Determination of the virulence of the pigmentation-deficient and pigmentation-/plasminogen activator-deficient strains of Yersinia pestis in non-human primate and mouse models of pneumonic plague.

The current human plague vaccine, a killed Yersinia pestis whole-cell preparation, does not protect against aerosol challenge and is reactogenic and antigenically undefined. Live attenuated Y. pestis, such as pigmentation-deficient (Pgm-) strains, have been used frequently as vaccines and are efficacious. They are used widely in plague research and assumed to be safe. However, they can cause serious adverse reactions, and their aerosol infectivity is not known. We tested the virulence of a defined Pgm- variant of the C092 strain of Y. pestis in mouse and non-human primate models of pneumonic plague. The ten-fold lower median lethal dose by the aerosol compared to the subcutaneous (s.c.) routes of the Pgm- strain in mice suggested that the Pgm- strain might be less attenuated by the former than by the latter route. After exposure of 16 African green monkeys to inhaled doses ranging from 1.1 x 10(4) to 8.1 x 10(7)cfu, eight died and eight survived. The terminal cultures collected from five of the non-survivors were all positive for Y. pestis. Two of the remaining three non-survivors were culture-negative but had pathologic and immunologic evidence of infection with Y. pestis, specimens could not be obtained nor the cause of death determined for the third one. The deaths were not dose-related, and there were some differences in the pathology associated with infection by the Pgm- strain compared to the wild-type (wt) strain. However, the Pgm- derivative was clearly virulent for monkeys by the aerosol route. A mutant of the Pgm- strain, which has a deletion in the plasminogen activator (Pla) virulence locus (pla), appeared to be more attenuated than was either the Pgm- single mutant (in NHPs and mice) or the Pla- single mutant strain (in mice) and has potential as a live vaccine.

Marburg virus vaccines: comparing classical and new approaches.

An effort to develop a safe and effective vaccine for Marburg virus (MBGV), one of the filoviruses known to cause high mortality rates in humans, led us to compare directly some of the merits of modern versus classical vaccine approaches for this agent. Prior work had established the MBGV-glycoprotein (GP), the only known virion surface antigen, as a candidate for inclusion in a vaccine. In this study, we vaccinated groups of Hartley guinea pigs with killed MBGV, live attenuated MBGV, soluble MBGV-GP expressed by baculovirus recombinants, MBGV-GP delivered as a DNA vaccine, or MBGV-GP delivered via an alphavirus RNA replicon. Serological responses were evaluated, and animals were challenged with a lethal dose of MBGV given either subcutaneously or via aerosol. Killed MBGV and replicon-delivered MBGV-GP were notably immunogenic and protective against MBGV, but results did not exclude any approach and suggested a role for DNA vaccines in immunological priming.

Onset and duration of protective immunity to IA/IB and IE strains of Venezuelan equine encephalitis virus in vaccinated mice.

Three vaccines developed for protection against IA/IB subtypes of Venezuelan equine encephalitis (VEE) virus were evaluated in mice for the ability to protect against systemic and mucosal challenges with a virulent virus of the IE subtype. The vaccines were the formaldehyde-inactivated C-84 and live attenuated TC-83 vaccines currently administered to people under investigational new drug (IND) status, and a new live attenuated vaccine candidate, V3526. V3526 was superior for inducing protection to VEE IA/IB within a week of vaccination, and protection persisted for at least a year. All three vaccines induced long-term clinical protection against peripheral or mucosal challenge with IE virus, with the mucosal immunity induced by attenuated vaccines lasting longer than that induced by the inactivated vaccine. These data show that the molecularly cloned V3526 vaccine induces equivalent or improved immunity to homologous and heterologous VEE viruses than the existing vaccines.

Combined effects of Venezuelan equine encephalitis IIIA virus and gamma irradiation in mice.

The combined effects of injury from exposure to ionizing radiation and the potential biological warfare agent Venezuelan equine encephalitis (VEE) virus remain largely unknown. To study these effects, 4- to 5-week-old B6D2F1/J female mice were given a sublethal whole-body 7 Gy dose of 60Co gamma-photon radiation followed 48 hours later by aerosol or intraperitoneal challenge with enzootic VEE IIIA virus. Survival was observed for 30 days. A single sublethal 7 Gy dose of gamma radiation reduced the LD50/30 of VEE IIIA virus, in intraperitoneal challenged mice by a factor of 10(4) from 1.1 x 10(6) plaque-forming units (pfu) to 1 x 10(2) pfu, and in aerosol challenged mice, by a factor of 5 from 70 pfu to 14 pfu. These findings further confirm there is a combined effect of exposure to ionizing radiation and biological warfare agents, which could be devastating to unprotected populations and thus should be investigated further.

