Enhanced Immunogenicity of HIV-1 Envelope gp140 Proteins Fused to APRIL

<div><p>Current HIV-1 vaccines based on the HIV-1 envelope glycoprotein spike (Env), the only relevant target for broadly neutralizing antibodies, are unable to induce protective immunity. Env immunogenicity can be enhanced by fusion to costimulatory molecules involved in B cell activation, such as APRIL and CD40L. Here, we found that Env-APRIL signaled through the two receptors, BCMA and TACI. In rabbits, Env-APRIL induced significantly higher antibody responses against Env compared to unconjugated Env, while the antibody responses against the APRIL component were negligible. To extend this finding, we tested Env-APRIL in mice and found minimal antibody responses against APRIL. Furthermore, Env-CD40L did not induce significant anti-CD40L responses. Thus, in contrast to the 4-helix cytokines IL-21 and GM-CSF, the TNF-superfamily members CD40L and APRIL induced negligible autoantibodies. This study confirms and extends previous work and shows that fusion of Env-based immunogens to APRIL can improve Env immunogenicity and might help in designing HIV vaccines that induce protective humoral immunity.</p></div

50% neutralization titers of sera from rabbits immunized with Env_wt and Env_rAPRIL.

<p>The categorization in neutralization sensitive viruses (tier 1 viruses) and neutralization resistant viruses (tier 2 viruses) is based on reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107683#pone.0107683-Seaman1" target="_blank">[51]</a>.</p><p>50% neutralization titers of sera from rabbits immunized with Env_wt and Env_rAPRIL.</p

Env_APRIL and Env_CD40L induce minimal anti-APRIL and anti-CD40L responses in mice and rabbits.

<p>(A) Reducing SDS-PAGE analysis of Env_wt, hAPRIL, rAPRIL and rCD40L proteins followed by western blot using an MAb against the His-tag. The migration of marker proteins is indicated. Env_wtmigrated at the expected apparent m.wt. of 140 kD, while the migration pattern of hAPRIL, rAPRIL and rCD40L was as expected based on their size of ∼17 kD. (B) Detection of hAPRIL by ELISA using Ab Aprily-5. (C) Midpoint titers of anti-rAPRIL and anti-gp140 Abs (left panel) and endpoint titers of anti-rAPRIL Abs (right panel) of week 10 sera from rabbits immunized with Env_wt or Env_rAPRIL (n = 12). (D) Midpoint titers of anti-rCD40L and anti-gp140 Abs (left panel) and endpoint titers of anti-rCD40L Abs (right panel) of week 12 sera from rabbits immunized with Env_wt or Env_rCD40L (n = 4). These 8 rabbits are described in reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107683#pone.0107683-Melchers2" target="_blank">[13]</a>. (E, F) Midpoint titers of anti-mAPRIL (E), anti-mCD40L (F) and anti-gp140 Abs (left panels) and endpoint titers of anti-mAPRIL (E), anti-mCD40L Abs (F) (right panels) of week 8 sera from mice immunized with Env_wt, Env_mAPRIL, Env_mCD40L (n = 4). The mice were from the study described in reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107683#pone.0107683-vanMontfort1" target="_blank">[18]</a>. All sera were tested in duplicate with the mean values shown.</p

Schematics, expression and activity of Env_APRIL.

<p>(A) Linear and (B) cartoon representation of Env_wt, Env_hAPRIL and Env_rAPRIL. The colors for hAPRIL (blue) and rAPRIL (green) are also used in the following figures. (C) The Env_wt, Env_hAPRIL and Env_rAPRIL proteins were expressed transiently in 293T cells and analyzed by reducing SDS-PAGE followed by western blotting using MAb PA1. The migration of marker proteins is indicated. Env_wt migrated at the expected apparent m.wt. of 140 kD, while Env_hAPRIL and Env_rAPRIL migrated slightly slower because of the addition of APRIL (∼17 kD). (D) Binding of Env_wt, Env_hAPRIL, Env_rAPRIL and mock supernatants to human BCMA-Fc and TACI-Fc by ELISA. (E) Signaling induced by Env_hAPRIL, Env_rAPRIL and controls in BCMA:Fas and TACI:Fas reporter cells as measured by a reduction of cell survival. Each condition was tested in duplicate and the results shown are representative for three independent experiments using proteins derived from three independent transfections.</p

Env_rAPRIL induces an enhanced Env-specific antibody response in rabbits.

<p>(A) Immunization scheme. Two groups (n = 12) of rabbits were immunized via gene gun immunization, one group receiving Env_wt and the other Env_rAPRIL. (B) Midpoint anti-gp120 IgG titers at week 6, 8, 10 and 12 as determined by ELISA. All sera were tested in duplicate with the mean values shown.*: p<0.05; **: p<0.01 (one-tailed Mann-Whitney test). (C) Total IgG, IgA and IgM midpoint titers in week 10 sera of rabbits immunized with Env_wt and Env_APRIL.</p

Schematics and expression of the Env_wt, Env_GM-CSF and truncated Env_GM-CSF variants Linear (A) and cartoon (B) representation of the original and truncated Env_GM-CSF proteins.

