuated vaccines typically offer many advantages over inactivated immunogens (e.g., single immunization, more efficient induction of mucosal immunity, longer duration of immunity). However, live arbovirus vaccines have the potential to be transmitted to secondary hosts, and may revert to a more virulent virus. This reversion to virulence could occur in either the vertebrate or the arthropod host. Thus, we examined VEE virus strains that contained attenuating mutations that might be included in a live-attenuated vaccine for their ability to infect and be transmitted by mosquitoes. In addition, we evaluated these vaccine candidates for reversion to a more virulent phenotype after intrathoracic inoculation of Aedes taeniorhynchus mosquitoes. Virus and virus assay. The strains of VEE virus evaluated are shown in Table 1. Potential vaccine candidate strains were provided by N. Davis and R. Johnston (University of North Carolina, Chapel Hill, NC).2,3,8 Plaque titers for specimens were determined on Vero cell monolayers grown in 6- or 12-well plastic cell culture plates. Serial 10-fold dilutions of each specimen were added to wells (0.1 ml/well). After a 1-hr absorption period, a nutrient overlay (Eagle’s basal medium with Earle’s salts, 7% heat-inactivated fetal bovine serum, 0.75% agarose, and antibiotics [100 units/ml of penicillin, 100 mg/ml of streptomycin, and 100 units/ml of nystatin]) was added to each well and the plates incubated at 358C for two days. Cells were then stained with 1 ml of the above medium, except that 5% neutral red was used in place of the fetal bovine serum and antibiotics. Plaques were enumerated the following day. Inoculation studies. Two- to 6-day-old female Ae. taeniorhynchus were inoculated intrathoracically9 with 0.3 ml of a suspension containing about 105 plaque-forming units (PFU)/ml (101.5 PFU/mosquito) of one of the strains of VEE virus. Mosquitoes were placed in 0.5-liter cardboard containers with netting over the open end and held in an incubator maintained at 268C with a 16:8 hr light:dark photoperiod. To determine the potential for replication of each of the VEE virus strains in mosquitoes, five mosquitoes were removed from each cage at selected times, triturated individually in 1 ml of diluent (10% heat-inactivated fetal bovine serum in medium 199 with Earle’s salts, antibiotics, and sodium bicarbonate), and frozen at 2708C until assayed for virus. To determine the ability of mosquitoes to transmit virus by bite, mosquitoes inoculated $ 7 days previously were allowed to feed individually on adult female Syrian hamsters. The hamsters were observed daily for 21 days. A sample of brain tissue was obtained from hamsters that died and were tested by plaque assay to confirm the presence of virus. Hamsters surviving $ 21 days were challenged with 104 50% lethal dose units (LD50) of the V3000 strain of VEE virus. Infection with this strain is nearly always fatal for MATERIALS AND METHODS Mosquitoes. Two laboratory strains of Ae. taeniorhynchus, Vero Beach and Medical and Veterinary Entomology Research Laboratory (MAVERL), were used during these studies. Both of these strains have been in colonies for more than 30 years and are derived from mosquitoes collected in the late 1950s in Florida. Mosquitoes were held at 268C with a 16:8 hr light:dark photoperiod and reared as described by Gargan and others.5 Aedes taeniorhynchus is considered to be a natural vector of VEE virus in the Americas,6 and both strains are competent vectors of the epizootic IAB strain of VEE virus7 (Turell MJ, unpublished data). 1041 1042 TURELL AND OTHERS TABLE 1 Strains of Venezuelan equine encephalitis virus used in this study Strain of virus* V3000 TC-83 V1000 V3014 V3038 V3040 V3042 V3043 V3520 V3526 Description Parent clone† Current live-attenuated IND vaccine Deletion 5493-5595 (nucleotides) E2-209 (Glu→Lys), E1-272 (Ala→Thr) E3-59 (Arg→Asp) E1-253 (Phe→Ser) E1-81 (Phe→Ile) Nt. #3 G→A E2-76 (Glu→Lys), E2-209 (Glu→Lys), E1-81 (Phe→Ile) PE-2 cleavage site deletion 1 E1-253 (Phe→Ser) * Potential vaccine candidate strains were provided by N. Davis and R. Johnston.2,3,8 † Derived from the Trinidad donkey strain. Syrian hamsters.3 Hamsters that died after mosquito feeding were considered to have been infected with a virulent strain of VEE virus. In contrast, hamsters that survived their initial mosquito exposure, but died upon challenge with the V3000 strain were considered not to have been infected by mosquito bite, while those that survived challenge were considered to have been infected with an avirulent strain of VEE virus by mosquito bite, and immunized by that infection. Oral exposure studies. We used a membrane feeder to feed female Ae. taeniorhynchus on a heparinized goose blood-virus suspension containing