The function and affinity maturation of HIV-1 gp120-specific monoclonal antibodies derived from colostral B cells.

Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants because of beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution, and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell-mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. We also identified divergent patterns of colostrum Env-specific B-cell lineage evolution with respect to crossreactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk immunoglobulin G (IgG) repertoire. Maternal vaccine strategies to specifically target this breast milk B-cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants.

Relationships between CD4 independence, neutralization sensitivity, and exposure of a CD4-induced epitope in a human immunodeficiency virus type 1 envelope protein.

A CD4-independent version of the X4 human immunodeficiency virus type 1 (HIV-1) HXBc2 envelope (Env) protein, termed 8x, mediates infection of CD4-negative, CXCR4-positive cells, binds directly to CXCR4 in the absence of CD4 due to constitutive exposure of a conserved coreceptor binding site in the gp120 subunit, and is more sensitive to antibody-mediated neutralization. To study the relationships between CD4 independence, neutralization sensitivity, and exposure of CD4-induced epitopes associated with the coreceptor binding site, we generated a large panel of Env mutants and chimeras between 8x and its CD4-dependent parent, HXBc2. We found that a frameshift mutation just proximal to the gp41 cytoplasmic domain in 8x Env was necessary but not sufficient for CD4 independence and led to increased exposure of the coreceptor binding site. In the presence of this altered cytoplasmic domain, single amino acid changes in either the 8x V3 (V320I) or V4/C4 (N386K) regions imparted CD4 independence, with other changes playing a modulatory role. The N386K mutation resulted in loss of an N-linked glycosylation site, but additional mutagenesis showed that it was the presence of a lysine rather than loss of the glycosylation site that contributed to CD4 independence. However, loss of the glycosylation site alone was sufficient to render Env neutralization sensitive, providing additional evidence that carbohydrate structures shield important neutralization determinants. Exposure of the CD4-induced epitope recognized by monoclonal antibody 17b and which overlaps the coreceptor binding site was highly sensitive to an R298K mutation at the base of the V3 loop and was often but not always associated with CD4 independence. Finally, while not all neutralization-sensitive Envs were CD4 independent, all CD4-independent Envs exhibited enhanced sensitivity to neutralization by HIV-1-positive human sera, indicating that the humoral immune response can exert strong selective pressure against the CD4-independent phenotype in vivo. Whether this can be used to advantage in designing more effective immunogens remains to be seen.

Differential CD4/CCR5 utilization, gp120 conformation, and neutralization sensitivity between envelopes from a microglia-adapted human immunodeficiency virus type 1 and its parental isolate.

Human immunodeficiency virus type 1 (HIV-1) infects and induces syncytium formation in microglial cells from the central nervous system (CNS). A primary isolate (HIV-1(BORI)) was sequentially passaged in cultured microglia, and the isolate recovered (HIV-1(BORI-15)) showed high levels of fusion and replicated more efficiently in microglia (J. M. Strizki, A. V. Albright, H. Sheng, M. O'Connor, L. Perrin, and F. González-Scarano, J. Virol. 70:7654-7662, 1996). The parent and adapted viruses used CCR5 as coreceptor. Recombinant viruses demonstrated that the syncytium-inducing phenotype was associated with four amino acid differences in the V1/V2 region of the viral gp120 (J. T. C. Shieh, J. Martin, G. Baltuch, M. H. Malim, and F. González-Scarano, J. Virol. 74:693-701, 2000). We produced luciferase-reporter, env-pseudotyped viruses using plasmids containing env sequences from HIV-1(BORI), HIV-1(BORI-15), and the V1/V2 region of HIV-1(BORI-15) in the context of HIV-1(BORI) env (named rBORI, rB15, and rV1V2, respectively). The pseudotypes were used to infect cells expressing various amounts of CD4 and CCR5 on the surface. In contrast to the parent recombinant, the rB15 and rV1V2 pseudotypes retained their infectability in cells expressing low levels of CD4 independent of the levels of CCR5, and they infected cells expressing CD4 with a chimeric coreceptor containing the third extracellular loop of CCR2b in the context of CCR5 or a CCR5 Delta4 amino-terminal deletion mutant. The VH-rB15 and VH-rV1V2 recombinant viruses were more sensitive to neutralization by a panel of HIV-positive sera than was VH-rBORI. Interestingly, the CD4-induced 17b epitope on gp120 was more accessible in the rB15 and rV1V2 pseudotypes than in rBORI, even before CD4 binding, and concomitantly, the rB15 and rV1V2 pseudotypes were more sensitive to neutralization with the human 17b monoclonal antibody. Adaptation to growth in microglia--cells that have reduced expression of CD4 in comparison with other cell types--appears to be associated with changes in gp120 that modify its ability to utilize CD4 and CCR5. Changes in the availability of the 17b epitope indicate that these affect conformation. These results imply that the process of adaptation to certain tissue types such as the CNS directly affects the interaction of HIV-1 envelope glycoproteins with cell surface components and with humoral immune responses.

