A V3 Loop-Dependent gp120 Element Disrupted by CD4 Binding Stabilizes the Human Immunodeficiency Virus Envelope Glycoprotein Trimer

Human immunodeficiency virus (HIV-1) entry into cells is mediated by a trimeric complex consisting of noncovalently associated gp120 (exterior) and gp41 (transmembrane) envelope glycoproteins. The binding of gp120 to receptors on the target cell alters the gp120-gp41 relationship and activates the membrane-fusing capacity of gp41. Interaction of gp120 with the primary receptor, CD4, results in the exposure of the gp120 third variable (V3) loop, which contributes to binding the CCR5 or CXCR4 chemokine receptors. We show here that insertions in the V3 stem or polar substitutions in a conserved hydrophobic patch near the V3 tip result in decreased gp120-gp41 association (in the unliganded state) and decreased chemokine receptor binding (in the CD4-bound state). Subunit association and syncytium-forming ability of the envelope glycoproteins from primary HIV-1 isolates were disrupted more by V3 changes than those of laboratory-adapted HIV-1 envelope glycoproteins. Changes in the gp120 β2, β19, β20, and β21 strands, which evidence suggests are proximal to the V3 loop in unliganded gp120, also resulted in decreased gp120-gp41 association. Thus, a gp120 element composed of the V3 loop and adjacent beta strands contributes to quaternary interactions that stabilize the unliganded trimer. CD4 binding dismantles this element, altering the gp120-gp41 relationship and rendering the hydrophobic patch in the V3 tip available for chemokine receptor binding.The entry of human immunodeficiency virus type 1 (HIV-1) is mediated by the viral envelope glycoproteins (9, 79). The HIV-1 envelope glycoproteins are synthesized as an ∼850-amino acid precursor, which trimerizes and is posttranslationally modified by carbohydrates to create a 160-kDa glycoprotein (gp160). The gp160 envelope glycoprotein precursor is proteolytically processed in the Golgi apparatus, resulting in a gp120 exterior envelope glycoprotein and a gp41 transmembrane envelope glycoprotein (16, 17, 66, 76). In the mature HIV-1 envelope glycoprotein trimer, the three gp120 subunits are noncovalently bound to three membrane-anchored gp41 subunits (32).HIV-1 entry involves the binding of gp120 in a sequential fashion to CD4 and one of the chemokine receptors, CCR5 or CXCR4 (1, 8, 15, 18, 25, 36). CD4 binding triggers the formation of an activated intermediate that is competent for binding to CCR5 or CXCR4 (29, 69, 73, 78). These chemokine receptors are G protein-coupled, 7-transmembrane segment receptors with relatively short N termini. The choice of chemokine receptors is dictated primarily by the sequence of a gp120 region, the third variable (V3) loop, that exhibits variability among HIV-1 strains and becomes exposed upon CD4 binding (4, 8, 10, 33, 37, 38, 49, 59, 75). X-ray crystal structures of CD4-bound HIV-1 gp120 have revealed that the gp120 “core” consists of a gp41-interactive inner domain, a surface-exposed and heavily glycosylated outer domain, and a conformationally flexible bridging sheet (38, 43, 79). In the CD4-bound state, the V3 loop projects 30 Å from the gp120 core, toward the chemokine receptor (38). The V3 loop in these structures consists of three elements: (i) conserved antiparallel β strands that contain a disulfide bond at the base of the loop; (ii) a conformationally flexible stem; and (iii) a conserved tip (37, 38). During the virus entry process, the base of the gp120 V3 loop and elements of the bridging sheet interact with the CCR5 N terminus, which is acidic and contains sulfotyrosine residues (12-14, 23, 24). Sulfotyrosine 14 of CCR5 is thought to insert into a highly conserved pocket near the V3 base, driving further conformational rearrangements that result in the rigidification of the V3 stem (37). The conserved β-turn at the tip of the V3 loop, along with some residues in the V3 stem, is believed to bind the “body” of CCR5, i.e., the extracellular loops and membrane-spanning helices. CCR5 binding is thought to induce further conformational changes in the HIV-1 envelope glycoproteins, leading to the fusion of the viral and target cell membranes by the gp41 transmembrane envelope glycoproteins.CCR5 binding involves two points of contact with the gp120 V3 loop: (i) the CCR5 N terminus with the V3 base and (ii) the CCR5 body with the V3 tip and distal stem (12-14, 23, 24, 37, 38). The intervening V3 stem can tolerate greater conformational and sequence variation, features that might decrease HIV-1 susceptibility to host antibodies (30). Despite amino acid variation, the length of the V3 loop is well conserved among naturally occurring group M (major group) HIV-1 strains (30, 42). This conserved length may be important for aligning the two CCR5-binding elements of the V3 loop. In addition to allowing optimal CCR5 binding, the conserved V3 length and orientation may be important for CCR5 binding to exert effects on the conformation of the HIV-1 envelope glycoproteins. We examine here the consequences of introducing extra amino acid residues into the V3 stem. The residues were introduced either into both strands of the V3 loop, attempting to preserve the symmetry of the structure, or into one of the strands, thereby kinking the loop. The effects of these changes on assembly, stability, receptor binding, and the membrane-fusing capacity of the HIV-1 envelope glycoproteins were assessed. In addition to effects on chemokine receptor binding, unexpected disruption of gp120-gp41 association was observed. Further investigation revealed a conserved patch in the tip of the V3 loop that is important for the association of gp120 with the trimeric envelope glycoprotein complex, as well as for chemokine receptor binding. Apparently, the V3 loop and adjacent gp120 structures contribute to the stability of the trimer in the unliganded HIV-1 envelope glycoproteins. These structures are known to undergo rearrangement upon CD4 binding, suggesting their involvement in receptor-induced changes in the virus entry process.     

Transitions to and from the CD4-Bound Conformation Are Modulated by a Single-Residue Change in the Human Immunodeficiency Virus Type 1 gp120 Inner Domain

Binding to the primary receptor CD4 induces conformational changes in the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein that allow binding to the coreceptor (CCR5 or CXCR4) and ultimately trigger viral membrane-cell membrane fusion mediated by the gp41 transmembrane envelope glycoprotein. Here we report the derivation of an HIV-1 gp120 variant, H66N, that confers envelope glycoprotein resistance to temperature extremes. The H66N change decreases the spontaneous sampling of the CD4-bound conformation by the HIV-1 envelope glycoproteins, thus diminishing CD4-independent infection. The H66N change also stabilizes the HIV-1 envelope glycoprotein complex once the CD4-bound state is achieved, decreasing the probability of CD4-induced inactivation and revealing the enhancing effects of soluble CD4 binding on HIV-1 infection. In the CD4-bound conformation, the highly conserved histidine 66 is located between the receptor-binding and gp41-interactive surfaces of gp120. Thus, a single amino acid change in this strategically positioned gp120 inner domain residue influences the propensity of the HIV-1 envelope glycoproteins to negotiate conformational transitions to and from the CD4-bound state.Human immunodeficiency virus type 1 (HIV-1), the cause of AIDS (6, 29, 66), infects target cells by direct fusion of the viral and target cell membranes. The viral fusion complex is composed of gp120 and gp41 envelope glycoproteins, which are organized into trimeric spikes on the surface of the virus (10, 51, 89). Membrane fusion is initiated by direct binding of gp120 to the CD4 receptor on target cells (17, 41, 53). CD4 binding creates a second binding site on gp120 for the chemokine receptors CCR5 and CXCR4, which serve as coreceptors (3, 12, 19, 23, 25). Coreceptor binding is thought to lead to further conformational changes in the HIV-1 envelope glycoproteins that facilitate the fusion of viral and cell membranes. The formation of an energetically stable six-helix bundle by the gp41 ectodomain contributes to the membrane fusion event (9, 10, 79, 89, 90).The energy required for viral membrane-cell membrane fusion derives from the sequential transitions that the HIV-1 envelope glycoproteins undergo, from the high-energy unliganded state to the low-energy six-helix bundle. The graded transitions down this energetic slope are initially triggered by CD4 binding (17). The interaction of HIV-1 gp120 with CD4 is accompanied by an unusually large change in entropy, which is thought to indicate the introduction of order into the conformationally flexible unliganded gp120 glycoprotein (61). In the CD4-bound state, gp120 is capable of binding CCR5 with high affinity; moreover, CD4 binding alters the quaternary structure of the envelope glycoprotein complex, resulting in the exposure of gp41 ectodomain segments (27, 45, 77, 92). The stability of the intermediate state induced by CD4 binding depends upon several variables, including the virus (HIV-1 versus HIV-2/simian immunodeficiency virus [SIV]), the temperature, and the nature of the CD4 ligand (CD4 on a target cell membrane versus soluble forms of CD4 [sCD4]) (30, 73). For HIV-1 exposed to sCD4, if CCR5 binding occurs within a given period of time, progression along the entry pathway continues. If CCR5 binding is impeded or delayed, the CD4-bound envelope glycoprotein complex decays into inactive states (30). In extreme cases, the binding of sCD4 to the HIV-1 envelope glycoproteins induces the shedding of gp120 from the envelope glycoprotein trimer (31, 56, 58). Thus, sCD4 generally inhibits HIV-1 infection by triggering inactivation events, in addition to competing with CD4 anchored in the target cell membrane (63).HIV-1 isolates vary in sensitivity to sCD4, due in some cases to a low affinity of the envelope glycoprotein trimer for CD4 and in other cases to differences in propensity to undergo inactivating conformational transitions following CD4 binding (30). HIV-1 isolates that have been passaged extensively in T-cell lines (the tissue culture laboratory-adapted [TCLA] isolates) exhibit lower requirements for CD4 than primary HIV-1 isolates (16, 63, 82). TCLA viruses bind sCD4 efficiently and are generally sensitive to neutralization compared with primary HIV-1 isolates. Differences in sCD4 sensitivity between primary and TCLA HIV-1 strains have been mapped to the major variable loops (V1/V2 and V3) of the gp120 glycoprotein (34, 42, 62, 81). Sensitivity to sCD4 has been shown to be independent of envelope glycoprotein spike density or the intrinsic stability of the envelope glycoprotein complex (30, 35).In general, HIV-1 isolates are more sensitive to sCD4 neutralization than HIV-2 or SIV isolates (4, 14, 73). The relative resistance of SIV to sCD4 neutralization can in some cases be explained by a reduced affinity of the envelope glycoprotein trimer for sCD4 (57); however, at least some SIV isolates exhibit sCD4-induced activation of entry into CD4-negative, CCR5-expressing target cells that lasts for several hours after exposure to sCD4 (73). Thus, for some primate immunodeficiency virus envelope glycoproteins, activated intermediates in the CD4-bound conformation can be quite stable.The HIV-1 envelope glycoprotein elements important for receptor binding, subunit interaction, and membrane fusion are well conserved among different viral strains (71, 91). Thus, these elements represent potential targets for inhibitors of HIV-1 entry. Understanding the structure and longevity of the envelope glycoprotein intermediates along the virus entry pathway is relevant to attempts at inhibition. For example, peptides that target the heptad repeat 1 region of gp41 exhibit major differences in potency against HIV-1 strains related to efficiency of chemokine receptor binding (20, 21), which is thought to promote the conformational transition to the next step in the virus entry cascade. The determinants of the duration of exposure of targetable HIV-1 envelope glycoprotein elements during the entry process are undefined.To study envelope glycoprotein determinants of the movement among the distinct conformational states along the HIV-1 entry pathway, we attempted to generate HIV-1 variants that exhibit improved stability. Historically, labile viral elements have been stabilized by selecting virus to replicate under conditions, such as high temperature, that typically weaken protein-protein interactions (38, 39, 76, 102). Thus, we subjected HIV-1 to repeated incubations at temperatures between 42°C and 56°C, followed by expansion and analysis of the remaining replication-competent virus fraction. In this manner, we identified an envelope glycoprotein variant, H66N, in which histidine 66 in the gp120 N-terminal segment was altered to asparagine. The resistance of HIV-1 bearing the H66N envelope glycoproteins to changes in temperature has been reported elsewhere (37). Here, we examine the effect of the H66N change on the ability of the HIV-1 envelope glycoproteins to negotiate conformational transitions, either spontaneously or in the presence of sCD4. The H66N phenotype was studied in the context of both CD4-dependent and CD4-independent HIV-1 variants.     

