The hypothesis that open conformers of HLA-C on target cells might directly exert an effect on th... more The hypothesis that open conformers of HLA-C on target cells might directly exert an effect on their infectability by human immunodeficiency virus (HIV) has been suggested previously. This was tested by exploiting the peculiar specificity of monoclonal antibody (mAb) L31 for HLA-C open conformers to show that normal levels of Env-driven fusion were restored in HLA-C transfectants of a major histocompatibility complex-deleted (fusion-incompetent) cell line. The physiological relevance of this finding is now confirmed in this report, where small interfering RNA (siRNA) technology was used to silence HLA-C expression in peripheral blood lymphocytes (PBLs) from 11 healthy donors. Infectability by HIV (strains IIIB and Bal and primary isolates) was significantly reduced (P50.016) in silenced cells compared with cells that maintained HLA-C expression in 10 of the 11 PBL donors. Normal infectability was resumed, together with HLA-C expression, when the effect of siRNA interference waned after several days in culture. Additional confirmation of the HLA-C effect was obtained in several assays employing HLA-C-positive and -negative cell lines, a number of HIV strains and also pseudoviruses. In particular, viruses pseudotyped with env genes from HIV strains AC10 and QH0692.42 were assayed on siRNA-silenced lymphocytes from three healthy donors: the differences in infection with pseudoviruses were even higher than those observed in infections with normal viruses.
Although selected chemokines act as natural inhibitors of human immunodeficiency virus (HIV) infe... more Although selected chemokines act as natural inhibitors of human immunodeficiency virus (HIV) infection, their inherent proinflammatory activity may limit a therapeutic use. To elucidate whether the antiviral and signaling functions of RANTES can be dissociated, several recombinant analogues mutated at the N terminus were generated and functionally compared with the wild-type (WT) molecule, as well as with three previously described mutants. Substitution of selected residues within the N-terminal region caused a marked loss of antiviral potency. By contrast, two unique analogues (C1.C5-RANTES and L-RANTES) exhibited an increased antiviral activity against different CXCR4-negative HIV-1 isolates grown in primary mononuclear cells or in macrophages. This enhanced HIV-blocking activity was associated with an increased binding affinity for CCR5. Both C1.C5-RANTES and L-RANTES showed a dramatically reduced ability to trigger intracellular calcium mobilization via CCR3 or CCR5, while potently antagonizing the action of the WT chemokine. By contrast, two previously described analogues (RANTES 3-68 and AOP-RANTES) maintained a WT ability to trigger CCR5-mediated signaling, while a third one (RANTES 9-68 ) showed a dramatic loss of antiviral activity. These data demonstrate that the antiviral and signaling functions of RAN-TES can be uncoupled, opening new perspectives for the development of chemokine-based therapeutic approaches for HIV infection.
Cross-reactive antibodies to HLA class I and HIV-1 gp120 were detected in the sera of HIV-1-posit... more Cross-reactive antibodies to HLA class I and HIV-1 gp120 were detected in the sera of HIV-1-positive individuals, and were found to share the same epitope specificity as a gp120-HLA class I cross-reactive monoclonal antibody (M38). The amino acid residues of HLA recognized by both M38 and the patient antibodies occur as a clustered pair in the HLA-C alpha 1 domain. These sequences (KYKR and RKLR) are shared by almost all HLA-C alleles and are available to antibody binding only on beta 2-microglobulin-dissociated HLA heavy chains expressed on activated cells. Similar to M38, the antibody-binding sites on HIV-1 gp120 were mapped to two noncontiguous stretches of amino acids (KYK and KAKR), which flank a hydrophobic area of the immunodominant C5 region involved in gp41 binding. Serum antibodies immunoaffinity purified on synthetic HLA and gp120 peptides representing the M38-reactive regions were shown to bind to both HLA and gp120 in Western blot, as well as to membrane-bound HLA heavy chains, and to exhibit selective complement-mediated lysis of activated T cells. No serum antibodies could be detected that bind to the gp120 C5 region (peptide IEPLGVAPT) flanked by the two HLA-like sequences.
