Neutralization Sensitivity of Human Immunodeficiency Virus Type 1 Primary Isolates to Antibodies and CD4-Based Reagents Is Independent of Coreceptor Usage

We have investigated whether the identity of the coreceptor (CCR5, CXCR4, or both) used by primary human immunodeficiency virus type 1 (HIV-1) isolates to enter CD4⁺ cells influences the sensitivity of these isolates to neutralization by monoclonal antibodies and CD4-based agents. Coreceptor usage was not an important determinant of neutralization titer for primary isolates in peripheral blood mononuclear cells. We also studied whether dualtropic primary isolates (able to use both CCR5 and CXCR4) were differentially sensitive to neutralization by the same antibodies when entering U87MG-CD4 cells stably expressing either CCR5 or CXCR4. Again, we found that the coreceptor used by a virus did not greatly affect its neutralization sensitivity. Similar results were obtained for CCR5- or CXCR4-expressing HOS cell lines engineered to express green fluorescent protein as a reporter of HIV-1 entry. Neutralizing antibodies are therefore unlikely to be the major selection pressure which drives the phenotypic evolution (change in coreceptor usage) of HIV-1 that can occur in vivo. In addition, the increase in neutralization sensitivity found when primary isolates adapt to growth in transformed cell lines in vitro has little to do with alterations in coreceptor usage.Human immunodeficiency virus type 1 (HIV-1) enters CD4⁺ T cells via an interaction with CD4 and coreceptor molecules, the most important of which yet identified are the chemokine receptors CXCR4 and CCR5 (4, 12, 23, 26, 28, 32). CXCR4 is used by T-cell line-tropic (T-tropic) primary isolates or T-cell line-adapted (TCLA) lab strains, whereas CCR5 is used by primary isolates of the macrophage-tropic (M-tropic) phenotype (4, 12, 23, 26, 28, 32). Most T-tropic isolates and some TCLA strains are actually dualtropic in that they can use both CXCR4 and CCR5 (and often other coreceptors such as CCR3, Bonzo/STRL33, and BOB/gpr15), at least in coreceptor-transfected cells (18, 24, 30, 54, 89). The M-tropic and T-tropic/dualtropic nomenclature has often been used interchangeably with the terms “non-syncytium-inducing” (NSI) and “syncytium-inducing” (SI), although it is semantically imprecise to do so.M-tropic viruses are those most commonly transmitted sexually (3, 33, 87, 106) and from mother to infant (2, 72, 81). If T-tropic strains are transmitted, or when they emerge, this is associated with a more rapid course of disease in both adults (17, 37, 46, 51, 52, 76, 78, 82, 92, 101) and children (6, 45, 84, 90). However, T-tropic viruses emerge in only about 40% of infected people, usually only several years after infection (76, 78). A well-documented, albeit anecdotal, study found that when a T-tropic strain was transmitted by direct transfer of blood, its replication was rapidly suppressed: the T-tropic virus was eliminated from the body, and M-tropic strains predominated (20). These results suggest that there is a counterselection pressure against the emergence of T-tropic strains during the early stages of HIV-1 infection in most people. But what is this pressure?Since the M-tropic and T-tropic phenotypes are properties mediated by the envelope glycoproteins whose function is to associate with CD4 and the coreceptors, a selection pressure differentially exerted on M- and T-tropic viruses could, in principle, act at the level of virus entry. In other words, neutralizing antibodies to the envelope glycoproteins, or the chemokine ligands of the coreceptors, could theoretically interfere more potently with the interactions of T-tropic strains with CXCR4 than with M-tropic viruses and CCR5. A differential effect of this nature could suppress the emergence of T-tropic viruses. Consistent with this possibility, neutralizing antibodies are capable of preventing the CD4-dependent association of gp120 with CCR5 (42, 94, 103), and chemokines can also prevent the coreceptor interactions of HIV-1 (8, 13, 23, 28, 70).Here, we explore whether the efficiency of HIV-1 neutralization is affected by coreceptor usage. Although earlier studies have not found T-tropic strains to be inherently more neutralization sensitive than M-tropic ones (20, 40, 44), previously available reagents and techniques may not have been adequate to fully address this question. One major problem is that even single residue changes can drastically affect both antibody binding to neutralization epitopes and the HIV-1 phenotype (25, 55, 62, 67, 83, 91), and so studies using relatively unrelated viruses and a fixed antibody (polyclonal or monoclonal) preparation have two variables to contend with: the viral phenotype (coreceptor use) and the antigenic structure of the virus and hence the efficiency of the antibody-virion interaction.We have used a new experimental strategy to explore whether coreceptor usage affects neutralization sensitivity in the absence of other confounding variables: the use of dualtropic viruses able to enter CD4⁺ cells via either CCR5 or CXCR4. By using a constant HIV-1 isolate or clone and the same monoclonal antibodies (MAbs) or CD4-based reagents as neutralizing agents, we can ensure that the only variable under study in the neutralization reaction is the nature of the coreceptor used for entry. Our major conclusion is that there is no strong association between coreceptor usage and neutralization sensitivity for primary HIV-1 isolates. Independent studies have reached the same conclusion (53a, 59). The emergence of T-tropic (SI) viruses in vivo may be unlikely to be due to escape from antibody-mediated selection pressure.     