HIV-1 gp120 Determinants Proximal to the CD4 Binding Site Shift Protective Glycans That Are Targeted by Monoclonal Antibody 2G12

HIV-1 R5 envelopes vary considerably in their capacities to exploit low CD4 levels on macrophages for infection and in their sensitivities to the CD4 binding site (CD4bs) monoclonal antibody (MAb) b12 and the glycan-specific MAb 2G12. Here, we show that nonglycan determinants flanking the CD4 binding loop, which affect exposure of the CD4bs, also modulate 2G12 neutralization. Our data indicate that such residues act via a mechanism that involves shifts in the orientation of proximal glycans, thus modulating the sensitivity of 2G12 neutralization and affecting the overall presentation and structure of the glycan shield.The trimeric envelope (Env) spikes on HIV-1 virions are comprised of gp120 and gp41 heterodimers. gp120 is coated extensively with glycans (9, 11, 15) that are believed to protect the envelope from neutralizing antibodies. The extents and locations of glycosylation are variable and evolving (15). Thus, while some glycans are conserved, others appear or disappear in a host over the course of infection. Such changes may result in exposure or protection of functional envelope sites and can result from selection by different environmental pressures in vivo, including neutralizing antibodies.We previously reported that HIV-1 R5 envelopes varied considerably in tropism and neutralization sensitivity (3, 4, 12-14). We showed that highly macrophage-tropic R5 envelopes were more frequently detected in brain than in semen, blood, and lymph node (LN) samples (12, 14). The capacity of R5 envelopes to infect macrophages correlated with their ability to exploit low levels of cell surface CD4 for infection (12, 14). Determinants within and proximal to the CD4 binding site (CD4bs) were shown to modulate macrophage infectivity (3, 4, 5, 12, 13) and presumably acted by altering the avidity of the trimer for cell surface CD4. These determinants include residues proximal to the CD4 binding loop, which is likely the first part of the CD4bs contacted by CD4 (1). We also observed that macrophage-tropic R5 envelopes were frequently more resistant to the glycan-specific monoclonal antibody (MAb) 2G12 than were non-macrophage-tropic R5 Envs (13).Here, we investigated the envelope determinants of 2G12 sensitivity by using two HIV-1 envelopes that we used previously to map macrophage tropism determinants (4), B33 from brain and LN40 from lymph node tissue of an AIDS patient with neurological complications. While B33 imparts high levels of macrophage infectivity and is resistant to 2G12, LN40 Env confers very inefficient macrophage infection and is 2G12 sensitive (12-14).     

HIV-1 Resistance to CCR5 Antagonists Associated with Highly Efficient Use of CCR5 and Altered Tropism on Primary CD4+ T Cells

We previously reported on a panel of HIV-1 clade B envelope (Env) proteins isolated from a patient treated with the CCR5 antagonist aplaviroc (APL) that were drug resistant. These Envs used the APL-bound conformation of CCR5, were cross resistant to other small-molecule CCR5 antagonists, and were isolated from the patient''s pretreatment viral quasispecies as well as after therapy. We analyzed viral and host determinants of resistance and their effects on viral tropism on primary CD4⁺ T cells. The V3 loop contained residues essential for viral resistance to APL, while additional mutations in gp120 and gp41 modulated the magnitude of drug resistance. However, these mutations were context dependent, being unable to confer resistance when introduced into a heterologous virus. The resistant virus displayed altered binding between gp120 and CCR5 such that the virus became critically dependent on the N′ terminus of CCR5 in the presence of APL. In addition, the drug-resistant Envs studied here utilized CCR5 very efficiently: robust virus infection occurred even when very low levels of CCR5 were expressed. However, recognition of drug-bound CCR5 was less efficient, resulting in a tropism shift toward effector memory cells upon infection of primary CD4⁺ T cells in the presence of APL, with relative sparing of the central memory CD4⁺ T cell subset. If such a tropism shift proves to be a common feature of CCR5-antagonist-resistant viruses, then continued use of CCR5 antagonists even in the face of virologic failure could provide a relative degree of protection to the T_CM subset of CD4⁺ T cells and result in improved T cell homeostasis and immune function.Entry of human immunodeficiency virus (HIV) into target cells is a complex, multistep process that is initiated by interactions between the viral envelope (Env) protein gp120 and the host cell receptor CD4, which trigger conformational changes in gp120 that form and orient the coreceptor binding site (9, 24). Upon binding to coreceptor, which is either CCR5 or CXCR4 for primary HIV isolates, Env undergoes further conformational changes resulting in insertion of the gp41 fusion peptide into the host cell membrane and gp41-mediated membrane fusion (8, 15, 26). Targeting stages of the HIV entry process with antiretroviral drugs is a productive method of inhibiting HIV replication, as demonstrated by the potent antiviral effects of small-molecule CCR5 antagonists and fusion inhibitors (23, 35, 49). As with other antiretroviral drugs, HIV can develop resistance to entry inhibitors, and a detailed understanding of viral and host determinants of resistance will be critical to the optimal clinical use of these agents.The coreceptor binding site that is induced by CD4 engagement consists of noncontiguous regions in the bridging sheet and V3 loop of gp120 (4, 18, 42, 43, 50). Interactions between gp120 and CCR5 occur in at least two distinct areas: (i) the bridging sheet and the stem of the V3 loop interact with sulfated tyrosine residues in the N′ terminus of CCR5, and (ii) the crown of the V3 loop is thought to engage the extracellular loops (ECLs), particularly ECL2, of CCR5 (10-12, 14, 18, 28). Small-molecule CCR5 antagonists bind to a hydrophobic pocket in the transmembrane helices of CCR5 and exert their effects on HIV by altering the position of the ECLs, making them allosteric inhibitors of HIV infection (13, 31, 32, 46, 52). The conformational changes in CCR5 that are induced by CCR5 antagonists vary to some degree with different drugs, as evidenced by differential binding of antibodies and chemokines to various drug-bound forms of CCR5 (47, 54).CCR5 antagonists are unusual among antiretroviral agents in that they bind to a host protein rather than a viral target, and therefore the virus cannot directly mutate the drug binding site to evade pharmacologic pressure. Nevertheless, HIV can escape susceptibility to CCR5 antagonists. One mechanism by which this occurs is the use of the alternative HIV coreceptor, CXCR4. In vivo, this has most often been manifest as the outgrowth of R5/X4-tropic HIV isolates that were present in the patient''s circulating viral swarm prior to therapy (17, 27, 55). A second mechanism of HIV resistance to CCR5 antagonists is the use of drug-bound CCR5 as a coreceptor for entry. Resistant viruses that utilize drug-bound CCR5 have been identified following in vitro passaging with multiple CCR5 antagonists (1, 2, 22, 33, 36, 51, 56). Recently, we identified a panel of viral Envs able to use aplaviroc (APL)-bound CCR5 that were isolated from a patient (21, 48). The Envs from this patient were cross resistant to the CCR5 antagonists AD101, TAK779, SCH-C, and maraviroc. Surprisingly, this antiretroviral-naïve patient harbored Envs resistant to aplaviroc prior to the initiation of therapy. In the present study, we have examined viral and host factors that contribute to aplaviroc resistance and examined the consequences of resistance for viral tropism. Aplaviroc resistance determinants were located within the V3 loop of gp120, although additional residues diffusely spread throughout the gp120 and gp41 proteins modulated the magnitude of drug resistance. The resistant virus displayed altered interactions between gp120 and CCR5 such that the virus became critically dependent upon the N′ terminus of drug-bound CCR5. This differential recognition of CCR5 in the presence of aplaviroc was also associated with increased dependence on a higher CCR5 receptor density for efficient virus infection and a tropism shift toward effector memory cells on primary CD4⁺ T cells.     

Influence of Novel CD4 Binding-Defective HIV-1 Envelope Glycoprotein Immunogens on Neutralizing Antibody and T-Cell Responses in Nonhuman Primates

The high-affinity in vivo interaction between soluble HIV-1 envelope glycoprotein (Env) immunogens and primate CD4 results in conformational changes that alter the immunogenicity of the gp120 subunit. Because the conserved binding site on gp120 that directly interacts with CD4 is a major vaccine target, we sought to better understand the impact of in vivo Env-CD4 interactions during vaccination. Rhesus macaques were immunized with soluble wild-type (WT) Env trimers, and two trimer immunogens rendered CD4 binding defective through distinct mechanisms. In one variant, we introduced a mutation that directly disrupts CD4 binding (368D/R). In the second variant, we introduced three mutations (423I/M, 425N/K, and 431G/E) that disrupt CD4 binding indirectly by altering a gp120 subdomain known as the bridging sheet, which is required for locking Env into a stable interaction with CD4. Following immunization, Env-specific binding antibody titers and frequencies of Env-specific memory B cells were comparable between the groups. However, the quality of neutralizing antibody responses induced by the variants was distinctly different. Antibodies against the coreceptor binding site were elicited by WT trimers but not the CD4 binding-defective trimers, while antibodies against the CD4 binding site were elicited by the WT and the 423I/M, 425N/K, and 431G/E trimers but not the 368D/R trimers. Furthermore, the CD4 binding-defective trimer variants stimulated less potent neutralizing antibody activity against neutralization-sensitive viruses than WT trimers. Overall, our studies do not reveal any potential negative effects imparted by the in vivo interaction between WT Env and primate CD4 on the generation of functional T cells and antibodies in response to soluble Env vaccination.The HIV-1 Envs mediate the entry of the virus into target cells and are the only virally encoded proteins exposed on the surface of the virus. HIV-1 Env is the sole target for neutralizing antibodies (Abs) and therefore is an important component of a vaccine designed to elicit protective antibody responses (4, 20). The viral spike is a trimer comprised of three heterodimers of the exterior envelope glycoprotein, gp120, noncovalently attached to the transmembrane protein, gp41. The gp120 subunit binds the primary receptor, CD4 (7), to form or to expose the gp120 coreceptor binding elements, which interact with the viral coreceptor, primarily CCR5 (1, 9, 12, 45). The highly conserved coreceptor binding site (CoRbs) overlaps the gp120 bridging sheet and also contains both proximal and distal elements of V3 (18, 32, 43, 45).In attempts to mimic the native trimeric structure of Env present on the virus, various forms of soluble Env trimers were designed (reviewed in reference 14). One design consists of cleavage-defective trimers derived from the primary R5 isolate YU2 that possess a heterologous trimerization motif derived from T4 bacteriophage fibritin (F; YU2 gp140-F) (3, 21, 34, 40, 50, 51). We recently demonstrated that the immunization of monkeys, but not rabbits, with gp140-F trimers resulted in the generation of Abs directed against the CoRbs of gp120 capable of cross-neutralizing HIV-2 (15). CoRbs-directed Abs (also referred to as CD4-induced, or CD4i, Abs) also were elicited in rabbits transgenic for human CD4 (15). Taken together, these data strongly suggest that Env interacts with high-affinity primate CD4 in vivo, resulting in the formation, or exposure, of a highly immunogenic gp120 determinant that overlaps the CoRbs. Early in infection, the frequency of HIV-1-infected individuals with significant antibody responses against the CoRbs is high (8, 33), and CoRbs-directed antibody responses are elicited abundantly in humans inoculated with Env-based immunogens (15). Collectively, these data suggest that the recognition of the HIV-1 CoRbs by naïve B cells is greatly increased when Env is presented in complex with high-affinity primate CD4, leading to a productive Ab response against this epitope (41). With rare exceptions, the majority of CoRbs-directed monoclonal antibodies (MAbs) do not neutralize HIV-1 primary viruses in vitro, bringing into question the utility of this region as a relevant neutralization target (23, 31, 47, 49). Strategies aimed to diminish vaccine-elicited B-cell responses to the CoRbs, and shift responses toward more accessible neutralization targets, represent one approach to improve the design of Env-based vaccine candidates. The selective manipulation of Env immunogens to decrease their CD4 binding capacity may reduce the elicitation of CoRbs-directed Abs and circumvent potential occlusion effects of the conserved CD4 binding site caused by CD4 itself.In addition to the potential effects of in vivo Env-CD4 interactions on the Ab repertoire elicited by Env-based immunogens, interactions between Env and CD4 also may have consequences on CD4⁺ T-cell responses. CD4 is an important costimulatory molecule expressed on several subsets of T cells and antigen-presenting cells, and interactions with Env were shown to alter the function of CD4-expressing T cells in a number of in vitro systems (13, 37, 44). The elimination of the Env-CD4 interaction in the context of vaccination may be beneficial to improve the elicitation of helper T-cell responses and effective neutralizing Ab responses. In vivo evaluation in subjects possessing high-affinity CD4 (i.e., rhesus macaques or humans) of CD4 binding-competent and CD4 binding-deficient Env immunogens so far have not been described.To address these questions, we designed Env trimer variants rendered CD4 binding defective through two distinct mechanisms. In the first variant, the interaction between CD4 and HIV-1 Env was directly disrupted by the introduction of a mutation (368D/R) in the CD4 binding loop of the gp120 outer domain (29). This alteration abolishes the initial binding of CD4 and most CD4 binding site (CD4bs)-directed MAbs (42) to variant forms of gp120 and would be expected to do the same in the soluble stable timer context. The aim of the second variant was to decrease the CD4 binding affinity while preserving the antigenicity of the CD4bs (48). This variant was generated in the soluble gp140-F trimers by the introduction of three point mutations, 423I/M, 425N/K, and 431G/E, in the β20 strand region of gp120. These mutations were suggested to favor a helix rather than the β20/21 antiparallel strands visible in the gp120 structure (23, 31, 47, 49). In the monomeric context, mutations in the β20 strand region of gp120 abolish binding by CoRbs-directed Abs, presumably because the bridging sheet cannot form (48). The introduction of the 423I/M, 425N/K, and 431G/E mutations in the trimer context therefore should disrupt the normally high-affinity gp120-CD4 interaction, while recognition by CD4bs Abs would not be affected. Indeed, a recent study provides a mechanistic basis for the impact of these mutations on CD4 binding (52). This study revealed that CD4 interacts with gp120 by a two-step binding mechanism in which the first step involves a direct, but low-affinity, CD4 interaction with the gp120 outer domain, while the second step requires a conformational change in gp120 to fully stabilize the high-affinity gp120-CD4 interaction.Here, we exploit this two-step model to generate novel CD4 binding-defective soluble trimers that, unlike the 368D/R trimers, possess a CD4bs surface that retains recognition by well-described CD4bs Abs. By immunizing rhesus macaques with the wild-type (WT) and CD4 binding-defective trimer variants, we demonstrate that similar levels of Env-specific Ab and T-cell responses were elicited in the three groups, suggesting that in vivo interactions between CD4 binding-competent (WT) Env and CD4 do not measurably affect T-cell responses against Env in this immunization regimen. However, the quality of the Ab response was markedly different between the groups. As hypothesized, CoRbs-directed Abs were elicited only in animals inoculated with WT trimers and not in those inoculated with 368D/R or 423I/M, 425N/K, and 431G/E trimers (hereafter referred to as 368 and 423/425/431 trimers, respectively). Importantly, we show that the 423/425/431 trimers retain the capacity to elicit binding and neutralizing CD4bs-directed Abs. In conclusion, the results generated in this study suggest that CD4 engagement by the WT soluble Env trimers did not impair the overall magnitude of the elicited Env-specific antibody or T-cell responses. Furthermore, our data provide new insights into the characteristics of Env that impact immunogenicity. The data also provide a potential path forward for the design of Env immunogens that have the capacity to elicit neutralizing Abs against the conserved gp120 CD4 binding surface while eliminating both the elicitation of nonneutralizing CoRbs-directed Abs and the potential occlusion of the CD4 binding surface of gp120 by the engagement of the primary virus receptor, CD4.     

