Unexpected coreceptor usage of primary human immunodeficiency virus type 1 isolates from viremic patients under highly active antiretroviral therapy.

Recently, combinations of antiretroviral drugs (highly active antiretroviral therapy [HAART]) have led to a dramatic reduction of human immunodeficiency virus type 1 (HIV-1)-related clinical symptoms. Success of treatment is defined as almost complete suppression of plasma viremia, although in a sizable fraction of patients this goal is not achieved. We characterized primary HIV-1 isolates from 2 cohorts of patients in which HAART failed in terms of viral suppression. One cohort showed clinical benefit and stable or increasing CD4+ T cell numbers despite high viral load. The second viremic cohort had no CD4+ T cell recovery and exhibited typical AIDS-related symptoms. Primary isolates from HAART patients with minor clinical symptoms used CXCR4 as the most relevant receptor on primary cells. Thus, for the first time, it is shown that patients improving clinically under HAART harbor relatively high viral loads with viruses preferring CXCR4 as coreceptor.

Chemoattractant factors and the control of human immunodeficiency virus replication.

Factors secreted by CD8(+) T cells have been described to suppress immunodeficiency virus replication. The research efforts to identify these factors led to the proposal of some candidate proteins as being responsible for the antiviral effects. Chemokines and IL-16 are secreted by CD8(+) T cells and inhibit HIV replication through different mechanisms. However, their antiviral properties cannot fully explain the inhibitory activities found in cell culture supernatants from CD8(+) T cells.

Folding, assembly, and intracellular trafficking of the human immunodeficiency virus type 1 envelope glycoprotein analyzed with monoclonal antibodies recognizing maturational intermediates.

Monoclonal antibodies (MAbs) that bind linear or conformational epitopes on monomeric or oligomeric human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins were screened for their recognition of maturational intermediates. On the basis of reactivities with gp160 at different times after pulse-labeling, the MAbs were sorted into groups that exhibited binding which was immediate and constant, immediate but transient, delayed, late, or very late. This grouping was consistent with the selectivity of the MAbs for structural features of gp160. Thus, a MAb to the V3 loop reacted with envelope proteins at all times, in accord with the relative conformational independence and accessibility of the epitope. Several MAbs that preferentially react with monomeric gp160 exhibited diminished binding after the pulse. A 10-min tag occurred before gp160 reacted with conformational MAbs that inhibited CD4 binding. The availability of epitopes for other conformational MAbs, including some that react equally with monomeric and oligomeric gp160 and some that react better with oligomeric forms, was half-maximal in 30 min and closely followed the kinetics of gp160 oligomerization. Remarkably, there was a 1- to 2-h delay before gp160 reacted with stringent oligomer-specific MAbs. After 4 h, approximately 20% of the gp160 was recognized by these MAbs. Epitopes recognized by monomerspecific or CD4-blocking MAbs but not by oligomer-dependent MAbs were present on gp160 molecules associated with the molecular chaperone BiP/GRP78. MAbs with a preference for monomers reacted with recombinant or HIV-1 envelope proteins in the endoplasmic reticulum, whereas the oligomer-specific MAbs recognized them in the Golgi complex. Additional information regarding gp160 maturation and intracellular trafficking was obtained by using brefeldin A, dithiothreitol, and a low temperature.

Calreticulin interacts with newly synthesized human immunodeficiency virus type 1 envelope glycoprotein, suggesting a chaperone function similar to that of calnexin.

The ubiquitous eukaryotic protein calreticulin has been detected in a wide variety of different cell types. Recently, calreticulin was found to bind in vitro to a number of proteins isolated from the endoplasmic reticulum. In addition, calreticulin has sequence similarities with the molecular chaperone calnexin. These data suggest that calreticulin might also act as a chaperone. We found that calreticulin associated transiently with a large number of newly synthesized cellular proteins. In cells expressing recombinant human immunodeficiency virus (HIV) envelope glycoprotein, gp160 bound transiently to calreticulin with a peak at 10 min after its synthesis. Binding of gp120 to calreticulin was not detected because proteolytic cleavage of gp160 occurs in the trans-Golgi. Nonglycosylated HIV envelope protein was not associated with calreticulin, suggesting a requirement for N-linked oligosaccharides on newly synthesized proteins as has been reported for calnexin. The in vivo binding kinetics of calnexin and calreticulin to gp160 were very similar. Sequential immunoprecipitations provided evidence for the existence of ternary complexes of gp160, calreticulin, and calnexin. The data suggested that most of the gp160 associated with calreticulin was also bound to calnexin but that only a portion of the gp160 associated with calnexin was also bound to calreticulin.