Neutralization Profiles of Primary Human Immunodeficiency Virus Type 1 Isolates in the Context of Coreceptor Usage

Most strains of human immunodeficiency virus type 1 (HIV-1) which have only been carried in vitro in peripheral blood mononuclear cells (primary isolates) can be neutralized by antibodies, but their sensitivity to neutralization varies considerably. To study the parameters that contribute to the differential neutralization sensitivity of primary HIV-1 isolates, we developed a neutralization assay with a panel of genetically engineered cell lines (GHOST cells) that express CD4, one of eight chemokine receptors which function as HIV-1 coreceptors, and a Tat-dependent green fluorescent protein reporter cassette which permits the evaluation and quantitation of HIV-1 infection by flow cytometry. All 21 primary isolates from several clades could grow in the various GHOST cell lines, and their use of one or more coreceptors could easily be defined by flow cytometric analysis. Ten of these primary isolates, three that were CXCR4 (X4)-tropic, three that were CCR5 (R5)-tropic, and four that were dual- or polytropic were chosen for study of their sensitivity to neutralization by human monoclonal and polyclonal antibodies. Viruses from the X4-tropic category of viruses were first tested since they have generally been considered to be particularly neutralization sensitive. It was found that the X4-tropic virus group contained both neutralization-sensitive and neutralization-resistant viruses. Similar results were obtained with R5-tropic viruses and with dual- or polytropic viruses. Within each category of viruses, neutralization sensitivity and resistance could be observed. Therefore, sensitivity to neutralization appears to be the consequence of factors that influence the antibody-virus interaction and its sequelae rather than coreceptor usage. Neutralization of various viruses by the V3-specific monoclonal antibody, 447-52D, was shown to be dependent not only on the presence of the relevant epitope but also on its presentation. An epitope within the envelope of a particular virus is not sufficient to render a virus sensitive to neutralization by an antibody that recognizes that epitope. Moreover, conformation-dependent factors may overcome the need for absolute fidelity in the match between an antibody and its core epitope, permitting sufficient affinity between the viral envelope protein and the antibody to neutralize the virus. The studies indicate that the neutralization sensitivity of HIV-1 primary isolates is a consequence of the complex interaction between virus, antibody, and target cell.     

Chemokine Coreceptor Usage by Diverse Primary Isolates of Human Immunodeficiency Virus Type 1