Crystallographic Definition of the Epitope Promiscuity of the Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2F5: Vaccine Design Implications

The quest to create a human immunodeficiency virus type 1 (HIV-1) vaccine capable of eliciting broadly neutralizing antibodies against Env has been challenging. Among other problems, one difficulty in creating a potent immunogen resides in the substantial overall sequence variability of the HIV envelope protein. The membrane-proximal region (MPER) of gp41 is a particularly conserved tryptophan-rich region spanning residues 659 to 683, which is recognized by three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, Z13, and 4E10. In this study, we first describe the variability of residues in the gp41 MPER and report on the invariant nature of 15 out of 25 amino acids comprising this region. Subsequently, we evaluate the ability of the bnMAb 2F5 to recognize 31 varying sequences of the gp41 MPER at a molecular level. In 19 cases, resulting crystal structures show the various MPER peptides bound to the 2F5 Fab′. A variety of amino acid substitutions outside the ⁶⁶⁴DKW⁶⁶⁶ core epitope are tolerated. However, changes at the ⁶⁶⁴DKW⁶⁶⁶ motif itself are restricted to those residues that preserve the aspartate''s negative charge, the hydrophobic alkyl-π stacking arrangement between the β-turn lysine and tryptophan, and the positive charge of the former. We also characterize a possible molecular mechanism of 2F5 escape by sequence variability at position 667, which is often observed in HIV-1 clade C isolates. Based on our results, we propose a somewhat more flexible molecular model of epitope recognition by bnMAb 2F5, which could guide future attempts at designing small-molecule MPER-like vaccines capable of eliciting 2F5-like antibodies.Eliciting broadly neutralizing antibodies (bnAbs) against primary isolates of human immunodeficiency virus type I (HIV-1) has been identified as a major milestone to attain in the quest for a vaccine in the fight against AIDS (12, 28). These antibodies would need to interact with HIV-1 envelope glycoproteins gp41 and/or gp120 (Env), target conserved regions and functional conformations of gp41/gp120 trimeric complexes, and prevent new HIV-1 fusion events with target cells (21, 57, 70, 71). Although a humoral response generating neutralizing antibodies against HIV-1 can be detected in HIV-1-positive individuals, the titers are often very low, and virus control is seldom achieved by these neutralizing antibodies (22, 51, 52, 66, 67). The difficulty in eliciting a broad and potent neutralizing antibody response against HIV-1 is thought to reside in the high degree of genetic diversity of the virus, in the heterogeneity of Env on the surface of HIV-1, and in the masking of functional regions by conformational covering, by an extensive glycan shield, or by the ability of some conserved domains to partition to the viral membrane (24, 25, 29, 30, 38, 39, 56, 68, 69). So far, vaccine trials using as immunogens mimics of Env in different conformations have primarily elicited antibodies with only limited neutralization potency across different HIV-1 clades although recent work has demonstrated more encouraging results (4, 12, 61).The use of conserved regions on gp41 and gp120 Env as targets for vaccine design has been mostly characterized by the very few anti-HIV-1 broadly neutralizing monoclonal antibodies (bnMAbs) that recognize them: the CD4 binding-site on gp120 (bnMAb b12), a CD4-induced gp120 coreceptor binding site (bnMAbs 17b and X5), a mannose cluster on the outer face of gp120 (bnMAb 2G12), and the membrane proximal external region (MPER) of gp41 (bnMAbs 2F5, Z13 and 4E10) (13, 29, 44, 58, 73). The gp41 MPER region is a particularly conserved part of Env that spans residues 659 to 683 (HXB2 numbering) (37, 75). Substitution and deletion studies have linked this unusually tryptophan-rich region to the fusion process of HIV-1, possibly involving a series of conformational changes (5, 37, 41, 49, 54, 74). Additionally, the gp41 MPER has been implicated in gp41 oligomerization, membrane leakage ability facilitating pore formation, and binding to the galactosyl ceramide receptor on epithelial cells for initial mucosal infection mediated by transcytosis (2, 3, 40, 53, 63, 64, 72). This wide array of roles for the gp41 MPER will put considerable pressure on sequence conservation, and any change will certainly lead to a high cost in viral fitness.Monoclonal antibody 2F5 is a broadly neutralizing monoclonal anti-HIV-1 antibody isolated from a panel of sera from naturally infected asymptomatic individuals. It reacts with a core gp41 MPER epitope spanning residues 662 to 668 with the linear sequence ELDKWAS (6, 11, 42, 62, 75). 2F5 immunoglobulin G binding studies and screening of phage display libraries demonstrated that the DKW core is essential for 2F5 recognition and binding (15, 36, 50). Crystal structures of 2F5 with peptides representing its core gp41 epitope reveal a β-turn conformation involving the central DKW residues, flanked by an extended conformation and a canonical α-helical turn for residues located at the N terminus and C terminus of the core, respectively (9, 27, 45, 47). In addition to binding to its primary epitope, evidence is accumulating that 2F5 also undergoes secondary interactions: multiple reports have demonstrated affinity of 2F5 for membrane components, possibly through its partly hydrophobic flexible elongated complementarity-determining region (CDR) H3 loop, and it has also been suggested that 2F5 might interact in a secondary manner with other regions of gp41 (1, 10, 23, 32, 33, 55). Altogether, even though the characteristics of 2F5 interaction with its linear MPER consensus epitope have been described extensively, a number of questions persist about the exact mechanism of 2F5 neutralization at a molecular level.One such ambiguous area of the neutralization mechanism of 2F5 is investigated in this study. Indeed, compared to bnMAb 4E10, 2F5 is the more potent neutralizing antibody although its breadth across different HIV-1 isolates is more limited (6, 35). In an attempt to shed light on the exact molecular requirements for 2F5 recognition of its primary gp41 MPER epitope, we performed structural studies of 2F5 Fab′ with a variety of peptides. The remarkable breadth of possible 2F5 interactions reveals a somewhat surprising promiscuity of the 2F5 binding site. Furthermore, we link our structural observations with the natural variation observed within the gp41 MPER and discuss possible routes of 2F5 escape from a molecular standpoint. Finally, our discovery of 2F5''s ability to tolerate a rather broad spectrum of amino acids in its binding, a spectrum that even includes nonnatural amino acids, opens the door to new ways to design small-molecule immunogens potentially capable of eliciting 2F5-like neutralizing antibodies.     

Utilization of Immunoglobulin G Fc Receptors by Human Immunodeficiency Virus Type 1: a Specific Role for Antibodies against the Membrane-Proximal External Region of gp41

Receptors (FcγRs) for the constant region of immunoglobulin G (IgG) are an important link between humoral immunity and cellular immunity. To help define the role of FcγRs in determining the fate of human immunodeficiency virus type 1 (HIV-1) immune complexes, cDNAs for the four major human Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa) were stably expressed by lentiviral transduction in a cell line (TZM-bl) commonly used for standardized assessments of HIV-1 neutralization. Individual cell lines, each expressing a different FcγR, bound human IgG, as evidence that the physical properties of the receptors were preserved. In assays with a HIV-1 multisubtype panel, the neutralizing activities of two monoclonal antibodies (2F5 and 4E10) that target the membrane-proximal external region (MPER) of gp41 were potentiated by FcγRI and, to a lesser extent, by FcγRIIb. Moreover, the neutralizing activity of an HIV-1-positive plasma sample known to contain gp41 MPER-specific antibodies was potentiated by FcγRI. The neutralizing activities of monoclonal antibodies b12 and 2G12 and other HIV-1-positive plasma samples were rarely affected by any of the four FcγRs. Effects with gp41 MPER-specific antibodies were moderately stronger for IgG1 than for IgG3 and were ineffective for Fab. We conclude that FcγRI and FcγRIIb facilitate antibody-mediated neutralization of HIV-1 by a mechanism that is dependent on the Fc region, IgG subclass, and epitope specificity of antibody. The FcγR effects seen here suggests that the MPER of gp41 could have greater value for vaccines than previously recognized.Fc receptors (FcRs) are differentially expressed on a variety of cells of hematopoietic lineage, where they bind the constant region of antibody (Ab) and provide a link between humoral and cellular immunity. Humans possess two classes of FcRs for the constant region of IgG (FcγRs) that, when cross-linked, are distinguished by their ability to either activate or inhibit cell signaling (69, 77, 79). The activating receptors FcγRI (CD64), FcγRIIa (CD32), and FcγRIII (CD16) signal through an immunoreceptor tyrosine-based activation motif (ITAM), whereas FcγRIIb (CD32) contains an inhibitory motif (ITIM) that counters ITAM signals and B-cell receptor signals. It has been suggested that a balance between activating and inhibitory FcγRs coexpressed on the same cells plays an important role in regulating adaptive immunity (23, 68). Moreover, the inhibitory FcγRIIb, being the sole FcγR on B cells, appears to play an important role in regulating self-tolerance (23, 68). The biologic role of FcγRs may be further influenced by differences in their affinity for immunoglobulin G (IgG); thus, FcγRI is a high-affinity receptor that binds monomeric IgG (mIgG) and IgG immune complexes (IC), whereas FcγRIIa, FcγRIIb, and FcγRIIIa are medium- to low-affinity receptors that preferentially bind IgG IC (10, 49, 78). FcγRs also exhibit differences in their relative affinity for the four IgG subclasses (10), which has been suggested to influence the balance between activating and inhibitory FcγRs (67).In addition to their participation in acquired immunity, FcγRs can mediate several innate immune functions, including phagocytosis of opsonized pathogens, Ab-dependent cell cytotoxicity (ADCC), antigen uptake by professional antigen-presenting cells, and the production of inflammatory cytokines and chemokines (26, 35, 41, 48, 69). In some cases, interaction of Ab-coated viruses with FcγRs may be exploited by viruses as a means to facilitate entry into FcγR-expressing cells (2, 33, 47, 84). Several groups have reported FcγR-mediated Ab-dependent enhancement (ADE) of HIV-1 infection in vitro (47, 51, 58, 63, 94, 96), whereas other reports have implicated FcγRs in efficient inhibition of the virus in vitro (19, 21, 29, 44-46, 62, 98) and possibly as having beneficial effects against HIV-1 in vivo (5, 27, 28, 42). These conflicting results are further complicated by the fact that HIV-1-susceptible cells, such as monocytes and macrophages, can coexpress more than one FcγR (66, 77, 79).HIV-1 entry requires sequential interactions between the viral surface glycoprotein, gp120, and its cellular receptor (CD4) and coreceptor (usually CCR5 or CXCR4), followed by membrane fusion that is mediated by the viral transmembrane glycoprotein gp41 (17, 106). Abs neutralize the virus by binding either gp120 or gp41 and blocking entry into cells. Several human monoclonal Abs that neutralize a broad spectrum of HIV-1 variants have attracted considerable interest for vaccine design. Epitopes for these monoclonal Abs include the receptor binding domain of gp120 in the case of b12 (71, 86), a glycan-specific epitope on gp120 in the case of 2G12 (13, 85, 86), and two adjacent epitopes in the membrane-proximal external region (MPER) of g41 in the cases of 2F5 and 4E10 (3, 11, 38, 93). At least three of these monoclonal Abs have been shown to interact with FcRs and to mediate ADCC (42, 43).A highly standardized and validated assay for neutralizing Abs against HIV-1 that quantifies reductions in luciferase (Luc) reporter gene expression after a single round of virus infection in TZM-bl cells has been developed (60, 104). TZM-bl (also called JC53BL-13) is a CXCR4-positive HeLa cell line that was engineered to express CD4 and CCR5 and to contain integrated reporter genes for firefly Luc and Escherichia coli β-galactosidase under the control of the HIV-1 Tat-regulated promoter in the long terminal repeat terminal repeat sequence (74, 103). TZM-bl cells are permissive to infection by a wide variety of HIV-1, simian immunodeficiency virus, and human-simian immunodeficiency virus strains, including molecularly cloned Env-pseudotyped viruses. Here we report the creation and characterization of four new TZM-bl cell lines, each expressing one of the major human FcγRs. These new cell lines were used to gain a better understanding of the individual roles that FcγRs play in determining the fate of HIV-1 IC. Two FcγRs that potentiated the neutralizing activity of gp41 MPER-specific Abs were identified.     

