Identification of membrane antigen C33 recognized by monoclonal antibodies inhibitory to human T-cell leukemia virus type 1 (HTLV-1)-induced syncytium formation: altered glycosylation of C33 antigen in HTLV-1-positive T cells.

We isolated four monoclonal antibodies (MAbs), M38, M101, M104, and C33, which were capable of inhibiting syncytium formation induced in a human T-cell line, MOLT-4-#8, by coculture with human T-cell leukemia virus type 1 (HTLV-1)-positive human T-cell lines. The MAbs had, however, no inhibitory activity on syncytium formation induced in a human osteosarcoma line, HOS, by HTLV-1-positive T-cell lines. They also did not inhibit syncytium formation induced in MOLT-4-#8 by human immunodeficiency virus type 1-positive MOLT-4. All MAbs reacted with various human cell lines of lymphoid and nonlymphoid origins, including HTLV-1-positive T-cell lines. Furthermore, they all reacted with a murine A9 clone containing human chromosome 11 fragment q23-pter. Two MAbs, M104 and C33, immunoprecipitated a membrane antigen with the same molecular size. The antigen (henceforth called C33 antigen) was about 40 to 55 kDa in HTLV-1-negative Jurkat, CEM, MOLT-4, and normal peripheral blood CD4-positive human T cells and about 40 to 75 kDa in HTLV-1-positive C91/PL, TCL-Kan, MT-2, and in fresh HTLV-1-transformed CD4-positive human T-cell lines. Pulse-chase experiments revealed that C33 antigen was synthesized as a 35-kDa precursor that was then processed to 41 to 50 kDa in MOLT-4 and to 44 to 70 kDa in C91/PL. In the presence of tunicamycin, a 28-kDa protein was synthesized. The conversion from 35 kDa to 41 to 50 kDa in MOLT-4 and to 44 to 70 kDa in C91/PL was inhibited by monensin. Treatment with N-glycanase alone, but not with sialidase and O-glycanase in combination, completely removed the sugar moiety of C33 antigen from both HTLV-1-negative Jurkat and HTLV-1-positive C91/PL. Therefore, C33 antigen has only N-linked carbohydrates, the modification of which appears to be substantially altered in the presence of the HTLV-1 genome.     

A putative G protein-coupled receptor, RDC1, is a novel coreceptor for human and simian immunodeficiency viruses

More than 10 G protein-coupled receptors (GPCRs) have been shown to act as coreceptors for infection of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). We have isolated HIV-1 variants infectious to primary brain-derived CD4-positive cells (BT-3 and BT-20/N) and U87/CD4 glioma cells that are resistant to T-cell line-tropic (T-tropic), macrophage-tropic (M-tropic), and T- and M-tropic (dualtropic) (X4, R5, and R5X4) HIV-1 strains. These primary brain-derived cells were also highly susceptible to HIV-2(ROD), HIV-2(SBL6669), and SIV(mndGB-1). A factor or coreceptor that determines the susceptibility of these brain-derived cells to these HIV and SIV strains has not been fully identified. To identify this coreceptor, we examined amino acid sequences of all known HIV and SIV coreceptors and noticed that tyrosine residues are well conserved in their extracellular amino-terminal domains. By this criterion, we selected 18 GPCRs as candidates of coreceptors for HIV and SIV strains infectious to these brain-derived cells. mRNA expression of an orphan GPCR, RDC1, was detected in the brain-derived cells, the C8166 T-cell line, and peripheral blood lymphocytes, all of which are susceptible to HIV-1 variants, but not in macrophages, which are resistant to them. When a CD4-expressing cell line, NP-2/CD4, which shows strict resistance to infection not only with HIV-1 but also with HIV-2 or SIV, was transduced with the RDC1 gene, the cells became highly susceptible to HIV-2 and SIV(mnd) strains but to neither M- nor T-tropic HIV-1 strains. The cells also acquired a low susceptibility to the HIV-1 variants. These findings indicate that RDC1 is a novel coreceptor for several HIV-1, HIV-2, and SIV strains which infect brain-derived cells.     

