Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1

Abstract

The transmembrane envelope (TM) protein gp41 of the human immunodeficiency virus—1 (HIV-1) plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb) directed against the membrane proximal external region (MPER) suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in HIV-1 immunopathogenesis through modulation of the cytokine release and active inhibition of immune responses.

Human Endogenous Retroviruses and Their Putative Role in the Development of Autoimmune Disorders Such as Multiple Sclerosis

“…Retroviral envelope proteins are hypothesized to both trigger and suppress an immune response. In this context, a peptide of 14 amino acids (LQARILAVERYLKD) located in the transmembrane (TM) glycoprotein gp41 of HIV-1 inhibits mitogen-induced and lymphokine-dependent T-lymphocyte proliferation (Denner et al, 1994 ; Mühle et al, 2017 ). It is also able to modulate cytokine levels as it increases IL-6 and IL-10 and decreases IL-2 and CXCL9 expression in human peripheral blood mononuclear cells (PBMCs) (Denner et al, 2013 ).…”

Absence of IL-10 production by human PBMCs co-cultivated with human cells expressing or secreting retroviral immunosuppressive domains

“…During previous experiments using recombinant gp41 produced in human cells and including methods to remove endotoxin, differences in the induction of cytokines by gp41 and LPS have been observed. For example proteins encoded by the genes serpine E1, IL1Ra, uPAR, PDGF-AA, thrombospondin-1, myeoloperoxidase and osteopontin have been induced by gp41 containing the ISU domain, but not by the LPS used [ 31 ]. However, under certain conditions these proteins have also been induced by defined LPS batches [ 45 – 51 ].…”

“…For example, they caused an increase of IL-10 and had an inhibitory effect on protein kinase C (PKC) [ 14 , 18 – 26 ]. Recombinant gp41 produced in bacteria [ 27 – 29 ] or human cells [ 30 , 31 ] also modulated cytokine expression of PBMCs from healthy donors. Single mutations in the ISU domain abrogated the ability of retroviral ISU domains to cause IL-10 release and to modulate gene expression [ 30 ].…”

“…Working with recombinant retroviral proteins produced in bacteria bears the risk that they are contaminated with endotoxin. Endotoxin induces similar, but not identical changes in the cytokine release by human PBMCs [ 31 ]. In addition, production of TM proteins in human cells and their release is not very effective and requires concentration of large amounts of culture fluid with the risk of endotoxin contamination.…”

The Hard Way towards an Antibody-Based HIV-1 Env Vaccine: Lessons from Other Viruses

“…If these vectors encode at the same time for soluble Env, the immune system will receive a continous boost immunization intended to augment induction of bnAbs. In vivo studies have to show if this approach is feasible for HIV-1 Env antigens or if Env-intrinsic properties like immunosuppressive domains present in gp41 [ 134 , 135 ] may limit the immunogenicity.…”