Molecular mimicry of major histocompatibility (MHC) antigens by viral glycoproteins has been sugg... more Molecular mimicry of major histocompatibility (MHC) antigens by viral glycoproteins has been suggested as one of the possible mechanisms of induction of an autoimmune response by human immunodeficiency viruses. A monoclonal antibody (M38) was previously shown to bind to both human immunodeficiency virus type 1 (HIV-1) gp120 and β-2 microglobulin-free HLA class I heavy chains encoded by an HLA C allele. Using HLA C recombinant proteins and synthetic peptides, the M38 class I binding site was mapped to a stretch of 44 amino acids of the al domain. The amino acid residues recognized are clustered in two non-contiguous regions at positions 66-69 (KYKR) and 79-82 (RKLR) shared by almost all HLA C alleles. On HIV-1 gp120, M38 binds to two non-contiguous sequences (KYK and KAKR) at positions 490-492 and 505-508 located at the edges of a large hydrophobic region that is apparently involved in binding the transmembrane glycoprotein gp41. The C-terminal gp120 M38-reactive region (KAKR) lies within the immunodominant sequence APTKAKRRVVQREKR, against which the majority of HIV-infected individuals produce antibodies. The results indicate that a functionally important region of HIV-1 gp120 shares similar amino acid sequence motifs with the antigen recognition site of most HLA class I C alleles. The molecular mimicry may be the basis for autoimmune responses in HIV infection.
A growing number of reports indicates that certain groups of individuals who almost certainly hav... more A growing number of reports indicates that certain groups of individuals who almost certainly have been exposed to human immunodeficiency virus (HIV), yet continue to exhibit no signs or symptoms of infection, often have subtle evidence of specific immunity. We studied such a high-risk (HR) cohort of persistently seronegative individuals with histories of long-term sexual exposure to an HIV-infected partner to look for evidence of both humoral and cellular immunity that might have been induced by exposure to the virus. Twenty-three heterosexual and four homosexual monogamous couples with discordant HIV status were included in the study. Twelve of the HR partners were studied for in vitro stimulation of peripheral blood mononuclear cells (PBMC) by HIV envelope-derived peptides. All 12 responded overwhelmingly to a peptide containing the fifth conserved region of gp120. By generating and cloning T cell lines specific for this peptide, we concluded that in these individuals the T cell response to the envelope is mainly focused on the carboxy-terminus region of gp120 and is characterized by an oligoclonal expansion of CD4+ T cells expressing the same TCR Eighteen HR partners and 37 HIV-1 seropositive subjects were tested for the presence of anti-CD4 antibodies (anti-CD4 Abs) using a recombinant CD4-based enzyme-linked immunosorbent assay (ELISA). Anti-CD4 Abs were detected in eight of the HR partners (six confirmed by Western blot) and in nine of the HIV-1 seropositive subjects (eight confirmed by Western blot). Results from binding competition assays with a panel of monoclonal anti-CD4 Abs suggested that the anti-CD4 Abs detected in the HR partners are directed toward epitopes that are induced by gp120 binding. Twenty-seven of the HR partners were tested for the presence of antibodies that cross-react with HLA class I and gp120 (anti-HLA Abs). Anti-HLA Abs were detected in 16 of the HR partner sera and in 4/94 sera from a control population of normal healthy blood donors. Taken together, the results suggest that in some individuals with a history of long-term exposure to HIV, specific immunity may develop in the absence of overt infection. The common trigger for these responses is gp120.
The double NF-KB site identified in the LTR of the human immunodeficiency virus-1 (HIV-1) has bee... more The double NF-KB site identified in the LTR of the human immunodeficiency virus-1 (HIV-1) has been demonstrated to be necessary for efficient viral transcription. In this report we present the characterisation of NF-KB subunits engaged in complexes binding to the HIV-1 NF-KB site in human 8e51 Tcells, that harbour a defective HIV-1. At least four different specific NF-KB complexes are present in the nucleus of these cells. With the use of specific antibodies we have determined the composition of each complex using electrophoretic mobility shift assays. The results show the presence of several NF-KB family members, with the transactivating RelA being engaged in multiple complexes. The importance of NF-KB complexes in viral functions has been established comparing the level of NF-KB DNA-binding complexes with syncytia-forming activity of 8e51 cells. In fact, 8e51 cells that had almost lost their syncytiaforming capacity were found to contain at least 10 times less active NF-KB DNA-binding complex than the actively fusing cells. The correlation is specific as the level of at least three other transcription factors did not change.