<p>The clade B JRFL gp140 protein (amino acids 31 to 681) contains several modifications for stabilization that have been previously described (see Materials and Methods). Codon optimized sequences encoding human GM-CSF1 (amino acids 13 to 118), GM-CSF2 (amino acids 19 to 114), and GM-CSF3 (amino acids 33 to 112) were inserted to the V1V2 domain of gp140. Env subdomains are indicated: 5 conserved domains (C1–C5); 5 variable domains (V1–V5); heptad repeats 1 and 2 (HR1, HR2); the trimerization domain (IZ) and the histidine tag, comprised of 8 histidine amino acids (HIS). The glycan assignments in Env are based on previous studies using gp120 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060126#pone.0060126-Cutalo1" target="_blank">[60]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060126#pone.0060126-Zhu1" target="_blank">[62]</a>. The composition of GM-CSF <i>N</i>-glycans is reported in ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060126#pone.0060126-Forno1" target="_blank">[63]</a>. C. Chimeric Env_GMCSF1/2/3 proteins expressed transiently in 293T cells were analyzed in reducing SDS-PAGE analysis followed by western blot.</p

Schematics and expression of truncated and cysteine modified Env_GM-CSF variants.

<p>Linear (A) and cartoon (B) rendering of the Env_GM-CSF and Env_{GM-CSF1/2/3-C88S} constructs. C. The resulting Env_GM-CSF1-C88S, Env_GM-CSF2-C88S, Env_GM-CSF3-C88S chimeric glycoproteins expressed transiently in 293T cells, were analyzed in reducing SDS-PAGE analysis followed by western blot.</p

The composition of the V1V2 domain of HIV-1 gp120 modulates the transition between the unliganded and CD4-bound states.

<p>Cartoon showing the CD4-induced conformational changes in Env and Env_GM-CSF variants. The thickness of the arrows represents direction of the equilibrium. A. Upon CD4 receptor binding wild type Env undergoes conformational changes that involve rearrangement of the V1V2 domain and expose the CD4i epitopes. B. Env lacking the V1V2 domain (Env_ΔV1V2) folds into the CD4-bound state and exposes CD4i epitopes constitutively. C. When the V1V2 domain is replaced with GM-CSF, the CD4i antibodies cannot bind, irrespective of the presence of CD4, indicating that intrinsic properties of the V1V2 allow the CD4-induced changes. D. Replacement of GM-CSF with smaller GM-CSF variants that were more similar in size to the V1V2, did not improve the exposure of the CD4i, suggesting that the size of the V1V2 is not a critical factor for allowing CD4-induced conformational changes to occur. E. The addition of flexible linkers between the Env and GM-CSF domains slightly improved the binding of CD4-IgG2 and CD4i antibodies.</p

Antigenicity and activity of Env_GM-CSF glycan variants. Recognition of Chimeric Env_GMCSF-N37Q, Env_GM-CSF-N47Q, Env_{GM-CSF-N37Q-N47Q} by HIV-Ig and 2G12 (A); b12 and CD4-IgG2 (B); and 48d (CD4i) in the absence and presence of sCD4 (C).

<p>Culture supernatant from mock transfected 293T cells was used as a negative control. D. Recognition of Env_GMCSF-N37Q, Env_GM-CSF-N47Q, Env_{GM-CSF-N37Q-N47Q} by the GMCSF antibody (clone 30-4). All ELISA results are representative for at least three independent experiments using proteins derived from three independent transfections. E. Survival of TF-1 cells in response to culture supernatant containing Env_GM-CSF, Env_GM-CSF variants or controls.</p

Chimeric Env_GM-CSF variants activate primary human monocytes.

<p>Monocytes were stimulated with chimeric Env_GM-CSF proteins and control and analyzed after 5 days for macrophage-like properties: (A) Percentages of living cells with macrophage-like morphology as analyzed by the SSC vs. FSC in flow cytometry. Data represents the mean of experiments with cells from 2 different donors. Expression of maturation markers CD206 and CD64 on cells incubated with (B) mock (gray line), rhGM-CSF (black line), (C) uncleaved Env (light gray histogram), Env_GM-CSF (black filled histogram) and Env_{GM-CSF-N1N2-C1C2} (gray filled histogram), (D) cleaved Env (light gray filled histogram), cleaved Env_GM-CSF (black filled histogram) and cleaved Env_{GM-CSF-N1N2-C1C2} (gray filled histogram). Fluorescence intensity (x-axis) is plotted against the percent of Max (y-axis), where the maximum y-axis value in absolute count becomes 100% of total. Data are representative of 5 independent experiments showing similar results. E. Percentages of Macrophage-like cells derived from monocytes deprived of human pooled serum and cultured with fetal calf serum. Data represents the mean of experiments with cells from 3 different donors.</p