The generation of broadly neutralizing antibodies against HIV-1 envelope glycoprotein is one of the key goals in HIV-1 vaccine development. HIV-1 Env presents a recalcitrant target in part due to the extensive glycosylation that hides antibody epitopes, and also because conserved domains which can serve as potential targets for such antibodies are physically sequestered [49
]. An additional factor that may contribute to low Env spike immunogenicity is the scarcity of envelope spikes on the virion surface, and their irregular, clustered distribution [1
Here, we tested the ability of lambda phage particles to act as a structural scaffold to display HIV-1 envelope spikes in a highly immunogenic context. To produce phage particles that displayed HIV-1 Env, we used a simple in vitro complementation system to decorate gpD-deficient phage particles with glycosylated envelope trimers that were translationally fused to gpD. This was achieved by producing recombinant Envgp140:gpD fusion protein (as well as gpD alone) in mammalian cells, and then comparing the biochemical properties of the resulting fusion protein to wild-type (WT) Envgp140 oligomers. Both native gel electrophoresis and gel filtration analysis demonstrated the purity and oligomeric integrity of our Envgp140:gpD fusion protein, and showed that it was essentially indistinguishable from WT gp140 protein.
With this material in hand, we then generated Env decorated phage particles by mixing gpD-deficient phage particles with purified gpD and Envgp140:gpD fusion proteins at various molar ratios, followed by CsCl-gradient purification. The effectiveness of the decoration reaction was assessed by measuring the titers of the resulting phage preparations before and after exposure to high concentrations of EDTA, which inactivates gpD-deficient capsids but has minimal effect on gpD-bearing particles [29
]. We were able to generate stable mosaic Env decorated phage particles using various molar ratios of soluble wild type gpD and gp140:gpD fusion protein (20:1, 5:1, and 1:1). Phage that were decorated with Env protein alone were not stable in the presence of EDTA. In contrast, phage decorated with a 1:1 ratio of gp140:gpD to wild type gpD, were stable in the presence of EDTA. Unexpectedly, however, when we measured the amount of gp140:gpD actually incorporated onto these mosaic phage capsids, we determined that only a fraction of the available binding sites for gpD on the phage capsid were in fact occupied.
The high density Env decorated phage capsids incorporated about 30 copies of Env trimers per particle, which occupied 90 (30 trimers × 3 molecules of gpD per trimer) of the 405 available gpD binding sites on the phage surface – or roughly 25% of the total. Since gp140:gpD was more abundant than wild type gpD on these mosaic particles (), we conclude that the Env-displaying phage capsids were EDTA stable even though most of the available binding sites for gpD were unoccupied.
Native HIV-1 virions display only 14 ± 7 spikes per virion (138) and have a diameter of approximately 145 nm (15). In contrast, the lambda phage head has a diameter of 50 nm and displays ~30 spikes per capsid in the case of phage particles prepared using a 1:1 ratio of wild-type gpD to gp140:gpD fusion protein. Thus, the expected density of Env spikes on the much smaller surface of the phage head will be roughly 18 to 20-fold greater than that on native HIV-1 particles.
We anticipated that the more dense, array of displayed envelope trimers on the surface of the phage capsid would result in enhanced humoral immune responses against the Env antigen. We therefore assessed the immunogenicity of our lambda phage particles displaying HIV-1 Env, using immunization experiments in rabbits that employed a very similar design and dose regimen to that described by Wyatt and colleagues in their studies of recombinant, oligomeric HIV-1 gp140 [35
]. In this experiment, the Env decorated phage particles elicited Env-binding antibody titers and virus neutralizing responses that were no higher than those in those animals that received adjuvanted, conventional oligomeric gp140 protein.
This unexpected result may be related to the fact that sequential immunizations with lambda phage particles displaying an exogenous antigen have been shown to result in progressively diminishing humoral immune response to the displayed antigen – with no immunologic boosting of the response [53
]. This has been attributed to a strong and immunodominant response to the phage-capsid, which is efficiently boosted upon sequential immunization, at the expense of the response to the exogenous antigen [53
]. Our experiments showed a similar boosting of the humoral response to the phage capsid (gpD coat protein) in those animals that received Env-displaying phage particles, suggesting that this may have contributed to the poor Env-specific response that was elicited by the Env-displaying particles. These findings are consistent with the phenomenon of carrier induced epitopic suppression (CIES) [54
]. For uncertain reasons, this phenomenon of CIES was less pronounced when Env-displaying phage particles were used to boost a heterologous gp140 protein prime. Neutralization titers elicited by the protein prime/phage boost group were comparable to those elicited by homologous prime-boost immunization with a high dose of adjuvanted, soluble gp140 - supporting the idea of phage being a poor prime but possibly effective boost.
Collectively, these results show that display of HIV-1 envelope spikes on the VLP-like scaffold provided by phage lambda capsids does not result in an improved humoral immune response as compared to adjuvanted, soluble oligomeric protein.