Caveolin-1 Modulates HIV-1 Envelope-Induced Bystander Apoptosis through gp41

Human immunodeficiency virus (HIV) envelope (Env)-mediated bystander apoptosis is known to cause the progressive, severe, and irreversible loss of CD4⁺ T cells in HIV-1-infected patients. Env-induced bystander apoptosis has been shown to be gp41 dependent and related to the membrane hemifusion between envelope-expressing cells and target cells. Caveolin-1 (Cav-1), the scaffold protein of specific membrane lipid rafts called caveolae, has been reported to interact with gp41. However, the underlying pathological or physiological meaning of this robust interaction remains unclear. In this report, we examine the interaction of cellular Cav-1 and HIV gp41 within the lipid rafts and show that Cav-1 modulates Env-induced bystander apoptosis through interactions with gp41 in SupT1 cells and CD4⁺ T lymphocytes isolated from human peripheral blood. Cav-1 significantly suppressed Env-induced membrane hemifusion and caspase-3 activation and augmented Hsp70 upregulation. Moreover, a peptide containing the Cav-1 scaffold domain sequence markedly inhibited bystander apoptosis and apoptotic signal pathways. Our studies shed new light on the potential role of Cav-1 in limiting HIV pathogenesis and the development of a novel therapeutic strategy in treating HIV-1-infected patients.HIV infection causes a progressive, severe, and irreversible depletion of CD4⁺ T cells, which is responsible for the development of AIDS (9). The mechanism through which HIV infection induces cell death involves a variety of processes (58). Among these processes, apoptosis is most likely responsible for T-cell destruction in HIV-infected patients (33), because active antiretroviral therapy has been associated with low levels of CD4⁺ T-cell apoptosis (7), and AIDS progression was shown previously to correlate with the extent of immune cell apoptosis (34). Importantly, bystander apoptosis of uninfected cells was demonstrated to be one of the major processes involved in the destruction of immune cells (58), with the majority of apoptotic CD4⁺ T cells in the peripheral blood and lymph nodes being uninfected in HIV patients (22).Binding to uninfected cells or the entry of viral proteins released by infected cells is responsible for the virus-mediated killing of innocent-bystander CD4⁺ T cells (2-4, 9, 65). The HIV envelope glycoprotein complex, consisting of gp120 and gp41 subunits expressed on an HIV-infected cell membrane (73), is believed to induce bystander CD4⁺ T-cell apoptosis (58). Although there is a soluble form of gp120 in the blood, there is no conclusive agreement as to whether the concentration is sufficient to trigger apoptosis (57, 58). The initial step in HIV infection is mediated by the Env glycoprotein gp120 binding with high affinity to CD4, the primary receptor on the target cell surface, which is followed by interactions with the chemokine receptor CCR5 or CXCR4 (61). This interaction triggers a conformational change in gp41 and the insertion of its N-terminal fusion peptide into the target membrane (30). Next, a prehairpin structure containing leucine zipper-like motifs is formed by the two conserved coiled-coil domains, called the N-terminal and C-terminal heptad repeats (28, 66, 70). This structure quickly collapses into a highly stable six-helix bundle structure with an N-terminal heptad repeat inside and a hydrophobic C-terminal heptad repeat outside (28, 66, 70). The formation of the six-helix bundle leads to a juxtaposition and fusion with the target cell membrane (28, 66, 70). The fusogenic potential of HIV Env is proven to correlate with the pathogenesis of both CXCR4- and CCR5-tropic viruses by not only delivering the viral genome to uninfected cells but also mediating Env-induced bystander apoptosis (71). Initial infection is dominated by the CCR5-tropic strains, with the CXCR4-tropic viruses emerging in the later stages of disease (20). Studies have shown that CXCR4-tropic HIV-1 triggers more depletion of CD4⁺ T cells than CCR5-tropic strains (36).Glycolipid- and cholesterol-enriched membrane microdomains, termed lipid rafts, are spatially organized plasma membranes and are known to have many diverse functions (26, 53). These functions include membrane trafficking, endocytosis, the regulation of cholesterol and calcium homeostasis, and signal transduction in cellular growth and apoptosis. Lipid rafts have also been implicated in HIV cell entry and budding processes (19, 46, 48, 51). One such organelle is the caveola, which is a small, flask-shaped (50 to 100 nm in diameter) invagination in the plasma membrane (5, 62). The caveola structure, which is composed of proteins known as caveolins, plays a role in various functions by serving as a mobile platform for many receptors and signal proteins (5, 62). Caveolin-1 (Cav-1) is a 22- to 24-kDa major coat protein responsible for caveola assembly (25, 47). This scaffolding protein forms a hairpin-like structure and exists as an oligomeric complex of 14 to 16 monomers (21). Cav-1 has been shown to be expressed by a variety of cell types, mostly endothelial cells, type I pneumocytes, fibroblasts, and adipocytes (5, 62). In addition, Cav-1 expression is evident in immune cells such as macrophages and dendritic cells (38, 39). However, Cav-1 is not expressed in isolated thymocytes (49). Furthermore, Cav-1 and caveolar structures are absent in human or murine T-cell lines (27, 41, 68). Contrary to this, there has been one report showing evidence of Cav-1 expression in bovine primary cell subpopulations of CD4⁺, CD8⁺, CD21⁺, and IgM⁺ cells with Cav-1 localized predominantly in the perinuclear region (38). That report also demonstrated a membrane region staining with Cav-1-specific antibody of human CD21⁺ and CD26⁺ peripheral blood lymphocytes (PBLs). Recently, the expression of Cav-1 in activated murine B cells, with a potential role in the development of a thymus-independent immune response, was also reported (56). It remains to be determined whether Cav-1 expression is dependent on the activation state of lymphocytes. For macrophages, however, which are one of the main cell targets for HIV infection, Cav-1 expression has been clearly documented (38).The scaffolding domain of Cav-1, located in the juxtamembranous region of the N terminus, is responsible for its oligomerization and binding to various proteins (5, 62, 64). It recognizes a consensus binding motif, ΦXΦXXXXΦ, ΦXXXXΦXXΦ, or ΦXΦXXXXΦXXΦ, where Φ indicates an aromatic residue (F, W, or Y) and X indicates any residue (5, 62, 64). A Cav-1 binding motif (WNNMTWMQW) has been identified in the HIV-1 envelope protein gp41 (42, 43). Cav-1 has been shown to associate with gp41 by many different groups under various circumstances, including the immunoprecipitation of gp41 and Cav-1 in HIV-infected cells (42, 43, 52). However, the underlying pathological or physiological functions of this robust interaction between Cav-1 and gp41 remain unclear.Here, we report that the interaction between Cav-1 and gp41 leads to a modification of gp41 function, which subsequently regulates Env-induced T-cell bystander apoptosis. Moreover, we show that a peptide containing the Cav-1 scaffold domain sequence is capable of modulating Env-induced bystander apoptosis, which suggests a novel therapeutic application for HIV-1-infected patients.     

Fundamental Difference in the Content of High-Mannose Carbohydrate in the HIV-1 and HIV-2 Lineages

The virus-encoded envelope proteins of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) typically contain 26 to 30 sites for N-linked carbohydrate attachment. N-linked carbohydrate can be of three major types: high mannose, complex, or hybrid. The lectin proteins from Galanthus nivalis (GNA) and Hippeastrum hybrid (HHA), which specifically bind high-mannose carbohydrate, were found to potently inhibit the replication of a pathogenic cloned SIV from rhesus macaques, SIVmac239. Passage of SIVmac239 in the presence of escalating concentrations of GNA and HHA yielded a lectin-resistant virus population that uniformly eliminated three sites (of 26 total) for N-linked carbohydrate attachment (Asn-X-Ser or Asn-X-Thr) in the envelope protein. Two of these sites were in the gp120 surface subunit of the envelope protein (Asn244 and Asn460), and one site was in the envelope gp41 transmembrane protein (Asn625). Maximal resistance to GNA and HHA in a spreading infection was conferred to cloned variants that lacked all three sites in combination. Variant SIV gp120s exhibited dramatically decreased capacity for binding GNA compared to SIVmac239 gp120 in an enzyme-linked immunosorbent assay (ELISA). Purified gp120s from six independent HIV type 1 (HIV-1) isolates and two SIV isolates from chimpanzees (SIVcpz) consistently bound GNA in ELISA at 3- to 10-fold-higher levels than gp120s from five SIV isolates from rhesus macaques or sooty mangabeys (SIVmac/sm) and four HIV-2 isolates. Thus, our data indicate that characteristic high-mannose carbohydrate contents have been retained in the cross-species transmission lineages for SIVcpz-HIV-1 (high), SIVsm-SIVmac (low), and SIVsm-HIV-2 (low).The envelope proteins of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) are heavily glycosylated. N-linked carbohydrate is attached to the nascent protein at the asparagine of the consensus sequence N-X-S or N-X-T, where X is any amino acid except a proline (31, 52, 53). The number of potential N-linked carbohydrate attachment sites in the surface subunit of Env (gp120) ranges from 18 to 33, with a median of 25 (34, 65). There are typically 3 or 4 potential N-linked sites in the ectodomain of the Env transmembrane protein (gp41) (34).N-linked glycosylation of a protein consists of the en bloc transfer of the carbohydrate core oligosaccharide (two N-acetylglucosamines, nine mannoses, and three glucoses) from dolichol to the asparagine of the N-linked attachment site (8, 60). Initially the attached carbohydrate is processed into the high-mannose type (8). In the Golgi complex, high-mannose carbohydrate may be further processed into complex or hybrid oligosaccharides (58). Incomplete processing of N-linked carbohydrate results in the production of high-mannose carbohydrate chains, which terminate in mannose (58). Fully processed complex carbohydrate chains terminate in galactose, N-acetylglucosamine, sialic acid, or glucose (33, 57). Hybrid carbohydrate chains have two branches from the core, one that terminates in mannose and one that terminates in a sugar of the complex type (63).Glycoproteins exist as a heterogeneous population, exhibiting heterogeneity with respect to the proportion of potential glycosylation sites that are occupied and to the oligosaccharide structure observed at each site. Factors that influence the type of carbohydrate chain that is attached at any one N-linked site are the accessibility of the carbohydrate chain to processing enzymes (49), protein sequences surrounding the site (5, 40), and the type of cell from which the protein is produced (19).The N-linked carbohydrate chains of HIV and SIV Env are critical for the proper folding and cleavage of the fusion-competent envelope spike (20, 59, 61). After Env is assembled, enzymatic removal of N-linked carbohydrate does not dramatically affect the functional conformation (2, 6, 7, 13, 24, 38). It is generally accepted that the carbohydrate attached to Env limits the ability of the underlying protein to be recognized by B cells (11, 48, 62). This carbohydrate also shields protein epitopes that would otherwise be the direct targets of antibodies that neutralize viral infection (41, 48, 62, 64). Furthermore, the high-mannose carbohydrates of HIV and SIV Env bind dynamically to an array of lectin proteins that are part of the host lymphoreticular system. The interaction of viral high-mannose carbohydrate with host lectin proteins has been associated with the enhancement (9, 16, 17, 43-45) or suppression (42, 56) of viral infection of CD4-positive T cells. The high-mannose carbohydrate of Env is also known to activate the release of immune-modulatory proteins from a subset of host antigen-presenting cells (12, 54).The plant lectin proteins from Galanthus nivalis (GNA) and Hippeastrum hybrid (HHA) specifically bind terminal α-1,3- and/or α-1,6-mannose of high-mannose oligosaccharides but not hybrid oligosaccharides (28, 55). GNA and HHA inhibit the replication of HIV-1 and SIVmac251, and uncloned, resistant populations of virus have been selected (3, 14). In this report, we define two N-linked sites in the external surface glycoprotein gp120 and one in the transmembrane glycoprotein gp41 whose mutation imparts high-level resistance to the inhibitory effects of GNA and HHA to cloned SIVmac239. Furthermore, using a GNA-binding enzyme-linked immunosorbent assay (ELISA), we show that assorted HIV-1 and SIVcpz gp120s consistently are considerably higher in mannose content than assorted gp120s from SIVmac, SIVsm, and HIV-2. These results shed new light on the impact of virus-host evolutionary dynamics on viral carbohydrate composition, and they may have important implications for the mechanisms by which long-standing natural hosts such as sooty mangabeys can resist generalized lymphoid activation and disease despite high levels of SIV replication.     