Determinants of CD4 independence for a human immunodeficiency virus type 1 variant map outside regions required for coreceptor specificity.

Although infection by human immunodeficiency virus (HIV) typically requires an interaction between the viral envelope glycoprotein (Env), CD4, and a chemokine receptor, CD4-independent isolates of HIV and simian immunodeficiency virus have been described. The structural basis and underlying mechanisms for this phenotype are unknown. We have derived a variant of HIV-1/IIIB, termed IIIBx, that acquired the ability to utilize CXCR4 without CD4. This virus infected CD4-negative T and B cells and fused with murine 3T3 cells that expressed human CXCR4 alone. A functional IIIBx env clone exhibited several mutations compared to the CD4-dependent HXBc2 env, including the striking loss of five glycosylation sites. By constructing env chimeras with HXBc2, the determinants for CD4 independence were shown to map outside the V1/V2 and V3 hypervariable loops, which determine chemokine receptor specificity, and at least partly within an area on the gp120 core that has been implicated in forming a conserved chemokine receptor binding site. We also identified a point mutation in the C4 domain that could render the IIIBx env clone completely CD4 dependent. Mutations in the transmembrane protein (TM) were also required for CD4 independence. Remarkably, when the V3 loop of a CCR5-tropic Env was substituted for the IIIBx Env, the resulting chimera was found to utilize CCR5 but remained CD4 independent. These findings show that Env determinants for chemokine receptor specificity are distinct from those that mediate CD4-independent use of that receptor for cell fusion and provide functional evidence for multiple steps in the interaction of Env with chemokine receptors. Combined with our observation that the conserved chemokine receptor binding site on gp120 is more exposed on the IIIBx gp120 (T. L. Hoffman, C. C. LaBranche, W. Zhang, G. Canziani, J. Robinson, I. Chaiken, J. A. Hoxie, and R. W. Doms, Proc. Natl. Acad. Sci. USA 96:6359-6364, 1999), the findings from this study suggest novel approaches to derive and design Envs with exposed chemokine receptor binding sites for vaccine purposes.

Stable exposure of the coreceptor-binding site in a CD4-independent HIV-1 envelope protein.

We recently derived a CD4-independent virus from HIV-1/IIIB, termed IIIBx, which interacts directly with the chemokine receptor CXCR4 to infect cells. To address the underlying mechanism, a cloned Env from the IIIBx swarm (8x) was used to produce soluble gp120. 8x gp120 bound directly to cells expressing only CXCR4, whereas binding of IIIB gp120 required soluble CD4. Using an optical biosensor, we found that CD4-induced (CD4i) epitopes recognized by mAbs 17b and 48d were more exposed on 8x than on IIIB gp120. The ability of 8x gp120 to bind directly to CXCR4 and to react with mAbs 17b and 48d in the absence of CD4 indicated that this gp120 exists in a partially triggered but stable state in which the conserved coreceptor-binding site in gp120, which overlaps with the 17b epitope, is exposed. Substitution of the 8x V3 loop with that from the R5 virus strain BaL resulted in an Env (8x-V3BaL) that mediated CD4-independent CCR5-dependent virus infection and a gp120 that bound to CCR5 in the absence of CD4. Thus, in a partially triggered Env protein, the V3 loop can change the specificity of coreceptor use but does not alter CD4 independence, indicating that these properties are dissociable. Finally, IIIBx was more sensitive to neutralization by HIV-positive human sera, a variety of anti-IIIB gp120 rabbit sera, and CD4i mAbs than was IIIB. The sensitivity of this virus to neutralization and the stable exposure of a highly conserved region of gp120 suggest new strategies for the development of antibodies and small molecule inhibitors to this functionally important domain.

CD4-independent utilization of the CXCR4 chemokine receptor by HIV-1 and HIV-2.

