Safety and immunogenicity of an inactivated eastern equine encephalitis virus vaccine

BackgroundEastern equine encephalitis virus (EEEV) is a mosquito borne alphavirus spread primarily in Atlantic and Gulf Coast regions of the United States. EEEV is the causative agent of a devastating meningoencephalitis syndrome, with approximately 30% mortality and significant morbidity. There is no licensed human vaccine against EEEV. An inactivated EEEV vaccine has been offered under investigational new drug (IND) protocols at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) since 1976.MethodsHealthy at-risk laboratory personnel received inactivated PE-6 strain EEEV (TSI-GSD 104) vaccine under two separate IND protocols. Protocol FY 99–11 (2002–2008) had a primary series consisting of doses on day 0, 7, and 28. Protocol FY 06–31 (2008–2016) utilized a primary series with doses on day 0 and 28, and month 6. Participants with an inadequate immune response, plaque reduction neutralization test with 80% cut-off (PRNT80) titer < 40, received booster vaccination. Volunteers with prior EEEV vaccination were eligible to enroll for booster doses based on annual titer evaluation.ResultsThe FY06-31 dosing schema resulted in significantly greater post-primary series immune response (PRNT80 ≥ 40) rates (84% vs 54%) and geometric mean titers (184.1 vs 39.4). The FY 06–31 dosing schema also resulted in significantly greater cumulative annual immune response rates from 1 to up to 7 years post vaccination (75% vs 59%) and geometric mean of titers (60.1 vs 43.0). The majority of probably or definitely related adverse events were mild and local; there were no probably or definitely related serious adverse events.ConclusionsInactivated PE-6 EEEV vaccine is safe and immunogenic in at-risk laboratory personnel. A prolonged primary series, with month 6 dose, significantly improved vaccine immunogenicity both post-primary series and longitudinally on annual titers. Despite decades of safe use under IND, full licensure is not planned due to manufacturing constraints, and ongoing development of alternatives.     

Molecular epidemiology of eastern equine encephalitis virus, New York

Perpetuation, overwintering, and extinction of eastern equine encephalitis virus (EEEV) in northern foci are poorly understood. We therefore sought to describe the molecular epidemiology of EEEV in New York State during current and past epizootics. To determine whether EEEV overwinters, is periodically reintroduced, or both, we sequenced the E2 and partial NSP3 coding regions of 42 EEEV isolates from New York State and the Eastern Seaboard of the United States. Our phylogenetic analyses indicated that derived subclades tended to contain southern strains that had been isolated before genetically similar northern strains, suggesting southern to northern migration of EEEV along the Eastern Seaboard. Strong clustering among strains isolated during epizootics in New York from 2003-2005, as well as from 1974-1975, demonstrates that EEEV has overwintered in this focus. This study provides molecular evidence for the introduction of southern EEEV strains to New York, followed by local amplification, perpetuation, and overwintering.     

A vaccine candidate for eastern equine encephalitis virus based on IRES-mediated attenuation

To develop an effective vaccine against eastern equine encephalitis (EEE), we engineered a recombinant EEE virus (EEEV) that was attenuated and capable of replicating only in vertebrate cells, an important safety feature for live vaccines against mosquito-borne viruses. The subgenomic promoter was inactivated with 13 synonymous mutations and expression of the EEEV structural proteins was placed under the control of an internal ribosomal entry site (IRES) derived from encephalomyocarditis virus (EMCV). We tested this vaccine candidate for virulence, viremia and efficacy in the murine model. A single subcutaneous immunization with 10(4) infectious units protected 100% of mice against intraperitoneal challenge with a highly virulent North American EEEV strain. None of the mice developed any signs of disease or viremia after immunization or following challenge. Our findings suggest that the IRES-based attenuation approach can be used to develop a safe and effective vaccine against EEE and other alphaviral diseases.     