We tested chemokine receptor subset usage by diverse, well-characterized primary viruses isolated from peripheral blood by monitoring viral replication with CCR1, CCR2b, CCR3, CCR5, and CXCR4 U87MG.CD4 transformed cell lines and STRL33/BONZO/TYMSTR and GPR15/BOB HOS.CD4 transformed cell lines. Primary viruses were isolated from 79 men with confirmed human immunodeficiency virus type 1 (HIV-1) infection from the Chicago component of the Multicenter AIDS Cohort Study at interval time points. Thirty-five additional well-characterized primary viruses representing HIV-1 group M subtypes A, B, C, D, and E and group O and three primary simian immunodeficiency virus (SIV) isolates were also used for these studies. The restricted use of the CCR5 chemokine receptor for viral entry was associated with infection by a virus having a non-syncytium-inducing phenotype and correlated with a reduced rate of disease progression and a prolonged disease-free interval. Conversely, broadening chemokine receptor usage from CCR5 to both CCR5 and CXCR4 was associated with infection by a virus having a syncytium-inducing phenotype and correlated with a faster rate of CD4 T-cell decline and progression of disease. We also observed a greater tendency for infection with a virus having a syncytium-inducing phenotype in men heterozygous for the defective CCR5 Δ32 allele (25%) than in those men homozygous for the wild-type CCR5 allele (6%) (P = 0.03). The propensity for infection with a virus having a syncytium-inducing phenotype provides a partial explanation for the rapid disease progression among some men heterozygous for the defective CCR5 Δ32 allele. Furthermore, we did not identify any primary viruses that used CCR3 as an entry cofactor, despite this CC chemokine receptor being expressed on the cell surface at a level commensurate with or higher than that observed for primary peripheral blood mononuclear cells. Whereas isolates of primary viruses of SIV also used STRL33/BONZO/TYMSTR and GPR15/BOB, no primary isolates of HIV-1 used these particular chemokine receptor-like orphan molecules as entry cofactors, suggesting a limited contribution of these other chemokine receptors to viral evolution. Thus, despite the number of chemokine receptors implicated in viral entry, CCR5 and CXCR4 are likely to be the physiologically relevant chemokine receptors used as entry cofactors in vivo by diverse strains of primary viruses isolated from blood.     

Evidence that Antibody-Mediated Neutralization of Human Immunodeficiency Virus Type 1 by Sera from Infected Individuals Is Independent of Coreceptor Usage

Human immunodeficiency virus type 1 (HIV-1) uses a variety of chemokine receptors as coreceptors for virus entry, and the ability of the virus to be neutralized by antibody may depend on which coreceptors are used. In particular, laboratory-adapted variants of the virus that use CXCR4 as a coreceptor are highly sensitive to neutralization by sera from HIV-1-infected individuals, whereas primary isolates that use CCR5 instead of, or in addition to, CXCR4 are neutralized poorly. To determine whether this dichotomy in neutralization sensitivity could be explained by differential coreceptor usage, virus neutralization by serum samples from HIV-1-infected individuals was assessed in MT-2 cells, which express CXCR4 but not CCR5, and in mitogen-stimulated human peripheral blood mononuclear cells (PBMC), where multiple coreceptors including CXCR4 and CCR5 are available for use. Our results showed that three of four primary isolates with a syncytium-inducing (SI) phenotype and that use CXCR4 and CCR5 were neutralized poorly in both MT-2 cells and PBMC. The fourth isolate, designated 89.6, was more sensitive to neutralization in MT-2 cells than in PBMC. We showed that the neutralization of 89.6 in PBMC was not improved when CCR5 was blocked by having RANTES, MIP-1α, and MIP-1β in the culture medium, indicating that CCR5 usage was not responsible for the decreased sensitivity to neutralization in PBMC. Consistent with this finding, a laboratory-adapted strain of virus (IIIB) was significantly more sensitive to neutralization in CCR5-deficient PBMC (homozygous Δ32-CCR5 allele) than were two of two SI primary isolates tested. The results indicate that the ability of HIV-1 to be neutralized by sera from infected individuals depends on factors other than coreceptor usage.Human immunodeficiency virus type 1 (HIV-1), the etiologic agent of AIDS, utilizes the HLA class II receptor, CD4, as its primary receptor to gain entry into cells (17, 30). Entry is initiated by a high-affinity interaction between CD4 and the surface gp120 of the virus (32). Subsequent to this interaction, conformational changes that permit fusion of the viral membrane with cellular membranes occur within the viral transmembrane gp41 (9, 58, 59). In addition to CD4, one or more recently described viral coreceptors are needed for fusion to take place. These coreceptors belong to a family of seven-transmembrane G-protein-coupled proteins and include the CXC chemokine receptor CXCR4 (3, 4, 24, 44), the CC chemokine receptors CCR5 (1, 12, 13, 18, 21, 23, 45) and, less commonly, CCR3 and CCR2b (12, 21), and two related orphan receptors termed BONZO/STRL33 and BOB (19, 34). Coreceptor usage by HIV-1 can be blocked by naturally occurring ligands, including SDF-1 for CXCR4 (4, 44), RANTES, MIP-1α, and MIP-1β in the case of CCR5 (13, 45), and eotaxin for CCR3 (12).The selective cellular tropisms of different strains of HIV-1 may be determined in part by coreceptor usage. For example, all culturable HIV-1 variants replicate initially in mitogen-stimulated human peripheral blood mononuclear cells (PBMC), but only a minor fraction are able to infect established CD4⁺ T-cell lines (43). This differential tropism is explained by the expression of CXCR4 together with CCR5 and other CC chemokine coreceptors on PBMC and the lack of expression of CCR5 on most T-cell lines (5, 10, 19, 35, 39, 50, 53). Indeed, low-passage field strains (i.e., primary isolates) of HIV-1 that fail to replicate in T-cell lines use CCR5 as their major coreceptor and are unable to use CXCR4 (1, 12, 18, 21, 23, 28). Because these isolates rarely produce syncytia in PBMC and fail to infect MT-2 cells, they are often classified as having a non-syncytium-inducing (NSI) phenotype. Primary isolates with a syncytium-inducing (SI) phenotype are able to use CXCR4 alone or, more usually, in addition to CCR5 (16, 20, 51). HIV-1 variants that have been passaged multiple times in CD4⁺ T-cell lines, and therefore considered to be laboratory adapted, exhibit a pattern of coreceptor usage that resembles that of SI primary isolates. Most studies have shown that the laboratory-adapted strain IIIB uses CXCR4 alone (3, 13, 20, 24, 51) and that MN and SF-2 use CXCR4 primarily and CCR5 to a lesser degree (11, 13). Sequences within the V3 loop of gp120 have been shown to be important, either directly or indirectly, for the interaction of HIV-1 with both CXCR4 (52) and CCR5 (12, 14, 54, 60). This region of gp120 contains multiple determinants of cellular tropism (43) and is a major target for neutralizing antibodies to laboratory-adapted HIV-1 but not to primary isolates (29, 46, 57).It has been known for some time that the ability of sera from HIV-1-infected individuals to neutralize laboratory-adapted strains of HIV-1 does not predict their ability to neutralize primary isolates in vitro (7). In general, the former viruses are highly sensitive to neutralization whereas the latter viruses are neutralized poorly by antibodies induced in response to HIV-1 infection (7, 43). Importantly, neutralizing antibodies generated by candidate HIV-1 subunit vaccines have been highly specific for laboratory-adapted viruses (26, 37, 38). In principle, the dichotomy in neutralization sensitivity between these two categories of virus could be related to coreceptor usage. To test this, we investigated whether the use of CXCR4 in the absence of CCR5 would render SI primary isolates highly sensitive to neutralization in vitro by sera from HIV-1-infected individuals. Two similar studies using human monoclonal antibodies and soluble CD4 have been reported (31a, 55).     