Epitope mapping of HLA-Cw4 indicates that the two monoclonal antibodies (mAbs) L31 and M38, speci... more Epitope mapping of HLA-Cw4 indicates that the two monoclonal antibodies (mAbs) L31 and M38, specific for beta 2-microglobulin ([32m)-free HLA-C heavy chains, react preferentially with the KYK motif, located in the binding groove (od domain). Transfection of HLA-Cw4 cDNA into a neuroblastoma cell line, which normally expresses negligible HLA class I, resulted in the constitutive surface expression of molecules displaying different reactivities with the two mAbs. This cellular system was used to determine whether L31 and M38 recognize distinct conformations of ~2m-free HLA-C proteins, and to investigate their mechanism of expression. Interferon-y greatly enhanced the expression of L31-reactive free chains, while abolishing that of M38-reactive molecules. The cytokineinduced expression of L31-reactive molecules was inhibited by anti-sense oligonucleotides specific for ~2m mRNA, while constitutive expression of L31-reactive molecules was only partially affected. Exogenous ~2m resulted in a reduction of constitutive L31 reactivity, and in a concomitant increase of M38 reactivity. These results indicate that: 1) at the cell surface, L31 and M38 react with two distinct conformations of HLA-Cw4 [32m-free heavy chains, of which the L31-reactive conformation is the least folded; 2) the expression of both conformers can be modulated by endogenous or exogenous ~2m; and (3) L31-reactive molecules exposed at the cell surface are likely to derive from the dissociation of empty HLA-Cw4/[~2m Complexes.
Cross-reactive antibodies to HLA class I and HIV-1 gp120 were detected in the sera of HIV-1-posit... more Cross-reactive antibodies to HLA class I and HIV-1 gp120 were detected in the sera of HIV-1-positive individuals, and were found to share the same epitope specificity as a gp120-HLA class I cross-reactive monoclonal antibody (M38). The amino acid residues of HLA recognized by both M38 and the patient antibodies occur as a clustered pair in the HLA-C alpha 1 domain. These sequences (KYKR and RKLR) are shared by almost all HLA-C alleles and are available to antibody binding only on beta 2-microglobulin-dissociated HLA heavy chains expressed on activated cells. Similar to M38, the antibody-binding sites on HIV-1 gp120 were mapped to two noncontiguous stretches of amino acids (KYK and KAKR), which flank a hydrophobic area of the immunodominant C5 region involved in gp41 binding. Serum antibodies immunoaffinity purified on synthetic HLA and gp120 peptides representing the M38-reactive regions were shown to bind to both HLA and gp120 in Western blot, as well as to membrane-bound HLA heavy chains, and to exhibit selective complement-mediated lysis of activated T cells. No serum antibodies could be detected that bind to the gp120 C5 region (peptide IEPLGVAPT) flanked by the two HLA-like sequences.
The hypothesis that open conformers of HLA-C on target cells might directly exert an effect on th... more The hypothesis that open conformers of HLA-C on target cells might directly exert an effect on their infectability by human immunodeficiency virus (HIV) has been suggested previously. This was tested by exploiting the peculiar specificity of monoclonal antibody (mAb) L31 for HLA-C open conformers to show that normal levels of Env-driven fusion were restored in HLA-C transfectants of a major histocompatibility complex-deleted (fusion-incompetent) cell line. The physiological relevance of this finding is now confirmed in this report, where small interfering RNA (siRNA) technology was used to silence HLA-C expression in peripheral blood lymphocytes (PBLs) from 11 healthy donors. Infectability by HIV (strains IIIB and Bal and primary isolates) was significantly reduced (P50.016) in silenced cells compared with cells that maintained HLA-C expression in 10 of the 11 PBL donors. Normal infectability was resumed, together with HLA-C expression, when the effect of siRNA interference waned after several days in culture. Additional confirmation of the HLA-C effect was obtained in several assays employing HLA-C-positive and -negative cell lines, a number of HIV strains and also pseudoviruses. In particular, viruses pseudotyped with env genes from HIV strains AC10 and QH0692.42 were assayed on siRNA-silenced lymphocytes from three healthy donors: the differences in infection with pseudoviruses were even higher than those observed in infections with normal viruses.
Although selected chemokines act as natural inhibitors of human immunodeficiency virus (HIV) infe... more Although selected chemokines act as natural inhibitors of human immunodeficiency virus (HIV) infection, their inherent proinflammatory activity may limit a therapeutic use. To elucidate whether the antiviral and signaling functions of RANTES can be dissociated, several recombinant analogues mutated at the N terminus were generated and functionally compared with the wild-type (WT) molecule, as well as with three previously described mutants. Substitution of selected residues within the N-terminal region caused a marked loss of antiviral potency. By contrast, two unique analogues (C1.C5-RANTES and L-RANTES) exhibited an increased antiviral activity against different CXCR4-negative HIV-1 isolates grown in primary mononuclear cells or in macrophages. This enhanced HIV-blocking activity was associated with an increased binding affinity for CCR5. Both C1.C5-RANTES and L-RANTES showed a dramatically reduced ability to trigger intracellular calcium mobilization via CCR3 or CCR5, while potently antagonizing the action of the WT chemokine. By contrast, two previously described analogues (RANTES 3-68 and AOP-RANTES) maintained a WT ability to trigger CCR5-mediated signaling, while a third one (RANTES 9-68 ) showed a dramatic loss of antiviral activity. These data demonstrate that the antiviral and signaling functions of RAN-TES can be uncoupled, opening new perspectives for the development of chemokine-based therapeutic approaches for HIV infection.
