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

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

Schematics and expression of Env_GM-CSF glycan mutants.

<p>Linear (A) and cartoon (B) presentation of Env_GM-CSF-N37Q, Env_GM-CSFN47Q and Env_{GM-CSF-N37Q-N47Q} variants. C. Expression of chimeric Env_GM-CSF-N37Q, Env_GM-CSF-N47Q, Env_{GM-CSF-N37Q-N47Q} proteins.</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