STRUCTURE–FUNCTION RELATIONSHIPS OF HIV-1 ENVELOPE SEQUENCE-VARIABLE REGIONS PROVIDE A PARADIGM FOR VACCINE DESIGN

Receptor-binding sites

It is now well-established that to trigger exposure of the HIV-1 gp41 fusion domain that initiates virus–cell fusion, gp120 must first bind to two cell-surface receptors: CD4 and CXC-chemokine receptor 4 (CXCR4) or CC-chemokine receptor 5 (CCR5)^14,15. The receptor-binding surfaces on gp120 form distinct structural and antigenic regions that are highly conformational and are formed by discontinuous amino acids in several regions of gp120^16,17(Box 1). The CD4 binding site (CD4bs) consists of many residues in the constant regions of gp120, but, until recently, only one of many CD4bs-specific monoclonal antibodies had been described with broad neutralizing activity¹⁸. Three more have recently been selected and characterized^19,20(Table 1). This suggests that the critical region within the large CD4bs may be difficult to target with vaccine constructs, a fact that is supported by disappointing attempts to induce neutralizing CD4bs-specific antibodies with various vaccine constructs ^6,7,9.

The chemokine receptor-binding region of gp120 consists of the invariant β2, β3, β20 and β21 strands (the bridging sheet), the V3 loop, and the stem of the V1/V2 double loop^16,17. This highly conformational, discontinuous region of gp120 is often known as the CD4-induced (CD4i) region, because, as its name implies, it is formed after gp120 has bound CD4 with high affinity. Many human monoclonal antibodies specific for CD4i have been described^21–24, indicating the strong immunogenicity of this region, but CD4i, when exposed on the virus particle, is sterically protected from most CD4i-specific antibodies during the process of infection owing presumably to its close proximity to the cell membrane²⁵. Consequently, CD4i-specific antibodies may not protect against infection by most HIV-1 strains.

The V3 loop

The V3 loop of gp120 is a component of the chemokine receptor-binding region, and is a major determinant of co-receptor usage^26–31. The length of the V3 loop is essentially constant at 34–35 amino acids. Although its amino acid sequence is highly variable, except in clade C viruses where its sequence is quite conserved³², sequence variation is restricted to only ~20% of the amino acid positions, and is located primarily in the two β-strands in the crown of the V3 loop (Figure 1B and 2D). The functional importance of V3 is illustrated by the fact that deletion of V3 completely abrogates virus infectivity³³. Nonetheless, the value of V3-specific antibodies in blocking virus infection has been controversial because some V3 antibodies, especially those elicited early after infection, have limited breadth³⁴, many neutralize only the most neutralization-sensitive “Tier 1” viruses^19,35, no single V3-specific monoclonal antibody neutralizes more than ~10–25% of the more resistant “Tier 2” viruses^19,36,37, and the accessibility of V3 is poor on many viruses^38,39. However, given its essential function in HIV infectivity, its structural conservation^40,41, its strong immunogenicity⁴², the existence of many V3-specific monoclonal antibodies with cross-clade neutralizing activity ^19,36,37,43, and the demonstration that polyclonal V3-specific antibodies can be induced that neutralize viruses from diverse HIV-1 subtypes⁴⁴, the V3 region remains a potentially important target for vaccine development.

The V2 loop

By contrast, V2 is not essential for infectivity³³, which suggests that the evolutionary constraints to preserve an invariant V2 structure are less pronounced than for V3. However, V2 participates in several Env functions, providing the motif that binds to α4β7 integrin (the receptor apparently involved in the homing of the virus to gut mucosal cells)⁴⁵, contributing to trimer formation ⁴⁶, and participating as part of the chemokine receptor surface. V2 also plays a crucial role in masking neutralizing epitopes of gp120: changes in V2 length and glycosylation patterns allow viruses to escape from neutralization mediated by antibodies specific for V3 and the CD4bs of gp120^47–50. Given these functions of the V2 loop, its newly recognized role as a component of QNEs^51–54 and its partial structural conservation (see below), V2 becomes an additional target for vaccine development.

V1, V4 and V5

The functions of the other variable regions of gp120 are poorly defined. V1 may have functional significance as it can serve as a neutralizing epitope, and several potent neutralizing V1-specific monoclonal antibodies have been developed in transgenic mice⁵⁵. The immunogenicity of V1 is affected by glycosylation⁵⁶ and mutations in V1 can affect virus-induced syncytium formation⁵⁷, but little more than this is known about its function. V4, by contrast, has no well-defined function, although it is a target for early autologous neutralizing antibodies⁵⁸, and V4-specific neutralizing antibodies have been described in immunized rabbits⁵⁹. V4 is involved in neutralization escape⁶⁰, and undergoes extreme variation in early infection⁶¹. The V5 region, the only variable region that does not form a disulphide-linked loop, participates in forming the CD4bs surface, but no polyclonal or monoclonal neutralizing V5-specific antibodies have been described; nonetheless V5 is involved in neutralization escape, although V5 peptides were not able to block neutralizing antibody activity in sera⁶². Current data, therefore, do not support these variable regions as targets for HIV-1 antibody-based vaccines.