Cross-reactive antibodies to HLA class I and HIV-1 gp120 were detected in the sera of HIV-1-posit... more Cross-reactive antibodies to HLA class I and HIV-1 gp120 were detected in the sera of HIV-1-positive individuals, and were found to share the same epitope specificity as a gp120-HLA class I cross-reactive monoclonal antibody (M38). The amino acid residues of HLA recognized by both M38 and the patient antibodies occur as a clustered pair in the HLA-C alpha 1 domain. These sequences (KYKR and RKLR) are shared by almost all HLA-C alleles and are available to antibody binding only on beta 2-microglobulin-dissociated HLA heavy chains expressed on activated cells. Similar to M38, the antibody-binding sites on HIV-1 gp120 were mapped to two noncontiguous stretches of amino acids (KYK and KAKR), which flank a hydrophobic area of the immunodominant C5 region involved in gp41 binding. Serum antibodies immunoaffinity purified on synthetic HLA and gp120 peptides representing the M38-reactive regions were shown to bind to both HLA and gp120 in Western blot, as well as to membrane-bound HLA heavy chains, and to exhibit selective complement-mediated lysis of activated T cells. No serum antibodies could be detected that bind to the gp120 C5 region (peptide IEPLGVAPT) flanked by the two HLA-like sequences.
Molecular mimicry of major histocompatibility (MHC) antigens by viral glycoproteins has been sugg... more Molecular mimicry of major histocompatibility (MHC) antigens by viral glycoproteins has been suggested as one of the possible mechanisms of induction of an autoimmune response by human immunodeficiency viruses. A monoclonal antibody (M38) was previously shown to bind to both human immunodeficiency virus type 1 (HIV-1) gp120 and β-2 microglobulin-free HLA class I heavy chains encoded by an HLA C allele. Using HLA C recombinant proteins and synthetic peptides, the M38 class I binding site was mapped to a stretch of 44 amino acids of the al domain. The amino acid residues recognized are clustered in two non-contiguous regions at positions 66-69 (KYKR) and 79-82 (RKLR) shared by almost all HLA C alleles. On HIV-1 gp120, M38 binds to two non-contiguous sequences (KYK and KAKR) at positions 490-492 and 505-508 located at the edges of a large hydrophobic region that is apparently involved in binding the transmembrane glycoprotein gp41. The C-terminal gp120 M38-reactive region (KAKR) lies within the immunodominant sequence APTKAKRRVVQREKR, against which the majority of HIV-infected individuals produce antibodies. The results indicate that a functionally important region of HIV-1 gp120 shares similar amino acid sequence motifs with the antigen recognition site of most HLA class I C alleles. The molecular mimicry may be the basis for autoimmune responses in HIV infection.
A growing number of reports indicates that certain groups of individuals who almost certainly hav... more A growing number of reports indicates that certain groups of individuals who almost certainly have been exposed to human immunodeficiency virus (HIV), yet continue to exhibit no signs or symptoms of infection, often have subtle evidence of specific immunity. We studied such a high-risk (HR) cohort of persistently seronegative individuals with histories of long-term sexual exposure to an HIV-infected partner to look for evidence of both humoral and cellular immunity that might have been induced by exposure to the virus. Twenty-three heterosexual and four homosexual monogamous couples with discordant HIV status were included in the study. Twelve of the HR partners were studied for in vitro stimulation of peripheral blood mononuclear cells (PBMC) by HIV envelope-derived peptides. All 12 responded overwhelmingly to a peptide containing the fifth conserved region of gp120. By generating and cloning T cell lines specific for this peptide, we concluded that in these individuals the T cell response to the envelope is mainly focused on the carboxy-terminus region of gp120 and is characterized by an oligoclonal expansion of CD4+ T cells expressing the same TCR Eighteen HR partners and 37 HIV-1 seropositive subjects were tested for the presence of anti-CD4 antibodies (anti-CD4 Abs) using a recombinant CD4-based enzyme-linked immunosorbent assay (ELISA). Anti-CD4 Abs were detected in eight of the HR partners (six confirmed by Western blot) and in nine of the HIV-1 seropositive subjects (eight confirmed by Western blot). Results from binding competition assays with a panel of monoclonal anti-CD4 Abs suggested that the anti-CD4 Abs detected in the HR partners are directed toward epitopes that are induced by gp120 binding. Twenty-seven of the HR partners were tested for the presence of antibodies that cross-react with HLA class I and gp120 (anti-HLA Abs). Anti-HLA Abs were detected in 16 of the HR partner sera and in 4/94 sera from a control population of normal healthy blood donors. Taken together, the results suggest that in some individuals with a history of long-term exposure to HIV, specific immunity may develop in the absence of overt infection. The common trigger for these responses is gp120.
