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.

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.

Streptococcus pneumononiae gyrase ATPase: development and validation of an assay for inhibitor discovery and characterization.

The rise in bacterial resistance to antibiotics demonstrates the medical need for new antibacterial agents. One approach to this problem is to identify new antibacterials that act through validated drug targets such as bacterial DNA gyrase. DNA gyrase uses the energy of ATP hydrolysis to introduce negative supercoils into plasmid and chromosomal DNA and is essential for DNA replication. Inhibition of the ATPase activity of DNA gyrase is the mechanism by which coumarin-class antibiotics such as novobiocin inhibit bacterial growth. Although ATPase inhibitors exhibit potent antibacterial activity against gram-positive pathogens, no gyrase ATPase activity from a gram-positive organism is described in the literature. To address this, we developed and optimized an enzyme-coupled phosphate assay and used this assay to characterize the ATPase kinetics of Streptococcus pneumoniae gyrase. The S. pneumoniae enzyme exhibits cooperativity with ATP and requires organic potassium salts. We also studied inhibition of the enzyme by novobiocin. Apparent inhibition constants for novobiocin increased linearly with ATP concentration, indicative of an ATP-competitive mechanism. Similar binding affinities were measured by isothermal titration calorimetry. These results reveal unique features of the S. pneumoniae DNA gyrase ATPase and demonstrate the utility of the assay for screening and kinetic characterization of ATPase inhibitors.