A Basic Patch on α-Adaptin Is Required for Binding of Human Immunodeficiency Virus Type 1 Nef and Cooperative Assembly of a CD4-Nef-AP-2 Complex

A critical function of the human immunodeficiency virus type 1 Nef protein is the downregulation of CD4 from the surfaces of infected cells. Nef is believed to act by linking the cytosolic tail of CD4 to the endocytic machinery, thereby increasing the rate of CD4 internalization. In support of this model, weak binary interactions between CD4, Nef, and the endocytic adaptor complex, AP-2, have been reported. In particular, dileucine and diacidic motifs in the C-terminal flexible loop of Nef have been shown to mediate binding to a combination of the α and σ2 subunits of AP-2. Here, we report the identification of a potential binding site for the Nef diacidic motif on α-adaptin. This site comprises two basic residues, lysine-297 and arginine-340, on the α-adaptin trunk domain. The mutation of these residues specifically inhibits the ability of Nef to bind AP-2 and downregulate CD4. We also present evidence that the diacidic motif on Nef and the basic patch on α-adaptin are both required for the cooperative assembly of a CD4-Nef-AP-2 complex. This cooperativity explains how Nef is able to efficiently downregulate CD4 despite weak binary interactions between components of the tripartite complex.CD4, a type I transmembrane glycoprotein that serves as a coreceptor for major histocompatibility complex class II (MHC-II) molecules, is expressed on the surfaces of helper T lymphocytes and cells of the monocyte/macrophage lineage (8). Primate immunodeficiency viruses gain access to these cells by virtue of the interaction of the viral envelope glycoprotein (Env) with a combination of CD4 and a chemokine receptor (63). This interaction causes a conformational change within the Env protein that promotes the fusion of the viral envelope with the plasma membrane. Upon the delivery of the viral genetic material into the cytoplasm of the host cells, one of the first virally encoded proteins to be expressed is Nef, an accessory factor that modulates specific signal transduction and protein-trafficking pathways in a manner that optimizes the intracellular environment for viral replication (reviewed in references 21, 39, and 65). Perhaps the best characterized function of Nef is the downregulation of CD4 from the surfaces of the host cells (6, 22, 29, 45). CD4 downregulation prevents superinfection (6, 41) and enhances virion release (19, 38, 48, 66, 76), thereby contributing to the establishment of a robust infective state (24, 72).The mechanism used by the Nef protein of human immunodeficiency virus type 1 (HIV-1) to downregulate CD4 has been the subject of extensive study, but only recently have the molecular details of this process begun to be unraveled. It is generally acknowledged that HIV-1 Nef accelerates the internalization of CD4 from the plasma membrane by linking the cytosolic tail of the receptor to the clathrin-associated endocytic machinery (1, 12, 20, 34, 40, 64). In support of this model, a hydrophobic pocket comprising W57 and L58 on the folded core domain of Nef binds with millimolar affinity to the cytosolic tail of CD4 (28) (all residues and numbers correspond to the NL4-3 variant of HIV-1 Nef used in this study). In addition, a dileucine motif (ENTSLL, residues 160 to 165) (10, 16, 26) and a diacidic motif (D174 and D175) (2) on the C-terminal flexible loop of Nef mediate an interaction of micromolar affinity with the clathrin-associated, heterotetrameric (α-β2-μ2-σ2) adaptor protein 2 (AP-2) complex (12, 20, 40, 49). These interactions draw CD4 into clathrin-coated pits that eventually bud inwards as clathrin-coated vesicles (11, 27). Internalized CD4 is subsequently delivered to endosomes and then to lysosomes for degradation (3, 23, 59, 64).Despite progress in the understanding of the mechanism of Nef-induced CD4 downregulation, several important aspects remain to be elucidated. Previous studies have shown that the Nef dileucine and diacidic motifs interact with a combination of the α and σ2 subunits of AP-2 (referred to as the α-σ2 hemicomplex) (12, 20, 40, 49), but the precise location of the Nef binding sites is unknown. It also remains to be determined whether Nef can actually bind CD4 and AP-2 at the same time. Indeed, the formation of a tripartite CD4-Nef-AP-2 complex in which Nef links the cytosolic tail of CD4 to AP-2 has long been hypothesized but has never been demonstrated experimentally. Given the relatively weak affinity of Nef for the CD4 tail (28) and AP-2 (12, 40), it is unclear how such a complex could assemble and function in CD4 downregulation.In this study, we have addressed these issues by using a combination of yeast hybrid, in vitro binding, and in vivo CD4 downregulation assays. We report the identification of a candidate binding site for the Nef diacidic motif on the AP-2 complex. This site, a basic patch comprising K297 and R340 on α-adaptin, is specifically required for Nef binding and Nef-induced CD4 downregulation. We also show that the Nef diacidic motif and the α-adaptin basic patch are required for the cooperative assembly of a tripartite complex composed of the CD4 cytosolic tail, Nef, and the α-σ2 hemicomplex. The cooperative manner in which this complex is formed explains how Nef is able to efficiently downregulate CD4 from the plasma membrane despite weak binary interactions between the components of this complex.     

Influence of Disulfide-Stabilized Structure on the Specificity of Helper T-Cell and Antibody Responses to HIV Envelope Glycoprotein gp120

CD4⁺ helper T cells specific for human immunodeficiency virus type 1 (HIV-1) are associated with control of viremia. Nevertheless, vaccines have had limited effectiveness thus far, in part because sequence variability and other structural features of the HIV envelope glycoprotein deflect the immune response. Previous studies indicated that CD4⁺ T-cell epitope dominance is controlled by antigen three-dimensional structure through its influence on antigen processing and presentation. In this work, three disulfide bonds in the outer domain of gp120 were individually deleted in order to destabilize the local three-dimensional structure and enhance the presentation of nearby weakly immunogenic epitopes. However, upon immunization of groups of BALB/c mice, the CD4⁺ T-cell response was broadly reduced for all three variants, and distinct epitope profiles emerged. For one variant, antibody titers were sharply increased, and the antibody exhibited significant CD4-blocking activity.The development of an effective vaccine against HIV has been hampered by an incomplete understanding of the correlates of protection against the virus. It is generally accepted that a robust antibody response and cytotoxic T-lymphocyte (CTL) response are required to control the disease and to prevent progression to AIDS (2, 17, 19, 20, 36, 38-42). Both of these arms of the immune system require help from CD4⁺ helper T cells (1, 27, 48). However, several important aspects of the CD4⁺ helper T-cell response remain poorly defined; these include the factors that determine epitope immunodominance in the CD4⁺ T-cell response, the relationship of specificity in the CD4⁺ T-cell response to specificity in the antibody and CD8⁺ responses, and the investment made by HIV (or any pathogen) to control the CD4⁺ T-cell response.Previous studies of mice showed that antigen structure modulates antigen processing and presentation of CD4⁺ helper T-cell epitopes (3-6, 9, 10, 23, 24, 43). Immunodominant CD4⁺ helper T-cell epitopes raised in response to immunization with the HIV envelope glycoprotein gp120 were found adjacent to flexible loops between elements of secondary structure (10). This was rationalized by the fact that flexible loops more readily conform to protease active sites and therefore are preferentially cleaved by proteases during antigen processing (10, 14, 15). Helper T-cell epitopes of gp120 in humans infected with HIV were also found flanking flexible loops (30). Dominant epitopes were located in the outer domain, an average of 12 residues C-terminal to flexible loops. In the less immunogenic inner domain, epitopes were found an average of five residues N-terminal to conserved regions of the protein, once again placing the epitopes C-terminal to flexible loops (30). These results suggested that antigen structure plays a significant role in the shaping of the helper T-cell response against HIV gp120 in both mice and humans.In reviewing previous studies mapping the helper T-cell response to gp120, we noted a marked absence of CD4⁺ T-cell responses to regions of the outer domain that coincided with the locations of highly conserved disulfide bonds (Fig. ​(Fig.1).1). Disulfide bonds have previously been shown to interfere with presentation of nearby helper T-cell epitopes (13, 26). Thus, we hypothesized that disulfide bonds stabilized these regions of the protein, protecting them from proteolysis. This resulted in the exclusion of these regions from presentation to helper T cells. We further hypothesized that the deletion of these disulfide bonds would result in the production of new helper T-cell epitopes by creating localized regions of flexibility that could now be processed and presented to T cells. The creation of new helper T-cell epitopes could also potentially lead to changes in the antibody response.Open in a separate windowFIG. 1.Gaps in helper T-cell epitope frequency in the outer domain of HIV gp120 coincide with the locations of disulfide bonds. The graph illustrates the frequencies of responses by residue for the combined profiles from immunized BALB/c and CBA mice (gray area) and for a group of seven HIV-infected human subjects (black line) (10, 30).For the present work, we constructed three disulfide-bond variants of gp120 by replacing paired cysteines in the outer domain with alanines. Characterization of the variants revealed that the proteins were structurally distinct from one another and from wild-type gp120. Groups of 10 BALB/c mice immunized with these proteins produced patterns of helper T-cell responses that were very different from each other and from that of a group of 10 BALB/c mice immunized with wild-type gp120. In general, the T-cell response was reduced in mice immunized with the variant proteins. For one of the variants, anti-gp120 antibody titers were increased and exhibited CD4-blocking activity.     

Epitope Mapping of Ibalizumab,a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients

Ibalizumab is a humanized monoclonal antibody that binds human CD4, the primary receptor for human immunodeficiency virus type 1 (HIV-1). With its unique specificity for domain 2 of CD4, this antibody potently and broadly blocks HIV-1 infection in vitro by inhibiting a postbinding step required for viral entry but without interfering with major histocompatibility complex class II (MHC-II)-mediated immune function. In clinical trials, ibalizumab has demonstrated anti-HIV-1 activity in patients without causing immunosuppression. Thus, a characterization of the ibalizumab epitope was conducted in an attempt to gain insight into the underlying mechanism of its antiviral activity as well as its safety profile. By studying mouse/human chimeric CD4 molecules and site-directed point mutants of CD4, amino acids L96, P121, P122, and Q163 in domain 2 were found to be important for ibalizumab binding, with E77 and S79 in domain 1 also contributing. All these residues appear to cluster on the interface between domains 1 and 2 of human CD4 on a surface opposite the site where gp120 and the MHC-II molecule bind on domain 1. Separately, the epitope of M-T441, a weakly neutralizing mouse monoclonal antibody that competes with ibalizumab, was localized entirely within domain 2 on residues 123 to 125 and 138 to 140. The results reported herein not only provide an appreciation for why ibalizumab has not had significant adverse immunological consequences in infected patients to date but also raise possible steric hindrance mechanisms by which this antibody blocks HIV-1 entry into a CD4-positive cell.The human immunodeficiency virus type 1 (HIV-1) epidemic continues to spread at the alarming rate of approximately 2.5 million new cases per year, despite intensive efforts from the scientific community. A safe and effective HIV-1 vaccine would be a key weapon to fight this epidemic; however, vaccine development has not yet proven successful. The extraordinary diversity of the virus, its capacity to evade adaptive immune responses, and the inability to induce broadly neutralizing antibodies against HIV-1 represent unprecedented challenges for vaccine development (3). Alternatively, the strategy of preexposure prophylaxis (PrEP) with antiretroviral drugs or even virus-specific immunoglobulins (Igs) (11) is gaining traction. Protection of rhesus macaques from challenge with simian immunodeficiency virus (SIV) has been observed after passive administration of anti-gp120 or anti-gp41 monoclonal antibodies, such as b12, 2G12, 2F5, and 4E10 (2, 20). However, the application of these antibodies as PrEP has been hindered due to their lack of potency or breadth or both. To this end, PrEP strategies could also consider antibodies to CCR5 (13) or CD4 (8, 12, 14), which have potent and broad inhibitory activities against HIV-1 without unwanted side effects.The CD4 molecule, a cell surface glycoprotein found primarily on T lymphocytes, is the primary receptor for the HIV-1 envelope gp160 glycoprotein (7, 18). A member of the immunoglobulin superfamily (19), CD4 consists of an extracellular segment composed of four tandem immunoglobulin-like domains (D1, D2, D3, and D4), a single transmembrane span, and a short C-terminal cytoplasmic tail (15, 24). It is worth noting that both human major histocompatibility complex (MHC) class II (26) and HIV-1 gp120 (16, 24) bind to the same surface on the first domain (D1) of the CD4 molecule.Ibalizumab (formerly known as TNX-355) is a humanized IgG4 monoclonal antibody that blocks HIV-1 entry by binding to human CD4 (8, 12, 14, 33). It was engineered from its mouse progenitor (5A8) by grafting the mouse complementary-determining region (CDR) onto a human IgG4 construct (4, 5). The IgG4 isotype was chosen to minimize the chances for CD4⁺ T-cell depletion by antibody- and complement-dependent cytotoxicity mediated by binding to Fc receptors. Ibalizumab or 5A8 blocks CD4-dependent virus entry and inhibits a broad spectrum of both laboratory-adapted and clinical HIV-1 isolates, including CCR5-tropic and CXCR4-tropic strains from multiple subtypes, with 50% inhibitory concentrations (IC₅₀s) of 0.0004 to 0.152 μg/ml (4, 5). In vivo, treatment with ibalizumab prominently reduced plasma viremia in rhesus macaques infected with SIV, because this monoclonal antibody has equal affinity for rhesus CD4 (22, 23). In HIV-1 patients, single as well as multiple doses of ibalizumab resulted in substantial reductions (∼10-fold) in viral loads and increases in CD4⁺ T-cell counts without evidence of serious adverse effects or immunologic impairments (12, 14).Efforts were made years ago to delineate the antibody binding site of 5A8 on human CD4 (hCD4) (5). Two stretches, amino acids (aa) 121 to 124 and aa 127 to 134, in domain 2 (D2) were found to be critical for binding. Since then, however, little work has been done to fine-map this epitope, and whether other parts of hCD4 are involved in the binding of this antibody has remained unexplored. The fact that an anti-hCD4-D2 antibody can noncompetitively, yet potently, block HIV-1 entry is intriguing, as viral gp120 binds to D1 of hCD4 (16, 24). Therefore, to gain a better understanding of the mechanism by which ibalizumab inhibits HIV-1 infection while avoiding undesired side effects, we sought to fine-map the epitope of this unique monoclonal antibody.     

Antibody Specificities Associated with Neutralization Breadth in Plasma from Human Immunodeficiency Virus Type 1 Subtype C-Infected Blood Donors

Defining the specificities of the anti-human immunodeficiency virus type 1 (HIV-1) envelope antibodies able to mediate broad heterologous neutralization will assist in identifying targets for an HIV-1 vaccine. We screened 70 plasmas from chronically HIV-1-infected individuals for neutralization breadth. Of these, 16 (23%) were found to neutralize 80% or more of the viruses tested. Anti-CD4 binding site (CD4bs) antibodies were found in almost all plasmas independent of their neutralization breadth, but they mainly mediated neutralization of the laboratory strain HxB2 with little effect on the primary virus, Du151. Adsorption with Du151 monomeric gp120 reduced neutralizing activity to some extent in most plasma samples when tested against the matched virus, although these antibodies did not always confer cross-neutralization. For one plasma, this activity was mapped to a site overlapping the CD4-induced (CD4i) epitope and CD4bs. Anti-membrane-proximal external region (MPER) (r = 0.69; P < 0.001) and anti-CD4i (r = 0.49; P < 0.001) antibody titers were found to be correlated with the neutralization breadth. These anti-MPER antibodies were not 4E10- or 2F5-like but spanned the 4E10 epitope. Furthermore, we found that anti-cardiolipin antibodies were correlated with the neutralization breadth (r = 0.67; P < 0.001) and anti-MPER antibodies (r = 0.6; P < 0.001). Our study suggests that more than one epitope on the envelope glycoprotein is involved in the cross-reactive neutralization elicited during natural HIV-1 infection, many of which are yet to be determined, and that polyreactive antibodies are possibly involved in this phenomenon.The generation of an antibody response capable of neutralizing a broad range of viruses remains an important goal of human immunodeficiency virus type 1 (HIV-1) vaccine development. Despite multiple efforts in the design of immunogens capable of inducing such humoral responses, little progress has been made (18, 20, 39). The sequence variability of the virus, as well as masking mechanisms exhibited by the envelope glycoprotein, has further hindered this pursuit (6, 22). It is known that while the majority of HIV-infected individuals mount a strong neutralization response against their own virus within the first 6 to 12 months of infection, breadth is observed in only a few individuals years later (5, 10, 15, 26, 33, 40, 41). However, very little is known about the specificities of the antibodies that confer this broad cross-neutralization. It is plausible that broadly cross-neutralizing (BCN) plasmas contain antibodies that target conserved regions of the envelope glycoprotein, as exemplified by a number of well-characterized broadly neutralizing monoclonal antibodies (MAbs). The b12 MAb recognizes the CD4 binding site (CD4bs), and 2G12 binds to surface glycans (7, 42, 44, 56). The 447-52D MAb recognizes the V3 loop, and 17b, E51, and 412d bind to CD4-induced (CD4i) epitopes that form part of the coreceptor binding site (13, 21, 51, 54). Finally, the MAbs 2F5, 4E10, and Z13e1 recognize distinct linear sequences in the gp41 membrane-proximal external region (MPER) (36, 57). The targets of these neutralizing MAbs provide a rational starting point for examining the complex nature of polyclonal plasma samples.Several groups have addressed the need to develop methodologies to elucidate the presence of certain neutralizing-antibody specificities (1, 8, 9, 29, 30, 43, 55). A number of these studies reported that the BCN antibodies in plasma can in some cases be adsorbed using gp120 immobilized on beads (1, 9, 29, 30, 43). Furthermore, the activities of some of these anti-gp120 neutralizing antibodies could be mapped to the CD4bs, as the D368R mutant gp120 failed to adsorb them (1, 29, 30, 43).Antibodies to CD4i epitopes are frequently found in HIV-1-infected individuals and are thought to primarily target the coreceptor binding site, which includes the bridging sheet and possibly parts of the V3 region. Decker and colleagues (8) showed that MAbs to HIV-1 CD4i epitopes can neutralize HIV-2 when pretreated with soluble CD4 (sCD4), indicating that the CD4i epitope is highly conserved among different HIV lineages. The poor accessibility of CD4i epitopes, however, has precluded this site from being a major neutralizing-antibody target (24), although a recent study suggested that some of the cross-neutralizing activity in polyclonal sera mapped to a CD4i epitope (30).Another site that has attracted considerable attention as a target for cross-neutralizing antibodies is the MPER, a linear stretch of 34 amino acids in gp41. Anti-MPER antibodies have been detected in the plasma of HIV-infected individuals by using chimeric viruses with HIV-1 MPER grafted into a simian immunodeficiency virus or an HIV-2 envelope glycoprotein (15, 55). These studies concluded that 2F5- and 4E10-like antibodies were rarely found in HIV-1-infected plasmas; however, other specificities within the MPER were recognized by around one-third of HIV-1-infected individuals (15). More recently, 4E10-like and 2F5-like antibodies (30, 43), as well as antibodies to novel epitopes within the MPER (1), have been shown to be responsible for neutralization breadth in a small number of plasma samples. The anti-MPER MAb 4E10 has been shown to react to autoantigens, leading to the suggestion that their rarity in human infection is due to the selective deletion of B cells with these specificities (17, 35). Furthermore, a recent study found an association between anti-MPER and anti-cardiolipin (CL) antibodies, although an association with neutralization was not examined (31).A recent study by Binley and coworkers used an array of methodologies to determine the antibody specificities present in subtype B and subtype C plasma samples with neutralization breadth (1). While antibodies to gp120, some of which mapped to the CD4bs, and to MPER were identified, most of the neutralizing activity in the BCN plasma could not be attributed to any of the known conserved envelope epitopes. Furthermore, it is not clear how common these specificities are among HIV-1-positive plasmas and whether they are only associated with BCN activity.In this study, we investigated a large collection of HIV-1-infected plasmas obtained from the South African National Blood Services. We aimed to determine if there is a relationship between the presence of certain antibody specificities, such as those against CD4i epitopes, MPER, or the CD4bs, and the neutralizing activities present in these plasmas. Furthermore, we evaluated the presence of various autoreactive antibodies and analyzed whether they might be associated with neutralization breadth.     

The Heptad Repeat 2 Domain Is a Major Determinant for Enhanced Human Immunodeficiency Virus Type 1 (HIV-1) Fusion and Pathogenicity of a Highly Pathogenic HIV-1 Env

Human immunodeficiency virus type 1 (HIV-1)-mediated depletion of CD4⁺ lymphocytes in an infected individual is the hallmark of progression to AIDS. However, the mechanism for this depletion remains unclear. To identify mechanisms of HIV-1-mediated CD4 T-cell death, two similar viral isolates obtained from a rapid progressor patient with significantly different pathogenic phenotypes were studied. One isolate (R3A) demonstrates enhanced pathogenesis in both in vivo models and relevant ex vivo lymphoid organ model systems compared to another isolate, R3B. The pathogenic determinants were previously mapped to the V5-gp41 envelope region, correlating functionally with enhanced fusion activity and elevated CXCR4 binding affinity. To further elucidate specific differences between R3A and R3B within the V5-gp41 domains that enhance CD4 depletion, R3A-R3B chimeras to study the V5-gp41 region were developed. Our data demonstrate that six residues in the ectodomain of R3A provide the major determinant for both enhanced Env-cell fusion and pathogenicity. Furthermore, three amino acid differences in the heptad repeat 2 (HR-2) domain of R3A determined its fusion activity and significantly elevated its pathogenic activity. The chimeric viruses with enhanced fusion activity, but not elevated CXCR4 affinity, correlated with high pathogenicity in the thymus organ. We conclude that the functional domain of a highly pathogenic HIV-1 Env is determined by mutations in the HR-2 region that contribute to enhanced fusion and CD4 T-cell depletion.Human immunodeficiency virus type 1 (HIV-1) is the causative agent for AIDS, which is characterized by a dramatic loss of CD4⁺ lymphocytes and impairment of the immune system against invading pathogens (13, 21, 22). Though much has been determined regarding interactions between HIV-1 virus and CD4⁺ target cells, the mechanisms by which the HIV-1 virus depletes CD4⁺ lymphocytes remain incompletely understood. Various studies have demonstrated that in an HIV-infected host, both infected and uninfected cells are prone to destruction, albeit by different pathways (15, 18, 29). Recently, our group and others have shown that while binding of CD4 and chemokine receptors contribute to syncytium formation in vitro, viral membrane fusion by the envelope glycoprotein plays an important role in depletion of both uninfected and infected cells by HIV-1 and simian-human immunodeficiency virus in vivo (1, 11, 12, 26, 29).HIV-1 entry into a cell is mediated by a multistep process that begins with high-affinity binding between viral envelope (gp120) and the cellular CD4 receptor (9, 14, 16). This binding causes a conformational change in the viral envelope, allowing for subsequent coreceptor binding (mainly CCR5 or CXCR4). Upon coreceptor binding, another conformational change is thought to take place that allows gp41 to engage the cell to form a fusion complex. Envelope proteins have been demonstrated to exist as a trimer, allowing for three gp41s to form a fusion assembly through noncovalent interactions. This fusion assembly is determined to exist in a six-helix bundle formation as the fusion event takes place, allowing for the virion to fuse to the host cell (5, 24).The envelope glycoprotein (Env) of HIV plays a significant role in viral pathogenesis, as seen in several in vitro and in vivo models of infection. The Env functions to mediate virus entry of cells and is also a major target for immune responses (31, 39). While the envelope initially forms as a precursor protein (gp160), subsequent cleavage by a cellular protease yields the surface subunit gp120 and the transmembrane gp41 although the gp120 and gp41 interact noncovalently (36). The gp120 protein is comprised of five variable (V1 to V5) and five conserved constant (C1 to C5) domains and binds CD4 and the coreceptors. The gp41 protein is comprised of an amino-terminal fusion domain and two heptad repeats (HR-1 and HR-2) in the ectodomain (extracellular domain), a single transmembrane domain, and a cytoplasmic tail (intracellular domain) (8, 10, 36, 37). Due to the discovery of fusion inhibitor peptides such as C34 (23, 24) and T20 (38), much is now known about the fusion complex formed by the HIV-1 fusion domain. Similar to other viral envelopes that carry a type 1 fusion complex (such as influenza and corona viruses), the ectodomain of HIV-1 Env carries two HRs that form a coiled-coiled structure. In order for HIV-cell fusion to occur, the HR-1 domains of the trimeric Env protein must interact with the cell surface. Following this initial interaction, HR-2 domains are thought to intertwine over the HR-1 coils to form a stable six-helix bundle, which represents the gp41 core structure. X-ray crystallographic studies show that the six-helix bundle core consists of the HR-1 and HR-2 peptides bound in an antiparallel manner. This structure brings the fusion peptide to the target cell membrane, allowing for the formation of a fusion pore and the entry of virions into the cell.HIV-1 Env expressed on the surface of infected cells can induce cell-cell fusion with adjacent uninfected cells to form multinucleated syncytia and single cell lysis in cell culture and apoptosis in primary cells. Various models (both ex vivo and in vivo) have been utilized to study HIV-1-induced depletion of CD4⁺ lymphocytes. Models such as SCID-human thymus-liver (SCID-hu thy/liv), tonsil histoculture, and human fetal thymus organ culture (HFTOC) have demonstrated significant use in the study of acute infection and pathogenesis in the appropriate lymphoid organ microenvironment as they retain the organ structure and do not require exogenous stimulation for productive viral infection to occur (2, 20, 28, 32). More importantly, tissue culture-adapted HIV-1 isolates such as HXB2 fail to replicate in the SCID-hu thy/liv or HFTOC models (30, 33). Organ models such as the SCID-hu thy/liv and HFTOC thus more accurately demonstrate infection, replication, and pathogenicity of primary HIV-1 strains.Here, HFTOC is used to investigate mechanisms by which an HIV-1 virus with a highly pathogenic viral Env is able to deplete CD4⁺ lymphocytes. Two viral isolates obtained from rapid progressor patient 3 of the ALIVE cohort (40) show significant sequence homology, particularly in the Env region, while they carry stark differences in pathogenic ability (26, 27). One isolate (denoted R3A) was found to demonstrate enhanced fusion in cell-cell fusion assays as well as enhanced pathogenesis in relevant ex-vivo/in vivo organ model systems compared to another isolate, R3B. To define the pathogenic determinants that differentiate R3A from R3B, this study demonstrates that the enhanced fusogenicity of R3A (governed by the ectodomain of the gp41), but not the elevated CXCR4 binding affinity, confers the pathogenic phenotype in HFTOC. We further demonstrate that three amino acid differences in the HR-2 domain allow for this enhanced fusion for R3A Env, defining a possible mechanism for a pathogenic HIV-1 envelope.     

