The functional HIV-1 viral spike is a trimer that consists of three gp120s, which associate non-covalently with the ectodomains of three gp41s. Despite extensive efforts by several groups worldwide, the trimeric spike has thus far resisted atomic-level determination. However, low resolution cryoelectron microscopy studies have provided important vaccine-relevant information about gp120–gp41 arrangements [14
], including structures of the viral spike prior to receptor encounter, intermediate states of the virus during entry and post-fusion states. This type of information can be used to assess antibody functionality on a molecular level that may be predictive of biological activity in more complex in vitro
and in vivo
assays of antibody activity. Additionally, we and others have obtained atomic-level structural information on individual gp120 and gp41 components. For gp41, only post-fusion structures have been determined. For gp120, the crystal structures of a number of states for a conserved core have been determined, including antibody-bound conformations, although the best characterization comes from the CD4-bound state.
Thus, structural biology has provided important information about the three-dimensional organization and chemical structure of the HIV-1 glycoproteins. This information and in particular an understanding of atomic-level structure can be used rationally to design proteins that have specific biological properties and functions that would be important in vaccine design, such as incorporation of conserved sequences, which are generally associated with functions that are essential to the virus, and properties of virus proteins, such as the ability to stimulate specific protective immune responses.
This concept is being applied to HIV-1 to provide a structural definition of the functional viral spike (a), which is used by the virus to enter host cells and is the target of all known virus-directed neutralizing antibodies. The ability to conduct atomic-level analysis of the spike facilitates immunogen designs that stabilize and help to present potential sites of neutralization more optimally to the immune system. Unfortunately, the same virus strategies noted above that allow the virus spike to evade an effective immune response also hinder structural analysis, and the entire HIV-1 spike has resisted and continues to resist atomic-level characterization.
Nevertheless, structural analysis and molecular modelling have facilitated the understanding of the antibody response against HIV. This knowledge includes (i) the capability to dissect the types of antibodies in sera and to ascertain what regions of the HIV Env are targeted [20
], (ii) the ability to isolate neutralizing antibodies from individual B-cells [21
], and (iii) the capacity to determine the atomic-level structure of neutralizing antibodies bound to the HIV Env [23
]. We have used this knowledge of the structure of the HIV Env to design protein probes that expose various regions of the HIV Env in specific conformations (). The concept underlying these probes is that one can use molecular biological tools and structural and functional knowledge to design and generate a molecule that preserves the antigenic structure of the viral surface that one wishes to study, but does not contain irrelevant antigenic regions of HIV-1. These kinds of probes can then be used to evaluate the regions of the HIV Env that are targeted by serum neutralizing antibodies. A key virus functional region, the CD4-binding site of gp120, has been studied in this way. Since CD4 is the primary cellular receptor for HIV, antibodies that bind to the CD4-binding site can block HIV infection of CD4+
T cells and thus function as neutralizing antibodies. To define the neutralizing antibodies to the CD4-binding site further, a specific protein probe was designed such that it exposed the CD4-binding site of gp120, while other regions of HIV were altered to be unrecognizable to HIV antibodies, e.g. by substitution with simian immunodeficiency virus (SIV) homologues or other non-HIV residues (). This epitope-specific probe, along with a knockout mutant was used to identify B-cells making antibodies to the CD4-binding site [25
]. Following the isolation of these B-cells by flow cytometry, one can then use PCR to amplify the genes encoding the antibody heavy and light chain variable regions (VH and VL) and subsequently express the full IgG monoclonal antibody in tissue culture (). Having these new informatics-designed monoclonal antibodies in hand, it is possible to verify and to study in detail their ability to neutralize HIV-l. Using this methodological approach, we recently isolated three CD4-binding site neutralizing monoclonal antibodies designated VRC01, VRC02 and VRC03 [26
]. Of all antibodies isolated with this method, the percentage of monoclonal antibodies that neutralize HIV-1 ranges from 25 to 75 per cent, while previous attempts to isolate such antibodies with gp140 probes were not successful [21
]. The crystal structure of VRC01 bound to HIV gp120 has provided an atomic-level footprint showing the precise region of HIV gp120 that is vulnerable to neutralizing antibodies [25
]. This structural information can be further used to make new vaccine immunogens that are designed to induce the immune system to generate antibodies similar to VRC01. It is important to recognize that the same probe used to isolate monoclonal antibodies will not necessarily serve as a vaccine to elicit them. For example, the RSC3 probe does not elicit CD4-binding site neutralizing antibodies and only serves as a starting point to design such immunogens. This observation underscores the important difference between antigenicity and immunogenicity.
Figure 2. Resurfaced stabilized core proteins derived from HIV-1 Env can serve as probes to define antibodies to the CD4-binding site and as prototype immunogens. By modifying surface exposed residues of the HIV-1 Env (left) and replacing them with alternative (more ...)
Figure 3. Strategy for isolation of new monoclonal antibodies based on HIV. Protein structure: rescue of antigen-specific B-cells. Selective probes (left) are labelled and used to identify B-cells that express broadly neutralizing antibodies by flow cytometry (middle). (more ...)
Another approach to the identification of relevant immunogens seeks to bypass the difficulties of using the entire viral spike and rather focuses on the functionally critical sites that the virus uses for entry. By definition, these sites cannot be altered without hindering spike function. We and others have used this approach to elicit antibodies against the highly conserved site of co-receptor binding [27
Unfortunately, while it is possible to prepare immunogens that will elicit antibodies to this site, because the site is only revealed after the engagement of the viral and target cell membranes, it is effectively inaccessible to protective antibodies that might be elicited by a vaccine [28
]. Thus, in choosing potential vaccine immunogens, one must verify that in addition to functional importance and sequence conservation, the site is vulnerable to the neutralizing antibody.
A third approach to identifying vaccine-relevant immunogens focuses on effective antibody responses [5
]. The underlying concept is that analysis of monoclonal antibodies selected for their ability to neutralize HIV-1 effectively, will facilitate an understanding of protective immune responses. After identifying the epitope recognized by these monoclonal antibodies, one can work backwards to create molecular mimics of the epitope with the goal of using these mimics to elicit the original template antibody. Unfortunately, many of the identified monoclonal antibodies that effectively neutralize HIV-1 appear to have unusual properties that make it difficult to elicit them. This situation suggests that one needs information about the frequency and elicitation pathway of the template antibody to succeed with this approach.