CXCR4 as a Functional Coreceptor for Human Immunodeficiency Virus Type 1 Infection of Primary Macrophages

The coreceptors used by primary syncytium-inducing (SI) human immunodeficiency virus type 1 isolates for infection of primary macrophages were investigated. SI strains using only CXCR4 replicated equally well in macrophages with or without CCR5 and were inhibited by several different ligands for CXCR4 including SDF-1 and bicyclam derivative AMD3100. SI strains that used a broad range of coreceptors including CCR3, CCR5, CCR8, CXCR4, and BONZO infected CCR5-deficient macrophages about 10-fold less efficiently than CCR5⁺ macrophages. Moreover, AMD3100 blocked infection of CCR5-negative macrophages by these strains. Our results therefore demonstrate that CXCR4, as well as CCR5, is used for infection of primary macrophages but provide no evidence for the use of alternative coreceptors.     

Continued Utilization of CCR5 Coreceptor by a Newly Derived T-Cell Line-Adapted Isolate of Human Immunodeficiency Virus Type 1

The differential use of CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) may be intimately involved in the transmission and progression of human immunodeficiency virus infection. Changes in coreceptor utilization have also been noted upon adaptation of primary isolates (PI) to growth in established T-cell lines. All of the T-cell line-adapted (TCLA) viruses studied to date utilize CXCR4 but not CCR5. This observation had been suggested as an explanation for the sensitivity of TCLA, but not PI, viruses to neutralization by recombinant gp120 antisera and V3-directed monoclonal antibodies, but recent studies have shown coreceptor utilization to be independent of neutralization sensitivity. Here we describe a newly isolated TCLA virus that is sensitive to neutralization but continues to utilize both CXCR4 and CCR5 for infection. This finding further divorces coreceptor specificity from neutralization sensitivity and from certain changes in cell tropism. That the TCLA virus can continue to utilize CCR5 despite the changes that occur upon adaptation and in the apparent absence of CCR5 expression in the FDA/H9 T-cell line suggests that the interaction between envelope protein and coreceptor may be mediated by multiple weak interactions along a diffuse surface.     

