Reactivity of primate sera to foamy virus Gag and Bet proteins.

In order to establish criteria for the serodiagnosis of foamy virus infections we investigated the extent to which sera from infected individuals of human and primate origin react with structural and non-structural virus proteins in immunoblot assays. Using lysates from infected cells as the source of virus antigen, antibodies were preferentially detected against the Gag proteins and the non-structural Bet protein. Both the Gag precursor molecules of 70 and 74K apparent M(r) and the cytoplasmic 60K M(r) Bet protein were found to be phosphorylated, the latter being synthesized in large amounts in infected cells. Rabbit antiserum raised against recombinant human foamy virus (HFV) Gag major capsid protein cross-reacted with foamy viruses of chimpanzee, gorilla, orang-utan, rhesus monkey and African green monkey origin. This was reflected by a broad cross-reactivity of the respective monkey sera to the Gag proteins of the various foamy virus isolates. Cross-reactivity of antisera against the Bet protein was restricted to viruses from man and the great apes. Recombinant Gag and Bet proteins expressed in prokaryotes or in insect cells were readily recognized by foamy virus-positive primate sera. Screening serum samples from chimpanzees with HFV Gag and Bet proteins expressed by recombinant baculoviruses revealed that 18 out of 35 (52%) were positive for Gag antibodies. Of these, 13 (72%) showed antibodies against the Bet protein, indicating that Bet antigen is of value in serological screening for foamy virus infections.

Formation of poliovirus-like particles by recombinant baculoviruses expressing the individual VP0, VP3, and VP1 proteins by comparison to particles derived from the expressed poliovirus polyprotein.

The cDNA sequences encoding the VP0, VP3, and VP1 structural proteins of poliovirus type 3 (strain P3/Leon/37) have been individually cloned and used to prepare recombinant baculoviruses based on Autographa californica nuclear polyhedrosis viruses (AcNPV). Expression of the proteins was monitored in virus-infected Spodoptera frugiperda cells. Both the individually expressed VP0 protein and VP0 derived from the complete poliovirus coding region were shown to incorporate myristic acid. A significant improvement in VP0 protein yield was obtained when the amino terminal glycine of VP0 was changed to alanine (VP0/ala), suggesting that the presence of glycine or its myristylation is unfavorable for VP0 expression. Even so, the expression levels of the poliovirus capsid proteins were low by comparison with those obtained for other foreign genes (e.g., lacZ). The reason does not appear to be due to protein instability or, from the studies undertaken with VP0, the sequences that flank the AUG codon. Using recombinant baculoviruses that express VP0, VP1, and VP3 (or VP0/ala, VP1, and VP3), poliovirus-like particles (VLPs) were isolated from infected S. frugiperda cells. Examination by electron microscopy of the VLPs purified by CsCl gradient centrifugation revealed structures corresponding in size, appearance, and antigenicity to those expected for poliovirus procapsids; however, the yields of particles were low when compared to those derived from a construct (AcLeon) that expresses the complete coding region of poliovirus type 3, indicating that procapsid synthesis from the P1 precursor is a more favored route.

Preparative separation of foreign antigens for highly efficient presentation to T cells in vitro.

A method is described for the separation and purification of proteins from complex mixtures of foreign antigens in a form suitable for stimulating T cells in vitro. The technique involves electrophoretic separation of proteins followed by elution, concentration and adsorption of the polypeptide subunits to latex microspheres. Alternatively, where a specific antibody is available, proteins may be affinity-purified from a heterogeneous mixture of antigens, using antibody-coated latex microspheres. Nanogram quantities of protein coupled to latex were shown to be highly efficient stimulators of antigen-specific T cells as tested by in vitro proliferation and cytokine release assays. The utility of this technique was demonstrated using poliovirus capsid proteins separated by SDS-polyacrylamide gel electrophoresis (PAGE) and coupled to latex microspheres for specificity analysis of T cell clones. Antigen reactivity of the T cell clones was confirmed using recombinant baculoviruses expressing individual poliovirus proteins. Furthermore, recombinant proteins coupled to latex microspheres were used for efficient stimulation and in vitro propagation of T cell clones specific for the simian immunodeficiency virus (SIV) envelope (env) protein. Although the technique is illustrated in this report using viral antigens, it has also proved to be an efficient method for the separation of bacterial antigens in studies of polyclonal T cell responses to Bordetella pertussis antigens.