Trimeric Env spikes

Crystal structures of gp120 monomers have been available for over a decade ¹⁷, but the crystal structure of the trimeric Env spike is not available, though it has been modeled using monomer structures and cryo-electron tomography^63–65. There are profound consequences to the oligomerization of the HIV-1 Env: trimer formation is a requirement for infectivity, results in the burial of neutralizing epitopes within oligomeric interfaces, and permits conformational epitope masking^66–68. The QNEs that form upon trimerization of gp120 have been defined by various human and macaque monoclonal antibodies and are exceptionally potent^51–54, a fact that reflects a critical function of the QNEs that have not yet been defined.

The V3 loop

Although the V3 loop is the least variable of the HIV-1 Env variable regions because, in part, it is the only one that is essentially constant in length, its sequence varies considerably across strains, and this variability is most pronounced in its “crown”, ~14 amino acids in the middle of the V3 loop (Figure 1B). While the specificity of some V3 antibodies restricts their immunochemical and biologic function^69,70, some V3-specific monoclonal and polyclonal antibodies from infected individuals are able to neutralize diverse HIV-1 strains^{19,36,43,71–74}. Furthermore, some individual V3-specific monoclonal antibodies can neutralize ~25–50% of Tier 1 and Tier 2 HIV-1 strains in which the correct epitope is present^{19,37,43,72,75}, polyclonal V3-specific responses to infection can neutralize Tier 1 and Tier 2 viruses from diverse clades ⁷⁴, and vaccine-induced immune responses targeted to V3 can induce broader neutralizing activity than that seen in many human HIV⁺ sera or in mixtures of human monoclonal antibodies⁴⁴.

Interestingly, crystallographic structures of the gp120 V3 loop crown from different virus strains in complex with various human monoclonal antibodies^{41,59,76–79}, and structures of the V3 loop freely arranged in situ on the core of gp120⁸⁰, all exhibit a V3 crown that forms variations of a common β-hairpin tertiary structure. Furthermore, the sequence variation of the V3 loop tends to cluster into a single continuous small zone when viewed in 3D space (Figure 1C and ⁴⁰). We hypothesize that the crown of the V3 loop might be organized into a folded (albeit highly flexible) globular domain, with the majority of its surface being structurally, and therefore antigenically, invariant. This structural perspective explains both the cross-reactivity of many V3-specific antibodies, which bind the conserved surfaces of V3, and the narrow specificity of some V3-specific monoclonal antibodies, which bind the small sequence-variable zone (Figure 1C and ^40,41). The discovery of conserved 3D structures in the sequence-variable V3 loop is consistent with the function of the V3 loop as a chemokine receptor-binding element: the receptor-binding surface of gp120 must be conserved to preserve virus infectivity, and these constraints apparently involve more of the V3 loop amino acid positions than they leave free to vary. This perspective of the V3 loop indicates which regions of V3 should be targeted with immunogens to elicit broadly neutralizing antibodies; it also reveals the region of V3 that induces only a strain-specific antibody response.

The V1/V2 double loop

V2 is variable in both sequence and length (Figures 2 and 3), and as a result is a profoundly more variable region than V3. V2 is immunogenic in ~20–45% of HIV-infected humans¹³ in contrast to V3, which induces antibodies in essentially all infected individuals⁴². Several human V2-specific monoclonal antibodies have been generated that cross-react with gp120 molecules from diverse HIV-1 isolates, and V2-specific monoclonal antibodies have also been produced from cells of infected chimpanzees and immunized rats^81–83; this indicates that V2 might also contain some structurally conserved elements. Interestingly, many V2-specific monoclonal antibodies interact with the same amino acid residues or with overlapping regions of V2 (Figure 2C and ^{13,52,81,83,84}). Published studies document the neutralizing activity of human and chimp V2-specific monoclonal antibodies. Although many of these antibodies have little or weak neutralizing activity, some are quite potent^83,85,86.

Little is known about the 3D structure of the V2 loop, but the biologic advantage conferred by the integrin α4β7 binding motif in V2 ⁴⁵ may constrain at least part of the V2 structure. This receptor-binding motif occurs in a region of V2 containing alternating and periodic conserved charged and hydrophobic amino acid residues typical of a folded domain. In addition, V2 contains a well-conserved disulphide bond common in small, loosely folded domains with high sequence variation in their 3D structural folds (such as the thioredoxin fold found in many proteins with known 3D structure)⁸⁷. It is also notable that the C-terminal portion of the stem of the V2 loop is located directly adjacent to the CD4bs in crystal structures of gp120 liganded to CD4^17,80, and that some data suggest that the presence or absence of V2 affects recognition and neutralization by the anti-CD4bs antibody IgG1b12^66,88. Further structural information is derived from a comparison of the length distributions of V1 and V2 (Figures 3A and 3B). The normal distribution exhibited by V1 is typical of random sampling from multiple optional structures, and therefore suggests that V1 is likely to be a dynamic, unfolded and disordered flexible loop that flickers between many conformations. The only other structural clues available for V1 come from the presence in the mid-region sequence observed in the most common length of repetitive glycosylation motifs (NXT/S shown as V1 residues 6–14, see Figure 2A).