Improved mucosal protection against Venezuelan equine encephalitis virus is induced by the molecularly defined, live-attenuated V3526 vaccine candidate.

The genetically engineered, live-attenuated Venezuelan equine encephalitis (VEE) virus vaccine candidate, V3526, was evaluated as a replacement for the TC-83 virus vaccine. Protection from lethal subcutaneous or aerosol challenge was evaluated in vaccinated mice clinically and immunohistochemically. Subcutaneous administration of V3526 induced systemic and mucosal protection more efficiently than did the TC-83 vaccine. The bronchial IgA responses induced in mice by subcutaneous administration of vaccines significantly corresponded to the ability to survive aerosol challenge with virulent virus. Furthermore, V3526 delivered by aerosol induced more complete mucosal protection than either vaccine administered subcutaneously. The ability of V3526 to induce protection in mice warrants its consideration for further testing as a potential vaccine candidate for human use.

Comparison of noninvasive sampling sites for early detection of Bacillus anthracis spores from rhesus monkeys after aerosol exposure.

Bacillus anthracis, a spore-forming bacterium, is the etiologic agent of anthrax. B. anthracis spores can be aerosolized, are relatively easy to produce, and are capable of producing high mortality when inhaled. The prompt use of postexposure antibiotics combined with vaccination greatly increases the survival rate. Rapid detection of exposure is critical to effective case management. Using common collection swabs, culture medium, and culturing equipment, we compared six different noninvasive sampling sites to determine which might best be used to rapidly detect the presence of B. anthracis spores on rhesus monkeys after aerosolization. The results indicate that the greatest number of spores were deposited in the nares, on the face, and on the haired portions of the head, suggesting that these locations are the most effective sampling sites when attempting to detect B. anthracis aerosol exposure.

Venezuelan equine encephalitis virus vaccines induce mucosal IgA responses and protection from airborne infection in BALB/c, but not C3H/HeN mice.

Immunization with either a live-attenuated (TC-83) or formalin-inactivated (C-84) vaccine for Venezuelan equine encephalitis (VEE) virus protected BALB/c mice from lethal VEE infection acquired subcutaneously or by aerosol. While vaccinated C3H/HeN mice were also protected from parenteral infection, neither vaccine protected these mice from an aerosol infection. The apparent vaccine failures in C3H/HeN mice could not be attributed to deficiencies in virus-neutralizing antibodies in serum, as these responses were typically of equal or higher titer than those observed in protected BALB/c mice before challenge. IgG subclass analysis offered no facile explanation: profiles of IgG2 alpha dominance were observed in C3H/HeN mice given either vaccine and in BALB/c mice given the live-attenuated vaccine, whereas BALB/c antibody responses shifted toward IgGl dominance after immunization with the killed C-84 vaccine. Data from immunized congenic mice showed that the H-2 genes from the C3H/He mice were not singularly responsible for the inability of these mice to resist aerosol infection with VEE virus. VEE virus-specific IgA responses were detected more frequently in respiratory and vaginal secretions obtained from the protected BALB/c mice.

Intranasal stimulation of long-lasting immunity against aerosol ricin challenge with ricin toxoid vaccine encapsulated in polymeric microspheres.

Intranasal (i.n.) immunization with ricin toxoid (RT) vaccine encapsulated in poly (lactide-co-glycolide) microspheres (RT-PLG-Ms) and poly (L-lactide) microspheres (RT-PLA-Ms) stimulated systemic and mucosal immune responses and protected mice from aerosolized ricin intoxication. High titers of anti-ricin IgG2a were stimulated in the serum of mice with one or two doses of RT-Ms 6 weeks postimmunization. However, in the lungs, no IgG2a or total IgG was elicited either with RT-Ms or with aqueous RT. At 6 weeks postimmunization, a single dose of the RT-Ms stimulated secretory IgA (sIgA) in the lungs of four of six mice, but a second immunizing dose did not enhance the stimulation. A single dose of aqueous RT vaccine failed to stimulate sIgA in the lungs, while, a second dose induced sIgA in 50% of the mice. One or two i.n. doses of RT-Ms protected most of the mice against lethal aerosol-delivered ricin toxin 6 weeks postimmunization. In contrast, protection was absent or marginal after one or two doses of aqueous RT vaccine. In both studies, the protection against lethal aerosol challenge was significantly better with one dose of RT-Ms than with two doses of aqueous vaccine, which may be attributed to the induction of sIgA in the lungs and the serum. Duration of the IgG2a and IgA in the serum, particularly that of IgG2a was much longer after the administration of RT-Ms than after the aqueous vaccine. The geometric mean IgG2a titers stimulated with two doses of RT-Ms remained high during 40 weeks postimmunization and were up to 25 times higher than the titers induced with aqueous RT vaccine. After 6 weeks, the IgG2a induced by two doses of aqueous vaccine was no longer detectable. Persistence of antibody response was predictive of efficacy. At 1 year postimmunization with two doses of RT-Ms, 100% of mice were protected against lethal ricin challenge. However, at the same time no protection was afforded by two doses of aqueous RT. The results of the present study consistently demonstrated the advantages of microencapsulated RT vaccine to stimulate effective and long-lasting protection by i.n. administration.