HIV entry is mediated by an interaction between CD4 and members of the chemokine receptor family of proteins. It is likely that CD4 induces conformational changes in the viral envelope glycoproteins that facilitate a subsequent interaction with the chemokine receptor. To understand these events, variants of HIV-2 and HIV-1 have been derived that are able to interact directly with CXCR4 in the absence of CD4. One HIV-2 variant. termed HIV-2/vcp, has an expanded host range that includes CXCR4+/CD4- lymphoid and nonlymphoid cell lines. In contrast to T-tropic isolates of HIV-1, HIV-2/vcp was shown to induce > 95% downregulation of CXCR4 on chronically infected cells and was able to superinfect HIV-1-infected cells. A variant of HIV-1/IIIB termed HIV-1/IIIBx was also derived that is both replication competent and fusogenic for a CD4-negative subclone of SupT1 cells, termed BC7. Infection of BC7 cells by HIV-1/IIIBx was resistant to anti-CD4 monoclonal antibodies but inhibited by the anti-CXCR4 mAb, 12G5. HIV-1/IIIBx was highly fusogenic on 3T3 cells expressing CXCR4 in the absence of CD4. In contrast to HIV-2/vcp, the host range of HIV-1/IIIBx was highly restricted and replication in several CD4+/CXCR4+ lymphoid cell lines was reduced compared to HIV-1/IIIB. In addition, HIV-1/IIIBx failed to downregulate CXCR4 on chronically infected cells. These studies indicate that HIV-1 and HIV-2 variants can be derived in vitro that utilize CXCR4 in the absence of CD4. Although the mechanism(s) for these changes remain unclear, possibilities include an increased avidity of the viral envelope glycoprotein for CXCR4 and/or the increased exposure of the chemokine receptor binding site. Further biochemical and molecular analysis of the envelope glycoproteins from these viruses should be helpful in addressing these and other possibilities.

An internalization signal in the simian immunodeficiency virus transmembrane protein cytoplasmic domain modulates expression of envelope glycoproteins on the cell surface.

A Tyr to Cys mutation at amino acid position 723 in the cytoplasmic domain of the simian immunodeficiency virus (SIV) transmembrane (TM) molecule has been shown to increase expression of envelope glycoproteins on the surface of infected cells. Here we show that Tyr-723 contributes to a sorting signal that directs the rapid endocytosis of viral glycoproteins from the plasma membrane via coated pits. On cells infected by SIVs with a Tyr at position 723, envelope glycoproteins were transiently expressed on the cell surface and then rapidly endocytosed. Similar findings were noted for envelope molecules expressed in the absence of other viral proteins. Immunoelectron microscopy demonstrated that these molecules were localized in patches on the cell surface and were frequently associated with coated pits. In contrast, envelope glycoproteins containing a Y723C mutation were diffusely distributed over the entire plasma membrane. To determine if an internalization signal was present in the SIV TM, chimeric molecules were constructed that contained the CD4 external and membrane spanning domains and a SIV TM cytoplasmic tail with a Tyr or other amino acids at SIV position 723. In Hela cells stably expressing these molecules, chimeras with a Tyr-723 were rapidly endocytosed, while chimeras containing other amino acids at position 723, including a Phe, were internalized at rates only slightly faster than a CD4 molecule that lacked a cytoplasmic domain. In addition, the biological effects of the internalization signal were evaluated in infectious viruses. A mutation that disrupted the signal and as a result, increased the level of viral envelope glycoprotein on infected cells, was associated with accelerated infection kinetics and increased cell fusion during viral replication. These results demonstrate that a Tyr-dependent motif in the SIV TM cytoplasmic domain can function as an internalization signal that can modulate expression of the viral envelope molecules on the cell surface and affect the biological properties of infectious viruses. The conservation of an analogous Tyr in all human and simian immunodeficiency viruses suggests that this signal may be present in other primate lentiviruses and could be important in the pathogenesis of these viruses in vivo.

A single amino acid change in the cytoplasmic domain of the simian immunodeficiency virus transmembrane molecule increases envelope glycoprotein expression on infected cells.

We have described a virus termed CP-MAC, derived from the BK28 molecular clone of simian immunodeficiency virus, that was remarkable for its ability to infect Sup-T1 cells with rapid kinetics, cell fusion, and CD4 down-modulation (C. C. LaBranche, M. M. Sauter, B. S. Haggarty, P. J. Vance, J. Romano, T. K. Hart, P. J. Bugelski, and J. A. Hoxie, J. Virol. 68:5509-5522, 1994 [Erratum 68:7665-7667]). Compared with BK28, CP-MAC exhibited a number of changes in its envelope glycoproteins, including a highly stable association between the external (SU) and transmembrane (TM) molecules, a more rapid electrophoretic mobility of TM, and, of particular interest, a marked increase in the level of envelope protein expression on the surface of infected cells. These changes were shown to be associated with 11 coding mutations in the env gene (5 in SU and 6 in TM). In this report, we demonstrate that a single amino acid mutation of a Tyr to a Cys at position 723 (Y723C) in the TM cytoplasmic domain of CP-MAC is the principal determinant for the increased expression of envelope glycoproteins on the cell surface. When introduced into the env gene of BK28, the Y723C mutation produced up to a 25-fold increase in the levels of SU and TM on chronically infected cells, as determined by fluorescence-activated cell sorter analysis with monoclonal and polyclonal antibodies. A similar effect was observed when a Tyr-to-Cys change was introduced at the analogous position (amino acid 721) in the SIVmac239 molecular clone, which, unlike BK28 does not contain a premature stop codon in its TM cytoplasmic tail. Substituting other amino acids, including Ala, Ile, and Ser, at this position produced increases in surface envelope glycoproteins that were similar to that observed for the Cys substitution, while a Tyr-to-Phe mutation produced a smaller increase. These results could not be accounted for by differences in the kinetics or efficiency of envelope glycoprotein processing or by shedding of SU from infected cells. However, immunoelectron microscopy demonstrated that the Y723C mutation in BK28 produced a striking redistribution of cell surface envelope molecules from localized patches to a diffuse pattern that covered the entire plasma membrane. This finding suggests that mutation of a Tyr residue in the simian immunodeficiency virus TM cytoplasmic domain may disrupt a structural element that can modulate envelope glycoprotein expression on the surface of infected cells.