serial dilutions of V3000, V3526, or TC-83 viruses. After a 30-min feeding period, unengorged mosquitoes were removed and discarded, while engorged mosquitoes were held as described above for inoculated mosquitoes. After 14 days, the mosquitoes were cold anesthetized and their legs and bodies were individually triturated in 1 ml of diluent and tested for the presence of virus as described above. Recovery of virus from the body, but not the legs, indicated that viral infection was limited to the midgut and had not disseminated to the hemocoel, while recovery of virus from both legs and body indicated that the mosquito had a disseminated infection.10 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. TABLE 2 Replication and transmission of selected strains of Venezuelan equine encephalitis virus in Aedes taeniorhynchus seven days after inoculation of approximately 101.5 plaque-forming units of virus Strain of virus V3000 TC-83 V1000 V3014 V3038 V3040 V3042 V3043 V3520 V3526 Mean 6 SE (no. tested)* 7.1 7.3 6.6 7.2 6.2 6.5 6.7 6.5 5.9 5.5 6 6 6 6 6 6 6 6 6 6 0.1 0.2 0.3 0.1 0.5 0.2 0.2 0.1 0.1 0.1 (21) (6) (9) (13) (5) (5) (10) (4) (15) (21) Transmission rate (no. tested)† 81% 64% 89% 93% 40% 100% 80% 75% 21% 18% (43)a (36)a (9)a (14)a (5)abc (5)a (10)a (4)ab (14)bc (33)c Case fatality rate (no. infected)‡ 94% 17% 100% 0% 100% 100% 100% 100% 0% 0% (35)a (23)c (8)a (13)c (2)ab (5)a (8)a (3)ab (3)bc (6)c * Mean logarithm10 plaque-forming units per mosquito. † Percentage of feeding Ae. taeniorhynchus that transmitted virus by bite as shown by death from mosquito-transmitted virus or by hamster survival after challenge with the V3000 parent strain. Numbers followed by the same letter are not significantly different at a 5 0.05 by either a chi-square test or a Fisher’s exact test. ‡ Percentage of infected hamsters that died. Numbers followed by the same letter are not significantly different at a 5 0.05 by either a chi-square test or a Fisher’s exact test. tion as shown by hamster survival after challenge with the V3000 parent strain (Table 2). Again, V3520 and V3526 viruses were transmitted significantly (x2 . 5.7, df 5 1, P # 0.017) less efficiently than were either the parent virus or the current vaccine virus (TC-83). Transmission rates for the parent and TC-83 viruses were not significantly different (x2 5 2.25, df 5 1, P 5 0.13). Although the strain containing the mutation at E2–209 (V3014) replicated to high titer and was efficiently transmitted by bite, none of the 13 hamsters infected with this RESULTS Mosquito inoculation studies. All of the strains of VEE virus tested replicated in Ae. taeniorhynchus (Table 2). However, two viruses (V3520 and V3526) grew to significantly lower titers (T $ 10.9, degrees of freedom [df] 5 $ 19, P , 0.001) than did either the parent or TC-83 strains (Table 2 and Figure 1). In general, viral titers increased rapidly and reached their highest levels about four days after inoculation, and then gradually decreased with increased incubation (Figure 1). Similarly, all of the strains were transmitted by bite after the mosquitoes had been infected by intrathoracic inocula- FIGURE 1. Replication of V3000 virus, the current live-attenuated Venezuelan equine encephalitis (VEE) virus vaccine strain (TC83), and three live-attenuated vaccine candidate strains of VEE in Aedes taeniorhynchus after intrathoracic inoculation of approximately 102 plaque-forming units (PFU) of virus. Five mosquitoes were sampled at each time point. POTENTIAL MOSQUITO TRANSMISSION OF VEE VIRUS VACCINE STRAINS TABLE 3 Replication of selected strains of Venezuelan equine encephalitis virus in Aedes taeniorhynchus 14 days after ingestion of a viremic blood meal from a membrane feeder* Strain of virus V3000 TC-83 V3526 PFU/ml ingested No. tested Infection rate† Dissemination rate‡ 108 107 106 108 107 106 108 107 106 46 47 11 47 51 29 54 44 21 22 19 0 2 4 3 80 32 5 2 0 0 0 0 0 19 0 0 * PFU 5 plaque-forming units. † Percentage of mosquitoes containing virus. ‡ Percentage of mosquitoes containing virus in their legs. strain by mosquito bite died (Table 2). Similarly, none of the nine hamsters infected with V3520 or V3526 by mosquito bite or 20 hamsters inoculated intraperitoneally with 105 PFU of V3526 died (Turell MJ, unpublished data). In contrast, four (17%) of the 23 hamsters infected with TC-83 virus died of their initial infection. This was essentially identical to the mortality rate (16%) of the 25 hamsters that received an intraperitoneal inoculation of 105 PFU of TC-83 vaccine (Turell MJ, unpublished data). Oral exposure studies. To test the oral susceptibility of a potential vector mosquito, groups of Ae. taeniorhynchus were fed blood-virus suspensions containing 104–108 PFU/ ml of V3000, V3526, or TC-83 viruses from a membrane feeder. There were no significant differences in mosquito susceptibility or dissemination rates between viral preparations unless mosquitoes received artificial blood meals containing $ 107 PFU/ml (Table 3). Mosquitoes were significantly more susceptible to V3526 virus than to TC-83 virus when artificial blood meal titers contained 107 PFU/ml (x2 5 11.2, df 5 1, P # 0.001), and to both TC-83 and V3000 viruses when the artificial blood meal contained 108 PFU/ml (x2 $ 31.1, df 5 1, P # 0.001). Differences in dissemination rates were only apparent at the greatest viral concentration tested. Again, the greatest rates occurred in mosquitoes ingesting V3526 virus (x2 . 