Sandra L. Bushmich

A recombinant West Nile virus envelope protein vaccine candidate produced in Spodoptera frugiperda expresSF+ cells

In this study, a recombinant truncated West Nile virus envelope protein antigen (rWNV-E) was produced in serum-free cultures of the expresSF+ insect cell line via baculovirus infection. This production system was selected based on its use in the production of candidate human and animal vaccine antigens. A defined fermentation and purification process for the rWNV-E antigen was established to control for purity and immunogenicity of each protein batch. The material formulated with aluminum hydroxide was stable for greater than 8months at 4 degrees C. The recombinant vaccine candidate was evaluated for immunogenicity and protective efficacy in several animal models. In mouse and hamster WNV challenge models, the vaccine candidate induced viral protection that correlated with anti-rWNV-E immunogenicity and WNV neutralizing antibody titers. The rWNV-E vaccine candidate was used to boost horses previously immunized with the Fort Dodge inactivated WNV vaccine and also to induce WNV neutralizing titers in naïve foals that were at least 14weeks of age. Furthermore, the vaccine candidate was found safe when high doses were injected into rats, with no detectable treatment-related clinical adverse effects. These observations demonstrate that baculovirus-produced rWNV-E can be formulated with aluminum hydroxide to produce a stable and safe vaccine which induces humoral immunity that can protect against WNV infection.

West Nile virus envelope protein: role in diagnosis and immunity.

Abstract: The role of antibodies to the West Nile virus envelope (E) protein in serodiagnosis and protection was examined. The E protein was expressed and purified in recombinant form. Antibodies to the E protein were detected in patients with West Nile virus infection. Passive immunization with rabbit anti‐E protein sera also partially protected mice from challenge with West Nile virus. The humoral response to the West Nile virus E protein is therefore useful as an aid in the diagnosis and may also play a role in immunity to infection.

Antibodies Targeting Linear Determinants of the Envelope Protein Protect Mice against West Nile Virus

The flavivirus envelope (E) protein mediates cellular attachment and fusion with host cell membranes and is recognized by virus-neutralizing antibodies. We raised antibodies against a broad range of epitopes by immunizing a horse with recombinant West Nile virus (WNV) E protein. To define epitopes recognized by protective antibodies, we selected, by affinity chromatography, immunoglobulins against immobilized linear peptides derived from parts of the E protein. Immunoglobulins binding 9 different peptides from domains I, II, and III of the E protein neutralized WNV in vitro. This indicates that multiple protective epitopes can be found in the E protein. Immunoglobulins recognizing 3 peptides derived from domains I and II of E protein protected mice against a lethal challenge with WNV. These immunoglobulins recognized the E proteins of related flaviviruses, demonstrating that antibodies targeting specific E protein epitopes could be developed for prevention and treatment of multiple flavivirus infections.

Viable Borrelia burgdorferi in the urine of two clinically normal horses

Diagnosticiansshould be aware of the possibilities of these concurrent viralinfections in raccoons with neurologic disorders.Acknowledgements We thank G. Moser, B. Lehman, andS. Hindman for technical assistance. This work was sup-ported by grants from the Pennsylvania Department of Ag-riculture and the Laboratory of Large Animal Pathology,University of Pennsylvania.References

Comparison of direct fluorescent antibody staining and real-time polymerase chain reaction for the detection of Borrelia burgdorferi in Ixodes scapularis ticks

Borrelia burgdorferi, the agent responsible for causing Lyme disease in humans and animals, is transmitted via the bite of infected Ixodes spp. ticks. Ticks removed from humans and animals are routinely tested by diagnostic laboratories to determine if they are infected with these bacteria. The objective of this study was to compare the efficacy of 2 commonly used methods, direct fluorescent antibody staining and real-time polymerase chain reaction (PCR), for the detection of B. burgdorferi in Ixodes scapularis ticks. One hundred and twenty-seven adult I. scapularis ticks collected in Connecticut, a Lyme disease endemic area, were tested, and results were compared. Results showed 24.8% ticks tested positive for Borrelia spp. by fluorescent antibody testing and 32.5% ticks were positive for B. burgdorferi by real-time PCR testing. When ticks were grouped into categories by level of engorgement (unengorged, partially engorged, and fully engorged), 95% of unengorged ticks, 90.5% of partially engorged, and 86.8% of engorged ticks tested were in agreement. Ten of the 127 ticks examined were too dehydrated to be tested by the fluorescent antibody technique; half of these tested positive by PCR. Real-time PCR appears to be the better of these 2 methods for the diagnosis of this bacterial infection in I. scapularis ticks.

Suspected clinical Lyme disease in horses: Serological and antigen testing differences between clinically ill and clinically normal horses from an endemic region

Abstract Equine Lyme disease is difficult to diagnose because of its nonspecific clinical signs and the high incidence of subclinical infection in endemic regions. In this study we compared serology, antigen presence, hematology, blood chemistries and clinical presentation of 22 horses from a highly endemic region that were clinically diagnosed with Lyme disease to that of 21 clinically normal horses from the same region. We found that horses clinically diagnosed with Lyme disease were more likely to have Borrelia burgdorferi spirochetal DNA in their blood and urine, have a higher percentage of positive immunoblots containing antibodies to certain B. burgdorferi proteins, and tend to have higher ELISA titers than healthy horses from the same region. These results may help to improve diagnostic testing for equine Lyme disease.

Direct detection of Lyme disease by digital microscopy

Studies were performed to test the hypothesis that an early direct diagnostic test for Lyme disease can be developed. This test involves automatic microscopic scanning of a relatively large volume of body fluids for the sparsely occurring Lyme disease causing agent, the spirochete bacterium Borrelia burgdorferi. Preliminary results indicate that segmentation, measurement, and classification techniques can be used to differentiate spirochetes from other materials in microscope images of blood smears stained with a fluorescent dye.<<ETX>>