Characterization of recombinant OspA in two different Borrelia vaccines with respect to immunological response and its relationship to functional parameters.

BACKGROUND: Prevention of Lyme disease in dogs in North America depends on effective vaccination against infection by the tick vector-born spirochete Borrelia burgdorferi. Most vaccines effectively prevent spirochete transmission to dogs during tick feeding based on immunization with the outer-surface lipoprotein A (OspA) of B. burgdorferi. More recently, vaccines containing additional OspC protein moieties have been introduced. These are designed to enhance protection by forming a second line of defense within the vertebrate host, where OspC expression replaces OspA as the dominant surface antigen. However, supportive data for demonstration of OspC mediated protection is still lacking. Since OspA immunogenicity is of paramount importance to protection against spirochete transmission; this study was designed to compare the immunogenicity of two commercially available vaccines against the Borrelia burgdorferi OspA. We further characterized OspA antigen fractions of these vaccines with respect to their biochemical and biophysical properties. RESULTS: Two groups of beagle dogs (n = 9) were administered either: (1) a nonadjuvanted/monovalent, recombinant OspA vaccine (Recombitek® Lyme) or (2) an adjuvanted, recombinant OspA /OspC chimeric fusion vaccine (Vanguard® crLyme). The onset of the anti-OspA antibody response elicited by the nonadjuvanted/monovalent OspA vaccine was significantly earlier than that for the bivalent OspA /OspC vaccine and serum borreliacidal activity was significantly greater at all post-vaccination time points. As expected, only dogs inoculated with the bivalent OspA/OspC vaccine mounted a humoral anti-OspC response. However, only three out of nine dogs in that group had a positive response. Comparison of the OspA vaccine structures revealed that the OspA in the nonadjuvanted/monovalent vaccine was primarily in the lipidated form, eluting (SEC-HPLC) at a high molecular weight, suggestive of micelle formation. Conversely, the OspA moiety of the OspA/OspC vaccine was found to be nonlipidated and eluted as the monomeric protein. CONCLUSIONS: We hypothesize that these structural differences may account for the superior immunogenicity of the nonadjuvanted monovalent recombinant OspA vaccine in dogs over the adjuvanted OspA fraction of the OspA/OspC vaccine.

Fifteen-month duration of immunity for the serovar Grippotyphosa fraction of a tetravalent canine leptospirosis vaccine.

Forty-four specific pathogen-free beagles, median age 65 days, received two subcutaneous doses of either a commercially available, five-way combination vaccine or the same vaccine in combination with a tetravalent Leptospira bacterin (Canicola, Grippotyphosa, Icterohaemorrhagiae, Pomona). They were subsequently challenged with a pathogenic strain L kirschneri serovar Grippotyphosa 470 days following completion of the vaccination protocol. Titres of agglutinating serum antibodies were determined at various time points before and after both vaccination and challenge, along with postchallenge reisolation of the challenge organisms from blood and urine, and evaluation of renal histopathology. Clinical signs of generalised leptospirosis were not observed in any of the dogs after challenge. In order to demonstrate efficacy, leptospirosis was defined as having at least one positive urine sample and a positive renal histopathology score; or, in the absence of renal pathology, multiple positive urine samples. Leptospiremia was not demonstrated in any of the vaccinated dogs versus 27 per cent of the controls; leptospiruria was noted in 5 per cent of the vaccinates compared with 76 per cent of controls; and renal lesions were observed in 15 per cent of the vaccinates and 65 per cent controls. Using these criteria, the vaccine was able to significantly prevent leptospirosis (P=0.0001) in the vaccinated animals. This study establishes duration of immunity of at least 15 months for the prevention of disease and renal excretion of leptospires for the Leptospira serovar Grippotyphosa fraction of a quadrivalent Leptospira vaccine.

Efficacy of a nonadjuvanted recombinant FeLV vaccine and two inactivated FeLV vaccines when subject to consistent virulent FeLV challenge conditions.