The differing length distributions in V1 and V2 reflect biological functions that suggest 3D structural order within V2, but, as noted, structural disorder within V1. Specifically, the V2 length distribution exhibits a sharp drop-off in length from the most frequent length to shorter lengths, but a more gradual drop-off as the length increases, suggesting a viral mechanism that affords very little tolerance of V2 lengths shorter than ~40 amino acids. This pattern of length variation is typical of either a folded globular domain, which requires a critical length below which the fold cannot assemble, or inter-monomer contacts, which require a critical length below which the loop cannot reach over from one monomer to another. In both cases, the observed length distribution requires some V2 structural order. The available structural data, together with the observed cross-reactive immunological patterns of V2 antibodies, suggest that a similar, but more challenging, approach to vaccine targeting, as envisioned for V3, might also be successful for V2, but that V1 is probably not a suitable vaccine target.

V2/V3 complexes in the envelope trimer

There is now evidence that V2 and V3 can, together, form complex epitopes comprised of contributions from both variable loops. These complex epitopes exist preferentially or exclusively in the trimeric form of the HIV Env spike, defining them as quaternary epitopes (Box 1). Antibodies specific for quaternary epitopes were first described in SHIV_89.6P-infected macaques; although they were difficult to characterize in polyclonal sera, their activity seemed to map to a discontinuous epitope formed by V2 and V3 present on the virus’ Env spikes⁸⁹. Similar antibodies were found in an HIV-infected chimpanzee^90,91. Remarkably, these antibodies were extremely potent as neutralizing reagents. The first human QNE-specific monoclonal antibody to be described, 2909, is extremely potent (IC₅₀ < 1.0 ng/ml) and targets both the V2 and V3 regions⁵¹. The epitope to which it binds is found only on the surface of virions and on env-transfected cells, and does not exist on the gp120 monomer or recombinant soluble Env trimers^52,68, which indicates that this epitope is formed only when gp120 monomers form trimers in lipid membranes. Monoclonal antibody 2909 is highly strain-specific although it tolerates extensive amino acid sequence changes in V3⁵². The strain-specificity is the result of a lysine, rather than the more common asparagine, at residue 160 in V2 (Figure 2C and position 3 in Figure 2B, and ^52,54). So, although it is narrow in its reactivity, this monoclonal antibody defined the first quaternary epitope in the HIV-1 Env and revealed both tolerance for extensive variation in V3 and specificity constraints imposed by a single position in V2.

Recently, several QNE-specific monoclonal antibodies were generated from macaques infected with SHIV_SF162P4 ⁵⁴. Similar to monoclonal antibody 2909, these antibodies have potent but strain-specific neutralizing activity, target V2 and V3, and are limited in breadth of reactivity by residues in V2. So, macaques and humans both make similar potent QNE-specific antibodies.

Notably, two clonally related human monoclonal antibodies (PG9 and PG16) have now been described that also target V2 and V3 and possess potent neutralizing activity (IC₅₀ < 1.0 ng/ml for many strains)⁵³. Both these monoclonal antibodies can be considered QNE-specific antibodies although PG9 reacts weakly with monomeric gp120 as well as with trimeric gp140 and seems to target mainly discontinuous residues in V2. Monoclonal antibody PG16, a true QNE antibody, recognizes only the trimeric form of gp120 and interacts with discontinuous residues found in V2 and the crown of V3. Unlike human monoclonal antibody 2909 and the macaque QNE-specific monoclonal antibodies, PG9 and PG16 have broad activity, neutralizing 73% and 79% of 162 diverse pseudoviruses, respectively. The crucial difference that distinguishes PG9 and PG16 from monoclonal antibody 2909, and accounts for their neutralization breadth, is their dependence on the asparagine-linked carbohydrate moiety present in most HIV-1 isolates at position 160 in V2 (Figure 2C and position 3 in Figure 2B). The neutralization breadth of PG9 and PG16 suggests that the epitopes they target are conserved and not masked in most strains of HIV-1.

The immunologic reactivity and neutralizing activity of the human QNE-specific monoclonal antibodies show that they can readily tolerate extensive amino acid changes in V3^52,53, and that broad or narrow activity is dependent on V2 variation. Furthermore, the broad neutralizing activity of PG9 and PG16 demonstrates that the quaternary epitopes recognized are composed of conserved elements in the V2 and V3 sequence-variable regions and are the among most accessible of all neutralizing epitopes present on the CD4-unliganded form of the trimeric Env spike. The modelled Env trimers^63–65, provide clues for the visualizing the 3D structures of V2/V3 contacts, which probably occur within the many possible unliganded forms of the trimer. The resolved 3D structure(s) of trimers in QNE bound conformations should ultimately be highly informative for vaccine design.

In summary, recent data reveal conserved targets for broadly reactive antibodies in the sequence-variable V2 and V3 loops of gp120 and on quaternary structures formed by these loops in the trimeric Env. These data explain the broad immunological and functional cross-reactivity of some of the monoclonal antibodies specific for these regions and provide a framework for conceptualizing their value in vaccine design.