Comparison of humoral immunity and induction of proliferating T lymphocytes in vaccinia virus-infected rabbits and rhesus macaques.

Vaccina virus (VV) infection induces specific antibodies and cytotoxic T cells in various animal species. Therefore, helper T cells also should be induced that stimulate the humoral and cellular immune responses. We determined such helper T-cell activity in 2 species after VV infection. Rabbits and rhesus macaques were infected with the Copenhagen strain of VV or with recombinant VV expressing retroviral proteins. Animals of both species developed antibodies and specific proliferative T-cell response. This reactivity could be enhanced by booster infection with VV. The proliferating macaque cells were CD4+ and major histocompatibility complex class II-restricted. These data confirm the broad immunogenicity of VV. Expression of additional polypeptides expressed from a recombinant VV does not lead to altered immune response to VV antigens. However, strength of the helper T-cell response, as well as clinical reactions, differed between macaques and rabbits. Infection with recombinant VV as delivery vectors offers the opportunity for combined vaccination against recombinant proteins and does not diminish cellular and humoral immune responses to VV itself.

Generation, characterization and cross-reactivities of monoclonal antibodies against the p24 core protein and the gp130 envelope glycoprotein of HIV-2ben.

The purpose of this study was to characterize antigenic determinants on structural polypeptides of human immunodeficiency virus type 2 (HIV-2ben). Therefore, three HIV-2-specific monoclonal antibodies (mAbs) against the p24 core protein (gag) and one mAb against the gp130 envelope glycoprotein (env) were produced. In addition to p24 the anti-core mAbs recognized the primary translation product of the viral gag gene p55 and an intermediate cleavage product p41. Core mAbs cross-reacted with another HIV-2 isolate (HIV-2rod), and several simian immunodeficiency viruses (SIVagm TYO7 and SIVmac), but not with SIVmnd and the HIV-1 isolates investigated (HIV-1han and HIV-1lai). The env mAb cross-reacted with HIV-2rod and SIVmac but not with SIVagm, SIVmnd or HIV-1. In competition assays and with epitope mapping possible binding sites for the mAbs were identified. The processing of HIV-2 core proteins is compared in retrovirus-infected T cell lines and during the expression by recombinant vaccinia virus. Finally, the mAb XIV DC10 which recognized a highly conserved epitope could be useful for an assay to detect HIV-1 and HIV-2 simultaneously. II D8 is the first mAb raised against HIV-2 env glycoprotein.

Characterization of monoclonal antibodies to the envelope proteins of an immunodeficiency virus of African green monkeys, SIVagmTYO-7.

Five monoclonal antibodies (mabs) specific for the envelope proteins of a simian immunodeficiency virus of African green monkeys (SIVagm) have been raised. Two mabs were directed against distinct epitopes on the transmembrane protein gp41. A conformational epitope on the gp130 was recognized by three mabs. This is the first report on mabs specific for SIVagm-gp130. Studies of the cross-reactivities revealed that the epitopes recognized by the env-directed mabs are conserved species-specifically in SIVagm isolates. Therefore, these mabs can be used to distinguish SIVagm strains from other virus groups.

Mapping of the human immunodeficiency virus type 2 envelope glycoprotein CD4 binding region and fusion domain with truncated proteins expressed by recombinant vaccinia viruses.

