Among the large number of antibodies generated during natural infection by HIV-1, most are highly strain specific or non-neutralizing. The non-neutralizing antibodies are often directed against the gp120 or gp41 regions that are occluded on the assembled trimeric spike and exposed only upon disassembly of the spike. The accessible surface of gp120 in the trimer is largely composed of variable, heavily glycosylated core and loop structures that surround the receptor-binding regions. Despite this barrier, broadly reactive, potently neutralizing sera have been found in select individuals living with HIV, demonstrating that humans are capable of making broadly neutralizing antibodies [17
]. Although a number of monoclonal antibodies against HIV-1 have now been identified, only a few display the combination of potent neutralization and breadth of reactivity that is desired for an efficacious vaccine response [20
AIDS vaccine research investigators have developed strategies for identifying broadly reactive sera, isolating individual B cells from these sera, and cloning potent and broadly neutralizing antibodies from the B cells. High throughput assays to measure HIV-1 neutralization in large panels of sera revealed that approximately 25% of individuals living with HIV make relatively broadly reactive neutralizing antibodies during the course of HIV-1 infection [18
]. Molecular probes used to isolate these antibodies were made by altering amino acids of the gp120 inner domain and exposing neutralization. These probes were designed to interact with antibodies specific for the highly conserved CD4 binding site of gp120 and are termed resurfaced stabilized cores, as the amino acid changes stabilize the desired gp120 structure and prevent the conformational flexibility that is normally present on the virus surface. Using peripheral blood B cells from a donor with potent serum neutralizing antibodies, three monoclonal antibodies were found to bind specifically to the CD4 binding site. One of these antibodies, designated VRC01, when cloned and expressed, neutralizes more than 90% of naturally circulating strains [23
]. The broad neutralization capability of VRC01 can be seen (a). Before VRC01 was identified, b12 was considered to be the best of the CD4 binding site neutralizing antibodies, but it was only able to neutralize 40% of clade B viruses [24
Figure 1 Broadly neutralizing antibodies against HIV-1. The VRC01 antibody is capable of near pan-neutralization of HIV-1, which it achieves by recognition of the initial contact site of the CD4 receptor on HIV-1 gp120. (a) Neutralization dendrogram of VRC01. (more ...)
Purified VRC01, produced in the laboratory and passively administered to nonhuman primates, has shown protection against SHIV in rectal and vaginal challenge models [25
]. The crystal structure of VRC01 in complex with an HIV-1 gp120 core shows that VRC01 binds directly over the site of initial attachment of CD4 to the virus, and this specificity may account for the potency and broad neutralization attributes of VRC01 (b) [26
If VRC01-like antibodies are to be elicited by a vaccine, it is important to understand how they are generated in infected individuals. Although there are large quantities of gp120 antigen in persons living with HIV-1, the human immune system takes several years to generate VRC01-like antibodies. While the delay in the evolution of broadly reactive antibodies is not yet well understood, we know that antibodies arise through a process that involves three steps: recombination of germline antibody genes to form unique B-cell receptors on naïve B cells, deletion of autoreactive B-cell clones, and antigen-driven affinity maturation. The third step of this process, somatic mutations leading to affinity maturation, is illustrated () [17
]. By using deep sequencing technology, it has been possible to study the precursor and maturation process of HIV-1 specific antibodies in an individual living with HIV. The VRC01-like antibodies identified to date have arisen most commonly from specific heavy chain genes, generally IGHV1-2*02. While this is a common precursor gene allele, extensive somatic maturation involving 70 to 90 changes in IGHV1-2*02 gives rise to mature broadly neutralizing VRC01-like antibodies. This maturation process focuses the developing antibodies onto a conserved site of HIV-1, a focusing that may be a common process in the generation of broadly neutralizing antibodies [27
]. More recently, additional CD4 binding site antibodies have been defined through deep sequencing and structure-based probes, both from the IGHV1-2*02 germline as well as closely related germlines [27
]. These additional antibodies will help to define more completely the alternative mechanisms used to generate this response and provide additional probes to define how alternative antibodies recognize the highly conserved CD4 binding site.
Figure 2 Maturation of broadly neutralizing HIV-1 immunity. VRC01-like antibodies mature from the human VH1-2*02 germline (grey ribbon at left) and acquire over 50 somatic mutations while evolving to effectively recognize HIV-1 gp120 (red ribbon at right). The (more ...)
How can all this information be used to develop a vaccine? The lengthy time required to generate VRC01-like antibodies suggests that the human immune system can generate such antibodies, but the process is not efficient. Whether this feature is due to the recessed nature of the immunogen, underlying immunodeficiency, or other factors is unclear. Analysis of germline and intermediate antibodies suggests at least one potential block exists at an early stage of B-cell receptor recognition. One approach then is to design modified gp120s that have high affinity for B cells expressing early, or germ line precursor, forms of VRC01. Such modified gp120s may help to stimulate the induction and appropriate maturation of VRC01-like antibodies, to guide the vaccine-induced elicitation of VRC01-like antibodies. The focus of current efforts to engage germline immunoglobulins is to remove structures that introduce potential clashes with the germline antibody. Based on knowledge of the mature VRC01-Env structure, attention has been focused on Loop D and the V5 region. These domains are responsible for VRC01 resistance in insensitive strains [25
], and the initial recognition of these sites by germline precursors may affect the ability to drive proteins down this pathway. To effect this change, modified forms of the resurfaced cores, the core gp120, trimers, and outer domain proteins are being evaluated with and without different adjuvants to engage the relevant B-cell receptors.