In a human lymphomyeloid progenitor cell line (KG-1) HIV-1 gp120 binds to chemokine-receptors CXC-R4 and CCR5, only in the presence of CD4.

A CD34+ human hematopoietic progenitor cell lines, KG-1, became susceptible to HIV-1 infection in the presence of a concurrent infection by human herpesvirus-6 (HHV-6). We have now analyzed the possible mechanism(s) underlying this phenomenon, in the light of the recent demonstration that at least two members of the chemokine receptor family, CXCR4 (LESTR/fusin) and CCR5 molecules, are the HIV-1-specific co-receptors, necessary, together with the high affinity receptor CD4, for the entry into target cells of HIV-1. Cytofluorimetric analysis demonstrated that in KG-1 cells, after HHV-6 infection, more than 40% of cell population became CD4 positive and only in KG-1 cells expressing the CD4+ phenotype, the exposure to r-gp120 masks a significant amount, not only of CD4, but also of both CXCR4 and CCR5 chemokine receptors. In fact, only when pre-infected by HHV-6, KG-1 cells, after exposure to r-gp120, exhibit a significant reduction in the percentage of CXCR4 or CCR5-positive cells.     

CCR5- and CXCR4-tropic HIV-1 are equally cytopathic for their T-cell targets in human lymphoid tissue

A rapid decline in T-cell counts and the progression to AIDS is often associated with a switch from CCR5-tropic (R5) HIV-1 to CXCR4-tropic (X4) HIV-1 or R5/X4 HIV-1 variants. Experimental infection with R5 HIV-1 causes less T-cell depletion than infection with X4 or R5/X4 variants in T-cell cultures, in ex vivo infected human lymphoid tissue and in SCID/hu mice, despite similar replication levels. Experimental genetic changes in those sequences in gp120 that transform R5 HIV-1 variants into otherwise isogenic X4 viruses make them highly cytopathic. Thus, it is now believed that R5 variants are less cytopathic for T cells than are X4 variants. However, it is not known why CCR5-mediated HIV-1 infection does not lead to a massive CD4+ T-cell depletion, as occurs in CXCR4-mediated HIV-1 infection. Here we demonstrate that R5 HIV-1 isolates are indeed highly cytopathic, but only for CCR5+/CD4+ T cells. Because these cells constitute only a small fraction of CD4+ T cells, their depletion does not substantially change the total CD4+ T-cell count. These results may explain why the clinical stage of HIV disease correlates with viral tropism.     

Distribution and functional analysis of a 120- to 130-kDa T-cell surface antigen

A monoclonal antibody (mAb), SPV-L14, was raised that detected a human T-cell surface antigen with a molecular weight (MW) of 120 kDa on resting and phytohemagglutinin-activated peripheral blood T lymphocytes (PBL). An additional band with a MW of 130 kDa could be precipitated with variable intensities from thymocytes, neoplastic T cells, and CD4+- or CD8+ T-cell clones. Based on their reactivity with SPV-L14 and a mAb directed against CD3, four subpopulations of CD2+ lymphocytes could be detected and their existence was confirmed at the clonal level. The majority (95%) of the CD3+ cells were SPV-L14+, whereas 5% were CD3+, SPV-L14-. Among cloned cell lines CD3-,SPV-L14- and CD3-,SPV-L14+ cells were found to exist. The CD3-,SPV-L14- and CD3-,SPV-L14+ clones were shown to have NK cell activity, indicating that the 120- to 130-kDa antigen is expressed heterogeneously on CD3- NK cell clones. In addition, neoplastic T cells representing these four subpopulations were shown to exist. Although the tissue distribution and the MW of the SPV-L14 target antigen strongly suggest that SPV-L14 reacts with an epitope on CD6, the SPV-L14 mAb did not react with resting or activated B cells or with malignant B cells. Blocking studies showed that SPV-L14 inhibited the proliferative response of PBL, induced by anti-CD3 mAb, but that SPV-L14 did not affect the proliferation induced by phytohemagglutinin. These results suggest that the 120- to 130-kDa MW antigen is associated with T-cell proliferation, depending on the mode of activation.     