Human immunodeficiency virus type 2 (HIV-2) is more closely related to certain simian immunodeficiency viruses than to HIV-1. The HIV-1 and HIV-2 envelope (env) glycoproteins share only approximately 40% amino acid (aa) sequence homology. Additionally, HIV-1 and HIV-2 seem to differ in pathogenicity and in host range. In order to identify the functional domains of the HIV-2 env glycoprotein, e.g., the CD4 binding region, the membrane anchor, and the fusion site, and to compare them to equivalent sites of HIV-1, a set of recombinant vaccinia viruses (VV) was constructed expressing N-terminal overlapping env proteins of 863 (full-length gp160), 708, 534 (full-length gp120), 438, 332, 198, and 488 aa (internal deletion of aa 333-707). Upon infection, only env proteins comprising the amino-terminal half of the transmembrane protein were expressed on the cell surface. Such VV constructs also induced syncytia in CD4-positive cells. The syncytia were smaller when the cytoplasmic domain of the transmembrane protein was removed. The CD4 binding site of HIV-2 was located between the carboxy terminus of gp120 (aa 512) and aa 438. Thus the amino-terminal half of the transmembrane protein of HIV-2 is sufficient for cell surface localization of the env protein and syncytia induction. These properties are shared with the HIV-1 env protein and demonstrate a functional conservation among HIV-1 and HIV-2 despite their genetic and phenotypic heterogeneity.

Virus-specific cytotoxic T-lymphocytes in HIV-2-infected cynomolgus macaques.

OBJECTIVE: Specific cytotoxic T-lymphocytes (CTL) are induced in humans or monkeys after infection with HIV-1 or SIVmac, respectively. Since, like HIV-1, HIV-2 causes AIDS, our objective was to determine the characteristics of the HIV-2-specific CTL response. DESIGN: Since it is rarely possible to study cellular immunity in individuals, because of the small number of HIV-2-infected patients available in Europe and the necessity for co-operation in the performance of sequential CTL assays, cynomolgus macaques were infected with HIV-2. Autologous transformed B-lymphoblastoid cell lines infected with recombinant vaccinia viruses were used as target cells for cytotoxicity assays. METHODS: Recombinant vaccinia viruses expressing HIV-2 genes were constructed to infect B-lymphoblastoid cell lines from macaques. These cells were used as target cells for cytotoxicity assays with peripheral blood mononuclear cells from HIV-2BEN-infected cynomolgus macaques. To characterize the effector cells, CD8+ cells were separated with immunomagnetic beads. Major histocompatibility complex (MHC) restriction of the cytotoxic cells was determined by incubation with matched or mismatched target cells. RESULTS: HIV-2BEN-infected cynomolgus macaques raised CTL against proteins of the three major viral structural genes, gag, pol and env. The cytotoxic cells were CD8+ and their activity was MHC class I-restricted. In contrast to SIVmac-infected macaques, env-specific lysis was mediated exclusively by CD8+ cells. CTL from individual animals recognized different viral proteins and the recognition pattern varied over time. CONCLUSIONS: Like HIV-1 and SIVmac, HIV-2 induces virus-specific CTL. The variation of antigen recognition between individual animals and over time indicates that sequential experiments are necessary to determine the complete spectrum of the CTL response of infected animals. HIV-2-infected macaques represent a suitable model for investigations into the cellular immune response against HIV.

Identification of a gag protein epitope conserved among all four groups of primate immunodeficiency viruses by using monoclonal antibodies.

Five monoclonal antibodies (MAbs) were raised against the gag proteins of simian immunodeficiency virus (SIV) from African green monkey (SIVagmTYO-7). Two MAbs reacted with the matrix protein p17 and the other three with the core protein p24. Studies on the cross-reactivity of the MAbs revealed that the anti-p24 MAbs detected an epitope shared by the viruses belonging to the human immunodeficiency virus type 2 (HIV-2)/SIVmac group and SIVagmTYO-7 and SIVagmTYO-5. The anti-p17 MAbs recognized an epitope present on all these viruses and on SIVagmTYO-1, HIV-1 and SIVmnd. This finding demonstrates for the first time that the matrix protein, p17 or p18, respectively, of all nine HIV and SIV isolates tested in this study expresses at least one conserved immunogenic epitope recognized serologically. By using synthetic peptides, this epitope was identified at the N terminus of p17. Furthermore, this epitope was analysed by multiple sequence alignments of the peptide with homologous sequences of HIV and SIV p17.