Coreceptor Usage of Human Immunodeficiency Virus Type 2 Primary Isolates and Biological Clones Is Broad and Does Not Correlate with Their Syncytium-Inducing Capacities

Entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by binding of the surface envelope glycoprotein to the CD4 molecule. Interaction of the resulting CD4-glycoprotein complex with α- or β-chemokine receptors, depending on the biological phenotype of the virus, then initiates the fusion process. Here, we show that primary HIV-2 isolates and biological clones, in contrast to those of HIV-1, may use a broad range of coreceptors, including CCR-1, CCR-3, CCR-5, and CXCR-4. The syncytium-inducing capacity of these viruses did not correlate with the ability to infect via CXCR-4 or any other coreceptor. One cell-free passage of the intermediate isolates in mitogen-stimulated, CD8⁺ cell-depleted peripheral blood mononuclear cells resulted in the outgrowth of variants with CCR-5 only, whereas the coreceptor usage of late and early isolates did not change. Since HIV-2 is less pathogenic in vivo than HIV-1, these data suggest that HIV pathogenicity in vivo is not directly related to the spectrum of coreceptors used in in vitro systems.     

A Cell Line-Based Neutralization Assay for Primary Human Immunodeficiency Virus Type 1 Isolates That Use either the CCR5 or the CXCR4 Coreceptor

We describe here a cell line-based assay for the evaluation of human immunodeficiency virus type 1 (HIV-1) neutralization. The assay is based on CEM.NKR cells, transfected to express the HIV-1 coreceptor CCR5 to supplement the endogenous expression of CD4 and the CXCR4 coreceptor. The resulting CEM.NKR-CCR5 cells efficiently replicate primary HIV-1 isolates of both R5 and X4 phenotypes. A comparison of the CEM.NKR-CCR5 cells with mitogen-activated peripheral blood mononuclear cells (PBMC) in neutralization assays with sera from HIV-1-infected individuals or specific anti-HIV-1 monoclonal antibodies shows that the sensitivity of HIV-1 neutralization is similar in the two cell types. The CEM.NKR-CCR5 cell assay, however, is more convenient to perform and eliminates the donor-to-donor variation in HIV-1 replication efficiency, which is one of the principal drawbacks of the PBMC-based neutralization assay. We suggest that this new assay is suitable for the general measurement of HIV-1 neutralization by antibodies.     

Intrinsic Human Immunodeficiency Virus Type 1 Resistance of Hematopoietic Stem Cells Despite Coreceptor Expression

Interactions of human immunodeficiency virus type 1 (HIV-1) with hematopoietic stem cells may define restrictions on immune reconstitution following effective antiretroviral therapy and affect stem cell gene therapy strategies for AIDS. In the present study, we demonstrated mRNA and cell surface expression of HIV-1 receptors CD4 and the chemokine receptors CCR-5 and CXCR-4 in fractionated cells representing multiple stages of hematopoietic development. Chemokine receptor function was documented in subsets of cells by calcium flux in response to a cognate ligand. Productive infection by HIV-1 via these receptors was observed with the notable exception of stem cells, in which case the presence of CD4, CXCR-4, and CCR-5, as documented by single-cell analysis for expression and function, was insufficient for infection. Neither productive infection, transgene expression, nor virus entry was detectable following exposure of stem cells to either wild-type HIV-1 or lentivirus constructs pseudotyped in HIV-1 envelopes of macrophage-tropic, T-cell-tropic, or dualtropic specificity. Successful entry into stem cells of a vesicular stomatitis virus G protein-pseudotyped HIV-1 construct demonstrated that the resistance to HIV-1 was mediated at the level of virus-cell membrane fusion and entry. These data define the hematopoietic stem cell as a sanctuary cell which is resistant to HIV-1 infection by a mechanism independent of receptor and coreceptor expression that suggests a novel means of cellular protection from HIV-1.     