5.1, df 5 1, P # 0.022). DISCUSSION A major environmental concern with the use of live-attenuated virus vaccines is the potential for spread of either the vaccine virus or a pathogenic revertant to susceptible hosts. This was documented by the isolation of TC-83, the current IND live-attenuated VEE virus vaccine, from fieldcollected mosquitoes after TC-83 was used to vaccinate equines during an outbreak of VEE in the early 1970s.11 Aedes taeniorhynchus was selected for these studies because it was incriminated as a vector in several outbreaks of VEE.6,12,13 In addition to being a competent laboratory vector,7,14 it is a common mosquito that readily feeds on humans throughout its range. Although female Ae. taeniorhynchus became infected when they ingested the V3526 strain, a viremia $ 108 PFU/ml was needed for the development of a disseminated infection in this potential mosquito vector. Be- 1043 cause no viremia was detected in any of six monkeys inoculated with V3526 (Pratt W, unpublished data), the viremia needed for mosquito transmission is at least 100,000-fold greater than the potential viremia that would have been observed in nonhuman primates, making dissemination of this strain unlikely in the natural setting. Other point mutations in VEE virus, such as the one at E2–209 (V3014), had almost no effect on replication in, or transmission by, Ae. taeniorhynchus, but these viruses remained avirulent after mosquito passage and protected hamsters challenged with 104 LD50 of virulent IAB virus. The V3526 construct, which contains a four-amino acid deletion in the PE2 furin cleavage site as well as a suppressor mutation in E1,8 proved to be more immunogenic in mice than V3520 that contains three independently attenuating point mutations. As expected, the deletion mutant was genetically stable, and showed no apparent phenotypic reversion on sequential passage in cell culture or in serial passage in mice inoculated intracerebrally (Ludwig G, unpublished data). For these reasons, V3526 was selected over V3520 and other potential candidates, and is currently being assessed in further preclinical studies. Both V3526 and V3520 replicated in Ae. taeniorhynchus after intrathoracic inoculation and both could be transmitted by bite to a hamster. However, the two vaccine candidates, V3526 and V3520, not only replicated less efficiently in mosquitoes than did either TC-83 or the parent virus, but also were transmitted less efficiently by mosquito bite. More importantly, both of these vaccine candidates remained avirulent after mosquito passage. Based on the low viremias in nonhuman primates, reduced ability to replicate in and be transmitted by a known vector of epizootic VEE virus, and evidence that V3526 does not revert to virulence after mosquito passage, it is unlikely that mosquito passage of this virus vaccine candidate poses a significant environmental danger. Acknowledgments: We thank N. Davis and R. Johnston (University of North Carolina, Chapel Hill, NC) for providing the various strains of VEE virus used. We also thank D. Kline (Medical and Veterinary Entomology Research Laboratory, Gainesville, FL) for providing the MAVERL strain of Ae. taeniorhynchus; J. Johnson, J. Robles, and B. Reynolds for assistance in rearing the mosquitoes; D. Dohm for his assistance in processing specimens; and M. Hevey, G. Korch, and K. Kenyon for their critical reading of 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’ addresses: Michael J. Turell, John Kondig, and Jonathan F. Smith, Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702– 5011. George V. Ludwig, Diagnostic Systems Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 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, MD 21702–5011. REFERENCES 1. Pittman PR, Makuch RS, Mangiafico JA, Cannon TL, Gibbs PH, Peters CJ, 1996. Long-term duration of detectable neutralizing antibodies after administration of live-attenuated 1044 2. 3. 4. 5. 6. 7. TURELL AND OTHERS VEE vaccine and following booster vaccination with inactivated VEE vaccine. 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