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Retroviral envelope protein p15E and antigenically related proteins have been implicated as potential mediators of immune dysfunction associated with retroviral infections and with neoplasia. Due to its extreme hydrophobicity, purified p15E has not been available in a nondenatured form or in sufficient quantities for detailed studies on the mechanisms of its immunosuppressive effects. Therefore, a plasmid was constructed to direct the synthesis in Escherichia coli of the major hydrophilic region of murine p15E. The purified recombinant p15E derivative, soluble under physiological conditions, inhibited by up to 60% (EC50 = 7.5 nM) the anti-CD3-driven proliferation of human T lymphocytes but had no effect on the proliferation of the transformed T-cell line Jurkat. The recombinant protein also inhibited, by up to an average of 92% (EC50 = 2.1 microM), the proliferation of the murine T-cell line CTLL-2. These data (i) provide direct evidence that a retroviral envelope protein can itself inhibit lymphoproliferative function and (ii) map the inhibitory activity to a specific region of p15E. The availability of soluble, recombinant p15E should facilitate studies of the pathogenesis of the immunosuppression accompanying retroviral infections and neoplastic diseases.

A synthetic heptadecapeptide, CKS-17, represents the highly conserved amino acid sequences occurring within the transmembrane envelope protein of many animal and human retroviruses. CKS-17 has been demonstrated to exhibit suppressive properties for numerous immune functions. We have recently shown that CKS-17 acts as an immunomodulatory epitope causing an imbalance of human type 1 and type 2 cytokine production and suppression of cell-mediated immunities. cAMP, an intracellular second messenger, plays an important role in regulation of cytokine biosynthesis--i.e., elevation of intracellular cAMP levels selectively inhibits type 1 cytokine production but has no effect or enhances type 2 cytokine production. Here, we demonstrate that CKS-17 induces dramatic rises in the intracellular cAMP levels of a human monocyte cell line and of human peripheral blood mononuclear cells in a time- and dose-dependent manner. A peptide corresponding to the reverse sequence of CKS-17, used as control, has no effect on intracellular cAMP levels. The cAMP-inducing ability of CKS-17 is significantly blocked by SQ-22536, an inhibitor of adenylate cyclase. These results indicate that CKS-17, a highly conserved component of the transmembrane proteins of immunosuppressive retroviruses, induces increased intracellular levels of cAMP via activation of adenylate cyclase and suggest that this retroviral envelope peptide may differentially modulate type 1 and type 2 cytokine production through elevation of intracellular cAMP levels.

The influence of a synthetic retroviral peptide, CKS-17, on T helper type 1 (Th1)- or Th2-related cytokines was investigated in human blood mononuclear cells. Cells were stimulated with staphylococcal enterotoxin A, anti-CD3 plus anti-CD28 monoclonal antibodies, or lipopolysaccharide to induce cytokine mRNA. mRNA was detected by a reverse transcription-polymerase chain reaction or Northern blot analysis. CKS-17 down-regulated stimulant-induced mRNA accumulation for interferon gamma (IFN-gamma), interleukin (IL)-2, and p40 heavy and p35 light chains of IL-12, a cytokine that mediates development of Th1 response. CKS-17 up-regulated stimulant-induced mRNA accumulation of IL-10 and did not suppress Th2-related cytokine (IL-4, IL-5, IL-6, or IL-13) mRNA expression. A reverse sequence of CKS-17 peptide, used as a control, showed no such action. Anti-human IL-10 monoclonal antibody blocked ability of CKS-17 to inhibit mRNA accumulation for IFN-gamma but not the CKS-17 suppressive activity of IL-12 p40 heavy chain mRNA. Thus, CKS-17-mediated suppression of IFN-gamma mRNA expression is dependent upon augmentation of IL-10 production by CKS-17. This conserved component of several retroviral envelope proteins, CKS-17, may act as an immunomodulatory epitope responsible for cytokine dysregulation that leads to suppression of cellular immunity.

A highly immunogenic epitope from a conserved COOH-terminal region of the human immunodeficiency virus (HIV) gp120 envelope protein has been identified with antisera from HIV-seropositive subjects and a synthetic peptide (SP-22) containing 15 amino acids from this region (Ala-Pro-Thr-Lys-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg). Peptide SP-22 absorbed up to 100% of anti-gp120 antibody reactivity from select HIV+ patient sera in immunoblot assays and up to 79% of serum anti-gp120 antibody reactivity in competition RIA. In RIA, 45% of HIV-seropositive subjects had antibodies that bound to peptide SP-22. Human anti-SP-22 antibodies that bound to and were eluted from an SP-22 affinity column reacted with gp120 in RIA and immunoblot assays but did not neutralize HIV or inhibit HIV-induced syncytium formation in vitro, even though these antibodies comprised 70% of all anti-gp120 antibodies in the test serum. In contrast, the remaining 30% of SP-22 nonreactive anti-gp120 antibodies did not react with gp120 in immunoblot assays but did not react in RIA and neutralized HIV in vitro. Thus, approximately 50% of HIV-seropositive patients make high titers of nonneutralizing antibodies to an immunodominant antigen on gp120 defined by SP-22. Moreover, the COOH terminus of gp120 contains the major antigen or antigens identified by human anti-gp120 antibodies in immunoblot assays.

Purified feline leukemia virus, UV light-inactivated feline leukemia virus, and a synthetic peptide (CKS-17) homologous to a well-conserved region of the transmembrane components of several human and animal retroviruses were each studied for their effects on IgG production by feline peripheral blood lymphocytes. Using a reverse hemolytic plaque assay, both the viable virus and the UV-inactivated feline leukemia virus, but not the CKS-17, activated B lymphocytes to secrete IgG. When staphylococcal protein A, a polyclonal B-cell activator, was used to stimulate IgG synthesis by feline lymphocytes, the viable virus, the UV-inactivated virus, and the CKS-17 peptide each strongly suppressed IgG secretion without compromising viability of the lymphocytes. These findings suggest that the immunosuppressive influences of feline leukemia virus on immunoglobulin synthesis may reside in a conserved portion of the envelope glycoprotein that includes the region homologous to CKS-17.