Stoichiometry of Monoclonal Antibody Neutralization of T-Cell Line-Adapted Human Immunodeficiency Virus Type 1

In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes are coexpressed. By the coexpression of Env glycoproteins that either can or cannot bind a neutralizing MAb in an env transcomplementation assay, virions were generated in which the proportion of MAb binding sites could be regulated. As the proportion of MAb binding sites in Env chimeric virus increased, MAb neutralization gradually increased. Virus neutralization by virion aggregation was minimal, as MAb binding to HIV-1 Env did not interfere with an AMLV Env-mediated infection by HIV-1(AMLV/HIV-1) pseudotypes of CD4(-) HEK293 cells. MAb neutralization of chimeric virions could be described as a third-order function of the proportion of Env antigen refractory to MAb binding. This scenario is consistent with the Env oligomer constituting the minimal functional unit and neutralization occurring incrementally as each Env oligomer binds MAb. Alternatively, the data could be fit to a sigmoid function. Thus, these data could not exclude the existence of a threshold for neutralization. However, results from MAb neutralization of chimeric virus containing wild-type Env and Env defective in CD4 binding was readily explained by a model of incremental MAb neutralization. In summary, the data indicate that MAb neutralization of T-cell line-adapted HIV-1 is incremental rather than all or none and that each MAb binding an Env oligomer reduces the likelihood of infection.     

Determinants of Entry Cofactor Utilization and Tropism in a Dualtropic Human Immunodeficiency Virus Type 1 Primary Isolate

Human immunodeficiency virus type 1 strain 89.6 is a dualtropic isolate that replicates in macrophages and transformed T cells, and its envelope mediates CD4-dependent fusion and entry with CCR5, CXCR-4, and CCR3. To map determinants of cofactor utilization by 89.6 and determine the relationship between cofactor use and tropism, we analyzed recombinants generated between 89.6 and T-cell-tropic (HXB) or macrophage-tropic (JRFL) strains. These chimeras showed that regions of 89.6 env outside V3 through V5 determine CXCR-4 utilization and T-cell line tropism as well as CCR5 utilization and macrophage tropism. However, the 89.6 env V3 domain also conferred on HXB the ability to use CCR5 for fusion and entry but not the ability to establish productive macrophage infection. CCR3 use was conferred on HXB by 89.6 env V3 or V3 through V5 sequences. While replacement of the 89.6 V3 through V5 region with HXB sequences abrogated CCR3 utilization, replacement of V3 or V4 through V5 separately did not. Thus, CCR3 use is determined by sequences within V3 through V5 and most likely can be conferred by either the V3 or the V4 through V5 domains. These results indicate that cofactor utilization and tropism in this dualtropic isolate are determined by complex interactions among multiple env segments, that distinct regions of the Env glycoprotein may be important for utilization of different chemokine receptors, and that determinants in addition to cofactor usage participate in postentry stages in the virus replication cycle that contribute to target cell tropism.     

Antibody Neutralization-Resistant Primary Isolates of Human Immunodeficiency Virus Type 1

Although typical primary isolates of human immunodeficiency virus type 1 (HIV-1) are relatively neutralization resistant, three human monoclonal antibodies and a small number of HIV-1⁺ human sera that neutralize the majority of isolates have been described. The monoclonal antibodies (2G12, 2F5, and b12) represent specificities that a putative vaccine should aim to elicit, since in vitro neutralization has been correlated with protection against primary viruses in animal models. Furthermore, a neutralization escape mutant to one of the antibodies (b12) selected in vitro remains sensitive to neutralization by the other two (2G12 and 2F5) (H. Mo, L. Stamatatos, J. E. Ip, C. F. Barbas, P. W. H. I. Parren, D. R. Burton, J. P. Moore, and D. D. Ho, J. Virol. 71:6869–6874, 1997), supporting the notion that eliciting a combination of such specificities would be particularly advantageous. Here, however, we describe a small subset of viruses, mostly pediatric, which show a high level of neutralization resistance to all three human monoclonal antibodies and to two broadly neutralizing sera. Such viruses threaten antibody-based antiviral strategies, and the basis for their resistance should be explored.     