A trans-receptor mechanism for infection of CD4-negative cells by human immunodeficiency virus type 1.

Chemokine receptors, particularly CCR5 and CXCR4, act as essential coreceptors in concert with CD4 for cellular entry by human immunodeficiency virus type 1 (HIV-1; reviewed in [1]). But infection of CD4(-) cells has also been encountered in various tissues in vivo, including astrocytes, neurons and microvascular endothelial cells of the brain [2] [3] [4] [5] [6], epithelial cells [5] [7], CD4(-) lymphocytes and thymocytes [8] [9], and cardiomyocytes [10]. Here, we present evidence for the infection of CD4(-) cell lines bearing coreceptors by well-known HIV-1 strains when co-cultured with CD4(+) cells. This process requires contact between the coreceptor-bearing and CD4(+) cells and supports the full viral replication cycle within the coreceptor-bearing target cell. Furthermore, CD4 provided in trans facilitates infection of primary human cells, such as brain-derived astrocytes. Although the pathobiological significance of infection of CD4(-) cells in vivo remains to be elucidated, this trans-receptor mechanism may facilitate generation of hidden reservoirs of latent virus that confound antiviral therapies and that contribute to specific AIDS-associated clinical syndromes.     

HIV-infected humans, but not chimpanzees, have circulating cytotoxic T lymphocytes that lyse uninfected CD4+ cells

It has been suggested that autoimmune phenomena contribute to the depletion of CD4+ T cells and the development of AIDS in HIV-1 infected humans based, in part, on observations that some HIV-1-infected humans have autoantibodies reactive with Ag expressed on uninfected CD4+ cells. In this study, 11 of 14 asymptomatic HIV-1-infected homosexuals and hemophiliacs, but none of 17 uninfected homosexuals or heterosexuals, were found to have cytotoxic lymphocytes in blood that can lyse uninfected CD4+ T cells from humans and chimpanzees but not human B lymphoblastoid cells or mouse T cells. The cytotoxic PBL were concluded to be CTL rather than NK cells, with the phenotype being CD3+, TCR-1 alpha beta+, CD8+, CD4-, CD16- based on findings that PBL-mediated lysis of uninfected CD4+ cells was 1) blocked by a mAb to CD3, which inhibits CTL but not NK activity; 2) diminished by treatment of PBL with a mAb to CD8 and C, but not by treatment with mAb to CD4 or CD16 and C; and 3) blocked by mAb WT31 directed against the TCR-1 alpha beta. In contrast, PBL from HIV-1-infected chimpanzees, which to date have not developed AIDS, lacked detectable CTL lytic for uninfected CD4+ cells.     

A human recombinant Fab identifies a human immunodeficiency virus type 1-induced conformational change in cell surface-expressed CD4.

To explore the role of the CD4 molecule in human immunodeficiency virus (HIV) infection following initial virus-CD4 binding, we have characterized CD4-specific antibodies raised by immunizing an HIV-1-infected human with human recombinant soluble CD4 (rsCD4). Fabs were selected from a human recombinant Fab library constructed from the bone marrow of this immunized individual. Here, we describe a human rsCD4-specific recombinant Fab clone selected by panning the library over complexes of human rsCD4 and recombinant HIV-1 envelope protein. While this Fab does not bind to CD4-positive T-cell lines or to human T lymphocytes, it recognizes cell surface-expressed CD4 following the incubation of these cells with a recombinant form of HIV-1 gp120 or with HIV-1 virions. The Fab is not HIV-1 envelope specific, since it does not bind to recombinant gp120 or to native cell surface-expressed HIV-1 envelope proteins. As confirmation of its CD4 specificity, we show that this Fab immunoprecipitates a 55-kDa protein, corresponding to the molecular mass of cellular CD4, from an H9 cell lysate. The specificity of this human Fab provides evidence for a virus-induced conformational change in cell surface-expressed on CD4. The characterization of this altered CD4 conformation and its effects on the host cell will be important in defining postbinding events in HIV infection.     

