Induction, via immunization with a monoclonal antiidiotypic antibody, of viral envelope specific Ab3 antisera that neutralize retrovirus infectivity.

We have developed five mouse monoclonal antiidiotypic antibodies to the 35/56 (Ab1) rat monoclonal that neutralizes retroviral infectivity by binding to the gp70f epitope of murine leukemia retrovirus. The anti-Id nature of these five Ab2s was evidenced by their inability to react with a panel of six other rat IgG2a kappa monoclonals isotype-matched to the 35/56 anti-gp70f mAb1, including two to the distinct epitopes "g" and "h" of gp70, or to normal rat IgG2a. On the basis of several competition assays four mAb2 were clearly either directed to the paratope of anti-gp70f mAb1 (.1C7, .1B, and .E) or not (.A, representing a noninternal image Ab2 alpha anti-Id). The P3E8 mAb2 was difficult to classify. Based on relative efficiency in these assays, .1C7 was chosen for further study, and upon injection was able to induce Ab3 responses in C57BL/6, BALB/c, and CBA mice. The fact that the Ab3 activity was detected in a competitive ELISA in which the hyperimmune antisera blocked the binding of Ab1 to Ab2, plus the ability to raise Ab3 neutralizing antibodies in three different mouse strains were consistent with .1C7 as an internal image Ab2 beta anti-Id. These results thus indicate the potential for internal-image monoclonal antiidiotypic antibody-based vaccines for retroviral diseases.

Isolation and characterization of COX12, the nuclear gene for a previously unrecognized subunit of Saccharomyces cerevisiae cytochrome c oxidase.

We have cloned and sequenced COX12, the nuclear gene for subunit VIb of Saccharomyces cerevisiae cytochrome c oxidase. This subunit, which was previously not found in cytochrome c oxidase purified from S. cerevisiae, has a deduced amino acid sequence which is 41% identical to the sequences of subunits VIb of bovine and human cytochrome c oxidases. The chromosomal copy of COX12 was replaced with a plasmid-derived copy of COX12, in which the coding region for the suspected cytochrome oxidase subunit was replaced with the yeast URA3 gene. The resulting Ura+ deletion strain grew poorly at room temperature and was unable to grow at 37 degrees C on ethanol/glycerol medium, whereas growth was normal at both temperatures on dextrose. This temperature-dependent, petite phenotype of the deletion strain was complemented to wild-type growth with a single copy plasmid carrying COX12. Cytochrome c oxidase activity in mitochondrial membranes from the cox12 deletion strain is decreased to 5-15% of that in membranes from the wild-type parent, and this activity is restored to normal when the cox12 deletion strain is complemented by the plasmid-borne COX12. Optical spectra of mitochondrial membranes from the cox12 deletion strain revealed that optically detectable cytochrome c oxidase is assembled at room temperature and at 37 degrees C, although the heme a + a3 absorption is diminished approximately 50%. The N-terminal amino acid sequence of the protein encoded by COX12 is identical to the N-terminal sequence of a subunit found in yeast cytochrome c oxidase purified by a new procedure (Taanman, J.-W., and Capaldi, R. A. (1992) J. Biol. Chem. 267, 22481-22485). We conclude that COX12 encodes a subunit of yeast cytochrome c oxidase which is essential during assembly for full cytochrome c oxidase activity but apparently can be removed after the oxidase is assembled, with retention of oxidase activity. This is the first instance in which deletion of a subunit of cytochrome c oxidase results in assembly of optically detectable cytochrome c oxidase but having markedly diminished activity.