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

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

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

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

Functional Analysis in Mouse Embryonic Stem Cells Reveals Wild-Type Activity for Three <i>Msh6</i> Variants Found in Suspected Lynch Syndrome Patients

<div><p>Lynch syndrome confers an increased risk to various types of cancer, in particular early onset colorectal and endometrial cancer. Mutations in mismatch repair (MMR) genes underlie Lynch syndrome, with the majority of mutations found in <i>MLH1</i> and <i>MSH2</i>. Mutations in <i>MSH6</i> have also been found but these do not always cause a clear cancer predisposition phenotype and <i>MSH6</i>-defective tumors often do not show the standard characteristics of MMR deficiency, such as microsatellite instability. In particular, the consequences of <i>MSH6</i> missense mutations are challenging to predict, which further complicates genetic counseling. We have previously developed a method for functional characterization of <i>MSH2</i> missense mutations of unknown significance. This method is based on endogenous gene modification in mouse embryonic stem cells using oligonucleotide-directed gene targeting, followed by a series of functional assays addressing the MMR functions. Here we have adapted this method for the characterization of <i>MSH6</i> missense mutations. We recreated three <i>MSH6</i> variants found in suspected Lynch syndrome families, MSH6-P1087R, MSH6-R1095H and MSH6-L1354Q, and found all three to behave like wild type MSH6. Thus, despite suspicion for pathogenicity from clinical observations, our approach indicates these variants are not disease causing. This has important implications for counseling of mutation carriers.</p> </div

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

Generation of <i>Msh6</i>^<i>mut/-</i> heterozygous ESC lines.

<p>(<b>A</b>) Southern blot analysis of the <i>Msh6</i>^<i>mut/-</i> and control cell lines, showing loss of one of the <i>Msh6</i> alleles in <i>Msh6</i>^<i>mut/-</i> cells. (<b>B</b> and <b>C</b>) Western blot analysis of mutant <i>Msh6</i> homozygous and heterozygous cell lines and controls. Whole cell lysates were analyzed for the presence of MSH6 and MSH2. γ-tubulin was used as a loading control. ‘-’ indicates a knockout allele. The relative percentages of MSH6 levels are indicated.</p

Functional analysis of <i>Msh6</i>^<i>mut/-</i> heterozygous ESC lines.

<p>(<b>A</b>) Black bars show the average percentage of unstable microsatellites (left Y-axis) as measured in 96 colonies for two different mononucleotide markers. Error bars show standard errors, measured over three independent clones per cell line. Grey bars show the average number of 6-TG resistant colonies per 10⁶ plated cells (right Y-axis). Error bars show standard errors, measured over three independent clones per cell line. (<b>B</b>) Survival of mutant and control cell lines exposed to MNNG. Error bars show standard errors from three independent experiments.</p

Generation of homozygous <i>Msh6</i> mutant ESC lines.

<p>Sequence analysis of (<b>A</b>) <i>Msh6</i>^<i>+/+</i>, <i>Msh6</i>^<i>PR/+</i> and <i>Msh6</i>^<i>PR/PR</i> genomic DNA, (<b>B</b>) <i>Msh6</i>^<i>+/+</i>, <i>Msh6</i>^<i>RH/+</i> and <i>Msh6</i>^<i>RH/RH</i> genomic DNA and (<b>C</b>) <i>Msh6</i>^<i>+/+</i>, <i>Msh6</i>^<i>LQ/+</i> and <i>Msh6</i>^<i>LQ/LQ</i> genomic DNA. Single letter amino acid codes are given below the sequence. (<b>D</b>) Whole cell lysates were analyzed for MSH6 and MSH2. γ-Tubulin was used as a loading control. ‘-‘ indicates a knockout allele. The relative percentages of MSH6 levels are indicated.</p

Functional analysis of <i>Msh6</i>^{<i>mut/mut</i>} ESC lines.

<p>(<b>A</b>) Black bars show the average percentage of unstable microsatellites (left Y-axis) as measured in 96 colonies for two or three different dinucleotide markers. Error bars show standard errors, measured over two to six independent clones per cell line. The grey bars show the average number of 6-TG-resistant colonies per 10⁶ plated cells (right Y-axis). Error bars show standard errors, measured over three to six independent clones per cell line. (<b>B</b>) Targeting efficiencies are shown in mutant and control cell lines for the 100% homologous (black bars) and the 99.4% homologous (white bars) <i>Rb</i> targeting constructs. Targeting efficiencies in <i>Msh2</i>^<i>+/+</i><i>Msh2</i>^<i>-/-</i> and <i>Msh6</i>^<i>-/-</i> ESCs are taken from de Wind et al. [10,18] and shown as controls. (<b>C</b>) Survival of mutant and control cell lines exposed to MNNG (n=2-6). (<b>D</b>) Survival of mutant and control cell lines exposed to 6-TG (n=2-5). Error bars show standard errors from independent experiments.</p