HIV-1 envelope glycoproteins with costimulatory domains for vaccine applications

Various immuno-evasive properties of the HIV-1 Env subunit limit the value of traditional vaccine strategies and urge us to explore different vaccine approaches. We have been working on a vaccine strategy in which we designed dual-functional fusion molecules of trimeric HIV-1 Env with a costimulatory molecule. First, we generated Env-GM-CSF trimers by replacing the V1V2 domain of Env with GM-CSF (chapter 2). This construct was further optimized in the study described in chapter 3. Another fusion molecule, Env-IL-21, was generated using a similar design strategy by replacement of the V1V2 domain with IL-21 and its activity was assessed in vitro (chapter 4). Next we studied both Env-GM-CSF and Env-IL-21 trimers in vivo by immunizing rabbits and mice (chapter 5). We found that both molecules induced a potent immune response against the cytokine inserts, irrespective of their location in the fusion protein. In chapter 6 we describe in vitro and in vivo studies on the bioactivity of Env trimers fused to APRIL. Furthermore, we describe a strategy to immunosilence this trimerization domain by covering its protein surface with glycans. Finally, in the last research chapter (chapter 8), we report on the in vitro and in vivo activity of APRIL fused to a next generation native-like Env trimer mimetic, BG505 SOSIP.664 gp140. We note that all our immunization were done with plasmids encoding Env-costimulatory fusion molecules. Different immunization approach may lead to different effects

Immunosilencing a highly immunogenic protein trimerization domain

Many therapeutic proteins and protein subunit vaccines contain heterologous trimerization domains, such as the widely used GCN4-based isoleucine zipper (IZ) and the T4 bacteriophage fibritin foldon (Fd) trimerization domains. We found that these domains induced potent anti-IZ or anti-Fd antibody responses in animals when fused to an HIV-1 envelope glycoprotein (Env) immunogen. To dampen IZ-induced responses, we constructed an IZ domain containing four N-linked glycans (IZN4) to shield the underlying protein surface. When fused to two different vaccine antigens, HIV-1 Env and influenza hemagglutinin (HA), IZN4 strongly reduced the antibody responses against the IZ, but did not affect the antibody titers against Env or HA. Silencing of immunogenic multimerization domains with glycans might be relevant for therapeutic proteins and protein vaccine

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

Autoantibodies induced by chimeric cytokine-HIV envelope glycoprotein immunogens

Cytokines are often used as adjuvants to increase the immunogenicity of vaccines because they can improve the immune response and/or direct it into a desired direction. As an alternative to codelivering Ags and cytokines separately, they can be fused into a composite protein, with the advantage that both moieties act on the same immune cells. The HIV-1 envelope glycoprotein (Env) spike, located on the outside of virus particles and the only relevant protein for the induction of neutralizing Abs, is poorly immunogenic. The induction of anti-Env Abs can be improved by coupling Env proteins to costimulatory molecules such as a proliferation inducing ligand (APRIL). In this study, we evaluated the immunogenicity of chimeric molecules containing uncleaved Env gp140 fused to the species-matched cytokines IL-21 or GM-CSF in rabbits and mice. Each cytokine was either fused to the C terminus of Env or embedded within Env at the position of the variable loops 1 and 2. The cytokine components of the chimeric Env-GM-CSF and Env-IL-21 molecules were functional in vitro, but none of the Env-cytokine fusion proteins resulted in improved Ab responses in vivo. Both the Env-GM-CSF and the Env-IL-21 molecules induced strong anticytokine Ab responses in both test species. These autoimmune responses were independent of the location of the cytokine in the chimeric Env molecules in that they were induced by cytokines inserted within the variable loops 1 and 2 of Env or fused to its C terminus. The induction of undesired autoimmune responses should be considered when using cytokines as costimulatory molecules in fusion protein

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

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

Chimeric HIV-1 envelope glycoproteins with potent intrinsic granulocyte-macrophage colony-stimulating factor (GM-CSF) activity

HIV-1 acquisition can be prevented by broadly neutralizing antibodies (BrNAbs) that target the envelope glycoprotein complex (Env). An ideal vaccine should therefore be able to induce BrNAbs that can provide immunity over a prolonged period of time, but the low intrinsic immunogenicity of HIV-1 Env makes the elicitation of such BrNAbs challenging. Co-stimulatory molecules can increase the immunogenicity of Env and we have engineered a soluble chimeric Env trimer with an embedded granulocyte-macrophage colony-stimulating factor (GM-CSF) domain. This chimeric molecule induced enhanced B and helper T cell responses in mice compared to Env without GM-CSF. We studied whether we could optimize the activity of the embedded GM-CSF as well as the antigenic structure of the Env component of the chimeric molecule. We assessed the effect of truncating GM-CSF, removing glycosylation-sites in GM-CSF, and adjusting the linker length between GM-CSF and Env. One of our designed Env(GM-CSF) chimeras improved GM-CSF-dependent cell proliferation by 6-fold, reaching the same activity as soluble recombinant GM-CSF. In addition, we incorporated GM-CSF into a cleavable Env trimer and found that insertion of GM-CSF did not compromise Env cleavage, while Env cleavage did not compromise GM-CSF activity. Importantly, these optimized Env(GM-CSF) proteins were able to differentiate human monocytes into cells with a macrophage-like phenotype. Chimeric Env(GM-CSF) should be useful for improving humoral immunity against HIV-1 and these studies should inform the design of other chimeric protein