Defucosylated anti-CCR4 monoclonal antibody exercises potent ADCC-mediated antitumor effect in the novel tumor-bearing humanized NOD/Shi-scid, IL-2Rγnull mouse model

Purpose  There are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities. Thus, the first aim of this study was to establish a human tumor-bearing mouse model in which human immune cells can engraft and mediate ADCC, but where the endogenous mouse immune cells cannot mediate ADCC. The second aim was to evaluate ADCC mediated in these humanized mice by the defucosylated anti-CC chemokine receptor 4 (CCR4) monoclonal antibody (mAb) which we have developed and which is now in phase I clinical trials. Experimental design  NOD/Shi-scid, IL-2Rγ^null (NOG) mice were the recipients of human immune cells, and CCR4-expressing Hodgkin lymphoma (HL) and cutaneous T-cell lymphoma (CTCL) cell lines were used as target tumors. Results  Humanized mice have been established using NOG mice. The chimeric defucosylated anti-CCR4 mAb KM2760 showed potent antitumor activity mediated by robust ADCC in these humanized mice bearing the HL or CTCL cell lines. KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in HL-bearing humanized mice. Conclusions  Anti-CCR4 mAb could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. In addition, using our humanized mice, we can perform the appropriate preclinical evaluation of many types of antibody based immunotherapy.     

Lysis of cells infected with HIV-1 by human lymphocytes targeted with monoclonal antibody heteroconjugates

The present study was undertaken to determine whether human PBL can be specifically focused to lyse cells infected with HIV-1 by mAb heteroconjugates that can bridge target and effector cells. A mAb directed against the central portion of HIV-1 glycoprotein gp110 was chemically cross-linked to a mAb directed against the CD3/TCR complex or to a mAb directed against the CD16 Fc gamma-R expressed on large granular lymphocytes (LGL). HIV-1-infected cells, but not uninfected cells, were found to be lysed to a greater extent by PBL in the presence of the gp110 X CD3 or the gp110 X CD16 antibody heteroconjugate than in the presence of the single antibodies or a mixture of the mAb comprising the heteroconjugates. Pretreatment of PBL with anti-CD3 or IL-2 augments their ability to lyse HIV-1-infected cells in the presence of the heteroconjugates. Lysis by anti-CD3-activated PBL in the presence of the gp110 X CD3 heteroconjugate was found to be mediated by CD8+-enriched T cells, whereas lysis by IL-2-treated PBL in the presence of the gp110 X CD16 heteroconjugate is mediated by PBL enriched for CD16+ cells, which are primarily LGL. Furthermore, PBL from asymptomatic, HIV-1-infected seropositive donors were found to be functional in lysing HIV-1-infected cells in the presence of the antibody heteroconjugates. Such antibody heteroconjugates, which can target T cells or LGL to lyse HIV-1-infected cells, may be of prophylactic or therapeutic value in HIV-1-infected individuals.     

Primary, syncytium-inducing human immunodeficiency virus type 1 isolates are dual-tropic and most can use either Lestr or CCR5 as coreceptors for virus entry.

A panel of primary syncytium-inducing (SI) human immunodeficiency virus type 1 isolates that infected several CD4+ T-cell lines, including MT-2 and C8166, were tested for infection of blood-derived macrophages. Infectivity titers for C8166 cells and macrophages demonstrated that primary SI strains infected macrophages much more efficiently than T-cell line-adapted HIV-1 strains such as LAI and RF. These primary SI strains were therefore dual-tropic. Nine biological clones of two SI strains, prepared by limiting dilution, had macrophage/C8166 infectivity ratios similar to those of their parental viruses, indicating that the dual-tropic phenotype was not due to a mixture of non-SI/macrophage-tropic and SI/T-cell tropic viruses. We tested whether the primary SI strains used either Lestr (fusin) or CCR5 as coreceptors. Infection of cat CCC/CD4 cells transiently expressing Lestr supported infection by T-cell line-adapted strains including LAI, whereas CCC/CD4 cells expressing CCR5 were sensitive to primary non-SI strains as well as to the molecularly cloned strains SF-162 and JR-CSF. Several primary SI strains, as well as the molecularly cloned dual-tropic viruses 89.6 and GUN-1, infected both Lestr+ and CCR5+ CCC/CD4 cells. Thus, these viruses can choose between Lestr and CCR5 for entry into cells. Interestingly, some dual-tropic primary SI strains that infected Lestr+ cells failed to infect CCR5+ cells, suggesting that these viruses may use an alternative coreceptor for infection of macrophages. Alternatively, CCR5 may be processed or presented differently on cat cells so that entry of some primary SI strains but not others is affected.     