Nonvascular delivery of St. Louis encephalitis and Venezuelan equine encephalitis viruses by infected mosquitoes (Diptera: Culicidae) feeding on a vertebrate host.

We determined whether mosquitoes infected with the viruses St. Louis encephalitis (SLE) or Venezuelan equine encephalitis inoculate virus extravascularly or directly into the vascular system. Infected mosquitoes fed on the distal 3rd of the tails of suckling mice. Significantly more mice whose tails were amputated at the midpoint within 10 min of mosquito feeding survived than did siblings whose tails remained intact. Even when tails were amputated 1-6 h after SLE virus-infected mosquitoes fed, the median time to death was significantly longer in mice with amputated tails (7.1 d) than in mice with intact tails (5.8 d). We concluded that mosquitoes inoculated virus primarily extravascularly, rather than directly into the vascular system, while feeding on a vertebrate host. Extravascular, rather than intravascular, delivery of pathogens by mosquitoes may affect disease pathogenesis and vaccine efficacy.

Dependence of ricin toxoid vaccine efficacy on the structure of poly(lactide-co-glycolide) microparticle carriers.

Biodegradable microparticles made of poly(lactide-co-glycolide) (PLG) were used for protracted and pulsed-release of the incorporated ricin toxoid (RT) vaccine to reduce the multiple immunization doses and the time required to induce complete protection against lethal aerosol-borne ricin challenge. The release rate of RT encapsulated in PLG microparticles was controlled by polymer selection and varying the preparation procedures, which allowed us to control microparticle size and the distribution of the vaccine in the polymeric matrix. PLG-microparticles in which RT vaccine was distributed heterogeneously in small pockets stimulated a rapid antibody response which was independent of the polymeric composition of the carriers. PLG-microparticles in which RT vaccine was distributed homogeneously throughout the polymeric matrix induced a slower antibody response, which depended on the polymeric composition of the carriers. Administration of RT in homogeneous microparticles made from 50/50 PLG or 100% polylactide stimulated two distinct anti-ricin IgG peaks, while RT in heterogeneous microparticles stimulated identical IgG peaks. An early (3 weeks) and long-lasting (1 year or longer) anti-ricin antibody response was evoked by a single administration of encapsulated RT vaccine when prepared by the above-mentioned conditions. In contrast, three administrations of the aqueous RT were required to stimulate similar antibody response. Reduction of immunization time from 6 to 4 weeks was achieved with RT encapsulated in small homogeneous microparticles but not with homogeneous large microparticles. These results demonstrated the usefulness of biodegradable microparticles to improve the efficacy of immunization with RT vaccine and probably many other vaccines as well.

Arbovirus isolations from mosquitoes collected during 1988 in the Senegal River basin.

During August and September 1988, we collected adult mosquitoes from 14 locations in the Senegal River basin to search for evidence of Rift Valley fever (RVF) viral activity one year after the 1987 outbreak, which occurred along the Senegal-Mauritania border. More than 62,000 specimens representing 18 species in seven genera were collected with carbon dioxide-baited, solid-state Army miniature light traps and sheep-baited traps. Twenty virus isolations from Culex, Aedes, and Anopheles mosquitoes were recovered from six locations: Fanaye Diery (11), Bode (four), Matam (two), Diongui (one), Ndialene (one), and Ngoui (one). Species yielding viral isolates were Anopheles pharoensis (eight), Culex tritaeniorhynchus (three), Cx. univitattus gr. (three), Cx. antennatus (two), Cx. poicillipes (two), Ae. hirsutus (one), and An. gambiae (one). Viruses were identified by complement fixation, and virus and plaque-reduction neutralization testing as Ngari (Bunyavirus, Bunyaviridae) (n = 15), Babanki (Alphavirus, Togaviridae) (n = 3), Bagaza (Flavivirus, Flaviviridae) (n = 1), and Bangui (Bunyavirus-like) (n = 1). No evidence of any RVF viral activity in the Senegal River Basin was detected in the mosquitoes tested.