Impact of naled on the mosquito vectors of eastern equine encephalitis virus

This letter questions the appropriateness of methodology used in a study by Howard and Oliver (J. Am. Mosq. Control Assoc. 13:315-325; 1988). Two independent data sets, collected for different purposes by 2 different groups, were subjected to statistical analysis to determine if the data sets differed. The experimental "design," as described by the authors, is an example of pseudoreplication, which arises when replicates are collected at a scale finer than the one for which conclusions of statistical testing are intended to be drawn. All of the components of a properly designed field experiment (control, replication, randomization, and interspersion) are missing from this study. The authors proceed to draw a series of conclusions from the data presented. Few, if any, of the conclusions can be supported by the evidence presented. The assertions put forward in this paper could have a severe negative impact on efforts to prevent transmission of arboviruses or other pathogens to humans and domestic animals.     

Envelope protein E1 as vaccine target for western equine encephalitis virus

Western equine encephalitis virus (WEEV) is a mosquito-borne RNA virus which causes lethal infection in humans and equines. There are no commercial vaccines or anti-WEEV drugs available for humans. We used replication-defective, human adenovirus serotype-5 (HAd5) as a delivery vector for developing WEEV vaccine. Our previous study found delivery of both E1 and E2 envelope proteins of WEEV by HAd5 vector offers complete protection against lethal challenge of WEEV. In this paper, we constructed a HAd5-vectored E1 vaccine, Ad5-E1. Mice given single-dose vaccination of Ad5-E1 were completely protected against both homologous and heterologous WEEV strains. The protection was rapid, which was achieved as early as day 7 after vaccination. In addition, Ad5-E1 induced a strong WEEV-specific T cell response. Our data suggest E1 is a potential target for developing single-dose, fast-acting, HAd5-vectored vaccine for WEEV.     

Susceptibility of the Aotus nancymaae owl monkey to eastern equine encephalitis

Eastern equine encephalitis virus (EEEV) is an arthropod-borne virus associated with life-threatening encephalitis in humans, equines, birds and many other domestic animals. To investigate the suitability of the Aotus nancymaae New World owl monkey as a viable animal model for EEE candidate vaccine testing we used clinical presentation, serology, viral isolation and PCR to evaluate pathogenesis and immunity in infected animals. Monkeys were inoculated subcutaneously (SQ) or intranasally (IN) with 10⁴ pfu of virulent EEEV and were initially followed for 45 days. While none of the animals displayed clinical signs of disease, all of the SC inoculated animals (n = 6) manifested a viremia averaging 3.2 days (±0.8 days). Likewise, serologic responses (IgM, IgG and PRNT) were observed in all SC infected animals. Interestingly, none of the IN inoculated animals (n = 6) became viremic or mounted an antibody response and no pathological abnormalities were observed in two animals that were necropsied on day 6 post-infection (p.i.) from each group. To determine if the antibodies produced by the SC inoculated animals were protective against homologous challenge, three animals from the SC group were serologically evaluated on day 253 p.i. and were administered an inoculum identical to initial challenge on day 270 p.i. A positive control group of four naïve animals was also infected as before. All of the naïve positive control animals manifested a similar viremia as observed initially, averaging 2.75 days (±0.5 days) while none of the previously challenged animals became viremic. On days 45 and 253 p.i. geometric mean PRNT titers in the SC group were 453 and 101, respectively. This study demonstrates that the Aotus nancymaae can be reproducibly infected with EEE virus and can serve as a suitable model for infection and immunogenicity for the evaluation of candidate vaccines against EEEV.     

Developing GIS-based eastern equine encephalitis vector-host models in Tuskegee,Alabama

Background

A site near Tuskegee, Alabama was examined for vector-host activities of eastern equine encephalomyelitis virus (EEEV). Land cover maps of the study site were created in ArcInfo 9.2^® from QuickBird data encompassing visible and near-infrared (NIR) band information (0.45 to 0.72 μm) acquired July 15, 2008. Georeferenced mosquito and bird sampling sites, and their associated land cover attributes from the study site, were overlaid onto the satellite data. SAS 9.1.4^® was used to explore univariate statistics and to generate regression models using the field and remote-sampled mosquito and bird data. Regression models indicated that Culex erracticus and Northern Cardinals were the most abundant mosquito and bird species, respectively. Spatial linear prediction models were then generated in Geostatistical Analyst Extension of ArcGIS 9.2^®. Additionally, a model of the study site was generated, based on a Digital Elevation Model (DEM), using ArcScene extension of ArcGIS 9.2^®.