CCR5 Antagonism Impacts Vaccination Response and Immune Profile in HIV-1 Infection

Maraviroc (MVC) is the first licensed antiretroviral therapeutic agent to target a host cell surface molecule, and successful HIV-1 entry blockade by this C-C chemokine receptor type 5 (CCR5)-antagonist potentiates immunomodulation. We hypothesized that MVC intensification impacts immunization responses, T-cell phenotype, function and delayed type hypersensitivity (DTH) in HIV-1⁺ subjects. A 24-wk, double-blinded, placebo-controlled study of the addition of MVC to suppressive antiretroviral therapy in HIV-1⁺ persons was performed. Subjects received DTH tests, intramuscular tetanus, meningococcal and oral cholera immunizations. Antibody titers, T-cell function and phenotype were assessed. Of 157 patients referred, 47 were randomized 1:1; MVC:placebo. MVC enhanced meningococcal neo-immunization, blunted cholera response and expedited lymphoproliferation to tetanus boost, without affecting recall humoral response. Anti-HIV-1 group-specific antigen (Gag) and tetanus toxoid (TTox) function improved significantly, HIV-1-associated CD8 T-cell skewing normalized, and the percentage of late-stage and major histocompatibility complex (MHC) class II expressing CD4 T-cells increased. Activated CD4⁺ CD38⁺ human leukocyte antigen (HLA)-DR⁺ T-cells declined, and costimulation shifted to coinhibition. DTH was unchanged. Maraviroc intensification, through antagonism of the cell surface molecule CCR5, favorably influences immune profiles of HIV-1⁺ patients, supporting its immunomodulatory use in HIV-1 infection and potentially in other immunologically relevant settings.     

A monoclonal antibody to beta 1 integrin (CD29) stimulates VLA- dependent adherence of leukocytes to human umbilical vein endothelial cells and matrix components

The leukocyte beta 1 integrin receptor very late activation antigen-4 (VLA-4) (alpha 4 beta 1, CD49d/CD29) binds to vascular cell adhesion molecule-1 (VCAM-1) expressed on cytokine-activated endothelium. A mAb designated 8A2 was identified that stimulated the binding of U937 cells to CHO cells transfected with VCAM-1 cDNA but not endothelial-leukocyte adhesion molecule or CD4 cDNA. mAb 8A2 also rapidly stimulated the adherence of peripheral blood lymphocytes (PBLs) to VCAM-1-transfected CHO cells or recombinant human tumor necrosis factor-treated human umbilical vein endothelial cells. mAb 8A2-stimulated binding of PBL was inhibited by mAbs to VLA-4 or VCAM-1. Surface expression of VLA-4 was not altered by mAb 8A2 treatment and monovalent Fab fragments of mAb 8A2 were active. Immunoprecipitation studies reveal that mAb 8A2 recognizes beta 1-subunit (CD29) of integrin receptors. In contrast to mAbs directed to VLA-4 alpha-subunit (alpha 4, CD49d), mAb 8A2 did not induce homotypic aggregation of PBL. Additionally, mAb 8A2 stimulated adherence of PBL and hematopoietic cell lines to purified matrix components laminin and fibronectin. This binding was blocked by mAbs to the VLA alpha-subunits alpha 6 (CD49f), or alpha 5 (CD49e) and alpha 4 (CD49d), respectively. We conclude that mAb 8A2 modulates the affinity of VLA-4 and other leukocyte beta 1 integrins, and should prove useful in studying the regulation of beta 1 integrin function.     

