Autoantibodies Induced by Chimeric Cytokine-HIV Envelope Glycoprotein Immunogens

Abstract

Cytokines are often used as adjuvants to increase the immunogenicity of vaccines as they can improve the immune response and/or direct it into a desired direction. As an alternative to co-delivering antigens 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 antibodies (NAbs), is poorly immunogenic. The induction of anti-Env Abs can be improved by coupling Env proteins to co-stimulatory molecules such as a proliferation inducing ligand (APRIL). Here, 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 (V1V2). 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 anti-cytokine 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 V1V2 of Env or fused to its Ct. The induction of undesired autoimmune responses should be considered when using cytokines as co-stimulatory molecules in fusion proteins.

Immunosilencing a Highly Immunogenic Protein Trimerization Domain

“…These rabbits were immunized with 30 g of Env-Fd protein in ISCOMATRIX TM adjuvant at weeks 0, 4, and 20. In previous vaccination experiments using similar protocols, five to six animals per group provided enough statistical power to detect significant differences between two groups in binding antibody measurements (35,37). Therefore, we used six New Zealand White rabbits for the experiments in Fig.…”

“…Therefore, we used six New Zealand White rabbits for the experiments in Fig. 5, A and B that were immunized in the abdominal dermis at week 0, 2, 4, 8 with 125 g endotoxin-free DNA encoding Env-IZ, Env-IZN4, HA-IZ, and Env-IZN4 using gene gun technology (35,37). All protocols dealing with animal manipulations were in accordance with guidelines published by FELASA (Federation of European Animal Science Association) and GV-SOLAS (German Society of Laboratory Animal Science) and were reviewed by the Harlan, MFD Diagnostics, or Davids Biotechnologie Animal Care Committees, as appropriate.…”

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

“…Here, we found that Env APRIL did not induce autoAbs targeting the APRIL domain in mice or rabbits and similar results were obtained with Env CD40L . These results were in stark contrast with the high anti-rGM-CSF and anti-rIL-21 responses observed in rabbits immunized with Env IL-21 and Env GM-CSF [20]. The anti-APRIL responses were>250-fold lower than those against GM-CSF and>25-fold lower than those against IL21, suggesting that the type of cytokine (trimeric TNF superfamily member vs. monomeric 4-helix bundle cytokine) influences the propensity to generate autoAbs.…”

“…We have previously reported on the strong induction of anti-cytokine responses with the chimeric proteins Env IL-21 and Env GM-CSF , which are similar in design to the Env APRIL described here [20]. Therefore, we looked for the presence of anti-APRIL Abs in sera from rabbits immunized with Env rAPRIL construct using a similar ELISA as we described previously for IL-21 and GM-CSF (see Materials and Methods and [20]).…”

“…Therefore, we looked for the presence of anti-APRIL Abs in sera from rabbits immunized with Env rAPRIL construct using a similar ELISA as we described previously for IL-21 and GM-CSF (see Materials and Methods and [20]). APRIL molecules were expressed efficiently and hAPRIL was recognized efficiently by the anti-hAPRIL Ab Aprily-5 in ELISA (Fig.…”