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Researchers subsequently isolated the gene that expresses HIS-62 and cloned it into a bacterial expression vector, pET19b. Further tests confirmed that the rHIS-62 induced protective immunity in BALB/c mice against intranasal challenge with a lethal dose of histoplasma. Currently, they are trying to identify the smallest fragment of rHIS-62 that can confer protective immunity, using 4 overlapping fragments of about 250 to 300 amino acids in length. Unfortunately, the hierarchy of proliferation response has been totally different for BALB/c mice immunized with whole H. capsulatum (fragment 3 highest, followed by 2, 4, and 1) than for mice immunized with rHIS-62 (fragment 1 highest, followed by 2, 3, and 4). This suggests that the fragments are being recognized and processed differently by the differently immunized mice.

When these proliferation experiments were repeated with Black-6 mice, which differ from BALB/c mice at the MHC locus, the mice immunized with whole organism did not respond to any of the four fragments. For mice immunized with rHIS-62, the hierarchy was fragment 2 highest, followed by 3, 4, and 1. Researchers do not know what the conformation of the antigen is in the organism, but they do know that adjuvants (which were used in these experiments) tend to linearize the antigen; this may explain at least part of the difference in how the fragments are being processed.

On the other hand, when mice were immunized with the four rHIS-62 fragments and challenged intranasally with a sublethal inoculum, fragment 3 produced the best protection in both strains of mice—about a log reduction in colony-forming units in the lungs and livers, and a 30- to 40-percent reduction in the spleens. Thus, there may be no correlation between the degree of stimulation and the degree of protection.

But while whole rHIS-62 had induced protective immunity against a lethal challenge in BALB/c mice, immunization with the fragments provided only 50 percent protection for fragment 4 and no protection at all for 1, 2, or 3. There are two possible explanations. First, hsp 60 has some adjuvant effect, at least in mycobacteria, but the region that induces it may not be on a particular fragment. Second, it may take all of the fragments working together to induce a strong proliferative response. This experiment is being repeated in Black-6 mice against intranasal challenge with a lethal dose of histoplasma. Researchers believe they will be able to demonstrated that fragment 4 of rHIS-62 can induce a protective immune response in mice.

Isolating Protective Immunogens for Coccidioides. Researchers are using a different approach for coccidioides, screening potential antigens with antisera from humans who have recovered from an infection. The genes for these antigens will be introduced into DNA vectors and used to immunize mice in hopes of getting a protective immune response. One of those antigens is the hsp 60 from coccidioides, which has about 90 percent identity with hsp 60 from histoplasma. Researchers have sequenced and cloned this gene and introduced it into a bacterial expression vector, pET21a or b. They have also inserted it into PCNV to see if it can induce genetic vaccination. An interesting question for future research is whether hsp 60 is crossprotective for histoplasma and cocci-



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