Envelope-Modified Single-Cycle Simian Immunodeficiency Virus Selectively Enhances Antibody Responses and Partially Protects against Repeated,Low-Dose Vaginal Challenge

Immunization of rhesus macaques with strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection elicits T-cell responses to multiple viral gene products and antibodies capable of neutralizing lab-adapted SIV, but not neutralization-resistant primary isolates of SIV. In an effort to improve upon the antibody responses, we immunized rhesus macaques with three strains of single-cycle SIV (scSIV) that express envelope glycoproteins modified to lack structural features thought to interfere with the development of neutralizing antibodies. These envelope-modified strains of scSIV lacked either five potential N-linked glycosylation sites in gp120, three potential N-linked glycosylation sites in gp41, or 100 amino acids in the V1V2 region of gp120. Three doses consisting of a mixture of the three envelope-modified strains of scSIV were administered on weeks 0, 6, and 12, followed by two booster inoculations with vesicular stomatitis virus (VSV) G trans-complemented scSIV on weeks 18 and 24. Although this immunization regimen did not elicit antibodies capable of detectably neutralizing SIV_mac239 or SIV_mac251_UCD, neutralizing antibody titers to the envelope-modified strains were selectively enhanced. Virus-specific antibodies and T cells were observed in the vaginal mucosa. After 20 weeks of repeated, low-dose vaginal challenge with SIV_mac251_UCD, six of eight immunized animals versus six of six naïve controls became infected. Although immunization did not significantly reduce the likelihood of acquiring immunodeficiency virus infection, statistically significant reductions in peak and set point viral loads were observed in the immunized animals relative to the naïve control animals.Development of a safe and effective vaccine for human immunodeficiency virus type 1 (HIV-1) is an urgent public health priority, but remains a formidable scientific challenge. Passive transfer experiments in macaques demonstrate neutralizing antibodies can prevent infection by laboratory-engineered simian-human immunodeficiency virus (SHIV) strains (6, 33, 34, 53, 59). However, no current vaccine approach is capable of eliciting antibodies that neutralize primary isolates with neutralization-resistant envelope glycoproteins. Virus-specific T-cell responses can be elicited by prime-boost strategies utilizing recombinant DNA and/or viral vectors (3, 10, 11, 16, 36, 73, 77, 78), which confer containment of viral loads following challenge with SHIV_89.6P (3, 13, 66, 68). Unfortunately, similar vaccine regimens are much less effective against SIV_mac239 and SIV_mac251 (12, 16, 31, 36, 73), which bear closer resemblance to most transmitted HIV-1 isolates in their inability to utilize CXCR4 as a coreceptor (18, 23, 24, 88) and inherent high degree of resistance to neutralization by antibodies or soluble CD4 (43, 55, 56). Live, attenuated SIV can provide apparent sterile protection against challenge with SIV_mac239 and SIV_mac251 or at least contain viral replication below the limit of detection (20, 22, 80). Due to the potential of the attenuated viruses themselves to cause disease in neonatal rhesus macaques (5, 7, 81) and to revert to a pathogenic phenotype through the accumulation of mutations over prolonged periods of replication in adult animals (2, 35, 76), attenuated HIV-1 is not under consideration for use in humans.As an experimental vaccine approach designed to retain many of the features of live, attenuated SIV, without the risk of reversion to a pathogenic phenotype, we and others devised genetic approaches for producing strains of SIV that are limited to a single cycle of infection (27, 28, 30, 38, 39, 45). In a previous study, immunization of rhesus macaques with single-cycle SIV (scSIV) trans-complemented with vesicular stomatitis virus (VSV) G elicited potent virus-specific T-cell responses (39), which were comparable in magnitude to T-cell responses elicited by optimized prime-boost regimens based on recombinant DNA and viral vectors (3, 16, 36, 68, 73, 78). Antibodies were elicited that neutralized lab-adapted SIV_mac251_LA (39). However, despite the presentation of the native, trimeric SIV envelope glycoprotein (Env) on the surface of infected cells and virions, none of the scSIV-immunized macaques developed antibody responses that neutralized SIV_mac239 (39). Therefore, we have now introduced Env modifications into scSIV that facilitate the development of neutralizing antibodies.Most primate lentiviral envelope glycoproteins are inherently resistant to neutralizing antibodies due to structural and thermodynamic properties that have evolved to enable persistent replication in the face of vigorous antibody responses (17, 46, 47, 64, 71, 75, 79, 83, 85). Among these, extensive N-linked glycosylation renders much of the Env surface inaccessible to antibodies (17, 48, 60, 63, 75). Removal of N-linked glycans from gp120 or gp41 by mutagenesis facilitates the induction of antibodies to epitopes that are occluded by these carbohydrates in the wild-type virus (64, 85). Consequently, antibodies from animals infected with glycan-deficient strains neutralize these strains better than antibodies from animals infected with the fully glycosylated SIV_mac239 parental strain (64, 85). Most importantly with regard to immunogen design, animals infected with the glycan-deficient strains developed higher neutralizing antibody titers against wild-type SIV_mac239 (64, 85). Additionally, the removal of a single N-linked glycan in gp120 enhanced the induction of neutralizing antibodies against SHIV_89.6P and SHIV_SF162 in a prime-boost strategy by 20-fold (50). These observations suggest that potential neutralization determinants accessible in the wild-type Env are poorly immunogenic unless specific N-linked glycans in gp120 and gp41 are eliminated by mutagenesis.The variable loop regions 1 and 2 (V1V2) of HIV-1 and SIV gp120 may also interfere with the development of neutralizing antibodies. Deletion of V1V2 from HIV-1 gp120 permitted neutralizing monoclonal antibodies to CD4-inducible epitopes to bind to gp120 in the absence of CD4, suggesting that V1V2 occludes potential neutralization determinants prior to the engagement of CD4 (82). A deletion in V2 of HIV-1 Env-exposed epitopes was conserved between clades (69), improved the ability of a secreted Env trimer to elicit neutralizing antibodies (9), and was present in a vaccine that conferred complete protection against SHIV_SF162P4 (8). A deletion of 100 amino acids in V1V2 of SIV_mac239 rendered the virus sensitive to monoclonal antibodies with various specificities (41). Furthermore, three of five macaques experimentally infected with SIV_mac239 with V1V2 deleted resisted superinfection with wild-type SIV_mac239 (51). Thus, occlusion of potential neutralization determinants by the V1V2 loop structure may contribute to the poor immunogenicity of the wild-type envelope glycoprotein.Here we tested the hypothesis that antibody responses to scSIV could be improved by immunizing macaques with strains of scSIV engineered to eliminate structural features that interfere with the development of neutralizing antibodies. Antibodies to Env-modified strains were selectively enhanced, but these did not neutralize the wild-type SIV strains. We then tested the hypothesis that immunization might prevent infection in a repeated, low-dose vaginal challenge model of heterosexual HIV-1 transmission. Indeed, while all six naïve control animals became infected, two of eight immunized animals remained uninfected after 20 weeks of repeated vaginal challenge. Relative to the naïve control group, reductions in peak and set point viral loads were statistically significant in the immunized animals that became infected.     

Alterations in the Immunogenic Properties of Soluble Trimeric Human Immunodeficiency Virus Type 1 Envelope Proteins Induced by Deletion or Heterologous Substitutions of the V1 Loop