Biological, molecular, and structural analysis of a cytopathic variant from a molecularly cloned simian immunodeficiency virus.

Some isolates of simian immunodeficiency virus (SIV) have been shown to infect Sup-T1 cells with slow kinetics and in the absence of cytopathic effects, including cell fusion or CD4 down-modulation (J. A. Hoxie, B. S. Haggarty, S. Bonser, J. Rackowski, H. Shan, and P. Kanki, J. Virol. 62:2557-2568, 1988). In the present study, we describe the isolation and characterization of a SIVmac variant, derived from the BK28 infectious molecular clone, that became highly cytopathic for Sup-T1 cells. This variant, termed CP-MAC, exhibited a number of differences from BK28, including (i) an altered tropism which largely restricted its host range to Sup-T1 cells, (ii) the ability to induce cell fusion and CD4 down-modulation, and (iii) a highly stable interaction of its external (SU) and transmembrane (TM) envelope glycoproteins. In addition, a marked increase in the level of surface envelope glycoproteins was observed both on CP-MAC-infected cells and on virions. The CP-MAC env gene was PCR amplified from infected cells, and sequence analysis identified five amino acid changes in SU and six in TM compared with BK28. The introduction of these changes into BK28 was shown to fully reconstitute the biological and morphological properties of CP-MAC. The limited number of mutations in CP-MAC should enable the molecular determinants to be more precisely defined and help to identify the underlying mechanisms responsible for the striking biological and structural alterations exhibited by this virus.

Deletion of carboxy-terminal residues of murine granulocyte-macrophage colony-stimulating factor results in a loss of biologic activity and altered glycosylation.

A deletion mutant of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) which differs in primary structure from native GM-CSF in the carboxy-terminal 11 amino acids was prepared. Four amino acid residues are mutated and the seven terminal residues including Cys-118 are deleted. Supernatants from COS-1 cells transfected with this deletion mutant (GM-CSF(del] showed a 3000-fold decrease in the ability to stimulate bone marrow stem cells to proliferate and differentiate into granulocytes and macrophages in vitro. Northern blot analysis using poly(A)+ RNA extracted from the transfected cells showed equal accumulations of GM-CSF and GM-CSF(del). Transfection with full-length GM-CSF followed by immunoprecipitation of metabolically labeled supernatant proteins with rabbit anti-rGM-CSF antiserum yielded predominantly the 23-kDa, fully glycosylated form and small amounts of both a 29-kDa form and the 18-kDa non-N-glycosylated form. Transfection of the GM-CSF(del) mutant and immunoprecipitation revealed a large, diffuse band on sodium dodecyl sulfate--polyacrylamide gel electrophoresis with a molecular weight of about 29 kDa. Digestion of the immunoprecipitated 29-kDa species with N-glycanase converted the 29-kDa form into two forms of about 23 and 18 kDa, suggesting that the increase in molecular weight of the deletion mutant protein resulted from hyperglycosylation. Adding tunicamycin to the culture medium of cells transfected with GM-CSF(del) also yielded a single non-N-glycosylated species of about 18 kDa, but secretion was at a significantly lower level than either the 29-kDa hyperglycosylated GM-CSF(del) protein from non-tunicamycin-treated cells or the 18-kDa non-N-glycosylated full-length GM-CSF from tunicamycin-treated cells. Since very recent scanning-deletion analysis indicates that there is a critical region for activity near Cys-118 and that Cys-118 is necessary for maximal activity, we conclude that the Cys-118 residue is necessary for proper glycosylation and maximal biologic activity of GM-CSF.