The purpose of this study was to compare the efficacy of three FeLV vaccines, under identical conditions in a laboratory challenge model that closely mimics natural infection. Four groups of cats (n = 20 per group) were administered two doses of vaccine, 21 days apart, starting at 9-10 weeks of age (Purevax® FeLV, Versifel® FeLV, Nobivac® feline 2-FeLV, and a placebo). Cats were challenged 3 weeks later with a virulent, heterologous FeLV isolate. FeLV antigenemia was determined at weekly intervals from 3 to 15 weeks postchallenge. Circulating proviral DNA was determined on terminal PBMC samples. Following challenge, persistent antigenemia developed in 15 (75%) placebo-vaccinated cats, 3 (15%) cats in the Versifel FeLV vaccinated group, and 1 cat (5%) each in the Purevax FeLV and the Nobivac FeLV vaccinated groups. The prevented fractions for three vaccine groups were 93%, 93%, and 80% respectively. The adjusted p-values for all vaccine group comparisons fail to approach statistical significance. There was excellent agreement between proviral FeLV DNA in circulating PBMCs and persistent antigenemia. It is shown that when cats are managed under the same conditions during a virulent challenge, via the normal route of infection, the tested vaccines all show a comparable degree of protection.

Demonstration of the ability of a canine Lyme vaccine to reduce the incidence of histological synovial lesions following experimentally-induced canine Lyme borreliosis.

Lyme disease in dogs can be effectively prevented by vaccination against antigens expressed by the spirochete Borrelia burgdorferi during transmission by the tick vector Ixodes sp. Lyme vaccine efficacy has traditionally been based on indicators of infection following wild-caught tick challenge whereas most other types of vaccine are required to demonstrate protection from clinical signs of disease. In this vaccination-challenge study we sought to demonstrate the ability of a nonadjuvanted, outer surface protein A (OspA) vaccine to protect from infection and to prevent synovial lesions consistent with Borreliosis. Thirty, purpose-bred beagles were randomly divided into vaccinated and unvaccinated groups. The vaccinated group was administered two subcutaneous doses of a nonadjuvanted, purified, Borrelia burgdorferi OspA vaccine at a 21- day interval. Dogs were challenged by wild-caught, B. burgdorferi-infected ticks (Ixodes scapularis). Clinical signs, serology, Borrelia isolation and PCR evaluated antemortem vaccine efficacy. Postmortem histopathological analysis of synovial tissue was compared to antemortem infection status. Borreliosis was demonstrated by Borrelia isolation from skin biopsies in 13 out of 15 unvaccinated dogs. All unvaccinated dogs' Western blot profiles were consistent with infection. Two of 15 vaccinated dogs had at least one positive spirochete culture which cleared 91days post-challenge, and Western blot profiles were consistent with vaccination alone. No dogs, vaccinated or unvaccinated, exhibited clinical signs consistent with borreliosis. Based on a histopathological cumulative joint scoring system (CJS), all unvaccinated dogs had synovial lesions indicative of Lyme disease. Only one of the vaccinated dogs had a CJS that was greater than the statistical cut off score for the absence of synovial lesions. There was high correlation between clinical histopathology and spirochete isolation. Infection with B burgdorferi may produce inconsistent clinical signs of lameness. Histopathological changes in joints from infected dogs are reliable indicators of borreliosis and correlate well with other indicators of infection. This model provides support that vaccination with a nonadjuvanted, purified OspA vaccine offers protection from Borrelia infection and the resulting synovial lesions that can lead to clinical signs of lameness.

Diversity of antibody responses to Borrelia burgdorferi in experimentally infected beagle dogs.

Lyme borreliosis (LB) is a common infection of domestic dogs in areas where there is enzootic transmission of the agent Borrelia burgdorferi. While immunoassays based on individual subunits have mostly supplanted the use of whole-cell preparations for canine serology, only a limited number of informative antigens have been identified. To more broadly characterize the antibody responses to B. burgdorferi infection and to assess the diversity of those responses in individual dogs, we examined sera from 32 adult colony-bred beagle dogs that had been experimentally infected with B. burgdorferi through tick bites and compared those sera in a protein microarray with sera from uninfected dogs in their antibody reactivities to various recombinant chromosome- and plasmid-encoded B. burgdorferi proteins, including 24 serotype-defining OspC proteins of North America. The profiles of immunogenic proteins for the dogs were largely similar to those for humans and natural-reservoir rodents; these proteins included the decorin-binding protein DbpB, BBA36, BBA57, BBA64, the fibronectin-binding protein BBK32, VlsE, FlaB and other flagellar structural proteins, Erp proteins, Bdr proteins, and all of the OspC proteins. In addition, the canine sera bound to the presumptive lipoproteins BBB14 and BB0844, which infrequently elicited antibodies in humans or rodents. Although the beagle, like most other domestic dog breeds, has a small effective population size and features extensive linkage disequilibrium, the group of animals studied here demonstrated diversity in antibody responses in measures of antibody levels and specificities for conserved proteins, such as DbpB, and polymorphic proteins, such as OspC.