Results

For total mosquito count data, a first-order trend ordinary kriging process was fitted to the semivariogram at a partial sill of 5.041 km, nugget of 6.325 km, lag size of 7.076 km, and range of 31.43 km, using 12 lags. For total adult Cx. erracticus count, a first-order trend ordinary kriging process was fitted to the semivariogram at a partial sill of 5.764 km, nugget of 6.114 km, lag size of 7.472 km, and range of 32.62 km, using 12 lags. For the total bird count data, a first-order trend ordinary kriging process was fitted to the semivariogram at a partial sill of 4.998 km, nugget of 5.413 km, lag size of 7.549 km and range of 35.27 km, using 12 lags. For the Northern Cardinal count data, a first-order trend ordinary kriging process was fitted to the semivariogram at a partial sill of 6.387 km, nugget of 5.935 km, lag size of 8.549 km and a range of 41.38 km, using 12 lags. Results of the DEM analyses indicated a statistically significant inverse linear relationship between total sampled mosquito data and elevation (R² = -.4262; p < .0001), with a standard deviation (SD) of 10.46, and total sampled bird data and elevation (R² = -.5111; p < .0001), with a SD of 22.97. DEM statistics also indicated a significant inverse linear relationship between total sampled Cx. erracticus data and elevation (R² = -.4711; p < .0001), with a SD of 11.16, and the total sampled Northern Cardinal data and elevation (R² = -.5831; p < .0001), SD of 11.42.

Conclusion

These data demonstrate that GIS/remote sensing models and spatial statistics can capture space-varying functional relationships between field-sampled mosquito and bird parameters for determining risk for EEEV transmission.

     

Weather factors in the prediction of western equine encephalitis epidemics in Manitoba.

Cases of western equine encephalitis in horses in 1987 in western USA and Manitoba, Canada were examined by backward trajectory analysis of winds. Culex tarsalis mosquitoes infected with western equine encephalitis virus could have been carried on southerly winds from Texas and Oklahoma to northern USA and from there to Manitoba. The presence of the Polar front over North Dakota and Minnesota at the end of July would have led to the landing of Cx. tarsalis in Montana and Wisconsin and prevented further carriage into Manitoba. Temperatures in southern Texas during the winter months (average daily maximum temperatures 19.7 degrees C and higher) would have permitted continuous transmission of western equine encephalitis virus by Cx. tarsalis in this area. Weather factors involved in outbreaks from 1975-88 were analysed to see if epidemics in Manitoba (23 or more cases in horses) could be predicted. The conditions for epidemics could be defined as follows: (a) the number of cases in horses in USA was 98 or more, (b) winds were southerly with speeds 45 kmh-1 or higher, and (c) counts of Cx. tarsalis females/light trap per day were 3.2 or higher. There were 3 or fewer cases in Manitoba, when the number of cases in USA was 27 or less, even when Cx. tarsalis counts were higher than 3.2. With Cx. tarsalis counts below 3 and/or unsuitable winds, or the Polar front further south, the number of cases in Manitoba was between 0 and 17, even when the number of cases in USA was from 38-172. Without information on the extent of infection further south, the weather variables would probably be more useful in excluding the possibility of an epidemic in Manitoba than in predicting one.     

Trajectory analysis of winds and eastern equine encephalitis in USA, 1980-5

Backward trajectories of winds were determined to identify possible sources of eastern equine encephalitis virus associated with isolation of virus from mosquitoes or birds or outbreaks in horses between 1980 and 1985 in Maryland, New Jersey, New York and Michigan, USA. The results of the trajectory analyses suggested that eastern equine encephalitis virus could have been carried by infected mosquitoes on surface winds at temperatures 13 degrees C or higher from North Carolina north-eastwards along the Atlantic Coast to Maryland and New Jersey and thence to upstate New York and from western Kentucky to Michigan. Landing of mosquitoes was associated with the presence of a cold front and rain leading to variations in the location and timing of outbreaks from year to year. The mosquito responsible was most likely to have been Culiseta melanura, but Coquillettidia perturbans and Aedes sollicitans could also have been involved. There may be a continual cycle of eastern equine encephalitis virus in mosquitoes and birds in south-eastern USA, from where the virus could be distributed by infected mosquitoes on the wind along the Gulf and Atlantic Coasts and up the Mississippi Valley.     

