AIDSWEEKLY Plus, 9 Sep 1996
Daniel J. DeNoon, Senior Editor

The immunization protocol apparently is suitable for human testing.

"This should be well tolerated in humans," said William M. Mitchell of Vanderbilt University, Nashville, Tennessee. "This is an active biologic metabolite of vitamin D3. It is currently used in clinical medicine and is an FDA-approved drug."

Mitchell described the mouse studies in a presentation to the XI International Conference on AIDS, held July 7-12, 1996, in Vancouver, British Columbia, Canada.

Mitchell and colleagues constructed a 7742-base-pair plasmid that contained the gene sequence for the entire HIV gp160 envelope precursor glycoprotein under the control of the cytomegalovirus (CMV) early promoter gene. The plasmid was dubbed pCMVgp160.

In order to reduce the amount of plasmid needed to stimulate an immune response, the researchers used a technique known as facilitated DNA vaccination.

"Typically naked DNA is used to transfect muscle cells," Mitchell said. "But if you look at the majority of the publications on genetic vaccination, in muscle with few exceptions they use massive amounts of DNA. We are trying to reduce the amount of DNA by facilitated transfection."

In mouse experiments, the researchers found that they could enormously decrease the amount of DNA plasmid needed - by about two logs - by administering it complexed with the facilitator dioctylgylcylspermine (DOGS) in a 5:1 DOGS:DNA ratio.

Balb/c mice received intramuscular injections of the pCMVgp160/DOGS vaccine at a dose of 1 (micro)g. They obtained anti-gp160 immunoglobulin titers from serum and parotid secretions at 2.5 weeks and also analyzed the binding of gp160 to mucosal surfaces by immunohistochemical analysis of tissue samples.

All of the mice seroconverted, and some 80 percent had serum anti-HIV antibody levels at titers considered protective.

"We also got mucosal immune responses that we believed, and still do, were common mucosal responses," Mitchell said. But these mucosal responses were considered too low to be protective.

"We really didn't believe this was good enough so we then turned to a trick that we first learned about ... several years ago," Mitchell said.

The "trick" involves the previously described ability of a vitamin D3 metabolite to shift immune responses to a protein antigen from a systemic to a mucosal response.

The vitamin D3 derivative is 1(alpha),25- dihydroxycholecalciferol or 1,25(OH)[2]D3. This hormone modulates a number of cellular functions through nonnuclear pathways, particularly the stimulation of intestinal calcium absorption and osteoclastic bone resorption, resulting in the elevation of plasma calcium. Studies also suggest that it promotes differentiation of a number of cells including skin keratinocytes, , macrophages, lymphocytes, and myeloid leukemia cells.

"Dihydroxycholecalciferol is a super steroid, an active metabolite of vitamin D3," Mitchell said.

"It has biological activities: calcium phosphorous homeostasis, but less well studied is the induction of differentiation - particularly temporal expression of genes in embryogenesis.

"It also binds to a cytoplasmic receptor which translocates to the nucleus and binds to DNA at specific sites just like estrogen receptor, and in fact the receptor is in a common class of receptors that includes estradiol, progesterone, cortisol, retinoic acid, and allosterone. The receptors are present on monocytes, perhaps on B cells, cardiac muscle, smooth muscle, T lymphocytes, myeloblasts, and GI epithelium."

Most relevant, perhaps, are the effects of this active form of vitamin D3 on the immune system.

"It depresses or down-regulates IL-2, upregulates IL-1, turns on class II MHA, downregulates gamma interferon, upregulates HLA DR, and so on," Mitchell said.

When 1,25(OH)[2]D3 was added to the pCMVgp160/DOGS immunizing mixture at a dose of 1 (micro)g in the mouse experiment, it greatly stimulated the induction of mucosal IgA responses to gp160.

"The inclusion of 1,25(OH)[2]D3 at 1 (micro)g concentrations in the genetic vaccine resulted in significant IgA titers (>1:1250 in seven animals and 1:250 in a single animal)," Mitchell and colleagues reported in their presentation abstract.

Mice immunized with the 1,25(OH)[2]D3/DOGS/pCMVgp160 vaccine exhibited specific binding of gp160 to mucosal surfaces from the lung, jejunum, colon, and vagina at the cervical os.

"We have shown that with one injection of the genetic immunogen with the DOGS-facilitated transfection (with the super-steroid) that we can induce mucosal antibody responses," Mitchell concluded. "We think it's a common mucosal immune response."

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