In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression.

Following the identification of the C-C chemokines RANTES, MIP-1alpha and MIP-1beta as major human immunodeficiency virus (HIV)-suppressive factors produced by CD8+ T cells, several chemokine receptors were found to serve as membrane co-receptors for primate immunodeficiency lentiretroviruses. The two most widely used co-receptors thus far recognized, CCR5 and CXCR4, are expressed by both activated T lymphocytes and mononuclear phagocytes. CCR5, a specific RANTES, MIP-1alpha and MIP-1 receptor, is used preferentially by non-MT2-tropic HIV-1 and HIV-2 strains and by simian immunodeficiency virus (SIV), whereas CXCR4, a receptor for the C-X-C chemokine SDF-1, is used by MT2-tropic HIV-1 and HIV-2, but not by SIV. Other receptors with a more restricted cellular distribution, such as CCR2b, CCR3 and STRL33, can also function as co-receptors for selected viral isolates. The third variable region (V3) of the gp120 envelope glycoprotein of HIV-1 has been fingered as a critical determinant of the co-receptor choice. Here, we document a consistent pattern of evolution of viral co-receptor usage and sensitivity to chemokine-mediated suppression in a longitudinal follow-up of children with progressive HIV-1 infection. Viral isolates obtained during the asymptomatic stages generally used only CCR5 as a co-receptor and were inhibited by RANTES, MIP-1alpha and MIP-1beta, but not by SDF-1. By contrast, the majority of the isolates derived after the progression of the disease were resistant to C-C chemokines, having acquired the ability to use CXCR4 and, in some cases, CCR3, while gradually losing CCR5 usage. Surprisingly, most of these isolates were also insensitive to SDF-1, even when used in combination with RANTES. An early acquisition of CXCR4 usage predicted a poor prognosis. In children who progressed to AIDS without a shift to CXCR4 usage, all the sequential isolates were CCR5-dependent but showed a reduced sensitivity to C-C chemokines. Discrete changes in the V3 domain of gp120 were associated with the loss of sensitivity to C-C chemokines and the shift in co-receptor usage. These results suggest an adaptive evolution of HIV-1 in vivo, leading to escape from the control of the antiviral C-C chemokines.

Tolerance and determinant hierarchy.

The overall T cell response to a multideterminant antigen consists of the sum of responses to a limited number of different determinants on the protein. Antigen-presenting cells (APCs) are crucial in delimiting the determinants on the protein to which a response will be mounted. This influence is apparent at two levels. First, the determinants that are generated and displayed by APCs in the thymus are pivotal in shaping the T cell repertoire that will be available for responding to antigen determinants in the periphery. Second, antigen processing affects the selection of determinants that become displayed by the various peripheral APC populations that are involved in inducing and promoting a T cell response. We have studied the effect of the display hierarchy on tolerance induction to individual determinants in transgenic mice expressing different serum levels of hen egg lysozyme. We have also analysed aspects of the processing machinery that contribute to shaping the hierarchy of determinant display on MHC class II molecules: proteolysis and reduction of disulfide bonds.

Epitope analysis of an HLA-B27-derived synthetic peptide: a possible approach to analyzing HLA class I antigens.

A monoclonal antibody, F3H7, was generated by immunizing mice with a synthetic peptide corresponding to residues 63-84 of the B*2705 allele of the HLA-B27 antigens. The reactive epitope and the contact residues on the peptide were localized by ELISA using a large panel of overlapping peptides as well as peptides with substituted amino acids. Residues corresponding to R75, D77 and L78 on the HLA-B27 protein appeared to be critical. The clarity of these results indicate that this is a potentially useful approach to the study of HLA class I epitopes.

Identification of antigenic genes in Trichinella spiralis by immunoscreening of cDNA libraries.

Genes encoding antigens of adult worm, newborn larvae and muscle larvae of Trichinella spiralis were identified by immunoscreening their corresponding cDNA libraries. The cDNA libraries of T. spiralis adult (3 day old, Ad3) and newborn larvae (NBL) were screened using the serum of a pig infected with 20,000 muscle larvae (ML) at 26 days post-infection (dpi). Fifty-two positive clones representing 18 unique genes were obtained from the Ad3 cDNA library. The deduced amino acid sequences of 8 cDNAs were sequence homologues of the serine protease-like protein family. In the screening of NBL cDNA library, 81 positive clones representing 8 unique genes were isolated and of these, 70 clones corresponded to an NBL stage-specific serine protease gene. The ML cDNA library was screened using pig anti-Trichinella serum obtained at 60 dpi and 18 positive clones representing 8 unique genes were identified. Ten clones encoded a protein that is identical to a T. spiralis serine protease inhibitor. In general, our results revealed that antigenic serine protease-like proteins were found during the two invasive stages, Ad and NBL when these libraries were screened using 26 dpi antiserum, whereas a serine protease inhibitor was found in abundance in the ML library when it is screened using 60 dpi antiserum. These data are consistent with serine proteases playing an important role during the invasive stages of Trichinella infections, but become inhibited or internally controlled when the parasite enters a more stable, non-developing environment.

Trichinella spiralis--a potential anti-tumor agent.

Murine forestomach carcinoma (cell line MFC), ascitic hepatoma (cell line H22) and sarcoma (cell line S180) solid tumor models were used to test the anti-tumor effect of Trichinella spiralis in vivo. Mice previously infected by oral administration of 400 viable T. spiralis larvae per mouse for 7 days were grafted with various solid tumor cell lines. Other groups of tumor-bearing mice were given caudal vein injection of crude extracts of adult and newborn larvae at 17.5, 35.0 or 70.0 mg kg(-1). These treatments to inhibit tumor growth were dose-dependent (p<0.05). The anti-proliferative activity of crude T. spiralis extract was examined in vitro at 0.035, 0.070 or 0.140 mg ml(-1) using MFC, H22, S180, human chronic myeloid leukemia cell line (K562) and hepatoma cell line (H7402), tumor cell proliferation in vitro was measured by methyl thiazolium stain and was inhibited in dose-dependent manner (p<0.05). At the same doses, crude T. spiralis extracts induced apoptosis of K562 and H7402 as detected by DNA fragmentation. Cell cycle analysis indicated that crude T. spiralis extracts, at 0.140 mg ml(-1), arrested the cell cycle of K562 and H7402 in G1 or S phase. It is concluded that T. spiralis contains anti-tumor active agent.

Expression cloning of new receptors used by simian and human immunodeficiency viruses.

Several members of the chemokine-receptor family serve, in conjunction with CD4, as receptors for the entry of human immunodeficiency virus type I (HIV-1) into cells. The principal receptor for entry of macrophage-tropic (M-tropic) HIV-1 strains is CCR5, whereas that for T-cell-line-tropic (T-tropic) strains is CXCR4. Unlike HIV-1, infection with either M-tropic or T-tropic strains of simian immunodeficiency virus (SIV) can be mediated by CCR5, but not CXCR4. SIV strains will also infect CD4+ cells that lack CCR5, which suggests that these strains use as yet unidentified receptors. Here we use an expression-cloning strategy to identify SIV receptors and have isolated genes encoding two members of the seven-transmembrane G-protein-coupled receptor family that are used not only by SIVs, but also by strains of HIV-2 and M-tropic HIV-1. Both receptors are closely related to the chemokine-receptor family and are expressed in lymphoid tissues. One of the receptors is also expressed in colon and may therefore be important in viral transmission. Usage of these new receptors following experimental infection of non-human primates with SIV strains may provide important insight into viral transmission and the mechanisms of SIV- and HIV-induced acquired immune-deficiency syndrome.