Immune interference in the setting of same-day administration of two similar inactivated alphavirus vaccines: Eastern equine and western equine encephalitis

We compared the effect on primary vaccination plaque-reduction neutralization 80% titers (PRNT80) responses of same-day administration (at different injection sites) of two similar investigational inactivated alphavirus vaccines, eastern equine encephalitis (EEE) vaccine (TSI-GSD 104) and western equine encephalitis (WEE) vaccine (TSI-GSD 210) to separate administration. Overall, primary response rate for EEE vaccine was 524/796 (66%) and overall primary response rate for WEE vaccine was 291/695 (42%). EEE vaccine same-day administration yielded a 59% response rate and a responder geometric mean titer (GMT) = 89 while separate administration yielded a response rate of 69% and a responder GMT = 119. WEE vaccine same-day administration yielded a 30% response rate and a responder GMT = 53 while separate administration yielded a response rate of 54% and a responder GMT = 79. EEE response rates for same-day administration (group A) vs. non-same-day administration (group B) were significantly affected by gender. A logistic regression model predicting response to EEE comparing group B to group A for females yielded an OR = 4.10 (95% CL 1.97–8.55; p = .0002) and for males yielded an OR = 1.25 (95% CL 0.76–2.07; p = .3768). WEE response rates for same-day administration vs. non-same-day administration were independent of gender. A logistic regression model predicting response to WEE comparing group B to group A yielded an OR = 2.14 (95% CL 1.22–3.73; p = .0077). We report immune interference occurring with same-day administration of two completely separate formalin inactivated viral vaccines in humans. These findings combined with the findings of others regarding immune interference would argue for a renewed emphasis on studying the immunological mechanisms of induction of inactivated viral vaccine protection.     

Chimeric Sindbis/eastern equine encephalitis vaccine candidates are highly attenuated and immunogenic in mice

We developed chimeric Sindbis (SINV)/eastern equine encephalitis (EEEV) viruses and investigated their potential for use as live virus vaccines against EEEV. One vaccine candidate contained structural protein genes from a typical North American EEEV strain, while the other had structural proteins from a naturally attenuated Brazilian isolate. Both chimeric viruses replicated efficiently in mammalian and mosquito cell cultures and were highly attenuated in mice. Vaccinated mice did not develop detectable disease or viremia, but developed high titers of neutralizing antibodies. Upon challenge with EEEV, mice vaccinated with >10(4) PFU of the chimeric viruses were completely protected from disease. These findings support the potential use of these SIN/EEEV chimeras as safe and effective vaccines.     

The use of an animal-baited net trap for collecting mosquitoes during western equine encephalitis investigations in Argentina

A large net trap was used to sample mosquito populations attracted to horses at three sites each in Santa Fe and Rio Negro Provinces, Argentina, during the austral summer of 1984. These provinces, as well as others in Argentina, were affected by a severe epizootic of western equine encephalitis (WEE) during 1982-83. Totals of 2,752 and 6,929 mosquitoes were collected in Santa Fe and Rio Negro Provinces during five and three trap nights, respectively. Culex mosquitoes of the subgenus Culex were predominant (45.8% of total) in the Santa Fe collections, although Aedes albifasciatus also was prevalent (21.7%). The latter species was predominant (95.7% of total) in the Rio Negro collections. The mosquito fauna was less complex (minimum of 6 species) in Rio Negro Province as compared to Santa Fe Province (minimum of 18 species). The advantages of the net trap indicate that this trap can become a useful tool in arbovirus ecology studies in other areas.