Evaluation of tyrosinase expression in canine and equine melanocytic tumors.

OBJECTIVE: To determine the tissue-restricted expression pattern of tyrosinase mRNA in canine and equine melanocytic tumors and relative tyrosinase and major histocompatibility complex (MHC) I mRNA expression in variants of melanocytic tumors. SAMPLE: 39 canine and 8 equine tumor samples and 10 canine and 6 equine normal tissue samples. PROCEDURES: RNA was isolated from formalin-fixed, paraffin-embedded tissues. Real-time PCR assays were designed to amplify canine and equine tyrosinase, S18 ribosomal RNA, and major histocompatibility complex I transcripts. Relative expression was determined by use of S18 as a reference and comparison with pigmented and nonpigmented normal tissues. RESULTS: High tyrosinase expression was found in all melanocytic tumors, compared with normal tissues, and expression had no correlation with presence or absence of tumor pigmentation. No significant difference in tyrosinase expression was found among histologic variants of melanocytic tumors. No correlation was found between MHC I and tyrosinase expression or tissue histologic classification. CONCLUSIONS AND CLINICAL RELEVANCE: In the present study, the methods used were highly sensitive and specific for detection of tyrosinase expression in equine and canine tumors, and overexpression of this transcript in melanomas was detected. This suggested that a DNA vaccine developed for use in dogs with melanoma that targets tyrosinase may be considered for use in other affected species, such as horses.

Safety and efficacy of a xenogeneic DNA vaccine encoding for human tyrosinase as adjunctive treatment for oral malignant melanoma in dogs following surgical excision of the primary tumor.

OBJECTIVE: To evaluate the safety and efficacy of a vaccine containing plasmid DNA with an insert encoding human tyrosinase (ie, huTyr vaccine) as adjunctive treatment for oral malignant melanoma (MM) in dogs. ANIMALS: 111 dogs (58 prospectively enrolled in a multicenter clinical trial and 53 historical controls) with stage II or III oral MM (modified World Health Organization staging scale, I to IV) in which locoregional disease control was achieved. PROCEDURES: 58 dogs received an initial series of 4 injections of huTyr vaccine (102 µg of DNA/injection) administered transdermally by use of a needle-free IM vaccination device. Dogs were monitored for adverse reactions. Surviving dogs received booster injections at 6-month intervals thereafter. Survival time for vaccinates was compared with that of historical control dogs via Kaplan-Meier survival analysis for the outcome of death. RESULTS: Kaplan-Meier analysis of survival time until death attributable to MM was determined to be significantly improved for dogs that received the huTyr vaccine, compared with that of historical controls. However, median survival time could not be determined for vaccinates because < 50% died of MM before the end of the observation period. No systemic reactions requiring veterinary intervention were associated with vaccination. Local reactions were primarily limited to acute wheal or hematoma formation, mild signs of pain at the injection site, and postvaccination bruising. CONCLUSIONS AND CLINICAL RELEVANCE: Results support the safety and efficacy of the huTyr DNA vaccine in dogs as adjunctive treatment for oral MM. IMPACT FOR HUMAN MEDICINE: Response to DNA vaccination in dogs with oral MM may be useful in development of plasmid DNA vaccination protocols for human patients with similar disease.

Molecular and pathological investigations of the central nervous system in Borrelia burgdorferi-infected dogs.

