Introduction
Major challenges in the development of an HIV vaccine have been the design of immunogens able to induce a strong and sustained immunity with a broad and cross-clade neutralizing activity.
In the course of natural infection, although HIV-1 is highly effective in evading the immune surveillance [
1‐
3], almost 20% of HIV-infected subjects are able to develop antibodies with a broad degree of neutralization activity, whose role in the disease control, however, is still debated (reviewed in [
4]. Such evidences, indeed, suggest that native antigens are able to elicit such bnAbs antibodies. To date, a number of human bnAbs targeting the HIV envelope glycoprotein in its trimeric status have been isolated from HIV-1 infected subjects [
5‐
14].
Trimeric envelope structures, either soluble or protruding from a membrane-surrounded particle, have been explored as vaccine models for eliciting broadly neutralizing antibodies (bnAbs) [
15‐
19].
Indeed, the native and functional HIV-1 envelope glycoprotein (Env) complex is present on the virus surface as a trimer, each of the monomers made of non-covalently loosely associated gp120 surface and gp41 transmembrane glycoproteins [
20‐
23]. However, recombinant soluble forms of fully cleaved and functional trimers, have been difficult to obtain for their high instability. On the other hand, the use of gp160 ectodomain (gp140) has led to the production of trimers that can mimic the native Env spike and have shown to be able to elicit neutralizing antibody responses in immunized animals [
24‐
27]. These gp140 trimers can be further stabilized by introduction of specific modification in order to strenghten intra- as well as inter-molecular bindings (gp140
SOSIP) [
28,
29].
We have recently used a similar strategy to present trimeric gp140 Env molecules on HIV Virus-Like Particles produced in both a transient baculovirus expression system [
19] and a stably transfected insect cell line [
18]. In particular, a gp120 Env molecule derived from a Ugandan HIV-1 isolate of the clade A (94UG018; GenBank accession number AF062521) [
30,
31] has previously been shown to induce high Ab titers with cross-clade neutralizing activity in immunized BALB/C mice [
32‐
34] and non-human primates [
35].
In the present study, the same gp14094UG018 presented on the surface of VLPs has been produced as recombinant soluble trimeric form of Env for evaluation in homologous prime-boost immunization schedules. An immunogenicity study has been performed in rabbits to evaluate the potency and broadness of specific humoral immune response as well as the mapping of the epitope recognition by the Abs elicited by such protein.
Discussion
In this study we evaluated the immunogenicity of a soluble trimeric gp14094UG018 derived from a clade A HIV-1 isolate administrated subcutaneously to rabbits. All four immunized animals developed high titers of specific anti-gp140 antibodies against the homologous gp14094UG018 protein, as well as against heterologous envelope glycoproteins of clade B (gp120IIIB and gp120W61D), clade A (gp140UG037) and clade C (gp140ZM96) which present a significant sequence divergence compared to the gp14094UG018. The highest binding activity was observed against the two gp140 UG037 and ZM96 molecules, effect that could be due to the highest sequence homology to the gp140 94UG018 molecule (UG037) and/or the presence of the ectodomain of gp41 which is substantially conserved across the clades (UG037 and ZM96). Overall, these results indicate that although rabbits were immunized with a clade A derived gp140, this immunization was able to elicit a binding activity to cross-clade Env antigens. This result was supported by the relevant sequence homology between the different Env molecules, especially in the constant regions.
All immunized animals’ sera were able to bind the C1, C2 and C5 regions with high affinity and the V2, V3 and V5 regions with lower affinity, presenting a direct correlation with the percentage of divergence between the gp14094UG018 immunogen and the peptides used as target in the binding assay. However, the differential binding efficacy to the same peptide by different sera is highly suggestive of distinct patterns of immune response elicited by the same gp14094UG018 in outbred animals, indicating that the specificity of individual responses to the same vaccine antigen is not totally predictable. A more in-depth epitope mapping analysis performed with individual epitopes covering the entire length of gp120 from the C1 to the C5 region confirmed the potency ranking observed with the peptide pools but showed that, for each region, specific peptides are recognized more efficiently than others. This further supports the concept that the immunogen is able to elicit distinct patterns of Abs focused on different epitopes. Further analysis have been planned to be conducted using peptides based on the sequence of 94UG018 protein, to verify whether additional Ab specificities are identified in immune sera elicited by the gp14094UG018.