Susceptibility of selected strains of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) to chikungunya virus.

The relative susceptibility of selected strains of Aedes aegypti (L.) and Aedes albopictus (Skuse) fed on a viremic monkey to infection with chikungunya virus was determined. Infection rates were consistently higher in 10 strains of Ae. albopictus tested than in 7 strains of Ae. aegypti tested, regardless of the geographic location from which the strains originated or the dose of virus ingested. Similarly, virus dissemination rates were higher in the Ae. albopictus strains compared with the Ae. aegypti strains. For nearly all (11 of 12) strains tested of both species, groups of mosquitoes with one or more females with a disseminated infection transmitted virus by bite to weanling mice. Based on these studies, Ae. albopictus appears to be a more competent laboratory vector of chikungunya virus than does Ae. aegypti.

Feeding patterns of mosquitoes collected in the Senegal River basin.

Mosquitoes were collected with light traps baited with dry ice at 16 locations in Senegal during the rainy season of 1988. Of 62,055 mosquitoes identified, 1,384 (2.2%), representing 25 species in 4 genera, were bloodfed. Mosquito bloodmeals were screened by enzyme-linked immunosorbent assay against antisera to humans, bovines, goats, sheep and chickens. Overall, 88% of the bloodmeals tested were identified. Bloodmeals from 5 species of Culex mosquitoes comprised 82% of the number tested. Culex antennatus (36.6%) and Cx. tritaeniorhyncus (33.5%) were the most abundant. All Culex except those of the Cx. univittatus group fed most often on humans. Aedes species fed almost equally on all mammals tested, while species of Anopheles preferred cattle over humans, goats and sheep.

Generation and transmission of Rift Valley fever viral reassortants by the mosquito Culex pipiens.

Reassortant viruses containing heterologous S and M genomic RNA segments were obtained from both mosquito and vertebrate hosts that had been coinfected with Egyptian and Senegalese strains of Rift Valley fever (RVF) virus. The origin of the S and M RNA segments in each plaque-cloned virus was determined with monoclonal antibodies capable of differentiating the nucleocapsid protein (S segment marker) or the G1 glycoprotein (M segment marker) of the parental strains. In the mosquito Culex pipiens, reassortants were detected after sequential ingestion of parental viruses by interrupted feeding on two infected hamster hosts, after feeding on a single host that had been infected with both parental strains, and from individual mosquitoes inoculated intrathoracically with both parental strains. Reassortant viruses replicated efficiently in mosquitoes and were readily transmissible by bite to hamsters. Replication of a second infecting strain of RVF virus was, however, completely inhibited if that virus was inoculated into a mosquito greater than or equal to 48 h after the first viral strain. Genetic reassortment may provide a mechanism for increased heterogeneity, and thus affect the epidemiology and evolution of RVF virus.

Experimental infection of vertebrates of the Pocomoke Cypress Swamp, Maryland with Keystone and Jamestown Canyon viruses.

Experimental studies were conducted to assess the susceptibility of white-tailed deer (Odocoileus virginianus), gray squirrels (Sciurus carolinensis), and cottontail rabbits (Sylvilagus floridanus) to Jamestown Canyon (JC) and/or Keystone (KEY) virus infection. Viremia occurred in 5 of 6 deer inoculated with JC virus; however, all deer developed KEY virus neutralizing antibody. Based on the observation that antibody elicited by primary infection of deer with either KEY or JC virus exhibited partial heterologous neutralization in vitro, cross-challenge experiments were performed in these animals. Keystone virus failed to infect deer 30 days post primary JC virus infection; however, deer became infected when challenged with KEY virus 80 days after the initial JC virus infection as indicated by a substantial increase in antibody titer. Similarly, JC virus failed to produce viremia in immune animals infected with KEY virus 80 days previously, although 2 of the 3 animals challenged had serological evidence of infection. Three field-collected cottontail rabbits with no evidence of KEY antibody were readily susceptible to KEY virus infection and developed viremias of 1-4 days' duration; rabbits with KEY virus antibody did not develop viremia upon KEY virus challenge. Eight antibody-negative field-collected gray squirrels became viremic following injection with KEY virus; however, a comparable group of squirrels did not become viremic when injected with JC virus.

Isolation of St. Louis encephalitis virus from overwintering Culex pipiens mosquitoes.

Two strains of St. Louis encephalitis virus were isolated from overwintering mosquitoes collected in Maryland and Pennsylvania during January and February 1977. There isolations from Culex pipiens constitute evidence that a mosquito-borne flavivirus can persist in a vector mosquito in temperate climates during the winter season.