Although neurological signs have been reported sporadically in dogs with systemic Lyme disease, it is unknown if neuroborreliosis occurs in dogs. The current study systematically evaluates canine brains for evidence of Borrelia burgdorferi infection. Twelve Beagles were experimentally challenged with B. burgdorferi-infected ticks at 18 weeks of age, and 2 unexposed dogs served as controls. One of the uninfected dogs and 6 infected dogs were each given 5 daily immunosuppressive doses of dexamethasone starting at 153 days post-infection. Eleven dogs were confirmed as infected by skin punch biopsy polymerase chain reaction (PCR) and serology. Neurological signs were not seen in any dogs through the end of the 190-day study. Whole blood, serum, cerebrospinal fluid (CSF), and brains from all dogs were collected. DNA was extracted from blood, CSF, and brain and evaluated by PCR for B. burgdorferi. Formalin-fixed brain tissue was examined by histopathology, immunohistochemistry, and PCR. Immunohistochemical staining for B. burgdorferi antigen was negative in all cases. The CSF analysis was normal, and PCR was uniformly negative for B. burgdorferi in all dogs. Six of the 11 (45%) infected dogs had mild to moderate lymphoplasmacytic choroid plexitis, which was more pronounced in the immunosuppressed dogs. The lack of B. burgdorferi DNA and immunohistochemical evidence of organisms, including within the choroid plexus lesions, suggests that B. burgdorferi does not have a direct role in the etiopathogenesis of canine central nervous system pathology.

Rabies challenge of captive striped skunks (Mephitis mephitis) following oral administration of a live vaccinia-vectored rabies vaccine.

Twenty-four adult striped skunks (Mephitis mephitis) were administered the raccoon product formulation of Rabies Vaccine, Live Vaccinia-Vectored (Raboral V-RG, Merial Limited, Athens, Georgia, USA), either by oral instillation or in vaccine-filled coated sachets either as single or multiple doses. A control group remained unvaccinated. Twenty-three of the skunks were challenged 116 days postvaccination with rabies virus (skunk isolate). Six of six naive skunks succumbed to challenge. Four of six skunks that received the vaccine by oral instillation survived challenge. The skunks that did not survive failed to seroconvert following vaccination. None of the skunks that accepted multiple doses of the vaccine offered in coated sachets survived challenge, nor were rabies virus-neutralizing antibodies (VNAs) detected in the sera. Likewise, none of the five skunks ingesting a single sachet developed VNA against rabies. However, in this group one skunk did survive rabies challenge. This preliminary study showed that the vaccinia-vectored oral rabies vaccine Raboral V-RG, as formulated for use in raccoons, is capable of protecting a percentage of skunks against rabies. However, although the fishmeal-coated sachets were readily consumed, subsequent challenge of these animals revealed poor vaccine delivery efficiency.

Protection from challenge following administration of a canarypox virus-vectored recombinant feline leukemia virus vaccine in cats previously vaccinated with a killed virus vaccine.

OBJECTIVE: To compare protection against FeLV challenge obtained following administration of 2 doses of an adjuvanted, chemically inactivated, whole FeLV (FeLV-k) vaccine with protection obtained following administration of 1 dose of an FeLV-k vaccine followed by 1 dose of a canarypox virus-vectored recombinant FeLV (rCP-FeLV) vaccine. DESIGN: Prospective study. ANIMALS: Thirty-two 9-week-old domestic shorthair cats. PROCEDURE: Cats received 2 doses of the FeLV-k vaccine SC, 21 days apart (n = 11); 1 dose of the FeLV-k vaccine SC and, 21 days later, 1 dose of the rCP-FeLV vaccine transdermally (11); or 2 doses of physiologic saline (0.9% NaCl) solution (control; 10). Four weeks after the second vaccine dose, all cats were challenged with FeLV by means of oronasal administration. Blood samples were collected at weekly intervals beginning 21 days after challenge, and serum was tested for FeLV antigen. RESULTS: All 10 control cats became persistently infected (ie, FeLV antigen detected in > or = 3 consecutive serum samples) following FeLV challenge, whereas only 1 of 11 cats that received 2 doses of the FeLV-k vaccine and none of the 11 cats that received 1 dose of the FeLV-k vaccine and 1 dose of the rCP-FeLV vaccine did. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that protection against FeLV challenge obtained following SC administration of a single dose of an FeLV-k vaccine followed, 21 days later, by transdermal administration of a single dose of an rCP-FeLV vaccine was similar to that obtained following SC administration of 2 doses of the FeLV-k vaccine 21 days apart.