Enhanced Exposure of Human Immunodeficiency Virus Type 1 Primary Isolate Neutralization Epitopes through Binding of CD4 Mimetic Compounds

N-(4-Chlorophenyl)-N′-(2,2,6,6-tetramethyl-piperidin-4-yl)-oxalamide (NBD-556) is a low-molecular-weight compound that reportedly blocks the interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and its receptor CD4. We investigated whether the enhancement of binding of anti-gp120 monoclonal antibodies (MAbs) toward envelope (Env) protein with NBD-556 are similar to those of soluble CD4 (sCD4) by comparing the binding profiles of the individual MAbs to Env-expressing cell surfaces. In flow cytometric analyses, the binding profiles of anti-CD4-induced epitope (CD4i) MAbs toward NBD-556-pretreated Env-expressing cell surfaces were similar to the binding profiles toward sCD4-pretreated cell surfaces. To investigate the binding position of NBD-556 on gp120, we induced HIV-1 variants that were resistant to NBD-556 and sCD4 in vitro. At passage 21 in the presence of 50 μM NBD-556, two amino acid substitutions (S375N in C3 and A433T in C4) were identified. On the other hand, in the selection with sCD4, seven mutations (E211G, P212L, V255E, N280K, S375N, G380R, and G431E) appeared during the passages. The profiles of the mutations after the selections with NBD-556 and sCD4 were very similar in their three-dimensional positions. Moreover, combinations of NBD-556 with anti-gp120 MAbs showed highly synergistic interactions against HIV-1. We further found that after enhancing the neutralizing activity by adding NBD-556, the contemporaneous virus became highly sensitive to antibodies in the patient''s plasma. These findings suggest that small compounds such as NBDs may enhance the neutralizing activities of CD4i and anti-V3 antibodies in vivo.Human immunodeficiency virus type 1 (HIV-1) replicates continuously in the face of a strong antibody (Ab) response, although Abs effectively control many viral infections (3). Neutralizing Abs (NAbs) are directed against the HIV-1 envelope (Env) protein, which is a heterodimer comprising an extensively glycosylated CD4-binding subunit (gp120) and an associated transmembrane protein (gp41). Env proteins are present on the virion surface as “spikes” composed of trimers of three gp120-gp41 complexes (20, 21, 29). These spikes resist neutralization through epitope occlusion within the oligomer, extensive glycosylation, extension of variable loops from the surface of the complex, and steric and conformational blocking of receptor binding sites (16, 18, 20).Ab access to conserved regions is further limited because viral entry is a stepwise process involving conformational changes that lead to only transient exposure of conserved domains such as the coreceptor binding site (4, 5). However, some early strains of HIV-1 appear to be highly susceptible to neutralization by Abs (1, 10). For instance, subtype A HIV-1 envelopes from the early stage of infection exhibit a broad range of neutralization sensitivities to both autologous and heterologous plasma (1), suggesting that at least a subset of the envelopes have some preserved and/or exposed neutralization epitopes. It is well known that the potential for neutralizing properties of particular Abs is enhanced after binding of soluble CD4 (sCD4), especially NAbs against CD4-induced epitopes (CD4i Abs) (27) and some anti-V3 Abs (22). CD4i Abs are detected in plasma samples from many patients at an early stage of HIV-1 infection (9). Consequently, we hypothesize that small compounds such as sCD4 can enhance the neutralizing activities of CD4i Abs and some anti-V3 Abs not only in vitro but also in vivo.In a previous report, two low-molecular-weight compounds that presumably interfere with viral entry of HIV-1 into cells were described (35). These two N-phenyl-N′-(2,2,6,6-tetramethyl-piperidin-4-yl)-oxalamide analogs, NBD-556 and NBD-557, comprise a novel class of HIV-1 entry inhibitors that block the interaction between gp120 and CD4. These compounds were found to be equally potent inhibitors of both X4 and R5 viruses in CXCR4- and CCR5-expressing cell lines, respectively (35). Schön et al. (25) also reported that NBD-556 binds to gp120 in a process characterized by a large favorable change in enthalpy that is partially compensated for by a large unfavorable entropy change, representing a thermodynamic signature similar to that observed for binding of sCD4 to gp120. In a recent study, Madani et al. (23) reported the following findings: (i) NBD-556 binds within the Phe43 cavity, a highly conserved and functionally important pocket formed as gp120 assumes the CD4-bound conformation; (ii) the NBD-556 phenyl ring projects into the Phe43 cavity; (iii) the enhancement of CD4-independent infection by NBD-556 requires the induction of conformational changes in gp120; and (iv) increased affinities of NBD-556 analogs toward gp120 improve the antiviral potency during infection of CD4-expressing cells. The latter two studies demonstrated that low-molecular-weight compounds such as NBDs can induce conformational changes in the HIV-1 gp120 glycoprotein similar to those observed upon sCD4 binding (23, 25). The authors of these studies concluded that their data supported the importance of gp120 residues near the Phe43 cavity in binding to NBD-556 and lent credence to the docked binding mode.In the present study, we investigated the binding position of NBD-556 on gp120 by inducing HIV-1 variants that were resistant to NBD-556 by exposing HIV-1_IIIB to increasing concentrations of the compound in vitro. We also induced sCD4-resistant HIV-1_IIIB variants and compared the profile of the sCD4-resistant mutations to that of the NBD-556-resistant mutations. We subsequently examined the virological properties of pseudotyped HIV-1 clones carrying the NBD-556 and sCD4 resistance-associated env gene mutations. Our findings provide a foundation for understanding the interaction of NBD-556 with the CD4-binding site of HIV-1 gp120. We also evaluated the anti-HIV-1 interactions between plasma NAbs and NBD-556 in vitro and considered the possibility of using the data as a key to opening the shield covering the conserved epitopes targeted by NAbs.(This study was presented in part at the 15th Conference on Retroviruses and Opportunistic Infection, Boston, MA, 3 to 6 February 2008 [Abstract 736].)     