CD4-independent infection of two CD4(-)/CCR5(-)/CXCR4(+) pre-T-cell lines by human and simian immunodeficiency viruses

The infection of CD4-negative cells by variants of tissue culture-adapted human immunodeficiency virus type 1 (HIV-1) or HIV-2 strains has been shown to be mediated by the CXCR4 coreceptor. Here we show that two in vitro-established CD4(-)/CCR5(-)/CXCR4(+) human pre-T-cell lines (A3 and A5) can be productively infected by wild-type laboratory-adapted T-cell-tropic HIV-1 and HIV-2 strains in a CD4-independent, CXCR4-dependent fashion. Despite the absence of CCR5 expression, A3 and A5 cells were susceptible to infection by the simian immunodeficiency viruses SIVmac239 and SIVmac316. Thus, at least in A3 and A5 cells, one or more of the chemokine receptors can efficiently support the entry of HIV and SIV isolates in the absence of CD4. These findings suggest that to infect cells of different compartments, HIV and SIV could have evolved in vivo to bypass CD4 and to interact directly with an alternative receptor.     

The HML-1 antigen of intestinal lymphocytes is an activation antigen.

The Ag recognized by the mAb HML-1 is expressed on more than 90% of human intestinal intraepithelial lymphocytes, whereas in other lymphoid tissues it is rarely or not expressed. In the present study, we have investigated the percentage of HML-1-positive cells in the human intestinal lamina propria and the coexpression of HML-1 with different T cell subset markers. In addition, we studied the inducibility of HML-1 on PBL which normally are HML-1-negative. Flow cytometric analysis of isolated intestinal lamina propria lymphocytes (LPL) showed that about 40% of the cells expressed HML-1, the majority belonging to the CD8-positive subpopulation. Virtually all LPL expressed CD45RO, whereas the percentage of CD29-positive cells was only about 50%, similar to PBL. There were only few cells expressing CD45RA or Leu-8 in the lamina propria. HML-1-positive cells were almost exclusively CD45RA-negative, but were found in both the CD29-positive and the CD29-negative subpopulation of LPL. In vitro stimulation of PBL showed that the expression of HML-1 was inducible on T cells by mitogens, phorbolester, Ag, and rIL-2. Expression of HML-1 was induced with a different time course and with differences in the response to the investigated stimuli compared with CD25. Activated HML-1-positive PBL were also predominantly CD45RA-negative. The findings show that HML-1 is an Ag, which is expressed in vivo on a specific subset of previously activated T cells in the unique environment of the intestinal mucosa, and which can be induced in vitro by different activation signals on PBL.     

Human immunodeficiency virus type 1 T-cell tropism is determined by events prior to provirus formation.

Different strains of human immunodeficiency virus type 1 (HIV-1) vary in the ability to replicate in cells that bear the HIV-1 receptor, CD4. The mechanism responsible for these cell tropism differences is unknown. We examined different isolates of HIV-1 with regard to replication in specific tumor-derived CD4-positive T-cell lines and normal peripheral blood lymphocytes. To investigate early events in the virus life cycle at low multiplicities of infection, we used a modification of the polymerase chain reaction method. Use of a molecularly cloned primary HIV-1 isolate, HIV-1 JR-CSF, restricted for replication in T-cell lines, demonstrated that little or no viral DNA or RNA was synthesized in nonpermissive cells after infection. However, transfection of proviral DNA resulted in efficient transient virus production from these cells. Therefore, we conclude that at least one block to infection for HIV-1 strains in nonpermissive T cells occurs at a point in entry or uncoating before provirus formation.     

V3 Recombinants Indicate a Central Role for CCR5 as a Coreceptor in Tissue Infection by Human Immunodeficiency Virus Type 1

Binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 to both CD4 and one of several chemokine receptors (coreceptors) permits entry of virus into target cells. Infection of tissues may establish latent viral reservoirs as well as cause direct pathologic effects that manifest as clinical disease such as HIV-associated dementia. We sought to identify the critical coreceptors recognized by HIV-1 tissue-derived strains as well as to correlate these coreceptor preferences with site of infection and dementia diagnosis. To reconstitute coreceptor use, we cloned HIV-1 envelope V3 sequences encoding the primary determinants of coreceptor specificity from 13 brain-derived and 6 colon-derived viruses into an isogenic (NL4-3) viral background. All V3 recombinants utilized the chemokine receptor CCR5 uniformly and efficiently as a coreceptor but not CXCR4, BOB/GPR15, or Bonzo/STRL33. Other receptors such as CCR3, CCR8, and US28 were inefficiently and variably used as coreceptors by various envelopes. CCR5 without CD4 present did not allow for detectable infection by any of the tested recombinants. In contrast to the pathogenic switch in coreceptor specificity frequently observed in comparisons of blood-derived viruses early after HIV-1 seroconversion and after onset of AIDS, the characteristics of these V3 recombinants suggest that CCR5 is a primary coreceptor for brain- and colon-derived viruses regardless of tissue source or diagnosis of dementia. Therefore, tissue infection may not depend significantly on viral envelope quasispeciation to broaden coreceptor range but rather selects for CCR5 use throughout disease progression.     

Comparison of the Antibody Repertoire Generated in Healthy Volunteers following Immunization with a Monomeric Recombinant gp120 Construct Derived from a CCR5/CXCR4-Using Human Immunodeficiency Virus Type 1 Isolate with Sera from Naturally Infected Individuals

We have characterized sera from healthy volunteers immunized with a monomeric recombinant gp120 (rgp120) derived from a CCR5/CXCR4 (R5X4)-using subtype B isolate of human immunodeficiency virus type (HIV-1), HIV-1_W61D, in comparison to sera from long-term HIV-1-infected individuals, using homologous reagents. Sera from vaccinees and HIV-1 positive subjects had similar binding titers to native monomeric rgp120_W61D and showed a similar titer of antibodies inhibiting the binding of soluble CD4 (sCD4) to rgp120_W61D. However, extensive peptide binding studies showed that the overall pattern of recognition of vaccinee and HIV-1-positive sera is different, with vaccinee sera displaying a wider and more potent recognition of linear V1/V2 and V3 domain epitopes. Neutralization of homologous HIV-1_W61D or heterologous HIV-1_M2424/4 peripheral blood mononuclear cell (PBMC)-derived virus lines by vaccinee sera could be achieved, but only after adaptation of the viruses to T-cell lines and was quickly lost on readaptation to growth in PBMC. Sera from HIV-positive individuals were able to neutralize both PBMC-grown and T-cell line-adapted viruses. Interestingly, rgp120_W61D was recognized by monoclonal antibodies previously shown to neutralize primary HIV-1 isolates. The use of very potent adjuvants and R5X4 rgp120 led to an antibody response equivalent in binding activity and inhibition of binding of sCD4 to gp120 to that of HIV-positive individuals but did not lead to the induction of antibodies capable of neutralizing PBMC-grown virus.     

HIV-1 entry into T-cells is not dependent on CD4 and CCR5 localization to sphingolipid-enriched,detergent-resistant,raft membrane domains

The contribution of raft domains to human immunodeficiency virus (HIV) 1 entry was assessed. In particular, we asked whether the CD4 and CCR5 HIV-1 receptors need to associate with sphingolipid-enriched, detergent-resistant membrane domains (rafts) to allow viral entry into primary and T-cell lines. Based on Triton X-100 solubilization and confocal microscopy, CD4 was shown to distribute partially to rafts. In contrast, CCR5 did not associate with rafts and localized in nonraft plasma membrane domains. HIV-1-receptor partitioning remained unchanged upon viral adsorption, suggesting that viral entry probably takes place outside rafts. To directly investigate this possibility, we targeted CD4 to nonraft domains of the membrane by preventing CD4 palmitoylation and interaction with p56(lck). Directed mutagenesis of both targeting signals significantly prevented association of CD4 with rafts, but did not suppress the HIV-1 receptor function of CD4. Collectively, these results strongly suggest that the presence of HIV-1 receptors in rafts is not required for viral infection. We show, however, that depleting plasma membrane cholesterol inhibits HIV-1 entry. We therefore propose that cholesterol modulates the HIV-1 entry process independently of its ability to promote raft formation.