Cats vaccinated intranasally (i.n.) with a temperature sensitive feline infectious peritonitis virus (ts-FIPV) vaccine were protected against an FIP-inducing challenge. Seventeen of 20 vaccinated cats (85%) survived a rigorous virulent FIPV challenge that caused FIP in 12 of 12 non-vaccinated cats (100%), 10 (83%) of which died. Intranasal vaccination stimulated serum IgG and serum and salivary IgA antibody responses (measured by ELISA), FIPV-neutralizing antibody (VN), and a cell-mediated immune (CMI) response as measured by lymphocyte proliferation. The serum antibody response to vaccination was not associated with protection. In fact, the IgG, IgA and VN titres were much higher in control cats than in vaccinated cats following challenge suggesting an immune-mediated pathogenesis. In contrast, stimulation of a mucosal IgA response to vaccination was related to protection. The in vitro proliferation of peripheral blood lymphocytes in response to virulent FIPV was observed in vaccinated cats, in vaccinated and challenged cats but not in non-vaccinated challenged cats.

Binding and signaling proteins based on Escherichia coli alkaline phosphatase (AP; EC 3.1.3.1) were designed for the detection of antibodies. Hybrid proteins were constructed by using wild-type AP and point mutants of AP [Asp-101 -> Ser (DlOlS) and Asp-153 -> Gly (D153G)]. The binding function of the hybrid proteins is provided by a peptide epitope inserted between amino acids 407 and 408 in AP. Binding of anti-epitope antibodies to the hybrid proteins modulates the enzyme activity of the hybrids; upon antibody binding, enzyme activity can increase to as much as 300%o of the level of activity in the absence of antibody or can decrease as much as 40%, depending on the presence or absence of the point mutations in AP. The fact that modulation is altered from inhibition to activation by single amino acid changes in the active site of AP suggests that the mechanism for modulation is due to structural alterations upon antibody binding. Modulation is a general phenomenon. The properties of the system are demonstrated by using two epitopes, one from the V3 loop of human immunodeficiency virus type 1 gpl20 protein and one from hepatitis C virus core protein, and corresponding monoclonal antibodies. The trend of modulation is consistent for all hybrids; those in wild-type AP are inhibited by antibody, while those in the AP mutants are activated by antibody. This demonstrates that modulation of enzyme activity of the AP-epitope hybrid proteins is not specific to either a particular epitope sequence or a particular antibody-epitope combination.Regulation of enzyme activity by binding a factor, small molecule, or protein is common (signal transduction, allosteric enzymes). While systems of this type are abundant in nature, construction of an artificial system presents a challenge; the surface of the enzyme must be modified to create a binding site for another protein, yet at the same time the catalytic activity of the enzyme must be maintained. In addition, the binding event must result in structural changes that alter the catalytic activity of the engineered enzyme.Here, we report a molecular sensor system based on the regulation of the enzyme activity of genetically engineered Escherichia coli alkaline phosphatase (AP; EC 3.1.3.1). A known linear peptide epitope sequence is introduced onto the surface of AP in a defined location, thus creating a binding site for an antibody. We show that the enzymatic activity of such a hybrid enzyme is reduced or increased upon binding of an antibody specific for the epitope. The direction of activity change depends on the presence or absence of point mutations in the vicinity of the AP active site.AP is a nonspecific phosphomonoesterase with a high catalytic turnover number (kcat). The AP homodimer (Mr = 94,000) contains 4 Zn2+ and 2 Mg2+ metal ions (1). Several point mutations in the vicinity of the active site have been shown to alter the enzymatic activity of AP. Alteration of the The publication costs of this article were defrayed in part by page charge payment. This article...

The characteristics of a temperature sensitive feline infectious peritonitis virus (TS-FIPV) were examined. TS-FIPV, unlike its parent strain, DF2 wild type FIPV (WT-FIPV), propagated at 31 degrees C (permissive temperature) but not at 39 degrees C (nonpermissive temperature). This temperature preference of TS-FIPV was also demonstrated in cats by the ability of the virus to replicate only at the lower temperature in the upper respiratory tract and not at systemic sites where higher temperatures (38-39 degrees C) prevail. Viral structural proteins and RNA were synthesized at 39 degrees C but some undefined maturational defect prevented the formation of infectious TS-FIPV at its nonpermissive temperature. TS-FIPV was more thermolabile than WT-FIPV which indicated alterations in the structural proteins of TS-FIPV, and a difference in the envelope protein of the two viruses was revealed by Western blot analysis. Plaque assay characterization showed that TS-FIPV produced small plaques in comparison to the large plaques of WT-FIPV. These unique characteristics possessed by TS-FIPV may account for its nonvirulent nature and ability to stimulate protective immune responses in cats.

An epitope from the HIV-1 gp120 protein V3 loop has been inserted onto the surface of bacterial alkaline phosphatase at different positions in the vicinity of the enzyme active site, creating hybrid proteins that can bind to an anti-gp120 monoclonal antibody. One of the hybrid proteins, API1, has a 13 amino acid V3 loop sequence inserted between residues 407 and 408 of alkaline phosphatase. The enzymatic activity of this protein is modulated upon antibody binding. API1 maintains the full activity of the wild type alkaline phosphatase but in the presence of the anti-gp120 antibody, the enzyme activity is inhibited by 40-50%. Thus, the hybrid enzyme can be used to detect the presence of antibody in solution. The concept of signalling proteins may have a wide application. Two models for the mechanism of modulation, steric hindrance and allosteric regulation, are discussed.

Here we present the refined crystal structures of three different conformational states of the Asp153-->Gly mutant (D153G) of alkaline phosphatase (AP), a metalloenzyme from Escherichia coli. The apo state is induced in the crystal over a 3 month period by metal depletion of the holoenzyme crystals. Subsequently, the metals are reintroduced in the crystalline state in a time-dependent reversible manner without physically damaging the crystals. Two structural intermediates of the holo form based on data from a 2 week (intermediate I) and a 2 month soak (intermediate II) of the apo crystals with Mg2+ and Zn2+ have been identified. The three-dimensional crystal structures of the apo (R = 18.1%), intermediate I (R = 19.5%), and intermediate II (R = 19.9%) of the D153G enzyme have been refined and the corresponding structures analyzed and compared. Large conformational changes that extend from the mutant active site to surface loops, located 20 A away, are observed in the apo structure with respect to the holo structure. The structure of intermediate I shows the recovery of the entire enzyme to an almost native-like conformation, with the exception of residues Asp 51 and Asp 369 in the active site and the surface loop (406-410) which remains partially disordered. In the three-dimensional structure of intermediate II, both Asp 51 and Asp 369 are essentially in a native-like conformation, but the main chain of residues 406-408 within the loop is still not fully ordered. The D153G mutant protein exhibits weak, reversible, time dependent metal binding in solution and in the crystalline state.(ABSTRACT TRUNCATED AT 250 WORDS)