Determinants for Sensitivity of Human Immunodeficiency Virus Coreceptor CXCR4 to the Bicyclam AMD3100

The bicyclam AMD3100 is a potent and selective inhibitor of the replication of human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2). It was recently demonstrated that the compound inhibited HIV entry through CXCR4 but not through CCR5. Selectivity of AMD3100 for CXCR4 was further indicated by its lack of effect on HIV-1 and HIV-2 infection mediated by the CCR5, CCR3, Bonzo, BOB, and US28, coreceptors. AMD3100 completely blocked HIV-1 infection mediated by a mutant CXCR4 bearing a deletion of most of the amino-terminal extracellular domain. In contrast, relative resistance to AMD3100 was conferred by different single amino acid substitutions in the second extracellular loop (ECL2) or in the adjacent membrane-spanning domain, TM4. Only substitutions of a neutral residue for aspartic acid and of a nonaromatic residue for phenylalanine (Phe) were associated with drug resistance. This suggests a direct interaction of AMD3100 with these amino acids rather than indirect effects of their mutation on the CXCR4 structure. The interaction of aspartic acids of ECL2 and TM4 with AMD3100 is consistent with the positive charge of bicyclams, which might block HIV-1 entry by preventing electrostatic interactions between CXCR4 and the HIV-1 envelope protein gp120. Other features of AMD3100 must account for its high antiviral activity, in particular the presence of an aromatic linker between the cyclam units. This aromatic group might engage in hydrophobic interactions with the Phe-X-Phe motifs of ECL2 or TM4. These results confirm the importance of ECL2 for the HIV coreceptor activity of CXCR4.     

Chemokine Receptor Utilization by Human Immunodeficiency Virus Type 1 Isolates That Replicate in Microglia

The role of human immunodeficiency virus (HIV) strain variability remains a key unanswered question in HIV dementia, a condition affecting around 20% of infected individuals. Several groups have shown that viruses within the central nervous system (CNS) of infected patients constitute an independently evolving subset of HIV strains. A potential explanation for the replication and sequestration of viruses within the CNS is the preferential use of certain chemokine receptors present in microglia. To determine the role of specific chemokine coreceptors in infection of adult microglial cells, we obtained a small panel of HIV type 1 brain isolates, as well as other HIV strains that replicate well in cultured microglial cells. These viruses and molecular clones of their envelopes were used in infections, in cell-to-cell fusion assays, and in the construction of pseudotypes. The results demonstrate the predominant use of CCR5, at least among the major coreceptors, with minor use of CCR3 and CXCR4 by some of the isolates or their envelope clones.     

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.