The double NF-KB site identified in the LTR of the human immunodeficiency virus-1 (HIV-1) has bee... more The double NF-KB site identified in the LTR of the human immunodeficiency virus-1 (HIV-1) has been demonstrated to be necessary for efficient viral transcription. In this report we present the characterisation of NF-KB subunits engaged in complexes binding to the HIV-1 NF-KB site in human 8e51 Tcells, that harbour a defective HIV-1. At least four different specific NF-KB complexes are present in the nucleus of these cells. With the use of specific antibodies we have determined the composition of each complex using electrophoretic mobility shift assays. The results show the presence of several NF-KB family members, with the transactivating RelA being engaged in multiple complexes. The importance of NF-KB complexes in viral functions has been established comparing the level of NF-KB DNA-binding complexes with syncytia-forming activity of 8e51 cells. In fact, 8e51 cells that had almost lost their syncytiaforming capacity were found to contain at least 10 times less active NF-KB DNA-binding complex than the actively fusing cells. The correlation is specific as the level of at least three other transcription factors did not change.
Epitope mapping of HLA-Cw4 indicates that the two monoclonal antibodies (mAbs) L31 and M38, speci... more Epitope mapping of HLA-Cw4 indicates that the two monoclonal antibodies (mAbs) L31 and M38, specific for beta 2-microglobulin ([32m)-free HLA-C heavy chains, react preferentially with the KYK motif, located in the binding groove (od domain). Transfection of HLA-Cw4 cDNA into a neuroblastoma cell line, which normally expresses negligible HLA class I, resulted in the constitutive surface expression of molecules displaying different reactivities with the two mAbs. This cellular system was used to determine whether L31 and M38 recognize distinct conformations of ~2m-free HLA-C proteins, and to investigate their mechanism of expression. Interferon-y greatly enhanced the expression of L31-reactive free chains, while abolishing that of M38-reactive molecules. The cytokineinduced expression of L31-reactive molecules was inhibited by anti-sense oligonucleotides specific for ~2m mRNA, while constitutive expression of L31-reactive molecules was only partially affected. Exogenous ~2m resulted in a reduction of constitutive L31 reactivity, and in a concomitant increase of M38 reactivity. These results indicate that: 1) at the cell surface, L31 and M38 react with two distinct conformations of HLA-Cw4 [32m-free heavy chains, of which the L31-reactive conformation is the least folded; 2) the expression of both conformers can be modulated by endogenous or exogenous ~2m; and (3) L31-reactive molecules exposed at the cell surface are likely to derive from the dissociation of empty HLA-Cw4/[~2m Complexes.
Cross-reactive antibodies to HLA class I and HIV-1 gp120 were detected in the sera of HIV-1-posit... more Cross-reactive antibodies to HLA class I and HIV-1 gp120 were detected in the sera of HIV-1-positive individuals, and were found to share the same epitope specificity as a gp120-HLA class I cross-reactive monoclonal antibody (M38). The amino acid residues of HLA recognized by both M38 and the patient antibodies occur as a clustered pair in the HLA-C alpha 1 domain. These sequences (KYKR and RKLR) are shared by almost all HLA-C alleles and are available to antibody binding only on beta 2-microglobulin-dissociated HLA heavy chains expressed on activated cells. Similar to M38, the antibody-binding sites on HIV-1 gp120 were mapped to two noncontiguous stretches of amino acids (KYK and KAKR), which flank a hydrophobic area of the immunodominant C5 region involved in gp41 binding. Serum antibodies immunoaffinity purified on synthetic HLA and gp120 peptides representing the M38-reactive regions were shown to bind to both HLA and gp120 in Western blot, as well as to membrane-bound HLA heavy chains, and to exhibit selective complement-mediated lysis of activated T cells. No serum antibodies could be detected that bind to the gp120 C5 region (peptide IEPLGVAPT) flanked by the two HLA-like sequences.
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Papers by Claudio Santis