Sera from animals immunized with gp14094UG018 showed a >50% neutralization efficacy against 3 out of 5 Tier 1 pseudoviruses, whereas poor or no neutralization was observed against Tier 2 pseudoviruses.
This soluble trimeric clade A gp140
94UG018 was able to induce a cross-clade neutralizing activity as demonstrated by the ability of the immune sera to neutralize Tier 1 pseudoviruses of different clades. Although the neutralization activity was limited to Tier 1 pseudoviruses, this result is in agreement with other immunogenicity studies performed with soluble HIV Envelope proteins of different clades with our data even suggesting improved neutralization in several instances [
39‐
42]. Moreover, it is prospected to re-evaluate the breadth of neutralization activity in A3R5 cells, which have been recently shown to be more sensitive to neutralization than the TZM-bl used in the present study [
43]. In particular, serum from the 49391 rabbit was the only one to show neutralization activity against four out of five Tier 1 with the highest activity and also to show very limited neutralization activity, although lower than the 50% threshold, against two of the Tier 2 pseudoviruses. This broader neutralization activity does not seem to be attributable to antibodies to the V3 domain, as serum from the 49391 rabbit, compared to the other sera, presents the weakest binding efficacy to V3 epitopes, considered both as pool or individual peptides. This observation is in contrast to previously reported data showing that broadly neutralizing activity, in sera from animals immunized with trimeric Clade A envelope molecules, is mostly associated to antibodies directed toward the envelope variable regions (V1, V2, V3) [
44]. Interestingly, our gp140
94UG018 seems to divert the immune response from variable region of envelope molecules. Further analyses will need to be performed since these results could be due to the high sequence divergence between the gp140
94UG018 and the gp120
IIIB peptides used in our assay.
Of note, all four immunized sera are characterized by a stronger binding efficacy to epitopes of the constant and V5 regions, which are known to be involved in the CD4bs of the HIV gp120 and are targets of few broadly cross-clade neutralizing monoclonal antibodies (bnAbs) [
11,
45,
46]. The poor or absent binding to C3 epitopes is most likely due to the extremely high divergence (>60%) of the C3 region sequence in the gp140
94UG018 which may severely affect the Ab-epitope recognition. Nevertheless, the strong binding to epitopes covering regions involved in the CD4bs may possibly suggest that such antibodies may play a relevant role in the neutralization activity of immunized sera, although the broadness of their activity is confined only to Tier 1 isolates. Indeed, the observed strong binding to C1 epitopes may possibly play a role in the insufficient pattern of neutralization, given that antibodies binding to C1-C4 domains have been reported to compete with CD4bs broadly cross-neutralizing antibodies for binding [
47]. Moreover, Abs specific to the C1 region have been reported to be associated with induction of antibody-dependent cellular cytotoxicity (ADCC) [
48], which plays a role in protection from HIV infection and disease progression, as shown also in the RV144 Thai vaccine trial [
49‐
51]. Overall, the results of the present study highlight that trimeric clade A gp140
94UG018 is a very effective immunogen capable of inducing significant cross-clade humoral immune responses in the rabbit model. The gp120 epitope mapping provides potential relevant insights to clarify the neutralization activity of the elicited immune sera confined to Tier 1 isolates. According to these observations, possible structural modifications of the clade A gp140
94UG018 can be envisaged (i.e. deletion of the C1 region) to improve the breadth of the neutralization activity.
Our results provide a rationale for the design and evaluation of immunogens to be used in HIV vaccine strategies. In particular, clade A gp14094UG018 shows promising characteristics for potential involvement in an effective HIV immunization regimen.
Competing interests
The authors declare that they have no competing interests of either financial or non-financial nature regarding the work described in the present manuscript and its publication.
Authors’ contributions
All authors conceived and designed the experiments. Performed the experiments: MLV, MT, LH, MJ, BG. Analyzed the data: GV, AF, GS, LB. Contributed reagents/materials/analysis tools: GS-J, MR. Wrote the paper: MLV, MT, LB. All authors complemented and approved the final version of the manuscript.