HIV-1 gp140 envelope immunogens express conserved epitopes that are targeted by broadly cross-reactive neutralizing antibodies, but they fail to elicit similar antibodies upon immunization. The poor immunogenicity of conserved epitopes on gp140 could be linked to the high immunogenicity of variable Env regions on such constructs. Previous studies have shown that the first hypervariable region (V1 loop) is immunogenic on soluble gp140s but elicits type-specific antibodies. To address issues related to the high immunogenicity of the V1 loop, two conceptually opposite approaches were tested. In the first approach, we eliminated the V1 loop from our gp140 construct and examined how V1 deletion altered the immunogenic properties of other Env regions. In the second approach, we took advantage of the high immunogenicity of the V1 loop and engrafted four diverse V1 loops onto a common gp140 Env “scaffold.” These four scaffolds were used as a cocktail of immunogens to elicit diverse anti-V1 antibodies, under the hypothesis that eliciting diverse anti-V1 antibodies would expand the neutralizing breadth of immune sera. Our study indicates that three of four heterologous V1 loops were immunogenic on the common Env backbone “scaffold,” but heterologous anti-V1 neutralizing responses were observed in only one case. Both types of V1 modification dampened the immunogenicity of the V3 loop, differentially altered the immunogenicity of the transmembrane gp41 subunit, and altered the relative immunogenicities of unknown Env regions, including potentially the CD4-binding site (CD4-bs) and trimer-specific targets, which elicited cross-reactive neutralizing antibodies but of limited breadth.An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will need to incorporate an envelope-derived immunogen capable of eliciting potent and broadly cross-reactive neutralizing antibody responses against diverse primary HIV-1 isolates. The target of anti-HIV neutralizing antibodies (NAbs), the viral envelope (Env) glycoprotein, is expressed as a single transmembrane polypeptide precursor (gp160) that is glycosylated and cleaved into an extracellular subunit (gp120) and a transmembrane subunit (gp41) during intracellular processing (10, 20, 21, 54). The functional Env form on virion surfaces is a trimer composed of three noncovalently associated gp120-gp41 heterodimers. Soluble forms of the trimeric Env have been generated by introducing stop codons immediately upstream of the transmembrane domain of gp41. These constructs are commonly referred to as gp140 proteins and have been tested extensively as immunogens to elicit anti-HIV-1 NAbs. Soluble gp140s express epitopes that are targets of NAbs, including cross-reactive NAbs such as b12, 4E10, and 2G12 (5, 17, 34, 45, 47, 49, 50, 52, 57). Immunization with gp140 immunogens nonetheless does not result in a broadly cross-reactive neutralizing antibody response (2, 3, 17, 18, 26, 56, 58).Epitope mapping analyses of the Abs elicited by soluble trimeric gp140 immunogens revealed that a large fraction of the gp140-induced neutralization response targets the first hypervariable region of gp120 (the V1 loop). In our hands, ∼40 to 70% of the neutralizing activity of sera from animals immunized with SF162 gp140 constructs is due to anti-V1 antibodies (17). In a study by Li et al. with YU2 gp140 (30) and a study by Wu et al. with HxB2/BaL gp145 (56), ∼10 to 80% of anti-YU2 neutralizing activity and 100% of anti-HxB2 neutralizing activity, respectively, were due to anti-V1 Abs. These anti-V1 Abs, however, are not cross-reactive. Previously, we also demonstrated that the diverse positionings of the V1 across heterologous strains limit access of broadly cross-reactive monoclonal antibodies (MAbs) to their targets (12).Here, taking into consideration the V1 loop''s high immunogenicity, we employed two opposing approaches aimed at the elicitation of cross-reactive neutralizing antibody responses to HIV-1. In the first approach, we deleted the V1 loop on our soluble trimeric gp140 construct (ΔV1SF162 gp140) and examined whether and how this modification altered the immunogenic properties of other Env regions. In the second approach, we substituted the V1 loop on our SF162 gp140 construct with the V1 loops from four heterologous HIV-1 viruses (89.6, YU2, JRFL, and HxB2) that differ in their amino acid compositions and in the number of potential N-linked glycosylation sites (PNGs). These four heterologous viruses display various neutralization phenotypes (7) and coreceptor utilization profiles (15, 35, 36, 48, 51). A total of four SF162 Env-based gp140 “scaffolds” expressing four different V1 loops were created and used as immunogens in a cocktail to test as a “proof of principle” the hypothesis that if diverse V1 loops are presented to the immune system simultaneously, the elicitation of anti-V1 NAbs with diverse specificities would broaden the overall neutralizing activity of immune sera. We also immunized animals with each of the four V1 chimeric scaffolds individually to ensure that all V1 loops were immunogenic when presented on the heterologous SF162 Env background.All immunogens (including wild-type [WT] SF162 gp140 and ΔV1SF162 gp140) elicited homologous anti-SF162 NAbs. All immunogens except the scaffold construct expressing the YU2 V1 also elicited heterologous NAbs against the sensitive lab-adapted strain HxB2. The heterologous YU2, 89.6, and HxB2 V1 loops, but not the JRFL V1 loop, were immunogenic on the background of the SF162 Env scaffold. However, only anti-V1 neutralizing activity against the HxB2 virus was observed. Although neither approach resulted in the development of broad anti-HIV-1 cross-neutralizing antibody responses, cross-neutralizing antibody responses of narrow breadth were elicited. These responses were not due to antibodies that target to variable regions of gp120 but were due to antibodies that target either epitopes of the CD4-binding site (CD4-bs) or epitopes that are not present on monomeric gp120. These observations have implications for guiding rational Env-based immunogen design and for potentially eliciting broadly cross-reactive NAb responses.     

Analysis of Human Immunodeficiency Virus Type 1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4⁺ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure—analysis of molecular variance and the Slatkin-Maddison test—to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4⁺ T cells but that proviruses in resting and activated CD4⁺ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4⁺ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4⁺ T cells has implications for eradication efforts.Successful treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART) reduces free virus in the blood to levels undetectable by the most sensitive clinical assays (18, 36). However, HIV-1 persists as a latent provirus in resting, memory CD4⁺ T lymphocytes (6, 9, 12, 16, 48) and perhaps in other cell types (45, 52). The latent reservoir in resting CD4⁺ T cells represents a barrier to eradication because of its long half-life (15, 37, 40-42) and because specifically targeting and purging this reservoir is inherently difficult (8, 25, 27).In addition to the latent reservoir in resting CD4⁺ T cells, patients on HAART also have a low amount of free virus in the plasma, typically at levels below the limit of detection of current clinical assays (13, 19, 35, 37). Because free virus has a short half-life (20, 47), residual viremia is indicative of active virus production. The continued presence of free virus in the plasma of patients on HAART indicates either ongoing replication (10, 13, 17, 19), release of virus after reactivation of latently infected CD4⁺ T cells (22, 24, 31, 50), release from other cellular reservoirs (7, 45, 52), or some combination of these mechanisms. Finding the cellular source of residual viremia is important because it will identify the cells that are still capable of producing virus in patients on HAART, cells that must be targeted in any eradication effort.Detailed analysis of this residual viremia has been hindered by technical challenges involved in working with very low concentrations of virus (13, 19, 35). Recently, new insights into the nature of residual viremia have been obtained through intensive patient sampling and enhanced ultrasensitive sequencing methods (1). In a subset of patients, most of the residual viremia consisted of a small number of viral clones (1, 46) produced by a cell type severely underrepresented in the peripheral circulation (1). These unique viral clones, termed predominant plasma clones (PPCs), persist unchanged for extended periods of time (1). The persistence of PPCs indicates that in some patients there may be another major cellular source of residual viremia (1). However, PPCs were observed in a small group of patients who started HAART with very low CD4 counts, and it has been unclear whether the PPC phenomenon extends beyond this group of patients. More importantly, it has been unclear whether the residual viremia generally consists of distinct virus populations produced by different cell types.Since the HIV-1 infection in most patients is initially established by a single viral clone (23, 51), with subsequent diversification (29), the presence of genetically distinct populations of virus in a single individual can reflect entry of viruses into compartments where replication occurs with limited subsequent intercompartmental mixing (32). Sophisticated genetic tests can detect such population structure in a sample of viral sequences (4, 39, 49). Using two complementary tests of population structure (14, 43), we analyzed viral sequences from multiple sources within individual patients in order to determine whether a source other than circulating resting CD4⁺ T cells contributes to residual viremia and viral persistence. Our results have important clinical implications for understanding HIV-1 persistence and treatment failure and for improving eradication strategies, which are currently focusing only on the latent CD4⁺ T-cell reservoir.     

Evaluation of CD4-CD4i Antibody Architectures Yields Potent,Broadly Cross-Reactive Anti-Human Immunodeficiency Virus Reagents

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) has several adaptations that allow the virus to evade antibody neutralization. Nevertheless, a few broadly cross-reactive neutralizing antibodies as well as reagents containing portions of CD4, the HIV receptor, have demonstrated partial efficacy in suppressing viral replication. One type of reagent designed for improved HIV neutralization fuses the CD4 D1-D2 domains to the variable regions of an antibody recognizing the CD4-induced (CD4i) coreceptor binding site on the gp120 portion of the HIV envelope spike. We designed, expressed, purified, and tested the neutralization potencies of CD4-CD4i antibody reagents with different architectures, antibody combining sites, and linkers. We found that fusing CD4 to the heavy chain of the CD4i antibody E51 yields a bivalent reagent including an antibody Fc region that expresses well, is expected to have a long serum half-life, and has comparable or greater neutralization activity than well-known broadly neutralizing anti-HIV antibodies. A CD4 fusion with the anti-HIV carbohydrate antibody 2G12 also results in a potent neutralizing reagent with more broadly neutralizing activity than 2G12 alone.The envelope spike of human immunodeficiency virus type 1 (HIV-1), a trimer of gp120/gp41 heterodimers, utilizes a number of strategies to avoid antibodies (Abs) elicited by the humoral immune response. These include variable loops, heavy glycosylation (36), conformational masking of key functional sites (19), and an architecture and surface density that reduce bivalent Ab engagement (18). Nevertheless, a small number of broadly cross-reactive neutralizing Abs have been found and extensively characterized (5, 32, 41). The targets of these Abs include the membrane proximal region of gp41 (24, 42), a cluster of high-mannose carbohydrates on gp120 (29), and the HIV receptor (CD4)-binding site (3, 28). A combination of several of these Abs has been evaluated in clinical trials as a passive immunotherapy to reduce viral rebound during an interruption of antiretroviral therapy (34).Several CD4-containing proteins have also been explored clinically as possible therapeutics for treating HIV-1: soluble CD4 (13, 16), a CD4-Fc fusion protein (7), and the tetravalent CD4-immunoglobulin G2 (CD4-IgG2; PRO 542) reagent (1, 17). In patients with advanced disease, CD4-IgG2 treatment led to a ∼0.5 log₁₀ mean reduction in viral load (17). In addition, D1D2-Igαtp, an approximately dodecameric CD4 reagent created as a chimeric IgG1/IgA fusion protein (2), exhibited very potent HIV neutralization activity and targeted HIV-infected cells for lysis by natural killer cells (14).Another approach to targeting gp120 is a fusion protein composed of CD4 linked to the variable regions of a CD4-induced (CD4i) Ab (11). CD4i Abs represent a potentially promising class of Abs because they bind to the conserved HIV-1 coreceptor binding site on gp120, which is exposed after a conformational change resulting from binding to CD4 (25, 27, 38). Examples of CD4i Abs include 17b (33), E51 (39), m9 (40), 412d (8), and 21c (38). These Abs are often broadly cross-reactive but generally show little neutralization potency in vivo due to limited steric accessibility when gp120 on the viral membrane is bound to CD4 on the surface of the target cell (20). Fusing CD4 to the combining site of a CD4i Ab solves the accessibility problem since the Ab epitope would be exposed by CD4 binding when the virion is not bound to the target cell. This class of reagent has two other favorable features: bivalent binding and targeting of functionally critical epitopes on gp120, the CD4 and coreceptor binding sites. One such reagent, sCD4-17b (referred to here as CD4-scFv_17b), contains the first two domains of CD4 linked to the single-chain fragment variable (scFv) form of the CD4i Ab 17b (Fig. ​(Fig.1)1) (11). This reagent was shown to potently neutralize multiple primary strains of HIV-1 (11), suggesting that CD4-CD4i Ab fusion proteins are promising candidates for passive immunization or gene therapy trials.Open in a separate windowFIG. 1.Schematic depiction of CD4-CD4i reagents and related molecules. V_H, variable domain of the IgG heavy chain (HC); V_L, variable domain of the IgG light chain (LC), C_H1, constant region 1 of the HC; C_L, constant region of the LC; Fc, C_H2 and C_H3 domains of dimerized HCs; CD4 D1-D2, N-terminal two domains of CD4; scFv, single-chain fragment variable (V_H and V_L domains of an IgG); CD4_HC, CD4 linked to the V_H domain of an IgG; CD4_LC, CD4 linked to the V_L domain of an IgG; CD4_HC,LC, CD4 linked to the V_H and V_L domains of an IgG.Critical properties for CD4-containing reagents include their breadth of neutralization activity, half-life, and, for reagents used in a gene therapy context, their expression level. We have undertaken a systematic effort to develop the optimal architecture for a CD4-CD4i Ab reagent by designing, constructing, and testing reagents with different CD4i Ab combining sites and including an Ab Fc region to increase valency and serum half-life (7). We varied the arrangements of the Ab combining sites; the lengths, attachments, and forms of the linking regions; and the ways in which CD4 was fused to the CD4i Ab (Fig. ​(Fig.1).1). CD4-CD4i Ab reagents were evaluated using in vitro neutralization assays across a broad range of clade A, B, and C HIV-1 strains. One promising reagent, a fusion of CD4 domains 1 and 2 (D1-D2) to the heavy chain of the E51 CD4i Ab, was expressed at high levels in mammalian cells and exhibited neutralization potencies that compared favorably with or exceeded those of known broadly neutralizing Abs such as 4E10, b12, 2G12, and 2F5.Since much of the activity of our CD4-CD4i reagents resulted from the CD4 component, we also explored the effects of attaching CD4 to an Ab with a different quaternary structure. The anticarbohydrate Ab 2G12 is unusual in that its heavy chains are involved in a domain swap creating a rigid (Fab)₂ unit in which the combining sites are separated by ∼35Å (6). This domain swapping tendency also leads to the formation of 2G12 dimers containing two (Fab)₂ units and two Fc regions, which form when the domain swapping occurs intermolecularly between two IgGs rather than intramolecularly between the two Fab arms of a single IgG (37). The 2G12 dimer is 50- to 80-fold more potent than monomeric 2G12 in neutralizing clade B 2G12-sensitive strains; however, neither form of 2G12 neutralizes clade C strains of HIV-1 (37). In order to assess the effects of adding CD4 to the 2G12 monomer and dimer architectures and to explore whether addition of CD4 would broaden the range of HIV-1 strains that are sensitive to 2G12, we constructed CD4-2G12 fusion proteins and tested their neutralization potencies (Fig. ​(Fig.1).1). We found that these hybrid reagents had potent neutralizing activities with both the CD4 and Ab combining site components apparently contributing to this behavior.